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This paragraph is excerpted from the American Technion Society Facebook page.
Professor Avi Schroeder and Dr. Josh Schiffman of the The University of Utah are working with elephants at Utah’s Hogle Zoo on a possible new tool to fight against lung, bone, breast, and other cancers. Dr. Schiffman found that p53, a cancer-suppressing protein, is far more prevalent in elephants, which rarely develop cancer. Prof. Schroeder is now working to manufacture the protein in nanoparticles to begin preclinical testing.
This article is excerpted from The Salt Lake Tribune, May 2, 2019.
Earth’s biggest, smallest, oddest life forms are getting new attention from scientists. A Utah author explores what they’re learning.
Published: May 2, 2019
Researchers have long ignored superlative life forms — the biggest, the tiniest, ones that can survive extremes — as outliers, Utah author Matthew D. LaPlante says.
But they’re now realizing the value of studying nature’s “oddballs,” he adds, which are helping scientists discover how to better fight disease and aging, understand the history of life on this planet and how we might reach others.
LaPlante’s new book, “Superlative: The Biology of Extremes” was released this week. On Friday at 7 p.m., the associate professor of journalistic writing at Utah State University will read from “Superlative” and talk about his work at The King’s English Bookshop, 1511 S. 1500 East, Salt Lake City. The event is free and open to the public.
The co-writer of several books on the intersection of scientific discovery and society, LaPlante now is working with Harvard geneticist David Sinclair on a book about human longevity. “Superlative” from BenBella Books is the first solo book by LaPlante, a former reporter for The Salt Lake Tribune.
As he surveys unusual life around the earth, there are stops in Utah — from Pando, the aspen clone in Sevier County believed to be the single most massive living organism known on Earth, to pop-up appearances by researchers at the University of Utah and elephants at Hogle Zoo in Salt Lake City.
Vast sequences of the genetic coding that humans share with elephants still perform similar functions in each species, LaPlante explains. And long after the two diverged, both developed the same genetic solution for the oxygen needs of a larger brain.
So there’s reason to believe that responses elephants have evolved — such as rarely developing cancer — might be spurred in humans.
The potential within a genome for such new traits to develop is at the heart of comparative genomics — and at the work of Utah pediatric oncologist Josh Schiffman.
This excerpt from “Superlative” explains how Schiffman began working with Hogle Zoo’s African elephants — the largest living land mammals — to fight cancer.
It all started in the summer of 2012, when [pediatric oncologist Josh] Schiffman’s beloved dog, Rhody, passed away [due] to histiocytosis, a condition that attacks the cells of skin and connective tissue. “It was the only time my wife has ever seen me cry,” he told me. “Rhody was like our first child.”
Schiffman had heard dogs like his had an elevated risk of cancer, but it wasn’t until after Rhody’s death that he learned just how elevated it was. Bernese mountain dogs who live to the age of ten have a 50 percent risk of dying from cancer.
“Suddenly it dawned on me there was this whole other world, this young field of comparative oncology,” he said, “and I was pulled into the idea of being a pioneer and maybe a leader to help move things along.”
Schiffman had long been intrigued by the fact that size doesn’t appear to correlate to cancer rates — a phenomenon known as “Peto’s Paradox,” named for Oxford University epidemiologist Richard Peto. But when Schiffman took his children on an outing to Utah’s Hogle Zoo — the same place I sometimes go to have lunch with my elephant friend, Zuri — everything came together.
A keeper named Eric Peterson had just finished giving a talk to a crowd of visitors, mentioning in passing that the zoo’s elephants have been trained to allow the veterinary staff to take small samples of blood from a vein behind their ears. As the crowd dispersed, an angular, excited man approached him.
“I’ve got a strange question,” Schiffman said.
“We’ve heard them all,” Peterson replied.
“OK then — how do I get me some of that elephant blood?” Schiffman asked.
Peterson contemplated calling security. Instead, after a bit of explanation from Schiffman, the zookeeper told the inquisitive doctor he’d look into it. Two and a half months later, the zoo’s institutional review board gave its blessing to Schiffman’s request.
Things moved fast after that.
(Steve Griffin | Tribune file photo) Lab specialists Lauren Donovan Cristhian Toruno, Lisa Abegglen and researcher Joshua Schiffman, from left, are testing the effects of elephant gene p53 (EP53) in human cancer cells at the Huntsman Cancer Institute.
Cancer develops in part because cells divide. During each division the cells must make a copy of their DNA, and once in a while, for various reasons, those copies include a mistake. The more cells divide, the greater the odds of an error, and the more prone an error is to be duplicated again and again.
And elephant cells? Those things are dividing like crazy. Based on the number of cell divisions elephants need to get from Zuri’s size when we met to the size she is now, in just a few short years, it stands to reason they should get lots of cancer. Yet they almost never do.
“Going from 300 pounds as a calf to more than 10,000 pounds, gaining three-plus pounds a day, they’re growing so quickly, so big and so fast — baby elephants really shouldn’t make it to adulthood,” Schiffman said. “They should have 100 times the cancer. Just by chance alone, elephants should be dropping dead all over the place.” Indeed, he said, they should probably die of cancer before they’re even old enough to reproduce. “They should be extinct!”
Already, comparative oncologists suspected the exceptionally low rate of cancer in elephants had something to do with p53, a gene whose human analog is a known cancer suppressor. Most humans have one copy — two alleles — of the gene. Those with an inherited condition known as Li–Fraumeni syndrome, however, have just one allele — and a nearly 100 percent chance of getting cancer. The logical conclusion is more p53 alleles mean a better chance of staving off cancer. And elephants, it turns out, have twenty of them.
The big find that came from Schiffman’s exploration of the elephant blood he got at the zoo, though, was not just that there were more of these genes in elephants, but that the genes behaved a little bit differently, too.
In humans, the gene’s first approach for suppressing tumor growth is to try to repair faulty cells — the sort that cause cancer. So, at first, Schiffman’s team assumed having more p53 genes meant elephants had bigger repair crews. With the goal of watching those crews in action, the researchers exposed the elephant cells to radiation, causing DNA damage. But they noticed that, instead of trying to fix what was broken, the elephant cells seemed to grow something of a conscience.
To understand this, it’s helpful to think about how you’d respond in a zombie apocalypse. Of course you’d fight long and hard to keep from being infected, right? But if a zombie was about to chomp down on your arm, and there was nothing you could do to stop it, and if you had but one bullet remaining in your gun —and a few moments to consider what you might do to your fellow humans as a part of the legion of the undead — what would you do?
That’s what elephant cells do, too. Under the directive of p53, mutated cells don’t put up a fight. Upon recognizing the inevitability of malignant mutation, they take their own lives in a process known as apoptosis.
And they don’t just do this for one kind of cancer. The p53 gene apparently programs cells to do this in response to all kinds of malignantly mutated cells in elephants—a finding that flies in the face of the conventional assumption that there is no one singular cure for the complex group of disorders we call cancer.
When I first met Schiffman in 2016, he was brimming with excitement about the potential elephants have to help us understand cancer. He was also very cautious not to suggest he was anywhere near a cure, nor that he ever would be.
Just a few years later, though, Schiffman was speaking openly about his intention to rid the world of cancer. And, to that end, what’s happening in his lab is encouraging, to say the least.
He and his team have been injecting cancer cells with a synthetic version of a p53 protein modeled on the DNA he’s drawn from Zuri and other elephants from around the world. Viewed on time-lapse video, the results are unmistakable and amazing.
Breast cancer. Gone.
bone cancer. Gone.
Lung cancer. Gone.
One by one, each type of cancer cell falls victim to zombie-cell hara-kiri, shriveling and then exploding, and leaving nothing behind to mutate. Schiffman is now working with Avi Schroeder, an expert in nanomedical delivery systems at Technion-Israel Institute of Technology, to create tiny delivery vehicles to take the synthetic elephant protein into mammalian tumors.
If this was all the benefit we ever derived from studying elephants, it would be plenty.
Researchers have classified a brand-new organ inside human body. Known as the mesentery, the new organ is found in our digestive systems, and was long thought to be made up of fragmented, separate structures. But recent research has shown that it’s actually one, continuous organ. The evidence for the organ’s reclassification is now published in The Lancet Gastroenterology & Hepatology. Although we now know about the structure of this new organ, its function is still poorly understood, and studying it could be the key to better understanding and treatment of abdominal and digestive disease.
J Calvin Coffey, a researcher from the University Hospital Limerick in Ireland, who first discovered that the mesentery was an organ. In 2012, Coffey and his colleagues showed through detailed microscopic examinations that the mesentery is actually a continuous structure. Over the past four years, they’ve gathered further evidence that the mesentery should actually be classified as its own distinct organ, and the latest paper makes it official. Mesentery is a double fold of peritoneum – the lining of the abdominal cavity – that holds our intestine to the wall of our abdomen. It was described by the Italian polymath Leanardo da Vinci in 1508, but it has been ignored throughout the centuries, until now. Although there are generally considered to be five organs in the human body, there are in fact now 79, including the mesentery. The heart, brain, liver, lungs and kidneys are the vital organs, but there are another 74 that play a role in keeping us healthy. The distinctive anatomical and functional features of mesentery have been revealed that justify designation of the mesentery as an organ. Accordingly, the mesentery should be subjected to the same investigatory focus that is applied to other organs and systems. This provides a platform from which to direct future scientific investigation of the human mesentery in health and disease.
A number of novel genes have been identified in association with a variety of endocrine phenotypes over the last few years. However, although mutations in a number of genes have been described in association with disorders such as
hypogonadotropic hypogonadism,
congenital hypopituitarism,
disorders of sex development, and
congenital hyperinsulinism,
these account for a minority of patients with these conditions, suggesting that many more genes remain to be identified.
How will these novel genes be identified? Monogenic disorders can arise as a result of genomic microdeletions or microduplications, or due to single point mutations that lead to a functional change in the relevant protein. Such disorders may also result from altered expression of a gene, and hence altered dosage of the protein. Candidate genes may be identified by utilizing naturally occurring or transgenic mouse models, and this approach has been particularly informative in the elucidation of the genetic basis of a number of disorders.
Other approaches include the identification of chromosomal rearrangements using conventional karyotyping techniques, as well as novel assays such as array comparative genomic hybridization (CGH) and single nucleotide polymorphism oligonucleotide arrays (SNP arrays). These molecular methods usually result in the identification of gross abnormalities as well as submicroscopic deletions and duplications, and eventually to the discovery of single gene defects that are associated with a particular phenotype.
However, there is no doubt that the major advances in novel gene identification will be made as a result of the sequencing of the genome of affected individuals and comparison with control data that are already available. Chip techniques allow hybridization of DNA or RNA to hundreds of thousands of probes simultaneously. Microarrays are being used for mutational analysisof human disease genes.
Complete sequencing of genomes or sequencing of exons that encode proteins (exome sequencing) is now possible, and will lead to the elucidation of the etiology of a number of human diseases in the next few years. High-throughput, high-density sequencing using microarray technology potentially offers the option of obtaining rapid, accurate, and relatively inexpensive sequence of large portions of the genome. One such technique is oligo-hybridization sequencing, which relies on the differential hybridization of target DNA to an array of oligonucleotide probes. This technique is ideally suited to the analysis of DNA from patients with defined disorders, such as disorders of sex development and retinal disease, but suffers from a relatively high false positive rate and failure to detect insertions and deletions.
