Cysteine-reactive Probes Research Articles

Solid-Phase Compatible Silane-Based Cleavable Linker Enables Custom Isobaric Quantitative Chemoproteomics.

Mass spectrometry-based chemoproteomics has emerged as an enabling technology for functional biology and drug discovery. To address limitations of established chemoproteomics workflows, including cumbersome reagent synthesis and low throughput sample preparation, here, we established the silane-based cleavable isotopically labeled proteomics (sCIP) method. The sCIP method is enabled by a high yielding and scalable route to dialkoxydiphenylsilane fluorenylmethyloxycarbonyl (DADPS-Fmoc)-protected amino acid building blocks, which enable the facile synthesis of customizable, isotopically labeled, and chemically cleavable biotin capture reagents. sCIP is compatible with both MS1- and MS2-based quantitation, and the sCIP-MS2 method is distinguished by its click-assembled isobaric tags in which the reporter group is encoded in the sCIP capture reagent and balancer in the pan cysteine-reactive probe. The sCIP-MS2 workflow streamlines sample preparation with early stage isobaric labeling and sample pooling, allowing for high coverage and increased sample throughput via customized low cost six-plex sample multiplexing. When paired with a custom FragPipe data analysis workflow and applied to cysteine-reactive fragment screens, sCIP proteomics revealed established and unprecedented cysteine-ligand pairs, including the discovery that mitochondrial uncoupling agent FCCP acts as a covalent-reversible cysteine-reactive electrophile.

N-Acryloylindole-alkyne (NAIA) enables imaging and profiling new ligandable cysteines and oxidized thiols by chemoproteomics

Cysteine has been exploited as the binding site of covalent drugs. Its high sensitivity to oxidation is also important for regulating cellular processes. To identify new ligandable cysteines which can be hotspots for therapy and to better study cysteine oxidations, we develop cysteine-reactive probes, N-acryloylindole-alkynes (NAIAs), which have superior cysteine reactivity owing to delocalization of π electrons of the acrylamide warhead over the whole indole scaffold. This allows NAIAs to probe functional cysteines more effectively than conventional iodoacetamide-alkyne, and to image oxidized thiols by confocal fluorescence microscopy. In mass spectrometry experiments, NAIAs successfully capture new oxidized cysteines, as well as a new pool of ligandable cysteines and proteins. Competitive activity-based protein profiling experiments further demonstrate the ability of NAIA to discover lead compounds targeting these cysteines and proteins. We show the development of NAIAs with activated acrylamide for advancing proteome-wide profiling and imaging ligandable cysteines and oxidized thiols.

Widespread, Reversible Cysteine Modification by Methylglyoxal Regulates Metabolic Enzyme Function.

Methylglyoxal (MGO), a reactive metabolite byproduct of glucose metabolism, is known to form a variety of posttranslational modifications (PTMs) on nucleophilic amino acids. For example, cysteine, the most nucleophilic proteinogenic amino acid, forms reversible hemithioacetal and stable mercaptomethylimidazole adducts with MGO. The high reactivity of cysteine toward MGO and the rate of formation of such modifications provide the opportunity for mechanisms by which proteins and pathways might rapidly sense and respond to alterations in levels of MGO. This indirect measure of alterations in glycolytic flux would thereby allow disparate cellular processes to dynamically respond to changes in nutrient availability and utilization. Here we report the use of quantitative LC-MS/MS-based chemoproteomic profiling approaches with a cysteine-reactive probe to map the proteome-wide landscape of MGO modification of cysteine residues. This approach led to the identification of many sites of potential functional regulation by MGO. We further characterized the role that such modifications have in a catalytic cysteine residue in a key metabolic enzyme and the resulting effects on cellular metabolism.

Comparison of Different Competitive Proteome Profiling Approaches in Target Identification of Covalent Inhibitors.

