A transcriptional evaluation of the melanoma and squamous cell carcinoma TIL compartment reveals an unexpected spectrum of exhausted and functional T cells.

Introduction: Significant heterogeneity exists within the tumor-infiltrating CD8 T cell population, and exhausted T cells harbor a subpopulation that may be replicating and may retain signatures of activation, with potential functional consequences in tumor progression. Dysfunctional immunity in the tumor microenvironment is associated with poor cancer outcomes, making exploration of these exhausted T cell subpopulations critical to the improvement of therapeutic approaches. Methods: To investigate mechanisms associated with terminally exhausted T cells, we sorted and performed transcriptional profiling of CD8+ tumor-infiltrating lymphocytes (TILs) co-expressing the exhaustion markers PD-1 and TIM-3 from large-volume melanoma tumors. We additionally performed immunologic phenotyping and functional validation, including at the single-cell level, to identify potential mechanisms that underlie their dysfunctional phenotype. Results: We identified novel dysregulated pathways in CD8+PD-1+TIM-3+ cells that have not been well studied in TILs; these include bile acid and peroxisome pathway-related metabolism and mammalian target of rapamycin (mTOR) signaling pathways, which are highly correlated with immune checkpoint receptor expression. Discussion: Based on bioinformatic integration of immunophenotypic data and network analysis, we propose unexpected targets for therapies to rescue the immune response to tumors in melanoma.

Design strategy and research progress of multifunctional nanoparticles in lung cancer therapy.

INTRODUCTION: Lung cancer is one of the cancer types with the highest mortality rate, exploring a more effective treatment modality that improves therapeutic efficacy while mitigating side effects is now an urgent requirement. Designing multifunctional nanoparticles can be used to overcome the limitations of drugs and conventional drug delivery systems. Nanotechnology has been widely researched, and through different needs, suitable nanocarriers can be selected to load anti-cancer drugs to improve the therapeutic effect. It is foreseeable that with the rapid development of nanotechnology, more and more lung cancer patients will benefit from nanotechnology. This paper reviews the merits of various multifunctional nanoparticles in the treatment of lung cancer to provide novel ideas for lung cancer treatment. AREAS COVERED: This review focuses on summarizing various nanoparticles for targeted lung cancer therapy and their advantages and disadvantages, using nanoparticles loaded with anti-cancer drugs, delivered to lung cancer sites, enhancing drug half-life, improving anti-cancer drug efficacy and reducing side effects. EXPERT OPINION: The delivery mode of nanoparticles with superior pharmacokinetic properties in the in vivo circulation enhances the half-life of the drug, and provides tissue-targeted selectivity and the ability to overcome biological barriers, bringing a revolution in the field of oncology.

Research on measurement indexes and evaluation for the collaborative efficiency of emergency information sharing in coastal cities of China.

BACKGROUND: The paper established indexes of coordination efficiency, the theoretical framework and operation mechanism of emergency information sharing for coastal cities of China. METHOD: First of all, we analyzed the operational relationship between the participants of emergency information sharing and information transmission, and based on the collaborative theory, constructed the emergency information sharing framework and operational mechanism suitable for the actual disaster prevention and reduction of coastal cities. Around the 3 dimensions of emergency information sharing mechanism construction, resource guarantee ability and collaborative driving force of emergency information sharing, the paper proposed the evaluation index system and the evaluation method. RESULTS: The empirical results showed that the efficiency of emergency information sharing in coastal cities in China was generally low, and the contribution rate of the construction level of emergency information sharing mechanism is higher than that of the resource guarantee ability and the collaborative driving force of emergency information sharing, but the efficiency of emergency information sharing in coastal cities in China was still at the bottom level. CONCLUSION: The research results provided theoretical basis and methods for the emergency management departments of coastal cities in China.

Arthroscopic Treatment of Calcific Tendinitis of Gemellus Superior and Gemellus Inferior: A Case Report and Literature Review.