It is often difficult to perform studies in humans, and so the generation of animal models may be valuable in understanding the etiology and pathogenesis of disease. A number of naturally occurring mouse models have led to the identification of corresponding candidate genes in humans, with mutations subsequently detected in human patients. More frequently, genes of interest are often deleted and lead to the generation of disease models.
In general, mouse models correlate well with human disease; however species-specific defects need to be taken into account. Additionally, the transgenic models could be used to manipulate a condition, with the potential for new therapies. The advent of conditional transgenesis has led to an exponential increase in our understanding of how the mutation of a single gene impacts on a single organ. Using technology such as inducible gene expression systems, the effect of switching on or switching off a gene at a particular stage in development can be determined.
Advances in genomics will also have a major impact on therapeutics.Micro RNAs (miRNA) are small non-coding RNAs that regulate gene expression by targeting mRNAs of protein coding genes or non-coding RNA transcripts. Micro RNAs also have an important role in developmental and physiological processes and can act as tumor suppressors or oncogenes in the ontogenesis of cancers. The use of small interfering RNA (siRNA)offers promise of novel therapies in a range of conditions, such as cystic fibrosis and Type II autosomal dominant IGHD. Elucidation of the genetic basis of disease also allows more direct targeting of therapy. For instance, children with permanent neonatal-onset diabetes mellitus (PNDM) due to mutations in SUR1 or KIR6.2 were previously treated with insulin but have now been shown to respond well to sulfonylureas,thereby allowing the cessation of insulin therapy.
Finally, we are now entering the era of pharmacogenetics when the response of an individual to various therapeutic agents may be determined by their genotype. For example, a polymorphism in the GH receptor that results in deletion of exon 3 may be associated with an improved response to GH. Thus the elucidation of the genetic basis of many disorders will aid their management, and permit the tailoring of therapy in individual patients.
This post is in continuation to Part 1 by the same title.
In part one I covered the basics of role of redox chemistry in immune reactions, the phagosome cauldron, and how bacteria bacteria, virus and parasites trigger the complex pathway of NO production and its downstream effects. While we move further in this post, the previous post can be accessed here.
Regulation of the redox immunomodulators—NO/RNS and ROS
In addition to eradicating pathogens, NO/RNS and ROS and their chemical interactions act as effective immunomodulators that regulate many cellular metabolic pathways and tissue repair and proinflammatory pathways. Figure 3 shows these pathways.
Figure 3. Schematic overview of interactive connections between NO and ROS-mediated metabolic pathways. Credit: (Wink et al., 2011)
Regulation of iNOS enzyme activity is critical to NO production. Factors such as the availability of arginine, BH4, NADPH, and superoxide affect iNOS activity and thus NO production. In the absence of arginine and BH4 iNOS becomes a O2_/H2O2 generator (Vásquez-Vivar et al., 1999). Hence metabolic pathways that control arginine and BH4 play a role in determining the NO/superoxide balance. Arginine levels in cells depend on various factors such as type of uptake mechanisms that determine its spatial presence in various compartments and enzymatic systems. As shown in Fig3 Arginine is the sole substrate for iNOS and arginase. Arginase is another key enzyme in immunemodulation. AG is also regulated by NOS and NOX activities. NOHA, a product of NOS, inhibits AG, and O2–increases AG activity. Importantly, high AG activity is associated with elevated ROS and low NO fluxes. NO antagonises NOX2 assembly that in turn leads to reduction in O2_ production. NO also inhibits COX2 activity thus reducing ROS production. Thus, as NO levels decline, oxidative mechanisms increase. Oxidative and nitrosative stress can also decrease intracellular GSH (reduced form) levels, resulting in a reduced antioxidant capability of the cell.
Immune-associated redox pathways regulate other important metabolic cell functions that have the potential for widespread impact on cells, organs, and organisms. These pathways, such as mediated via methionine and polyamines, are critical for DNA stabilization, cell proliferation, and membrane channel activity, all of which are also involved in immune-mediated repair processes.
NO levels dictate the immune signaling pathway
NO/RNS and ROS actively control innate and adaptive immune signaling by participating in induction, maintenance, and/or termination of proinflammatory and anti-inflammatory signaling. As in pathogen eradication, the temporal and spatial concentration profiles of NO are key factors in determining immune-mediated processes.
Brune and coworkers (Messmer et al., 1994) first demonstrated that p53 expression was associated with the concentrations of NO that led to apoptosis in macrophages. Subsequent studies linked NO concentration profiles with expression of other key signaling proteins such as HIF-1α and Akt-P (Ridnour et al., 2008; Thomas et al., 2008). Various levels of NO concentrations trigger different pathways and expectedly this concentration-dependent profile varies with distance from the NO source.NO is highly diffucible and this characteristic can result in 1000 fold reduction in concentration within one cell length distance travelled from the source of production. Time course studies have also shown alteration in effects of same levels of NO over time e.g. NO-mediated ERK-P levels initially increased rapidly on exposure to NO donors and then decreased with continued NO exposure (Thomas et al., 2004), however HIF-1α levels remained high as long as NO levels were elevated. Thus some of the important factors that play critical role in NO effects are: distance from source, NO concentrations, duration of exposure, bioavailability of NO, and production/absence of other redox molecules.
Figure and legend credits: (Wink et al., 2011)
Fig 4: The effect of steady-state flux of NO on signal transduction mechanisms.
This diagram represents the level of sustained NO that is required to activate specific pathways in tumor cells. Similar effects have been seen on endothelial cells. These data were generated by treating tumor or endothelial cells with the NO donor DETANO (NOC-18) for 24 h and then measuring the appropriate outcome measures (for example, p53 activation). Various concentrations of DETANO that correspond to cellular levels of NO are: 40–60 μM DETANO = 50 nM NO; 80–120 μM DETANO = 100 nM NO; 500 μM DETANO = 400 nM NO; and 1 mM DETANO = 1 μM NO. The diagram represents the effect of diffusion of NO with distance from the point source (an activated murine macrophage producing iNOS) in vitro (Petri dish) generating 1 μM NO or more. Thus, reactants or cells located at a specific distance from the point source (i.e., iNOS, represented by star) would be exposed to a level of NO that governs a specific subset of physiological or pathophysiological reactions. The x-axis represents the different zone of NO-mediated events that is experienced at a specific distance from a source iNOS producing >1 μM. Note: Akt activation is regulated by NO at two different sites and by two different concentration levels of NO.
Species-specific NO production
The relationship of NO and immunoregulation has been established on the basis of studies on tumor cell lines or rodent macrophages, which are readily available sources of NO. However in humans the levels of protein expression for NOS enzymes and the immune induction required for such levels of expression are quite different than in rodents (Weinberg, 1998). This difference is most likely due to the human iNOS promotor rather than the activity of iNOS itself. There is a significant mismatch between the promoters of humans and rodents and that is likely to account for the notable differences in the regulation of gene induction between them. The combined data on rodent versus human NO and O2– production strongly suggest that in general, ROS production is a predominant feature of activated human macrophages, neutrophils, and monocytes, and the equivalent murine immune cells generate a combination of O2– and NO and in some cases, favor NO production. These differences may be crucial to understanding how immune responses are regulated in a species-specific manner. This is particularly useful, as pathogen challenges change constantly.
The next post in this series will cover the following topics:
The impact of NO signaling on an innate immune response—classical activation
NO and proinflammatory genes
NO and regulation of anti-inflammatory pathways
NO impact on adaptive immunity—immunosuppression and tissue-restoration response
Coronary Artery Disease – Medical Devices Solutions: From First-In-Man Stent Implantation, via Medical Ethical Dilemmas to Drug Eluting Stents August 13, 2012
Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production July 19, 2012
Curator and Research Study Originator: Aviva Lev-Ari, PhD, RN
Macrovascular Disease – Therapeutic Potential of cEPCs: Reduction Methods for CV Risk
July 2, 2012
An Investigation of the Potential of circulating Endothelial Progenitor Cells (cEPCs) as a Therapeutic Target for Pharmacological Therapy Design for Cardiovascular Risk Reduction: A New Multimarker Biomarker Discovery
Inhibition of ET-1, ETA and ETA-ETB, Induction of NO production, stimulation of eNOS and Treatment Regime with PPAR-gamma agonists (TZD): cEPCs Endogenous Augmentation for Cardiovascular Risk Reduction – A Bibliography
Advances in Separations Technology for the “OMICs” and Clarification of Therapeutic Targets
Curator, Reporter, EAW: Larry H Bernstein, MD, FCAP
Article 1.5 Advances in Separations Technology for the “OMICs” and Clarification of Therapeutic Targets
This discussion is a continuation of an earlier piece on the technologic framework for , proteomics, nutrigenomics, and translational medicine. The last decade has seen the emergence of a genomic science that is changing the trajectory of biological sciences and medicine. It has not resolved all of our problems by any means, but it has begun to redraw the map, which began with the elucidation of major metabolic pathways in the first half of the 20th century, was then captured by the transformation of genetics with the discovery of the “Watson-Crick Model”, and then later was recharged with the discovery of the Toll-like receptor and the drawing of “signaling pathways”. What we have seen in an unraveling of protein-genome interactions, small peptide regulators, and dynamic changes in pathway dominance, bloackage, and reentry, depending on genetic, dietary, and environmental conditions, mostly expressed in what we refer to as “oxidative stress”.
Unraveling the multitude of nutrigenomic, proteomic, and metabolomic patterns that arise from the ingestion of foods or their bioactive food components will not be simple but is likely to provide insights into a tailored approach to diet and health. The use of new and innovative technologies, such as microarrays, RNA interference, and nanotechnologies, will provide needed insights into molecular targets for specific bioactive food components and how they harmonize to influence individual phenotypes. A challenging aspect of omic technologies is the refined analysis of quantitative dynamics in biological systems.
In recent years, nutrition research has moved from classical epidemiology and physiology to molecular biology and genetics. The new era of nutrition research translates empirical knowledge to evidence-based molecular science. Following this trend, Nutrigenomics has emerged as a novel and multidisciplinary research field in nutritional science that aims to elucidate how diet can influence human health. It is already well known that bioactive food compounds can interact with genes affecting transcription factors, protein expression and metabolite production. The study of these complex interactions requires the development of advanced analytical approaches combined with bioinformatics. The Institute of Medicine recently convened a workshop to review the state of the various domains of nutritional genomics research and policy and to provide guidance for further development and translation of this knowledge into nutrition practice and policy. Nutritional genomics holds the promise to revolutionize both clinical and public health nutrition practice and facilitate the establishment of
genome-informed nutrient and food-based dietary guidelines for disease prevention and healthful aging,
individualized medical nutrition therapy for disease management, and
better targeted public health nutrition interventions (including micronutrient fortification and supplementation) that maximize benefit and minimize adverse outcomes within genetically diverse human populations.
