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The relationship of stress hypermetabolism to essential protein needs

Curator: Larry H. Bernstein, MD, FCAP

 

 

The relationship of stress hypermetabolism to essential protein needs

A Second Look at the Transthyretin Nutrition Inflammatory Conundrum

Subtitle: Transthyretin and the Systemic Inflammatory Response

Larry H. Bernstein, MD, FACP, Clinical Pathologist, Biochemist, and Transfusion Physician
President, Triplex, Trumbull, CT 06611, USA

 

Brief introduction

Transthyretin  (also known as prealbumin) has been widely used as a biomarker for identifying protein-energy malnutrition (PEM) and for monitoring the improvement of nutritional status after implementing a nutritional intervention by enteral feeding or by parenteral infusion. This has occurred because transthyretin (TTR) has a rapid removal from the circulation in 48 hours and it is readily measured by immunometric assay. Nevertheless, concerns have been raised about the use of TTR in the ICU setting, which prompted a review of the  benefit of using this test in acute and chronic care. TTR is easily followed in the underweight and the high risk populations in an ambulatory setting, which has a significant background risk of chronic diseases. It is sensitive to the systemic inflammatory response syndrome (SIRS), and needs to be understood in the context of acute illness to be used effectively. There are a number of physiologic changes associated with SIRS and the injury/repair process that affect TTR. The most important point is that in the context of an ICU setting, the contribution of TTR is significant in a complex milieu.  A much better understanding of the significance of this program has emerged from studies of nitrogen and sulfur in health and disease.

Transthyretin protein structure

Transthyretin protein structure (Photo credit: Wikipedia)

Age-standardised disability-adjusted life year...

Age-standardised disability-adjusted life year (DALY) rates from Protein-energy malnutrition by country (per 100,000 inhabitants). (Photo credit: Wikipedia)

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The systemic inflammatory response syndrome C-reactive protein and transthyretin conundrum.
Larry H Bernstein
Clin Chem Lab Med 2007; 45(11):0
ICID: 939932
Article type: Editorial

The Transthyretin Inflammatory State Conundrum
Larry H. Bernstein
Current Nutrition & Food Science, 2012, 8, 00-00

Keywords: Tranthyretin (TTR), systemic inflammatory response syndrome (SIRS), protein-energy malnutrition (PEM), C- reactive protein, cytokines, hypermetabolism, catabolism, repair.

Transthyretin has been widely used as a biomarker for identifying protein-energy malnutrition (PEM) and for monitoring the improvement of nutritional status after implementing a nutritional intervention by enteral feeding or by parenteral infusion. This has occurred because transthyretin (TTR) has a rapid removal from the circulation in 48 hours and it is readily measured by immunometric assay. Nevertheless, concerns have been raised about the use of TTR in the ICU setting, which prompts a review of the actual benefit of using this test in a number of settings. TTR is easily followed in the underweight and the high risk populations in an ambulatory setting, which has a significant background risk of chronic diseases. It is sensitive to the systemic inflammatory response syndrome (SIRS), and needs to be understood in the context of acute illness to be used effectively.

There are a number of physiologic changes associated with SIRS and the injury/repair process that affect TTR and  in the context of an ICU setting, the contribution of TTR is essential.  The only consideration is the timing of initiation since the metabolic burden is sufficiently high that a substantial elevation is expected in the first 3 days post admission, although the level of this biomarker is related to the severity of injury. Despite the complexity of the situation, TTR is not to be considered a test “for all seasons”. In the context of age, prolonged poor meal intake, chronic or acute illness, TTR needs to be viewed in a multivariable lens, along with estimated lean body mass, C-reactive protein, the absolute lymphocyte count, presence of neutrophilia, and perhaps procalcitonin if there is remaining uncertainty. Furthermore, the reduction of risk of associated complication requires a systematized approach to timely identification, communication, and implementation of a suitable treatment plan.

The most important point is that in the context of an ICU setting, the contribution of TTR is significant in a complex milieu.

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Title: The Automated Malnutrition Assessment
Accepted 29 April 2012. http://www.nutritionjrnl.com. Nutrition (2012), doi:10.1016/j.nut.2012.04.017.
Authors: Gil David, PhD; Larry Howard Bernstein, MD; Ronald R Coifman, PhD
Article Type: Original Article

Keywords: Network Algorithm; unsupervised classification; malnutrition screening; protein energy malnutrition (PEM); malnutrition risk; characteristic metric; characteristic profile; data characterization; non-linear differential diagnosis

We have proposed an automated nutritional assessment (ANA) algorithm that provides a method for malnutrition risk prediction with high accuracy and reliability.  The problem of rapidly identifying risk and severity of malnutrition is crucial for minimizing medical and surgical complications. These are not easily performed or adequately expedited. We characterized for each patient a unique profile and mapped similar patients into a classification. We also found that the laboratory parameters were sufficient for the automated risk prediction.

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Title: The Increasing Role for the Laboratory in Nutritional Assessment
Article Type: Editorial
Section/Category: Clinical Investigation
Accepted 22 May 2012. http://www.elsevier.com/locate/clinbiochem.
Clin Biochem (2012), doi:10.1016/j.clinbiochem.2012.05.024
Keywords: Protein Energy Malnutrition; Nutritional Screening; Laboratory Testing
Author: Dr. Larry Howard Bernstein, MD

The laboratory role in nutritional management of the patient has seen remarkable growth while there have been dramatic changes in technology over the last 25 years, and it is bound to be transformative in the near term. This editorial is an overview of the importance of the laboratory as an active participant in nutritional care.

The discipline emerged divergently along separate paths with unrelated knowledge domains in physiological chemistry, pathology, microbiology, immunology and blood cell recognition, and then cross-linked emerging into clinical biochemistry, hematology-oncology, infectious diseases, toxicology and therapeutics, genetics, pharmacogenomics, translational genomics and clinical diagnostics.

In reality, the more we learn about nutrition, the more we uncover of metabolic diversity of individuals, the family, and societies in adapting and living in many unique environments and the basic reactions, controls, and responses to illness. This course links metabolism to genomics and individual diversity through metabolomics, which will be enlightened by chemical and bioenergetic insights into biology and translated into laboratory profiling.

Vitamin deficiencies were discovered as clinical entities with observed features as a result of industrialization (rickets and vitamin D deficiency) and mercantile trade (scurvy and vitamin C)[2].  Advances in chemistry led to the isolation of each deficient “substance”.  In some cases, a deficiency of a vitamin and what is later known as an “endocrine hormone” later have confusing distinctions (vitamin D, and islet cell insulin).

The accurate measurement and roles of trace elements, enzymes, and pharmacologic agents was to follow within the next two decades with introduction of atomic absorption, kinetic spectrophotometers, column chromatography and gel electrophoresis.  We had fully automated laboratories by the late 1960s, and over the next ten years basic organ panels became routine.   This was a game changer.

Today child malnutrition prevalence is 7 percent of children under the age of 5 in China, 28 percent in sub-Saharan African, and 43 percent in India, while under-nutrition is found mostly in rural areas with 10 percent of villages and districts accounting for 27-28 percent of all Indian underweight children. This may not be surprising, but it is associated with stunting and wasting, and it has not receded with India’s economic growth. It might go unnoticed viewed alongside a growing concurrent problem of worldwide obesity.

The post WWII images of holocaust survivors awakened sensitivity to nutritional deprivation.

In the medical literature, Studley [HO Studley.  Percentage of weight loss. Basic Indicator of surgical risk in patients with chronic peptic ulcer.  JAMA 1936; 106(6):458-460.  doi:10.1001/jama.1936.02770060032009] reported the association between weight loss and poor surgical outcomes in 1936.  Ingenbleek et al [Y Ingenbleek, M De Vissher, PH De Nayer. Measurement of prealbumin as index of protein-calorie malnutrition. Lancet 1972; 300[7768]: 106-109] first reported that prealbumin (transthyretin, TTR) is a biomarker for malnutrition after finding very low TTR levels in African children with Kwashiorkor in 1972, which went unnoticed for years.  This coincided with the demonstration by Stanley Dudrick  [JA Sanchez, JM Daly. Stanley Dudrick, MD. A Paradigm ShiftArch Surg. 2010; 145(6):512-514] that beagle puppies fed totally through a catheter inserted into the superior vena cava grew, which method was then extended to feeding children with short gut.  Soon after Bistrian and Blackburn [BR Bistrian, GL Blackburn, E Hallowell, et al. Protein status of general surgical patients. JAMA 1974; 230:858; BR Bistrian, GL Blackburn, J Vitale, et al. Prevalence of malnutrition in general medicine patients, JAMA, 1976, 235:1567] showed that malnourished hospitalized medical and surgical patients have increased length of stay, increased morbidity, such as wound dehiscence and wound infection, and increased postoperative mortality, later supported by many studies.

Michael Meguid,MD, PhD, founding editor of Nutrition [Elsevier] held a nutrition conference “Skeleton in the Closet – 20 years later” in Los Angeles in 1995, at which a Beckman Prealbumin Roundtable was held, with Thomas Baumgartner and Michael M Meguid as key participants.  A key finding was that to realize the expected benefits of a nutritional screening and monitoring program requires laboratory support. A Ross Roundtable, chaired by Dr. Lawrence Kaplan, resulted in the first Standard of Laboratory Practice Document of the National Academy of Clinical Biochemists on the use of the clinical laboratory in nutritional support and monitoring. Mears then showed a real benefit to a laboratory interactive program in nutrition screening based on TTR [E Mears. Outcomes of continuous process improvement of a nutritional care program incorporating serum prealbumin measurements. Nutrition 1996; 12 (7/8): 479-484].

A later Ross Roundtable on Quality in Nutritional Care included a study of nutrition screening and time to dietitian intervention organized by Brugler and Di Prinzio that showed a decreased length of hospital stay with $1 million savings in the first year (which repeated), which included reduced cost for dietitian evaluations and lower complication rates.

Presentations were made at the 1st International Transthyretin Congress in Strasbourg, France by Mears [E Mears.  The role of visceral protein markers in protein calorie malnutrition. Clin Chem Lab Med 2002; 40:1360-1369] on the impact of TTR in screening for PEM in a public hospital in Louisiana, and by Potter [MA Potter, G Luxton. Prealbumin measurement as a screening tool for patients with protein calorie malnutrition in emergency hospital admissions: a pilot study.  Clin Invest Med. 1999; 22(2):44-52] that indicated a 17% in-hospital mortality rate in a Canadian hospital for patients with PCM compared with 4% without PCM (p < 0.02), while only 42% of patients with PCM received nutritional supplementation. Cost analysis of screening with prealbumin level projected a saving of $414 per patient screened.  Ingenbleek and Young [Y Ingenbleek, VR Young.  Significance of transthyretin in protein metabolism.  Clin Chem Lab Med. 2002; 40(12):1281–1291.  ISSN (Print) 1434-6621, DOI: 10.1515/ CCLM.2002.222, December 2002. published online: 01/06/2005] tied the TTR to basic effects reflected in protein metabolism.

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Transthyretin as a marker to predict outcome in critically ill patients.
Arun Devakonda, Liziamma George, Suhail Raoof, Adebayo Esan, Anthony Saleh, Larry H Bernstein
Clin Biochem 2008; 41(14-15):1126-1130
ICID: 939927
Article type: Original article

TTR levels correlate with patient outcomes and are an accurate predictor of patient recovery in non-critically ill patients, but it is uncertain whether or not TTR level correlates with level of nutrition support and outcome in critically ill patients. This issue has been addressed only in critically ill patients on total parenteral nutrition and there was no association reported with standard outcome measures. We revisit this in all patients admitted to a medical intensive care unit.

Serum TTR was measured on the day of admission, day 3 and day 7 of their ICU stay. APACHE II and SOFA score was assessed on the day of admission. A registered dietician for their entire ICU stay assessed the nutritional status and nutritional requirement. Patients were divided into three groups based on initial TTR level and the outcome analysis was performed for APACHE II score, SOFA score, ICU length of stay, hospital length of stay, and mortality.

TTR showed excellent concordance with the univariate or multivariate classification of patients with PEM or at high malnutrition risk, and followed for seven days in the ICU, it is a measure of the metabolic burden.  TTR levels decline from day 1 to day 7 in spite of providing nutritional support. Twenty-five patients had an initial TTR serum concentration more than 17 mg/dL (group 1), forty-eight patients had mild malnutrition with a concentration between 10 and 17 mg/dL (group 2), Forty-five patients had severe malnutrition with a concentration less than 10 mg/dL (group 3).  Initial TTR level had inverse correlation with ICU length of stay, hospital length of stay, and APACHE II score, SOFA score; and predicted mortality, especially in group 3.

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A simplified nutrition screen for hospitalized patients using readily available laboratory and patient
information.
Linda Brugler, Ana K Stankovic, Madeleine Schlefer, Larry Bernstein
Nutrition 2005; 21(6):650-658
ICID: 825623
Article type: Review article
The role of visceral protein markers in protein calorie malnutrition.
Linda Brugler, Ana Stankovic, Larry Bernstein, Frederick Scott, Julie O’Sullivan-Maillet
Clin Chem Lab Med 2002; 40(12):1360-1369
ICID: 636207
Article type: Original article

The Automated Nutrition Score is a data-driven extension of continuous quality improvement.

Larry H Bernstein
Nutrition 2009; 25(3):316-317
ICID: 939934

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Transthyretin: its response to malnutrition and stress injury. clinical usefulness and economic implications.
LH Bernstein, Y Ingenbleek
Clin Chem Lab Med 2002; 40(12):1344-1348
ICID: 636205
Article type: Original article

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THE NUTRITIONALLY-DEPENDENT ADAPTIVE DICHOTOMY (NDAD) AND STRESS HYPERMETABOLISM
Yves Ingenbleek  MD  PhD  and  Larry Bernstein MD
J CLIN LIGAND ASSAY  (out of print)

The acute reaction to stress is characterized by major metabolic, endocrine and immune alterations. According to classical descriptions, these changes clinically present as a succession of 3 adaptive steps – ebb phase, catabolic flow phase, and anabolic flow phase. The ebb phase, shock and resuscitation, is immediate, lasts several hours, and is characterized by hypokinesis, hypothermia, hemodynamic instability and reduced basal metabolic rate. The catabolic flow phase, beginning within 24 hours and lasting several days, is characterized by catabolism with the flow of gluconeogenic substrates and ketone bodies in response to the acute injury. The magnitude of the response depends on the acuity and the severity of the stress. The last, a reparative anabolic flow phase, lasts weeks and is characterized by the accretion of amino acids (AAs) to rebuilding lean body mass.

The current opinion is that the body economy is reset during the course of stress at novel thresholds of metabolic priorities. This is exemplified mainly by proteolysis of muscle, by an effect on proliferating gut mucosa and lymphoid tissue as substrates are channeled to support wound healing, by altered syntheses of liver proteins with preferential production of acute phase proteins (APPs) and local repair in inflamed tissues (3). The first two stages demonstrate body protein breakdown exceeding the rate of protein synthesis, resulting in a negative nitrogen (N) balance, muscle wasting and weight loss. In contrast, the last stage displays reversed patterns, implying progressive recovery of endogenous N pools and body weight.

These adaptive alterations undergo continuing elucidation. The identification of cytokines, secreted by activated macrophages/monocytes or other reacting cells, has provided further insights into the molecular mechanisms controlling energy expenditure, redistribution of protein pools, reprioritization of syntheses and secretory processes.

The free fraction of hormones bound to specific binding-protein(s) [BP(s)] manifests biological activities, and any change in the BP blood level modifies the effect of the hormone on the end target organ.  The efficacy of these adaptive responses may be severely impaired in protein-energy malnourished (PEM) patients. This is especially critical with respect to changes of the circulating levels of transthyretin (TTR), retinol-binding protein (RBP) and corticosteroid-binding globulin (CBG) conveying thyroid hormones (TH), retinol and cortisol, respectively.  This reaction is characterized by cytokine mediated autocrine, paracrine and endocrine changes. Among the many inducing molecules identified, interleukins 1 and 6 (Il-1, Il-6) and tumor necrosis factor a (TNF) are associated with enhanced production of 3 counterregulatory hormonal families (cortisol, catecholamines and glucagon). Growth hormone (GH) and TH also have roles in these metabolic adjustments.

There is overproduction of cortisol mediated by several cytokines acting on both the adrenal cortex (10) and on the pituitary through hypothalamic CRH with loss of feedback regulation of ACTH production (11). Hypercortisolemia is a major finding observed after surgery (12), sepsis (13), and medical insults, usually correlated with severity of insult and of complications. Rising cortisol values parallel hyperglycemic trends, as an effect of both gluconeogenesis and insulin resistance. Working in concert with TNF, glucocorticoids govern the breakdown of muscle mass, which is regarded as the main factor responsible for the negative N balance.

Under normal conditions, GH exerts both lipolytic and anabolic influences in the whole body economy under the dual control of the hypothalamic hormones somatocrinin (GHRH) and somatostatin (SRIH). GH secretion is usually depressed by rising blood concentrations of glucose and free fatty acids (FFAs) but is paradoxicaly elevated despite hyperglycemia in stressed patients.

The oversecretion of counterregulatory hormones working in concert generates subtle equilibria between glycogenolytic/glycolytic/gluconeogenic adaptive processes. The net result is the neutralization of the main hypoglycemic and anabolic activities of insulin and the development of a persisting and controlled hyperglycemic tone in the stressed body. The molecular mechanisms whereby insulin resistance occurs in the course of stress refer to
cytokine-  and  hormone-induced  phosphorylation abnormalities affecting receptor signaling. The insulin-like anabolic processes of GH are mediated by IGF1 working as relay agent. The expected high IGF1 surge associated with GH oversecretion is not observed in severe stress as plasma values are usually found at the lower limit of normal or even in the subnormal range.  The end result of this dissociation between high GH and low IGF1 levels is to favor the proteolysis of muscle mass to release AAs for gluconeogenesis and the breakdown of adipose tissue to provide ketogenic substrates.

The acute stage of stress is associated with the onset of a low T3 syndrome typically delineated by the drop of both total (TT3) and free (FT3) triiodothyronine plasma levels in the subnormal range. In contrast, both total (TT4) and free (FT4) thyroxine values usually remain within normal ranges with declining trends observed for TT4 and rising tendencies for FT4 (44). This last free compound is regarded as the sensor reflecting the actual thyroid status and governing the release of TSH whereas FT3 works as the active hormonal mediator at nuclear receptor level. The maintenance of an euthyroid sick syndrome is compatible with the down-regulation of most metabolic and energetic processes in healthy tissues. These inhibitory effects , negatively affecting all functional steps of the hypothalamo-pituitary-thyroid axis concern TSH production, iodide uptake, transport and organification into iodotyrosyl residues, peroxidase coupling activity as well as thyroglobulin synthesis and TH leakage. Taken together, the above-mentioned data indicate that the development of hyperglycemia and of insulin-resistance in healthy tissues – mainly in the muscle mass – are hallmarks resulting from the coordinated activities of the counterregulatory hormones.

A growing body of recent data suggest that the stressed territory, whatever the causal agent – bacterial or viral sepsis, auto-immune disorder, traumatic or toxic shock, burns, cancer – manifest differentiated metabolic and immune reactions. The amplitude, duration and efficacy of these responses are reportedly impaired along several ways in PEM patients. These last detrimental effects are accompanied by a number of medical, social and economical consequences, such as extended length of hospital stay and increased complication / mortality rates. It is therefore mandatory to correctly identify and follow up the nutritional status of hospitalized patients. Such approaches are prerequisite to timely and scientifically grounded nutritional and pharmacological mediated interventions.

Contrary to the rest of the body, energy requirements of the inflamed territory are primarily fulfilled by anaerobic glycolysis, an effect triggered by the inhibition of key-enzymes of carbohydrate metabolism, notably pyruvate-dehydrogenase. This non-oxidative combustion of glucose reveals low conversion efficiency but offers the major advantage to maintain, in the context of hyperglycemia, fuel provision to poorly irrigated and/or edematous tissues. The depression of the 5’-monodeiodinating activity (5’-DA) plays a pivotal role in these adaptive changes, yielding inactive reverse T3 (rT3) as index of impaired T4 to T3 conversion rates, but at the same time there is an augmented supply of bioactive T3 molecules and local overstimulation of thyro-dependent processes characterized by thyroid down-regulation.  The same differentiated evolutionary pattern applies to IGF1. In spite of lowered plasma total concentrations, the proportion of IGF1 released in free form may be substantially increased owing to the proteolytic degradation of IGFBP-3 in the intravascular compartment. The digestion of  BP-3 results from the surge of several proteases occurring the course of stress, yielding biologically active IGF1 molecules available for the repair of damaged tissues. In contrast, healthy receptors oppose a strong resistance to IGF1 ligands freed in the general circulation, likely induced by an acquired phosphorylation defect very similar in nature to that for the insulin transduction pathway.

PEM is the generic denomination of a broad spectrum of nutritional disorders, commonly found in hospital settings, and whose extreme poles are identified as marasmus and kwashiorkor. The former condition is usually regarded as the result of long-lasting starvation leading to the loss of lean body mass and fat reserves but relatively well-preserved liver function and immune capacities. The latter condition is typically the consequence of (sub)acute deprivation predominantly affecting the protein content of staplefood, an imbalance causing hepatic steatosis, fall of visceral proteins, edema and increased vulnerability to most stressful factors. PEM may be hypometabolic or hypermetabolic, usually coexists with other diseased states and is frequently associated with complications. Identification of PEM calls upon a large set of clinical and analytical disciplines comprising anthropometry, immunology, hematology and biochemistry.

CBG, TTR and RBP share in common the transport of specific ligands exerting their metabolic effects at nuclear receptor level. Released from their specific BPs in free form, cortisol, FT4 and retinol immediately participe to the strenghtening of the positive and negative responses to stressful stimuli. CBG is a relatively weak responder to short-term nutritional influences (73)  although long-lasting PEM is reportedly capable of causing its significant diminution (74). The dramatic drop of CBG in the course of stress appears as the combined effect of Il-6-induced posttranscriptional blockade of its liver synthesis (75) and peripheral overconsumption by activated neutrophils (61). The divergent alterations outlined by CBG and total cortisolemia result in an increased disposal of free ligand reaching proportions considerably higher than the 4 % recorded under physiological conditions.

The appellation of negative APPs that was once given to the visceral group of carrier-proteins. The NDAD concept takes the opposite view, defending the opinion that their suppressed synthesis releases free ligands which positively contribute to strengthen all aspects of the stress reaction, justifying the ABR denomination. This implies that the role played by ABRs should no longer be interpreted in terms of concentrations but in terms of functionality.

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THE OXIDATIVE STRESS OF HYPERHOMOCYSTEINEMIA RESULTS FROM REDUCED BIOAVAILABILITY OF SULFUR-CONTAINING REDUCTANTS.
Yves Ingenbleek. The Open Clinical Chemistry Journal, 2011, 4, 34-44.

Vegetarian subjects consuming subnormal amounts of methionine (Met) are characterized by subclinical protein malnutrition causing reduction in size of their lean body mass (LBM) best identified by the serial measurement of plasma transthyretin (TTR). As a result, the transsulfuration pathway is depressed at cystathionine-β-synthase (CβS) level triggering the upstream sequestration of homocysteine (Hcy) in biological fluids and promoting its conversion to Met. Maintenance of beneficial Met homeostasis is counterpoised by the drop of cysteine (Cys) and glutathione (GSH) values downstream to CβS causing in turn declining generation of hydrogen sulfide (H2S) from enzymatic sources. The biogenesis of H2S via non-enzymatic reduction is further inhibited in areas where earth’s crust is depleted in elemental sulfur (S8) and sulfate oxyanions. Combination of subclinical malnutrition and S8-deficiency thus maximizes the defective production of Cys, GSH and H2S reductants, explaining persistence of unabated oxidative burden. The clinical entity increases the risk of developing cardiovascular diseases (CVD) and stroke in underprivileged plant-eating populations regardless of Framingham criteria and vitamin-B status. Although unrecognized up to now, the nutritional disorder is one of the commonest worldwide, reaching top prevalence in populated regions of Southeastern Asia. Increased risk of hyperhomocysteinemia and oxidative stress may also affect individuals suffering from intestinal malabsorption or westernized communities having adopted vegan dietary lifestyles.

Metabolic pathways: Met molecules supplied by dietary proteins are submitted to TM processes allowing to release Hcy which may in turn either undergo Hcy – Met RM pathways or be irreversibly committed into TS decay. Impairment of CbS activity, as described in protein malnutrition, entails supranormal accumulation of Hcy in body fluids, stimulation of activity and maintenance of Met homeostasis. This last beneficial effect is counteracted by decreased concentration of most components generated downstream to CbS, explaining the depressed CbS- and CbL-mediated enzymatic production of H2S along the TS cascade. The restricted dietary intake of elemental S further operates as a limiting factor for its non-enzymatic reduction to H2S which contributes to downsizing a common body pool. Combined protein- and S-deficiencies work in concert to deplete Cys, GSH and H2S from their body reserves, hence impeding these reducing molecules to properly face the oxidative stress imposed by hyperhomocysteinemia.

see also …

McCully, K.S. Vascular pathology of homocysteinemia: implications for the pathogenesis of arteriosclerosis. Am. J. Pathol., 1996, 56, 111-128.

Cheng, Z.; Yang, X.; Wang, H. Hyperhomocysteinemia and endothelial dysfunction. Curr. Hypertens. Rev., 2009, 5,158-165.

Loscalzo, J. The oxidant stress of hyperhomocyst(e)inemia. J. Clin.Invest., 1996, 98, 5-7.

Ingenbleek, Y.; Hardillier, E.; Jung, L. Subclinical protein malnutrition is a determinant of hyperhomocysteinemia. Nutrition, 2002, 18, 40-46.

Ingenbleek, Y.; Young, V.R. The essentiality of sulfur is closely related to nitrogen metabolism: a clue to hyperhomocysteinemia. Nutr. Res. Rev., 2004, 17, 135-153.

Hosoki, R.; Matsuki, N.; Kimura, H. The possible role of hydrogen sulfide as an endogenous smooth muscle relaxant in synergy with nitric oxide. Biochem. Biophys. Res. Commun., 1997, 237, 527-531.

Tang, B.; Mustafa, A.; Gupta, S.; Melnyk, S.; James S.J.; Kruger, W.D. Methionine-deficient diet induces post-transcriptional downregulation of cystathionine-􀀁-synthase. Nutrition, 2010, 26, 1170-1175.

Yves Ingenbleek. Plasma Transthyretin Reflects the Fluctuations of Lean Body Mass in Health and Disease. Chapter 20. In S.J. Richardson and V. Cody (eds.), Recent Advances in Transthyretin Evolution, Structure and Biological Functions, DOI: 10.1007/978‐3‐642‐00646‐3_20, # Springer‐Verlag Berlin Heidelberg 2009.

Transthyretin (TTR) is a 55-kDa protein secreted mainly by the choroid plexus and the liver. Whereas its intracerebral production appears as a stable secretory process allowing even distribution of intrathecal thyroid hormones, its hepatic synthesis is influenced by nutritional and inflammatory circumstances working concomitantly. Both morbid conditions are governed by distinct pathogenic mechanisms leading to the reduction in size of lean body mass (LBM). The liver production of TTR integrates the dietary and stressful components of any disease spectrum, explaining why it is the sole plasma protein whose evolutionary patterns closely follow the shape outlined by LBM fluctuations. Serial measurement of TTR therefore provides unequalled information on the alterations affecting overall protein nutritional status. Recent advances in TTR physiopathology emphasize the detecting power and preventive role played by the protein in hyperhomocysteinemic states, acquired metabolic disorders currently ascribed to dietary restriction in water-soluble vitamins. Sulfur (S)-deficiency is proposed as an additional causal factor in the sizeable proportion of hyperhomocysteinemic patients characterized by adequate vitamin intake but experiencing varying degrees of nitrogen (N)-depletion. Owing to the fact that N and S coexist in plant and animal tissues within tightly related concentrations, decreasing LBM as an effect of dietary shortage and/or excessive hypercatabolic losses induces proportionate S-losses. Regardless of water-soluble vitamin status, elevation of homocysteine plasma levels is negatively correlated with LBM reduction and declining TTR plasma levels. These findings occur as the result of impaired cystathionine-b-synthase activity, an enzyme initiating the transsulfuration pathway and whose suppression promotes the upstream accumulation and remethylation of homocysteine molecules. Under conditions of N- and S-deficiencies, the maintenance of methionine homeostasis indicates high metabolic priority.

Schematically, the human body may be divided into two major compartments, namely fat mass (FM) and FFM that is obtained by substracting
FM from body weight (BW). The fat cell mass sequesters about 80% of the total body lipids, is poorly hydrated and contains only small quantities of lean tissues and nonfat constituents. FFM comprises the sizeable part of lean tissues and minor mineral compounds among which are Ca, P, Na, and Cl pools totaling about 1.7 kg or 2.5% of BW in a healthy man weighing 70 kg. Subtraction of mineral mass from FFM provides LBM, a composite aggregation of organs and tissues with specific functional properties. LBM is thus nearly but not strictly equivalent to FFM. With extracellular mineral content subtracted, LBM accounts for most of total body proteins (TBP) and of TBN assuming a mean 6.25 ratio between protein and N content.

