Modification Of Cysteine Residues Research Articles

Efficient and irreversible antibody-cysteine bioconjugation using carbonylacrylic reagents.

There is considerable interest in the development of chemical methods for the precise, site-selective modification of antibodies for therapeutic applications. In this protocol, we describe a strategy for the irreversible and selective modification of cysteine residues on antibodies, using functionalized carbonylacrylic reagents. This protocol is based on a thiol-Michael-type addition of native or engineered cysteine residues to carbonylacrylic reagents equipped with functional compounds such as cytotoxic drugs. This approach is a robust alternative to the conventional maleimide technique; the reaction is irreversible and uses synthetically accessible reagents. Complete conversion to the conjugates, with improved quality and homogeneity, is often achieved using a minimal excess (typically between 5 and 10 equiv.) of the carbonylacrylic reagent. Potential applications of this method cover a broad scope of cysteine-tagged antibodies in various formats (full-length IgGs, nanobodies) for the site-selective incorporation of cytotoxic drugs without loss of antigen-binding affinity. Both the synthesis of the carbonylacrylic reagent armed with a synthetic molecule of interest and the subsequent preparation of the chemically defined, homogeneous antibody conjugate can be achieved within 48 h and can be easily performed by nonspecialists. Importantly, the conjugates formed are stable in human plasma. The use of liquid chromatography-mass spectrometry (LC-MS) analysis is recommended for monitoring the progression of the bioconjugation reactions on protein and antibody substrates with accurate resolution.

Bioorthogonal Profiling of a Cancer Cell Proteome Identifies a Large Set of 3-Bromopyruvate Targets beyond Glycolysis.

3-Bromopyruvate (3BP) is a potential anticancer agent viewed as a glycolytic inhibitor that preferentially kills cancer cells through inhibition of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), resulting in severe energy depletion. We previously identified four cysteine residues in GAPDH that are alkylated by 3BP, resulting in its inactivation. However, we also showed that addition of excess pyruvate, the final product of glycolysis, was unable to rescue cells from 3BP treatment. This result indicates that GAPDH may not be the only relevant target and is consistent with the chemical reactivity of 3BP that should result in the modification of cysteine residues in many different proteins. To directly test this hypothesis, we first synthesized a probe of 3BP activity bearing an alkyne functionality, termed AO3BP, and then demonstrated that this probe could modify a variety of proteins in living cells. Subsequent competition of AO3BP labeling with pretreatment by 3BP identified 62 statistically significant proteins of various functions as targets of 3BP, confirming that 3BP labeling is indeed widespread. We conclude that 3BP's cytotoxic impact on cancer cells does not only result from selective inhibition of glycolysis but rather from a more widespread effect on cellular proteins that could be driven by the pharmacokinetics of the 3BP. These pleiotropic consequences should be considered when thinking about the potential toxicity of this highly reactive compound.

104 - Interactions of hydrogen sulfide with AhpE from Mycobacterium tuberculosis: formation and reactions of a model peroxiredoxin persulfide

Hydrogen sulfide (H2S) has been proposed to be a signaling agent. However, its mechanisms of action are usually unknown. H2S can modify cysteine residues to persulfides (RSSH), poorly characterized species proposed to mediate the signaling effects of H2S. The successful intracellular pathogen Mycobacterium tuberculosis (Mt), the causative agent of tuberculosis disease, is able to produce H2S and to import it from the host, although the effects of H2S on Mt physiology and virulence are still unknown. In this work, we utilized the one-cysteine peroxiredoxin alkyl hydroperoxide reductase E from Mt (MtAhpE-S-) as a model to study the formation and reactions of protein persulfides. The peroxidatic cysteine in MtAhpE is oxidized by different peroxides to a stable sulfenic acid (MtAhpE-SOH), which reacts with H2S to form a persulfide derivative (MtAhpE-SS-). The formation of MtAhpE-SS- was evidenced by treatment with iodoacetamide and detection of the alkyl derivative (MtAhpE-SS-R) by mass spectrometry. The kinetics of persulfide formation reaction was determined (103 M-1s-1 at pH 7.4, 25oC) by two independent competition assays. In addition, MtAhpE catalyzed the reduction of H2O2 by H2S indicating that H2S was able to complete the catalytic cycle of MtAhpE. However, H2S is not expected to be the preferred reducing substrate in vivo, since it would not compete with mycothiol/mycoredoxin-1. Reactivity of MtAhpE-SS- was compared with that of MtAhpE-S-: MtAhpE-SS- reacted faster in non-specific nucleophilic reactions (such as with the disulfide 4,4’-dithiodipyridine) but lost the specific fast reactivity with hydroperoxides (H2O2 and peroxynitrite). Computational simulations indicated that persulfidation of peroxidatic cysteine leads to a disruption of the hydrogen bond network at the active site, which is required for fast hydroperoxide reduction. Overall, the data indicate that persulfuration is a posttranslational modification that can alter the reactivities of cysteine residues in different directions depending on their specific roles and protein microenvironments.