Competitive proteome profiling is a powerful approach for the identification of targets of small molecules. This approach usually employs an inhibitor-derived probe or a cysteine-reactive probe such as an IA-alkyne in a comparison between inhibitor-treated and untreated samples, thus enabling distinction between genuine targets and nonspecific labeling. We have developed an active probe derived from an EGFR inhibitor, afatinib, and a cysteine reactive probe, an alkyne-containing α,β-unsaturated amide, to compare their characterization of cellular targets. In both approaches, myosin heavy chain 9 (MYH9) was identified as an off-target. Subsequent functional validation experiments suggested that MYH9 might be involved in the function of afatinib.

Enhancing Cysteine Chemoproteomic Coverage through Systematic Assessment of Click Chemistry Product Fragmentation.

Mass spectrometry-based chemoproteomics has enabled functional analysis and small molecule screening at thousands of cysteine residues in parallel. Widely adopted chemoproteomic sample preparation workflows rely on the use of pan cysteine-reactive probes such as iodoacetamide alkyne combined with biotinylation via copper-catalyzed azide-alkyne cycloaddition (CuAAC) or "click chemistry" for cysteine capture. Despite considerable advances in both sample preparation and analytical platforms, current techniques only sample a small fraction of all cysteines encoded in the human proteome. Extending the recently introduced labile mode of the MSFragger search engine, here we report an in-depth analysis of cysteine biotinylation via click chemistry (CBCC) reagent gas-phase fragmentation during MS/MS analysis. We find that CBCC conjugates produce both known and novel diagnostic fragments and peptide remainder ions. Among these species, we identified a candidate signature ion for CBCC peptides, the cyclic oxonium-biotin fragment ion that is generated upon fragmentation of the N(triazole)-C(alkyl) bond. Guided by our empirical comparison of fragmentation patterns of six CBCC reagent combinations, we achieved enhanced coverage of cysteine-labeled peptides. Implementation of labile searches afforded unique PSMs and provides a roadmap for the utility of such searches in enhancing chemoproteomic peptide coverage.

Discovery of Non-Cysteine-Targeting Covalent Inhibitors by Activity-Based Proteomic Screening with a Cysteine-Reactive Probe.

Covalent inhibitors of enzymes are increasingly appreciated as pharmaceutical seeds, yet discovering non-cysteine-targeting inhibitors remains challenging. Herein, we report an intriguing experience during our activity-based proteomic screening of 1601 reactive small molecules, in which we monitored the ability of library molecules to compete with a cysteine-reactive iodoacetamide probe. One epoxide molecule, F8, exhibited unexpected enhancement of the probe reactivity for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a rate-limiting glycolysis enzyme. In-depth mechanistic analysis suggests that F8 forms a covalent adduct with an aspartic acid in the active site to displace NAD+, a cofactor of the enzyme, with concomitant enhancement of the probe reaction with the catalytic cysteine. The mechanistic underpinning permitted the identification of an optimized aspartate-reactive GAPDH inhibitor. Our findings exemplify that activity-based proteomic screening with a cysteine-reactive probe can be used for discovering covalent inhibitors that react with non-cysteine residues.

Comparative reactivity profiling of cysteine-specific probes by chemoproteomics

Cysteines, as one of the most intrinsically nucleophilic amino acids, play important roles in proteins involved in diverse biological processes. They are also targets of covalent drugs for treating cancers and other diseases. Understanding the cysteine reactivity towards different types of electrophilic reactive groups will contribute to design of cysteine-reactive probes and facilitate the development of cysteine-based covalent drugs. In this study, we systematically evaluated the cysteinome reactivity toward two common electrophilic probes that are based on nucleophilic substitution and Michael addition, respectively, using chemical proteomic strategies. Our profiling results showed that each probe had its own preferential reactivity towards different cysteines and the engagement of the ligand fragments could only be distinguished by the probe with the matching reactive group. Our study highlighted the importance of choosing proper cysteine-reactive probes for screening covalent ligands and provided informative guidance for covalent drug development.