SM accounts for 45% of TBN whereas the remaining 55% is in nonmuscle lean tissues. The LBM of the reference man contains 98% of total
body potassium (TBK) and the bulk of total body sulfur (TBS). TBK and TBS reach equal intracellular amounts (140 g each) and share distribution patterns (half in SM and half in the rest of cell mass).  The body content of K and S largely exceeds that of magnesium (19 g), iron (4.2 g) and zinc (2.3 g). The average hydration level of LBM in healthy subjects of all age is 73% with the proportion of the intracellular/extracellular fluid spaces being 4:3. SM is of particular relevance in nutritional studies due to its capacity to serve as a major reservoir of amino acids (AAs) and as a dispenser of gluconeogenic substrates. An indirect estimate of SM size consists in the measurement of urinary creatinine, end-product of the nonenzymatic hydrolysis of phosphocreatine which is limited to muscle cells.

During ageing, all the protein components of the human body decrease regularly. This shrinking tendency is especially well documented for SM  whose absolute amount is preserved until the end of the fifth decade, consistent with studies showing unmodified muscle structure, intracellular K content and working capacit. TBN and TBK are highly correlated in healthy subjects and both parameters manifest an age-dependent curvilinear decline
with an accelerated decrease after 65 years.  The trend toward sarcopenia is more marked and rapid in elderly men than in elderly women decreasing strength and functional capacity. The downward SM slope may be somewhat prevented by physical training or accelerated by supranormal cytokine status as reported in apparently healthy aged persons suffering low-grade inflammation. 2002) or in critically ill patients whose muscle mass undergoes proteolysis and contractile dysfunction.

The serial measurement of plasma TTR in healthy children shows that BP values are low in the neonatal period and rise linearly with superimposable concentrations in both sexes during infant growth consistent with superimposable N accretion and protein synthesis rates. Starting from the sixties, TTR values progressively decline showing steeper slopes in elderly males. The lowering trend seems to be initiated by the attenuation of androgen influences and trophic stimuli with increasing age. The normal human TTR trajectory from birth to death has been well documented by scientists belonging to the Foundation for Blood Research. TTR is the first plasma protein to decline in response to marginal protein restricion, thus working as an early signal warning that adaptive mechanisms maintaining homeostasis are undergoing decompensation.

TTR was proposed as a marker of protein nutritional status following a clinical investigation undertaken in 1972 on protein-energy malnourished (PEM) Senegalese children (Ingenbleek et al. 1972). By comparison with ALB and transferrin (TF) plasma values, TTR revealed a much higher degree of sensitivity to changes in protein status that has been attributed to its shorter biological half-life (2 days) and to its unusual Trp richness (Ingenbleek et al. 1972, 1975a). Transcription of the TTR gene in the liver is directed by CCAAT/enhancer binding protein (C/EBP) bound to hepatocyte nuclear factor 1 (HNF1) under the control of several other HNFs. The mechanism responsible for the suppressed TTR synthesis in PEM-states is a restricted AA and energy supply working as limiting factors (Ingenbleek and Young 2002). The rapidly turning over TTR protein is highly responsive to any change in protein flux and energy supply, being clearly situated on the cutting edge of the equipoise.

LBM shrinking may be the consequence of either dietary restriction reducing protein syntheses to levels compatible with survival or that of cytokine-induced tissue proteolysis exceeding protein synthesis and resulting in a net body negative N balance. The size of LBM in turn determines plasma TTR concentrations whose liver production similarly depends on both dietary provision and inflammatory conditions. In animal cancer models, reduced TBN pools were correlated with decreasing plasma TTR values and provided the same predictive ability. In kidney patients, LBM is proposed as an excellent predictor of outcome working in the same direction as TTR plasma levels.  High N intake, supposed to preserve LBM reserves, reduces significantly the mortality rate of kidney patients and is positively correlated with the alterations of TTR plasma concentrations appearing as the sole predictor of final outcome. It is noteworthy that most SELDI or MALDI workers interested in defining protein nutritional status have chosen TTR as a biomarker, showing that there exists a large consensus considering the BP as the most reliable indicator of protein depletion in most morbid circumstances.

Total homocysteine (tHcy) is a S-containing AA not found in customary diets but endogenously produced in the body of mammals by the enzymatic transmethylation of methionine (Met), one of the eight IAAs supplied by staplefoods. tHcy may either serve as precursor substrate for the synthesis of new Met molecules along the remethylation (RM) pathway or undergo irreversible kidney leakage through a cascade of derivatives defining the transsulfuration (TS) pathway. Hcy is thus situated at the crossroad of RM and TS pathways that are regulated by three water-soluble vitamins (pyridoxine, B6; folates, B9; cobalamins, B12).

Significant positive correlations are found between tHcy and plasma urea and plasma creatinine, indicating that both visceral and muscular tissues undergo proteolytic degradation throughout the course of rampant inflammatory burden. In healthy individuals, tHcy plasma concentrations maintain positive correlations with LBM and TTR from birth until the end of adulthood. Starting from the onset of normal old age, tHcy values become disconnected from LBM control and reveal diverging trends with TTR values. Of utmost importance is the finding that, contrary to all protein
components which are downregulated in protein-depleted states, tHcy values are upregulated.  Hyperhomocysteinemia is an acquired clinical entity characterized by mild or moderate elevation in tHcy blood values found in apparently healthy individuals (McCully 1969). This distinct morbid condition appears as a public health problem of increasing importance in the general population, being regarded as an independent and graded risk factor for vascular pathogenesis unrelated to hypercholesterolemia, arterial hypertension, diabetes and smoking.

Studies grounded on stepwise multiple regression analysis have concluded that the two main watersoluble vitamins account for only 28% of tHcy variance whereas vitamins B6, B9, and B12, taken together, did not account for more than 30–40% of variance. Moreover, a number of hyperhomocysteinemic conditions are not responsive to folate and pyridoxine supplementation. This situation prompted us to search for other causal factors which might fill the gap between the public health data and the vitamin triad deficiencies currently incriminated. We suggest that S – the forgotten element – plays central roles in nutritional epidemiology (Ingenbleek and Young 2004).

Aminoacidemia studies performed in PEM children, adult patients and elderly subjects have reported that the concentrations of plasma IAAs invariably display lowering trends as the morbid condition worsens. The depressed tendency is especially pronounced in the case of tryptophan and for the so-called branched-chain AAs (BCAAs, isoleucine, leucine, valine) the decreases in which are regarded as a salient PEM feature following the direction outlined by TTR (Ingenbleek et al. 1986). Met constitutes a notable exception to the above described evolutionary profiles, showing unusual stability in chronically protein depleted states.

Maintenance of normal methioninemia is associated with supranormal tHcy blood values in PEMadults (Ingenbleek et al. 1986) and increased tHcy leakage in the urinary output of PEM children. In contrast, most plasma and urinary S-containing compounds produced along the TS pathway downstream to CbSconverting step (Fig. 20.1) display significantly diminished values. This is notably the case for cystathionine (Ingenbleek et al. 1986), glutathione, taurine, and sulfaturia. Such distorted patterns are reminiscent of abnormalities defining homocystinuria, an inborn disease of Met metabolism characterized by CbS refractoriness to pyridoxine stimuli, thereby promoting the upstream retention of tHcy in biological fluids. It
was hypothesized more than 20 years ago (Ingenbleek et al. 1986) that PEM is apparently able to similarly depress CbS activity, suggesting that the enzyme is a N-status sensitive step working as a bidirectional lockgate, overstimulated by high Met intake (Finkelstein and Martin 1986) and downregulated under N-deprivation conditions (Ingenbleek et al. 2002). Confirmation that N dietary deprivation may inhibit CbS activity has recently provided. The tHcy precursor pool is enlarged in biological fluids, boosting Met remethylation processes along the RM pathway, consistent with studies showing overstimulation of Met-synthase activity in conditions of protein restriction. In other words, high tHcy plasma concentrations observed in PEM states are the dark side of adaptive mechanisms for maintaining Met homeostasis. This is consistent with the unique role played by Met in the preservation of N body stores.

The classical interpretation that strict vegans, who consume plenty of folates in their diet and manifest nevertheless higher tHcy plasma concentrations than omnivorous counterparts, needs to be revisited. On the basis of hematological and biochemical criteria, cobalamin deficiency is one of the most prevalent vitamin-deficiencies wordwide, being often incriminated as deficient in vegan subjects. It seems, however, likely that its true causal impact on rising tHcy values is substantially overestimated in most studies owing to the modest contribution played by cobalamins on tHcy
variance analyses. In contrast, there exists a growing body of converging data indicating that the role played by the protein component is largely underscored in vegan studies. It is worth recalling that S is the main intracellular anion coexisting with N within a constant mean S:N ratio (1:14.5) in animal tissues and dietary products of animal origin (Ingenbleek 2006). The mean S:N ratio found in plant items ranges from 1:20 to 1:35, a proportion that does not optimally meet human tissue requirements (Ingenbleek 2006), paving the way for borderline S and N deficiencies.

A recent Taiwanese investigation on hyperhomocysteinemic nuns consuming traditional vegetarian regimens consisting of mainly rice, soy products,
vegetables and fruits with few or no dairy items illustrates such clinical misinterpretation (Hung et al. 2002). The authors reported that folates and cobalamins, taken together, accounted for only 28.6% of tHcy variance in the vegetarian cohort whereas pyridoxine was inoperative (Hung et al. 2002). The daily vegetable N and Met intakes were situated highly significantly (p < 0.001) below the recommended allowances for humans (FAO/WHO/United Nations University 1985), causing a stage of unrecognized PEM documented by significantly depressed BCAA plasma
concentrations. Met levels escaped the overall decline in IAAs levels, emphasizing that efficient homeostatic mechanisms operate at the expense of an acquired hyperhomocysteinemic state. The diagnosis of subclinical PEM was missed because the authors ignored the exquisitely sensitive TTR detecting power. A proper PEM identification would have allowed the authors to confirm the previously described TTR–tHcy relationship that was established in Western Africa from comparable field studies involving country dwellers living on plant products.

The concept that acute or chronic stressful conditions may exert similar inhibitory effects on CbS activity and thereby promote hyperhomocysteinemic states is founded on previous studies showing that hypercatabolic states are characterized by increased urinary N and S losses maintaining tightly correlated depletion rates (Cuthbertson 1931; Ingenbleek and Young 2004; Sherman and Hawk 1900) which reflect the S:N ratio found in tissues undergoing cytokine induced proteolysis. This has been documented in coronary infarction and in acute pancreatitis where tHcy elevation evolves too rapidly to allow for a nutritional vitamin B-deficit explanation.  tHcy is considered stable in plasma and the two investigations report unaltered folate and cobalamin plasma concentrations.

The clinical usefulness of TTR as a nutritional biomarker, described in the early seventies (Ingenbleek et al. 1972) has been substantially disregarded by the scientific community for nearly four decades. This long-lasting reluctance expressed by many investigators is largely due to the fact that protein malnutrition and stressful disorders of various causes have combined inhibitory effects on hepatic TTR synthesis. Declining TTR plasma concentrations may result from either dietary protein and energy restrictions or from cytokine-induced transcriptional blockade (Murakami et al. 1988) of its hepatic synthesis. The proposed marker was therefore seen as having high sensitivity but poor specificity. Recent advances in protein metabolism settle the controversy by throwing further light on the relationships between TTR and the N-components of body composition.

The developmental patterns of LBM and TTR exhibit striking similarities. Both parameters rise from birth to puberty, manifest gender dimorphism during full sexual maturity then decrease during ageing. Uncomplicated PEM primarily affects both visceral and structural pools of LBM with distinct kinetics, reducing protein synthesis to levels compatible with prolonged survival. In acute or chronic stressful disorders, LBM undergoes muscle proteolysis exceeding the upregulation of protein syntheses in liver and injured areas, yielding a net body negative N balance. These adaptive responses are well identified by the measurement of TTR plasma concentrations which therefore appear as a plasma marker for LBM fluctuations.
Attenuation of stress and/or introduction of nutritional rehabilitation restores both LBM and TTR to normal values following parallel slopes. TTR fulfills, therefore, a unique position in assessing actual protein nutritional status, monitoring the efficacy of dietetic support and predicting the patient’s outcome (Bernstein and Pleban 1996).

see also…

Acosta PB, Yannicelli S, Ryan AS, Arnold G, Marriage BJ, Plewinska M, Bernstein L, Fox J, Lewis V, Miller M, Velazquez A (2005) Nutritional therapy improves growth and protein status of children with a urea cycle enzyme defect. Mol Genet Metab 86:448–455.

Arroyave G, Wilson D, Be´har M, Scrimshaw NS (1961) Serum and urinary creatinine in children with severe protein malnutrition. Am J Clin Nutr 9:176–179.

Bates CJ, Mansoor MA, van der Pols J, Prentice A, Cole TJ, Finch S (1997) Plasma total homocysteine in a representative sample of 972 British men and women aged 65 and over. Eur J Clin Nutr 51:691–697.

Battezzatti A, Bertoli S, San Romerio A, Testolin G (2007) Body composition: An important determinant of homocysteine and methionine concentrations in healthy individuals. Nutr Metab Cardiovasc Dis 17:525–534.

Bernstein LH, Bachman TE, Meguid M, Ament M, Baumgartner T, Kinosian B, Martindale R, Spiekerman M (1995) Prealbumin in nutritional care Consensus Group. Measurement of visceral protein status in assessing protein and energy malnutrition: Standard of care. Nutrition 11:169–171

Bernstein LH, Ingenbleek Y (2002) Transthyretin: Its response to malnutrition and stress injury. Clinical usefulness and economical implications. Clin Chem Lab Med 40:1344–1348.

Boorsook H, Dubnoff JW (1947) The hydrolysis of phosphocreatine and the origin of creatinine. J Biol Chem 168:493–510.

Briend A, Garenne M, Maire B, Fontaine O, Dieng F (1989) Nutritional status, age and survival: The muscle mass hypothesis. Eur J Clin Nutr 43:715–726

Gray GE, Landel AM, Meguid MM (1994) Taurine-supplemented total parenteral nutrition and taurine status of malnourished cancer patients. Nutrition 10:11–15

Heymsfield SB, McManus C, Stevens V, Smith J (1982) Muscle mass: Reliable indicator of protein-energy malnutrition and outcome. Am J Clin Nutr 35:1192–1199

Ingenbleek Y (2006) The nutritional relationship linking sulfur to nitrogen in living organisms. J Nutr 136:S1641–S1651
Ingenbleek Y (2008) Plasma transthyretin indicates the direction of both nitrogen balance and retinoid status in health and disease. Open Clin Chem J 1:1–12
Ingenbleek Y, Bernstein LH (1999a) The stressful condition as a nutritionally dependent adaptive dichotomy. Nutrition 15:305–320
Ingenbleek Y, Bernstein LH (1999b) The nutritionally dependent adaptive dichotomy (NDAD) and stress hypermetabolism. J Clin Ligand Assay 22:259–267
Ingenbleek Y, Carpentier YA (1985) A prognostic inflammatory and nutritional index scoring critically ill patients. Internat J Vitam Nutr Res 55:91–101

Ingenbleek Y, Young VR (1994) Transthyretin (prealbumin) in health and disease: Nutritional implications. Annu Rev Nutr 14:495–533
Ingenbleek Y, Young VR (2002) Significance of transthyretin in protein metabolism. Clin Chem Lab Med 40:1281–1291
Ingenbleek Y, Young VR (2004) The essentiality of sulfur is closely related to nitrogen metabolism. Nutr Res Rev 17:135–151

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Simple representation of the toll-like recepto...

Sepsis, Multi-organ Dysfunction Syndrome, and Septic Shock: A Conundrum of Signaling Pathways Cascading Out of Control

Curator and Author: Larry H Bernstein, MD, FCAP

What is Septic Shock?
Scripps Research Professor Wolfram Ruf and colleagues have identified a key connection between the signaling pathways and the immune system spiraling out of control involving the coagulation system and vascular endothelium that, if disrupted may be a target for sepsis. (Science Daily, Feb 29, 2008). It may be caused by a bacterial infection that enters the bloodstream, but we now recognize the same cascade not triggered by bacterial invasion. These invading bacteria produce endotoxins and other toxins that trigger a widespread inflammatory response of the innate immune system–a response that is necessary, as it turns out, because without the inflammation, the body cannot fight off the bacterial infection. During sepsis, the inflammation triggers widespread coagulation in the bloodstream. This coagulation can block blood vessels in vital organs, starving the organs of oxygen and damaging them. The organs can be further damaged when the blood starts to flow again because the lining of the blood vessels remain leaky due to inflammatory cytokines and damage by intravascular coagulation.
What is the Pathogenesis of Sepsis?
The acute respiratory distress syndrome (ARDS) has been defined as a severe form of acute lung injury featuring pulmonary inflammation and increased capillary leak. ARDS is associated with a high mortality rate and accounts for 100,000 deaths annually in the United States. ARDS may arise in a number of clinical situations, especially in patients with sepsis. A well-described pathophysiological model of ARDS is one form of the acute lung inflammation mediated by neutrophils, cytokines, and oxidant stress. Neutrophils are major effect cells at the frontier of innate immune responses, and they play a critical role in host defense against invading microorganisms. The tissue injury appears to be related to proteases and toxic reactive oxygen radicals released from activated neutrophils. In addition, neutrophils can produce cytokines and chemokines that enhance the acute inflammatory response. Neutrophil accumulation in the lung plays a pivotal role in the pathogenesis of acute lung injury during sepsis. Directed movement of neutrophils is mediated by a group of chemoattractants, especially CXC chemokines. Local lung production of CXC chemokines is intensified during experimental sepsis induced by cecal ligation and puncture (CLP). Under these conditions of stimulation, activation of MAPKs (p38, p42/p44) occurs in sham neutrophils but not in CLP neutrophils, while under the same conditions phosphorylation of p38 and p42/p44 occurs in both sham and CLP alveolar macrophages. These data indicate that, under septic conditions, there is impaired signaling in neutrophils and enhanced signaling in alveolar macrophages, resulting in CXC chemokine production, and C5a appears to play a pivotal role in this process. As a result, CXC chemokines increase in lung, setting the stage for neutrophil accumulation in lung during sepsis.
Uncontrolled activation of the coagulation cascade following lung injury contributes to the development of lung inflammation and fibrosis in acute lung injury/acute respiratory distress syndrome (ALI/ARDS) and fibrotic lung disease. This article reviews our current understanding of the mechanisms leading to the activation of the coagulation cascade in response to lung injury and the evidence that excessive procoagulant activity is of pathophysiological significance in these disease settings. This is consistent with a pneumonia or lung injury preceding sepsis. On the other hand, it is not surprising that abdominal, cardiac bypass, and post cardiac revascularization may also lead to events resembling sepsis and/or cardiovascular collapse. The tissue factor-dependent extrinsic pathway is the predominant mechanism by which the coagulation cascade is locally activated in the lungs of patients with ALI/ARDS and pulmonary fibrosis. The cellular effects mediated via activation of proteinase-activated receptors (PARs) may be of particular importance in influencing inflammatory and fibroproliferative responses in experimental models involving direct injury to the lung. In this regard, studies in PAR1 knockout mice have shown that this receptor plays a major role in orchestrating the interplay between coagulation, inflammation and lung fibrosis.
The activation of the coagulation cascade is one of the earliest events initiated following tissue injury. The prime function of this complex and highly regulated proteolytic system is to generate insoluble, crosslinked fibrin strands, which bind and stabilize weak platelet hemostatic plugs, formed at sites of tissue injury. The formation of this provisional clot is critically dependent on the action of thrombin, and is generated following the stepwise activation of coagulation proteinases via the extrinsic and intrinsic systems. Under normal circumstances, blood is not exposed to tissue factor (TF). However, upon tissue injury, exposure of plasma to TF expressed on non-vascular cells or on activated endothelial cells results in the formation of the TF-activated factor VII (FVIIa) complex. The TF–FVIIa complex subsequently catalyses the initial activation of FX to activated factor X (FXa) and FIX to activated factor IX. FXa in association with activated factor V catalyses the conversion of prothrombin to thrombin. Sustained coagulation is achieved when thrombin synthesized through the initial TF–FVIIa–FXa complex catalyses the activation of FXI, FIX, FVIII and FX. In this manner, the intrinsic pathway is activated.
The systemic inflammatory response syndrome (SIRS) is the massive inflammatory reaction resulting from systemic mediator release that may lead to multiple organ dysfunction. I introduce an analysis of the roles of cytokines, cytokine production, and the relationship of cytokine production to the development of SIRS. The article postulates a three-stage development of SIRS, in which stage 1 is a local production of cytokines in response to an injury or infection. Stage 2 is the protective release of a small amount of cytokines into the body’s circulation. Stage 3 is the massive systemic reaction where cytokines turn destructive by compromising the integrity of the capillary walls and flooding end organs. While cytokines are generally viewed as a destructive development in the patient that generally leads to multiple organ dysfunction, cytokines also protect the body when localized. It will be necessary to study the positive effects of cytokines while also studying their role in causing SIRS. It will also be important to investigate the relationship between cytokines and their blockers in SIRS.
Monocyte/macrophage- and neutrophil-mediated inflammatory responses can be stimulated through a variety of receptors, including G protein-linked 7-transmembrane receptors (e.g., FPR1; MIM 136537), Fc receptors (see MIM 146790), CD14 (MIM 158120) and Toll-like receptors (e.g., TLR4; MIM 603030), and cytokine receptors (e.g., IFNGR1; MIM 107470). Engagement of these receptors can also prime myeloid cells to respond to other stimuli. Myeloid cells express receptors belonging to the Ig superfamily, such as TREM1, or to the C-type lectin superfamily. Depending on their transmembrane and cytoplasmic sequence structure, these receptors have either activating (e.g., KIR2DS1; MIM 604952) or inhibitory functions (e.g., KIR2DL1; MIM 604936).[supplied by OMIM].
TREM-1 associates with and signals via the adapter protein 12DAP12/12TYROBP, which contains an ITAM. To mediate activation, TREM-1 associates with the transmembrane adapter molecule 12DAP12. In sharp contrast to the effect by Ad-FDAP12, transgene expression in the liver of soluble form of extracellular domain of TREM-1 as an antagonist of 12DAP12 signaling, remarkably inhibited zymosan A-induced granuloma formation at every time point examined.
For signal transduction, 01TREM-1 couples to the ITAM-containing adapter DNAX activation protein of 12 kDa (23DAP12 ). MARV and EBOV activate TREM-1 on human neutrophils, resulting in 12DAP12 phosphorylation, TREM-1 shedding, mobilization of intracellular calcium, secretion of proinflammatory cytokines, and phenotypic changes. TREM-1 is the best-characterized member of a growing family of 12DAP12-associated receptors that regulate the function of myeloid cells in innate and adaptive responses. TREM-1 (triggering receptor expressed on myeloid cells), a recently discovered receptor of the immunoglobulin superfamily, activates neutrophils and monocytes/macrophages by signaling through the adapter protein 12DAP12. 522Granulocyte TREM-1 expression was high at baseline and immediately down-regulated upon LPS exposure along with an increase in soluble TREM-1.
DIC is primarily a laboratory diagnosis, based on the combination of elevated fibrin-related markers (FRM), with decreased procoagulant factors and platelets. Non-overt DIC is observed in most patients with sepsis, whereas overt DIC is less frequent. Consumption coagulopathy is a bleeding disorder caused by low levels of platelets and procoagulant factors associated with massive coagulation activation. Treatment with drotrecogin alfa (activated) improves survival and other outcome parameters in severe sepsis, including a subgroup of patients fulfilling the laboratory criteria of overt DIC. No randomized trials demonstrating effective therapies in consumption coagulopathy have been published.
Sepsis is a complex syndrome characterized by simultaneous activation of inflammation and coagulation manifested as systemic inflammatory response syndrome (SIRS)/sepsis symptoms through release of proinflammatory cytokines, procoagulants, and adhesion molecules from immune cells and/or damaged endothelium. Conventional treatments have focused on source control, antimicrobials, vasopressors, and fluid resuscitation; however, a new treatment paradigm exists: that of treating the host response to infection with adjunct therapies including early goal-directed therapy, drotrecogin alfa (activated), and immunonutrition. The drotrecogin alfa (activated) has been shown to reduce mortality in the severely septic patient when combined with traditional treatment. Therapies targeting improved oxygen and blood flow and reduction of apoptosis and free radicals are under investigation. Ultimately, intervention timing may be the most important factor in reducing severe sepsis mortality.

Cell Signaling in Sepsis
Recent data have shown stable patterns of activation among peripheral blood mononuclear cells and neutrophils in healthy human subjects. Although polymorphisms in Toll-like receptors play a contributory role in determining cellular activation, other factors are involved as well. In addition, circulating and locally released mediators of inflammation, including cytokines, complement fragments, and components of activated coagulation and fibrinolytic systems, that are generated in increased amounts during severe infection also interact with membrane-based receptors, leading to activation of intracellular path ways capable of further accelerating proinflammatory cascades. Circulating and organ-specific cell populations are activated to produce proinflammatory mediators during sepsis. Neutrophils and PBMCs bear TLR2 and TLR4, as well as other receptors, such as protein —coupled receptor, that induce increased generation of cytokines and other immunoregulatory proteins, as well as enhance release of proinflammatory mediators, including reactive oxygen species.
The expression of cytokines such as TNF-α and IL-1β is increased in sepsis, and engagement of TNF-α with type I(p55) and type II(p75) TNF receptors or IL-1β with IL-1 receptors belonging to the TLR/IL-1 receptor family produces activation of kinases (including Src, p38, extracellular signal—regulated kinase, and phosphoinositide 3–kinase) and transcriptional factors (such as nuclear factor [NF]–κB) important for further up-regulation of inflammatory proteins.
Genetic polymorphisms lead to alterations in TLR conformation (a small percentage of the variability in humans when their cells are exposed to bacterial products) that are accompanied by decreased cellular activation after exposure to bacterial products. The stable variability in cellular activation that is present among the genetically heterogeneous human population, only a limited number of studies have examined how such patterns may correlate with clinical outcome. A number of studies have examined the transcriptional factor NF-κB and kinases, including p38 and Akt, and provide insights into how heterogeneity in cell signaling may contribute to subsequent clinical course.
Increased activation of the mitogen-activated protein kinase protein 38, Akt, and nuclear factor (NF)–κB in neutrophils and other cell populations obtained at early time points in the clinical course of sepsis-induced acute lung injury or after accidental trauma is associated with a more-severe clinical course, suggesting that a proinflammatory cellular phenotype contributes to organ system dysfunction in such settings. Identification of patients with cellular phenotypes characterized by increased activation of NF-κB, Akt, and protein 38, as well as discrete patterns of gene activation, may permit identification of patients with sepsis who are likely to have a worse clinical outcome, thereby permitting early institution of therapies that modulate deleterious signaling pathways before organ system dysfunction develops, reducing morbidity and improving survival.

NF-kB

The transcriptional regulatory factor NF-κB is a central participant in modulating the expression of many immuno regulatory mediators involved in the acute inflammatory response [30–35]. NF-κB/rel transcription factors function as dimers held latently in the cytoplasm of cells by inhibitory IκB proteins. Signaling pathways initiated by engagement of TLRs, such as TLR 2 and TLR 4, by microbial products and other inflammatory mediators lead to nuclear accumulation of NF-κB and enhanced transcription of genes responsible for the expression of cytokines, chemokines, adhesion molecules, and other mediators of the inflammatory response associated with infection. Association of NF-κB with the inhibitory protein κB-α in the cytoplasm blocks the nuclear localization sequence of NF-κB, inhibiting its movement into the nucleus. Phosphorylation events, in addition to those involving IKKα/β and IκB-α, and involving NF-κB subunits (such as p 65) and nuclear coactivator proteins (such as TATA box binding protein or cAMP-responsive element—binding protein) are mediated by p 38, Akt, and other kinases and play an important role in regulating the transcriptional activity of NF-κB.

Studies have shown that greater nuclear accumulation of NF-κB is accompanied by higher mortality and worse clinical course in patients with sepsis. These clinical series demonstrated that persistent activation of NF-κB was found in nonsurvivors, with surviving patients having lower nuclear concentrations of NF-κB at early time points in their septic course than did nonsurvivors as well as more rapid return of nuclear accumulation of NF-κB.  Although studies of patients with sepsis have generally shown that nuclear concentrations of NF-κB are higher in non survivors than in survivors, an unresolved issue is whether such changes occur early and, therefore, define the subsequent course of sepsis or whether pathophysiological changes that result in poor clinical outcome also produce NF-κB activation as a secondary event, so that such changes in NF-κB are simply associated with more severe organ system dysfunction but do not contribute directly to outcome. A study of surgical patients without sepsis supports the hypothesis that neutrophil phenotypes defined by NF-κB activation patterns predict clinical outcome [54]. In that clinical series of patients undergoing repair of aortic aneurysms, higher preoperative levels of NF-κB in peripheral neutrophils were associated with death and with the development of postoperative organ dysfunction.

NF-κB

NF-κB (Photo credit: Wikipedia)

Stable high and low responder phenotypes in the healthy population, implies that the presence of a preexistent high responder neutrophil phenotype, as characterized by increased nuclear translocation of NF-κB after stimulation with TLR 2 or TLR 4 ligands, would be associated with more severe pulmonary inflammatory response and clinical course in response to infection. Conversely, persons whose neutrophils have diminished activation of NF-κB after stimulation would be expected to have less-intense neutrophil-driven inflammation, as well as organ dysfunction. In addition, Nuclear levels of nuclear factor (NF)–κB are significantly increased in neutrophils obtained within 24h of initiation of mechanical ventilation in patients whose clinical course from sepsis-induced acute lung injury is more severe (as defined by death or ventilation for >14 days—that is, ⩽14 ventilator-free days [VFD]), compared with patients with a less-severe course (as defined by mechanical ventilation for <14 days, or >14 VFD).  Baseline nuclear concentrations of NF-κB were lower in healthy volunteers than in patients with sepsis-induced acute lung injury, regardless of subsequent clinical course, demonstrating baseline activation of NF-κB in association with sepsis. *P <.05, vs. volunteers. †P< .05, vs. >14VFD.

