Global Methods to Monitor the Thiol–Disulfide State of Proteins In Vivo

Cysteines play an important role in protein biochemistry. The unique chemical property and high reactivity of the free thiol group makes reduced cysteine a versatile component of catalytic centers and metal binding sites in many cytosolic proteins and oxidized cystine a stabilizing component in many secreted proteins. Moreover, cysteines readily react with reactive oxygen and nitrogen species to form reversible oxidative thiol modifications. As a result, these reversible thiol modifications have found a use as regulatory nano-switches in an increasing number of redox sensitive proteins. These redox-regulated proteins are able to adjust their activity quickly in response to changes in their redox environment. Over the past few years, a number of techniques have been developed that give insight into the global thiol–disulfide state of proteins in the cell. They have been successfully used to find substrates of thiol–disulfide oxidoreductases and to discover novel redoxregulated proteins. This review will provide an overview of the current techniques, focus on approaches to quantitatively describe the extent of thiol modification in vivo, and summarize their applications.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63278/1/ars.2006.8.763.pd

Protein Thiol Modifications Visualized In Vivo

Thiol-disulfide interconversions play a crucial role in the chemistry of biological systems. They participate in the major systems that control the cellular redox potential and prevent oxidative damage. In addition, thiol-disulfide exchange reactions serve as molecular switches in a growing number of redox-regulated proteins. We developed a differential thiol-trapping technique combined with two-dimensional gel analysis, which in combination with genetic studies, allowed us to obtain a snapshot of the in vivo thiol status of cellular proteins. We determined the redox potential of protein thiols in vivo, identified and dissected the in vivo substrate proteins of the major cellular thiol-disulfide oxidoreductases, and discovered proteins that undergo thiol modifications during oxidative stress. Under normal growth conditions most cytosolic proteins had reduced cysteines, confirming existing dogmas. Among the few partly oxidized cytosolic proteins that we detected were proteins that are known to form disulfide bond intermediates transiently during their catalytic cycle (e.g., dihydrolipoyl transacetylase and lipoamide dehydrogenase). Most proteins with highly oxidized thiols were periplasmic proteins and were found to be in vivo substrates of the disulfide-bond-forming protein DsbA. We discovered a substantial number of redox-sensitive cytoplasmic proteins, whose thiol groups were significantly oxidized in strains lacking thioredoxin A. These included detoxifying enzymes as well as many metabolic enzymes with active-site cysteines that were not known to be substrates for thioredoxin. H(2)O(2)-induced oxidative stress resulted in the specific oxidation of thiols of proteins involved in detoxification of H(2)O(2) and of enzymes of cofactor and amino acid biosynthesis pathways such as thiolperoxidase, GTP-cyclohydrolase I, and the cobalamin-independent methionine synthase MetE. Remarkably, a number of these proteins were previously or are now shown to be redox regulated

Hsp33 confers bleach resistance by protecting elongation factor Tu against oxidative degradation in Vibrio cholerae

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90122/1/j.1365-2958.2012.07982.x.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/90122/2/MMI_7982_sm_FigS1-S5.pd

Umfrage der UB Mannheim - Ergebnisse der Auswertung : "Sagen Sie uns Ihre Meinung" vom 13.3. – 8.4.2012 in Zusammenarbeit mit dem Lehrstuhl für ABWL, Public & Nonprofit Management (Prof. Helmig)

"Sagen Sie uns Ihre Meinung" – unter diesem Motto führte die UB Mannheim im Frühjahr 2012 eine große Umfrage zu ihren Services durch. Ziel der Umfrage war es, die aktuelle Zufriedenheit der NutzerInnen mit der Bibliothek abzufragen und die Erwartungen hinsichtlich zukünftiger Dienstleistungen zu ermitteln. Dieser Bericht präsentiert die wesentlichen Ergebnisse und Schlussfolgerungen aus Sicht der UB Mannheim

CoSMoS: Conserved Sequence Motif Search in the proteome

BACKGROUND: With the ever-increasing number of gene sequences in the public databases, generating and analyzing multiple sequence alignments becomes increasingly time consuming. Nevertheless it is a task performed on a regular basis by researchers in many labs. RESULTS: We have now created a database called CoSMoS to find the occurrences and at the same time evaluate the significance of sequence motifs and amino acids encoded in the whole genome of the model organism Escherichia coli K12. We provide a precomputed set of multiple sequence alignments for each individual E. coli protein with all of its homologues in the RefSeq database. The alignments themselves, information about the occurrence of sequence motifs together with information on the conservation of each of the more than 1.3 million amino acids encoded in the E. coli genome can be accessed via the web interface of CoSMoS. CONCLUSION: CoSMoS is a valuable tool to identify highly conserved sequence motifs, to find regions suitable for mutational studies in functional analyses and to predict important structural features in E. coli proteins

Label-free and redox proteomic analyses of the triacylglycerol-accumulating Rhodococcus jostii RHA1