For metabolomics, gas and liquid chromatography coupled to mass spectrometry are well suited for coping with high sample numbers in reliable measurement times with respect to both technical accuracy and the identification and quantitation of small-molecular-weight metabolites. This potential is a prerequisite for the analysis of dynamic systems. Thus, metabolomics is a key technology for systems biology. The bioavailability of bioactive food constituents as well as dose-effect correlations are key information to understand the impact of food on defined health outcomes. Both strongly depend on appropriate analytical tools to identify and quantify minute amounts of individual compounds in highly complex matrices–food or biological fluids–and to monitor molecular changes in the body in a highly specific and sensitive manner. Based on these requirements, mass spectrometry has become the analytical method of choice with broad applications throughout all areas of nutrition research.
Dynamic Construct of the –Omics
Metabolomics is a term that encompasses several types of analyses, including
metabolic fingerprinting, which measures a subset of the whole profile with little differentiation or quantitation of metabolites;
metabolic profiling, the quantitative study of a group of metabolites, known or unknown, within or associated with a particular metabolic pathway; and
target isotope-based analysis, which focuses on a particular segment of the metabolome by analyzing only a few selected metabolites that comprise a specific biochemical pathway.
Any unifying concept of the metabolome was incomplete or debatable in the first 30 years of the 20th century. It was only known that insulin is anabolic and that insulin deficiency (or resistance) would have consequences in the point of entry into the citric acid cycle, which generates 28-32 ATPs. In fat catabolism, triglycerides are hydrolyzed to break them into fatty acids and glycerol. In the liver the glycerol can be converted into glucose via dihydroxyacetone phosphate and glyceraldehyde-3-phosphate by way of gluconeogenesis. In the case of this cycle there is a tie in with both catabolism and anabolism.
For bypass of the Pyruvate Kinase reaction of Glycolysis, cleavage of 2 ~P bonds is required. The free energy change associated with cleavage of one ~P bond of ATP is insufficient to drive synthesis of phosphoenolpyruvate (PEP), since PEP has a higher negative DG of phosphate hydrolysis than ATP. The two enzymes that catalyze the reactions for bypass of the Pyruvate Kinase reaction are the following:
Pyruvate Carboxylase (Gluconeogenesis) catalyzes pyruvate + HCO3- + ATP — oxaloacetate + ADP + Pi
PEP Carboxykinase (Gluconeogenesis) catalyzes: oxaloacetate + GTP —- phosphoenolpyruvate + GDP + CO2
Many high throughput methods have been employed to get some insight into the whole process and several examples of successful research. Proteomics and metabolomics need to encompass large numbers of experiments and linked data. Due to the nature of the proteins, as well as due to the properties of various metabolites, experimental approaches require the use of comprehensive high throughput methods and a sufficiency of analysed tissue or body fluids.
An important and revolutionary aspect of ‘The 2010 Project’ is that it implicitly endorses the allocation of resources to attempts to assign function to genes that have no known function. This represents a significant departure from the common practice of defining and justifying a scientific goal based on the biological phenomena. The rationale for endorsing this radical change is that for the first time it is feasible to envision a whole-systems approach to gene and protein function. I shall not discuss the emerging field of bioinformatics that makes this possible. In this review, the end-of-the line “detector will be considered having been covered. The entire focus proceeds to a discussion of separation methods. Separation methods have always been tricky, time consuming, and a multiple step process that depended on using anionic and cationic resins as intermediate steps in bulk separation, and then molecular size separation. Therapeutic Targets will be identified as they are seen.
Affinity Chromatography The rapid development of biotechnology and biomedicine requires more reliable and efficient separation technologies for the isolation and purification of biopolymers such as therapeutic proteins, antibodies, enzymes and nucleic acids. In particular, monoclonal antibodies are centrally important as therapeutics for the treatment of cancer and other diseases, leading to recombinant monoclonal antibodies that dominate today’s biopharmaceutical pipeline. The large-scale production of therapeutic biopolymers requires
a manufacturing process that delivers reliability and in high-yield, as well as
an effective purification process affording extremely pure products.
Because of its high selectivity, affinity chromatography has been used extensively to isolate a variety of biopolymers. The retention of solutes is based on specific, reversible interactions found in biological systems, such as the binding of an enzyme with an inhibitor or an antibody with an antigen. These interactions are exploited in affinity chromatography by immobilizing an affinity ligand onto a support, and using this as a stationary phase. Non-porous particles having an average diameter of 2.1 mm were prepared by co-polymerization of styrene, methyl methacrylate and glycidyl methacrylate, which was abbreviated as P(S–MMA–GMA). The particles were mechanically stable due to the presence of benzene rings in the backbone of polymer chains, and could withstand high pressures when a column packed with these particles was operated in the HPLC mode.
The polymer particles were advantaged by immobilization of ligands via the epoxy groups on the particle surface that were introduced by one of the monomers, glycidyl methacrylate. As a model system, Cibacron Blue 3G-A was covalently immobilized onto the non-porous copolymer beads. The dye-immobilized P(S–MMA–GMA) particles were slurry packed into a 1.0 cm30.46 cm I.D. column. This affinity column was effective for the separation of turkey egg white lysozyme from a protein mixture. The bound lysozyme could be eluted to yield a sharp peak by using a phosphate buffer containing 1 M NaCl. For a sample containing up to 8 mg of lysozyme, the retained portion of proteins could be completely eluted without any slit peak. Due to the use of a shorter column, the analysis time was shorter in comparison with other affinity systems reported in the literature. The retention time could be reduced significantly by increasing the flow-rate, while the capacity factor remained at the same level. CH Chen, WC Lee. Affinity chromatography of proteins on non-porous copolymerized particles of styrene, methyl methacrylate and glycidyl methacrylate. Journal of Chromatography A 2001; 921: 31–37.
Affinity separation membranes, consisting of electrospun nanofibers, have been developed recently. Affinity ligands are attached to the surface of the constituent fibers, offering a potential solution to some of the problems of traditional, column-based, affinity chromatography. Electrospun fibers are good candidates for use in affinity separation because of their
unique characteristics of high surface area to volume ratio, resulting in
high ligand loading, and
their large porosity, resulting in
high throughput operation.
A number of polymers have been used for electrospun fiber mesh-based affinity membrane separations including poly (ether-urethane-urea), cellulose, poly(ethylene terephthalate, polysulphone, and polyacrlonitrile. Typically, very thin electrospun fiber meshes are produced by electrostatically collecting negatively charged fibers on a collector electrode. These very thin 2D electrospun fiber mesh mats provide excellent solution permeability as compared to 3D column packed with affinity beads. M Miyauchi, J Miao, TJ Simmons, JS Dordick and RJ Linhardt. Flexible Electrospun Cellulose Fibers as an Affinity Packing Material for the Separation of Bovine Serum Albumin. J Chromatograph Separat Techniq 2011; 2:2 http://dx.doi.org/10.4172/2157-7064.1000110
Dye Affinity Chromatography Biomimetic Dyes Affinity adsorbents based on immobilized triazine dyes offer important advantages circumventing many of the problems associated with biological ligands. The main drawback of dyes is their moderate selectivity for proteins. Rational attempts to tackle this problem are realized through the biomimetic dye concept according to which new dyes, the biomimetic dyes, are designed to mimic natural ligands. Biomimetic dyes are expected to exhibit increased affinity and purifying ability for the targeted proteins.
Biocomputing offers a powerful approach to biomimetic ligand design. The successful exploitation of contemporary computational techniques in molecular design requires the knowledge of the three-dimensional structure of the target protein, or at least, the amino acid sequence of the target protein and the three-dimensional structure of a highly homologous protein. From such information one can then design, on a graphics workstation,
the model of the protein and also
a number of suitable synthetic ligands which mimic natural biological ligands of the protein.
There are several examples of enzyme purifications
Interactions between Cibacron Blue F3GA (CB F3GA), as a model of triazine dye, and 2-hydroxypropyl-b-cyclodextrin (HP-b-CD), as a model of cyclodextrin, were investigated by monitoring the spectral shift that accompanies the binding phenomena. Matrix analysis of the difference spectral titration of CB F3GA with HP-b-CD revealed only two absorbing species, indicating a host–guest ratio of 1:1. The dissociation constant for this HP-b-CD–CB F3GA complex, K , was found d to be 0.43 mM. The data for HP-b-CD forming inclusion complexes with CB F3GA were used to develop the concept of competitive elution by inclusion complexes in dye-affinity chromatography. When this concept was applied to the elution of L-lactate dehydrogenase from a CB F3GA affinity matrix, it was shown to be an effective elution strategy. It provided a 15-fold purification factor with 89% recovery and sharp elution profile (0.8 column volumes for 80% recovery), which is as good as that obtained by specific elution with NADH (16-fold, 78% recovery and 1.8 column volumes). In addition, the new elution strategy showed a better purification factor and sharper elution profile than traditional non-specific. JA Lopez-Mas, SA Streitenberger, F Garcıa-Carmona, AA Sanchez-Ferrer. Cyclodextrin biospecific-like displacement in dye-affinity chromatography. Journal of Chromatography A 2001; 911: 47–53.
Affinity chromatography uses biospecific binding usually between an antibody and an antigen, an enzyme and a substrate or other pairs of key-lock type of matching molecules. Due to its high selectivity, it is able to purify proteins and other macromolecules from very dilute solutions. In this work, a general rate model for affinity chromatography was used for scale-up studies. Parameters for the model were estimated from existing correlations, or from experimental results obtained on a small column with the same packing material. As anexample, Affi-Gel with 4.5mol cm−3 Cibacron Blue F-3GA as immobilized ligands covalently attached to cross-linked 6% agarose was used for column packing. Cibacron Blue F-3GA was also used as a soluble ligand in the elution stage. Satisfactory scale-up predictions were obtained for a 98.2 ml column and a 501 ml column based on a few experimental data obtained on a 7.85 ml small column. T. Gu, K.-H. Hsu and M.-J. Syu, “Scale-Up of Affinity Chromatography for Purification of Enzymes and Other Proteins.” Enzyme and Microbial Technology 2003; 33:433-437.
Affinity Column with AAAA as a Model Sense Ligand The degeneracy of antisense peptides was studied by high-performance affinity chromatography. A model sense peptide (AAAA) and its antisense peptides (CGGG, GGGG, RGGG, SGGG) were designed and synthesized according to the degeneracy of genetic codes. An affinity column with AAAA as the ligand was prepared. The affinity chromatographic behaviors of antisense peptides on the column were evaluated. The results indicated that model antisense peptides have clear retention on the immobilized AAAA affinity column. RGGG showed the strongest affinity interaction. R Zhao, X Yu, H Liu, L Zhai, S Xiong, et al. Study on the degeneracy of antisense peptides using affinity chromatography. Journal of Chromatography A 2001; 913: 421–428.
Frontal AC for Biomolecular Interactions Frontal affinity chromatography is a method for quantitative analysis of biomolecular interactions. We reinforced it by incorporating various merits of a contemporary liquid chromatography system. As a model study, the interaction between an immobilized Caenorhabditis elegans galectin (LEC-6) and fluorescently labeled oligosaccharides (pyridylaminated sugars) was analyzed. LEC-6 was coupled to N-hydroxysuccinimide-activated Sepharose 4 Fast Flow (100 mm diameter), and packed into a miniature column (e.g., 1034.0 mm, 0.126 ml). The volume of the elution front (V) determined graphically for each sample was compared with that obtained in the presence of an excess amount of hapten saccharide, lactose (V ); and the dissociation constant, K , was calculated according to the literature. This system also proved to be useful for an inverse confirmation; that is, application of galectins to an immobilized glycan column (in the present case, asialofetuin was immobilized on Sepharose 4 Fast Flow), and the elution profiles were monitored by fluorescence based on tryptophan. The newly constructed system proved to be extremely versatile. It enabled rapid (analysis time 12 min/ cycle) and sensitive (20 nM for pyridylaminated derivatives, and 1 mg/ml for protein) analyses of lectin–carbohydrate interactions. J Hirabayashi, Y Arata, K Kasai. Reinforcement of frontal affinity chromatography for effective analysis of lectin–oligosaccharide interactions. Journal of Chromatography A 2000; 890:261–271.