BACKGROUND: Tendon calcification is a common disease, and it could happen in the tendons of the shoulder, wrist, etc. However, tendon calcification in the superior and inferior gemellus is rare, and in this region is likely to be misdiagnosed. CASE PRESENTATION: Here, our case report first reported a 53-year-old female patient with an unusual case of calcific tendinitis of the gemellus superior and gemellus inferior muscles. The patient presented with severe pain in the right hip and lower extremities, not relieved using nonsteroidal anti-inflammatory drugs (NSAIDs). The preoperative physical examination indicated an abnormality in the hip joint. Preoperative imaging confirmed the results of the physical examination and showed a right hip lesion. We did not make a definite diagnosis preoperatively but considered that the patient might have an osteochondroma. However, surgical findings indicated that the lesion was not in the hip capsule on subsequent arthroscopic surgery, which suggested that the preoperative diagnosis might be wrong. We opened the posterior capsule and found a "toothpaste-like" lesion in the superior and inferior gemellus muscles' tendon. We thought this might be the calcified tendon. Then the arthroscopic surgery was finished to remove the lesion, and the removed tissue was sent to the pathology department for pathological examination. The pathological report confirmed the diagnosis of the calcified tendon. Postoperative follow-up was conducted. The effect of the operation was noticeable. Postoperative symptoms were relieved. CONCLUSIONS: Calcification of the tendons of the superior and inferior gemellus muscles can be easily misdiagnosed, and the disease can be treated minimally with arthroscopy.

Atypical Iron-Sulfur Cluster Binding, Redox Activity and Structural Properties of Chlamydomonas reinhardtii Glutaredoxin 2.

Glutaredoxins (GRXs) are thioredoxin superfamily members exhibiting thiol-disulfide oxidoreductase activity and/or iron-sulfur (Fe-S) cluster binding capacities. These properties are determined by specific structural factors. In this study, we examined the capacity of the class I Chlamydomonas reinhardtii GRX2 recombinant protein to catalyze both protein glutathionylation and deglutathionylation reactions using a redox sensitive fluorescent protein as a model protein substrate. We observed that the catalytic cysteine of the CPYC active site motif of GRX2 was sufficient for catalyzing both reactions in the presence of glutathione. Unexpectedly, spectroscopic characterization of the protein purified under anaerobiosis showed the presence of a [2Fe-2S] cluster despite having a presumably inadequate active site signature, based on past mutational analyses. The spectroscopic characterization of cysteine mutated variants together with modeling of the Fe-S cluster-bound GRX homodimer from the structure of an apo-GRX2 indicate the existence of an atypical Fe-S cluster environment and ligation mode. Overall, the results further delineate the biochemical and structural properties of conventional GRXs, pointing to the existence of multiple factors more complex than anticipated, sustaining the capacity of these proteins to bind Fe-S clusters.

Discovery of a Redox Thiol Switch: Implications for Cellular Energy Metabolism.

The redox-based modifications of cysteine residues in proteins regulate their function in many biological processes. The gas molecule H2S has been shown to persulfidate redox sensitive cysteine residues resulting in an H2S-modified proteome known as the sulfhydrome. Tandem Mass Tags (TMT) multiplexing strategies for large-scale proteomic analyses have become increasingly prevalent in detecting cysteine modifications. Here we developed a TMT-based proteomics approach for selectively trapping and tagging cysteine persulfides in the cellular proteomes. We revealed the natural protein sulfhydrome of two human cell lines, and identified insulin as a novel substrate in pancreatic beta cells. Moreover, we showed that under oxidative stress conditions, increased H2S can target enzymes involved in energy metabolism by switching specific cysteine modifications to persulfides. Specifically, we discovered a Redox Thiol Switch, from protein S-glutathioinylation to S-persulfidation (RTSGS). We propose that the RTSGS from S-glutathioinylation to S-persulfidation is a potential mechanism to fine tune cellular energy metabolism in response to different levels of oxidative stress.

PACT-mediated PKR activation acts as a hyperosmotic stress intensity sensor weakening osmoadaptation and enhancing inflammation.