Tetrahydrobiopterin modulates ubiquitin conjugation to UBC13/UBE2N and proteasome activity by S-nitrosation

Nitric Oxide (NO) is an intracellular signalling mediator, which affects many biological processes via the posttranslational modification of proteins through S-nitrosation. The availability of NO and NOS-derived reactive oxygen species (ROS) from enzymatic uncoupling are determined by the NO synthase cofactor Tetrahydrobiopterin (BH4). Here, using a global proteomics “biotin-switch” approach, we identified components of the ubiquitin-proteasome system to be altered via BH4-dependent NO signalling by protein S-nitrosation. We show S-nitrosation of ubiquitin conjugating E2 enzymes, in particular the catalytic residue C87 of UBC13/UBE2N, leading to impaired polyubiquitylation by interfering with the formation of UBC13~Ub thioester intermediates. In addition, proteasome cleavage activity in cells also seems to be altered by S-nitrosation, correlating with the modification of cysteine residues within the 19S regulatory particle and catalytic subunits of the 20S complex. Our results highlight the widespread impact of BH4 on downstream cellular signalling as evidenced by the effect of a perturbed BH4-dependent NO-Redox balance on critical processes within the ubiquitin-proteasome system (UPS). These studies thereby uncover a novel aspect of NO associated modulation of cellular homeostasis.

Retraction: Modification of Cysteine Residue in p65 Subunit of Nuclear Factor-κB (NF-κB) by Picroliv Suppresses NF-κB–Regulated Gene Products and Potentiates Apoptosis

Retraction: Modification of Cysteine Residue in p65 Subunit of Nuclear Factor-κB (NF-κB) by Picroliv Suppresses NF-κB–Regulated Gene Products and Potentiates Apoptosis

Speciation of reactive sulfur species and their reactions with alkylating agents: do we have any clue about what is present inside the cell?

Buffered aqueous solutions of inorganic, organic, cysteine, GSH and GAPDH polysulfide species were used. Additional experiments in human plasma and serum revealed that monobromobimane can extract sulfide from the endogenous sulfur pool by shifting speciation equilibria, suggesting caution should be exercised when interpreting experimental results using this tool. In the majority of cases, the speciation of alkylated polysulfide/thiol derivatives depended on the experimental conditions. Alkylation perturbed sulfur speciation in both a concentration- and time-dependent manner and strong alkylating agents cleaved polysulfur chains. Moreover, the labelling of sulfenic acids with dimedone also affected cysteine speciation, suggesting that part of the endogenous pool of products previously believed to represent sulfenic acid species may represent polysulfides. We highlight methodological caveats potentially arising from these pitfalls and conclude that current derivatization strategies often fail to adequately capture physiological speciation of sulfur species. This article is part of a themed section on Chemical Biology of Reactive Sulfur Species. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.4/issuetoc.

Regulation of antigen 85C activity by reversible S-glutathionylation.