Immobilized Metal Ion Affinity New immobilized metal ion affinity chromatography (IMAC) matrices containing a high concentration of metal–chelate moieties and completely coated with inert flexible and hydrophilic dextrans are here proposed to improve the purification of polyhistidine (poly-His) tagged proteins. The purification of an interesting recombinant multimeric enzyme (a thermoresistant b-galactosidase from Thermus sp. strain T2) has been used to check the performance of these new chromatographic media.
IMAC supports with a high concentration (and surface density) of metal chelate groups promote a rapid adsorption of poly-His tagged proteins during IMAC. However, these supports also favor the promotion of undesirable multi-punctual adsorptions and problems may arise for the simple and effective purification of poly-His tagged proteins. For example, desorption of the pure enzyme from the support may become quite difficult (e.g., it is not fully desorbed from the support even using 200 mM of imidazole).
The coating of these IMAC supports with dextrans greatly reduces these undesired multi-point adsorptions. However, this dextran coating of chromatographic matrices seems to allow the formation of strong one-point adsorptions that involve small areas of the protein and support surface, but the dextran coating seems to have dramatic effects for the prevention of weak or strong multipoint interactions that should involve a high geometrical congruence between the enzyme and the support surface. C Mateo , G Fernandez-Lorente , BCC Pessela , A Vian, et al. Affinity chromatography of polyhistidine tagged enzymes. New dextran-coated immobilized metal ion affinity chromatography matrices for prevention of undesired multipoint adsorptions. Journal of Chromatography A 2001; 915:97–106. The underlying principle of immobilized metal ion affinity chromatography (IMAC) of proteins is the coordination between the electron donor groupings on a protein surface (histidine, tryptophan, cysteine) and chelated (iminodiacetate; IDA) transition metal ions [IDA-M(II)]. This principle of immobilized metal ion affinity (IMA) has been presented by now in some detail. The practice of IMAC in the purification of proteins has had its empirical phase. There is now a need, from the body of data, to establish somewhat more detailed ground rules that would allow for the use of IMAC in a more predictive manner. Immobilized metal ion affinity chromatography (IMAC) has been explored as a probe into the topography of histidyl residues of a protein molecule. An evaluation of the chromatographic behavior of selected model proteins-
thioredoxin
ubiquitin
calmodulin
lysozyme
cytochrome c
myoglobin
on immobilized transition metal ions
Co2+
Ni2+
Cu2+
Zn2
-allows establishment of the following facets of the histidyl side chain distribution:
either interior or surface;
when localized on the surface, accessible or unaccessible for coordination;
single or multiple;
When multiple, either distant or vicinal.
Moreover, proteins displaying single histidyl side chains on their surfaces may, in some instances, be resolved by IMAC; apparently, the microenvironments of histidyl residues are sufficiently diverse to result in different affinities for the immobilized metal ions. IMAC, previously introduced as an approach to the fractionation of proteins, has become also, upon closer examination, a facile probe into the topography of histidyl residues. This is possible because of the inherent versatility of IMAC; an appropriate metal ion (M2+) can be selected to suit the analytical purpose and a particular chromatographic protocol can be applied (isocratic pH, falling pH, and imidazole elution). We now report that IMAC may be exploited as an analytical tool in addition to its use as a protein purification technique. IMAC can be used to ascertain several facets of the status of a histidyl residue(s) in a protein molecule:
localization (interior vs. surface)
coordination potential as defined by the steric accessibility and the state of protonation
A novel, two-step preparative technique is described for the purification of authentic recombinant human prolactin (rhPRL) secreted into the periplasm of transformed Escherichia coli cells. The first step is based on immobilized metal ion affinity chromatography of periplasmic extract, using Ni(II) as a relatively specific ligand for hPRL in this system. It gives superior resolution and yield than established ion-exchange chromatography. Size-exclusion chromatography is used for further purification to .99.5% purity. The methodology is reproducible, leading to 77% recovery. Identity and purity of the rhPRL were demonstrated using sodium dodecylsulphate–polyacrylamide electrophoresis, isoelectric focusing, mass spectrometry (matrix-assisted laser desorption ionization time-of-flight), radioimmunoassay, RP-HPLC and high-performance size-exclusion chromatography. In the Nb2 bioassay, the hormone showed a bioactivity of 40.9 IU/mg.
Adenosine Affinity Ligand for Glutamine Synthase Glutamine synthetase has been purified from both procaryotic and eucaryotic sources using various types of affinity chromatography. For example, ADP-agarose has been used to purify glutamine synthetase from photosynthetic bacteria, while the related “Blue” chromatography media (e.g. Affigel Blue) have been used to purify glutamine synthetases from a variety of sources. In addition, 2’,5’-ADPSepharose 4B has been used to purify glutamine synthetase from procaryotes, plants and insects. However, this latter affinity ligand resembles NADP more than ADP, particularly with respect to the position of the phosphate moieties. This is reflected in the more general use of this affinity ligand in the purification of NADPH-dependent enzymes. In the present report, we characterize the ability of glutamine synthetase to be purified by three different adenosine-affinity ligands: 5’-ADP-agarose (an ADP analogue), 2’,5’-ADP-Sepharose 4B (an NADP analogue) and 3’,5’-ADP-agarose (a cyclic AMP analogue). We report conditions for the successful purification of insect flight muscle glutamine synthetase using each of these three different affinity ligands. The enzyme bound most strongly to the
ADP analogue (S-ADP-agarose),
followed by the NADPH analogue (2’,5’-ADP-Sepharose 4B), and least strongly to
the cyclic AMP analogue (3’J’-ADP-agarose).
In all cases, binding was strongest in the presence of Mn2+ when compared to Mg”. These results suggest that the binding of glutamine synthetase to adenosine-affinity media is related to the participation of Mn. ADP in the y-glutamyl transferase reaction that is catalyzed by glutamine synthetase. M Dowton, IR Kennedy. Purification of glutamine synthetase by adenosine-affinity chromatography. Journal of Chromatography A 1994; 664: 280-283
Aptamer Based Stationary Phase An anti-adenosine aptamer was evaluated as a stationary phase in packed capillary liquid chromatography. Using an 21 aqueous mobile phase containing 20 mM Mg , adenosine was strongly retained on the column. A gradient of increasing 21 Ni (to 18 mM), which is presumed to complex with nitrogen atoms in adenosine involved in binding to the aptamer, eluted adenosine in a narrow zone. The adenosine assay, which required no sample preparation, was used on microdialysis samples. Total analysis times were short so samples could be injected every 5 min. Q Deng, CJ Watson, RT Kennedy. Aptamer affinity chromatography for rapid assay of adenosine in microdialysis samples collected in vivo. Journal of Chromatography A 2003; 1005:123–130.
We will realize the full power of proteomics only when we can measure and compare the proteomes of many individuals to identify biomarkers of human health and disease and track the blood-based proteome of an individual over time. Because the human proteome contains an estimated 20,000 proteins – plus splicing and post-translational variants – that span a concentration range of ,12 logs, identifying and quantifying valid biomarkers is a great technical challenge. Proteomic measurements demand
extreme sensitivity
specificity
dynamic range
accurate quantification.
We describe a new class of DNA-based aptamers enabled by a versatile chemistry technology that endows nucleotides with protein-like functional groups. These modifications greatly expand the repertoire of targets accessible to aptamers. The resulting technology provides efficient, large-scale selection of exquisite protein-binding reagents selected specifically for use in highly multiplexed proteomics arrays. Aptamers are a class of nucleic acid-based molecules discovered twenty years ago, and have since been employed in diverse applications including
therapeutics
catalysis
proteomics
Aptamers are short single-stranded oligonucleotides, which fold into diverse and intricate molecular structures that bind with high affinity and specificity to
proteins
peptides
small molecules.
Aptamers are selected in vitro from enormously large libraries of randomized sequences by the process of Systematic Evolution of Ligands by EXponential enrichment (SELEX). A SELEX library with 40 random sequence positions has 440 (,1024) possible combinations and a typical selection screens 1014–1015 unique molecules. This is on the order of 105 times larger than standard peptide or protein combinatorial molecular libraries.
The interrogation of proteomes (‘‘proteomics’’) in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology and medicine. We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 mL of serum or plasma).
Our current assay measures 813 proteins with low limits of detection (1 pM median), 7 logs of overall dynamic range (,100 fM–1 mM), and 5% median coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding signature of DNA aptamer concentrations, which is quantified on a DNA microarray.
Our assay takes advantage of the dual nature of aptamers as both folded protein-binding entities with defined shapes and unique nucleotide sequences recognizable by specific hybridization probes.
This is a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine. L Gold, D Ayers, J Bertino, Christopher Bock, et al. Aptamer-Based Multiplexed Proteomic Technology for Biomarker Discovery. PlosONE 2010; 5 (12): e15004
Biomarker Discovery, Diagnostics, and Therapeutics Progression from health to disease is accompanied by complex changes in protein expression in both the circulation and affected tissues. Large-scale comparative interrogation of the human proteome can offer insights into disease biology as well as lead to
the discovery of new biomarkers for diagnostics
new targets for therapeutics
can identify patients most likely to benefit from treatment.
Although genomic studies provide an increasingly sharper understanding of basic biological and pathobiological processes, they ultimately only offer a prediction of relative disease risk, whereas proteins offer an immediate assessment of “real-time” health and disease status. We have recently developed a new proteomic technology, based on modified aptamers, for biomarker discovery that is capable of simultaneously measuring more than a thousand proteins from small volumes of biological samples such as plasma, tissues, or cells. Our technology is enabled by SOMAmers (Slow Off-rate Modified Aptamers), a new class of protein binding reagents that contain chemically modified nucleotides that greatly expand the physicochemical diversity of nucleic acid-based ligands. Such modifications introduce functional groups that are absent in natural nucleic acids but are often found in protein-protein, small molecule-protein, and antibody-antigen interactions. The use of these modifications expands the range of possible targets for SELEX (Systematic Evolution of Ligands by EXponential Enrichment), results in improved binding properties, and facilitates selection of SOMAmers with slow dissociation rates. Our assay works by transforming protein concentrations in a mixture into a corresponding DNA signature, which is then quantified on current commercial DNA microarray platforms. In essence, we take advantage of the dual nature of SOMAmers as
both folded binding entities with defined shapes and
unique nucleic acid sequences recognizable by specific hybridization probes.