Modulation of intracellular signaling cascades involving kinases, such as p 38 or Akt, or transcriptional factors, such as NF-κB, through specific inhibitory approaches has shown their pathophysiological importance in experimental models. However, the role of specific intra cellular pathways in contributing to clinical outcomes in patients with sepsis remains incompletely determined, primarily because such alterations in cellular activation patterns have not been examined at early time points before the onset of multiple organ dysfunction. Recent information shows that alterations in p38, Akt, and NF-κB among neutrophils and other cell populations not only precedes the development of organ system dysfunction but also has predictive value in identifying patients with a more severe subsequent clinical course.

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Abraham E.  Alterations in Cell Signaling in Sepsis. Clin Infect Dis 2005: 41 (Supplement 7): S459-S464. doi: 10.1086/431997

Yang KY, Arcaroli JJ, Abraham E. Early alterations in neutrophil activation are associated with outcome in acute lung injury. Am J Respir Crit Care Med 2003; 167:1567-74.

Abraham E. Neutrophils and Acute Lung Injury. Crit Care Med 2003; 31:195-9.

Abraham E, Carmody A, Shenkar R, Arcaroli J. Neutrophils as early immunologic effectors in hemorrhage- or endotoxemia-induced acute lung injury. Am J Physiol Lung Cell Mol Physiol 2000; 279:1137-45.

Sepsis Bundles

The Institute for Healthcare Improvement (IHI) has highlighted sepsis as an area of focus and has identified several deficiencies that may cause suboptimal care of patients with severe sepsis.

These deficiencies include inconsistency in the early diagnosis of severe sepsis and septic shock, frequent inadequate volume resuscitation without defined endpoints, late or inadequate use of antibiotics, frequent failure to support the cardiac output when depressed, frequent failure to control hyperglycemia adequately, frequent failure to use low tidal volumes and pressures in acute lung injury, and frequent failure to treat adrenal inadequacy in refractory shock.

To address these deficiencies, the Surviving Sepsis Campaign and IHI have revised and added to the Surviving Sepsis Guidelines and created 2 sepsis treatment bundles (resuscitation and management) to guide therapy for patients with severe sepsis.

“Implicit in the use of the bundles is the need to adopt all the elements contained in the bundle,” the authors write. “One cannot choose to apply only selected items from the bundle and expect to achieve comparable benefit. The IHI sepsis website provides tools to screen patients for severe sepsis, as well as to measure success with adherence to implementing the bundles (http://www.ihi.org/IHI/Topics/CriticalCare/Sepsis/).” (The authors are employees of Eli Lilly and Co, the maker of drotrecogin alfa (activated). South Med J. 2007;100:594-600.

The sepsis resuscitation bundle, which should be accomplished as soon as possible and scored during the first 6 hours

Prealbumin (Transthyretin)

Discharge prealbumin and the change in prealbumin were positively correlated with protein and energy intake and inversely correlated with markers of inflammation, particularly CRP and IL-6. When all covariates were included in a multivariable regression analysis, the markers of inflammation predominantly accounted for the variance in prealbumin change (56%), whereas discharge protein intake accounted for 6%.

These authors propose an updated approach that incorporates current understanding of the systemic inflammatory response to help guide assessment, diagnosis, and treatment. An appreciation of a continuum of inflammatory response in relation to malnutrition syndromes is described. This discussion serves to highlight a research agenda to address deficiencies in diagnostics, biomarkers, and therapeutics of inflammation in relation to malnutrition.

Procalcitonin

The most frequent indication for antibiotic prescriptions in the northwestern hemisphere is lower respiratory tract infections (LRTIs),which range in severity from self-limited acute bronchitis to severe acute exacerbation of chronic obstructive pulmonary disease (COPD), and to life-threatening bacterial community-acquired pneumonia (CAP).4 Clinical signs and symptoms, as well as commonly used laboratory markers, are unreliable in distinguishing viral from bacterial LRTI. As many as 75% of patients with LRTI are treated with antibiotics, despitethe predominantly viral origin of their infection. An approach to estimate the probability of bacterial origin in LRTI is the measurement of serum procalcitonin (PCT).

In patients with LRTIs, a strategy of PCT guidance compared with standard guidelines resulted in similar rates of adverse outcomes, as well as lower rates of antibiotic exposure and antibiotic-associated adverse effects. (Trial Registration isrctn.org Identifier: ISRCTN95122877)

Neutrophil CD64

Despite improvements in the treatment of sepsis in recent years, there have been few diagnostic innovations which improve the sensitivity and specificity of diagnosis or facilitate therapeutic monitoring. The clinical reliance on the CBC and leukocyte differential with associated band count to indicate myeloid left shift of immaturity is not accurate, and it is not comparable to the measurement of the metamyeloctes and myelocytes. Only the introduction of a test which measures procalcitonin (PCT), an acute phase marker which is claimed to be more specific for bacterial infections than for viral infections, can be cited as a new diagnostic for the evaluation of patients with suspected infection. A need still persists for improved diagnostic indictors of infection or sepsis, as well as better tests to facilitate monitoring of therapy in the treatment of infection, so that use of antibiotics might be less empirical.

Studies have indicated that quantitative neutrophil CD64 expression is a sensitive and specific laboratory indicator of sepsis or the presence of a systemic acute inflammatory response.  Neutrophil CD64 is a highly sensitive marker for neonatal sepsis. Prospective studies incorporating CD64 into a sepsis scoring system are warranted. Studies have indicated that quantitative neutrophil CD64 (high affinity Fc receptor) expression is a worth­while candidate for evaluation as a more sensitive and specific laboratory indi­cator of sepsis or the presence of a systemic acute inflammatory response than available diagnostics . Neutrophil (PMN) CD64 is one of many activa­tion-related antigenic changes manifested by neutrophils during the normal pathophysiological acute inflammatory or innate immune response. PMN expression of CD64 is up-regulated under the influence of inflammatory relat­ed cytokines such as interleukin 12 (IL-12), interferon gamma (IFN-y) and granulocyte colony stimulating factor (G-CSF).

The first commercially available assay for PMN CD64, developed by Trillium Diagnostics, LLC is a fluorescence based, no wash flow cytometric assay, namely the Leuko64. The assay kit contains a cocktail of monoclonal antibodies includ­ing two monoclonal antibodies to CD64 and a monoclonal antibody to CD163, red cell lysis buffer, fluorescence quantitation beads, and a software program for automated analysis of the flow cytometric data that reports PMN CD64 as a CD64 index. The PMN CD64 index is designed so that normal inactivated PMNs yield values of < 1.00 and blood samples from individuals with docu­mented infection or sepsis typically show values > 1.50. Using clinical flow cytometers, the assay can be completed within 30 minutes. While this initial assay format was developed for multiparameter flow cytometers, a new version of the assay has been developed to give nearly identical results on the CD4000 and Sapphire (manufactured by Abbott Diagnostics, Santa Clara, CA) blood cell counters, which are equipped with laser light sources and fluorescence detection capabilities. If these blood cell counters are available in diagnostic haematology laboratories, the Leuko64 assay can be utilised on a 24 hour basis, in contrast to the more typical daytime operation hours of flow cytometric diagnostic laboratories.

Leukocare and Trillium Diagnostics entered an agreement to develop and market Leukocare’s method for detecting inflammatory activity using circulating cell-free DNA. Trillium aims to create a cf-DNA test as a “simple and cost effective” tool that healthcare professionals can use to obtain clinically relevant data on patients who are suspected of having sepsis. The companies said that they expect to finish developing the assay and market it in two years.

B Casserly, R Read, MM Levy. Multimarker Panels  in Sepsis. Crit Care Clin 27 (2011) 391–405 doi:10.1016/j.ccc.2010.12.011 criticalcare.theclinics.com

Dennis RA, Johnson LE, Roberson PK, Heif M, Bopp MM, et al.  Changes in prealbumin, nutrient intake, and systemic inflammation in elderly recuperative care patients.  J Am Geriatr Soc. 2008; 56(7):1270-5. Epub 2008 Jun 10. PMID: 18547360

Jensen GL, Bistrian B, Roubenoff R, Heimburger DC.  Malnutrition Syndromes: A Conundrum vs Continuum.

Bernstein LH. The systemic inflammatory response syndrome C-reactive protein and transthyretin conundrum. Clinical Chemistry Laboratory Medicine 2007; 45(11):1566–1567, ISSN (Online) 14374331, ISSN (Print) 14346621, DOI: 10.1515/CCLM.2007.334.

Schuetz P, Christ-Crain M, Thomann R, Falconnier C, Wolbers M, et al.  for the ProHOSP Study Group. Effect of Procalcitonin-Based Guidelines vs Standard Guidelines on Antibiotic Use in Lower Respiratory Tract Infections: The ProHOSP Randomized Controlled Trial.  JAMA  2009; 302(10): 1059

Bhandari V, Wang C, Rinder C, Rinder H. Hematologic Profile of Sepsis in Neonates: Neutrophil CD64 as a Diagnostic Marker. Pediatrics 2007; 31:4005.   (ISSN Numbers: Print, 0031-4005; Online, 1098-4275). doi:10.1542/peds.2007-1308

Davis BH.  Neutrophil CD64 expression in infection and sepsis. CLI Ocober 2006.

Chapter 1 Statement of Inferential    Second Opinion

Realtime Clinical Expert Support

Gil David and Larry Bernstein have developed, in consultation with Prof. Ronald Coifman, in the Yale University Applied Mathematics Program, a software system that is the equivalent of an intelligent Electronic Health Records Dashboard that provides empirical medical reference and suggests quantitative diagnostics options.

Keywords: Entropy, Maximum Likelihood Function, separatory clustering, peripheral smear, automated hemogram, Anomaly, classification by anomaly, multivariable and multisyndromic, automated second opinion

Abbreviations: Akaike Information Criterion, AIC;  Bayes Information Criterion, BIC, Systemic Inflammatory Response Syndrome, SIRS.

Background: The current design of the Electronic Medical Record (EMR) is a linear presentation of portions of the record by services, by diagnostic method, and by date, to cite examples.  This allows perusal through a graphical user interface (GUI) that partitions the information or necessary reports in a workstation entered by keying to icons.  This requires that the medical practitioner finds the history, medications, laboratory reports, cardiac imaging and EKGs, and radiology in different workspaces.  The introduction of a DASHBOARD has allowed a presentation of drug reactions, allergies, primary and secondary diagnoses, and critical information about any patient the care giver needing access to the record.  The advantage of this innovation is obvious.  The startup problem is what information is presented and how it is displayed, which is a source of variability and a key to its success.

Intent: We are proposing an innovation that supercedes the main design elements of a DASHBOARD and utilizes the conjoined syndromic features of the disparate data elements.  So the important determinant of the success of this endeavor is that it facilitates both the workflow and the decision-making process with a reduction of medical error. Continuing work is in progress in extending the capabilities with model datasets, and sufficient data because the extraction of data from disparate sources will, in the long run, further improve this process.  For instance, the finding of  both ST depression on EKG coincident with an elevated cardiac biomarker (troponin), particularly in the absence of substantially reduced renal function. The conversion of hematology based data into useful clinical information requires the establishment of problem-solving constructs based on the measured data.

The most commonly ordered test used for managing patients worldwide is the hemogram that often incorporates the review of a peripheral smear.  While the hemogram has undergone progressive modification of the measured features over time the subsequent expansion of the panel of tests has provided a window into the cellular changes in the production, release or suppression of the formed elements from the blood-forming organ to the circulation.  In the hemogram one can view data reflecting the characteristics of a broad spectrum of medical conditions.

Progressive modification of the measured features of the hemogram has delineated characteristics expressed as measurements of size, density, and concentration, resulting in many characteristic features of classification. In the diagnosis of hematological disorders proliferation of marrow precursors, the domination of a cell line, and features of suppression of hematopoiesis provide a two dimensional model.  Other dimensions are created by considering the maturity of the circulating cells.  The application of rules-based, automated problem solving should provide a valid approach to the classification and interpretation of the data used to determine a knowledge-based clinical opinion. The exponential growth of knowledge since the mapping of the human genome enabled by parallel advances in applied mathematics that have not been a part of traditional clinical problem solving.  As the complexity of statistical models has increased the dependencies have become less clear to the individual.  Contemporary statistical modeling has a primary goal of finding an underlying structure in studied data sets.  The development of an evidence-based inference engine that can substantially interpret the data at hand and convert it in real time to a “knowledge-based opinion” could improve clinical decision-making by incorporating multiple complex clinical features as well as duration of onset into the model.

An example of a difficult area for clinical problem solving is found in the diagnosis of SIRS and associated sepsis.  SIRS (and associated sepsis) is a costly diagnosis in hospitalized patients.   Failure to diagnose sepsis in a timely manner creates a potential financial and safety hazard.  The early diagnosis of SIRS/sepsis is made by the application of defined criteria (temperature, heart rate, respiratory rate and WBC count) by the clinician.   The application of those clinical criteria, however, defines the condition after it has developed and has not provided a reliable method for the early diagnosis of SIRS.  The early diagnosis of SIRS may possibly be enhanced by the measurement of proteomic biomarkers, including transthyretin, C-reactive protein and procalcitonin.  Immature granulocyte (IG) measurement has been proposed as a more readily available indicator of the presence of granulocyte precursors (left shift).  The use of such markers, obtained by automated systems in conjunction with innovative statistical modeling, provides a promising approach to enhance workflow and decision making.   Such a system utilizes the conjoined syndromic features of disparate data elements with an anticipated reduction of medical error.  This study is only an extension of our approach to repairing a longstanding problem in the construction of the many-sided electronic medical record (EMR).  In a classic study carried out at Bell Laboratories, Didner found that information technologies reflect the view of the creators, not the users, and Front-to-Back Design (R Didner) is needed.

Costs would be reduced, and accuracy improved, if the clinical data could be captured directly at the point it is generated, in a form suitable for transmission to insurers, or machine transformable into other formats.  Such data capture, could also be used to improve the form and structure of how this information is viewed by physicians, and form a basis of a more comprehensive database linking clinical protocols to outcomes, that could improve the knowledge of this relationship, hence clinical outcomes.

How we frame our expectations is so important that it determines the data we collect to examine the process.   In the absence of data to support an assumed benefit, there is no proof of validity at whatever cost.   This has meaning for hospital operations, for nonhospital laboratory operations, for companies in the diagnostic business, and for planning of health systems.

In 1983, a vision for creating the EMR was introduced by Lawrence Weed,  expressed by McGowan and Winstead-Fry (J J McGowan and P Winstead-Fry. Problem Knowledge Couplers: reengineering evidence-based medicine through interdisciplinary development, decision support, and research. Bull Med Libr Assoc. 1999 October; 87(4): 462–470.)   PMCID: PMC226622    Copyright notice

They introduce Problem Knowledge Couplers as a clinical decision support software tool that  recognizes that functionality must be predicated upon combining unique patient information, but obtained through relevant structured question sets, with the appropriate knowledge found in the world’s peer-reviewed medical literature.  The premise of this is stated by LL WEED in “Idols of the Mind” (Dec 13, 2006): “ a root cause of a major defect in the health care system is that, while we falsely admire and extol the intellectual powers of highly educated physicians, we do not search for the external aids their minds require”.  HIT use has been focused on information retrieval, leaving the unaided mind burdened with information processing.

The data presented has to be comprehended in context with vital signs, key symptoms, and an accurate medical history.  Consequently, the limits of memory and cognition are tested in medical practice on a daily basis.  We deal with problems in the interpretation of data presented to the physician, and how through better design of the software that presents this data the situation could be improved.  The computer architecture that the physician uses to view the results is more often than not presented as the designer would prefer, and not as the end-user would like.  In order to optimize the interface for physician, the system would have a “front-to-back” design, with the call up for any patient ideally consisting of a dashboard design that presents the crucial information that the physician would likely act on in an easily accessible manner.  The key point is that each item used has to be closely related to a corresponding criterion needed for a decision.  Currently, improved design is heading in that direction.  In removing this limitation the output requirements have to be defined before the database is designed to produce the required output.  The ability to see any other information, or to see a sequential visualization of the patient’s course would be steps to home in on other views.  In addition, the amount of relevant information, even when presented well, is a cognitive challenge unless it is presented in a disease- or organ-system structure.  So the interaction between the user and the electronic medical record has a significant effect on practitioner time, ability to minimize errors of interpretation, facilitate treatment, and manage costs.  The reality is that clinicians are challenged by the need to view a large amount of data, with only a few resources available to know which of these values are relevant, or the need for action on a result, or its urgency. The challenge then becomes how fundamental measurement theory can lead to the creation at the point of care of more meaningful actionable presentations of results.  WP Fisher refers to the creation of a context in which computational resources for meeting the challenges will be incorporated into the electronic medical record.  The one which he chooses is a probabilistic conjoint (Rasch) measurement model, which uses scale-free standard measures and meets data quality standards. He illustrates this by fitting a set of data provided by Bernstein (19)(27 items for the diagnosis of acute myocardial infarction (AMI) to a Rasch multiple rating scale model testing the hypothesis that items work together to delineate a unidimensional measurement continuum. The results indicated that highly improbable observations could be discarded, data volume could be reduced based on internal, and increased ability of the care provider to interpret the data.

 

Classified data a separate issue from automation

 Feature Extraction. This further breakdown in the modern era is determined by genetically characteristic gene sequences that are transcribed into what we measure.  Eugene Rypka contributed greatly to clarifying the extraction of features in a series of articles, which set the groundwork for the methods used today in clinical microbiology.  The method he describes is termed S-clustering, and will have a significant bearing on how we can view hematology data.  He describes S-clustering as extracting features from endogenous data that amplify or maximize structural information to create distinctive classes.  The method classifies by taking the number of features with sufficient variety to map into a theoretic standard. The mapping is done by a truth table, and each variable is scaled to assign values for each: message choice.  The number of messages and the number of choices forms an N-by N table.  He points out that the message choice in an antibody titer would be converted from 0 + ++ +++ to 0 1 2 3.

Even though there may be a large number of measured values, the variety is reduced by this compression, even though there is risk of loss of information.  Yet the real issue is how a combination of variables falls into a table with meaningful information.  We are concerned with accurate assignment into uniquely variable groups by information in test relationships. One determines the effectiveness of each variable by its contribution to information gain in the system.  The reference or null set is the class having no information.  Uncertainty in assigning to a classification is only relieved by providing sufficient information.  One determines the effectiveness of each variable by its contribution to information gain in the system.  The possibility for realizing a good model for approximating the effects of factors supported by data used for inference owes much to the discovery of Kullback-Liebler distance or “information”, and Akaike found a simple relationship between K-L information and Fisher’s maximized log-likelihood function. A solid foundation in this work was elaborated by Eugene Rypka.  Of course, this was made far less complicated by the genetic complement that defines its function, which made  more accessible the study of biochemical pathways.  In addition, the genetic relationships in plant genetics were accessible to Ronald Fisher for the application of the linear discriminant function.    In the last 60 years the application of entropy comparable to the entropy of physics, information, noise, and signal processing, has been fully developed by Shannon, Kullback, and others,  and has been integrated with modern statistics, as a result of the seminal work of Akaike, Leo Goodman, Magidson and Vermunt, and unrelated work by Coifman. Dr. Magidson writes about Latent Class Model evolution:

The recent increase in interest in latent class models is due to the development of extended algorithms which allow today’s computers to perform LC analyses on data containing more than just a few variables, and the recent realization that the use of such models can yield powerful improvements over traditional approaches to segmentation, as well as to cluster, factor, regression and other kinds of analysis.

Perhaps the application to medical diagnostics had been slowed by limitations of data capture and computer architecture as well as lack of clarity in definition of what are the most distinguishing features needed for diagnostic clarification.  Bernstein and colleagues had a series of studies using Kullback-Liebler Distance  (effective information) for clustering to examine the latent structure of the elements commonly used for diagnosis of myocardial infarction (CK-MB, LD and the isoenzyme-1 of LD),  protein-energy malnutrition (serum albumin, serum transthyretin, condition associated with protein malnutrition (see Jeejeebhoy and subjective global assessment), prolonged period with no oral intake), prediction of respiratory distress syndrome of the newborn (RDS), and prediction of lymph nodal involvement of prostate cancer, among other studies.   The exploration of syndromic classification has made a substantial contribution to the diagnostic literature, but has only been made useful through publication on the web of calculators and nomograms (such as Epocrates and Medcalc) accessible to physicians through an iPhone.  These are not an integral part of the EMR, and the applications require an anticipation of the need for such processing.

Gil David et al. introduced an AUTOMATED processing of the data available to the ordering physician and can anticipate an enormous impact in diagnosis and treatment of perhaps half of the top 20 most common causes of hospital admission that carry a high cost and morbidity.  For example: anemias (iron deficiency, vitamin B12 and folate deficiency, and hemolytic anemia or myelodysplastic syndrome); pneumonia; systemic inflammatory response syndrome (SIRS) with or without bacteremia; multiple organ failure and hemodynamic shock; electrolyte/acid base balance disorders; acute and chronic liver disease; acute and chronic renal disease; diabetes mellitus; protein-energy malnutrition; acute respiratory distress of the newborn; acute coronary syndrome; congestive heart failure; disordered bone mineral metabolism; hemostatic disorders; leukemia and lymphoma; malabsorption syndromes; and cancer(s)[breast, prostate, colorectal, pancreas, stomach, liver, esophagus, thyroid, and parathyroid].

Extension of conditions and presentation to the electronic medical record (EMR)

We have published on the application of an automated inference engine to the Systemic Inflammatory Response (SIRS), a serious infection, or emerging sepsis.  We can report on this without going over previous ground.  Of considerable interest is the morbidity and mortality of sepsis, and the hospital costs from a late diagnosis.  If missed early, it could be problematic, and it could be seen as a hospital complication when it is not. Improving on previous work, we have the opportunity to look at the contribution of a fluorescence labeled flow cytometric measurement of the immature granulocytes (IG), which is now widely used, but has not been adequately evaluated from the perspective of diagnostic usage.  We have done considerable work on protein-energy malnutrition (PEM), to which the automated interpretation is currently in review.  Of course, the

cholesterol, lymphocyte count, serum albumin provide the weight of evidence with the primary diagnosis (emphysema, chronic renal disease, eating disorder), and serum transthyretin would be low and remain low for a week in critical care.  This could be a modifier with age in providing discriminatory power.

Chapter  3           References

The Cost Burden of Disease: U.S. and Michigan. CHRT Brief. January 2010. @www.chrt.org

The National Hospital Bill: The Most Expensive Conditions by Payer, 2006. HCUP Brief #59.

Rudolph RA, Bernstein LH, Babb J: Information-Induction for the diagnosis of

myocardial infarction. Clin Chem 1988;34:2031-2038.

Bernstein LH (Chairman). Prealbumin in Nutritional Care Consensus Group.

Measurement of visceral protein status in assessing protein and energy malnutrition: standard of care. Nutrition 1995; 11:169-171.

Bernstein LH, Qamar A, McPherson C, Zarich S, Rudolph R. Diagnosis of myocardial infarction: integration of serum markers and clinical descriptors using information theory. Yale J Biol Med 1999; 72: 5-13.

Kaplan L.A.; Chapman J.F.; Bock J.L.; Santa Maria E.; Clejan S.; Huddleston D.J.; Reed R.G.; Bernstein L.H.; Gillen-Goldstein J. Prediction of Respiratory Distress Syndrome using the Abbott FLM-II amniotic fluid assay. The National Academy of Clinical Biochemistry (NACB) Fetal Lung Maturity Assessment Project.  Clin Chim Acta 2002; 326(8): 61-68.

Bernstein LH, Qamar A, McPherson C, Zarich S. Evaluating a new graphical ordinal logit method (GOLDminer) in the diagnosis of myocardial infarction utilizing clinical features and laboratory data. Yale J Biol Med 1999; 72:259-268.

Bernstein L, Bradley K, Zarich SA. GOLDmineR: Improving models for classifying patients with chest pain. Yale J Biol Med 2002; 75, pp. 183-198.

Ronald Raphael Coifman and Mladen Victor Wickerhauser. Adapted Waveform Analysis as a Tool for Modeling, Feature Extraction, and Denoising. Optical Engineering, 33(7):2170–2174, July 1994.

R. Coifman and N. Saito. Constructions of local orthonormal bases for classification and regression. C. R. Acad. Sci. Paris, 319 Série I:191-196, 1994.

Chapter 4           Clinical Expert System

Realtime Clinical Expert Support and validation System

We have developed a software system that is the equivalent of an intelligent Electronic Health Records Dashboard that provides empirical medical reference and suggests quantitative diagnostics options. The primary purpose is to gather medical information, generate metrics, analyze them in realtime and provide a differential diagnosis, meeting the highest standard of accuracy. The system builds its unique characterization and provides a list of other patients that share this unique profile, therefore utilizing the vast aggregated knowledge (diagnosis, analysis, treatment, etc.) of the medical community. The main mathematical breakthroughs are provided by accurate patient profiling and inference methodologies in which anomalous subprofiles are extracted and compared to potentially relevant cases. As the model grows and its knowledge database is extended, the diagnostic and the prognostic become more accurate and precise. We anticipate that the effect of implementing this diagnostic amplifier would result in higher physician productivity at a time of great human resource limitations, safer prescribing practices, rapid identification of unusual patients, better assignment of patients to observation, inpatient beds, intensive care, or referral to clinic, shortened length of patients ICU and bed days.

The main benefit is a real time assessment as well as diagnostic options based on comparable cases, flags for risk and potential problems as illustrated in the following case acquired on 04/21/10. The patient was diagnosed by our system with severe SIRS at a grade of 0.61 .

The patient was treated for SIRS and the blood tests were repeated during the following week. The full combined record of our system’s assessment of the patient, were derived from the further Hematology tests.  Following treatment, the SIRS risk as a major concern was eliminated and the system provides a positive feedback for the treatment of the physician.

 

Method for data organization and classification via characterization metrics.

Our database organized to enable linking a given profile to known profiles. This is achieved by associating a patient to a peer group of patients having an overall similar profile, where the similar profile is obtained through a randomized search for an appropriate weighting of variables. Given the selection of a patients’ peer group, we build a metric that measures the dissimilarity of the patient from its group. This is achieved through a local iterated statistical analysis in the peer group.

We then use this characteristic metric to locate other patients with similar unique profiles, for each of whom we repeat the procedure described above. This leads to a network of patients with similar risk condition. Then, the classification of the patient is inferred from the medical known condition of some of the patients in the linked network. Given a set of points (the database) and a newly arrived sample (point), we characterize the behavior of the newly arrived sample, according to the database. Then, we detect other points in the database that match this unique characterization. This collection of detected points defines the characteristic neighborhood of the newly arrived sample. We use the characteristic neighbor hood in order to classify the newly arrived sample. This process of differential diagnosis is repeated for every newly arrived point.   The medical colossus we have today has become a system out of control and beset by the elephant in the room – an uncharted complexity. We offer a method that addresses the complexity and enables rather than disables the practitioner.  The method identifies outliers and combines data according to commonality of features.

Summary and Perspectives: Impairments in Pathological States: Endocrine Disorders, Stress Hypermetabolism and Cancer

Author and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/11/09/summary-and-perspectives-impairments-in-pathological-states-endocrine-disorders-stress-hypermetabolism-cancer/

This summary is the last of a series on the impact of transcriptomics, proteomics, and metabolomics on disease investigation, and the sorting and integration of genomic signatures and metabolic signatures to explain phenotypic relationships in variability and individuality of response to disease expression and how this leads to  pharmaceutical discovery and personalized medicine.  We have unquestionably better tools at our disposal than has ever existed in the history of mankind, and an enormous knowledge-base that has to be accessed.  I shall conclude here these discussions with the powerful contribution to and current knowledge pertaining to biochemistry, metabolism, protein-interactions, signaling, and the application of the -OMICS to diseases and drug discovery at this time.

The Ever-Transcendent Cell

Deriving physiologic first principles By John S. Torday | The Scientist Nov 1, 2014
http://www.the-scientist.com/?articles.view/articleNo/41282/title/The-Ever-Transcendent-Cell/

Both the developmental and phylogenetic histories of an organism describe the evolution of physiology—the complex of metabolic pathways that govern the function of an organism as a whole. The necessity of establishing and maintaining homeostatic mechanisms began at the cellular level, with the very first cells, and homeostasis provides the underlying selection pressure fueling evolution.

While the events leading to the formation of the first functioning cell are debatable, a critical one was certainly the formation of simple lipid-enclosed vesicles, which provided a protected space for the evolution of metabolic pathways. Protocells evolved from a common ancestor that experienced environmental stresses early in the history of cellular development, such as acidic ocean conditions and low atmospheric oxygen levels, which shaped the evolution of metabolism.

The reduction of evolution to cell biology may answer the perennially unresolved question of why organisms return to their unicellular origins during the life cycle.

As primitive protocells evolved to form prokaryotes and, much later, eukaryotes, changes to the cell membrane occurred that were critical to the maintenance of chemiosmosis, the generation of bioenergy through the partitioning of ions. The incorporation of cholesterol into the plasma membrane surrounding primitive eukaryotic cells marked the beginning of their differentiation from prokaryotes. Cholesterol imparted more fluidity to eukaryotic cell membranes, enhancing functionality by increasing motility and endocytosis. Membrane deformability also allowed for increased gas exchange.