Mycobacterium tuberculosis is the causative agent of many strains of tuberculosis, as it is composed of an impenetrable, complex cell wall. The proteins active in the synthesis of the cell wall are mycolyl transferase antigens 85A, 85B, and 85C, encoded by genes fbpA, fbpB, and fbpC. Antigen 85C contains one cysteine residue. S-Glutathionylation is the formation of a mixed disulfide between a protein cysteine residue and glutathione (GSH), an abundant antioxidant molecule. It is a post-translational modification of cysteine residues which can occur under oxidative stress or physiological conditions. It is a known mechanism to regulate enzyme activity, signaling pathways, and the progression of diseases. By S-glutathionylation, the lone cysteine residue in antigen 85C is modified by biotinylated GSH ethyl ester to form a mixed disulfide. This modification results in a decrease in enzyme activity by 90%, representing a decrease in ability of the protein to synthesize the bacterial cell wall. Both the modification and the enzymatic activity of the protein are concentration dependent and can be reversed upon addition of a thiol reducing agent. The results provide a potential strategy for inhibiting the synthesis of the cell wall of M. tuberculosis by promoting oxidation of the lone cysteine residue. To our knowledge, this is a novel finding to demonstrate the modification of antigen 85C and the regulation of its activity by a physiological molecule. © 2018 IUBMB Life, 70(11):1111-1114, 2018.

Stimulation of the ATPase activity of Hsp90 by zerumbone modification of its cysteine residues destabilizes its clients and causes cytotoxicity.

Hsp90 is an ATP-dependent molecular chaperone that assists folding and conformational maturation/maintenance of many proteins. It is a potential cancer drug target because it chaperones oncoproteins. A prokaryotic homolog of Hsp90 (HtpG) is essential for thermo-tolerance in some bacteria and virulence of zoonotic pathogens. To identify a new class of small molecules which target prokaryotic and eukaryotic Hsp90s, we studied the effects of a naturally occurring cyclic sesquiterpene, zerumbone, which inhibits proliferation of a wide variety of tumor cells, on the activity of Hsp90. Zerumbone enhanced the ATPase activity of cyanobacterial Hsp90 (Hsp90SE), yeast Hsp90, and human Hsp90α. It also enhanced the catalytic efficiency of Hsp90SE by greatly increasing kcat Mass analysis showed that zerumbone binds to cysteine side chains of Hsp90SE covalently. Mutational studies identified 3 cysteine residues (one per each domain of Hsp90SE) that are involved in the enhancement, suggesting the presence of allosteric sites in the middle and C-terminal domains of Hsp90SE Treatment of cyanobacterial cells with zerumbone caused them to become very temperature-sensitive, a phenotype reminiscent of cyanobacterial Hsp90 mutants, and also decreased the cellular level of linker polypeptides that are clients for Hsp90SE Zerumbone showed cellular toxicity on cancer-derived mammalian cells by inducing apoptosis. In addition, zerumbone inhibited the binding of Hsp90/Cdc37 to client kinases. Altogether, we conclude that modification of cysteine residues of Hsp90 by zerumbone enhances its ATPase activity and inhibits physiological Hsp90 function. The activation of Hsp90 may provide new strategies to inhibit its chaperone function in cells.

Biolabeling with cobaltocinium tags

Abstract A label for amino and thiol functionalities of peptides and proteins based on the activated cobaltocinium hexafluorophosphate succinimide ester (CoS) is presented. Despite the known selectivity of a succinimide ester towards amines, CoS also modifies cysteine residues under the same reaction conditions. The derivatized biomolecules were investigated using liquid chromatography with subsequent electrospray-mass spectrometric detection (LC/ESI-MS). In combination with their remarkable stability under physiological conditions, easy handling and good spectroscopic properties, cobaltocinium ions provide all requirements for a powerful labeling reagent. Furthermore, in direct comparison to the isoelectronic well-established ferrocene reagents, the higher redox potential and the chemical stability of the cobaltocinium moiety add to the benefits as a derivatizing agent for bioanalysis.

