Archive for the ‘Sepsis’ Category

H2S-mediated protein sulfhydration in stress reveals metabolic reprogramming

Larry H. Bernstein, MD, FCAP, Curator




Quantitative H2S-mediated protein sulfhydration reveals metabolic reprogramming during the Integrated Stress Response

” data-author-inst=”CaseWesternReserveUniversityUnitedStates”>Bo-JhihGuan, 

Ilya Bederman
Department of Pediatrics, Case Western Reserve University, Cleveland, United States
No competing interests declared

” data-author-inst=”CaseWesternReserveUniversityUnitedStates”>IlyaBederman, 

Mithu Majumder
Department of Pharmacology, Case Western Reserve University, Cleveland, United States
No competing interests declared

” data-author-inst=”CaseWesternReserveUniversityUnitedStates”>MithuMajumder, et al.
eLife 2015;10.7554/eLife.10067

The sulfhydration of cysteine residues in proteins is an important mechanism involved in diverse biological processes. We have developed a proteomics approach to quantitatively profile the changes of sulfhydrated cysteines in biological systems. Bioinformatics analysis revealed that sulfhydrated cysteines are part of a wide range of biological functions. In pancreatic β cells exposed to endoplasmic reticulum (ER) stress, elevated H2S promotes the sulfhydration of enzymes in energy metabolism and stimulates glycolytic flux. We propose that transcriptional and translational reprogramming by the Integrated Stress Response (ISR) in pancreatic β cells is coupled to metabolic alternations triggered by sulfhydration of key enzymes in intermediary metabolism.
Posttranslational modification is a fundamental mechanism in the regulation of structure and function of proteins. The covalent modification of specific amino acid residues influences diverse biological processes and cell physiology across species. Reactive cysteine residues in proteins have high nucleophilicity and low pKa values and serve as a major target for oxidative modifications, which can vary depending on the subcellular environment, including the type and intensity of intracellular or environmental cues. Oxidative environments cause different post-translational cysteine modifications, including disulfide bond formation (-S-S-), sulfenylation (-S-OH), nitrosylation (-S-NO), glutathionylation (-S-SG), and sulfhydration (-S-SH) (also called persulfidation) (Finkel, 2012; Mishanina et al., 2015). In the latter, an oxidized cysteine residue included glutathionylated, 60 sulfenylated and nitrosylated on a protein reacts with the sulfide anion to form a cysteine persulfide. The reversible nature of this modification provides a mechanism to fine tune biological processes in different cellular redox states. Sulfhydration coordinates with other post-translational protein modifications such as phosphorylation and nitrosylation to regulate cellular functions (Altaany et al., 2014; Sen et al., 2012). Despite great progress in bioinformatics and advanced mass spectroscopic techniques (MS), identification of different cysteine-based protein modifications has been slow compared to other post-translational modifications. In the case of sulfhydration, a small number of proteins have been identified, among them the glycolytic enzyme glyceraldehyde phosphate dehydrogenase, GAPDH (Mustafa et al., 2009). Sulfhydrated GAPDH at Cys150 exhibits an increase in its catalytic activity, in contrast to the inhibitory effects of nitrosylation or glutathionylation of the same cysteine residue (Mustafa et al., 2009; Paul and Snyder, 2012). The biological significance of the Cys150 modification by H2S is not well-studied, but H2S could serve as a biological switch for protein function acting via oxidative modification of specific cysteine residues in response to redox homeostasis (Paul and Snyder, 2012). Understanding the physiological significance of protein sulfhydration requires the development of genome-wide innovative experimental approaches. Current methodologies based on the modified biotin switch technique do not allow detection of a broad spectrum of sulfhydrated proteins (Finkel, 2012). Guided by a previously reported strategy (Sen et al., 2012), we developed an experimental approach that allowed us to quantitatively evaluate the sulfhydrated proteome and the physiological consequences of H2S synthesis during chronic ER stress. The new methodology allows a quantitative, close-up view of the integrated cellular response to environmental and intracellular cues, and is pertinent to our understanding of human disease development.
The ER is an organelle involved in synthesis of proteins followed by various modifications. Disruption of this process results in the accumulation of misfolded proteins, causing ER stress (Tabas and Ron, 2011; Walter and Ron, 2011), which is associated with development of many diseases ranging from metabolic dysfunction to neurodegeneration (Hetz, 2012). ER stress induces transcriptional, translational, and metabolic reprogramming, all of which are interconnected through the transcription factor Atf4. Atf4 increases expression of genes promoting adaptation to stress via their protein products. One such gene is the H2S-producing enzyme, γ-cystathionase (CTH), previously shown to be involved in the signaling pathway that negatively regulates the activity of the protein tyrosine phosphatase 1B (PTP1B) via sulfhydration (Krishnan et al., 2011). We therefore hypothesized that low or even modest levels of reactive oxygen species (ROS) during ER stress may reprogram cellular metabolism via H2S-mediated protein sulfhydration (Figure 1A).