Acidification of the oceans by atmospheric carbon dioxide generated high intracellular calcium ion concentrations in primitive aquatic eukaryotes, which had to be lowered to prevent toxic effects, namely the aggregation of nucleotides, proteins, and lipids. The early cells achieved this by the evolution of calcium channels composed of cholesterol embedded within the cell’s plasma membrane, and of internal membranes, such as that of the endoplasmic reticulum, peroxisomes, and other cytoplasmic organelles, which hosted intracellular chemiosmosis and helped regulate calcium.

As eukaryotes thrived, they experienced increasingly competitive pressure for metabolic efficiency. Engulfed bacteria, assimilated as mitochondria, provided more bioenergy. As the evolution of eukaryotic organisms progressed, metabolic cooperation evolved, perhaps to enable competition with biofilm-forming, quorum-sensing prokaryotes. The subsequent appearance of multicellular eukaryotes expressing cellular growth factors and their respective receptors facilitated cell-cell signaling, forming the basis for an explosion of multicellular eukaryote evolution, culminating in the metazoans.

Casting a cellular perspective on evolution highlights the integration of genotype and phenotype. Starting from the protocell membrane, the functional homolog for all complex metazoan organs, it offers a way of experimentally determining the role of genes that fostered evolution based on the ontogeny and phylogeny of cellular processes that can be traced back, in some cases, to our last universal common ancestor.  ….

As eukaryotes thrived, they experienced increasingly competitive pressure for metabolic efficiency. Engulfed bacteria, assimilated as mitochondria, provided more bioenergy. As the evolution of eukaryotic organisms progressed, metabolic cooperation evolved, perhaps to enable competition with biofilm-forming, quorum-sensing prokaryotes. The subsequent appearance of multicellular eukaryotes expressing cellular growth factors and their respective receptors facilitated cell-cell signaling, forming the basis for an explosion of multicellular eukaryote evolution, culminating in the metazoans.

Casting a cellular perspective on evolution highlights the integration of genotype and phenotype. Starting from the protocell membrane, the functional homolog for all complex metazoan organs, it offers a way of experimentally determining the role of genes that fostered evolution based on the ontogeny and phylogeny of cellular processes that can be traced back, in some cases, to our last universal common ancestor.

Given that the unicellular toolkit is complete with all the traits necessary for forming multicellular organisms (Science, 301:361-63, 2003), it is distinctly possible that metazoans are merely permutations of the unicellular body plan. That scenario would clarify a lot of puzzling biology: molecular commonalities between the skin, lung, gut, and brain that affect physiology and pathophysiology exist because the cell membranes of unicellular organisms perform the equivalents of these tissue functions, and the existence of pleiotropy—one gene affecting many phenotypes—may be a consequence of the common unicellular source for all complex biologic traits.  …

The cell-molecular homeostatic model for evolution and stability addresses how the external environment generates homeostasis developmentally at the cellular level. It also determines homeostatic set points in adaptation to the environment through specific effectors, such as growth factors and their receptors, second messengers, inflammatory mediators, crossover mutations, and gene duplications. This is a highly mechanistic, heritable, plastic process that lends itself to understanding evolution at the cellular, tissue, organ, system, and population levels, mediated by physiologically linked mechanisms throughout, without having to invoke random, chance mechanisms to bridge different scales of evolutionary change. In other words, it is an integrated mechanism that can often be traced all the way back to its unicellular origins.

The switch from swim bladder to lung as vertebrates moved from water to land is proof of principle that stress-induced evolution in metazoans can be understood from changes at the cellular level.

http://www.the-scientist.com/Nov2014/TE_21.jpg

A MECHANISTIC BASIS FOR LUNG DEVELOPMENT

The switch from swim bladder to lung as vertebrates moved from water to land is proof of principle that stress-induced evolution in metazoans can be understood from changes at the cellular level.

http://www.the-scientist.com/Nov2014/TE_21.jpg

A MECHANISTIC BASIS FOR LUNG DEVELOPMENT: Stress from periodic atmospheric hypoxia (1) during vertebrate adaptation to land enhances positive selection of the stretch-regulated parathyroid hormone-related protein (PTHrP) in the pituitary and adrenal glands. In the pituitary (2), PTHrP signaling upregulates the release of adrenocorticotropic hormone (ACTH) (3), which stimulates the release of glucocorticoids (GC) by the adrenal gland (4). In the adrenal gland, PTHrP signaling also stimulates glucocorticoid production of adrenaline (5), which in turn affects the secretion of lung surfactant, the distension of alveoli, and the perfusion of alveolar capillaries (6). PTHrP signaling integrates the inflation and deflation of the alveoli with surfactant production and capillary perfusion.  THE SCIENTIST STAFF

From a cell-cell signaling perspective, two critical duplications in genes coding for cell-surface receptors occurred during this period of water-to-land transition—in the stretch-regulated parathyroid hormone-related protein (PTHrP) receptor gene and the β adrenergic (βA) receptor gene. These gene duplications can be disassembled by following their effects on vertebrate physiology backwards over phylogeny. PTHrP signaling is necessary for traits specifically relevant to land adaptation: calcification of bone, skin barrier formation, and the inflation and distention of lung alveoli. Microvascular shear stress in PTHrP-expressing organs such as bone, skin, kidney, and lung would have favored duplication of the PTHrP receptor, since sheer stress generates radical oxygen species (ROS) known to have this effect and PTHrP is a potent vasodilator, acting as an epistatic balancing selection for this constraint.

Positive selection for PTHrP signaling also evolved in the pituitary and adrenal cortex (see figure on this page), stimulating the secretion of ACTH and corticoids, respectively, in response to the stress of land adaptation. This cascade amplified adrenaline production by the adrenal medulla, since corticoids passing through it enzymatically stimulate adrenaline synthesis. Positive selection for this functional trait may have resulted from hypoxic stress that arose during global episodes of atmospheric hypoxia over geologic time. Since hypoxia is the most potent physiologic stressor, such transient oxygen deficiencies would have been acutely alleviated by increasing adrenaline levels, which would have stimulated alveolar surfactant production, increasing gas exchange by facilitating the distension of the alveoli. Over time, increased alveolar distension would have generated more alveoli by stimulating PTHrP secretion, impelling evolution of the alveolar bed of the lung.

This scenario similarly explains βA receptor gene duplication, since increased density of the βA receptor within the alveolar walls was necessary for relieving another constraint during the evolution of the lung in adaptation to land: the bottleneck created by the existence of a common mechanism for blood pressure control in both the lung alveoli and the systemic blood pressure. The pulmonary vasculature was constrained by its ability to withstand the swings in pressure caused by the systemic perfusion necessary to sustain all the other vital organs. PTHrP is a potent vasodilator, subserving the blood pressure constraint, but eventually the βA receptors evolved to coordinate blood pressure in both the lung and the periphery.

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Summary – Volume 4, Part 2: Translational Medicine in Cardiovascular Diseases


Summary – Volume 4, Part 2:  Translational Medicine in Cardiovascular Diseases

Author and Curator: Larry H Bernstein, MD, FCAP

 

We have covered a large amount of material that involves

  • the development,
  • application, and
  • validation of outcomes of medical and surgical procedures

that are based on translation of science from the laboratory to the bedside, improving the standards of medical practice at an accelerated pace in the last quarter century, and in the last decade.  Encouraging enabling developments have been:

1. The establishment of national and international outcomes databases for procedures by specialist medical societies

Stent Design and Thrombosis: Bifurcation Intervention, Drug Eluting Stents (DES) and Biodegrable Stents
Curator: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/08/06/stent-design-and-thrombosis-bifurcation-intervention-drug-eluting-stents-des-and-biodegrable-stents/

On Devices and On Algorithms: Prediction of Arrhythmia after Cardiac Surgery and ECG Prediction of an Onset of Paroxysmal Atrial Fibrillation
Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC
https://pharmaceuticalintelligence.com/2013/05/07/on-devices-and-on-algorithms-arrhythmia-after-cardiac-surgery-prediction-and-ecg-prediction-of-paroxysmal-atrial-fibrillation-onset/

Mitral Valve Repair: Who is a Patient Candidate for a Non-Ablative Fully Non-Invasive Procedure?
Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC and Article Curator: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/11/04/mitral-valve-repair-who-is-a-candidate-for-a-non-ablative-fully-non-invasive-procedure/

Cardiovascular Complications: Death from Reoperative Sternotomy after prior CABG, MVR, AVR, or Radiation; Complications of PCI; Sepsis from Cardiovascular Interventions
Author, Introduction and Summary: Justin D Pearlman, MD, PhD, FACC and Article Curator: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/07/23/cardiovascular-complications-of-multiple-etiologies-repeat-sternotomy-post-cabg-or-avr-post-pci-pad-endoscopy-andor-resultant-of-systemic-sepsis/

Survivals Comparison of Coronary Artery Bypass Graft (CABG) and Percutaneous Coronary Intervention (PCI) /Coronary Angioplasty
Larry H. Bernstein, MD, Writer And Aviva Lev-Ari, PhD, RN, Curator
https://pharmaceuticalintelligence.com/2013/06/23/comparison-of-cardiothoracic-bypass-and-percutaneous-interventional-catheterization-survivals/

Revascularization: PCI, Prior History of PCI vs CABG
Curator: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/04/25/revascularization-pci-prior-history-of-pci-vs-cabg/

Outcomes in High Cardiovascular Risk Patients: Prasugrel (Effient) vs. Clopidogrel (Plavix); Aliskiren (Tekturna) added to ACE or added to ARB
Reporter and Curator: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2012/08/27/outcomes-in-high-cardiovascular-risk-patients-prasugrel-effient-vs-clopidogrel-plavix-aliskiren-tekturna-added-to-ace-or-added-to-arb/

Endovascular Lower-extremity Revascularization Effectiveness: Vascular Surgeons (VSs), Interventional Cardiologists (ICs) and Interventional Radiologists (IRs)
Curator: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2012/08/13/coronary-artery-disease-medical-devices-solutions-from-first-in-man-stent-implantation-via-medical-ethical-dilemmas-to-drug-eluting-stents/

and more

2. The identification of problem areas, particularly in activation of the prothrombotic pathways, infection control to an extent, and targeting of pathways leading to progression or to arrythmogenic complications.

Cardiovascular Complications: Death from Reoperative Sternotomy after prior CABG, MVR, AVR, or Radiation; Complications of PCI; Sepsis from Cardiovascular Interventions Author, Introduction and Summary: Justin D Pearlman, MD, PhD, FACC and Article Curator: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/07/23/cardiovascular-complications-of-multiple-etiologies-repeat-sternotomy-post-cabg-or-avr-post-pci-pad-endoscopy-andor-resultant-of-systemic-sepsis/

Anticoagulation genotype guided dosing
Larry H. Bernstein, MD, FCAP, Author and Curator
https://pharmaceuticalintelligence.com/2013/12/08/anticoagulation-genotype-guided-dosing/

Stent Design and Thrombosis: Bifurcation Intervention, Drug Eluting Stents (DES) and Biodegrable Stents
Curator: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/08/06/stent-design-and-thrombosis-bifurcation-intervention-drug-eluting-stents-des-and-biodegrable-stents/

The Effects of Aprotinin on Endothelial Cell Coagulant Biology
Co-Author (Kamran Baig, MBBS, James Jaggers, MD, Jeffrey H. Lawson, MD, PhD) and Curator
https://pharmaceuticalintelligence.com/2013/07/20/the-effects-of-aprotinin-on-endothelial-cell-coagulant-biology/

Outcomes in High Cardiovascular Risk Patients: Prasugrel (Effient) vs. Clopidogrel (Plavix); Aliskiren (Tekturna) added to ACE or added to ARB
Reporter and Curator: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2012/08/27/outcomes-in-high-cardiovascular-risk-patients-prasugrel-effient-vs-clopidogrel-plavix-aliskiren-tekturna-added-to-ace-or-added-to-arb/

Pharmacogenomics – A New Method for Druggability  Author and Curator: Demet Sag, PhD
https://pharmaceuticalintelligence.com/2014/04/28/pharmacogenomics-a-new-method-for-druggability/

Advanced Topics in Sepsis and the Cardiovascular System at its End Stage    Author: Larry H Bernstein, MD, FCAP
https://pharmaceuticalintelligence.com/2013/08/18/advanced-topics-in-Sepsis-and-the-Cardiovascular-System-at-its-End-Stage/

3. Development of procedures that use a safer materials in vascular management.

Stent Design and Thrombosis: Bifurcation Intervention, Drug Eluting Stents (DES) and Biodegrable Stents
Curator: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/08/06/stent-design-and-thrombosis-bifurcation-intervention-drug-eluting-stents-des-and-biodegrable-stents/

Biomaterials Technology: Models of Tissue Engineering for Reperfusion and Implantable Devices for Revascularization
Author and Curator: Larry H Bernstein, MD, FACP and Curator: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/05/05/bioengineering-of-vascular-and-tissue-models/

Vascular Repair: Stents and Biologically Active Implants
Author and Curator: Larry H Bernstein, MD, FACP and Curator: Aviva Lev-Ari, RN, PhD
https://pharmaceuticalintelligence.com/2013/05/04/stents-biologically-active-implants-and-vascular-repair/

Drug Eluting Stents: On MIT’s Edelman Lab’s Contributions to Vascular Biology and its Pioneering Research on DES
Author: Larry H Bernstein, MD, FACP and Curator: Aviva Lev-Ari, PhD, RN
http://PharmaceuticalIntelligence.com/2013/04/25/Contributions-to-vascular-biology/

MedTech & Medical Devices for Cardiovascular Repair – Curations by Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2014/04/17/medtech-medical-devices-for-cardiovascular-repair-curation-by-aviva-lev-ari-phd-rn/

4. Discrimination of cases presenting for treatment based on qualifications for medical versus surgical intervention.

Treatment Options for Left Ventricular Failure – Temporary Circulatory Support: Intra-aortic balloon pump (IABP) – Impella Recover LD/LP 5.0 and 2.5, Pump Catheters (Non-surgical) vs Bridge Therapy: Percutaneous Left Ventricular Assist Devices (pLVADs) and LVADs (Surgical)
Author: Larry H Bernstein, MD, FCAP And Curator: Justin D Pearlman, MD, PhD, FACC
https://pharmaceuticalintelligence.com/2013/07/17/treatment-options-for-left-ventricular-failure-temporary-circulatory-support-intra-aortic-balloon-pump-iabp-impella-recover-ldlp-5-0-and-2-5-pump-catheters-non-surgical-vs-bridge-therapy/

Coronary Reperfusion Therapies: CABG vs PCI – Mayo Clinic preprocedure Risk Score (MCRS) for Prediction of in-Hospital Mortality after CABG or PCI
Writer and Curator: Larry H. Bernstein, MD, FCAP and Curator: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/06/30/mayo-risk-score-for-percutaneous-coronary-intervention/

ACC/AHA Guidelines for Coronary Artery Bypass Graft Surgery Reporter: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/11/05/accaha-guidelines-for-coronary-artery-bypass-graft-surgery/

Mitral Valve Repair: Who is a Patient Candidate for a Non-Ablative Fully Non-Invasive Procedure?
Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC and Article Curator: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/11/04/mitral-valve-repair-who-is-a-candidate-for-a-non-ablative-fully-non-invasive-procedure/ 

5.  This has become possible because of the advances in our knowledge of key related pathogenetic mechanisms involving gene expression and cellular regulation of complex mechanisms.

What is the key method to harness Inflammation to close the doors for many complex diseases?
Author and Curator: Larry H Bernstein, MD, FCAP
https://pharmaceuticalintelligence.com/2014/03/21/what-is-the-key-method-to-harness-inflammation-to-close-the-doors-for-many-complex-diseases/

CVD Prevention and Evaluation of Cardiovascular Imaging Modalities: Coronary Calcium Score by CT Scan Screening to justify or not the Use of Statin
Curator: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2014/03/03/cvd-prevention-and-evaluation-of-cardiovascular-imaging-modalities-coronary-calcium-score-by-ct-scan-screening-to-justify-or-not-the-use-of-statin/

Richard Lifton, MD, PhD of Yale University and Howard Hughes Medical Institute: Recipient of 2014 Breakthrough Prizes Awarded in Life Sciences for the Discovery of Genes and Biochemical Mechanisms that cause Hypertension
Curator: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2014/03/03/richard-lifton-md-phd-of-yale-university-and-howard-hughes-medical-institute-recipient-of-2014-breakthrough-prizes-awarded-in-life-sciences-for-the-discovery-of-genes-and-biochemical-mechanisms-tha/

Pathophysiological Effects of Diabetes on Ischemic-Cardiovascular Disease and on Chronic Obstructive Pulmonary Disease (COPD)
Curator:  Larry H. Bernstein, MD, FCAP
https://pharmaceuticalintelligence.com/2014/01/15/pathophysiological-effects-of-diabetes-on-ischemic-cardiovascular-disease-and-on-chronic-obstructive-pulmonary-disease-copd/

Atherosclerosis Independence: Genetic Polymorphisms of Ion Channels Role in the Pathogenesis of Coronary Microvascular Dysfunction and Myocardial Ischemia (Coronary Artery Disease (CAD))
Reviewer and Co-Curator: Larry H Bernstein, MD, CAP and Curator: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/12/21/genetic-polymorphisms-of-ion-channels-have-a-role-in-the-pathogenesis-of-coronary-microvascular-dysfunction-and-ischemic-heart-disease/

Notable Contributions to Regenerative Cardiology  Author and Curator: Larry H Bernstein, MD, FCAP and Article Commissioner: Aviva Lev-Ari, PhD, RD
https://pharmaceuticalintelligence.com/2013/10/20/notable-contributions-to-regenerative-cardiology/

As noted in the introduction, any of the material can be found and reviewed by content, and the eTOC is identified in attached:

http://wp.me/p2xfv8-1W

 

This completes what has been presented in Part 2, Vol 4 , and supporting references for the main points that are found in the Leaders in Pharmaceutical Intelligence Cardiovascular book.  Part 1 was concerned with Posttranslational Modification of Proteins, vital for understanding cellular regulation and dysregulation.  Part 2 was concerned with Translational Medical Therapeutics, the efficacy of medical and surgical decisions based on bringing the knowledge gained from the laboratory, and from clinical trials into the realm opf best practice.  The time for this to occur in practice in the past has been through roughly a generation of physicians.  That was in part related to the busy workload of physicians, and inability to easily access specialty literature as the volume and complexity increased.  This had an effect of making access of a family to a primary care provider through a lifetime less likely than the period post WWII into the 1980s.

However, the growth of knowledge has accelerated in the specialties since the 1980’s so that the use of physician referral in time became a concern about the cost of medical care.  This is not the place for or a matter for discussion here.  It is also true that the scientific advances and improvements in available technology have had a great impact on medical outcomes.  The only unrelated issue is that of healthcare delivery, which is not up to the standard set by serial advances in therapeutics, accompanied by high cost due to development costs, marketing costs, and development of drug resistance.

I shall identify continuing developments in cardiovascular diagnostics, therapeutics, and bioengineering that is and has been emerging.

1. Mechanisms of disease

REPORT: Mapping the Cellular Response to Small Molecules Using Chemogenomic Fitness Signatures 

Science 11 April 2014:
Vol. 344 no. 6180 pp. 208-211
http://dx.doi.org/10.1126/science.1250217

Abstract: Genome-wide characterization of the in vivo cellular response to perturbation is fundamental to understanding how cells survive stress. Identifying the proteins and pathways perturbed by small molecules affects biology and medicine by revealing the mechanisms of drug action. We used a yeast chemogenomics platform that quantifies the requirement for each gene for resistance to a compound in vivo to profile 3250 small molecules in a systematic and unbiased manner. We identified 317 compounds that specifically perturb the function of 121 genes and characterized the mechanism of specific compounds. Global analysis revealed that the cellular response to small molecules is limited and described by a network of 45 major chemogenomic signatures. Our results provide a resource for the discovery of functional interactions among genes, chemicals, and biological processes.

Yeasty HIPHOP

Laura Zahn
Sci. Signal. 15 April 2014; 7(321): ec103.   http://dx.doi.org/10.1126/scisignal.2005362

In order to identify how chemical compounds target genes and affect the physiology of the cell, tests of the perturbations that occur when treated with a range of pharmacological chemicals are required. By examining the haploinsufficiency profiling (HIP) and homozygous profiling (HOP) chemogenomic platforms, Lee et al.(p. 208) analyzed the response of yeast to thousands of different small molecules, with genetic, proteomic, and bioinformatic analyses. Over 300 compounds were identified that targeted 121 genes within 45 cellular response signature networks. These networks were used to extrapolate the likely effects of related chemicals, their impact upon genetic pathways, and to identify putative gene functions

Key Heart Failure Culprit Discovered

A team of cardiovascular researchers from the Cardiovascular Research Center at Icahn School of Medicine at Mount Sinai, Sanford-Burnham Medical Research Institute, and University of California, San Diego have identified a small, but powerful, new player in thIe onset and progression of heart failure. Their findings, published in the journal Nature  on March 12, also show how they successfully blocked the newly discovered culprit.
Investigators identified a tiny piece of RNA called miR-25 that blocks a gene known as SERCA2a, which regulates the flow of calcium within heart muscle cells. Decreased SERCA2a activity is one of the main causes of poor contraction of the heart and enlargement of heart muscle cells leading to heart failure.

Using a functional screening system developed by researchers at Sanford-Burnham, the research team discovered miR-25 acts pathologically in patients suffering from heart failure, delaying proper calcium uptake in heart muscle cells. According to co-lead study authors Christine Wahlquist and Dr. Agustin Rojas Muñoz, developers of the approach and researchers in Mercola’s lab at Sanford-Burnham, they used high-throughput robotics to sift through the entire genome for microRNAs involved in heart muscle dysfunction.

Subsequently, the researchers at the Cardiovascular Research Center at Icahn School of Medicine at Mount Sinai found that injecting a small piece of RNA to inhibit the effects of miR-25 dramatically halted heart failure progression in mice. In addition, it also improved their cardiac function and survival.

“In this study, we have not only identified one of the key cellular processes leading to heart failure, but have also demonstrated the therapeutic potential of blocking this process,” says co-lead study author Dr. Dongtak Jeong, a post-doctoral fellow at the Cardiovascular Research Center at Icahn School of  Medicine at Mount Sinai in the laboratory of the study’s co-senior author Dr. Roger J. Hajjar.

Publication: Inhibition of miR-25 improves cardiac contractility in the failing heart.Christine Wahlquist, Dongtak Jeong, Agustin Rojas-Muñoz, Changwon Kho, Ahyoung Lee, Shinichi Mitsuyama, Alain Van Mil, Woo Jin Park, Joost P. G. Sluijter, Pieter A. F. Doevendans, Roger J. :  Hajjar & Mark Mercola.     Nature (March 2014)    http://www.nature.com/nature/journal/vaop/ncurrent/full/nature13073.html

 

“Junk” DNA Tied to Heart Failure

Deep RNA Sequencing Reveals Dynamic Regulation of Myocardial Noncoding RNAs in Failing Human Heart and Remodeling With Mechanical Circulatory Support

Yang KC, Yamada KA, Patel AY, Topkara VK, George I, et al.
Circulation 2014;  129(9):1009-21.
http://dx.doi.org/10.1161/CIRCULATIONAHA.113.003863              http://circ.ahajournals.org/…/CIRCULATIONAHA.113.003863.full

The myocardial transcriptome is dynamically regulated in advanced heart failure and after LVAD support. The expression profiles of lncRNAs, but not mRNAs or miRNAs, can discriminate failing hearts of different pathologies and are markedly altered in response to LVAD support. These results suggest an important role for lncRNAs in the pathogenesis of heart failure and in reverse remodeling observed with mechanical support.

Junk DNA was long thought to have no important role in heredity or disease because it doesn’t code for proteins. But emerging research in recent years has revealed that many of these sections of the genome produce noncoding RNA molecules that still have important functions in the body. They come in a variety of forms, some more widely studied than others. Of these, about 90% are called long noncoding RNAs (lncRNAs), and exploration of their roles in health and disease is just beginning.

The Washington University group performed a comprehensive analysis of all RNA molecules expressed in the human heart. The researchers studied nonfailing hearts and failing hearts before and after patients received pump support from left ventricular assist devices (LVAD). The LVADs increased each heart’s pumping capacity while patients waited for heart transplants.

In their study, the researchers found that unlike other RNA molecules, expression patterns of long noncoding RNAs could distinguish between two major types of heart failure and between failing hearts before and after they received LVAD support.

“The myocardial transcriptome is dynamically regulated in advanced heart failure and after LVAD support. The expression profiles of lncRNAs, but not mRNAs or miRNAs, can discriminate failing hearts of different pathologies and are markedly altered in response to LVAD support,” wrote the researchers. “These results suggest an important role for lncRNAs in the pathogenesis of heart failure and in reverse remodeling observed with mechanical support.”

‘Junk’ Genome Regions Linked to Heart Failure

In a recent issue of the journal Circulation, Washington University investigators report results from the first comprehensive analysis of all RNA molecules expressed in the human heart. The researchers studied nonfailing hearts and failing hearts before and after patients received pump support from left ventricular assist devices (LVAD). The LVADs increased each heart’s pumping capacity while patients waited for heart transplants.

“We took an unbiased approach to investigating which types of RNA might be linked to heart failure,” said senior author Jeanne Nerbonne, the Alumni Endowed Professor of Molecular Biology and Pharmacology. “We were surprised to find that long noncoding RNAs stood out.

In the new study, the investigators found that unlike other RNA molecules, expression patterns of long noncoding RNAs could distinguish between two major types of heart failure and between failing hearts before and after they received LVAD support.

“We don’t know whether these changes in long noncoding RNAs are a cause or an effect of heart failure,” Nerbonne said. “But it seems likely they play some role in coordinating the regulation of multiple genes involved in heart function.”

Nerbonne pointed out that all types of RNA molecules they examined could make the obvious distinction: telling the difference between failing and nonfailing hearts. But only expression of the long noncoding RNAs was measurably different between heart failure associated with a heart attack (ischemic) and heart failure without the obvious trigger of blocked arteries (nonischemic). Similarly, only long noncoding RNAs significantly changed expression patterns after implantation of left ventricular assist devices.

Comment

Decoding the noncoding transcripts in human heart failure

Xiao XG, Touma M, Wang Y
Circulation. 2014; 129(9): 958960,  http://dx.doi.org/10.1161/CIRCULATIONAHA.114.007548 

Heart failure is a complex disease with a broad spectrum of pathological features. Despite significant advancement in clinical diagnosis through improved imaging modalities and hemodynamic approaches, reliable molecular signatures for better differential diagnosis and better monitoring of heart failure progression remain elusive. The few known clinical biomarkers for heart failure, such as plasma brain natriuretic peptide and troponin, have been shown to have limited use in defining the cause or prognosis of the disease.1,2 Consequently, current clinical identification and classification of heart failure remain descriptive, mostly based on functional and morphological parameters. Therefore, defining the pathogenic mechanisms for hypertrophic versus dilated or ischemic versus nonischemic cardiomyopathies in the failing heart remain a major challenge to both basic science and clinic researchers. In recent years, mechanical circulatory support using left ventricular assist devices (LVADs) has assumed a growing role in the care of patients with end-stage heart failure.3 During the earlier years of LVAD application as a bridge to transplant, it became evident that some patients exhibit substantial recovery of ventricular function, structure, and electric properties.4 This led to the recognition that reverse remodeling is potentially an achievable therapeutic goal using LVADs. However, the underlying mechanism for the reverse remodeling in the LVAD-treated hearts is unclear, and its discovery would likely hold great promise to halt or even reverse the progression of heart failure.

 

Efficacy and Safety of Dabigatran Compared With Warfarin in Relation to Baseline Renal Function in Patients With Atrial Fibrillation: A RE-LY (Randomized Evaluation of Long-term Anticoagulation Therapy) Trial Analysis

Circulation. 2014; 129: 951-952     http://dx.doi.org/10.1161/​CIR.0000000000000022

In patients with atrial fibrillation, impaired renal function is associated with a higher risk of thromboembolic events and major bleeding. Oral anticoagulation with vitamin K antagonists reduces thromboembolic events but raises the risk of bleeding. The new oral anticoagulant dabigatran has 80% renal elimination, and its efficacy and safety might, therefore, be related to renal function. In this prespecified analysis from the Randomized Evaluation of Long-Term Anticoagulant Therapy (RELY) trial, outcomes with dabigatran versus warfarin were evaluated in relation to 4 estimates of renal function, that is, equations based on creatinine levels (Cockcroft-Gault, Modification of Diet in Renal Disease (MDRD), Chronic Kidney Disease Epidemiology Collaboration [CKD-EPI]) and cystatin C. The rates of stroke or systemic embolism were lower with dabigatran 150 mg and similar with 110 mg twice daily irrespective of renal function. Rates of major bleeding were lower with dabigatran 110 mg and similar with 150 mg twice daily across the entire range of renal function. However, when the CKD-EPI or MDRD equations were used, there was a significantly greater relative reduction in major bleeding with both doses of dabigatran than with warfarin in patients with estimated glomerular filtration rate ≥80 mL/min. These findings show that dabigatran can be used with the same efficacy and adequate safety in patients with a wide range of renal function and that a more accurate estimate of renal function might be useful for improved tailoring of anticoagulant treatment in patients with atrial fibrillation and an increased risk of stroke.

Aldosterone Regulates MicroRNAs in the Cortical Collecting Duct to Alter Sodium Transport.