The inability of cells to adapt to increased environmental tonicity can lead to inflammatory gene expression and pathogenesis. The Rel family of transcription factors TonEBP and NF-κB p65 play critical roles in the switch from osmoadaptive homeostasis to inflammation, respectively. Here we identified PACT-mediated PKR kinase activation as a marker of the termination of adaptation and initiation of inflammation in Mus musculus embryonic fibroblasts. We found that high stress-induced PACT-PKR activation inhibits the interaction between NF-κB c-Rel and TonEBP essential for the increased expression of TonEBP-dependent osmoprotective genes. This resulted in enhanced formation of TonEBP/NF-κB p65 complexes and enhanced proinflammatory gene expression. These data demonstrate a novel role of c-Rel in the adaptive response to hyperosmotic stress, which is inhibited via a PACT/PKR-dependent dimer redistribution of the Rel family transcription factors. Our results suggest that inhibiting PACT-PKR signaling may prove a novel target for alleviating stress-induced inflammatory diseases.

Cells are sensitive to changes in their environment. For example, maintaining normal salt levels in the blood, also called tonicity, is essential for the health of individual cells and the organism as a whole. Tonicity controls the movement of water in and out of the cell: high levels of salt inside the cell draw water in, while high levels of salt outside the cell draw water out. If salt levels in the environment surrounding the cells become too high, too much water will be drawn out, causing the cells to shrink. Changes in tonicity can cause the cell to become stressed. Initially, cells adapt to this stress by switching on sets of genes that help restore fluid balance and allow the cell to regain its normal shape and size. If the increase in tonicity exceeds tolerable stress levels and harms the cell, this initiates an inflammatory response which ultimately leads to cell death. However, it remained unclear how cells switch from adapting to responding with inflammation. Now, Farabaugh et al. have used an experimental system which mimics high salt to identify the mechanism that allows cells to switch between these two responses. The experiments showed that when salt levels are too high, cells switch on a stress sensing protein called PACT, which activates another protein called PKR. When PACT was deleted from mouse cells, this led to a decrease in the activity of inflammatory genes, and prevented the cells from self-destructing. Other proteins that are involved in the adaptive and inflammatory response are the NF-κB family of proteins and TonEBP. Farabaugh et al. found that under low intensity stress, when salt levels outside the cell are slightly too high, a family member of NF-κB works with TonEBP to switch on adaptive genes. But, if salt levels continue to rise, PACT activates and turns on PKR. This blocks the interaction between NF-κB and TonEBP, allowing another family member of NF-κB to interact with TonEBP instead. This switches the adaptive response off and the inflammatory response on. There are many diseases that involve changes in tonicity, including diabetes, cancer, inflammatory bowel disease, and dry eye syndrome. Understanding the proteins involved in the adaptive and inflammatory response could lead to the development of drugs that help to protect cells from stress-induced damage.

Digital Analysis of Immunostaining of ZW10 Interacting Protein in Human Lung Tissues.

The purpose of this study is to introduce a methodology of the immunostaining of human lung tissues, followed by whole-slide digital scanning and image analysis. Digital scanning is a fast way to scan a stack of slides and produce digital images with high quality. It can produce concordant results with conventional light microscopy (CLM) by pathologists. Furthermore, the availability of digital images makes it possible that the same slide can be concurrently observed by multiple people. Moreover, digital images of slides can be stored in a database, which means the long-term deterioration of glass slides is avoided. The limitations of this technique are as follows. First, it needs high-quality prepared tissue and the original immunohistochemistry (IHC) slides without any damage or excess sealant residue. Second, tumor or nontumor areas should be specified by experienced pathologists before the analysis using software, in order to avoid any confusion about the tumor or nontumor areas during scoring. Third, the operator needs to control the color reproduction throughout the digitization process in whole-slide imaging.

Protein Changes in Response to Lead Stress of Lead-Tolerant and Lead-Sensitive Industrial Hemp Using SWATH Technology.