SVM-SulfoSite: A support vector machine based predictor for sulfenylation sites

Protein S-sulfenylation, which results from oxidation of free thiols on cysteine residues, has recently emerged as an important post-translational modification that regulates the structure and function of proteins involved in a variety of physiological and pathological processes. By altering the size and physiochemical properties of modified cysteine residues, sulfenylation can impact the cellular function of proteins in several different ways. Thus, the ability to rapidly and accurately identify putative sulfenylation sites in proteins will provide important insights into redox-dependent regulation of protein function in a variety of cellular contexts. Though bottom-up proteomic approaches, such as tandem mass spectrometry (MS/MS), provide a wealth of information about global changes in the sulfenylation state of proteins, MS/MS-based experiments are often labor-intensive, costly and technically challenging. Therefore, to complement existing proteomic approaches, researchers have developed a series of computational tools to identify putative sulfenylation sites on proteins. However, existing methods often suffer from low accuracy, specificity, and/or sensitivity. In this study, we developed SVM-SulfoSite, a novel sulfenylation prediction tool that uses support vector machines (SVM) to identify key determinants of sulfenylation among five feature classes: binary code, physiochemical properties, k-space amino acid pairs, amino acid composition and high-quality physiochemical indices. Using 10-fold cross-validation, SVM-SulfoSite achieved 95% sensitivity and 83% specificity, with an overall accuracy of 89% and Matthew’s correlation coefficient (MCC) of 0.79. Likewise, using an independent test set of experimentally identified sulfenylation sites, our method achieved scores of 74%, 62%, 80% and 0.42 for accuracy, sensitivity, specificity and MCC, with an area under the receiver operator characteristic (ROC) curve of 0.81. Moreover, in side-by-side comparisons, SVM-SulfoSite performed as well as or better than existing sulfenylation prediction tools. Together, these results suggest that our method represents a robust and complementary technique for advanced exploration of protein S-sulfenylation.

Indirubin, a bis-indole alkaloid binds to tubulin and exhibits antimitotic activity against HeLa cells in synergism with vinblastine.

Indirubin, a bis-indole alkaloid used in traditional Chinese medicine has shown remarkable anticancer activity against chronic myelocytic leukemia. The present work was aimed to decipher the underlying molecular mechanisms responsible for its anticancer attributes. Our findings suggest that indirubin inhibited the proliferation of HeLa cells with an IC50 of 40 μM and induced a mitotic block. At concentrations higher than its IC50, indirubin exerted a moderate depolymerizing effect on the interphase microtubular network and spindle microtubules in HeLa cells. Studies with goat brain tubulin indicated that indirubin bound to tubulin at a single site with a dissociation constant of 26 ± 3 μM and inhibited the in vitro polymerization of tubulin into microtubules in the presence of glutamate as well as microtubule-associated proteins. Molecular docking analysis and molecular dynamics simulation studies indicate that indirubin stably binds to tubulin at the interface of the α-β tubulin heterodimer. Further, indirubin stabilized the binding of colchicine on tubulin and promoted the cysteine residue modification by 5,5'-dithiobis-2-nitrobenzoic acid, indicating towards alteration of tubulin conformation upon binding. In addition, we found that indirubin synergistically enhanced the anti-mitotic and anti-proliferative activity of vinblastine, a known microtubule-targeted agent. Collectively our studies indicate that perturbation of microtubule polymerization dynamics could be one of the possible mechanisms behind the anti-cancer activities of indirubin.

P-130 - The conserved cysteine residue of type III intermediate filaments serves as a structural element and redox sensor

Glial fibrillary acidic protein (GFAP) along with vimentin, desmin and peripherin constitutes the type III class of intermediate filament proteins, which are expressed in a cell type-dependent manner. GFAP and vimentin play a role in organelle positioning, cell migration and mechanical resistance and possess a conserved cysteine residue (cys328 in vimentin and cys294 in GFAP). We previously showed that cys328 of vimentin is critical for stress sensing, since mutants lacking this residue are resistant to vimentin reorganization induced by oxidants and electrophilic lipids. We have observed that GFAP cys294 is also a target for lipoxidation, which induces a drastic filament rearrangement in cells. Therefore, the conserved cysteine residues of type III intermediate filaments emerge as important players in cellular responses to oxidative damage. Interestingly, cysteine to serine mutations compromise assembly and copolymerization of GFAP and vimentin, showing that these residues are important for the structure of the intermediate filament network. This raises the hypothesis that modifications of cysteine residues of intermediate filaments may contribute to cellular dysfunction under pathophysiological conditions.