Robert S Edinger, Claudia Coronnello, Andrew J Bodnar, William A Laframboise, Panayiotis V Benos, Jacqueline Ho, John P Johnson, Michael B Butterworth

Journal of the American Society of Nephrology (Impact Factor: 8.99). 04/2014;     http://dx. DO.org/I:10.1681/ASN.2013090931

Source: PubMed

ABSTRACT A role for microRNAs (miRs) in the physiologic regulation of sodium transport in the kidney has not been established. In this study, we investigated the potential of aldosterone to alter miR expression in mouse cortical collecting duct (mCCD) epithelial cells. Microarray studies demonstrated the regulation of miR expression by aldosterone in both cultured mCCD and isolated primary distal nephron principal cells.

Aldosterone regulation of the most significantly downregulated miRs, mmu-miR-335-3p, mmu-miR-290-5p, and mmu-miR-1983 was confirmed by quantitative RT-PCR. Reducing the expression of these miRs separately or in combination increased epithelial sodium channel (ENaC)-mediated sodium transport in mCCD cells, without mineralocorticoid supplementation. Artificially increasing the expression of these miRs by transfection with plasmid precursors or miR mimic constructs blunted aldosterone stimulation of ENaC transport.

Using a newly developed computational approach, termed ComiR, we predicted potential gene targets for the aldosterone-regulated miRs and confirmed ankyrin 3 (Ank3) as a novel aldosterone and miR-regulated protein.

A dual-luciferase assay demonstrated direct binding of the miRs with the Ank3-3′ untranslated region. Overexpression of Ank3 increased and depletion of Ank3 decreased ENaC-mediated sodium transport in mCCD cells. These findings implicate miRs as intermediaries in aldosterone signaling in principal cells of the distal kidney nephron.

 

2. Diagnostic Biomarker Status

A prospective study of the impact of serial troponin measurements on the diagnosis of myocardial infarction and hospital and 6-month mortality in patients admitted to ICU with non-cardiac diagnoses.

Marlies Ostermann, Jessica Lo, Michael Toolan, Emma Tuddenham, Barnaby Sanderson, Katie Lei, John Smith, Anna Griffiths, Ian Webb, James Coutts, John hambers, Paul Collinson, Janet Peacock, David Bennett, David Treacher

Critical care (London, England) (Impact Factor: 4.72). 04/2014; 18(2):R62.   http://dx.doi.org/:10.1186/cc13818

Source: PubMed

ABSTRACT Troponin T (cTnT) elevation is common in patients in the Intensive Care Unit (ICU) and associated with morbidity and mortality. Our aim was to determine the epidemiology of raised cTnT levels and contemporaneous electrocardiogram (ECG) changes suggesting myocardial infarction (MI) in ICU patients admitted for non-cardiac reasons.
cTnT and ECGs were recorded daily during week 1 and on alternate days during week 2 until discharge from ICU or death. ECGs were interpreted independently for the presence of ischaemic changes. Patients were classified into 4 groups: (i) definite MI (cTnT >=15 ng/L and contemporaneous changes of MI on ECG), (ii) possible MI (cTnT >=15 ng/L and contemporaneous ischaemic changes on ECG), (iii) troponin rise alone (cTnT >=15 ng/L), or (iv) normal. Medical notes were screened independently by two ICU clinicians for evidence that the clinical teams had considered a cardiac event.
Data from 144 patients were analysed [42% female; mean age 61.9 (SD 16.9)]. 121 patients (84%) had at least one cTnT level >=15 ng/L. A total of 20 patients (14%) had a definite MI, 27% had a possible MI, 43% had a cTNT rise without contemporaneous ECG changes, and 16% had no cTNT rise. ICU, hospital and 180 day mortality were significantly higher in patients with a definite or possible MI.Only 20% of definite MIs were recognised by the clinical team. There was no significant difference in mortality between recognised and non-recognised events.At time of cTNT rise, 100 patients (70%) were septic and 58% were on vasopressors. Patients who were septic when cTNT was elevated had an ICU mortality of 28% compared to 9% in patients without sepsis. ICU mortality of patients who were on vasopressors at time of cTNT elevation was 37% compared to 1.7% in patients not on vasopressors.
The majority of critically ill patients (84%) had a cTnT rise and 41% met criteria for a possible or definite MI of whom only 20% were recognised clinically. Mortality up to 180 days was higher in patients with a cTnT rise.

 

Prognostic performance of high-sensitivity cardiac troponin T kinetic changes adjusted for elevated admission values and the GRACE score in an unselected emergency department population.

Moritz BienerMatthias MuellerMehrshad VafaieAllan S JaffeHugo A Katus,Evangelos Giannitsis

Clinica chimica acta; international journal of clinical chemistry (Impact Factor: 2.54). 04/2014;   http://dx.doi.org/10.1016/j.cca.2014.04.007

Source: PubMed

ABSTRACT To test the prognostic performance of rising and falling kinetic changes of high-sensitivity cardiac troponin T (hs-cTnT) and the GRACE score.
Rising and falling hs-cTnT changes in an unselected emergency department population were compared.
635 patients with a hs-cTnT >99th percentile admission value were enrolled. Of these, 572 patients qualified for evaluation with rising patterns (n=254, 44.4%), falling patterns (n=224, 39.2%), or falling patterns following an initial rise (n=94, 16.4%). During 407days of follow-up, we observed 74 deaths, 17 recurrent AMI, and 79 subjects with a composite of death/AMI. Admission values >14ng/L were associated with a higher rate of adverse outcomes (OR, 95%CI:death:12.6, 1.8-92.1, p=0.01, death/AMI:6.7, 1.6-27.9, p=0.01). Neither rising nor falling changes increased the AUC of baseline values (AUC: rising 0.562 vs 0.561, p=ns, falling: 0.533 vs 0.575, p=ns). A GRACE score ≥140 points indicated a higher risk of death (OR, 95%CI: 3.14, 1.84-5.36), AMI (OR,95%CI: 1.56, 0.59-4.17), or death/AMI (OR, 95%CI: 2.49, 1.51-4.11). Hs-cTnT changes did not improve prognostic performance of a GRACE score ≥140 points (AUC, 95%CI: death: 0.635, 0.570-0.701 vs. 0.560, 0.470-0.649 p=ns, AMI: 0.555, 0.418-0.693 vs. 0.603, 0.424-0.782, p=ns, death/AMI: 0.610, 0.545-0.676 vs. 0.538, 0.454-0.622, p=ns). Coronary angiography was performed earlier in patients with rising than with falling kinetics (median, IQR [hours]:13.7, 5.5-28.0 vs. 20.8, 6.3-59.0, p=0.01).
Neither rising nor falling hs-cTnT changes improve prognostic performance of elevated hs-cTnT admission values or the GRACE score. However, rising values are more likely associated with the decision for earlier invasive strategy.

 

Troponin assays for the diagnosis of myocardial infarction and acute coronary syndrome: where do we stand?

Arie Eisenman

ABSTRACT: Under normal circumstances, most intracellular troponin is part of the muscle contractile apparatus, and only a small percentage (< 2-8%) is free in the cytoplasm. The presence of a cardiac-specific troponin in the circulation at levels above normal is good evidence of damage to cardiac muscle cells, such as myocardial infarction, myocarditis, trauma, unstable angina, cardiac surgery or other cardiac procedures. Troponins are released as complexes leading to various cut-off values depending on the assay used. This makes them very sensitive and specific indicators of cardiac injury. As with other cardiac markers, observation of a rise and fall in troponin levels in the appropriate time-frame increases the diagnostic specificity for acute myocardial infarction. They start to rise approximately 4-6 h after the onset of acute myocardial infarction and peak at approximately 24 h, as is the case with creatine kinase-MB. They remain elevated for 7-10 days giving a longer diagnostic window than creatine kinase. Although the diagnosis of various types of acute coronary syndrome remains a clinical-based diagnosis, the use of troponin levels contributes to their classification. This Editorial elaborates on the nature of troponin, its classification, clinical use and importance, as well as comparing it with other currently available cardiac markers.

Expert Review of Cardiovascular Therapy 07/2006; 4(4):509-14.   http://dx.doi.org/:10.1586/14779072.4.4.509 

 

Impact of redefining acute myocardial infarction on incidence, management and reimbursement rate of acute coronary syndromes.

Carísi A Polanczyk, Samir Schneid, Betina V Imhof, Mariana Furtado, Carolina Pithan, Luis E Rohde, Jorge P Ribeiro

ABSTRACT: Although redefinition for acute myocardial infarction (AMI) has been proposed few years ago, to date it has not been universally adopted by many institutions. The purpose of this study is to evaluate the diagnostic, prognostic and economical impact of the new diagnostic criteria for AMI. Patients consecutively admitted to the emergency department with suspected acute coronary syndromes were enrolled in this study. Troponin T (cTnT) was measured in samples collected for routine CK-MB analyses and results were not available to physicians. Patients without AMI by traditional criteria and cTnT > or = 0.035 ng/mL were coded as redefined AMI. Clinical outcomes were hospital death, major cardiac events and revascularization procedures. In-hospital management and reimbursement rates were also analyzed. Among 363 patients, 59 (16%) patients had AMI by conventional criteria, whereas additional 75 (21%) had redefined AMI, an increase of 127% in the incidence. Patients with redefined AMI were significantly older, more frequently male, with atypical chest pain and more risk factors. In multivariate analysis, redefined AMI was associated with 3.1 fold higher hospital death (95% CI: 0.6-14) and a 5.6 fold more cardiac events (95% CI: 2.1-15) compared to those without AMI. From hospital perspective, based on DRGs payment system, adoption of AMI redefinition would increase 12% the reimbursement rate [3552 Int dollars per 100 patients evaluated]. The redefined criteria result in a substantial increase in AMI cases, and allow identification of high-risk patients. Efforts should be made to reinforce the adoption of AMI redefinition, which may result in more qualified and efficient management of ACS.

International Journal of Cardiology 03/2006; 107(2):180-7. · 5.51 Impact Factor   http://www.sciencedirect.com/science/article/pii/S0167527305005279

 

3. Biomedical Engineerin3g

Safety and Efficacy of an Injectable Extracellular Matrix Hydrogel for Treating Myocardial Infarction 

Sonya B. Seif-Naraghi, Jennifer M. Singelyn, Michael A. Salvatore,  et al.
Sci Transl Med 20 February 2013 5:173ra25  http://dx.doi.org/10.1126/scitranslmed.3005503

Acellular biomaterials can stimulate the local environment to repair tissues without the regulatory and scientific challenges of cell-based therapies. A greater understanding of the mechanisms of such endogenous tissue repair is furthering the design and application of these biomaterials. We discuss recent progress in acellular materials for tissue repair, using cartilage and cardiac tissues as examples of application with substantial intrinsic hurdles, but where human translation is now occurring.

 Acellular Biomaterials: An Evolving Alternative to Cell-Based Therapies

J. A. Burdick, R. L. Mauck, J. H. Gorman, R. C. Gorman,
Sci. Transl. Med. 2013; 5, (176): 176 ps4    http://stm.sciencemag.org/content/5/176/176ps4

Acellular biomaterials can stimulate the local environment to repair tissues without the regulatory and scientific challenges of cell-based therapies. A greater understanding of the mechanisms of such endogenous tissue repair is furthering the design and application of these biomaterials. We discuss recent progress in acellular materials for tissue repair, using cartilage and cardiac tissues as examples of applications with substantial intrinsic hurdles, but where human translation is now occurring.


Instructive Nanofiber Scaffolds with VEGF Create a Microenvironment for Arteriogenesis and Cardiac Repair

Yi-Dong Lin, Chwan-Yau Luo, Yu-Ning Hu, Ming-Long Yeh, Ying-Chang Hsueh, Min-Yao Chang, et al.
Sci Transl Med 8 August 2012; 4(146):ra109.   http://dx.doi.org/ 10.1126/scitranslmed.3003841

Angiogenic therapy is a promising approach for tissue repair and regeneration. However, recent clinical trials with protein delivery or gene therapy to promote angiogenesis have failed to provide therapeutic effects. A key factor for achieving effective revascularization is the durability of the microvasculature and the formation of new arterial vessels. Accordingly, we carried out experiments to test whether intramyocardial injection of self-assembling peptide nanofibers (NFs) combined with vascular endothelial growth factor (VEGF) could create an intramyocardial microenvironment with prolonged VEGF release to improve post-infarct neovascularization in rats. Our data showed that when injected with NF, VEGF delivery was sustained within the myocardium for up to 14 days, and the side effects of systemic edema and proteinuria were significantly reduced to the same level as that of control. NF/VEGF injection significantly improved angiogenesis, arteriogenesis, and cardiac performance 28 days after myocardial infarction. NF/VEGF injection not only allowed controlled local delivery but also transformed the injected site into a favorable microenvironment that recruited endogenous myofibroblasts and helped achieve effective revascularization. The engineered vascular niche further attracted a new population of cardiomyocyte-like cells to home to the injected sites, suggesting cardiomyocyte regeneration. Follow-up studies in pigs also revealed healing benefits consistent with observations in rats. In summary, this study demonstrates a new strategy for cardiovascular repair with potential for future clinical translation.

Manufacturing Challenges in Regenerative Medicine

I. Martin, P. J. Simmons, D. F. Williams.
Sci. Transl. Med. 2014; 6(232): fs16.   http://dx.doi.org/10.1126/scitranslmed.3008558

Along with scientific and regulatory issues, the translation of cell and tissue therapies in the routine clinical practice needs to address standardization and cost-effectiveness through the definition of suitable manufacturing paradigms.

 

 

 

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Ubiquinin-Proteosome pathway, autophagy, the mitochondrion, proteolysis and cell apoptosis


Original description - :Cartoon representation...

Original description – :Cartoon representation of ubiquitin protein, highlighting the secondary structure. α-helices are coloured in blue and the β-sheet in green. The normal attachment point for a further ubiquitin molecule in polyubiquitin chain formation, lysine 48, is shown in pink. :Image was created using PyMOL (Photo credit: Wikipedia)

Ubiquinin-Proteosome pathway, autophagy, the mitochondrion, proteolysis and cell apoptosis

Larry H Bernstein, MD, FACP, Curator, Reporter, AEW

The work reviewed follows a seminal contribution by two Israeli and an American molecular biologists who shared the Nobel Prize in Chemistry in 2004.

The Royal Swedish Academy of Sciences awarded the Nobel Prize in Chemistry for 2004 “for the discovery of ubiquitin-mediated protein degradation” jointly to Aaron Ciechanover Technion – Israel Institute of Technology, Haifa, Israel, Avram Hershko Technion – Israel Institute of Technology, Haifa, Israel and Irwin Rose – University of California, Irvine, USA.

Aaron Ciechanover, born 1947 (57 years) in Haifa, Israel (Israeli citizen) received a Doctor’s degree in medicine in 1975 at Hebrew University of Jerusalem, and in biology in 1982 at the Technion (Israel Institute of Technology), Haifa. He is a Distinguished Professor at the Center for Cancer and Vascular Biology, and the Rappaport Faculty of Medicine and Research Institute at the Technion, Haifa,
Israel.

Avram Hershko, born 1937 (67 years) in Karcag, Hungary (Israeli citizen) earned the Doctor’s degree in medicine in 1969 at the Hadassah and the Hebrew University Medical School, Jerusalem.  He is a Distinguished Professor at the Rappaport Family Institute for Research in Medical Sciences at the Technion (Israel Institute of Technology), Haifa, Israel.

Irwin Rose, born 1926 (78 years) in New York, USA (American citizen) achieved a Doctor’s degree in 1952 at the University of Chicago, USA. Specialist at the Department of Physiology and Biophysics, College of Medicine, University of California, Irvine, USA.

Proteins labelled for destruction
Proteins build up all living things: plants, animals and therefore us humans. In the past few decades biochemistry has come a long way towards explaining how the cell produces all its various proteins. But as to the breaking down of proteins, not so many researchers were interested. Aaron Ciechanover, Avram Hershko and Irwin Rose went against the stream and at the beginning of the 1980s discovered one of the cell’s most important cyclical processes, regulated protein degradation. For this, they are being rewarded
with the 2004 Nobel Prize in Chemistry.

The label consists of a molecule called ubiquitin. This fastens to the protein to be destroyed, accompanies it to the proteasome where it is recognised as the key in a lock, and signals that a protein is on the way for disassembly. Shortly before the protein is squeezed into the proteasome, its ubiquitin label is disconnected for re-use.

Aaron Ciechanover, Avram Hershko and Irwin Rose have brought us to realise that the cell functions as a highly-efficient checking station where proteins are built up and broken down at a furious rate. The degradation is not indiscriminate but takes place through a process that is controlled in detail so that the proteins to be broken down at any given moment are given a molecular label, a ‘kiss of death’, to be dramatic. The labelled proteins are then fed into the cells’ “waste disposers”, the so called proteasomes, where they are chopped into small pieces and destroyed.

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Thanks to the work of the three Laureates it is now possible to understand at  molecular level how the cell controls a number of central processes by breaking down certain proteins and not others. Examples of processes governed by ubiquitin-mediated protein degradation are cell division, DNA repair, quality control of newly-produced proteins, and important parts of the immune defence. When the degradation does not work correctly, we fall ill. Cervical cancer and cystic fibrosis are two examples. Knowledge of
ubiquitin-mediated protein degradation offers an opportunity to develop drugs against these diseases and others.

Aaron Ciechanover and Ronen Ben-Saadon. N-terminal ubiquitination: more protein substrates join in. TRENDS in Cell Biology 2004; 14 (3):103-106.

The ubiquitin–proteasome system (UPS) is involved in selective targeting of innumerable cellular proteins through a complex pathway that plays important roles in a broad array of processes. An important step in the proteolytic cascade is specific recognition of the substrate by one of many ubiquitin ligases, E3s, which is followed by generation of the polyubiquitin degradation signal. For most substrates, it is believed that the first ubiquitin moiety is conjugated, through its C-terminal Gly76 residue, to an 1-NH2 group of an internal Lys residue. Recent findings indicate that, for several proteins, the first ubiquitin moiety is fused linearly to the a-NH2 group of the N-terminal residue.

The ubiquitin–proteasome system (UPS). Ubiquitin is first activated to a high-energy intermediate by E1. It is then transferred to a member of the E2 family of enzymes. From E2 it can be transferred directly to the substrate (S, red) that is bound specifically to a member of the ubiquitin ligase family of proteins, E3

  • (a). This occurs when the E3 belongs to the RING finger family of ligases. In the case of a HECT-domain-containing ligase
  • (b), the activated ubiquitin is transferred first to the E3 before it is conjugated to the E3-bound substrate . Additional ubiquitin moieties are added successively to the previously conjugated moiety to generate a polyubiquitin chain.
  • The polyubiquitinated substrate binds to the 26S proteasome complex (comprising 19S and 20S sub-complexes): the substrate is degraded to short peptides, and free and reusable ubiquitin is released through the activity of de-ubiquitinating enzymes (DUBs).

Ubiquitination on an internal lysine and on the N-terminal residue of the target substrate.

  • (a) The first ubiquitin moiety is conjugated, through its C-terminal Gly76 residue, to the 1-NH2 group of an internal lysine residue of the target substrate (Kn).
  • (b) The first ubiquitin moiety is conjugated to a free a-NH2 group of the N-terminal residue, X.
  • In both cases, successive addition of activated ubiquitin moieties to internal Lys48 on the previously conjugated ubiquitin moiety leads to the synthesis of a  polyubiquitin chain that serves as the degradation signal for the 26S proteasome

 

A UPS Autophagy Review

Summary: This discussion is another in a series discussing mitochondrial metabolism, energetics and regulatory function, and dysfunction, and the process leading to apoptosis and a larger effect on disease, with a specific targeting of neurodegeneration. Why neurological and muscle damage are more sensitive than other organs is not explained easily, but recall in the article on mitochondrial oxidation-reduction reactions and repair that there are organ specific differences in the rates of organelle mutation errors and in the rates of repair. In addition, consider the effect of iron-binding in the function of the cell, and Ca2+ binding in the creation of the mechanic work or signal transmission carried out by the neuromuscular system. We target the previously mentioned role of ubiquitin-proteosome, and interaction with autophagy, mitophagy, and disease.

Keywords: autophagy, ubiquitin-proteosome, UPS, protein degradation, defective organelle removal, selective degradation, E3, neurodegenerative disease, mitochondria, mitophagy, proteolysis, ribosomes, apoptosis, Ca++, rapamycin, TORC1, atg1p kinase, ubiqitization, trafficking pathways, unfolded protein response (UPS), p52/sequestrome, IC3, nitrogen starvation, acetaldehyde dehydrogenase (Ald6p), Ut1hp, toxisomes, Pex3/14 proteins, Bax, E3 Ligase, TRAP1, TNF-a, NFkB.

Ubiquitin-Proteosome Pathway
Three recent papers, describing three apparently independent biological processes, highlight the role of the ubiquitin-proteasome system as a major, however selective, proteolytic and regulatory pathway. Using specific inhibitors to the proteasome, Rock et al. (1994) demonstrate a role for this protease in the degradation of the major bulk of cellular proteins. They also showed that antigen processing requires the ubiquitin-activating enzyme, El. This indicates that antigen processing is both ubiquitin dependent and proteasome dependent. Furthermore, inhibitors to the proteasome prevent tumor necrosis factor a (TNFa)-induced activation of mature NFKB and its entry into the nucleus. The two studies clearly demonstrate that the ubiquitin-proteasome system is involved not only in complete destruction of its protein substrates, but also in limited proteolysis and posttranslational processing in which biologically active peptides or fragments are generated. In addition, the unstable c-Jut but not the stable v-Jun, is multiubiquitinated and degraded. The escape of the oncogenic v-Jun from ubiquitin-dependent degradation suggests a novel route to malignant transformation. Presented here is a review of the components, mechanisms of action, and cellular physiology of the ubiquitin-proteasome pathway.

Experimental evidence implicates the ubiquitin system in the degradation of

  • mitotic cyclins,
  • oncoproteins,
  • the tumor suppressor protein p53,
  • several cell surface receptors,
  • transcriptional regulators, and
  • mutated and damaged proteins.

Some of the proteolytic processes occur throughout the cell cycle, whereas others are tightly programmed and occur following cell cycle-dependent posttranslational modifications of the components involved. Signaling and degradation of other proteins (cell surface receptors, for example) may occur only following structural changes or modification(s) in the target molecule that results from ligand binding. Cell cycle-and modification-dependent degradation, as well the ability of the system to destroy completely or only partially its protein substrates, reflects the complexity involved in regulated intracellular protein degradation.

Enzymes of the System
The reaction occurs in two distinct steps:

  1. signaling of the protein by covalent attachment of multiple ubiquitin molecules and
  2. degradation of the targeted protein with the release of free and reutilizable ubiquitin.

Conjugation of ubiquitin to proteins destined for degradation proceeds, in general, in a three-step mechanism.

  1. Initially, the C-terminal Gly of ubiquitin is activated by ATP to a high energy thiol ester intermediate in a reaction catalyzed by the ubiquitin-activating enzyme, El.
  2. Following activation, E2 (ubiquitin carrier protein or ubiquitin-conjugating enzyme [USC]) transfers ubiquitin from El to the substrate that is bound to a ubiquitin-protein ligase, E3.
  3. Here an isopeptide bond is formed between the activated C-terminal Gly of ubiquitin and an c-NH2 group of a Lys residue of the substrate.

As E3 enzymes specifically synthesized by processive transfer of ubiquitin moieties to Lys-48 of the previous (and already conjugated) ubiquitin molecule. In many cases, E2 transfers activated ubiquitin directly to the protein substrate. Thus, E2 enzymes also play an important role in substrate recognition, although, in most cases, this modification is of the monoubiquitin type.

The Ubiquitin-Mediated Proteolytic Pathway
(1) Activation of ubiquitin by El and E2.
(2) Binding of the protein substrate to E3.
(3) EP dependent but EM independent monoubiquitination.
(4) EP-dependent but EM independent polyubiquitination?
(5) Ed-dependent polyubiquitination.
(6) Degradation of ubiquitin-protein conjugate by the 26s protease.
(7) “Correction” function of C-terminal hydrolase(s).
(6) Release of ubiquitin from terminal proteolytic products by &terminal hydrolase(s).

It is essential for the system that ubiquitin recycles. This function is carried out by ubiquitin C-terminal hydrolases (isopeptidases). In protein degradation, hydrolase(s) is required to release ubiquitin from isopeptide linkage with Lys residues of the protein substrate at the final stage of the proteolytic process. A ubiquitin C-terminal hydrolytic activity is also required to disassemble polyubiquitin chains linked to the protein substrate, following or during the degradative process. A “proofreading” function has been proposed for hydrolases to release free protein from “incorrectly” ubiquitinated proteins. Another possibility is that ubiquitin C-terminal hydrolases are required for trimming polyubitin chains.

Hydrolases are probably required for the processing of biosynthetic precursors of ubiquitin, since most ubiquitin genes are arranged either in linear polyubiquitin arrays or are fused to ribosomal proteins. Yet another hydrolase may be required for the removal of extra amino acid residues that are encoded by certain genes at the C-termini of some polyubiquitin molecules. Ubiquitin C-terminal hydrolases may have other functions as well. High energy El-ubiquitin and E2-ubiquitin thiol esters may react with intracellular nucleophiles (such as glutathione or polyamines). Such reactions may lead to rapid depletion of free ubiquitin unless such side products are rapidly cleaved.

Recognition of Substrates
Short-lived proteins contain a region enriched with Pro, Glu, Ser, and Thr (PEST region). However, it has not been shown that this region indeed serves as a consensus proteolysis targeting signal. An interesting problem involves the evolution of the N-end rule pathway and its physiological roles. Proteins that are derived from processing of polyproteins (Sindbis virus RNA polymerase, for example) may contain destabilizing N-termini and thus are proteolyzed via the N-end rule pathway.

Using a “synthetic lethal” screen, Ota and Varshavsky attempted to isolate a mutant that requires the N-end rule pathway for viability. They characterized an extragenic suppressor of the mutation and found that it encodes a protein with a strong correlation to protein phosphotyrosine phosphatase. The target protein or the connection between dephosphorylation of phosphotyrosine and the N-end rule pathway is still obscure. In an additional study, these researchers have shown that a missense mutation in SLNI, a member of a two-component signal transduction system in yeast, is lethal in the absence, but not in the presence, of the N-end rule pathway. Further studies are required to isolate the target protein and identify the signal transduction pathway.

Two recent studies have shed light on the role of the ubiquitin system and the proteasome in the process. Michalek et al. (1993) have shown that a mutant cell that harbors a thermolabile El cannot present peptides derived from ovalbumin following inactivation of the enzyme. In contrast, presentation of a minigene-expressed antigene peptide or presentation of exogenous similar peptide was not perturbed at the nonpermissive temperature. The important conclusion of the researchers is that the processing of the protein to peptides requires the complete ubiquitin pathway. In a complementary study, Rock et al. (1994) have shown that inhibitors that block the chymotryptic activity of the proteasome also block antigen presentation, most probably by inhibiting proteolysis of the antigen (ovalbumin). Thus, it appears that processing of MHC restricted class I antigens requires both ubiquitination and subsequent degradation by the proteasome. It is likely that the proteasome catalyzes processing of these antigens as part of the 26s protease complex.
Ciechanover A. The Ubiquitin-Proteasome Proteolytic Pathway. Cell 1994; 79:13-21.
Regulation of autophagy
The protein content of the cell is determined by the balance between protein synthesis and protein degradation. At constant intracellular protein concentration, i.e. at steady state, rates of protein synthesis and degradation are equal. Although turnover of protein results in energy dissipation, regulation at the level of protein degradation effectively controls protein levels.
Intracellular proteins to be degraded in the lysosomes can get access to these organelles by the following processes:

  • macroautophagy,
  • microautophagy,
  • crinophagy and selective,
  • chaperonin mediated, direct uptake of proteins.

Overview of the involvement of signal transduction in the regulation of macroautophagic proteolysis by amino acids and cell swelling.

  1. Amino acids (AA) stimulate a protein kinase cascade via a plasma membrane receptor.
  2. Receptor activation results in activation of PtdIns 3-kinase (PI3K), possibly via a heterotrimeric Gái3 protein.
  3. followed by activation of PKC-æ, PKB/Akt, p70S6 kinase (p70S6k) and finally phosphorylation of ribosomal protein S6 (S6P).
  4. The GDP-bound form of Gái3 is required for autophagic sequestration, whereas the GTP-bound form is inhibitory.
  5. The constitutively formed phosphatidylinositol 3-phosphate (PI3P) is also required for autophagic sequestration. Therefore,

inhibition of PtdIns 3-kinase activity by

  • wortmannin (W),
  • LY294002 (LY) or
  • 3-methyladenine (3MA) prevents autophagic sequestration.

Activation of PKC-æ and PKB/Akt is mediated by the 3,4- and 3,4,5-phosphate forms of phosphatidylinositol (PI3,4P2 and PI3,4,5P3) that are produced upon activation of PtdIns 3-kinase.

As a result of this, the first step of the macroautophagic pathway is

  • inhibited by components of the cascade that are downstream of PtdIns 3-kinase.
  • inhibition of this downstream cascade by rapamycin (RAPA) accelerates autophagic sequestration.
  • cell swelling potentiates the effect of amino acids via a change in the receptor owing to membrane stretch.

Furthermore, the site of action of the different effectors of the cytoskeleton (okadaic acid, cytochalasin, nocodazole, vinblastin and colchicine) are indicated.

  • AVi,
  • initial autophagic vacuole;
  • AVd,
  • mature degradative autophagic vacuole,
  • ER, endoplasmic reticulum.