Hemp is a Pb-tolerant and Pb-accumulating plant and the study of its tolerance mechanisms could facilitate the breeding of hemp with enhanced Pb tolerance and accumulation. In the present study, we took advantage of sequential window acquisition of all theoretical mass spectra (SWATH) technology to study the difference in proteomics between the leaves of Pb-tolerant seed-type hemp variety Bamahuoma (BM) and the Pb-sensitive fiber-type hemp variety Yunma 1 (Y1) under Pb stress (3 g/kg soil). A total of 63 and 372 proteins differentially expressed under Pb stress relative to control conditions were identified with liquid chromatography electro spray ionization tandem mass spectrometry in BM and Y1, respectively; with each of these proteins being classified into 14 categories. Hemp adapted to Pb stress by: accelerating adenosine triphosphate (ATP) metabolism; enhancing respiration, light absorption and light energy transfer; promoting assimilation of intercellular nitrogen (N) and carbon (C); eliminating reactive oxygen species; regulating stomatal development and closure; improving exchange of water and CO2 in leaves; promoting intercellular transport; preventing aggregation of unfolded proteins; degrading misfolded proteins; and increasing the transmembrane transport of ATP in chloroplasts. Our results provide an important reference protein and gene information for future molecular studies into the resistance and accumulation of Pb in hemp.

SMAR1 promotes immune escape of Tri-negative Breast Cancer through a mechanism involving T-bet/PD-1 Axis.

This study was aimed at investigating the specific molecular mechanisms involved in the regulation of immune escape of triple-negative breast cancer (TNBC) by SMRI, so as to provide a new clinical treatment target for the disease. Mouse original 4T1 breast cancer cells were inoculated subcutaneously in BALB/C to establish TNBC mouse model. CD8+T cells with immunological effects were selected from mouse thymus glands for primary culture. The CD8+positive T cells were infected with lentivirus interference vectors, and the proliferation of CD8+ T cells were determined by trypan blue staining and flow cytometry. CD8+T cells and 4T1 cells were cultured together so as to determine the cytotoxic effects of SMAR1-downregulated CD8+ T cells on tumor cells and the expression of cytokines (IFN-γ, TNF-α, IL-2, IL-4 and IL-6). The expressions of SMAR1, T-bet and PD-1 were assayed by Western blot. SMAR1-downregulated CD8+T cells were injected into 4T1 tumor-bearing mice through the caudal vein, and the growth of tumor in mice was monitored. Following the infection of CD8+T cells with SMAR1-downregulated lentiviral system, cell apoptosis level was decreased significantly (control vs. sh-SMAR1: 32.23 ± 12.4 % vs. 18.28 ± 8.93 %, p < 0.05). Results from trypan blue staining experiments showed that the proliferation of CD8+ T cells in the SMAR1-downregulated group was significantly increased; SMAR1-downregulated CD8+ T cells promoted the production of IFN-γ, TNF-α, IL-2, IL-4 and IL-6 in 4T1 breast cancer cells (p < 0.05). Western blot showed that SMAR1 down-regulation led to significant upregulation of T-bet, while PD-1 was downregulated, when compared to the control group (p < 0.05). The downregulation of SMAR1 was associated with significant reduction in tumor size in mice (p < 0.05). SMAR1 downregulation enhances the tumor killing effect of CD8+T cells by activating T-bet and down-regulating PD-1.

Hydrogen sulfide modulates eukaryotic translation initiation factor 2α (eIF2α) phosphorylation status in the integrated stress-response pathway.

Hydrogen sulfide (H2S) regulates various physiological processes, including neuronal activity, vascular tone, inflammation, and energy metabolism. Moreover, H2S elicits cytoprotective effects against stressors in various cellular models of injury. However, the mechanism of the signaling pathways mediating the cytoprotective functions of H2S is not well understood. We previously uncovered a heme-dependent metabolic switch for transient induction of H2S production in the trans-sulfuration pathway. Here, we demonstrate that increased endogenous H2S production or its exogenous administration modulates major components of the integrated stress response promoting a metabolic state primed for stress response. We show that H2S transiently increases phosphorylation of eukaryotic translation initiation factor 2 (eIF2α) resulting in inhibition of general protein synthesis. The H2S-induced increase in eIF2α phosphorylation was mediated at least in part by inhibition of protein phosphatase-1 (PP1c) via persulfidation at Cys-127. Overexpression of a PP1c cysteine mutant (C127S-PP1c) abrogated the H2S effect on eIF2α phosphorylation. Our data support a model in which H2S exerts its cytoprotective effect on ISR signaling by inducing a transient adaptive reprogramming of global mRNA translation. Although a transient increase in endogenous H2S production provides cytoprotection, its chronic increase such as in cystathionine ß-synthase deficiency may pose a problem.