Chemoproteomic Profiling by Cysteine Fluoroalkylation Reveals Myrocin G as an Inhibitor of the Nonhomologous End Joining DNA Repair Pathway.

Chemoproteomic profiling of cysteines has emerged as a powerful method for screening the proteome-wide targets of cysteine-reactive fragments, drugs, and natural products. Herein, we report the development and an in-depth evaluation of a tetrafluoroalkyl benziodoxole (TFBX) as a cysteine-selective chemoproteomic probe. We show that this probe features numerous key improvements compared to the traditionally used cysteine-reactive probes, including a superior target occupancy, faster labeling kinetics, and broader proteomic coverage, thus enabling profiling of cysteines directly in live cells. In addition, the fluorine "signature" of probe 7 constitutes an additional advantage resulting in a more confident adduct-amino acid site assignment in mass-spectrometry-based identification workflows. We demonstrate the utility of our new probe for proteome-wide target profiling by identifying the cellular targets of (-)-myrocin G, an antiproliferative fungal natural product with a to-date unknown mechanism of action. We show that this natural product and a simplified analogue target the X-ray repair cross-complementing protein 5 (XRCC5), an ATP-dependent DNA helicase that primes DNA repair machinery for nonhomologous end joining (NHEJ) upon DNA double-strand breaks, making them the first reported inhibitors of this biomedically highly important protein. We further demonstrate that myrocins disrupt the interaction of XRCC5 with DNA leading to sensitization of cancer cells to the chemotherapeutic agent etoposide as well as UV-light-induced DNA damage. Altogether, our next-generation cysteine-reactive probe enables broader and deeper profiling of the cysteinome, rendering it a highly attractive tool for elucidation of targets of electrophilic small molecules.

Sweetly profiling the cysteinome.

A new sugar-based cysteine-reactive probe, combined with competitive activity-based protein profiling (ABPP), enables site-centric target deconvolution of itaconate in native proteomes, shedding light on a novel mechanism of action for this important immunoregulatory metabolite in inflammatory macrophages.

Ethanol Induced Disordering of Pancreatic Acinar Cell Endoplasmic Reticulum: An ER Stress/Defective Unfolded Protein Response Model

Background & AimsHeavy alcohol drinking is associated with pancreatitis, whereas moderate intake lowers the risk. Mice fed ethanol long term show no pancreas damage unless adaptive/protective responses mediating proteostasis are disrupted. Pancreatic acini synthesize digestive enzymes (largely serine hydrolases) in the endoplasmic reticulum (ER), where perturbations (eg, alcohol consumption) activate adaptive unfolded protein responses orchestrated by spliced X-box binding protein 1 (XBP1). Here, we examined ethanol-induced early structural changes in pancreatic ER proteins.MethodsWild-type and Xbp1+/- mice were fed control and ethanol diets, then tissues were homogenized and fractionated. ER proteins were labeled with a cysteine-reactive probe, isotope-coded affinity tag to obtain a novel pancreatic redox ER proteome. Specific labeling of active serine hydrolases in ER with fluorophosphonate desthiobiotin also was characterized proteomically. Protein structural perturbation by redox changes was evaluated further in molecular dynamic simulations.ResultsEthanol feeding and Xbp1 genetic inhibition altered ER redox balance and destabilized key proteins. Proteomic data and molecular dynamic simulations of Carboxyl ester lipase (Cel), a unique serine hydrolase active within ER, showed an uncoupled disulfide bond involving Cel Cys266, Cel dimerization, ER retention, and complex formation in ethanol-fed, XBP1-deficient mice.ConclusionsResults documented in ethanol-fed mice lacking sufficient spliced XBP1 illustrate consequences of ER stress extended by preventing unfolded protein response from fully restoring pancreatic acinar cell proteostasis during ethanol-induced redox challenge. In this model, orderly protein folding and transport to the secretory pathway were disrupted, and abundant molecules including Cel with perturbed structures were retained in ER, promoting ER stress-related pancreas pathology.