Chemical‐proteomic strategies to investigate reactive cysteines

Cysteine residues regulate the activity of diverse proteins via post translational modification by endogenous electrophiles and reactive oxygen species. These modified cysteine residues do not conform to a conserved sequence or structural motif, rendering the global identification of regulatory cysteines a considerable challenge. To identify regulatory cysteines in human proteomes, we have developed chemical‐proteomic tools to monitor cysteine reactivity both in vitro as well as directly in living cells. These global profiling methods have been applied to monitor the susceptibility of cysteines to S‐nitrosation and to identify redox‐active disulfides induced by epidermal‐growth factor (EGF) stimulation. In order to identify cysteine residues hypersensitive to S‐nitrosation, we developed a chemical‐proteomic platform that ranks cysteines in the human proteome by sensitivity to a variety of transnitrosating agents. Our proteomic studies identified several previously unannotated cysteines, which we proceeded to demonstrate were important regulators of protein activity. To identify cysteine oxidation events triggered by EGF stimulation, we developed a caged cysteine‐reactive probe to enable profiling of cysteine reactivity directly in living cells. We identified several redox‐active disulfides that were formed upon EGF stimulation for future functional characterization. Overall, our studies aim to unearth novel functional cysteine residues in the proteome, with the long‐term goal of identifying new modes of protein regulation through reactive cysteines.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of Protein Dynamics in Selectivity of Thiadiazolidinone Inhibition of RGS Proteins

BackgroundRegulator of G‐protein Signaling (RGS) proteins play a critical role in GPCR (G‐protein Coupled Receptor) signaling by binding to active, GTP‐bound Gα subunits and accelerating GTP hydrolysis, thus terminating signaling. There are multiple RGS isoforms with signature tissue distributions, so by targeting RGS proteins in an isoform‐specific manner, better tissue specificity of pharmacological effects may be achieved than by global application of a GPCR agonist. Thiadiazolidinones (TDZDs) are a series of compounds that inhibit RGS proteins by covalent modification of cysteine residues. They are very potent against RGS4, but also act less potently on several other cysteine‐containing RGS proteins. RGS4, RGS8, and RGS19, three proteins that are inhibited by TDZDs, share a cysteine on the α4 helix of the RGS homology domain. RGS19 has only this cysteine, while RGS4 and RGS8 have additional cysteines, including a shared one on the α6‐α7 interhelical loop. Interestingly, these shared cysteines are not exposed to solvent in crystal structures.HypothesisDifferences in potency due to covalent modification of the shared cysteine at the α4 helix of RGS proteins is driven by differences in flexibility.ApproachThe rate of covalent modification at individual cysteines can be determined by treating proteins with iodoacetamide, digestion, and detection of adduct by mass spectrometry. In addition, the effect of modification at different cysteines on protein function can be evaluated by measuring TDZD inhibition of binding between Gα and cysteine mutants of RGS proteins.ResultsDeuterium exchange and molecular dynamics studies have shown differential flexibility in key regions of these RGS proteins, which may be necessary for TDZD access to buried cysteines. RGS4 is more potently inhibited by CCG‐50014 (IC50: 81 nM) than RSG19 (IC50: 1.1 μM) or RGS8 (IC50: 1.8 μM). However, when RGS4 or RGS8 are mutated to contain only the α4 helical cysteine, the potency of TDZD inhibition is drastically reduced to below that of RGS19 (RGS4 IC50: 8.5 μM, RGS8 IC50 >100 μM). In accordance with previous molecular simulation data showing this α4 cysteine to be least solvent accessible in RGS4, it had a lower degree of covalent adduct formation by iodoacetamide than other cysteines.ConclusionThese results indicate that while additional cysteines in RGS4 may be responsible for its high potency of inhibition by TDZDs, differences in the rate of covalent modification and subsequent potency of inhibition at the conserved α4 cysteine is driven by differences in RGS protein flexibility.Support or Funding InformationNIH T32 GM092715NSF 1507588This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hydrogen Sulfide inhibits store‐operated calcium influx by selectively targeting STIM1‐Orai3 interactions.

