Persulfide Reactivity in the Detection of Protein <i>S</i>‑Sulfhydration

Hydrogen sulfide (H₂S) has emerged as a new member of the gaseous transmitter family of signaling molecules and appears to play a regulatory role in the cardiovascular and nervous systems. Recent studies suggest that protein cysteine <i>S</i>-sulfhydration may function as a mechanism for transforming the H₂S signal into a biological response. However, selective detection of <i>S</i>-sulfhydryl modifications is challenging since the persulfide group (RSSH) exhibits reactivity akin to other sulfur species, especially thiols. A modification of the biotin switch technique, using <i>S</i>-methyl methanethiosulfonate (MMTS) as an alkylating reagent, was recently used to identify a large number of proteins that may undergo <i>S</i>-sulfhydration, but the underlying mechanism of chemical detection was not fully explored. To address this key issue, we have developed a protein persulfide model and analogue of MMTS, <i>S</i>-4-bromobenzyl methanethiosulfonate (BBMTS). Using these new reagents, we investigated the chemistry in the modified biotin switch method and examined the reactivity of protein persulfides toward different electrophile/nucleophile species. Together, our data affirm the nucleophilic properties of the persulfide sulfane sulfur and afford new insights into protein <i>S</i>-sulfhydryl chemistry, which may be exploited in future detection strategies

Light-Mediated Sulfenic Acid Generation from Photocaged Cysteine Sulfoxide

S-Sulfenylation is a post-translational modification with a crucial role in regulating protein function. However, its analysis has remained challenging due to the lack of facile sulfenic acid models. We report the first photocaged cysteine sulfenic acid with efficient photodeprotection and demonstrate its utility by generating sulfenic acid in a thiol peroxidase after illumination in vitro. These caged sulfoxides should be promising for site-specific incorporation of Cys sulfenic acid in living cells via genetic code expansion

Design, Synthesis and Evaluation of Fe-S Targeted Adenosine 5′-Phosphosulfate Reductase Inhibitors

<div><p>Adenosine 5′-phosphosulfate reductase (APR) is an iron-sulfur enzyme that is vital for survival of <i>Mycobacterium tuberculosis</i> during dormancy and is an attractive target for the treatment of latent tuberculosis (TB) infection. The 4Fe-4S cluster is coordinated to APR by sulfur atoms of four cysteine residues, is proximal to substrate, adenosine 5′-phopsphosulfate (APS), and is essential for catalytic activity. Herein, we present an approach for the development of a new class of APR inhibitors. As an initial step, we have employed an improved solid-phase chemistry method to prepare a series of <i>N</i>⁶-substituted adenosine analogues and their 5′-phosphates as well as adenosine 5′-phosphate diesters bearing different Fe and S binding groups, such as thiols or carboxylic and hydroxamic acid moieties. Evaluation of the resulting compounds indicates a clearly defined spacing requirement between the Fe-S targeting group and adenosine scaffold and that smaller Fe-S targeting groups are better tolerated. Molecular docking analysis suggests that the S atom of the most potent inhibitor may establish a favorable interaction with an S atom in the cluster. In summary, this study showcases an improved solid-phase method that expedites the preparation of adenosine and related 5′-phosphate derivatives and presents a unique Fe-S targeting strategy for the development of APR inhibitors.</p></div

Reactivity, Selectivity, and Stability in Sulfenic Acid Detection: A Comparative Study of Nucleophilic and Electrophilic Probes

The comparative reaction efficiencies of currently used nucleophilic and electrophilic probes toward cysteine sulfenic acid have been thoroughly evaluated in two different settings(i) a small molecule dipeptide based model and (ii) a recombinant protein model. We further evaluated the stability of corresponding thioether and sulfoxide adducts under reducing conditions which are commonly encountered during proteomic protocols and in cell analysis. Powered by the development of new cyclic and linear C-nucleophiles, the unsurpassed efficiency in the capture of sulfenic acid under competitive conditions is achieved and thus holds great promise as highly potent tools for activity-based sulfenome profiling

A Chemical Approach for the Detection of Protein Sulfinylation

Protein sulfinic acids are formed by the reaction of reactive oxygen species with protein thiols. Sulfinic acid formation has long been considered an irreversible state of oxidation and is associated with high cellular oxidative stress. Increasing evidence, however, indicates that cysteine is oxidized to sulfinic acid in cells to a greater extent, and is more controlled, than first thought. The discovery of sulfiredoxin has demonstrated that cysteine sulfinic acid can be reversed, pointing to a vast array of potential implications for redox biology. Identification of the site of protein sulfinylation is crucial in clarifying the physiological and pathological effects of post-translational modifications. Currently, the only methods for detection of sulfinic acids involve mass spectroscopy and the use of specific antibodies. However, these methodologies are not suitable for proteomic studies. Herein, we report the first probe for detection of protein sulfinylation, NO-Bio, which combines a C-nitroso warhead for rapid labeling of sulfinic acid with a biotin handle. Based on this new tool, we developed a selective two-step approach. In the first, a sulfhydryl-reactive compound is introduced to selectively block free cysteine residues. Thereafter, the sample is treated with NO-Bio to label sulfinic acids. This new technology represents a rapid, selective, and general technology for sulfinic acid detection in biological samples. As proof of our concept, we also evaluated protein sulfinylation levels in various human lung tumor tissue lysates. Our preliminary results suggest that cancer tissues generally have higher levels of sulfinylation in comparison to matched normal tissues. A new ability to monitor protein sulfinylation directly should greatly expand the impact of sulfinic acid as a post-translational modification