Aptamers and Smart Drug delivery Targeting In this review, the strategies for using functional nucleic acids in creating smart drug delivery devices will be explained, as their has been very recent progress in controlled drug release based on molecular gating achieved with aptamers. Aptamers are functional nucleic acid sequences which can bind specific targets. An artificial combinatorial methodology can identify aptamer sequences for any target molecule, from ions to whole cells. Drug delivery systems seek to increase efficacy and reduce side-effects by concentrating the therapeutic agents at specific disease sites in the body. This is generally achieved by specific targeting of inactivated drug molecules. Aptamers which can bind to various cancer cell types selectively and with high affinity have been exploited in a variety of drug delivery systems for therapeutic purposes. Recent progress in selection of cell-specific aptamers has provided new opportunities in targeted drug delivery. Especially functionalization of nanoparticles with such aptamers has drawn major attention in the biosensor and biomedical areas.
Nucleic acids are recognized as attractive building materials in nanomachines because of their unique molecular recognition properties and structural features. An active controlled delivery of drugs once targeted to a disease site is a major research challenge. Stimuli-responsive gating is one way of achieving controlled release of nanoparticle cargoes. Recent reports incorporate the structural properties of aptamers in controlled release systems of drug delivering nanoparticles.
Nanoparticle-encapsulated drug delivery aims to deliver the active therapeutic ingredients to the disease site in stable compartments in order to reduce premature release. This ensures that the effects of drug are maximized and the side effects are reduced. An encapsulated nanoparticle system requires a specific targeting mechanism and at the same time the retention of drugs inside the container should be high. The balance between specificity of targeting and the extent of premature leakage determines the success of a given delivery system.
Nanotechnology research approaches in drug delivery include a wide variety of nanomaterials ranging from soft hydrogels to solid polymeric particles. Large surface area, high drug loading efficiency and potential combination with other organic/inorganic materials are the main properties of hollow nanostructures that are attractive for biomedical applications.
Packaging of small-molecule drugs
improves their availability
compatibility
reduces toxicity
Controlling the drug release profile is the main challenge in drug delivery development when the drug is to be successfully targeted to a specific site. Stimuli-responsive materials have been created by using biological, physical and chemical properties of materials for heat-activated, light-activated or pH-activated delivery. Nucleic acids are utilized to construct rationally designed nanostructures at molecular levels for nanotechnology applications. Integration of the properties of nucleic acids can offer many opportunities for drug delivery systems, including stimuli-responsive nanogates for nanocarriers and molecular sensors. Favorable drug release kinetics can be achieved at the target sites by aptamer-based capping systems.
Activity Based Profiling Powerful strategies for the gel-free analysis of proteomes have emerged, including isotope-coded affinity tagging (ICAT) for quantitative proteomics and multidimensional protein identification technology (MudPIT) for comprehensive proteomics, both of which utilize liquid chromatography (LC) and MS for protein separation and detection, respectively. Nonetheless, these methods, like 2DE-MS, still focus on measuring changes in protein abundance and, therefore, provide only an indirect estimate of dynamics in protein function. Indeed, several important forms of post-translational regulation, including protein–protein and protein–small-molecule interactions, may elude detection by abundance-based proteomic methods. To facilitate the analysis of protein function, several proteomic methods have been introduced to characterize the activity of proteins on a global scale. These include large-scale yeast two-hybrid screens and epitope tagging immunoprecipitation experiments, which aim to construct comprehensive maps of protein–protein interactions, and protein microarrays, which aim to provide an assay platform for the rapid assessment of protein activities. A chemical proteomic strategy referred to as activity-based protein profiling (ABPP) has emerged that utilizes active site-directed probes to profile the functional state of enzyme families directly in complex proteomes.
Recent advances in genomic and proteomic technologies have begun to address the challenge of assigning molecular and cellular functions to the numerous protein products encoded by prokaryotic and eukaryotic genomes. In particular, chemical strategies for proteome analysis have emerged that enable profiling of protein activity on a global scale. Herein, we highlight these chemical proteomic methods and their application to the discovery and characterization of disease-related enzyme activities.
Cells with fundamental metabolic alterations commonly arise during tumorigenesis, and it is these types of changes that help to establish a biochemical foundation for disease progression and malignancy. A seminal example of this was discovered in the 1920s when Otto Warburg found that cancer cells consume higher levels of glucose and secrete most of the glucose carbon as lactate rather than oxidizing it completely. Since then, studies by multiple groups have uncovered a diverse array of metabolic changes in cancer, including alterations in
glycolytic pathways
the citric acid cycle
glutaminolysis
lipogenesis
lipolysis
proteolysis
These in turn modulate the levels of cellular building blocks
lipids, nucleic acids and amino acids,
cellular energetics,
oncogenic signaling molecules
the extracellular environment to confer protumorigenic and malignant properties.
Despite these advances, our current understanding of cancer metabolism is far from complete and would probably benefit from experimental strategies that are capable of profiling enzymatic pathways on a global scale. To this end, conventional genomic and proteomic methods, which comparatively quantify the expression levels of transcripts and proteins, respectively, have yielded many useful insights. These platforms are, however, limited in their capacity to identify changes in protein activity that are caused by posttranslational mechanisms.
Annotating biochemical pathways in cancer is further complicated by the potential for enzymes to carry out distinct metabolic activities in tumor cells that might not be mirrored in normal physiology. In addition, a substantial proportion of the human proteome remains functionally uncharacterized, and it is likely that at least some of these poorly understood proteins also have roles in tumorigenesis. These challenges require new proteomic technologies that can accelerate the assignment of protein function in complex biological systems, such as cancer cells and tumors.
Metabolomics has emerged as a powerful approach for investigating enzyme function in living systems. Metabolomic experiments in the context of enzyme studies typically start with
the extraction of metabolites from control and enzyme-disrupted biological systems,
followed by metabolite detection and comparative data analysis.
For example, lipophilic metabolites can be enriched from cells or tissues by organic extraction. Mass spectrometry (MS) has become a primary analytical method for surveying metabolites in complex biological samples, with upfront separation accomplished by liquid chromatography (LC–MS) or gas chromatography (GC–MS). MS experiments can be carried out using
targeted or untargeted approaches,
depending on whether the objective is
to profile and quantitate known metabolites or
to broadly scan for metabolites across a large mass range, respectively.
As metabolomic experiments generate a large amount of data, powerful software tools are needed for identification and quantitation of ions in LC–MS data sets (see the figure; the mass to charge ratio (m/z) is indicated). One such program is XCMS95, which
aligns,
quantifies and
statistically ranks ions that are altered between two sets of metabolomic data.
This program can be used to rapidly identify metabolomic signatures of various disease states or to assess metabolic networks that are regulated by an enzyme using pharmacological or genetic tools that modulate enzyme function. Additional databases assist in metabolite structural characterization, such as HMDB96,97, METLIN98,99 and LIPID MAPS100. In this Review, we discuss one such proteomic platform, termed activity based protein profiling (ABPP), and its implementation in the discovery and functional characterization of deregulated enzymatic pathways in cancer. We discuss the evidence that, when coupled with other large scale profiling methods, such as metabolomics and proteomics, ABPP can provide a compelling, systems level understanding of biochemical networks that are important for the development and progression of cancer.
Large-scale profiling methods have uncovered numerous gene and protein expression changes that correlate with tumorigenesis. However, determining the relevance of these expression changes and which biochemical pathways they affect has been hindered by our incomplete understanding of the proteome and its myriad functions and modes of regulation. Activity-based profiling platforms enable both the discovery of cancer-relevant enzymes and selective pharmacological probes to perturb and characterize these proteins in tumour cells. When integrated with other large-scale profiling methods, activity-based proteomics can provide insight into the metabolic and signaling pathways that support cancer pathogenesis and illuminate new strategies for disease diagnosis and treatment.
Representative activity-based probes and their application to cancer research
enzyme class applications in cancer
Serine hydrolases increased KIAA1363 and MAGL
aggressive human cancer lines
uPA and tPA serine protease aggressive cancers
RBBP9 activity in pancreatic carcinoma
Metalloproteinases neprilysin activity in melanoma cell lines
Cysteine proteases cathepsin cysteine protease in pancreatic islet tumours
Kinases Inhibitor selectivity profiling of kinase inhibitors
Caspases visualization of apoptosis in colon tumour-bearing mice treated with Apomab
Deubiquitylases Identified increased carboxy-terminal hydrolase UCHL3 and UCH37 activity in HPV cervical carcinomas
Cytochrome P450s Identified the aromatase inhibitor anastrazole as an inducer of CYP1A2
Serine hydrolases KIaa1363 and MaGL regulate lipid metabolic pathways that support cancer pathogenesis. Activity-based protein profiling (ABPP) identified
KIAA1363 and
monoacylglycerol (MAG) lipase (MAGL)
as being increased in aggressive human cancer cells from multiple tumour types. Pharmacological and/or RNA interference ablation of KIAA1363 and MAGL coupled with metabolomic analysis revealed specific roles for KIAA1363 and MAGL in cancer metabolism. Disruption of KIAA1363 by the small-molecule inhibitor AS115 lowered monoalkylglycerol ether (MAGE), alkyl lysophosphatidic acid (alkyl LPA) and alkyl lysophosphatidyl choline (alkyl LPC) levels in cancer cells. Disruption of MAGL by the small-molecule inhibitor JZL184 raised MAG levels and reduced free fatty acid, lysophosphatidic acid (LPA) and prostaglandin E2 (PGE2) levels in cancer cells. Disruption of KIAA1363 and MAGL leads to impairments in cancer cell aggressiveness and tumour growth, PAF, platelet-activating factor.
At a glance
• Activity-based protein profiling (ABPP) facilitates the discovery of deregulated enzymes in cancer. • Competitive ABPP yields selective inhibitors for functional characterization of cancer enzymes. • ABPP can be integrated with metabolomics to map deregulated enzymatic pathways in cancer. • ABPP can be integrated with other proteomic methods to map proteolytic pathways in cancer. • ABPP probes can be used to image tumour development in living animals.
Activity-based protein profiling (ABPP), the use of active site-directed chemical probes to monitor enzyme function in complex biological systems, is emerging as a powerful post-genomic technology. ABPP probes have been developed for several enzyme classes and have been used to inventory enzyme activities en masse for a range of (patho)physiological processes.
ABPP uses active site–directed, small molecule–based covalent probes to report on the functional state of enzyme activities directly in native biological systems. ABPP probes are designed or selected to target a subset of the proteome based on shared principles of binding and/or reactivity and have been successfully developed for many enzyme classes, including
serine
cysteine,
aspartyl
metallo hydrolases
kinases
glycosidases
histone deacetylases and
oxidoreductases.
These probes have been shown to selectively label active enzymes but not their inactive precursor (zymogen) or inhibitor-bound forms, thus allowing researchers to capture functional information that is beyond the scope of standard proteomic methods. By presenting specific examples, we show here that ABPP provides researchers with a distinctive set of chemical tools to embark on the assignment of functions to many of the uncharacterized enzymes that populate eukaryotic and prokaryotic proteomes.
Vinyl-methylester UL from HSV-1 Deubiquitinating enzyme (DUB)
Aryl 2-deoxy-2-fluoro glycoside Cfx from C. fimi Glycosidase (β-1-4-glycanase) Fluorophosphonate SAE Serine hydrolase
Examples of enzymes assigned to specific mechanistic classes by ABPP
ABPP can also be implemented as a direct assay for inhibitor discovery, allowing researchers to develop potent and selective pharmacological probes for uncharacterized enzymes.