The rate of proteolysis , an important determinant of the intracellular protein content, and part of its degradation occurs in the lysosomes and is mediated by macroautophagy. In liver, macroautophagy is very active and almost completely accounts for starvation-induced proteolysis. Factors inhibiting this process include

  • amino acids,
  • cell swelling and
  • insulin.

In the mechanisms controlling macroautophagy, protein phosphorylation plays an important role.

  • Activation of a signal transduction pathway, ultimately
  • leading to phosphorylation of ribosomal protein S6,
  • accompanies Inhibition of macroautophagy.

Components of this pathway may include

  • a heterotrimeric Gi3-protein,
  • phosphatidylinositol 3-kinase and
  • p70S6 kinase.

Selectivity of Autophagy
It has been assumed for a long time that macroautophagy is a non-selective process, in which macromolecules are randomly degraded in the same ratio as they occur in the cytoplasm . However, recent observations strongly suggest that this may not always be the case, and that macroautophagy can be selective. Lysosomal protein degradation can selectively occur via ubiquitin-dependent and -independent pathways. In the perfused liver, although autophagic breakdown of protein and RNA (mainly ribosomal RNA) is sensitive to inhibition by amino acids and insulin, glucagon accelerates proteolysis but has no effect on RNA degradation.

Another example of selective autophagy is the degradation of superfluous peroxisomes in hepatocytes from clofibrate-treated rats. When hepatocytes from these rats, in which the number of peroxisomes is greatly increased, are incubated in the absence of amino acids to ensure maximal flux through the macroautophagic pathway, peroxisomes are degraded at a relative rate that exceeds that of any other component in the liver cell. The accelerated degradation of peroxisomes was sensitive to inhibition by 3-methyladenine, a specific autophagic sequestration inhibitor. Interestingly, the accelerated removal of peroxisomes was prevented by long-chain but not short-chain fatty acids. Since long-chain fatty acids are substrates for peroxisomal â-oxidation, this indicates that these organelles are removed by autophagy when they are functionally redundant.  Our hypothesis is that acylation (palmitoylation?) of a peroxisomal membrane protein protects the peroxisome against autophagic sequestration.

Under normal conditions macroautophagy may be largely unselective and serves, for example, to produce amino acids for gluconeogenesis and the synthesis of essential proteins in starvation. When cell structures are functionally redundant or when they become damaged, the autophagic system is able to recognize this and is able to degrade the structure concerned. As yet, nothing is known about the recognition signals. A possibility is that ubiquitination of membrane proteins is required to mark the structure to be degraded for autophagic sequestration.

Ubiquitin may be involved in macroautophagy
Ubiquitin not only contributes to extralysosomal proteolysis but is also involved in autophagic protein degradation. Thus, in fibroblasts ubiquitin–protein conjugates can be found in the lysosomes, as shown by immunohistochemistry and immunogold electron microscopy. Free ubiquitin can also be found inside lysosomes. Accumulations of ubiquitin–protein conjugates in filamentous, presumably lysosomal, structures are also found in a large number of neurodegenerative diseases. Mallory bodies in the liver of alcoholics also contain ubiquitin–protein conjugates.

This presence of ubiquitin–protein conjugates in filamentous inclusions in neurons and other cells can be caused by a defect in the extralysosomal ubiquitin-dependent proteolytic pathway. However, it is also possible that these filamentous inclusions represent an attempt of the cell to get rid of unwanted material (proteins, organelles) via autophagy. Direct evidence that ubiquitin may be involved in the control of macroautophagy came from experiments with CHO cells with a temperature-sensitive mutation in the ubiquitin-activating enzyme E1. Wild-type cells increased their rate of proteolysis in response to stress (amino acid depletion, increased temperature). This was prevented by the acidotropic agent ammonia or by the autophagic sequestration inhibitor 3-methyladenine, indicating that the accelerated proteolysis occurred by autophagy. In the mutant cells, there was no such increase in proteolysis in response to stress at the restrictive temperature.

Autophagy and carcinogenesis
In cancer development, cell growth is mainly induced by inhibition of protein degradation, since differences in the rate of protein synthesis between tumorigenic cells and their normal counterparts are rather small. A striking example of how reduced autophagic proteolysis can contribute to cell growth can be found in the development of liver carcinogenesis. This decrease in autophagic flux results from a decrease in the rate of autophagic sequestration and is already detectable in the early preneoplastic stage. Autophagic flux is then hardly inhibitable by amino acids nor is it inducible by catabolic stimuli
and declines in the more advanced stage of cancer development to a rate of less than 20% of that seen in normal hepatocytes. The fact that the addition of 3-methyladenine to hepatocytes from normal rats increased hepatocyte viability to the same level as observed for the tumour cells strongly suggests that the fall in autophagic proteolysis contributes to the rapid growth rate of these cells and gives them a selective advantage over the normal hepatocytes.

Underlying control mechanisms for autophagy are gradually being unravelled. It is perhaps not surprising that protein phosphorylation and signal transduction are key elements in these mechanisms. The discovery of an amino acid receptor in the plasma membrane of the hepatocyte with a signal transduction pathway coupled to it may have important repercussions, not only for the control of macroautophagy but also for the control of other pathways.

It remains to be seen whether the details of the mechanisms controlling the process in yeast are similar to those in mammalian cells. For example, it is not known whether amino acids are able to control the process as they do in mammalian cells.

Blommaart EFC, Luiken JJFP, Meijer AJ. Autophagic proteolysis: control and specificity. Histochemical Journal (1997); 29:365–385.
A Novel Type of Selective Autophagy
Eukaryotic cells use autophagy and the ubiquitin–proteasome system (UPS) as their major protein degradation pathways. Whereas the UPS is required for the rapid degradation of proteins when fast adaptation is needed, autophagy pathways selectively remove protein aggregates and damaged or excess organelles. However, little is known about the targets and mechanisms that provide specificity to this process. Here we show that mature ribosomes are rapidly degraded by autophagy upon nutrient starvation in Saccharomyces cerevisiae. Surprisingly, this degradation not only occurs by a nonselective mechanism, but also involves a novel type of selective autophagy, which we term ‘ribophagy’. A genetic screen revealed that selective degradation of ribosomes requires catalytic activity of the Ubp3p/Bre5p ubiquitin protease. Although Ubp3p and Bre5p cells strongly accumulate 60S ribosomal particles upon starvation, they are proficient in starvation sensing and in general trafficking and autophagy pathways. Moreover, ubiquitination of several ribosomal subunits and/or ribosome associated proteins was specifically enriched in Ubp3p cells, suggesting that the regulation of ribophagy by ubiquitination may be direct. Interestingly, Ubp3p cells are sensitive to rapamycin and nutrient starvation, implying that selective degradation of ribosomes is functionally important in vivo. Taken together, our results suggest a link between ubiquitination and the regulated degradation of mature ribosomes by autophagy.
Kraft C, Deplazes A, Sohrmann M,Peter M. Mature ribosomes are selectively degraded upon starvation by an autophagy pathway requiring the Ubp3p/Bre5p ubiquitin protease. Nature Cell Biology 2008; 10(5): 603-609. DOI: 10.1038/ncb1723.  www.nature.com/naturecellbiology

Mitochondrial Failure and Protein Degradation

Progressive mitochondrial failure is tightly associated with the the development of most age-related human diseases including neurodegenerative diseases, cancer, and type 2 diabetes.

This tight connection results from the double-edged sword of mitochondrial respiration, which is responsible for generating both ATP and ROS, as well as from risks that are inherent to mitochondrial biogenesis. To prevent and treat these diseases, a precise understanding of the mechanisms that maintain functional mitochondria is necessary. Mitochondrial protein quality control is one of the mechanisms that protect mitochondrial integrity, and increasing evidence implicates the cytosolic ubiquitin/proteasome system (UPS) as part of this surveillance network. In this review, we will discuss our current understanding of UPS-dependent mitochondrial protein degradation, its roles in diseases progression, and insights into future studies.

While mitochondria have their own genome, about 99% of the roughly 1000 mitochondrial proteins are encoded in the nuclear genome. Most mitochondrial proteins are therefore

  • synthesized in the cytoplasm,
  • unfolded,
  • transported across one or both mitochondrial membranes,
  • then refolded and/or assembled into complexes (Tatsuta, 2009).

Failure of this complex series of events generates unfolded or misfolded proteins within mitochondria, often disrupting critical functions.

Mitochondrial oxidative phosphorylation generates usable cellular energy in the form of ATP, but also produces reactive oxygen species (ROS) . ROS tend to react quickly, so their predominant sites of damage are mitochondrial macromolecules that are localized nearby the source of ROS production.

Exposure to oxidative stress facilitates misfolding and aggregation of these mitochondrial proteins, leading to disassembly of protein complexes and eventual loss of mitochondrial integrity.

The clearance of misfolded and aggregated proteins is constantly needed to maintain functional mitochondria.
There are several systems promoting this turnover.

  1. Mitophagy, a selective mitochondrial autophagy, mediates a bulk removal of damaged mitochondria.
  2. mitochondria intrinsically contain proteases in each of their compartments and these proteases recognize misfolded mitochondrial proteins and mediate their degradation.

Accumulating evidence shows that the ubiquitin proteasome system (UPS) plays an important role in mitochondrial protein degradation. At various cellular sites, the UPS is involved in protein degradation. With the help of ubiquitin E1–E2–E3 enzyme cascades, target proteins destined for destruction are marked by conjugation of K48-linked poly-ubiquitin chain. This poly-ubiquitinated protein is then targeted to the proteasome for degradation.

Cells treated with proteasome inhibitors exhibit elevated levels of ubiquitinated mitochondrial proteins, suggesting the potentially important roles of the proteasome on mitochondrial protein degradation. Studies have also identified mitochondrial substrates of the UPS.

  • Fzo1, an outer mitochondrial membrane (OMM) protein involved in mitochondrial fusion, is partially dependent on the proteasome for its degradation in yeast.
  • The F box protein Mdm30 mediates ubiquitination of Fzo1 by Skp1-Cullin-F-boxMdm30 ligase, which leads to proteasomal degradation.

The UPS has also been implicated in mitochondrial protein degradation in higher organisms. In mammals,

  • the OMM proteins mitofusin 1 and 2 (Mfn1/2; the mammalian orthologs of Fzo1) and Mcl1 are polyubiquitinated and degraded by the proteasome.
  • VDAC1, Tom20 and Tom70 were also suggested as targets of proteasomal degradation as they are stabilized by proteasome inhibition.
  •  inactivation of the proteasome also induces accumulation of intermembrane space (IMS) proteins and, consistent with this, the proteasome plays a role in degradation of the IMS protein, Endonuclease G.

Turnover of some inner mitochondrial membrane (IMM) proteins is also dependent upon the proteasome. Uncoupling proteins (UCPs) 2 and 3 exhibit an unusually short half-life compared with other IMM proteins, and Brand and colleagues showed that inactivation of the proteasome prevents their turnover in vivo and in a reconstituted in vitro system. Finally, mitochondrial matrix proteins can also be degraded by the proteasome.

Cdc48/p97 is involved in many cellular processes through its role in protein degradation and is targeted to different subcellular sites by adaptor proteins. For example, Cdc48/p97 is recruited to the endoplasmic reticulum with the help of two adaptor proteins, Npl4 and Ufd1. This implies the existence of specific adaptors that recruit Cdc48/p97 to mitochondria. Consistent with this notion, the authors recently identified a mitochondrial adaptor protein for Cdc48, which we named Vms1 (VCP/Cdc48-associated mitochondrial stress responsive 1). Vms1 interacts with Cdc48/p97 and Npl4, but not with Ufd1, which indicates that the Cdc48/p97–Npl4–Ufd1 complex functions in ER protein degradation while the Vms1–Cdc48/p97–Npl4 complex acts in mitochondria. In agreement with this notion, overexpression of Cdc48 or Npl4 rescues the Vms1 mutant phenotype while Ufd1 has no effect.

Normally, Vms1 is cytoplasmic. Upon mitochondrial stress, however, Vms1 recruits Cdc48 and Npl4 to mitochondria. In agreement with the role of Cdc48/p97 in OMM protein degradation, loss of the Vms1 system results in accumulation of ubiquitin-conjugated proteins in purified mitochondria as well as stabilization of Fzo1 under mitochondrial stress conditions. Accumulation of damaged and misfolded mitochondrial proteins disturbs the normal physiology of the mitochondria, leading to mitochondrial dysfunction. As expected, the Vms1 mutants progressively lose mitochondrial respiratory activity, eventually leading to cell death. The VMS1 gene is broadly conserved in eukaryotes, implying an important functional role in a wide range of organisms. The C. elegans Vms1 homolog exhibits a similar pattern of mitochondrial stress responsive translocation and is required for normal lifespan. Additionally, mammalian Vms1 also forms a stable complex with p97. Combining these observations, the authors conclude that Vms1 is a conserved component of the UPS-dependent mitochondrial protein quality control system.

The UPS regulates mitochondrial dynamics and initiation of mitophagy
The UPS regulates mitochondrial dynamics. Major proteins involved in mitochondrial fission or fusion (e.g. Mfn1/2, Drp1 and Fis1) are degraded by the UPS.  MITOL, a mitochondrial E3 ubiquitin ligase, is required for Drp1-dependent mitochondrial fission as depletion or inactivation of MITOL blocks mitochondrial fragmentation. Moreover, knockdown of USP30, an OMM-localized deubiquitinating enzyme, induces an elongated mitochondrial morphology, suggesting a defect in fission. Through this regulatory process, the UPS controls mitochondrial dynamics. Parkin, an E3 ligase involved in mitophagy, utilizes the UPS to enhance mitochondrial fission through degradation of components of the fusion machinery. By facilitating fragmentation of damaged mitochondria, which is essential for initiation of mitophagy, Parkin stimulates mitophagy. The underlying mechanisms linking the UPS to the regulation of mitochondrial dynamics remain unclear.

Accumulation of aberrant proteins and human diseases
In neurodegenerative diseases wherein aberrant pathological proteins accumulate throughout the cell, including sites in mitochondria. Amyloid precursor protein (APP), a protein associated with Alzheimer’s disease, accumulates within mitochondria and is implicated in blockade of mitochondrial protein import. A, a neurotoxic APP cleavage product, can also facilitate the formation of the mitochondrial permeability transition pore (mPTP) by binding to mPTP components VDAC1, CypD and ANT, which provokes cell death.
-Synuclein, a protein associated with the development of Parkinson’s disease, is targeted to the IMM where it binds to the mitochondrial respiratory complex I and impairs its function. -Synuclein interferes with mitochondrial dynamics as its unique interaction with the mitochondrial membrane disturbs the fusion process. Finally, in Huntington’s disease, increased association of the mutant huntingtin protein with mitochondria can impair mitochondrial trafficking. Moreover, accumulation of mutant huntingtin protein disrupts cristae structure and it facilitates mitochondrial fragmentation by activation of Drp1. These examples demonstrate the crucial importance of prompt removal of dysfunctional and/or aberrant proteins in maintaining functional mitochondria.

UPS-mediated mitochondrial protein degradation.
Misfolded and/or damaged mitochondrial proteins destined for proteasomal degradation in the cytosol are recruited to the outer mitochondrial membrane (OMM) from each mitochondrial compartment by unknown mechanisms. Upon reaching the OMM, these proteins are presented to the proteasome through a series of events. They are K48 polyubiquitinated by the cytoplasmic (e.g. Parkin) or mitochondrial ubiquitin E3 ligases. For proteasomal degradation, polyubiquitinated mitochondrial substrate proteins need to be retrotranslocated to the cytoplasm, probably, either by the proteasome per se or by the help of UPS components such as Vms1, Cdc48/p97 and Npl4. Following dislocation to the cytoplasm, these substrate proteins are degraded by the proteasome.

Treatment of diseases that arise from defects in protein quality control will depend on greater depth in our understanding of this process, which could contribute to the development of novel therapeutic approaches. For instance, both mutant SOD1, a misfolded mitochondrial protein associated with the onset of amyotrophic lateral sclerosis, and polyglutamine expanded ataxin-3, a pathogenic protein causing Machado-Joseph disease, are ubiquitinated by MITOL and then degraded by the proteasome. Facilitating the proteasomal degradation of these aberrant proteins might therefore efficiently control diseases progression and, eventually, cure the diseases. Answering these questions would partially unveil the mysterious physiology of mitochondria, which, in turn, would facilitate the development of therapeutics to prevent and cure devastating human diseases.

Heo JM, Rutter J. Ubiquitin-dependent mitochondrial protein degradation. The International Journal of Biochemistry & Cell Biology 2011; 43:1422– 1426. http://www.elsevier.com/locate/biocel
UPS Inhibitors and Apoptotic Machinery
Over the past decade, the promising results of UPSIs (UPS inhibitors) in eliciting apoptosis in various cancer cells, and the approval of the first UPSI (Bortezomib/Velcade/PS-341) for the treatment of multiple myeloma have raised interest in assessing the death program activated upon proteasomal blockage. Several reports indicate that UPSIs stimulate apoptosis in malignant cells by operating at multiple levels, possibly by inducing different types of cellular stress. Normally cellular stress signals converge on the core elements of the apoptotic machinery to trigger the cellular demise. In addition to eliciting multiple stresses, UPSIs can directly operate on the core elements of the apoptotic machinery to control their abundance. Alterations in the relative levels of anti and pro-apoptotic factors can render cancer cells more prone to die in response to other anti-cancer treatments. Aim of the present review is to discuss those core elements of the apoptotic machinery that are under the control of the UPS.

The UPS (Ubquitin-Proteasome System)
To fulfill the protein-degradation process two branches, operating at different levels, principally comprise the UPS.

  • The first branch is formed by the enzymatic activities responsible for delivering the substrate to the degradative machinery: the targeting branch.
  • The second branch is represented by the proteolytic machinery, which ultimately fragments the protein substrate into small oligopeptides.

Oligopeptides are further digested to single amino acids by cytosolic proteases.
It is important to remember that conjugation of ubiquitin to a specific protein is not sufficient to determine its degradation. In fact, mono-ubiquitination or poly-monoubiquitination and in certain cases also poly-ubiquitination of proteins are post-translational modifications related to various cellular functions including DNA repair or membrane trafficking . To deliver polypeptides for proteasomal degradation poly-ubiquitin chains of more than 4 ubiquitins must be assembled through lysine-48 linkages.

There are 3 catalytic sites for each polyubiquitin chain. These sites show specific requirements in terms of substrate specificities and catalytic activities, and they are identified as

  1. trypsin-like, which prefer to cleave after hydrophobic bonds, chymotrypsin-like, which cleave at basic residues and
  2. postglutamyl peptide hydrolase-like or
  3. caspase-like activities, which cut after acidic amino acid.

Each proteasome active site uses the side chain hydroxyl group of an NH2-terminal threonine as the catalytic nucleophile, a mechanism that distinguishes the proteasome from other cellular proteases. The presence of substrate proteolysis small size peptides ranging from 3 to 22 residues are generated. Alternative catalytic sites guarantees the efficient processing of several different substrates.

UPS Inhibitors
By UPS inhibitors (UPSI) we mean small molecules that share the ability to target and inhibit specific activities of the UPS, causing the accumulation of poly-ubiquitinated proteosomal substrates. UPSIs are heterogeneous compounds and among them bortezomib is the only one used in clinical practice.

PR-171, a modified peptide related to the natural product epoxomicin, is composed of two key elements:

  1. a peptide portion that selectively binds with high affinity in the substrate binding pocket(s) of the proteasome and
  2. an epoxyketone pharmacophore that stereospecifically interacts with the catalytic threonine residue and irreversibly inhibits enzyme activity.

In comparison to bortezomib, PR-171 exhibits equal potency, but greater selectivity, for the chymotrypsin-like activity of the proteasome. In cell culture PR-171 is more cytotoxic than bortezomib. In mice PR-171 is well tolerated and shows stronger anti-tumor activity when compared with bortezomib . Clinical studies are in progress to test the safety of PR-171 at different dose levels on some hematological cancers.

Cell Death by UPSI
In vitro experiments have unambiguously established that incubation of neoplastic cells with UPSIs including bortezomib triggers their death. Apoptosis or type I cell death relies on the timed activation of caspases, a group of cysteine proteases, which cleave selected cellular substrates after aspartic residues. Two main apoptotic pathways keep in check caspase activation.

The turnover of a large number of cellular proteins is under the control of the UPS. Thus in principle any proteosomal substrate could contribute directly or indirectly to the cell death phenotype. This is perfectly exemplified by two master regulators of cell life and death, p53 and NFkB.  UPSIs cause

  • NF-kB inhibition through reduced IkB degradation and,
  • in opposition; they promote stabilization and accumulation of p53.

c-FLIP is the most important element of the extrinsic pathway under the direct control of the UPS. Two different FLIP isoforms exist:

  1. c-FLIPL (Long) and
  2. c-FLIPS (Short).

c-FLIPL is highly homologus to caspase-8 and contains two tandem repeat Death Effector Domains (DED) and a catalytically inactive caspase-like domain. Both FLIPs can be degraded by the UPS; however they display distinct half-lives and the unique C terminus of c-FLIPS possesses a destabilizing function. The regulation of c-FLIP levels in response to UPSIs is rather controversial. Some reports indicate that UPSIs can reduce c-FLIP levels and in this manner synergize with TRAIL to promote apoptosis.

UPSIs activate multiple cellular responses and different stress signals that ultimately cause cell death. For this reason they represent broad inducers of apoptosis. In addition, since many of the available UPSIs alter the proteolytic activity of the proteasome, they represent non-specific modulators of the expression/activity of various components of the apoptotic machinery. Paradoxically they can simultaneously favor the accumulation of pro- and anti-apoptotic factors.
Brancolini C. Inhibitors of the Ubiquitin-Proteasome System and the Cell Death Machinery: How Many Pathways are Activated? Current Molecular Pharmacology, 2008; 1:24-37.

Mitochondrial Quality Control
The PINK1–Parkin pathway plays a critical role in mitochondrial quality control by selectively targeting damaged mitochondria for autophagy. The AAA-type ATPase p97 acts downstream of PINK1 and Parkin to segregate fusion-incompetent mitochondria for turnover. [Tanaka et al. (2010. J. Cell Biol. doi: 10.1083/jcb.201007013)]. p97 acts by targeting the mitochondrial fusion-promoting factor mitofusin for degradation through an endoplasmic reticulum–associated degradation (ERAD)-like mechanism.

Pallanck LJ. Culling sick mitochondria from the herd. J Cell Biol 2012;191(7):1225–1227. http://www.jcb.org/cgi/doi/10.1083/jcb.201011068

PINK1 and Parkin and Parkinson’s Disease

Studies of the familial Parkinson disease-related proteins PINK1 and Parkin have demonstrated that these factors promote the fragmentation and turnover of mitochondria following treatment of cultured cells with mitochondrial depolarizing agents. Whether PINK1 or Parkin influence mitochondrial quality control under normal physiological conditions in dopaminergic neurons, a principal cell type that degenerates in Parkinson disease, remains unclear. To address this matter, we developed a method to purify and characterize neural subtypes of interest from the adult Drosophila brain.

Using this method, we find that dopaminergic neurons from Drosophila parkin mutants accumulate enlarged, depolarized mitochondria, and that genetic perturbations that promote mitochondrial fragmentation and turnover rescue the mitochondrial depolarization and neurodegenerative phenotypes of parkin mutants. In contrast, cholinergic neurons from parkin mutants accumulate enlarged depolarized mitochondria to a lesser extent than dopaminergic neurons, suggesting that a higher rate of mitochondrial damage, or a deficiency in alternative mechanisms to repair or eliminate damaged mitochondria explains the selective vulnerability of dopaminergic neurons in Parkinson disease.

Our study validates key tenets of the model that PINK1 and Parkin promote the fragmentation and turnover of depolarized mitochondria in dopaminergic neurons. Moreover, our neural purification method provides a foundation to further explore the pathogenesis of Parkinson disease, and to address other neurobiological questions requiring the analysis of defined neural cell types.

Burmana JL, Yua S, Poole AC, Decala RB , Pallanck L. Analysis of neural subtypes reveals selective mitochondrial dysfunction in dopaminergic neurons from parkin mutants.

Autophagy in Parkinson’s Disease.
Parkinson’s disease is a common neurodegenerative disease in the elderly. To explore the specific role of autophagy and the ubiquitin-proteasome pathway in apoptosis, a specific proteasome inhibitor and macroautophagy inhibitor and stimulator were selected to investigate pheochromocytoma (PC12) cell lines transfected with human mutant (A30P) and wildtype (WT) -synuclein.

The apoptosis ratio was assessed by flow cytometry. LC3, heat shock protein 70 (hsp70) and caspase-3 expression in cell culture were determined by Western blot. The hallmarks of apoptosis and autophagy were assessed with transmission electron microscopy. Compared to the control group or the rapamycin (autophagy stimulator) group, the apoptosis ratio in A30P and WT cells was significantly higher after treatment with inhibitors of the proteasome and macroautophagy. The results of Western blots for caspase-3 expression were similar to those of flow cytometry; hsp70 protein was significantly higher in the proteasome inhibitor group than in control, but in the autophagy inhibitor and stimulator groups, hsp70 was similar to control. These findings show that inhibition of the proteasome and autophagy promotes apoptosis, and the macroautophagy stimulator rapamycin reduces the apoptosis ratio. And inhibiting or stimulating autophagy has less impact on hsp70 than the proteasome pathway.

In conclusion, either stimulation or inhibition of macroautophagy, has less impact on hsp70 than on the proteasome pathway. This study found that rapamycin decreased apoptotic cells in A30P cells independent of caspase-3 activity. Although several lines of evidence recently demonstrated crosstalk between autophagy and caspase-independent apoptosis, we could not confirm that autophagy activation protects cells from caspase-independent cell death. Undoubtedly, there are multiple connections between the apoptotic and autophagic processes.

Inhibition of autophagy may subvert the capacity of cells to remove damaged organelles or to remove misfolded proteins, which would favor apoptosis. However, proteasome inhibition activated macroautophagy and accelerated apoptosis. A likely explanation is inhibition of the proteasome favors oxidative reactions that trigger apoptosis, presumably through

1. a direct effect on mitochondria, and
2. the absence of NADPH2 and ATP

which may deinhibit the activation of caspase-2 or MOMP. Another possibility is that aggregated proteins induced by proteasome inhibition increase apoptosis.

Yang F, Yanga YP, Maoa CJ, Caoa BY, et al. Role of autophagy and proteasome degradation pathways in apoptosis of PC12 cells overexpressing human -synuclein. Neuroscience Letters 2009; 454:203–208. doi:10.1016/j.neulet.2009.03.027. http://www.elsevier.com/locate/neulet

Parkin-dependent Ubiquitination of Endogenous Bax 

Autosomal recessive loss-of-function mutations within the PARK2 gene functionally inactivate the E3 ubiquitin ligase parkin, resulting in neurodegeneration of catecholaminergic neurons and a familial form of Parkinson disease. Current evidence suggests both a mitochondrial function for parkin and a neuroprotective role, which may in fact be interrelated. The antiapoptotic effects of Parkin have been widely reported, and may involve fundamental changes in the threshold for apoptotic cytochrome c release, but the substrate(s) involved in Parkin dependent protection had not been identified. Here, we demonstrate the Parkin-dependent ubiquitination of endogenous Bax comparing primary cultured neurons from WT and Parkin KO mice and using multiple Parkin-overexpressing cell culture systems. The direct ubiquitination of purified Bax was also observed in vitro following incubation with recombinant parkin. The authors found that Parkin prevented basal and apoptotic stress induced translocation of Bax to the mitochondria. Moreover, an engineered ubiquitination-resistant form of Bax retained its apoptotic function, but Bax KO cells complemented with lysine-mutant Bax did not manifest the antiapoptotic effects of Parkin that were observed in cells expressing WT Bax. These data suggest that Bax is the primary substrate responsible for the antiapoptotic effects of Parkin, and provide mechanistic insight into at least a subset of the mitochondrial effects of Parkin.

Johnson BN, Berger AK, Cortese GP, and LaVoie MJ. The ubiquitin E3 ligase Parkin regulates the proapoptotic function of Bax. PNAS 2012, pp 6. http://www.pnas.org/cgi/doi/10.1073/pnas.1113248109
Parkin Promotes Mitochondrial Loss in Autophagy
Parkin, an E3 ubiquitin ligase implicated in Parkinson’s disease, promotes degradation of dysfunctional mitochondria by autophagy. Using proteomic and cellular approaches, we show that upon translocation to mitochondria, Parkin activates the ubiquitin–proteasome system (UPS) for widespread degradation of outer membrane proteins. This is evidenced by an increase in K48-linked polyubiquitin on mitochondria, recruitment of the 26S proteasome and rapid degradation of multiple outer membrane proteins. The degradation of proteins by the UPS occurs independently of the autophagy pathway, and inhibition of the 26S proteasome completely abrogates Parkin-mediated mitophagy in HeLa, SH-SY5Y and mouse cells. Although the mitofusins Mfn1 and Mfn2 are rapid degradation targets of Parkin, degradation of additional targets is essential for mitophagy. These results indicate that remodeling of the mitochondrial outer membrane proteome is important for mitophagy, and reveal a causal link between the UPS and autophagy, the major pathways for degradation of intracellular substrates.

Chan NC, Salazar AM, Pham AH, Sweredoski MJ, et al. Broad activation of the ubiquitin–proteasome system by Parkin is critical for mitophagy. Human Molecular Genetics 2011; 20(9): 1726–1737. doi:10.1093/hmg/ddr048.