The bacterium Rhodococcus jostii RHA1 synthesizes large amounts of triacylglycerols (TAG) under conditions of nitrogen starvation. To better understand the molecular mechanisms behind this process, we performed proteomic studies in this oleaginous bacterium. Upon nitrogen starvation, we observed a re-routing of the carbon flux towards the formation of TAG. Under these conditions, the cellular lipid content made up more than half of the cell?s dry weight. On the proteome level, this coincided with a shift towards non-glycolytic carbohydrate-metabolizing pathways. These pathways (Entner-Doudoroff and pentose-phosphate shunt) contribute NADPH and precursors of glycerol-3-phosphate and acetyl-CoA to lipogenesis. The expression of proteins involved in the degradation of branched-chain-amino acids and the methyl malonyl-CoA pathway probably provided propionyl-CoA for the biosynthesis of odd-numbered fatty acids, which make up almost 30% of RHA1 fatty acid composition. Additionally, lipolytic and glycerol-degrading enzymes increased in abundance, suggesting a dynamic cycling of cellular lipids. Conversely, abundance of proteins involved in consuming intermediates of lipogenesis decreased. Furthermore, we identified another level of lipogenesis regulation through redox-mediated thiol modification in R. jostii. Enzymes affected included acetyl-CoA carboxylase and a β-ketoacyl-[ACP] synthase II (FabF). An integrative metabolic model for the oleaginous RHA1 strain is proposed based on our results.Fil: Dávila Costa, José Sebastián. Universidad Nacional de la Patagonia "San Juan Bosco"; ArgentinaFil: Herrero, O. Marisa. Universidad Nacional de la Patagonia "san Juan Bosco". Facultad de Ciencias Naturales - Sede Comodoro; ArgentinaFil: Alvarez, Hector Manuel. Universidad Nacional de la Patagonia "san Juan Bosco". Facultad de Ciencias Naturales - Sede Comodoro; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Nacional Patagónico; ArgentinaFil: Leichert, Lars. Ruhr-Universitat Bochum; Alemani

Does the Transcription Factor NemR Use a Regulatory Sulfenamide Bond to Sense Bleach?

Reactive chlorine species (RCS), such as hypochlorous acid (i.e., bleach), are antimicrobial oxidants produced by the innate immune system. Like many redox-regulated transcription factors, the Escherichia coli repressor NemR responds to RCS by using the reversible oxidation of highly conserved cysteines to alter its DNA-binding affinity. However, earlier work showed that RCS response in NemR does not depend on any commonly known oxidative cysteine modifications. We have now determined the crystal structure of NemR, showing that the regulatory cysteine, Cys106, is in close proximity to a highly conserved lysine (Lys175). We used crystallographic, biochemical, and mass spectrometric analyses to analyze the role of this lysine residue in RCS sensing. Based on our results, we hypothesize that RCS treatment of NemR results in the formation of a reversible Cys106-Lys175 sulfenamide bond. This is, to our knowledge, the first description of a protein whose function is regulated by a cysteine?lysine sulfenamide thiol switch, constituting a novel addition to the biological repertoire of functional redox switches. Antioxid. Redox Signal. 23, 747?754.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140309/1/ars.2015.6346.pd

The mitochondrial oxidoreductase CHCHD4 is present in a semi-oxidized state in vivo.

Disulfide formation in the mitochondrial intermembrane space is an essential process catalyzed by a disulfide relay machinery. In mammalian cells, the key enzyme in this machinery is the oxidoreductase CHCHD4/Mia40. Here, we determined the in vivo CHCHD4 redox state, which is the major determinant of its cellular activity. We found that under basal conditions, endogenous CHCHD4 redox state in cultured cells and mouse tissues was predominantly oxidized, however, degrees of oxidation in different tissues varied from 70% to 90% oxidized. To test whether differences in the ratio between CHCHD4 and ALR might explain tissue-specific differences in the CHCHD4 redox state, we determined the molar ratio of both proteins in different mouse tissues. Surprisingly, ALR is superstoichiometric over CHCHD4 in most tissues. However, the levels of CHCHD4 and the ratio of ALR over CHCHD4 appear to correlate only weakly with the redox state, and although ALR is present in superstoichiometric amounts, it does not lead to fully oxidized CHCHD4

An increase in surface hydrophobicity mediates chaperone activity in N-chlorinated RidA

Under physiological conditions, Escherichia coli RidA is an enamine/imine deaminase, which promotes the release of ammonia from reactive enamine/imine intermediates. However, when modified by hypochlorous acid (HOCl), it turns into a potent chaperone-like holdase that can effectively protect E. coli\u27s proteome during oxidative stress. However, it is unknown, which residues need to be chlorinated for activation. Here, we employ a combination of LC-MS/MS analysis, a chemo-proteomic approach, and a mutagenesis study to identify residues responsible for RidA\u27s chaperone-like function. Through LC-MS/MS of digested RidAHOCl, we obtained direct evidence of the chlorination of one arginine residue. To overcome the instability of the N-chloramine modification, we established a chemoproteomic approach using 5-(dimethylamino) naphthalene-1-sulfinic acid (DANSO$_{2}$H) as a probe to label N-chlorinated lysines. Using this probe, we were able to detect the N-chlorination of six additional lysine residues. Moreover, using a mutagenesis study to genetically probe the role of single arginine and lysine residues, we found that the removal of arginines R105 and/or R128 led to a substantial reduction of RidA$_{HOCl\u27s}$ chaperone activity. These results, together with structural analysis, confirm that the chaperone activity of RidA is concomitant with the loss of positive charges on the protein surface, leading to an increased overall protein hydrophobicity. Molecular modelling of RidAHOCl and the rational design of a RidA variant that shows chaperone activity even in the absence of HOCl further supports our hypothesis. Our data provide a molecular mechanism for HOCl-mediated chaperone activity found in RidA and a growing number of other HOCl-activated chaperones