Examples of enzymes assigned to specific mechanistic classes by ABPP.
Probe Leu-Asp-αCA probe selectively labeled Upβ
Substrate the endogenous Upβ substrate, N-carbamoyl-β-alanine
Substrate mimicry of an ABPP probe.
Multidimensional profiling strategy for the annotation of the cancer-related enzyme KIAA1363. ABPP using fluorophosphonate probes identified KIAA1363 as a highly elevated enzyme activity in aggressive cancer cells. Competitive ABPP was then used to develop a selective KIAA1363 inhibitor (AS115). Metabolomic analysis of cancer cells treated with AS115 determined a role for this enzyme in the regulation of MAGE lipids in cancer cells. Biochemical studies confirmed that KIAA1363 acts as 2-acetyl MAGE hydrolase in a metabolic network that bridges the platelet activating factor and lysophosphatidic acid classes of signaling lipids. Assignment of enzyme mechanism by ABPP
There are multiple levels of annotation for enzymes. The most basic level is assignment to a specific mechanistic class based on the general chemical reaction catalyzed by the enzyme (for example, hydrolase, kinase, oxidoreductase and others). Additional annotation involves determining the endogenous substrates and products for the enzyme. Finally, complete annotation requires an understanding of how the specific chemical transformation(s) catalyzed by an enzyme integrate into larger metabolic and signaling pathways to influence cell physiology and behavior.
Many of the predicted enzymes uncovered by genome sequencing projects can be assigned to a mechanistic class or ascribed a putative biochemical function based on sequence homology to well-characterized enzymes. But some enzymes have insufficient sequence relatedness for class assignment or have a function different from that predicted by sequence comparisons. ABPP has facilitated class annotation for several such uncharacterized enzymes.
A principal goal of modern biomedical research is to discover, assemble, and experimentally manipulate molecular pathways in cells and organisms to reveal new disease mechanisms.
Toward this end, complete genome sequences for numerous bacteria and higher organisms, including humans, have laid the fundamental groundwork for understanding the molecular basis of life in its many forms. However, the information content of DNA sequences is limited and, on its own, cannot describe most physiological and pathological processes.
Unlike oligonucleotides, proteins are a very diverse group of biomolecules that display a wide range of chemical and biophysical features, including
membrane-binding,
hetero/homo-oligomerization, and
posttranslational modification.
The biochemical complexity intrinsic to protein science intimates that several complementary analytical strategies will be needed to achieve the ultimate goal of proteomics – a comprehensive characterization of the expression, modification state, interaction map, and activity of all proteins in cells and tissues.
A powerful LC-MS strategy for proteomics involves the use of isotope-coded affinity tags (ICAT). This approach enables the comparison of protein expression in proteomes by treating samples with isotopically distinct forms of a chemical labeling reagent. ICAT methods provide superior resolving power compared to gel-based methods and improve access to membrane-associated proteins. More recently, isotope-free MS methods for quantitative proteomics have emerged.
Reverse protein microarrays have also been described in which proteomes themselves are arrayed and the antibodies used for detection in a format analogous to Western blotting. In addition to increasing the throughput of proteomic experiments by integrating the protein separation and detection steps, microarrays consume much less material than conventional proteomic methods. Still, the general application of microarrays for proteomics is currently limited by the availability of high-quality capture reagents (e.g., antibodies, aptamers, etc).
These approaches, by measuring protein abundance provide, like genomics, only an indirect assessment of protein activity and may fail to detect important posttranslational events that regulate protein function, such as protein–protein or protein–small-molecule interactions. To address these limitations, complementary strategies for the functional analysis of proteins have been introduced. Prominent among these functional proteomic efforts is the use of chemistry for the design of active site-directed probes that measure enzyme activity in samples of high biological complexity.
Many post-translational modes of enzyme regulation share a common mechanistic foundation – they perturb the active site such that catalytic power and/or substrate recognition is impaired. Accordingly, it was hypothesized that chemical probes capable of reporting on the integrity of enzyme active sites directly in cells and tissues might serve as effective functional proteomic tools. These activity based protein profiling (ABPP) probes consist of at least two general elements:
a reactive group for binding and covalently modifying the active sites of many members of a given enzyme class or classes
a reporter tag for the detection, enrichment, and identification of probe-labeled proteins
ABPP probes have been successfully developed for more than a dozen enzyme classes, including
all major classes of proteases
kinases
phosphatases
glycosidases
GSTs
oxidoreductases.
Post-translational regulation of enzyme activity. Many enzymes are produced as inactive precursors, or zymogens, which require proteolytic processing for activation. Enzyme activity can be further regulated by interactions with endogenous protein inhibitors. The field of proteomics aims to develop and apply technologies for the characterization of protein function on a global scale. Toward this end, synthetic chemistry has played a major role by providing new reagents to profile segments of the proteome based on activity rather than abundance. Small molecule probes for activity-based protein profiling have been created for more than a dozen enzyme classes and used to discover several enzyme activities elevated in disease states. These innovations have inspired complementary advancements in analytical chemistry, where new platforms have been introduced to augment the information content achievable in chemical proteomics experiments. Here, we will review these analytical platforms and discuss how they have exploited the versatility of chemical probes to gain unprecedented insights into the function of proteins in biological samples of high complexity.
Advanced analytical platforms utilize a range of separation and detection strategies, including LC-MS, CELIF, and antibody microarrays, to achieve an unprecedented breadth and depth of proteome coverage in ABPP investigations. The complementary strengths and weaknesses of each of these methods suggest that the selection of an appropriate analytical platform should be guided by the specific experimental question being addressed. SA Sieber and BF Cravatt. Analytical platforms for activity-based protein profiling – exploiting the versatility of chemistry for functional proteomics. Chem. Commun. 2006, 2311–2319. http://www.rsc.org/chemcomm
Diagnostic Therapeutics in Activity Based Probes Activity-based chemical proteomics-an emerging field involving a combination of organic synthesis, biochemistry, cell biology, biophysics and bioinformatics-allows the detection, visualisation and activity quantification of whole families or selected sub-sets of proteases based upon their substrate specificity. This approach can be applied for drug target/lead identification and validation, the fundamentals of drug discovery. The activity-based probes discussed in this review contain three key features;
a ‘warhead’ (binds irreversibly but selectively to the active site),
a ‘tag’ (allowing enzyme ‘handling’, with a combination of fluorescent, affinity and/or radio labels),
a linker region between warhead and tag.
From the design and synthesis of the linker arise some of the latest developments discussed here; not only can the physical properties (e.g., solubility, localisation) of the probe be tuned, but the inclusion of a cleavable moiety allows selective removal of tagged enzyme from affinity beads etc. Heal WP, Wickramasinghe SR, Tate EW. Activity based chemical proteomics: profiling proteases as drug targets. Curr Drug Discov Technol 2008; 5(3):200-12. PMID: 18690889
The genomic revolution has created a wealth of information regarding the fundamental genetic code that defines the inner workings of a cell. However, it has become clear that analyzing genome sequences alone will not lead to new therapies to fight human disease. Rather, an understanding of protein function within the context of complex cellular networks will be required to facilitate the discovery of novel drug targets and, subsequently, new therapies directed against them. The past ten years has seen a dramatic increase in technologies that allow large-scale, systems-based methods for analysis of global biological processes and disease states.
In the field of proteomics, several well-established methods persist as a means to resolve and analyze complex mixtures of proteins derived from cells and tissues. However, the resolving power of these methods is often challenged by the diverse and dynamic nature of the proteome. The field of activity-based proteomics, or chemical proteomics, has been established in an attempt to focus proteomic efforts on subsets of physiologically important protein targets. This new approach to proteomics is centered around the use of small molecules termed activity-based probes (ABPs) as a means to tag, enrich, and isolate, distinct sets of proteins based on their enzymatic activity. Berger AB, Vitorino PM, Bogyo M. Activity-based protein profiling: applications to biomarker discovery, in vivo imaging and drug discovery. Am J Pharmacogenomics. 2004;4(6):371-81.
Recent advances in global genomic and proteomic methods have led to a greater understanding of how genes and proteins function in complex networks within a cell. One of the major limitations in these methodologies is their inability to provide information on the dynamic, post-translational regulation of enzymatic proteins. In particular proteases are often synthesized as inactive zymogens that need to be activated in order to carry out specific biological processes. Thus, methods that allow direct monitoring of protease activity in the context of a living cell or whole animal will be required to begin to understand the systems-wide functional roles of proteases. In this review, we discuss the development and applications of activity based probes (ABPs) to study proteases and their role in pathological processes. Specifically we focus on application of this technique for biomarker discovery, in vivo imaging and drug screening.
Proteases, in particular, are known for their multilayered post-translational activity regulation that can lead to a significant difference between protease abundance levels and their enzyme activity. To address these issues, the field of activity-based proteomics has been established in order to characterize protein activity and monitor the functional regulation of enzymes in complex proteomes.
As a result of the recent enormous technological progress, experimental structure determination has become an integral part of the development of drugs against disease-related target proteins. The post-translational modification of proteins is an important regulatory process in living organisms; one such example is lytic processing by peptidases. Many different peptidases represent disease targets and are being used in structure-based drug design approaches. The development of drugs such as aliskiren and tipranavir, which inhibit renin and HIV protease, respectively, testifies to the success of this approach.
Presenilin is the catalytic component of γ-secretase, a complex aspartyl protease and a founding member of intramembrane-cleaving proteases. γ-Secretase is involved in the pathogenesis of Alzheimer’s disease and a top target for therapeutic intervention. However, the protease complex processes a variety of transmembrane substrates, including the Notch receptor, raising concerns about toxicity. Nevertheless, γ-secretase inhibitors and modulators have been identified that allow Notch processing and signaling to continue, and promising compounds are entering clinical trials.
Molecular and biochemical studies offer a model for how this protease hydrolyzes transmembrane domains in the confines of the lipid bilayer. Progress has also been made toward structure elucidation of presenilin and the γ-secretase complex by electron microscopy as well as by studying cysteine-mutant presenilins. The signal peptide peptidase (SPP) family of proteases are distantly related to presenilins. However, the SPPs work as single polypeptides without the need for cofactors and otherwise appear to be simple model systems for presenilin in the γ-secretase complex.
Critical clues to the identity of γ-secretase included: (1) Genes encoding the multi-pass membrane proteins presenilin-1 and presenilin-2 are, like APP, associated with familial, early-onset Alzheimer’s disease. The disease-causing missense mutations were found to alter how γ-secretase cuts APP, leading to increased proportions of longer, more aggregation-prone forms of Aβ. (2) Knockout of presenilin genes eliminates γ-secretase cleavage of APP. (3) Peptidomimetics that inhibit γ-secretase contain moieties typically found in aspartyl protease inhibitors. These findings led to the identification of two conserved transmembrane aspartates in the multi-pass presenilins that are critical for γ-secretase cleavage of APP, evidence that presenilins are aspartyl proteases. Presenilin is endoproteolytically cleaved into two polypeptides, an N-terminal fragment (NTF) and a C-terminal fragment (CTF), the formation of which is
regulated
metabolically stable
part of a high-molecular weight complex
suggesting that the NTF-CTF heterodimer is the biologically active form. NTF and CTF each contribute one of the essential and conserved aspartates, and transition-state analogue inhibitors of γ-secretase, compounds designed to interact with the active site of the protease, bind directly to presenilin NTF and CTF. Presenilins are also required for Notch signaling (Levitan and Greenwald, 1995), a pathway essential for cell differentiation during development and beyond.
