Bioorthogonal Chemical Reporters for Analyzing Protein Sulfenylation in Cells

Protein sulfenylation (RSOH), the redox‐based modification of cysteine thiol side chains by hydrogen peroxide (H2O2), is an important mechanism in signal transduction. Likewise, dysregulated protein sulfenylation contributes to a range of human pathologies, including cancer. Efforts to elucidate the diverse roles of protein sulfenylation in physiology and disease have been hampered by the lack of techniques to probe these modifications in native environments. To address this problem, selective chemical reporters have been developed for the detection and identification of sulfenylated proteins directly in cells. In the approach described here, a cyclic β‐diketone warhead is functionalized with an azide or alkyne chemical handle. An orthogonally functionalized biotin or fluorescent reporter is then appended to the probe post‐homogenization via click chemistry for downstream analysis. These bi‐functional probes are exquisitely selective for protein sulfenyl modifications, non‐toxic, and do not perturb intracellular redox balance. These reagents have been utilized to investigate sulfenylation in vitro and to identify intracellular protein targets of H2O2 during cell signaling. These methods provide a facile way to detect protein sulfenic acids and to study the biological role of cysteine oxidation with regard to physiological and pathological events. Curr. Protoc. Chem. Biol. 4:101‐122 © 2012 by John Wiley & Sons, Inc.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/152599/1/cpch110219.pd

Chemical Dissection of an Essential Redox Switch in Yeast

SummarySaccharomyces cerevisiae responds to elevated levels of hydrogen peroxide in its environment via a redox relay system comprising the thiol peroxidase Gpx3 and transcription factor Yap1. In this signaling pathway, a central unresolved question is whether cysteine sulfenic acid modification of Gpx3 is required for Yap1 activation in cells. Here we report that cell-permeable chemical probes, which are selective for sulfenic acid, inhibit peroxide-dependent nuclear accumulation of Yap1, trap the Gpx3 sulfenic acid intermediate, and block formation of the Yap1-Gpx3 intermolecular disulfide directly in cells. In addition, we present electrostatic calculations that show cysteine oxidation is accompanied by significant changes in charge distribution, which might facilitate essential conformational rearrangements in Gpx3 during catalysis and intermolecular disulfide formation with Yap1

3‘-Phosphoadenosine-5‘-phosphosulfate Reductase in Complex with Thioredoxin: A Structural Snapshot in the Catalytic Cycle

The crystal structure of Escherichia coli 3‘-phosphoadenosine-5‘-phosphosulfate (PAPS) reductase in complex with E. coli thioredoxin 1 (Trx1) has been determined to 3.0 Å resolution. The two proteins are covalently linked via a mixed disulfide that forms during nucleophilic attack of Trx's N-terminal cysteine on the Sγ atom of the PAPS reductase S-sulfocysteine (E-Cys-Sγ-SO_3^-), a central intermediate in the catalytic cycle. For the first time in a crystal structure, residues 235−244 in the PAPS reductase C-terminus are observed, depicting an array of interprotein salt bridges between Trx and the strictly conserved glutathione-like sequence, Glu^(238)Cys^(239)Gly^(240)Leu^(241)His^(242). The structure also reveals a Trx-binding surface adjacent to the active site cleft and regions of PAPS reductase associated with conformational change. Interaction at this site strategically positions Trx to bind the S-sulfated C-terminus and addresses the mechanism for requisite structural rearrangement of this domain. An apparent sulfite-binding pocket at the protein−protein interface explicitly orients the S-sulfocysteine Sγ atom for nucleophilic attack in a subsequent step. Taken together, the structure of PAPS reductase in complex with Trx highlights the large structural rearrangement required to accomplish sulfonucleotide reduction and suggests a role for Trx in catalysis beyond the paradigm of disulfide reduction

Redox-Based Probes for Protein Tyrosine Phosphatases

No AbstractPeer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/83769/1/4423_ftp.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/83769/2/anie_201007871_sm_miscellaneous_information.pd

Empirical entropic contributions in computational docking: Evaluation in APS reductase complexes

The results from reiterated docking experiments may be used to evaluate an empirical vibrational entropy of binding in ligand–protein complexes. We have tested several methods for evaluating the vibrational contribution to binding of 22 nucleotide analogues to the enzyme APS reductase. These include two cluster size methods that measure the probability of finding a particular conformation, a method that estimates the extent of the local energetic well by looking at the scatter of conformations within clustered results, and an RMSD-based method that uses the overall scatter and clustering of all conformations. We have also directly characterized the local energy landscape by randomly sampling around docked conformations. The simple cluster size method shows the best performance, improving the identification of correct conformations in multiple docking experiments. © 2008 Wiley Periodicals, Inc. J Comput Chem, 2008Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/60220/1/20936_ftp.pd