Protein Kinase R Mediates the Inflammatory Response Induced by Hyperosmotic Stress.

High extracellular osmolarity results in a switch from an adaptive to an inflammatory gene expression program. We show that hyperosmotic stress activates the protein kinase R (PKR) independently of its RNA-binding domain. In turn, PKR stimulates nuclear accumulation of nuclear factor κB (NF-κB) p65 species phosphorylated at serine-536, which is paralleled by the induction of a subset of inflammatory NF-κB p65-responsive genes, including inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), and IL-1ß. The PKR-mediated hyperinduction of iNOS decreases cell survival in mouse embryonic fibroblasts via mechanisms involving nitric oxide (NO) synthesis and posttranslational modification of proteins. Moreover, we demonstrate that the PKR inhibitor C16 ameliorates both iNOS amplification and disease-induced phenotypic breakdown of the intestinal epithelial barrier caused by an increase in extracellular osmolarity induced by dextran sodium sulfate (DSS) in vivo Collectively, these findings indicate that PKR activation is an essential part of the molecular switch from adaptation to inflammation in response to hyperosmotic stress.

Adverse Outcomes Associated with Cigarette Smoke Radicals Related to Damage to Protein-disulfide Isomerase.

Identification of factors contributing to the development of chronic obstructive pulmonary disease (COPD) is crucial for developing new treatments. An increase in the levels of protein-disulfide isomerase (PDI), a multifaceted endoplasmic reticulum resident chaperone, has been demonstrated in human smokers, presumably as a protective adaptation to cigarette smoke (CS) exposure. We found a similar increase in the levels of PDI in the murine model of COPD. We also found abnormally high levels (4-6 times) of oxidized and sulfenilated forms of PDI in the lungs of murine smokers compared with non-smokers. PDI oxidation progressively increases with age. We begin to delineate the possible role of an increased ratio of oxidized PDI in the age-related onset of COPD by investigating the impact of exposure to CS radicals, such as acrolein (AC), hydroxyquinones (HQ), peroxynitrites (PN), and hydrogen peroxide, on their ability to induce unfolded protein response (UPR) and their effects on the structure and function of PDIs. Exposure to AC, HQ, PN, and CS resulted in cysteine and tyrosine nitrosylation leading to an altered three-dimensional structure of the PDI due to a decrease in helical content and formation of a more random coil structure, resulting in protein unfolding, inhibition of PDI reductase and isomerase activity in vitro and in vivo, and subsequent induction of endoplasmic reticulum stress response. Addition of glutathione prevented the induction of UPR, and AC and HQ induced structural changes in PDI. Exposure to PN and glutathione resulted in conjugation of PDI possibly at active site tyrosine residues. The findings presented here propose a new role of PDI in the pathogenesis of COPD and its age-dependent onset.

Quantitative H2S-mediated protein sulfhydration reveals metabolic reprogramming during the integrated stress response.

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 ß 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 ß cells is coupled to metabolic alternations triggered by sulfhydration of key enzymes in intermediary metabolism.

DNMT1-associated long non-coding RNAs regulate global gene expression and DNA methylation in colon cancer.

The cancer epigenome exhibits global loss of DNA methylation, which contributes to genomic instability and aberrant gene expression by mechanisms that are yet to be fully elucidated. We previously discovered over 3300 long non-coding (lnc)RNAs in human cells and demonstrated that specific lncRNAs regulate gene expression via interactions with chromatin-modifying complexes. Here, we tested whether lncRNAs could also associate with DNA methyltransferases to regulate DNA methylation and gene expression. Using RIP-seq, we identified a subset of lncRNAs that interact with the DNA methyltransferase DNMT1 in a colon cancer cell line, HCT116. One lncRNA, TCONS_00023265, which we named DACOR1 (DNMT1-associated Colon Cancer Repressed lncRNA 1), shows high, tissue-specific expression in the normal colon (including colon crypts) but was repressed in a panel of colon tumors and patient-derived colon cancer cell lines. We identified the genomic occupancy sites of DACOR1, which we found to significantly overlap with known differentially methylated regions (DMRs) in colon tumors. Induction of DACOR1 in colon cancer cell lines significantly reduced their ability to form colonies in vitro, suggesting a growth suppressor function. Consistent with the observed phenotype, induction of DACOR1 led to the activation of tumor-suppressor pathways and attenuation of cancer-associated metabolic pathways. Notably, DACOR1 induction resulted in down-regulation of Cystathionine ß-synthase, which is known to lead to increased levels of S-adenosyl methionine-the key methyl donor for DNA methylation. Collectively, our results demonstrate that deregulation of DNMT1-associated lncRNAs contributes to aberrant DNA methylation and gene expression during colon tumorigenesis.