Diverse Redoxome Reactivity Profiles of Carbon Nucleophiles

Targeted covalent inhibitors have emerged as a powerful approach in the drug discovery pipeline. Key to this process is the identification of signaling pathways (or receptors) specific to (or overexpressed in) disease cells. In this context, fragment-based ligand discovery (FBLD) has significantly expanded our view of the ligandable proteome and affords tool compounds for biological inquiry. To date, such covalent ligand discovery has almost exclusively employed cysteine-reactive small-molecule fragments. However, functional cysteine residues in proteins are often redox-sensitive and can undergo oxidation in cells. Such reactions are particularly relevant in diseases, like cancer, which are linked to excessive production of reactive oxygen species. Once oxidized, the sulfur atom of cysteine is much less reactive toward electrophilic groups used in the traditional FBLD paradigm. To address this limitation, we recently developed a novel library of diverse carbon-based nucleophile fragments that react selectively with cysteine sulfenic acid formed in proteins via oxidation or hydrolysis reactions. Here, we report analysis of sulfenic acid-reactive C-nucleophile fragments screened against a colon cancer cell proteome. Covalent ligands were identified for >1280 <i>S</i>-sulfenylated cysteines present in “druggable” proteins and orphan targets, revealing disparate reactivity profiles and target preferences. Among the unique ligand–protein interactions identified was that of a pyrrolidinedione nucleophile that reacted preferentially with protein tyrosine phosphatases. Fragment-based covalent ligand discovery with C-nucleophiles affords an expansive snapshot of the ligandable “redoxome” with significant implications for covalent inhibitor pharmacology and also affords new chemical tools to investigate redox-regulation of protein function

Iron–Sulfur Cluster Engineering Provides Insight into the Evolution of Substrate Specificity among Sulfonucleotide Reductases

Assimilatory sulfate reduction supplies prototrophic organisms with reduced sulfur that is required for the biosynthesis of all sulfur-containing metabolites, including cysteine and methionine. The reduction of sulfate requires its activation <i>via</i> an ATP-dependent activation to form adenosine-5′-phosphosulfate (APS). Depending on the species, APS can be reduced directly to sulfite by APS reductase (APR) or undergo a second phosphorylation to yield 3′-phosphoadenosine-5′-phosphosulfate (PAPS), the substrate for PAPS reductase (PAPR). These essential enzymes have no human homologue, rendering them attractive targets for the development of novel antibacterial drugs. APR and PAPR share sequence and structure homology as well as a common catalytic mechanism, but the enzymes are distinguished by two features, namely, the amino acid sequence of the phosphate-binding loop (P-loop) and an iron–sulfur cofactor in APRs. On the basis of the crystal structures of APR and PAPR, two P-loop residues are proposed to determine substrate specificity; however, this hypothesis has not been tested. In contrast to this prevailing view, we report here that the P-loop motif has a modest effect on substrate discrimination. Instead, by means of metalloprotein engineering, spectroscopic, and kinetic analyses, we demonstrate that the iron–sulfur cluster cofactor enhances APS reduction by nearly 1000-fold, thereby playing a pivotal role in substrate specificity and catalysis. These findings offer new insights into the evolution of this enzyme family and extend the known functions of protein-bound iron–sulfur clusters

Reengineering Redox Sensitive GFP to Measure Mycothiol Redox Potential of <i>Mycobacterium tuberculosis</i> during Infection