The highly conserved role of γ-secretase in Notch signalling and its importance in development led to genetic screens in Caenorhabditis elegans that identified three other integral membrane proteins besides presenilin that modify Notch signaling. Designed inhibitors have proven to be useful tools in understanding the mechanism of γ-secretase and substrate recognition – affinity labelling with transition-state analogue inhibitors showed binding at the interface between the presenilin NTF and CTF subunits, consistent with the active site residing at this interface, with each presenilin subunit contributing one of the essential aspartates. The concept of presenilin as the catalytic component for γ-secretase was considerably strengthened when
signal peptide peptidase (SPP) was found to be a similar intramembrane aspartyl protease
SPP is exploited by the hepatitis C virus for the maturation of its core protein, suggesting that this protease may be a suitable target for antiviral therapy
SPP was identified by affinity labeling with a peptidomimetic inhibitor, and the protein sequence displayed similarities with presenilin.
SPP contains two conserved aspartates, each predicted to lie in the middle of a transmembrane domain, and the aspartate-containing sequences resemble those found in presenilins.
SPP appears to be less complicated than γ-secretase.
Expression of human SPP in yeast reconstituted the protease activity, suggesting that the protein has activity on its own and does not require other mammalian protein cofactors.
Aspartyl I-CLiPs are found in all forms of life and play essential roles in biology and disease. How these enzymes carry out hydrolysis in the membrane is a fascinating question that is not entirely resolved, but evidence suggests an initial substrate docking site and a lateral gate into a pore where water and the active site aspartates reside. Designed inhibitors have been critical in elucidating these mechanisms, but inhibitors targeting γ-secretase for the treatment of Alzheimer’s disease must avoid interfering with Notch signaling.
MS Wolfe. Structure, Mechanism and Inhibition of γ-Secretase and Presenilin-Like Proteases. Biol Chem. 2010 August; 391(8): 839–847. doi: 10.1515/BC.2010.086. PMCID: PMC2997569. NIHMSID: NIHMS254540 Study Suggests Expanding the Genetic Alphabet May Be Easier than Previously Thought Genomics Monday, June 4, 2012 A new study led by scientists at The Scripps Research Institute suggests that the replication process for DNA—the genetic instructions for living organisms that is composed of four bases (C, G, A and T)—is more open to unnatural letters than had previously been thought.
An expanded “DNA alphabet” could carry more information than natural DNA, potentially coding for a much wider range of molecules and enabling a variety of powerful applications, from precise molecular probes and nanomachines to useful new life forms. The new study, which appears in the June 3, 2012 issue of Nature Chemical Biology, solves the mystery of how a previously identified pair of artificial DNA bases can go through the DNA replication process almost as efficiently as the four natural bases. “We now know that the efficient replication of our unnatural base pair isn’t a fluke, and also that the replication process is more flexible than had been assumed,” said Floyd E. Romesberg, principal developer of the new DNA bases.
Adding to the DNA Alphabet Romesberg and his lab have been trying to find a way to extend the DNA alphabet since the late 1990s. In 2008, they developed the efficiently replicating bases NaM and 5SICS, which come together as a complementary base pair within the DNA helix, much as, in normal DNA, the base adenine (A) pairs with thymine (T), and cytosine (C) pairs with guanine (G).
The following year, Romesberg and colleagues showed that NaM and 5SICS could be efficiently transcribed into RNA. But these bases’ lack the ability to form the hydrogen bonds that join natural base pairs in DNA. Such bonds had been thought to be an absolute requirement for successful DNA replication‑—a process in which a large enzyme, DNA polymerase, moves along a single, unwrapped DNA strand and stitches together the opposing strand, one complementary base at a time.
An early structural study of a very similar base pair in double-helix DNA added to Romesberg’s concerns. The data strongly suggested that NaM and 5SICS do not even approximate the edge-to-edge geometry of natural base pairs—termed the Watson-Crick geometry, after the co-discoverers of the DNA double-helix. Instead, they join in a looser, overlapping, “intercalated” fashion. “Their pairing resembles a ‘mispair,’ such as two identical bases together, which normally wouldn’t be recognized as a valid base pair by the DNA polymerase.” Yet in test after test, the NaM-5SICS pair was efficiently replicable.
Edge to Edge The NaM-5SICS pair maintain an abnormal, intercalated structure within double-helix DNA—but remarkably adopt the normal, edge-to-edge, “Watson-Crick” positioning when gripped by the polymerase during the crucial moments of DNA replication. “The DNA polymerase apparently induces this unnatural base pair to form a structure that’s virtually indistinguishable from that of a natural base pair.” NaM and 5SICS, lacking hydrogen bonds, are held together in the DNA double-helix by “hydrophobic” forces, which cause certain molecular structures to be repelled by water molecules, and thus to cling together in a watery medium. “It’s very possible that these hydrophobic forces have characteristics that enable the flexibility and thus the replicability of the NaM-5SICS base pair.”
An Arbitrary Choice? The finding suggests that NaM-5SICS and potentially other, hydrophobically bound base pairs could some day be used to extend the DNA alphabet. It also hints that Evolution’s choice of the existing four-letter DNA alphabet—on this planet—may have been somewhat arbitrary. “It seems that life could have been based on many other genetic systems.” Source: The Scripps Research Institute
DNA damage response (DDR) network
Eukaryotic cells have evolved an intricate system to resolve DNA damage to prevent its transmission to daughter cells. This system, collectively known as the DNA damage response (DDR) network, includes many proteins that detect DNA damage, promote repair, and coordinate progression through the cell cycle. Because defects in this network can lead to cancer, this network constitutes a barrier against tumorigenesis. The modular BRCA1 carboxyl-terminal (BRCT) domain is frequently present in proteins involved in the DDR, can exist either as an individual domain or as tandem domains (tBRCT), and can bind phosphorylated peptides. We performed a systematic analysis of protein-protein interactions involving tBRCT in the DDR.
We identified 23 proteins containing conserved BRCT domains and generated a human protein-protein interaction network for seven proteins with tBRCT. This study also revealed previously unknown components in DNA damage signaling, such as COMMD1 and the target of rapamycin complex mTORC2. Additionally, integration of tBRCT domain interactions with DDR phosphoprotein studies and analysis of kinase-substrate interactions revealed signaling subnetworks that may aid in understanding the involvement of tBRCT in disease and DNA repair.
Mitochondria have various essential functions in metabolism and in determining cell fate during apoptosis. In addition, mitochondria are also important nodes in a number of signaling pathways. For example, mitochondria can modulate signals transmitted by second messengers such as calcium. Because mitochondria are also major sources of reactive oxygen species (ROS), they can contribute to redox signaling—for example, by the production of ROS such as hydrogen peroxide that can reversibly modify cysteine residues and thus the activity of target proteins. Mitochondrial ROS production is thought to play a role in hypoxia signaling by stabilizing the oxygen-sensitive transcription factor hypoxia-inducible factor–1α. New evidence has extended the mechanism of mitochondrial redox signaling in cellular responses to hypoxia in interesting and unexpected ways. Hypoxia altered the microtubule-dependent transport of mitochondria so that the organelles accumulated in the perinuclear region, where they increased the intranuclear concentration of ROS. The increased ROS in turn enhanced the expression of hypoxia-sensitive genes such as VEGF (vascular endothelial growth factor) not by reversibly oxidizing a protein, but by oxidizing DNA sequences in the hypoxia response element of the VEGF promoter. This paper and other recent work suggest a new twist on mitochondrial signaling: that the redistribution of mitochondria within the cell can be a component of regulatory pathways.
A challenge in the treatment of lung cancer is the lack of early diagnostics. Here, we describe the application of monoclonal antibody proteomics for discovery of a panel of biomarkers for early detection (stage I) of non-small cell lung cancer (NSCLC). We produced large monoclonal antibody libraries directed against the natural form of protein antigens present in the plasma of NSCLC patients. Plasma biomarkers associated with the presence of lung cancer were detected via high throughput ELISA. Differential profiling of plasma proteomes of four clinical cohorts, totaling 301 patients with lung cancer and 235 healthy controls, identified 13 lung cancer-associated (p < 0.05) monoclonal antibodies. The monoclonal antibodies recognize five different cognate proteins identified using immunoprecipitation followed by mass spectrometry. Four of the five antigens were present in non-small cell lung cancer cells in situ.
Nitric oxide (NO), reactive nitrogen species (RNS) and reactive oxygen species (ROS) perform dual roles as immunotoxins and immunomodulators. An incoming immune signal initiates NO and ROS production both for tackling the pathogens and modulating the downstream immune response via complex signaling pathways. The complexity of these interactions is a reflection of involvement of redox chemistry in biological setting (fig. 1)
Fig 1. Image credit: (Wink et al., 2011)
Previous studies have highlighted the role of NO in immunity. It was shown that macrophages released a substance that had antitumor and antipathogen activity and required arginine for its production (Hibbs et al., 1987, 1988). Hibbs and coworkers further strengthened the connection between immunity and NO by demonstrating that IL2 mediated immune activation increased NO levels in patients and promoted tumor eradication in mice (Hibbs et al., 1992; Yim et al., 1995).
In 1980s a number of authors showed the direct evidence that macrophages made nitrite, nitrates and nitrosamines. It was also shown that NO generated by macrophages could kill leukemia cells (Stuehr and Nathan, 1989). Collectively these studies along with others demonstrated the important role NO plays in immunity and lay the path for further research in understanding the role of redox molecules in immunity.
NO is produced by different forms of nitric oxide synthase (NOS) enzymes such as eNOS (endothelial), iNOS (inducible) and nNOS (neuronal). The constitutive forms of eNOS generally produce NO in short bursts and in calcium dependent manner. The inducible form produces NO for longer durations and is calcium independent. In immunity, iNOS plays a vital role. NO production by iNOS can occur over a wide range of concentrations from as little as nM to as much as µM. This wide range of NO concentrations provide iNOS with a unique flexibility to be functionally effective in various conditions and micro-environements and thus provide different temporal and concentration profiles of NO, that can be highly efficient in dealing with immune challenges.
Redox reactions in immune responses
NO/RNS and ROS are two categories of molecules that bring about immune regulation and ‘killing’ of pathogens. These molecules can perform independently or in combination with each other. NO reacts directly with transition metals in heme or cobalamine, with non-heme iron, or with reactive radicals (Wink and Mitchell, 1998). The last reactivity also imparts it a powerful antioxidant capability. NO can thus act directly as a powerful antioxidant and prevent injury initiated by ROS (Wink et al., 1999). On the other hand, NO does not react directly with thiols or other nucleophiles but requires activation with superoxide to generate RNS. The RNS species then cause nitrosative and oxidative stress (Wink and Mitchell, 1998).