Cysteine-reactive probes and their use in chemical proteomics.

Proteomic profiling using bioorthogonal chemical probes that selectively react with certain amino acids is now a widely used method in life sciences to investigate enzymatic activities, study posttranslational modifications and discover novel covalent inhibitors. Over the past two decades, researchers have developed selective probes for several different amino acids, including lysine, serine, cysteine, threonine, tyrosine, aspartate and glutamate. Among these amino acids, cysteines are particularly interesting due to their highly diverse and complex biochemical role in our cells. In this feature article, we focus on the chemical probes and methods used to study cysteines in complex proteomes.

Identifying Functional Cysteine Residues in the Mitochondria.

The mitochondria are dynamic organelles that regulate oxidative metabolism and mediate cellular redox homeostasis. Proteins within the mitochondria are exposed to large fluxes in the surrounding redox environment. In particular, cysteine residues within mitochondrial proteins sense and respond to these redox changes through oxidative modifications of the cysteine thiol group. These oxidative modifications result in a loss in cysteine reactivity, which can be monitored using cysteine-reactive chemical probes and quantitative mass spectrometry (MS). Analysis of cell lysates treated with cysteine-reactive probes enable the identification of hundreds of cysteine residues, however, the mitochondrial proteome is poorly represented (<10% of identified peptides), due to the low abundance of mitochondrial proteins and suppression of mitochondrial peptide MS signals by highly abundant cytosolic peptides. Here, we apply a mitochondrial isolation and purification protocol to substantially increase coverage of the mitochondrial cysteine proteome. Over 1500 cysteine residues from ∼450 mitochondrial proteins were identified, thereby enabling interrogation of an unprecedented number of mitochondrial cysteines. Specifically, these mitochondrial cysteines were ranked by reactivity to identify hyper-reactive cysteines with potential catalytic and regulatory functional roles. Furthermore, analyses of mitochondria exposed to nitrosative stress revealed previously uncharacterized sites of protein S-nitrosation on mitochondrial proteins. Together, the mitochondrial cysteine enrichment strategy presented herein enables detailed characterization of protein modifications that occur within the mitochondria during (patho)physiological fluxes in the redox environment.

Quantitative Metaproteomics and Activity-Based Probe Enrichment Reveals Significant Alterations in Protein Expression from a Mouse Model of Inflammatory Bowel Disease.

Tandem mass spectrometry based shotgun proteomics of distal gut microbiomes is exceedingly difficult due to the inherent complexity and taxonomic diversity of the samples. We introduce two new methodologies to improve metaproteomic studies of microbiome samples. These methods include the stable isotope labeling in mammals to permit protein quantitation across two mouse cohorts as well as the application of activity-based probes to enrich and analyze both host and microbial proteins with specific functionalities. We used these technologies to study the microbiota from the adoptive T cell transfer mouse model of inflammatory bowel disease (IBD) and compare these samples to an isogenic control, thereby limiting genetic and environmental variables that influence microbiome composition. The data generated highlight quantitative alterations in both host and microbial proteins due to intestinal inflammation and corroborates the observed phylogenetic changes in bacteria that accompany IBD in humans and mouse models. The combination of isotope labeling with shotgun proteomics resulted in the total identification of 4434 protein clusters expressed in the microbial proteomic environment, 276 of which demonstrated differential abundance between control and IBD mice. Notably, application of a novel cysteine-reactive probe uncovered several microbial proteases and hydrolases overrepresented in the IBD mice. Implementation of these methods demonstrated that substantial insights into the identity and dysregulation of host and microbial proteins altered in IBD can be accomplished and can be used in the interrogation of other microbiome-related diseases.