3‘-Phosphoadenosine-5‘-phosphosulfate Reductase in Complex with Thioredoxin: A Structural Snapshot in the Catalytic Cycle

The crystal structure of Escherichia coli 3‘-phosphoadenosine-5‘-phosphosulfate (PAPS) reductase in complex with E. coli thioredoxin 1 (Trx1) has been determined to 3.0 Å resolution. The two proteins are covalently linked via a mixed disulfide that forms during nucleophilic attack of Trx's N-terminal cysteine on the Sγ atom of the PAPS reductase S-sulfocysteine (E-Cys-Sγ-SO_3^-), a central intermediate in the catalytic cycle. For the first time in a crystal structure, residues 235−244 in the PAPS reductase C-terminus are observed, depicting an array of interprotein salt bridges between Trx and the strictly conserved glutathione-like sequence, Glu^(238)Cys^(239)Gly^(240)Leu^(241)His^(242). The structure also reveals a Trx-binding surface adjacent to the active site cleft and regions of PAPS reductase associated with conformational change. Interaction at this site strategically positions Trx to bind the S-sulfated C-terminus and addresses the mechanism for requisite structural rearrangement of this domain. An apparent sulfite-binding pocket at the protein−protein interface explicitly orients the S-sulfocysteine Sγ atom for nucleophilic attack in a subsequent step. Taken together, the structure of PAPS reductase in complex with Trx highlights the large structural rearrangement required to accomplish sulfonucleotide reduction and suggests a role for Trx in catalysis beyond the paradigm of disulfide reduction

A randomised controlled trial to assess the effectiveness of a single session of nurse administered massage for short term relief of chronic non-malignant pain

Background: Massage is increasingly used to manage chronic pain but its benefit has not been clearly established. The aim of the study is to determine the effectiveness of a single session of nurse-administered massage for the short term relief of chronic non-malignant pain and anxiety. Methods: A randomised controlled trial design was used, in which the patients were assigned to a massage or control group. The massage group received a 15 minute manual massage and the control group a 15 minute visit to talk about their pain. Adult patients attending a pain relief unit with a diagnosis of chronic pain whose pain was described as moderate or severe were eligible for the study. An observer blind to the patients' treatment group carried out assessments immediately before (baseline), after treatment and 1, 2, 3 and 4 hours later. Pain was assessed using 100 mm visual analogue scale and the McGill Pain Questionnaire. Pain Relief was assessed using a five point verbal rating scale. Anxiety was assessed with the Spielberger short form State-Trait Anxiety Inventory. Results: 101 patients were randomised and evaluated, 50 in the massage and 51 in the control group. There were no statistically significant differences between the groups at baseline interview. Patients in the massage but not the control group had significantly less pain compared to baseline immediately after and one hour post treatment. 95% confidence interval for the difference in mean pain reduction at one hour post treatment between the massage and control groups is 5.47 mm to 24.70 mm. Patients in the massage but not the control group had a statistically significant reduction in anxiety compared to baseline immediately after and at 1 hour post treatment. Conclusion: Massage is effective in the short term for chronic pain of moderate to severe intensity

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

‘Trying to pin down jelly’ - exploring intuitive processes in quality assessment for meta-ethnography

Background: Studies that systematically search for and synthesise qualitative research are becoming more evident in health care, and they can make an important contribution to patient care. However, there is still no agreement as to whether, or how we should appraise studies for inclusion. We aimed to explore the intuitive processes that determined the ‘quality’ of qualitative research for inclusion in qualitative research syntheses. We were particularly interested to explore the way that knowledge was constructed. Methods: We used qualitative methods to explore the process of quality appraisal within a team of seven qualitative researchers funded to undertake a meta-ethnography of chronic non-malignant musculoskeletal pain. Team discussions took place monthly between October 2010 and June 2012 and were recorded and transcribed. Data was coded and organised using constant comparative method. The development of our conceptual analysis was both iterative and collaborative. The strength of this team approach to quality came from open and honest discussion, where team members felt free to agree, disagree, or change their position within the safety of the group. Results: We suggest two core facets of quality for inclusion in meta-ethnography - (1) Conceptual clarity; how clearly has the author articulated a concept that facilitates theoretical insight. (2) Interpretive rigour; fundamentally, can the interpretation ‘be trusted?’ Our findings showed that three important categories help the reader to judge interpretive rigour: (ii) What is the context of the interpretation? (ii) How inductive is the interpretation? (iii) Has the researcher challenged their interpretation? Conclusions: We highlight that methods alone do not determine the quality of research for inclusion into a meta-ethnography. The strength of a concept and its capacity to facilitate theoretical insight is integral to meta-ethnography, and arguably to the quality of research. However, we suggest that to be judged ‘good enough’ there also needs to be some assurance that qualitative findings are more than simply anecdotal. Although our conceptual model was developed specifically for meta-ethnography, it may be transferable to other research methodologies