Insight into protein S-nitrosylation in Chlamydomonas reinhardtii.

AIMS: Protein S-nitrosylation, a post-translational modification (PTM) consisting of the covalent binding of nitric oxide (NO) to a cysteine thiol moiety, plays a major role in cell signaling and is recognized to be involved in numerous physiological processes and diseases in mammals. The importance of nitrosylation in photosynthetic eukaryotes has been less studied. The aim of this study was to expand our knowledge on protein nitrosylation by performing a large-scale proteomic analysis of proteins undergoing nitrosylation in vivo in Chlamydomonas reinhardtii cells under nitrosative stress. RESULTS: Using two complementary proteomic approaches, 492 nitrosylated proteins were identified. They participate in a wide range of biological processes and pathways, including photosynthesis, carbohydrate metabolism, amino acid metabolism, translation, protein folding or degradation, cell motility, and stress. Several proteins were confirmed in vitro by western blot, site-directed mutagenesis and activity measurements. Moreover, 392 sites of nitrosylation were also identified. These results strongly suggest that S-nitrosylation could constitute a major mechanism of regulation in C. reinhardtii under nitrosative stress conditions. INNOVATION: This study constitutes the largest proteomic analysis of protein nitrosylation reported to date. CONCLUSION: The identification of 381 previously unrecognized targets of nitrosylation further extends our knowledge on the importance of this PTM in photosynthetic eukaryotes. The data have been deposited to the ProteomeXchange repository with identifier PXD000569.

Aging-dependent changes in rat heart mitochondrial glutaredoxins--Implications for redox regulation.

Clinical and animal studies have documented that hearts of the elderly are more susceptible to ischemia/reperfusion damage compared to young adults. Recently we found that aging-dependent increase in susceptibility of cardiomyocytes to apoptosis was attributable to decrease in cytosolic glutaredoxin 1 (Grx1) and concomitant decrease in NF-κB-mediated expression of anti-apoptotic proteins. Besides primary localization in the cytosol, Grx1 also exists in the mitochondrial intermembrane space (IMS). In contrast, Grx2 is confined to the mitochondrial matrix. Here we report that Grx1 is decreased by 50-60% in the IMS, but Grx2 is increased by 1.4-2.6 fold in the matrix of heart mitochondria from elderly rats. Determination of in situ activities of the Grx isozymes from both subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria revealed that Grx1 was fully active in the IMS. However, Grx2 was mostly in an inactive form in the matrix, consistent with reversible sequestration of the active-site cysteines of two Grx2 molecules in complex with an iron-sulfur cluster. Our quantitative evaluations of the active/inactive ratio for Grx2 suggest that levels of dimeric Grx2 complex with iron-sulfur clusters are increased in SSM and IFM in the hearts of elderly rats. We found that the inactive Grx2 can be fully reactivated by sodium dithionite or exogenous superoxide production mediated by xanthine oxidase. However, treatment with rotenone, which generates intramitochondrial superoxide through inhibition of mitochondrial respiratory chain Complex I, did not lead to Grx2 activation. These findings suggest that insufficient ROS accumulates in the vicinity of dimeric Grx2 to activate it in situ.

Glutaredoxin s12: unique properties for redox signaling.