In summary, sulfhydration of specific cysteines in proteins is a key function of H2S (Kabil and Banerjee, 2010; Paul and Snyder, 2012; Szabo et al., 2013). Thus, the development of tools that can quantitatively measure genome-wide protein sulfhydration in physiological or pathological conditions is of central importance. However, a significant challenge in studies of the biological significance of protein sulfhydration is the lack of an approach to selectively detect sulfhydrated cysteines from other modifications (disulfide bonds, glutathionylated thiols and sulfienic acids) in complex biological samples. In this study, we introduced the BTA approach that allowed the quantitative assessment of changes in the sulfhydration of specific cysteines in the proteome and in individual proteins. BTA is superior to other reported methodologies that aimed to profile cysteine modifications, such as the most commonly used, a modified biotin switch technique (BST). BST was originally designed to study protein nitrosylation and postulated to differentiate free thiols and persulfides (Mustafa et al., 2009). A key advantage of BTA over the existing methodologies, is that the experimental approach has steps to avoid false-positive and negative results, as target proteins for sulfhydration. BST is commonly generating such false targets for cysteine modifications (Forrester et al., 2009; Sen et al., 2012). Using mutiple validations, our data support the specificity and reliability of the BTA assay for analysis of protein sulfhydration both in vitro and in vivo. With this approach, we found that ATF4 is the master regulator of protein sulfhydration in pancreatic β cells during ER stress, by means of its function as a transcription factor. A large number of protein targets have been discovered to undergo sulfhydration in β cells by the BTA approach. Almost 1,000 sulfhydrated cysteine- containing peptides were present in the cells under the chronic ER stress condition of treatment with Tg for 18 h. Combined with the isotopic-labeling strategy, almost 820 peptides on more than 500 proteins were quantified in the 405 cells overexpressing ATF4. These data show the potential of the BTA method for further systematic studies of biological events. To our knowledge, the current dataset encompasses most known sulfhydrated cysteine residues in proteins in any organism. Our bioinformatics analyses revealed sulfhydrated cysteine residues located on a variety of structure-function domains, suggesting the possibility of regulatory mechanism(s) mediated by protein sulfhydration. Structure and sequence analysis revealed consensus motifs that favor sulfhydration; an arginine residue and alpha-helix dipoles are both contributing to stabilize sulfhydrated cysteine thiolates in the local environment.
Pathway analyses showed that H2S-mediated sulfhydration of cysteine residues is that part of the ISR with the highest enrichment in proteins involved in energy metabolism. The metabolic flux revealed that H2S promotes aerobic glycolysis associated with decreased oxidative phosphorylation in mitochondria during ER stress in β cells. The TCA cycle revolves by the action of the respiratory chain that requires oxygen to operate. In response to ER stress, mitochondrial function and cellular respiration are down-regulated to limit oxygen demand and to sustain mitochondria. When ATP production from the TCA cycle becomes limited and glycolytic flux increases, there is a risk of accumulation of lactate from pyruvate. One way to escape accumulation of lactate is the mitochondrial conversion of pyruvate to oxalacetic acid (OAA) by pyruvate carboxylase. This latter enzyme was found to be sulfhydrated, consistent with the notion that sulfhydration is linked to metabolic reprogramming towards glycolysis.
The switch of energy production from mitochondria to glycolysis is known as a signature of hypoxic conditions. This metabolic switch has also been observed in many cancer cells characterized as the Warburg effect, which contributes to tumor growth. The Warburg effect provides advantages to cancer cell survival via the rapid ATP production through glycolysis, as well as the increased conversion of glucose into anabolic biomolecules (amino acid, nucleic acid and lipid biosynthesis) and reducing power (NADPH) for regeneration of antioxidants. This metabolic response of tumor cells contributes to tumor growth and metastasis (Vander Heiden et al., 2009). By analogy, the aerobic glycolysis trigged by increased H2S production could give β cells the capability to acquire ATP and nutrients to adapt their cellular metabolism towards maintaining ATP levels in the ER (Vishnu et al., 2014), increasing synthesis of glycerolphospholipids, glycoproteins and protein (Krokowski et al., 2013b), all important components of the ISR. Similar to hypoxic conditions, a phenotype associated with most tumors, the decreased mitochondria function in β cells during ER stress, can also be viewed as an adaptive response by limiting mitochondria ROS and mitochondria-mediated apoptosis. We therefore view that the H2S-mediated increase in glycolysis is an adaptive mechanism for survival of β cells to chronic ER stress, along with the improved ER function and insulin production and folding, both critical factors controlling hyperglycemia in diabetes. Future work should determine which are the key proteins targeted by H2S and thus contributing to metabolic reprogramming of β cells, and if and how insulin synthesis and secretion is affected by sulfhydration of these proteins during ER stress.
Abnormal H2S metabolism has been reported to occur in various diseases, mostly through the deregulation of gene expression encoding for H2S-generating enzymes (Wallace and Wang, 2015). An increase of their levels by stimulants is expected to have similar effects on sulfhydration of proteins like the ATF4- induced CTH under conditions of ER stress. It is the levels of H2S under oxidative conditions that influence cellular functions. In the present study, ER stress in β cells induced elevated Cth levels, whereas CBS was unaffected. The deregulated oxidative modification at cysteine residues by H2S may be a major contributing factor to disease development. In this case, it would provide a rationale for the design of therapeutic agents that would modulate the activity of the involved enzymes.