TRAP1 and TBP7 Interaction in Refolding of Damaged Proteins
TRAP1 is a mitochondrial antiapoptotic heat shock protein. The information available on the TRAP1 pathway describes just a few well-characterized functions of this protein in mitochondria. However, our group’s use of mass spectrometry analysis identified TBP7, an AAA-ATPase of the 19S proteasomal subunit, as a putative TRAP1-interacting protein. Surprisingly, TRAP1 and TBP7 co-localize in the endoplasmic reticulum (ER), as demonstrated by biochemical and confocal/electron microscopy analyses, and directly interact, as confirmed by FRET analysis. This is the first demonstration of TRAP1 presence in this cellular compartment. TRAP1 silencing by shRNAs, in cells exposed to thapsigargin-induced ER stress, correlates with up-regulation of BiP/Grp78, thus suggesting a role of TRAP1 in the refolding of damaged proteins and in ER stress protection. Consistently, TRAP1 and/or TBP7 interference enhanced stress-induced cell death and increased intracellular protein ubiquitination. These experiments led us to hypothesize an involvement of TRAP1 in protein quality control for mistargeted/misfolded mitochondria-destined proteins, through interaction with the regulatory proteasome protein TBP7. Remarkably, the expression of specific mitochondrial proteins decreased upon TRAP1 interference as a consequence of increased ubiquitination. The proposed TRAP1 network has an impact in vivo, since it is conserved in human colorectal cancers, is controlled by ER-localized TRAP1 interacting with TBP7 and provides a novel model of ER-mitochondria crosstalk.

Amoroso MR, Matassa DS, Laudiero G, Egorova AV. TRAP1 AND THE PROTEASOME REGULATORY PARTICLE TBP7/Rpt3 INTERACT IN THE ENDOPLASMIC RETICULUM AND CONTROL CELLULAR UBIQUITINATION OF SPECIFIC MITOCHONDRIAL PROTEINS. Cell Death and Differentiation 2012; pp? DOI : 10.1038/cdd.2011.128

VMS1 and Mitochondrial Protein Degradation
We show that Ydr049 (renamed VCP/Cdc48-associated mitochondrial stress-responsive—Vms1), a member of an unstudied pan-eukaryotic protein family, translocates from the cytosol to mitochondria upon mitochondrial stress. Cells lacking Vms1 show progressive mitochondrial failure, hypersensitivity to oxidative stress, and decreased chronological life span. Both yeast and mammalian Vms1 stably interact with Cdc48/VCP/p97, a component of the ubiquitin/proteasome system with a well-defined role in endoplasmic reticulum-associated protein degradation (ERAD), wherein misfolded ER proteins are degraded in the cytosol. We show that oxidative stress triggers mitochondrial localization of Cdc48 and this is dependent on Vms1. When this system is impaired by mutation of Vms1,

  • ubiquitin-dependent mitochondrial protein degradation,
  • mitochondrial respiratory function,and
  • cell viability are compromised.

We demonstrate that Vms1 is a required component of an evolutionarily conserved system for mitochondrial protein degradation, which is
necessary to maintain

  • mitochondrial,
  • cellular, and
  • organismal viability.

Heo JM, Livnat-Levanon N, Taylor EB, Jones KT. A Stress-Responsive System
for Mitochondrial Protein Degradation. Molecular Cell 2010; 40:465–480.
DOI 10.1016/j.molcel.2010.10.021

Mitochondrial Protein Degradation
The biogenesis of mitochondria and the maintenance of mitochondrial functions depends on an autonomous proteolytic system in the organelle which is highly conserved throughout evolution. Components of this system include processing

  • peptidases and
  • ATP-dependent proteases, as well as
  • molecular chaperone proteins and
  • protein complexes with apparently regulatory functions.

While processing peptidases mediate maturation of nuclear-encoded mitochondrial preproteins, quality control within various subcompartments of mitochondria is ensured by ATP-dependent proteases which selectively remove non-assembled or misfolded polypeptides. Moreover, these proteases appear to control the activity- or steady-state levels of specific regulatory proteins and thereby ensure mitochondrial genome integrity, gene expression and protein assembly.

Kaser M and Langer T. Protein degradation in mitochondria. CELL & DEVELOPMENTAL BIOLOGY 2000; 11:181–190. doi: 10.1006/10.1006/scdb.2000.0166.

RING finger E3s

Ubiquitin-ligases or E3s are components of the ubiquitin proteasome system (UPS) that coordinate the transfer of ubiquitin to the target protein. A major class of ubiquitin-ligases consists of RING-finger domain proteins that include the substrate recognition sequences in the same polypeptide; these are known as single-subunit RING finger E3s. We are studying a particular family of RING finger E3s, named ATL, that contain a transmembrane domain and the RING-H2 finger domain; none of the member of the family contains any other previously described domain. Although the study of a few members in A. thaliana and O. sativa has been reported, the role of this family in the life cycle of a plant is still vague.

To provide tools to advance on the functional analysis of this family we have undertaken a phylogenetic analysis of ATLs in twenty-four plant genomes. ATLs were found in all the 24 plant species analyzed, in numbers ranging from 20–28 in two basal species to 162 in soybean. Analysis of ATLs arrayed in tandem indicates that sets of genes are expanding in a species-specific manner. To
get insights into the domain architecture of ATLs we generated 75 pHMM LOGOs from 1815 ATLs, and unraveled potential protein-protein interaction regions by means of yeast two-hybrid assays. Several ATLs were found to interact with DSK2a/ubiquilin through a region at the amino-terminal end, suggesting that this is a widespread interaction that may assist in the mode of action of ATLs; the region was traced to a distinct sequence LOGO. Our analysis provides significant observations on the evolution and expansion of the ATL family in addition to information on the domain structure of this class of ubiquitin-ligases that may be involved in plant adaptation to environmental stress.

Aguilar-Hernandez V, Aguilar-Henonin L, Guzman P. Diversity in the Architecture of ATLs, a Family of Plant Ubiquitin-Ligases, Leads to Recognition and Targeting of Substrates in Different Cellular Environments. PLoS ONE 2011; 6(8): e23934. doi:10.1371/journal.pone.0023934
UPS Proteolytic Function Inadequate in Proteinopathies
Proteinopathies are a family of human disease caused by toxic aggregation-prone proteins and featured by the presence of protein aggregates in the affected cells. The ubiquitin-proteasome system (UPS) and autophagy are two major intracellular protein degradation pathways. The UPS mediates the targeted degradation of most normal proteins after performing their normal functions as well as the removal of abnormal, soluble proteins. Autophagy is mainly responsible for degradation of defective organelles and the bulk degradation of cytoplasm during starvation. The collaboration between the UPS and autophagy appears to be essential to protein quality control in the cell.

UPS proteolytic function often becomes inadequate in proteinopathies which may lead to activation of autophagy, striving to remove abnormal proteins especially the aggregated forms. HADC6, p62, and FoxO3 may play an important role in mobilizing this proteolytic consortium. Benign measures to enhance proteasome function are currently lacking; however, enhancement of autophagy via pharmacological intervention and/or lifestyle change has shown great promise in alleviating bona fide proteinopathies in the cell and animal models. These pharmacological interventions are expected to be applied clinically to treat human proteinopathies in the near future.

Zheng Q, Li J, Wang X. Interplay between the ubiquitin-proteasome system and
autophagy in proteinopathies. Int J Physiol Pathophysiol Pharmacol 2009;1:127-142. http://www.ijppp.org/IJPPP904002

Ubiquitin-associated Protein-Protein Interactions

Applicability of in vitro biotinylated ubiquitin for evaluation of endogenous ubiquitin conjugation and analysis of ubiquitin-associated protein-protein interactions has been investigated. Incubation of rat brain mitochondria with biotinylated ubiquitin followed by affinity chromatography on avidin-agarose, intensive washing, tryptic digestion of proteins bound to the affinity sorbent and their mass spectrometry analysis resulted in reliable identification of 50 proteins belonging to mitochondrial and extramitochondrial compartments. Since all these proteins were bound to avidin-agarose only after preincubation of the mitochondrial fraction with biotinylated ubiquitin, they could therefore be referred to as specifically bound proteins. A search for specific
ubiquitination signature masses revealed several extramitochondrial and intramitochondrial ubiquitinated proteins representing about 20% of total number of proteins bound to avidin-agarose. The interactome analysis suggests that the identified non-ubiquitinated proteins obviously form tight complexes either with ubiquitinated proteins or with their partners and/or mitochondrial membrane components. Results of the present study demonstrate that the use of biotinylated ubiquitin may be considered as the method of choice for in vitro evaluation of endogenous ubiquitin-conjugating machinery in particular
subcellular organelles and changes in ubiquitin/organelle associated interactomes. This may be useful for evaluation of changes in interactomes induced by protein ubiquitination.

Buneeva OA, Medvedeva MV, Kopylov AT, Zgoda VG, Medvedev AE. Use of Biotinylated Ubiquitin for Analysis of Rat Brain Mitochondrial Proteome and Interactome. Int J Mol Sci 2012; 13: 11593-11609; doi:10.3390/ijms130911593
IL-6 regulation on mitochondrial remodeling/dysfunction

Muscle protein turnover regulation during cancer cachexia is being rapidly defined, and skeletal muscle mitochondria function appears coupled to processes regulating muscle wasting. Skeletal muscle oxidative capacity and the expression of proteins regulating mitochondrial biogenesis and dynamics are disrupted in severely cachectic ApcMin/+ mice. It has not been determined if these changes occur at the onset of cachexia and are necessary for the progression of muscle wasting. Exercise and anti-cytokine therapies have proven effective in preventing cachexia development in tumor bearing mice, while their effect on mitochondrial content, biogenesis and dynamics is not well understood.

The purposes of this study were to

1) determine IL-6 regulation on mitochondrial remodeling/dysfunction during the progression of cancer cachexia and
2) to determine if exercise training can attenuate mitochondrial dysfunction and the induction of proteolytic pathways during IL-6 induced cancer cachexia.

ApcMin/+ mice were examined during the progression of cachexia, after systemic interleukin (IL)-6r antibody treatment, or after IL-6 over-expression with or without exercise. Direct effects of IL-6 on mitochondrial remodeling were examined in cultured C2C12 myoblasts.

Mitochondrial content was not reduced during the initial development of cachexia, while muscle PGC-1α and fusion (Mfn1, Mfn2) protein expression was repressed.

With progressive weight loss mitochondrial content decreased, PGC-1α and fusion proteins were further suppressed, and fission protein (FIS1) was induced.

IL-6 receptor antibody administration after the onset of cachexia

  • improved mitochondrial content,
  • PGC-1α,
  • Mfn1/Mfn2 and
  • FIS1 protein expression.

IL-6 over-expression in pre-cachectic mice

  • accelerated body weight loss and muscle wasting, without reducing mitochondrial content,
  • while PGC-1α and Mfn1/Mfn2 protein expression was suppressed
  • and FIS1 protein expression induced.

Exercise normalized these IL-6 induced effects. C2C12 myotubes administered IL-6 had

  • increased FIS1 protein expression,
  • increased oxidative stress, and
  • reduced PGC-1α gene expression
  • without altered mitochondrial protein expression.

Altered expression of proteins regulating mitochondrial biogenesis and fusion are early events in the initiation of cachexia regulated by IL-6, which precede the loss of muscle mitochondrial content. Furthermore, IL-6 induced mitochondrial remodeling and proteolysis can be rescued with moderate exercise training even in the presence of high circulating IL-6 levels.

White JP, Puppa MJ, Sato S, Gao S. IL-6 regulation on skeletal muscle mitochondrial remodeling during cancer cachexia in the ApcMin/+ mouse. Skeletal Muscle 2012; 2:14-30.
http://www.skeletalmusclejournal.com/content/2/1/14

Starvation-induced Autophagy
Upon starvation cells undergo autophagy, a cellular degradation pathway important in the turnover of whole organelles and long lived proteins. Starvation-induced protein degradation has been regarded as an unspecific bulk degradation process. We studied global protein dynamics during amino acid starvation-induced autophagy by quantitative mass spectrometry and were able to record nearly 1500 protein profiles during 36 h of starvation. Cluster analysis of the recorded protein profiles revealed that cytosolic proteins were degraded rapidly, whereas proteins annotated to various complexes and organelles were degraded later at different time periods. Inhibition of protein degradation pathways identified the lysosomal/autophagosomal system as the main degradative route.

Thus, starvation induces degradation via autophagy, which appears to be selective and to degrade proteins in an ordered fashion and not completely arbitrarily as anticipated so far.

Kristensen AR, Schandorff S, Høyer-Hansen M, Nielsen MO, et al. Ordered Organelle Degradation during Starvation-induced Autophagy. Molecular & Cellular Proteomics 2008; 7:2419–2428.

Skeletal Muscle Macroautophagy
Skeletal muscles are the agent of motion and one of the most important tissues responsible for the control of metabolism. Coordinated movements are allowed by the highly organized structure of the cytosol of muscle fibers (or myofibers), the multinucleated and highly specialized cells of skeletal muscles involved in contraction. Contractile proteins are assembled into repetitive structures, the basal unit of which is the sarcomere, that are well packed into the myofiber cytosol. Myonuclei are located at the edge of the myofibers, whereas the various organelles such as mitochondria and sarcoplasmic reticulum are embedded among the myofibrils. Many different changes take place in the cytosol of myofibers during catabolic conditions:

  • proteins are mobilized
  • organelles networks are reorganized for energy needs
  • the setting of myonuclei can be modified.

Further,

  • strenuous physical activity,
  • improper dietary regimens and
  • aging

lead to mechanical and metabolic damages of myofiber organelles, especially mitochondria, and contractile proteins. During aging the protein turnover is slowed down, therefore it is easier to accumulate aggregates of dysfunctional proteins. Therefore, a highly dynamic tissue such as skeletal muscle requires a rapid and efficient system for the removal of altered organelles, the elimination of protein aggregates, and the disposal of toxic products.

The two major proteolytic systems in muscle are the ubiquitin-proteasome and the autophagy-lysosome pathways. The proteasome system requires

  • the transcription of the two ubiquitin ligases (atrogin-1 and MuRF1) and
  • the ubiquitination of the substrates.

Therefore, the ubiquitin-proteasome system can provide the rapid elimination of single proteins or small aggregates. Conversely, the autophagic system is able to degrade entire organelles and large proteins aggregates. In the autophagy-lysosome system, double-membrane vesicles named autophagosomes are able to engulf a portion of the cytosol and fuse with lysosomes, where their content is completely degraded by lytic enzymes.

The autophagy flux can be biochemicaly monitored following LC3 lipidation and p62 degradation. LC3 is the mammalian homolog of the yeast Atg8 gene, which is lipidated when recruited for the double-membrane commitment and growth. p62 (SQSTM-1) is a polyubiquitin-binding protein involved in the proteasome system and that can either reside free in the cytosol and nucleus or occur within autophagosomes and lysosomes. The GFP-LC3 transgenic mouse model allows easy detection of autophagosomes by simply monitoring the presence of bright GFP-positive puncta inside the myofibrils and beneath the plasma membrane of the myofibers, thus investigate the activation of autophagy in skeletal muscles with different contents of slow and fast-twitching myofibers and in response to stimuli such as fasting. For example, in the fast-twiching extensor digitorum longus muscle few GFP-LC3 dots were observed before starvation, while many small GFP-LC3 puncta appeared between myofibrils and in the perinuclear regions after 24 h starvation. Conversely, in the slow-twitching soleus muscle, autophagic puncta were almost absent in standard condition and scarcely induced after 24 h starvation.
Autophagy in Muscle Homeostasis
The autophagic flux was found to be increased during certain catabolic conditions, such as fasting, atrophy , and denervation , thus contributing to protein breakdown. Food deprivation is one of the strongest stimuli known to induce autophagy in muscle. Indeed skeletal muscle, after the liver, is the most responsive tissue to autophagy activation during food deprivation. Since muscles are the biggest reserve of amino acids in the body, during fasting autophagy has the vital role to maintain the amino acid pool by digesting muscular protein and organelles. In mammalian cells, mTORC1, which consists of

  • mTOR and
  • Raptor,

is the nutrient sensor that negatively regulates autophagy.

During atrophy, protein breakdown is mediated by atrogenes, which are under the forkhead box O (FoxO) transcription factors control, and activation of autophagy seems to aggravate muscle loss during atrophy. In vivo and in vitro studies demonstrated that several genes coding for components of the autophagic machinery, such as

  • LC3,
  • GABARAP,
  • Vps34,
  • Atg12 and
  • Bnip3,

are controlled by FoxO3 transcription factor. FoxO3 is able to regulate independently the ubiquitin-proteasome system and the autophagy-lysosome machinery in vivo and in vitro. Denervation is also able to induce autophagy in skeletal muscle, although at a slower rate than fasting. This effect is mediated by RUNX1, a transcription factor upregulated during autophagy; the lack of RUNX1 results in excessive autophagic flux in denervated muscle and leads to atrophy. The generation of Atg5 and Atg7 muscle-specific knockout mice have shown that with suppression of autophagy both models display muscle weakness and atrophy and a significant reduction of weight, which is correlated with the important loss of muscle tissue due to an atrophic condition. An unbalanced autophagy flux is highly detrimental for muscle, as too much induces atrophy whereas too little leads to muscle weakness and degeneration. Muscle wasting associated with autophagy inhibition becomes evident and symptomatic only after a number of altered proteins and dysfunctional organelles are accumulated, a condition that becomes evident after months or even years. On the other hand, the excessive increase of autophagy flux is able to induce a rapid loss of muscle mass (within days or weeks).
Alterations of autophagy are involved in the pathogenesis of several myopathies and dystrophies.

The maintenance of muscle homeostasis is finely regulated by the balance between catabolic and anabolic process. Macroautophagy (or autophagy) is a catabolic process that provides the degradation of protein aggregation and damaged organelles through the fusion between autophagosomes and lysosomes. Proper regulation of the autophagy flux is fundamental for the homeostasis of skeletal muscles during physiological situations and in response to stress. Defective as well as excessive autophagy is harmful for muscle health and has a pathogenic role in several forms of muscle diseases.
Grumati P, Bonaldo P. Autophagy in Skeletal Muscle Homeostasis and in Muscular Dystrophies. Cells 2012, 1, 325-345; doi:10.3390/cells1030325. ISSN 2073-4409. http://www.mdpi.com/journal/cells

Parkinson’s Disease Mutations
Mutations in parkin, a ubiquitin ligase, cause early-onset familial Parkinson’s disease (AR-JP). How Parkin suppresses Parkinsonism remains unknown. Parkin was recently shown to promote the clearance of impaired mitochondria by autophagy, termed mitophagy. Here, we show that Parkin promotes mitophagy by catalyzing mitochondrial ubiquitination, which in turn recruits ubiquitin-binding autophagic components, HDAC6 and p62, leading to mitochondrial clearance.

During the process, juxtanuclear mitochondrial aggregates resembling a protein aggregate-induced aggresome are formed. The formation of these “mito-aggresome” structures requires microtubule motor-dependent transport and is essential for efficient mitophagy. Importantly, we show that AR-JP–causing Parkin mutations are defective in supporting mitophagy due to distinct defects at

  • recognition,
  • transportation, or
  • ubiquitination of impaired mitochondria,

thereby implicating mitophagy defects in the development of Parkinsonism. Our results show that impaired mitochondria and protein aggregates are processed by common ubiquitin-selective autophagy machinery connected to the aggresomal pathway, thus identifying a mechanistic basis for the prevalence of these toxic entities in Parkinson’s disease.
Lee JY,Nagano Y, Taylor JP,Lim KL, and Yao TP. Disease-causing mutations in Parkin impair mitochondrial ubiquitination, aggregation, and HDAC6-dependent mitophagy. J Cell Biol 2010; 189(4):671-679. http://www.jcb.org/cgi/doi/10.1083/jcb.201001039

Drosophila Parkin Requires PINK1

Loss of the E3 ubiquitin ligase Parkin causes early onset Parkinson’s disease, a neurodegenerative disorder of unknown etiology. Parkin has been linked to multiple cellular processes including

  • protein degradation,
  • mitochondrial homeostasis, and
  • autophagy;

however, its precise role in pathogenesis is unclear. Recent evidence suggests that Parkin is recruited to damaged mitochondria, possibly affecting

  • mitochondrial fission and/or fusion,
  • to mediate their autophagic turnover.

The precise mechanism of recruitment and the ubiquitination target are unclear. Here we show in Drosophila cells that PINK1 is required to recruit Parkin to dysfunctional mitochondria and promote their degradation. Furthermore, PINK1 and Parkin mediate the ubiquitination of the profusion factor Mfn on the outer surface of mitochondria. Loss of Drosophila PINK1 or parkin causes an increase in Mfn abundance in vivo and concomitant elongation of mitochondria. These findings provide a molecular mechanism by which the PINK1/Parkin pathway affects mitochondrial fission/fusion as suggested by previous genetic interaction studies. We hypothesize that Mfn ubiquitination may provide a mechanism by which terminally damaged mitochondria are labeled and sequestered for degradation by autophagy.

Ziviani E, Tao RN, and Whitworth AJ. Drosophila Parkin requires PINK1 for mitochondrial translocation and ubiquitinates Mitofusin. PNAS 2010. Pp6 http://www.pnas.org/cgi/doi/10.1073/pnas.0913485107

Dynamin-related protein 1 (Drp1) in Parkinson’s
Mutations in Parkin, an E3 ubiquitin ligase that regulates protein turnover, represent one of the major causes of familial Parkinson’s disease (PD), a neurodegenerative disorder characterized by the loss of dopaminergic neurons and impaired mitochondrial functions. The underlying mechanism by which pathogenic parkin mutations induce mitochondrial abnormality is not fully understood. Here we demonstrate that Parkin interacts with and subsequently ubiquitinates dynamin-related protein 1 (Drp1), for promoting its proteasome-dependent degradation. Pathogenic mutation or knockdown of Parkin inhibits the ubiquitination and degradation of Drp1, leading to an increased level of Drp1 for mitochondrial fragmentation. These results identify Drp1 as a novel substrate of Parkin and suggest a potential mechanism linking abnormal Parkin expression to mitochondrial dysfunction in the pathogenesis of PD.

Wang H, Song P, Du L, Tian W. Parkin ubiquitinates Drp1 for proteasome-dependent degradation: implication of dysregulated mitochondrial dynamics in Parkinson’s disease.
JBC Papers in Press. Published on February 3, 2011 as Manuscript M110.144238. http://www.jbc.org/cgi/doi/10.1074/jbc.M110.144238

Pink1, Parkin, and DJ-1 Form a Complex
Mutations in the genes PTEN-induced putative kinase 1 (PINK1), PARKIN, and DJ-1 cause autosomal recessive forms of Parkinson disease (PD), and the Pink1/Parkin pathway regulates mitochondrial integrity and function. An important question is whether the proteins encoded by these genes function to regulate activities of other cellular compartments. A study in mice, reported by Xiong et al. in this issue of the JCI, demonstrates that Pink1, Parkin, and DJ-1 can form a complex in the cytoplasm, with Pink1 and DJ-1 promoting the E3 ubiquitin ligase activity of Parkin to degrade substrates via the proteasome (see the related article, doi:10.1172/ JCI37617).

This protein complex in the cytosol may or may not be related to the role of these proteins in regulating mitochondrial function or oxidative stress in vivo.
Three models for the role of the PPD complex. In this issue of the JCI, Xiong et al. report that Pink1, Parkin, and DJ-1 bind to each other and form a PPD E3 ligase complex in which Pink1 and DJ-1 modulate Parkin-dependent ubiquitination and subsequent degradation of substrates via the proteasome. Previous work suggests that the Pink1/Parkin pathway regulates mitochondrial integrity and promotes mitochondrial fission in Drosophila.

(A) Parkin and DJ-1 may be recruited to the mitochondrial outer membrane during stress and interact with Pink1. These interactions may facilitate the ligase activity of Parkin, thereby facilitating the turnover of molecules that regulate mitochondrial dynamics and mitophagy. The PPD complex may have other roles in the cytosol that result in degradative ubiquitination and/or relay information from mitochondria to other cellular compartments.
(B) Alternatively, Pink1 may be released from mitochondria after cleavage to interact with DJ-1 and Parkin in the cytosol.
A and B differ in the site of action of the PPD complex and the cleavage status of Pink1.
The complex forms on the mitochondrial outer membrane potentially containing full-length Pink1 in A, and in the cytosol with cleaved Pink1 in B.
Lack of DJ-1 function results in phenotypes that are distinct from the mitochondrial phenotypes observed in null mutants of Pink1 or Parkin in Drosophila. Thus, although the PPD complex is illustrated here as regulating mitochondrial fission, the role of DJ-1 in vivo remains to be clarified.
(C) It is also possible that the action occurs in the cytosol and is independent of the function of Pink1/Parkin in regulating mitochondrial integrity and function.

The Xiong et al. study offers an entry point for explorations of the role of Pink1, Parkin, and DJ-1 in the cytoplasm. It remains to be shown whether Parkin, in complex with Pink1 and DJ-1, carries out protein degradation in vivo.

Li H, and Guo M. Protein degradation in Parkinson disease revisited: it’s complex. commentaries. J Clin Invest.  doi:10.1172/JCI38619. http://www.jci.org

Xiong, H., et al. Parkin, PINK1, and DJ-1 form a ubiquitin E3 ligase complex promoting unfolded protein degradation. J. Clin. Invest. 2009; 119:650–660.

 Mitochondrial Ubiquitin Ligase, MITOL, protects neuronal cells

Nitric oxide (NO) is implicated in neuronal cell survival. However, excessive NO production mediates neuronal cell death, in part via mitochondrial dysfunction. Here, we report that the mitochondrial ubiquitin ligase, MITOL, protects neuronal cells from mitochondrial damage caused by accumulation of S-nitrosylated microtubule associated protein 1B-light chain 1 (LC1). S-nitrosylation of LC1 induces a conformational change that serves both to activate LC1 and to promote its ubiquination by MITOL, indicating that microtubule
stabilization by LC1 is regulated through its interaction with MITOL. Excessive NO production can inhibit MITOL, and MITOL inhibition resulted in accumulation of S-nitrosylated LC1 following stimulation of NO production by calcimycin and N-methyl-D-aspartate. LC1 accumulation under these conditions resulted in mitochondrial dysfunction and neuronal cell death. Thus, the balance between LC1 activation by S-nitrosylation and down-regulation by MITOL is critical for neuronal cell survival. Our findings may contribute significantly to an understanding of the mechanisms of neurological diseases caused by nitrosative stress-mediated mitochondrial dysfunction.

Yonashiro R, Kimijima Y, Shimura T, Kawaguchi K, et al. Mitochondrial ubiquitin ligase MITOL blocks S-nitrosylated MAP1B-light chain 1-mediated mitochondrial dysfunction and neuronal cell death. PNAS; 2012. pp 6. http://www.pnas.org/cgi/doi/10.1073/pnas.1114985109

Ubiquitin–Proteasome System in Neurodegeneration
A common histopathological hallmark of most neurodegenerative diseases is the presence of aberrant proteinaceous inclusions inside affected neurons. Because these protein aggregates are detected using antibodies against components of the ubiquitin–proteasome system (UPS), impairment of this machinery for regulated proteolysis has been suggested to be at the root of neurodegeneration. This hypothesis has been difficult to prove in vivo owing to the lack of appropriate tools. The recent report of transgenic mice with ubiquitous expression of a UPS-reporter protein should finally make it possible to test in vivo the role of the UPS in neurodegeneration.

Hernandez F, Dıaz-Hernandez M, Avila J and Lucas JJ. Testing the ubiquitin–proteasome hypothesis of neurodegeneration in vivo. TRENDS in Neurosciences 2004; 27(2): 66-68.

ALP in Parkinson’s
The ubiquitin-proteasome system (UPS) and autophagy-lysosome pathway (ALP) are the two most important mechanisms that normally repair or remove abnormal proteins. Alterations in the function of these systems to degrade misfolded and aggregated proteins are being increasingly recognized as playing a pivotal role in the pathogenesis of many neurodegenerative disorders such as Parkinson’s disease. Dysfunction of the UPS has been already strongly implicated in the pathogenesis of this disease and, more recently, growing interest has been shown in identifying the role of ALP in neurodegeneration. Mutations of a-synuclein and the increase of intracellular concentrations of non-mutant a-synuclein have been associated with Parkinson’s disease phenotype.

The demonstration that a-synuclein is degraded by both proteasome and autophagy indicates a possible linkage between the dysfunction of the UPS or ALP and the occurrence of this disorder.The fact that mutant a-synucleins inhibit ALP functioning by tightly binding to the receptor on the lysosomal membrane for autophagy pathway further supports the assumption that impairment of the ALP may be related to the development of Parkinson’s disease.

In this review, we summarize the recent findings related to this topic and discuss the unique role of the ALP in this neurogenerative disorder and the putative therapeutic potential through ALP enhancement.

Pan Y, Kondo S, Le W, Jankovic J. The role of autophagy-lysosome pathway in
neurodegeneration associated with Parkinson’s disease. Brain 2008; 131: 1969-1978. doi:10.1093/brain/awm318.

Ubiquitin-Proteasome System in Parkinson’s

There is growing evidence that dysfunction of the mitochondrial respiratory chain and failure of the cellular protein degradation machinery, specifically the ubiquitin-proteasome system, play an important role in the pathogenesis of Parkinson’s disease. We now show that the corresponding pathways of these two systems are linked at the transcriptomic level in Parkinsonian substantia nigra. We examined gene expression in medial and lateral substantia nigra (SN) as well as in frontal cortex using whole genome DNA oligonucleotide microarrays. In this study, we use a hypothesis-driven approach in analysing microarray data to describe the expression of mitochondrial and ubiquitin-proteasomal system (UPS) genes in Parkinson’s disease (PD).