AIMS: Cysteines (Cys) made acidic by the protein environment are generally sensitive to pro-oxidant molecules. Glutathionylation is a post-translational modification that can occur by spontaneous reaction of reduced glutathione (GSH) with oxidized Cys as sulfenic acids (-SOH). The reverse reaction (deglutathionylation) is strongly stimulated by glutaredoxins (Grx) and requires a reductant, often GSH. RESULTS: Here, we show that chloroplast GrxS12 from poplar efficiently reacts with glutathionylated substrates in a GSH-dependent ping pong mechanism. The pK(a) of GrxS12 catalytic Cys is very low (3.9) and makes GrxS12 itself sensitive to oxidation by H(2)O(2) and to direct glutathionylation by nitrosoglutathione. Glutathionylated-GrxS12 (GrxS12-SSG) is temporarily inactive until it is deglutathionylated by GSH. The equilibrium between GrxS12 and glutathione (E(m(GrxS12-SSG))= -315 mV, pH 7.0) is characterized by K(ox) values of 310 at pH 7.0, as in darkened chloroplasts, and 69 at pH 7.9, as in illuminated chloroplasts. INNOVATION: Based on thermodynamic data, GrxS12-SSG is predicted to accumulate in vivo under conditions of mild oxidation of the GSH pool that may occur under stress. Moreover, GrxS12-SSG is predicted to be more stable in chloroplasts in the dark than in the light. CONCLUSION: These peculiar catalytic and thermodynamic properties could allow GrxS12 to act as a stress-related redox sensor, thus allowing glutathione to play a signaling role through glutathionylation of GrxS12 target proteins.

Mechanisms of altered redox regulation in neurodegenerative diseases--focus on S--glutathionylation.

SIGNIFICANCE: Neurodegenerative diseases are characterized by progressive loss of neurons. A common feature is oxidative stress, which arises when reactive oxygen species (ROS) and/or reactive nitrogen species (RNS) exceed amounts required for normal redox signaling. An imbalance in ROS/RNS alters functionality of cysteines and perturbs thiol-disulfide homeostasis. Many cysteine modifications may occur, but reversible protein mixed disulfides with glutathione (GSH) likely represents the common steady-state derivative due to cellular abundance of GSH and ready conversion of cysteine-sulfenic acid and S-nitrosocysteine precursors to S-glutathionylcysteine disulfides. Thus, S-glutathionylation acts in redox signal transduction and serves as a protective mechanism against irreversible cysteine oxidation. Reversal of protein-S-glutathionylation is catalyzed specifically by glutaredoxin which thereby plays a critical role in cellular regulation. This review highlights the role of oxidative modification of proteins, notably S-glutathionylation, and alterations in thiol homeostatic enzyme activities in neurodegenerative diseases, providing insights for therapeutic intervention. RECENT ADVANCES: Recent studies show that dysregulation of redox signaling and sulfhydryl homeostasis likely contributes to onset/progression of neurodegeneration. Oxidative stress alters the thiol-disulfide status of key proteins that regulate the balance between cell survival and cell death. CRITICAL ISSUES: Much of the current information about redox modification of key enzymes and signaling intermediates has been gleaned from studies focused on oxidative stress situations other than the neurodegenerative diseases. FUTURE DIRECTIONS: The findings in other contexts are expected to apply to understanding neurodegenerative mechanisms. Identification of selectively glutathionylated proteins in a quantitative fashion will provide new insights about neuropathological consequences of this oxidative protein modification.

Biochemical characterization of glutaredoxins from Chlamydomonas reinhardtii: kinetics and specificity in deglutathionylation reactions.

Protein deglutathionylation is mainly catalyzed by glutaredoxins (GRXs). We have analyzed the biochemical properties of four of the six different GRXs of Chlamydomonas reinhardtii. Kinetic parameters were determined for disulfide and dehydroascorbate reduction but also for deglutathionylation of artificial and protein substrates. The results indicate that GRXs exhibit striking differences in their catalytic properties, mainly linked to the class of GRX considered but also to the pK(a) of the N-terminal catalytic cysteine. Furthermore, glutathionylated proteins were found to exhibit distinct reactivities with GRXs. These results suggest that glutathionylation may allow a fine tuning of cell metabolism under stress conditions.