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Blood-Gut Barrier

Larry H Bernstein, MD, FCAP, Curator



Blood-Gut Barrier

Scientists identify a barrier in mice between the intestine and its blood supply, and suggest how Salmonella sneaks through it.

By Ruth Williams | November 12, 2015

Salmonella invading an immune cell   WIKIMEDIA, NIAID

A person’s gut is full of microbes—some beneficial, some not. Friend or foe, these bacteria must be prevented from accessing the rest of the body, where they could cause harm. In a paper published in Science today (November 12), researchers describe a barrier in mice between the intestine and the adjacent blood vessels that restricts the size of particles that can pass through. The team also shows, however, that Salmonella bacteria can suppress a chemical pathway critical to barrier function, enabling the bugs to invade.

“First of all, [the authors] are really defining and showing the existence of this barrier and in what way it resembles the brain [barrier],” said immunologist Bana Jabri of the University of Chicago who was not involved in the study. “Then they show a pathogen that apparently has evolved to modulate that barrier to its own favor.”

The intestine is lined with epithelial cells covered in mucus. These cells provide both a physical barrier against microbial intrusion—they are tightly packed together—as well as biochemical one—they secrete antimicrobial proteins. In addition, the mucus itself “acts like a rip tide,” continuously washing the bacteria away from the epithelial shore, said gastroenterologist and immunologist Andrew Macpherson of the University of Bern in Switzerland who also did not participate in the work.

Aside from keeping the bacteria at bay, the epithelial cells’ job is to absorb digested food molecules. These nutrients enter the blood vessels adjacent to the intestine and ultimately reach the liver via the hepatic portal vein. What puzzled Maria Rescigno of the European Institute of Oncology in Milan, however, was why commensal bacteria, if they did beat the odds and enter the body, tended to end up in the nearby lymph nodes but were not found in the liver.

“Why would bacteria get to the lymphatics but not into the blood vessels which are very close to the epithelium?” she asked. And she began wondering: “is there something else other than just the epithelial barrier?”

For clues, Rescigno and her colleagues turned to probably the best-characterized barrier in the body: the blood-brain barrier. “We wondered whether the endothelium of the blood vessels [in the gut] would resemble the endothelium of the blood vessels in the brain,” she said.

There were indeed similarities. Endothelial cells of the blood-brain barrier have characteristically strong connections, called tight junctions and adherens junctions, and are surrounded by astrocytes—essential for barrier function. Endothelial cells of mouse intestinal blood vessels on the other hand had their own distinctive tight and adherens junctions, and were surrounded by enteric glial cells, “similar to the astrocytes” said Rescigno. These features also appeared to be present in human intestinal blood vessels, Rescigno’s team showed.

Furthermore the intestinal blood vessels of mice exhibited barrier function: while 4 kilodalton particles injected into the vessels could pass across the endothelium freely, 70 kilodalton particles could not. The blood-brain barrier by comparison prevents passage of any particles bigger than 500 Daltons.

Although this barrier appears to prevent most bacteria from entering the bloodstream, certain pathogenic species—including some Salmonella—are capable of establishing infections in the blood, liver and other organs. Rescigno and colleagues therefore investigated how Salmonella bacteria manage to squeeze through.

It turned out that infection of cultured endothelium with Salmonella suppressed the cells’ Wnt/β-catenin signaling pathway—known to be essential for blood-brain barrier function. Furthermore, the forced overexpression of β-catenin in endothelial cells of live mice, prevented Salmonella bacteria present in the animals’ guts from propagating around their bodies.

Prior to this study, the gut epithelium was considered the extent of the intestinal boundary, explained Macpherson, so “highlighting this vascular barrier is actually a rather important step forward.”

Depending on the species and serotype of Salmonella as well as on the health of the patient, some infections will lead to an acute illness limited to the gastrointestinal tract, while others will disseminate systemically and become chronic. If it were possible to develop treatments that close the vascular barrier, suggested Jabri, it might be possible to give such a drug alongside antibiotics. “In people who get acute infection one could address this barrier problem immediately,” she said, “so that [the infection] could not become chronic.”

I. Spadoni et al., “A gut-vascular barrier controls the systemic dissemination of bacteria,”Science, 350:830-34, 2015.


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