Although a number of genes showed up-regulation, we found an overall decrease in expression affecting the majority of mitochondrial and UPS sequences. The down-regulated genes include genes that encode subunits of complex I and the Parkinson’s-disease-linked UCHL1. The observed changes in expression were very similar for both medial and lateral SN and also affected the PD cerebral cortex. As revealed by “gene shaving” clustering analysis, there was a very significant correlation between the transcriptomic profiles of both systems including in control brains.

Therefore, the mitochondria and the proteasome form a higher-order gene regulatory network that is severely perturbed in Parkinson’s disease. Our quantitative results also suggest that Parkinson’s disease is a disease of more than one cell class, i.e. that it goes beyond the catecholaminergic neuron and involves glia as well.

Duke DC, Moran LB, Kalaitzakis ME, Deprez M, et al. Transcriptome analysis reveals link between proteasomal and mitochondrial pathways in Parkinson’s disease. Neurogenetics 2006; 7:139-148.
Bax Degradation a Novel Mechanism for Survival in Bcl-2 overexpressed cancer cells
The authors previously reported that proteasome inhibitors were able to overcome Bcl-2-mediated protection from apoptosis, and now show that inhibition of the proteasome activity in Bcl-2-overexpressing cells accumulates the proapoptotic Bax protein to mitochondrial cytoplasm, where it interacts to Bcl-2 protein. This event was followed by release of mitochondrial cytochrome c into the cytosol and activation of caspase-mediated apoptosis. In contrast, proteasome inhibition did not induce any apparent changes in Bcl-2 protein levels. In addition, treatment with a proteasome inhibitor increased levels of ubiquitinated forms of Bax protein, without any effects on Bax mRNA expression. They also established a cell-free Bax degradation assay in which an in vitro-translated, 35S-labeled Bax protein can be degraded by a tumor cell protein extract, inhibitable by addition of a proteasome inhibitor or depletion of the proteasome or ATP. The Bax degradation activity can be reconstituted in the proteasome-depleted supernatant by addition of a purified 20S proteasome or proteasome-enriched fraction. Finally, by using tissue samples of human prostate adenocarcinoma, they demonstrated that increased levels of Bax degradation correlated well with decreased levels of Bax protein and increased Gleason scores of prostate cancer. These studies strongly suggest that ubiquitin-proteasome-mediated Bax degradation is a novel survival mechanism in human cancer cells and that selective targeting of this pathway should provide a unique approach for treatment of human cancers, especially those overexpressing Bcl-2.
In the current study, These investigators report that

  • (i) proteasome inhibition results in Bax accumulation before release of cytochrome c and induction of apoptosis, which is associated with the ability of proteasome inhibitors to overcome Bcl-2-mediated antiapoptotic function;
  • (ii) Bax is regulated by an ATP-ubiquitin-proteasome-dependent degradation pathway; and
  • (iii) decreased levels of Bax protein correlate with increased levels of Bax degradation in aggressive human prostate cancer.

Li B and Dou QP. Bax degradation by the ubiquitin-proteasome-dependent pathway: Involvement in tumor survival and progression. PNAS 2000; 97(8): 3851-3855. http://www.pnas.org

p97 and DBeQ, ATP-competitive p97 inhibitor
A major limitation to current studies on the biological functions of p97/Cdc48 is that there is no method to rapidly shut off its ATPase activity. Given the range of cellular processes in which Cdc48 participates, it is difficult to determine whether any particular phenotype observed in the existing mutants is due to a direct or indirect effect. Moreover, inhibition of p97 activity in animal cells by siRNA or expression of a dominant-negative version is challenged by its high abundance and is not suited to evaluating proximal phenotypic effects of p97 loss of function.

A specific small-molecule inhibitor of p97 would provide an important tool to investigate diverse functions of this essential ATPase associated with diverse cellular activities (AAA) ATPase and to evaluate its potential to be a therapeutic target in human disease. Cancer cells may be particularly sensitive to killing by suppression of protein degradation mechanisms, because they may exhibit a heightened dependency on these mechanisms to clear an elevated burden of quality-control substrates. For example, some cancers produce high levels of a specific protein that is a prominent quality-control substrate (e.g., Ig light chains in multiple myeloma) or produce high levels of reactive oxygen species, which can result in excessive protein damage via oxidation. Therefore, a specific p97 inhibitor would be a valuable research tool to investigate p97 function in cells.

We carried out a high-throughput screen to identify inhibitors of p97 ATPase activity. Dual-reporter cell lines that simultaneously express p97-dependent and p97-independent proteasome substrates were used to stratify inhibitors that emerged from the screen. N2,N4-dibenzylquinazoline-2,4-diamine (DBeQ) was identified as a selective,potent, reversible, and ATP-competitive p97 inhibitor.

DBeQ blocks multiple processes that have been shown by RNAi to depend on p97, including degradation of ubiquitin fusion degradation and endoplasmic reticulum-associated degradation pathway reporters, as well as autophagosome maturation. DBeQ also potently inhibits cancer cell growth and is more rapid than a proteasome inhibitor at mobilizing the executioner caspases-3 and -7.

Simultaneous inhibition of proteasome and histone deacetylase 6 (HDAC6) [which is required for autophagy results in synergistic killing of multiple myeloma cells]. Interestingly, more than one dozen human clinical trials (www.clinicaltrials.gov) combine bortezomib with the broad-spectrum HDAC inhibitor vorinostat, which is active toward HDAC6. Targeting p97
may provide an alternative route to achieving the same objective. Our results provide a rationale for targeting p97 in cancer therapy. Future work will provide molecular insight into how inhibition of p97 activity by DBeQ results in apoptosis and could strengthen the rationale for a p97-targeted cancer therapeutic.

Chou TF, Brown SJ, Minond D, Nordin BE, et al. Reversible inhibitor of p97, DBeQ, impairs both ubiquitin-dependent and autophagic protein clearance pathways. PNAS 2011; pp 6 http://www.pnas.org/cgi/doi/10.1073/pnas.1015312108

The causes of various neurodegenerative diseases, particularly sporadic cases, remain unknown, but increasing evidence suggests that these diseases may share similar molecular and cellular mechanisms of pathogenesis. One prominent feature common to most neurodegenerative diseases is the accumulation of misfolded proteins in the form of insoluble protein aggregates or inclusion bodies. Although these aggregates have different protein compositions, they all contain ubiquitin and proteasome subunits, implying a failure of the ubiquitin-proteasome system (UPS) in the removal of misfolded proteins.

A direct link between UPS dysfunction and neurodegeneration has been
provided by recent findings that genetic mutations in UPS components cause several rare, familial forms of neurodegenerative diseases. Furthermore, it is becoming increasingly clear that oxidative stress, which results from aging or exposure to environmental toxins, can directly damage UPS components, thereby contributing to the pathogenesis of sporadic forms of neurodegenerative diseases.

Aberrations in the UPS often result in defective proteasome-mediated protein degradation, leading to accumulation of toxic proteins and eventually to neuronal cell death. Interestingly, emerging evidence has begun to suggest that impairment in substrate-specific components of the UPS, such as E3 ubiquitin-protein ligases, may cause aberrant ubiquitination and neurodegeneration in a proteasome-independent manner. This provides an overview of the molecular components of the UPS and their impairment in familial and sporadic forms of neurodegenerative diseases, and summarizes present knowledge about the pathogenic mechanisms of UPS dysfunction in neurodegeneration.

Molecular mechanisms of protein ubiquitination and degradation by the UPS. Ubiquitination involves a highly specific enzyme cascade in which

  • ubiquitin (Ub) is first activated by the ubiquitinactivating enzyme (E1),
  • then transferred to an ubiquitin-conjugating enzyme (E2), and
  • finally covalently attached to the substrate by an ubiquitin-protein ligase (E3).

Ubiquitination is a reversible posttranslational modification in which the removal of Ub is mediated by a deubiquitinating enzyme (DUB).

  • Substrate proteins can be either monoubiquitinated or polyubiquitinated through successive conjugation of Ub moieties to an internal lysine residue in Ub.
  • K48-linked poly-Ub chains are recognized by the 26S proteasome, resulting in degradation of the substrate and recycling of Ub.
  • Monoubiquitination or K63-linked polyubiquitination plays a number of regulatory roles in cells that are proteasome-independent.

Parkin

Loss-of-function mutations in parkin, a 465-amino-acid RING-type E3 ligase, were first identified as the cause for autosomal recessive juvenile Parkinsonism (AR-JP) and subsequently found to account for ~50% of all recessively transmitted early-onset PD cases. Interestingly, patients with parkin mutations do not exhibit Lewy body pathology.

Possible pathogenic mechanisms by which impaired UPS components cause neurodegeneration. Genetic mutations or oxidative stress from aging and/or exposure to environmental toxins have been shown to impair the ubiquitination machinery (particularly E3 ubiquitin-protein ligases) and deubiquitinating enzymes (DUBs), resulting in abnormal ubiquitination. Depending on the type of ubiquitination affected, the impairment could cause neurodegeneration through two different mechanisms.

In the first model, aberrant K48-linked polyubiquitination resulting from impaired E3s or DUBs alters protein degradation by the proteasome, leading to accumulation of toxic proteins and subsequent neurodegeneration. The proteasomes could be directly damaged by oxidative stress or might be inhibited by protein aggregation, which exacerbates the neurotoxicity.

In the second model, aberrant monoubiquitination or K63-linked polyubiquitination resulting from impaired E3s or DUBs alters crucial non-proteasomal functions, such as gene transcription and protein trafficking, thereby causing neurodegeneration without protein aggregation.

These two models are not mutually exclusive because a single E3 or DUB enzyme, such as parkin or UCH-L1, could regulate more than one type of ubiquitination. In addition, abnormal ubiquitination and neurodegeneration could also result from mutation or oxidative stress-induced structural changes in the protein substrates that alter their recognition and degradation by the UPS.

Lian Li and Chin LS. IMPAIRMENT OF THE UBIQUITIN-PROTEASOME SYSTEM: A COMMON PATHOGENIC MECHANISM IN NEURODEGENERATIVE DISORDERS. In The Ubiquitin Proteasome System…Chapter 23. (Eds: Eds: Mario Di Napoli and Cezary Wojcik) 553-577 © 2007 Nova Science Publishers, Inc. ISBN 978-1-60021-749-4.

filedesc Schematic diagram of the ubiquitylati...

filedesc Schematic diagram of the ubiquitylation system. Created by Roger B. Dodd (Photo credit: Wikipedia)

 

Current Noteworthy Work

Nassif M and Hetz C.  Autophagy impairment: a crossroad between neurodegeneration and tauopathies.  BMC Biology 2012; 10:78. http://www.biomedcentral.com/1741-7007/10/78

Impairment of protein degradation pathways such as autophagy is emerging as a consistent and transversal pathological phenomenon in neurodegenerative diseases, including Alzheimer´s, Huntington´s, and Parkinson´s disease. Genetic inactivation of autophagy in mice has demonstrated a key role of the pathway in maintaining protein homeostasis in the brain, triggering massive neuronal loss and the accumulation of abnormal protein inclusions.  A paper in Molecular Neurodegeneration from Abeliovich´s group now suggests a role for phosphorylation of Tau and the activation of glycogen synthase kinase 3β (GSK3β) in driving neurodegeneration in autophagy-deficient neurons. We discuss the implications of this study for understanding the factors driving neurofibrillary tangle formation in Alzheimer´s disease and tauopathies.

Cajee UF, Hull R and Ntwasa M. Modification by Ubiquitin-Like Proteins: Significance in Apoptosis and Autophagy Pathways. Int. J. Mol. Sci. 2012, 13, 11804-11831; doi:10.3390/ijms130911804

Ubiquitin-like proteins (Ubls) confer diverse functions on their target proteins. The modified proteins are involved in various biological processes, including DNA replication, signal transduction, cell cycle control, embryogenesis, cytoskeletal regulation,
metabolism, stress response, homeostasis and mRNA processing. Modifiers such as SUMO, ATG12, ISG15, FAT10, URM1, and UFM have been shown to modify proteins thus conferring functions related to programmed cell death, autophagy and regulation of
the immune system. Putative modifiers such as Domain With No Name (DWNN) have been identified in recent times but not fully characterized. In this review, we focus on cellular processes involving human Ubls and their targets.

Aloy P. Shaping the future of interactome networks. (A report of the third Interactome Networks Conference, Hinxton, UK, 29 August-1 September 2007). Genome Biology 2007; 8:316 (doi:10.1186/gb-2007-8-10-316)

Complex systems are often networked, and biology is no exception. Following on from the genome sequencing projects,
experiments show that proteins in living organisms are highly connected, which helps to explain how such great complexity
can be achieved by a comparatively small set of gene products. At a recent conference on interactome networks held outside
Cambridge, UK, the most recent advances in research on cellular networks were discussed. This year’s conference focused on
identifying the strengths and weaknesses of currently resolved interaction networks and the techniques used to determine
them – reflecting the fact that the field of mapping interaction networks is maturing.

Peroutka RJ, Orcutt SJ, Strickler JE, and Butt TR. SUMO Fusion Technology for Enhanced Protein Expression and Purification in Prokaryotes and Eukaryotes. Chapter 2. in T.C. Evans, M.-Q. Xu (eds.), Heterologous Gene Expression in E. coli, Methods in Molecular Biology 705:15-29. DOI 10.1007/978-1-61737-967-3_2, © Springer Science+Business Media, LLC 2011

The preparation of sufficient amounts of high-quality protein samples is the major bottleneck for structural proteomics. The use of recombinant proteins has increased significantly during the past decades. The most commonly used host, Escherichia coli, presents many challenges including protein misfolding, protein degradation, and low solubility. A novel SUMO fusion technology appears to enhance protein expression and solubility (www.lifesensors.com). Efficient removal of the SUMO tag by SUMO protease in vitro facilitates the generation of target protein with a native N-terminus. In addition to its physiological relevance in eukaryotes, SUMO can be used as a powerful biotechnology tool for enhanced functional protein expression in prokaryotes and eukaryotes.

Juang YC, Landry MC, et al. OTUB1 Co-opts Lys48-Linked Ubiquitin Recognition to Suppress E2 Enzyme Function. Molecular Cell 2012; 45: 384–397. DOI 10.1016/j.molcel.2012.01.011

Ubiquitylation entails the concerted action of E1, E2, and E3 enzymes. We recently reported that OTUB1, a deubiquitylase, inhibits the DNA damage response independently of its isopeptidase activity. OTUB1 does so by blocking ubiquitin transfer by UBC13, the cognate E2 enzyme for RNF168. OTUB1 also inhibits E2s of the UBE2D and UBE2E families. Here we elucidate the structural mechanism by which OTUB1 binds E2s to inhibit ubiquitin transfer. OTUB1 recognizes ubiquitin-charged E2s through contacts with both donor ubiquitin and the E2 enzyme. Surprisingly, free ubiquitin associates with the canonical distal ubiquitin-binding site on OTUB1 to promote formation of the inhibited E2 complex. Lys48 of donor ubiquitin lies near the OTUB1 catalytic site and the C terminus of free ubiquitin, a configuration that mimics the products of Lys48-linked ubiquitin chain cleavage. OTUB1 therefore co-opts Lys48-linked ubiquitin chain recognition to suppress ubiquitin conjugation and the DNA damage response.

Hunter T. The Age of Crosstalk: Phosphorylation, Ubiquitination, and Beyond. Molecular Cell  2007; 28:730-738. DOI 10.1016/ j.molcel.2007.11.019.

Crosstalk between different types of posttranslational modification is an emerging theme in eukaryotic biology. Particularly prominent are the multiple connections between phosphorylation and ubiquitination, which act either positively or negatively in both directions to regulate these processes.

Tu Y, Chen C, et al. The Ubiquitin Proteasome Pathway (UPP) in the regulation of cell cycle control and DNA damage repair and its implication in tumorigenesis. Int J Clin Exp Pathol 2012;5(8):726-738. www.ijcep.com /ISSN:1936-2625/IJCEP1208018

Accumulated evidence supports that the ubiquitin proteasome pathway (UPP) plays a crucial role in protein
metabolism implicated in the regulation of many biological processes such as cell cycle control, DNA damage
response, apoptosis, and so on. Therefore, alterations for the ubiquitin proteasome signaling or functional impairments
for the ubiquitin proteasome components are involved in the etiology of many diseases, particularly in cancer
development.The authors discuss the ubiquitin proteasome pathway in the regulation of cell cycle control and DNA
damage response, the relevance for the altered regulation of these signaling pathways in tumorigenesis, and finally
assess and summarize the advancement for targeting the ubiquitin proteasome pathway in cancer therapy.

Cebollero E , Reggiori F  and Kraft C.  Ribophagy: Regulated Degradation of Protein Production Factories. Int J Cell Biol. 2012; 2012: 182834. doi:  10.1155/2012/182834 (online).

During autophagy, cytosol, protein aggregates, and organelles are sequestered into double-membrane vesicles called autophagosomes and delivered to the lysosome/vacuole for breakdown and recycling of their basic components. In all eukaryotes this pathway is important for adaptation to stress conditions such as nutrient deprivation, as well as to regulate intracellular homeostasis by adjusting organelle number and clearing damaged structures. For a long time, starvation-induced autophagy has been viewed as a nonselective transport pathway; however, recent studies have revealed that autophagy is able to selectively engulf specific structures, ranging from proteins to entire organelles. In this paper, we discuss recent findings on the mechanisms and physiological implications of two selective types of autophagy: ribophagy, the specific degradation of ribosomes, and reticulophagy, the selective elimination of portions of the ER.

Lee JH, Yu WH,…, Nixon RA.  Lysosomal Proteolysis and Autophagy Require Presenilin 1 and Are Disrupted by Alzheimer-Related PS1 Mutations. Cell 2010; 141, 1146–1158. DOI 10.1016/j.cell.2010.05.008.

Macroautophagy is a lysosomal degradative pathway essential for neuron survival. Here, we show that macroautophagy requires the Alzheimer’s disease (AD)-related protein presenilin-1 (PS1). In PS1 null blastocysts, neurons from mice hypomorphic for PS1 or
conditionally depleted of PS1, substrate proteolysis and autophagosome clearance during macroautophagy are prevented as a result of a selective impairment of autolysosome acidification and cathepsin activation. These deficits are caused by failed PS1-dependent
targeting of the v-ATPase V0a1 subunit to lysosomes. N-glycosylation of the V0a1 subunit, essential for its efficient ER-to-lysosome delivery, requires the selective binding of PS1 holoprotein to the unglycosylated subunit and the  sec61alpha/ oligosaccharyltransferase complex. PS1 mutations causing early-onset AD produce a similar lysosomal/autophagy phenotype in
fibroblasts from AD patients. PS1 is therefore essential for v-ATPase targeting to lysosomes, lysosome acidification, and proteolysis during autophagy. Defective lysosomal proteolysis represents a basis for pathogenic protein accumulations and neuronal cell death in AD and suggests previously unidentified therapeutic targets.

Pohl C and Jentsch S. Midbody ring disposal by autophagy is a post-abscission event of cytokinesis. nature cell biology 2009; 11 (1): 65-70.  DOI: 10.1038/ncb1813.

At the end of cytokinesis, the dividing cells are connected by an intercellular bridge, containing the midbody along with a single,
densely ubiquitylated, circular structure called the midbody ring (MR). Recent studies revealed that the MR serves as a target
site for membrane delivery and as a physical barrier between the prospective daughter cells. The MR materializes in telophase,
localizes to the intercellular bridge during cytokinesis, and moves asymmetrically into one cell after abscission. Daughter
cells rarely accumulate MRs of previous divisions, but how these large structures finally disappear remains unknown.
Here, we show that MRs are discarded by autophagy, which involves their sequestration into autophagosomes and delivery to
lysosomes for degradation. Notably, autophagy factors, such as the ubiquitin adaptor p62 and the ubiquitin-related protein Atg8 , associate with the MR during abscission, suggesting that autophagy is coupled to cytokinesis. Moreover, MRs accumulate in cells of patients with lysosomal storage disorders, indicating that defective MR disposal is characteristic of these diseases. Thus our findings suggest that autophagy has a broader role than previously assumed, and that cell renovation by clearing from superfluous large macromolecular assemblies, such as MRs, is an important autophagic function.

 

Hanai JI, Cao P, Tanksale P, Imamura S, et al. The muscle-specific ubiquitin ligase atrogin-1/MAFbx mediates statin-induced muscle toxicity. The Journal of Clinical Investigation  2007; 117(12):3930-3951.    http://www.jci.org

Statins inhibit HMG-CoA reductase, a key enzyme in cholesterol synthesis, and are widely used to treat hypercholesterolemia.
These drugs can lead to a number of side effects in muscle, including muscle fiber breakdown; however, the mechanisms of muscle injury by statins are poorly understood. We report that lovastatin induced the expression of atrogin-1, a key gene involved in skeletal muscle atrophy, in humans with statin myopathy, in zebrafish embryos, and in vitro in murine skeletal muscle cells. In cultured mouse myotubes, atrogin-1 induction following lovastatin treatment was accompanied by distinct morphological changes, largely absent in
atrogin-1 null cells. In zebrafish embryos, lovastatin promoted muscle fiber damage, an effect that was closely mimicked by knockdown of zebrafish HMG-CoA reductase. Moreover, atrogin-1 knockdown in zebrafish embryos prevented lovastatin-induced muscle injury. Finally, overexpression of PGC-1α, a transcriptional coactivator that induces mitochondrial biogenesis and protects against the development of muscle atrophy, dramatically prevented lovastatin-induced muscle damage and abrogated atrogin-1 induction both in fish and in cultured mouse myotubes. Collectively, our human, animal, and in vitro findings shed light on the molecular mechanism of statin-induced myopathy and suggest that atrogin-1 may be a critical mediator of the muscle
damage induced by statins.

Inami Y, Waguri S, Sakamoto A, Kouno T, et al.  Persistent activation of Nrf2 through p62 in hepatocellular carcinoma cells. J. Cell Biol. 2011; 193(2): 275–284. http://www.jcb.org/cgi/doi/10.1083/jcb.201102031

Macroautophagy (hereafter referred to as autophagy) is a cellular degradation system in which cytoplasmic components, including
organelles, are sequestered by double membrane structures called autophagosomes and the sequestered materials are
degraded by lysosomal hydrolases for supply of amino acids and for cellular homeostasis. Although autophagy has generally been considered nonselective, recent studies have shed light on another indispensable role for basal autophagy in cellular homeostasis, which is mediated by selective degradation of a specific substrate(s).  p62 is a ubiquitously expressed cellular protein that is conserved in metazoa but not in plants and fungi, and recently it has been known as one of the selective substrates for autophagy.
This protein is localized at the autophagosome formation site  and directly interacts with LC3, an autophagosome localizing protein . Subsequently, the p62 is incorporated into the autophagosome and then degraded. Therefore, impaired autophagy is accompanied by
accumulation of p62 followed by the formation of p62 and ubiquitinated protein aggregates because of the nature of both self- oligomerization and ubiquitin binding of p62.

 

Kima K, Khayrutdinov BI, Leeb CK, et al. Solution structure of the Zβ domain of human DNA-dependent activator of IFN-regulatory factors and its binding modes to B- and Z-DNAs. PNAS 2010; Early Edition ∣ pp 6. www.pnas.org/cgi/doi/10.1073/pnas.1014898107

The DNA-dependent activator of IFN-regulatory factors (DAI), also known as DLM-1/ZBP1, initiates an innate immune response by binding to foreign DNAs in the cytosol. For full activation of the immune response, three DNA binding domains at the N terminus are required: two Z-DNA binding domains (ZBDs), Zα and Zβ, and an adjacent putative B-DNA binding domain. The crystal structure of the Zβ domain of human DAI (hZβDAI) in complex with Z-DNA revealed structural features distinct from other known Z-DNA binding proteins, and it was classified as a group II ZBD. To gain structural insights into the DNA binding mechanism of hZβDAI, the solution structure of the free hZβDAI was solved, and its bindings to B- and Z-DNAs were analyzed by NMR spectroscopy. Compared to the Z-DNA–bound structure, the conformation of free hZβDAI has notable alterations in the α3 recognition helix, the “wing,” and Y145, which are critical in Z-DNA recognition. Unlike some other Zα domains, hZβDAI appears to have conformational flexibility, and structural adaptation is required for Z-DNA binding. Chemical-shift perturbation experiments revealed that hZβDAI also binds weakly to B-DNA via a different binding mode. The C-terminal domain of DAI is reported to undergo a conformational change on B-DNA binding; thus, it is possible that these changes are correlated. During the innate immune response, hZβDAI is likely to play an active role in binding to DNAs in both B and Z conformations in the recognition of foreign DNAs.

 

Epicrisis

This extensive review leaves little left unopened. We have seen the central role that the UPS system plays in normal organelle proteolysis in concert with autophagy. Impaired ubiquitination occurs from aging, and/or toxins, under oxidative stress involving E3s or DUBs.

This leads to altered gene transcripton, altered protein trafficking, and plays a role in neurodegenative disease, muscle malfunction, and cancer as well.

English: A cartoon representation of a lysine ...

English: A cartoon representation of a lysine 48-linked diubiquitin molecule. The two ubiquitin chains are shown as green cartoons with each chain labelled. The components of the linkage are indicated and shown as orange sticks. Image was created using PyMOL from PDB id 1aar. (Photo credit: Wikipedia)

Different forms of protein ubiquitylation

Different forms of protein ubiquitylation (Photo credit: Wikipedia)

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A Protease for ‘Middle-down’ Proteomics

Author and Reporter: Ritu Saxena, Ph.D.

Neil Kelleher and his research team at Northwestern University have developed a method for enzymatic proteolysis large peptides for mass spectrometry–based proteomics using a protease OmpT. The method was published in a recent issue of the journal Nature. http://www.ncbi.nlm.nih.gov/pubmed/22706673

Proteomics is defined as the study of the structure and function of proteins. Proteomic technologies will play an important role in drug discovery, diagnostics and molecular medicine because is the link between genes, proteins and disease. As researchers study defective proteins that cause particular diseases, their findings will help develop new drugs that either alter the shape of a defective protein or mimic a missing one. http://www.ama-assn.org/ama/pub/physician-resources/medical-science/genetics-molecular-medicine/current-topics/proteomics.page Proteomics, although refers to the study of the structure and function of proteins, it is often specifically used for protein purification and mass spectrometry.

‘Bottom-up’ and ‘Top-down’ are the two main strategies for proteomic studies using mass spectrometry. In Bottom-up proteomics referred to as the more common method, proteins are broken down into smaller pieces through enzymatic digestion followed by characterization into amino acid sequences and post translational modifications prior to analysis by mass spectrometry. By identifying and sequencing these smaller pieces, researchers can then determine the identity of the protein they make up. In Top-down proteomics, on the other hand, the process of proteolysis is skipped and it focuses on complete characterization of intact proteins and their post-translational modifications (PTMs).

“Although both the top-down and bottom-up approaches continue to mature, they each have limitations. The tryptic peptides used in the bottom-up approach are the primary unit of measurement, but their relatively small size (typically ~8–25 residues long) leads to problems such as sample complex­ity, difficulties in assigning peptides to specific gene products rather than protein groups, and loss of single and combinato­rial PTM information. The top-down approach handles these issues by characterizing intact proteins, but its success declines in the high-mass region. Therefore, a hybrid approach based on 2–20 kDa peptides could unite positive aspects of both bottom-up and top-down proteomics” says Kelleher et al in the research article.

The hybrid approach, referred to as ‘middle-down’ proteomics would enable the analysis of complex mixtures pre-sorted by protein size. Previously research efforts ‘middle-down’ proteomics included exploring the restricted proteolysis with enzyme alternatives to Trypsin and chemical methods (such as microwave-assisted acid hydrolysis), However, these methods generated peptides that were marginally longer than those produced by trypsin digestion. For the current study, Kelleher adds “We established an OmpT-based middle-down platform to analyze complex mixtures pre-sorted by protein size. After inte­grating the data from the middle-down workflow that was applied to ~20–100-kDa proteins fractionated from the HeLa cell proteome, we identified 3,697 unique peptides (average size: 6.3 kDa) from 1,038 unique proteins (26% average sequence coverage) at an esti­mated 1% false discovery rate”.

OmpT, a protease derived from Escherichia coli K12 outer membrane belongs to the novel omptin protease family10 and is known to cleave between two consecutive basic amino acid residues (Lys/Arg-Lys/Arg). The authors developed OmpT into an efficient rea­gent to generate >2-kDa peptides for middle-down proteomics, thus, utilizing OmpT to achieve robust, yet restricted, proteolysis of a complex genome. http://www.ncbi.nlm.nih.gov/pubmed/22706673

Researcher Kelleher and his team have been in news earlier for their work on ‘top-down’ proteomics when his team developed a new method that could separate and identify thousands of protein molecules quickly. In the first large-scale demonstration of the top-down method, the researchers were able to identify more than 3,000 protein forms created from 1,043 genes from human HeLa cells. The study was published in last year in the October issue of the journal Nature. http://www.ncbi.nlm.nih.gov/pubmed?term=22037311

Thus, Kelleher and his group was able to demonstrate that OmpT-based proteomic approach has a robust and restricted proteolysis capacity making it an attractive option for mass-spectrometry-based analysis of primary structure of protein.

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