Hydrogen sulfide (H2S) is an endogenous signaling molecule that posttranslationally modifies cysteine residues on target proteins involved in regulating the physiology and pathophysiology of neurodegeneration, blood pressure, metabolism, and inflammation. We recently determined that H2S impinges on Ca2+ signaling‐regulated processes by inhibiting store operated Ca2+ entry (SOCE). The SOCE machinery consists of the plasma membrane‐localized Orai channels and ER‐localized STIM proteins. Interestingly, H2S inhibits SOCE mediated by STIM1‐Orai3 and not STIM1‐Orai1 or Orai2 interactions. The current study tested the hypothesis that H2S achieves selectively and disrupts STIM1‐Orai3 interactions by targeting cysteines unique to Orai3. The approach was to transiently transfect either CFP‐ or mCherry‐tagged Orai3 into HEK293 cells stably expressing STIM1‐YFP and measure SOCE by Ca2+ imaging. Two cysteines (C226 and C232) are present in Orai3 that are absent in the Orai1 and Orai2 sequences. Mutation of either of these cysteines to serine, alone or in combination, abolished SOCE inhibition by the H2S donor GYY4137. Residues C226 and C232 in Orai3 are predicted to form an extracellular intermolecular disulfide. To determine the requirement for disulfide formation, cells were pretreated with DTT. When performed prior to GYY4137 treatment, disulfide reduction also abolished inhibition of SOCE by GYY4137. SOCE is activated upon ER Ca2+ store depletion when STIM interacts with and gates open Orai channels. We postulated that H2S inhibited SOCE by inhibiting STIM1‐Orai3 interactions. To test this, cells were treated with GYY4137 both before and after store depletion. GYY4137 treatment decreased SOCE only when cells were pretreated with GYY4137. These data suggest that H2S modification of Orai3 inhibits the initiation of STIM1‐Orai3 interactions but cannot disrupt them once formed. We next visualized the interaction between STIM1‐YFP and Orai3‐mCherry using confocal microscopy and measured the rate of YFP‐mCherry puncta formation upon store depletion. Pre‐treatment with GYY4137 caused a decrease in puncta formation rate. Taken together, these data are consistent with a model in which H2S impairs SOCE by modifying a disulfide bridge on Orai3 resulting in decreased STIM1‐Orai3 interactions.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Are heme proteins major targets for homocysteine‐induced modifications?

The prime cause of homocysteine (Hcy) toxicity towards proteins has been attributed to the modification of protein by homocysteine thiolactone (HTL). But the effects of Hcy (the parent molecule) on protein structure and function are still scarce. Hcy also has the ability to modify proteins, which could in turn affect the functionality and proper folding of the modified protein. Hcy is a highly pro‐oxidizing agent, thus Hcy‐induced oxidative stress has been commonly used to explain the toxicity of Hcy on endothelial cells and other tissues. Moreover, Hcy is known to modify cysteine residues in proteins in a process termed as S‐homocysteinylation. However, the structural and functional consequences of such modification are not yet clearly understood. The present study aims at investigating possible effects of Hcy on proteins structure and function. Using RNase‐A, Lyz and horseradish peroxidise (HRP) as models, we studied the effects of Hcy on the enzyme function and conformational status of these proteins. Enzyme activity measurements after overnight incubation with Hcy led to gradual decrease in enzyme function of HRP in a concentration dependent manner but not in Lyz and RNase‐A. Conformational analyses revealed no gross structural alterations in all proteins studied. However, certain alteration in the heme‐Trp distance was observed in case of HRP as evident from Trp fluorescence measurements. The loss of function in HRP was due to disruption of heme redox state in the presence of Hcy. Similar findings were obtained for three other common heme metalloprotein (Cytochrome c, Cyt c; Hemoglobin, Hb; and Myoglobin, Myo), wherein Hcy induced certain alterations in the heme environment rendering it reduced with further disruption of the heme‐Trp distance. In addition, Hcy was found to alter the heme redox states without direct interaction with the proteins (in case of Cyt c and Myo). The findings suggest that heme and heme containing proteins could be major targets for Hcy‐induced modifications and involved in Hcy‐induced cytotoxicity.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Differential Protein Dynamics of Regulators of G-Protein Signaling: Role in Specificity of Small-Molecule Inhibitors