Cysteine-reactive chemical probes based on a modular 4-aminopiperidine scaffold

Tri-substituted 4-aminopiperidine provides a modular and versatile scaffold for the generation of cysteine-reactive probes for diverse proteins.

A Competitive Chemical-Proteomic Platform To Identify Zinc-Binding Cysteines

Zinc ions (Zn(2+)) play vital catalytic, structural, and regulatory roles in protein function and are commonly chelated to cysteine residues within the protein framework. Current methods to identify Zn(2+)-binding cysteines rely on computational studies based on known Zn(2+)-chelating motifs, as well as high-resolution structural data. These available approaches preclude the global identification of putative Zn(2+)-chelating cysteines, particularly on poorly characterized proteins in the proteome. Herein, we describe an experimental platform that identifies metal-binding cysteines on the basis of their reduced nucleophilicity upon treatment with metal ions. As validation of our platform, we utilize a peptide-based cysteine-reactive probe to show that the known Zn(2+)-chelating cysteine in sorbitol dehydrogenase (SORD) demonstrates an expected loss in nucleophilicity in the presence of Zn(2+) ions and a gain in nucleophilicity upon treatment with a Zn(2+) chelator. We also identified the active-site cysteine in glutathione S-transferase omega-1 (GSTO1) as a potential Zn(2+)-chelation site, albeit with lower metal affinity relative to SORD. Treatment of recombinant GSTO1 with Zn(2+) ions results in a dose-dependent decrease in GSTO1 activity. Furthermore, we apply a promiscuous cysteine-reactive probe to globally identify putative Zn(2+)-binding cysteines across ∼900 cysteines in the human proteome. This proteomic study identified several well-characterized Zn(2+)-binding proteins, as well as numerous uncharacterized proteins from functionally distinct classes. This platform is highly versatile and provides an experimental tool that complements existing computational and structural methods to identify metal-binding cysteine residues.

Novel Inhibitors for PRMT1 Discovered by High-Throughput Screening Using Activity-Based Fluorescence Polarization

Protein arginine methyltransferases (PRMTs) catalyze the posttranslational methylation of arginine using S-adenosylmethionine (SAM) as a methyl-donor. The PRMT family is widely expressed and has been implicated in biological functions such as RNA splicing, transcriptional control, signal transduction, and DNA repair. Therefore, specific inhibitors of individual PRMTs have potentially significant research and therapeutic value. In particular, PRMT1 is responsible for >85% of arginine methyltransferase activity, but currently available inhibitors of PRMT1 lack specificity, efficacy, and bioavailability. To address this limitation, we developed a high-throughput screening assay for PRMT1 that utilizes a hyper-reactive cysteine within the active site, which is lacking in almost all other PRMTs. This assay, which monitors the kinetics of the fluorescence polarization signal increase upon PRMT1 labeling by a rhodamine-containing cysteine-reactive probe, successfully identified two novel inhibitors selective for PRMT1 over other SAM-dependent methyltransferases.

Alternating Access to the Transmembrane Domain of the ATP-binding Cassette Protein Cystic Fibrosis Transmembrane Conductance Regulator (ABCC7)

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a member of the ATP-binding cassette (ABC) protein family, most members of which act as active transporters. Actively transporting ABC proteins are thought to alternate between "outwardly facing" and "inwardly facing" conformations of the transmembrane substrate pathway. In CFTR, it is assumed that the outwardly facing conformation corresponds to the channel open state, based on homology with other ABC proteins. We have used patch clamp recording to quantify the rate of access of cysteine-reactive probes to cysteines introduced into two different transmembrane regions of CFTR from both the intracellular and extracellular solutions. Two probes, the large [2-sulfonatoethyl]methanethiosulfonate (MTSES) molecule and permeant Au(CN)(2)(-) ions, were applied to either side of the membrane to modify cysteines substituted for Leu-102 (first transmembrane region) and Thr-338 (sixth transmembrane region). Channel opening and closing were altered by mutations in the nucleotide binding domains of the channel. We find that, for both MTSES and Au(CN)(2)(-), access to these two cysteines from the cytoplasmic side is faster in open channels, whereas access to these same sites from the extracellular side is faster in closed channels. These results are consistent with alternating access to the transmembrane regions, however with the open state facing inwardly and the closed state facing outwardly. Our findings therefore prompt revision of current CFTR structural and mechanistic models, as well as having broader implications for transport mechanisms in all ABC proteins. Our results also suggest possible locations of both functional and dysfunctional ("vestigial") gates within the CFTR permeation pathway.