<div><p><i>Mycobacterium tuberculosis (Mtb)</i> survives under oxidatively hostile environments encountered inside host phagocytes. To protect itself from oxidative stress, <i>Mtb</i> produces millimolar concentrations of mycothiol (MSH), which functions as a major cytoplasmic redox buffer. Here, we introduce a novel system for real-time imaging of mycothiol redox potential (<i>E_MSH</i>) within <i>Mtb</i> cells during infection. We demonstrate that coupling of <i>Mtb</i> MSH-dependent oxidoreductase (mycoredoxin-1; Mrx1) to redox-sensitive GFP (roGFP2; Mrx1-roGFP2) allowed measurement of dynamic changes in intramycobacterial <i>E_MSH</i> with unprecedented sensitivity and specificity. Using Mrx1-roGFP2, we report the first quantitative measurements of <i>E_MSH</i> in diverse mycobacterial species, genetic mutants, and drug-resistant patient isolates. These cellular studies reveal, for the first time, that the environment inside macrophages and sub-vacuolar compartments induces heterogeneity in <i>E_MSH</i> of the <i>Mtb</i> population. Further application of this new biosensor demonstrates that treatment of <i>Mtb</i> infected macrophage with anti-tuberculosis (TB) drugs induces oxidative shift in <i>E_MSH</i>, suggesting that the intramacrophage milieu and antibiotics cooperatively disrupt the MSH homeostasis to exert efficient <i>Mtb</i> killing. Lastly, we analyze the membrane integrity of <i>Mtb</i> cells with varied <i>E_MSH</i> during infection and show that subpopulation with higher <i>E_MSH</i> are susceptible to clinically relevant antibiotics, whereas lower <i>E_MSH</i> promotes antibiotic tolerance. Together, these data suggest the importance of MSH redox signaling in modulating mycobacterial survival following treatment with anti-TB drugs. We anticipate that Mrx1-roGFP2 will be a major contributor to our understanding of redox biology of <i>Mtb</i> and will lead to novel strategies to target redox metabolism for controlling <i>Mtb</i> persistence.</p></div

Heterogeneity in intrabacterial <i>E_MSH</i> modulates drug tolerance.

<p>(A) THP-1 cells infected with <i>Mtb</i> H37Rv were treated with INH (0.5 µg/ml), CFZ (0.5 µg/ml), RIF (0.5 µg/ml) and ETH (5 µg/ml). At indicated time points, intracellular bacteria were fixed with NEM, released from macrophages, and stained with Pi. Pi status and <i>E_MSH</i> of bacterial cells were determined using multi-parameter flow cytometric analysis. Pie charts display the percentage of Pi+ve and Pi-ve cells in each subpopulation. (B) THP-1 cells infected with <i>Mtb</i> H37Rv were treated with INH (0.5 µg/ml), CFZ (0.5 µg/ml), with or without rapamycin (200 nM) immediately after infection. At 24 h p.i. redox heterogeneity within <i>Mtb</i> cells was analyzed by flow cytometry. (C) In a parallel experiment, infected macrophages were lysed and released bacilli were stained with Pi. (D) Exponentially grown culture of <i>Mtb</i> H37Rv was treated with INH (0.5 µg/ml), CFZ (0.5 µg/ml), RIF (0.5 µg/ml) and ETH (5 µg/ml) in the presence or absence of 5 mM DTT (added at 0 and 2^nd day, post-antibiotic treatment) and number of bacilli were counted by plating for CFU. Error bars represent standard deviations from the mean. C: represents untreated control in each panel. Error bars represent standard deviations from the mean. * p<0.05, ** p<0.001, ns: not significant. Data are representative of at least three independent experiments.</p

Sub-vacuolar compartments are the source of redox heterogeneity within <i>Mtb</i> population during infection.

<p>THP-1 cells were infected with H37Rv expressing Mrx1-roGFP2 (moi: 10). At 24 h p.i. infected cells were treated by NEM-PFA. Cells were then stained for EEA1 and LC3 and analyzed by confocal microscopy for measuring ratiometric sensor response in <i>Mtb</i> co-localized within early endosomes and autophagosomes, respectively. In case of lysosomes, the cells were first pre-treated with Lysotracker followed by NEM-PFA fixation. (A) False color ratio confocal image of <i>Mtb</i> (∼80) inside THP-1 at 24 h p.i. <i>E_MSH</i> of bacilli was measured using <i>in vitro</i> calibration curve (SI <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003902#ppat.1003902.s005" target="_blank">Figure S5A</a>). (B) Representative bacilli for each subpopulation are shown. Co-localization of <i>Mtb</i> H37Rv in (C) endosomes, (D) lysosomes, and (E) autophagosomes. The co-localization is demonstrated in the merged images, where green indicates bacteria, red indicates phagosomal markers, and yellow indicates a positive correlation. False color ratio images were generated as described in SI <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003902#s4" target="_blank"><i>Materials and Methods</i></a>. Small dashed line boxes indicate co-localized bacilli and large solid line boxes represent the enlarged view of the one of the co-localized bacilli. Numbers represent <i>E_MSH</i> in millivolts. <i>E_MSH</i> of co-localized bacilli (≥50) is calculated and distribution is shown in scatter plot. In each panel, scatter plot depicts quantification of microscopy data. Each point on the plot represents a bacterium. Bar represents mean values. p-values were calculated by one way ANOVA followed by Tukey's HSD statistical test (* p<0.01). Percentage of bacilli in each subpopulation is represented as a stacked bar graph in every panel. Color bar corresponds to the 405/488 nm ratios ranging from 0 to 1. Data shown is the representative of at least three independent experiments.</p