Small-molecule inhibitor selectivity may be influenced by variation in dynamics among members of a protein family. Regulator of G-protein Signaling (RGS) proteins are a family that plays a key role in G-Protein Coupled Receptor (GPCR) signaling by binding to active Gα subunits and accelerating GTP hydrolysis, thereby terminating activity. Thiadiazolidinones (TDZDs) inhibit the RGS-Gα interaction by covalent modification of cysteine residues in RGS proteins. Some differences in specificity may be explained by differences in the complement of cysteines among RGS proteins. However, key cysteines shared by RGS proteins inhibited by TDZDs are not exposed on the protein surface, and differences in potency exist among RGS proteins containing only buried cysteines. We hypothesize that differential exposure of buried cysteine residues among RGS proteins partially drives TDZD selectivity. Hydrogen-deuterium exchange (HDX) studies and molecular dynamics (MD) simulations were used to probe the dynamics of RGS4, RGS8, and RGS19, three RGS proteins inhibited at a range of potencies by TDZDs. When these proteins were mutated to contain a single, shared cysteine, RGS19 was found to be most potently inhibited. HDX studies revealed differences in α4 and α6 helix flexibility among RGS isoforms, with particularly high flexibility in RGS19. This could cause differences in cysteine exposure and lead to differences in potency of TDZD inhibition. MD simulations of RGS proteins revealed motions that correspond to solvent exposure observed in HDX, providing further evidence for a role of protein dynamics in TDZD selectivity.

Active and dynamic mitochondrial S-depalmitoylation revealed by targeted fluorescent probes

The reversible modification of cysteine residues by thioester formation with palmitate (S-palmitoylation) is an abundant lipid post-translational modification (PTM) in mammalian systems. S-palmitoylation has been observed on mitochondrial proteins, providing an intriguing potential connection between metabolic lipids and mitochondrial regulation. However, it is unknown whether and/or how mitochondrial S-palmitoylation is regulated. Here we report the development of mitoDPPs, targeted fluorescent probes that measure the activity levels of “erasers” of S-palmitoylation, acyl-protein thioesterases (APTs), within mitochondria of live cells. Using mitoDPPs, we discover active S-depalmitoylation in mitochondria, in part mediated by APT1, an S-depalmitoylase previously thought to reside in the cytosol and on the Golgi apparatus. We also find that perturbation of long-chain acyl-CoA cytoplasm and mitochondrial regulatory proteins, respectively, results in selective responses from cytosolic and mitochondrial S-depalmitoylases. Altogether, this work reveals that mitochondrial S-palmitoylation is actively regulated by “eraser” enzymes that respond to alterations in mitochondrial lipid homeostasis.

Abstract 21314: Glutaredoxin-1: An Endothelial Protector in Hyperlipidemia

Background: Hypercholesterolemia (HC) dominates the risk factors for atherosclerosis. Sustained oxidative stress is a central mechanism driving endothelial dysfunction (ED) in HC. But the underlying redox mechanism remains unclear. Protein S-glutathionylation, a reversible oxidative modification of cysteine residues is tightly controlled by a specific de-glutathionylation enzyme: glutaredoxin-1 (Glrx-1), which thereby plays a critical role in redox regulation of cellular function. Methods and Results: The protein levels of Glrx1 in aorta and lung tissues are inversely correlated with circulating cholesterol in WT and ApoE -/- mice. Overexpression of Glrx1 in ApoE -/- mice attenuates HC-induced ED as measured by flow-mediated dilation of femoral artery using a new vascular imaging method with optical coherence tomography technique, and impaired eNOS function as evidenced by increased eNOS monomer to dimer ratio and suppressed phosphorylation of vasodilator-stimulated phosphoprotein in the aorta. In HAECs oxidized LDL promotes downregulation of Glrx1 without altering its mRNA level. The consequent elevated PrS-SG inactivates Akt/eNOS and stimulates degradation of Rac1, a key regulator of eNOS. All of these deteriorate effects of oxLDL are prevented by adenoviral overexpression of Glrx1. Conclusions: HC-induced downregulation of Glrx1 promotes ED by redox inactivation of eNOS and its upstream regulators: Akt1 and Rac1.

Chemical Biology of H2S Signaling through Persulfidation.

Signaling by H2S is proposed to occur via persulfidation, a posttranslational modification of cysteine residues (RSH) to persulfides (RSSH). Persulfidation provides a framework for understanding the physiological and pharmacological effects of H2S. Due to the inherent instability of persulfides, their chemistry is understudied. In this review, we discuss the biologically relevant chemistry of H2S and the enzymatic routes for its production and oxidation. We cover the chemical biology of persulfides and the chemical probes for detecting them. We conclude by discussing the roles ascribed to protein persulfidation in cell signaling pathways.