The Pore of the Voltage-Gated Proton Channel

In classical tetrameric voltage-gated ion channels four voltage-sensing domains (VSDs), one from each subunit, control one ion permeation pathway formed by four pore domains. The human Hv1 proton channel has a different architecture, containinga VSD, but lacking a pore domain. Since its location is not known, we searched for the Hv permeation pathway. We find that mutation of the S4 segment's third arginine R211 (R3) compromises proton selectivity, enabling conduction of a metal cation and even of the large organic cation guanidinium, reminiscent of Shaker's omega pore. In the open state, R3 appears to interact with an aspartate (D112) that is situated in the middle of S1 and is unique to Hv channels. The double mutation of both residues further compromises cation selectivity. We propose that membrane depolarization reversibly positions R3 next to D112 inthe transmembrane VSD to form the ion selectivity filter in the channel's open conformation.

What site-directed labeling studies tell us about the mechanism of rhodopsin activation and G-protein binding

Rhodopsin is the photoreceptor protein responsible for dim-light vision in mammals. Due to extensive biophysical, structural and computational analysis of this membrane protein, it is presently the best understood G-protein coupled receptor (GPCR). Here I briefly review one approach that has been extensively used to identify dynamic and structural changes in rhodopsin--the use of site-directed labeling methods (SDL) coupled with electron paramagnetic resonance (EPR) and fluorescence spectroscopy. These SDL studies involve introducing individual cysteine residues into the receptor, then labeling them with cysteine-reactive probes for subsequent analysis by the appropriate spectroscopy. I will give a brief overview of how SDL methods are carried out and how the data is analyzed. Then, I will discuss how SDL studies were carried out on rhodopsin, and how they were used to identify a key structural change that occurs in rhodopsin upon activation--movement of transmembrane helix 6 (TM6). I will also briefly discuss how the SDL studies of rhodopsin compare with SDL studies of other GPCRs, and compare the SDL data with early and recent crystal structures of rhodopsin. Finally, I will discuss why the TM6 movement is required for rhodopsin to couple with the G-protein transducin, and speculate how this mechanism might be a universal method used by all GPCRs to bind G-proteins and perhaps other proteins involved in visual signal transduction, such as arrestin and rhodopsin kinase.

Site‐Specific Chemical Modification of Proteins with a Prelabelled Cysteine Tag Using the Artificially Split Mxe GyrA Intein

The selective modification of proteins with a synthetic probe is of central interest for many aspects of protein chemistry. We have recently reported a new approach in which a short cysteine-containing tag (CysTag) fused to one part of a split intein is first modified with a sulfhydryl-reactive probe. In a second step, protein trans-splicing is used to link the labelled CysTag to a target protein that has been expressed in fusion with the complementary split intein fragment. Here, we present the generation and biochemical characterisation of the artificially split Mycobacterium xenopi GyrA intein. We show that this split intein is active without a renaturation step and that it provides a significant improvement for the CysTag protein-labelling approach in terms of product yields and target protein tolerance. Two proteins with multiple cysteine residues, human growth hormone and a multidomain nonribosomal peptide synthetase, were site-specifically modified with high yields. Our approach combines the benefits of the plethora of commercially available cysteine-reactive probes with a straightforward route for their site-specific incorporation even into complex and cysteine-rich proteins.