The variety of functions achieved by NO can be understood if one looks at certain chemical concepts. NO and NO2 are lipophilic and thus can migrate through cells, thus widening potential target profiles. ONOO-, a RNS, reacts rapidly with CO2 that shortens its half life to <10 ms. The anionic form and short half life limits its mobility across membranes. When NO levels are higher than superoxide levels, the CO2-OONO–intermediate is converted to NO2 and N2O3 and changes the redox profile from an oxidative to a nitrosative microenvironment. The interaction of NO and ROS determines the bioavailability of NO and proximity of RNS generation to superoxide source, thus defining a reaction profile. The ROS also consumes NO to generate NO2 and N2O3 as well as nitrite in certain locations. The combination of these reactions in different micro-environments provides a vast repertoire of reaction profiles for NO/RNS and ROS entities.
The Phagosome ‘cauldron’
The phagosome provides an ‘isolated’ environment for the cell to carry out foreign body ‘destruction’. ROS, NO and RNS interact to bring about redox reactions. The concentration of NO in a phagosome can depend on the kind of NOS in the vicinity and its activity and other localised cellular factors. NO and is metabolites such as nitrites and nitrates along with ROS combine forces to kill pathogens in the acidic environment of the phagosome as depicted in the figure 2 below.
Fig 2. The NO chemistry of the phagosome. (image credit: (Wink et al., 2011)
This diagram depicts the different nitrogen oxide and ROS chemistry that can occur within the phagosome to fight pathogens. The presence of NOX2 in the phagosomes serves two purposes: one is to focus the nitrite accumulation through scavenging mechanisms, and the second provides peroxide as a source of ROS or FA generation. The nitrite (NO2−) formed in the acidic environment provides nitrosative stress with NO/NO2/N2O3. The combined acidic nature and the ability to form multiple RNS and ROS within the acidic environment of the phagosome provide the immune response with multiple chemical options with which it can combat bacteria.
Bacteria
There are various ways in which NO combines forces with other molecules to bring about bacterial killing. Here are few examples
E.coli: It appears to be resistant to individual action of NO/RNS and H2O2 /ROS. However, when combined together, H2O2 plus NO mediate a dramatic, three-log increase in cytotoxicity, as opposed to 50% killing by NO alone or H2O2 alone. This indicates that these bacteria are highly susceptible to their synergistic action.
Staphylococcus: The combined presence of NO and peroxide in staphylococcal infections imparts protective effect. However, when these bacteria are first exposed to peroxide and then to NO there is increased toxicity. Hence a sequential exposure to superoxide/ROS and then NO is a potent tool in eradicating staphylococcal bacteria.
Mycobacterium tuberculosis: These bacterium are sensitive to NO and RNS, but in this case, NO2 is the toxic species. A phagosome microenvironment consisting of ROS combined with acidic nitrite generates NO2/N2O3/NO, which is essential for pathogen eradication by the alveolar macrophage. Overall, NO has a dual function; it participates directly in killing an organism, and/or it disarms a pathway used by that organism to elude other immune responses.
Parasites
Many human parasites have demonstrated the initiation of the immune response via the induction of iNOS, that then leads to expulsion of the parasite. The parasites include Plasmodia(malaria), Leishmania(leishmaniasis), and Toxoplasma(toxoplasmosis). Severe cases of malaria have been related with increased production of NO. High levels of NO production are however protective in these cases as NO was shown to kill the parasites (Rockett et al., 1991; Gyan et al., 1994). Leishmania is an intracellualr parasite that resides in the mamalian macrophages. NO upregulation via iNOS induction is the primary pathway involved in containing its infestation. A critical aspect of NO metabolism is that NOHA inhibits AG activity, thereby limiting the growth of parasites and bacteria including Leishmania, Trypanosoma, Schistosoma, Helicobacter, Mycobacterium, and Salmonella, and is distinct from the effects of RNS. Toxoplasma gondii is also an intracellular parasite that elicits NO mediated response. INOS knockout mice have shown more severe inflammatory lesions in the CNS that their wild type counterparts, in response to toxoplasma exposure. This indicates the CNS preventative role of iNOS in toxoplasmosis (Silva et al., 2009).
Virus
Viral replication can be checked by increased production of NO by induction of iNOS (HIV-1, coxsackievirus, influenza A and B, rhino virus, CMV, vaccinia virus, ectromelia virus, human herpesvirus-1, and human parainfluenza virus type 3) (Xu et al., 2006). NO can reduce viral load, reduce latency and reduce viral replication. One of the main mechanisms as to how NO participates in viral eradication involves the nitrosation of critical cysteines within key proteins required for viral infection, transcription, and maturation stages. For example, viral proteases or even the host caspases that contain cysteines in their active site are involved in the maturation of the virus. The nitrosative stress environment produced by iNOS may serve to protect against some viruses by inhibiting viral infectivity, replication, and maturation.
Coronary Artery Disease – Medical Devices Solutions: From First-In-Man Stent Implantation, via Medical Ethical Dilemmas to Drug Eluting Stents August 13, 2012
Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production July 19, 2012
Curator and Research Study Originator: Aviva Lev-Ari, PhD, RN
Macrovascular Disease – Therapeutic Potential of cEPCs: Reduction Methods for CV Risk
July 2, 2012
An Investigation of the Potential of circulating Endothelial Progenitor Cells (cEPCs) as a Therapeutic Target for Pharmacological Therapy Design for Cardiovascular Risk Reduction: A New Multimarker Biomarker Discovery
Inhibition of ET-1, ETA and ETA-ETB, Induction of NO production, stimulation of eNOS and Treatment Regime with PPAR-gamma agonists (TZD): cEPCs Endogenous Augmentation for Cardiovascular Risk Reduction – A Bibliography
Cancer remains the second leading cause of death by disease. Hundreds of new medicines to treat cancer are now being developed for lessening the burden of cancer to patients, their families and society.
Biopharmaceutical researchers are now working on 981 medicines for cancer. Many are high-tech weapons to fight the disease, while some involve innovative research into using existing medicines in new ways, the report says.
Recent developments in early detection and a steady stream of new and improved treatments suggesting that cancer is a manageable chronic disease (not a deadly one any more). Families and patients alike are with increasing expectations from the industry for more and better treatment options and America’s biopharmaceutical research companies are responding to that.
America’s biopharmaceutical research companies are working on many new cutting-edge approaches to fight cancer. They include:
• A medicine that interferes with the metabolism of cancer cells by depriving them of the energy provided by glucose.
• A medicine for acute myeloid leukemia (AML) that inhibits cancer cells with a mutation found in about a third of AML sufferers.
• A therapy that uses nanotechnology to target the delivery of medicines to cancer cells, potentially overcoming some limitations of existing treatments.
Systemic sclerosis (SSc) is a type of autoimmune disease when the body’s immune system attacks and destroys body’s healthy tissue. It is characterised by lesions in the vessels and accumulation of collagen in the tissues. Although the pathogenesis of this disease is not clear, but one of the suggestions is that the endothelium fails to produce NO upon cold stimulation. Physiologically, NO acts as a vasodilator and its deficiency has been implicated in diseases such as hypertension and atherosclerosis.
In the body NO is generated when L-arginine is converted to L-citruline in the presence of NO synthase (NOS) enzyme, molecular oxygen, NADPH, and other cofactors. Principally, three isoenzymes of NOS are present in the body to catalyse the production of NO in various anatomic locations and under various physiological conditions. Three distinct genes encode for the three types of NOS i.e. endothelial (eNOS or NOS-3), neuronal (nNOS or NOS-1), and inducible (iNOS or NOS-2) NOS.
The inducible type 2 NOS (iNOS) may act as an immunoregulator. Several reports have provided evidence for the existence of a NO pathway in human mononuclear cells.
It is not well established if NO production increases or decreases in SSc patients. In one study by Allanore et al, NO production was shown to be reduced in plasma and PBMC supernatants, and iNOS synthesis in PBMCs.
The authors of this study investigated NO metabolites in plasma and PBMC supernatants, and iNOS synthesis in PBMC to see if the level of NO production by peripheral blood mononuclear cells (PBMC) was low in SSc, as this might contribute to the vasodilatory abnormalities observed in this disease.
Eighteen patients with SSc were compared with two control groups: 16 patients with rheumatoid arthritis (RA) and 23 patients with mechanical sciatica.The NO metabolites nitrite and nitrates were determined by flurometeric and spectrometeric assays respectively. iNOS expression was determined by using monoclonal anti‐NOS2 antibody and FACS analyses.
The data suggested a decrease in plasma NO concentration and iNOS production in PBMC in patients with systemic sclerosis as compared with patients with rheumatoid arthritis and sciatica. Subgroup analysis showed no difference between limited and diffuse SSc forms. Total plasma nitrite concentrations in five healthy volunteers were similar to those in patients with sciatica, which is consistent with this group being an appropriate control group.
Thus the authors suggest that low NO production in Ssc patients might be involved in the tendency towards vasospam.
However in other studies authors have shown an increase in NO production in SSc patients. Takagi et al set out to investigate this discrepancy in NO production in SSc patients. They sought to determine whether increased NO levels are associated with various clinical subsets of SSc patients, and to assess the contribution of fibroblasts in skin lesions to NO synthesis.
In this study Takagi et al measured the levels of serum NO metabolites in SSc patients and determined the contribution of the excessive production of NO synthase (NOS)-2 by skin fibroblasts to NO synthesis. Serum NO levels of 45 patients with SSc were significantly higher than those of 20 healthy volunteers. In addition, some clinical features of SSc (the extent of skin fibrosis, short disease duration, and the complication of active fibrosing alveolitis) were all correlated positively with the levels of NO metabolites in SSc patients. RT PCR was used to determine NOS-2 mRNA expression levels in cultured fibroblasts derived from SSc patients.
The authors showed that serum NO levels were significantly elevated in patients with SSc as compared to healthy normal controls. They also demonstrated that NOS-2 was produced spontaneously by cultured SSc fibroblasts, suggesting that increased serum NO levels might reflect in part the elevated expression of NOS-2 by fibroblasts derived from SSc patients.
The discrepancy in NO production could be explained by disease stage, severity of tissue fibrosis and various circumstances of endothelial damage. Takagi et al found increased NO production in early stages of SSc with tissue fibrosis and not in later stages of the disease. Hence they suggest that NO levels may be a sensitive marker of the early stages of the development of severe tissue fibrosis in SSc patients, although a longitudinal and prospective study is needed to confirm this.
NO is known to have dual functions in the body, both beneficial and cyototoxic. Generally it depends upon the concentration and the duration of NO production. Similarily in SSC, the dual functions of NO seem to be both beneficial (as a vasodilator) and harmful (as a cytotoxic effector) in regard to the clinical manifestations of SSc. One of the limitations of these studies is that they did not investigate the absolute concentrations of NO production but only its metaoblites were measured. This might be due the fact that NO is a short-lived molecule (half-life < 5 s) and it is challenging to quantify NO production at single cell level. Such techniques (e.g.DAR 4M AM flurophore) have been developed but are challenging to apply to various experimental set ups. DAR 4M AM has been used to quantify NO production online in single cells. In SSc determination of absolute NO concentrations at cellular or tissue level at various stages of the disease process will go a long way in solving the discrepancy of NO production in SSc patients.
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2012pharmaceutical
Amandeep Kaur
Aashir Awan, Phd
Abhisar Anand
Adina Hazan
Alan F. Kaul, PharmD., MS, MBA, FCCP
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Arav Gandhi
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David Orchard-Webb, PhD
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Demet Sag, Ph.D., CRA, GCP
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Ethan Coomber
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