Structures of the glucocorticoid-bound adhesion receptor GPR97-Go complex.

Adhesion G-protein-coupled receptors (GPCRs) are a major family of GPCRs, but limited knowledge of their ligand regulation or structure is available1-3. Here we report that glucocorticoid stress hormones activate adhesion G-protein-coupled receptor G3 (ADGRG3; also known as GPR97)4-6, a prototypical adhesion GPCR. The cryo-electron microscopy structures of GPR97-Go complexes bound to the anti-inflammatory drug beclomethasone or the steroid hormone cortisol revealed that glucocorticoids bind to a pocket within the transmembrane domain. The steroidal core of glucocorticoids is packed against the 'toggle switch' residue W6.53, which senses the binding of a ligand and induces activation of the receptor. Active GPR97 uses a quaternary core and HLY motif to fasten the seven-transmembrane bundle and to mediate G protein coupling. The cytoplasmic side of GPR97 has an open cavity, where all three intracellular loops interact with the Go protein, contributing to the high basal activity of GRP97. Palmitoylation at the cytosolic tail of the Go protein was found to be essential for efficient engagement with GPR97 but is not observed in other solved GPCR complex structures. Our work provides a structural basis for ligand binding to the seven-transmembrane domain of an adhesion GPCR and subsequent G protein coupling.

Nucleophilic covalent ligand discovery for the cysteine redoxome.

With an eye toward expanding chemistries used for covalent ligand discovery, we elaborated an umpolung strategy that exploits the 'polarity reversal' of sulfur when cysteine is oxidized to sulfenic acid, a widespread post-translational modification, for selective bioconjugation with C-nucleophiles. Here we present a global map of a human sulfenome that is susceptible to covalent modification by members of a nucleophilic fragment library. More than 500 liganded sulfenic acids were identified on proteins across diverse functional classes, and, of these, more than 80% were not targeted by electrophilic fragment analogs. We further show that members of our nucleophilic fragment library can impair functional protein-protein interactions involved in nuclear oncoprotein transport and DNA damage repair. Our findings reveal a vast expanse of ligandable sulfenic acids in the human proteome and highlight the utility of nucleophilic small molecules in the fragment-based covalent ligand discovery pipeline, presaging further opportunities using non-traditional chemistries for targeting proteins.

A modification-centric assessment tool for the performance of chemoproteomic probes.

Chemoproteomics has emerged as a key technology to expand the functional space in complex proteomes for probing fundamental biology and for discovering new small-molecule-based therapies. Here we report a modification-centric computational tool termed pChem to provide a streamlined pipeline for unbiased performance assessment of chemoproteomic probes. The pipeline starts with an experimental setting for isotopically coding probe-derived modifications that can be automatically recognized by pChem, with masses accurately calculated and sites precisely localized. pChem exports on-demand reports by scoring the profiling efficiency, modification homogeneity and proteome-wide residue selectivity of a tested probe. The performance and robustness of pChem were benchmarked by applying it to eighteen bioorthogonal probes. These analyses reveal that the formation of unexpected probe-derived modifications can be driven by endogenous reactive metabolites (for example, bioactive aldehydes and glutathione). pChem is a powerful and user-friendly tool that aims to facilitate the development of probes for the ever-growing field of chemoproteomics.

Mining for protein S-sulfenylation in Arabidopsis uncovers redox-sensitive sites.

Hydrogen peroxide (H2O2) is an important messenger molecule for diverse cellular processes. H2O2 oxidizes proteinaceous cysteinyl thiols to sulfenic acid, also known as S-sulfenylation, thereby affecting the protein conformation and functionality. Although many proteins have been identified as S-sulfenylation targets in plants, site-specific mapping and quantification remain largely unexplored. By means of a peptide-centric chemoproteomics approach, we mapped 1,537 S-sulfenylated sites on more than 1,000 proteins in Arabidopsis thaliana cells. Proteins involved in RNA homeostasis and metabolism were identified as hotspots for S-sulfenylation. Moreover, S-sulfenylation frequently occurred on cysteines located at catalytic sites of enzymes or on cysteines involved in metal binding, hinting at a direct mode of action for redox regulation. Comparison of human and Arabidopsis S-sulfenylation datasets provided 155 conserved S-sulfenylated cysteines, including Cys181 of the Arabidopsis MITOGEN-ACTIVATED PROTEIN KINASE4 (AtMAPK4) that corresponds to Cys161 in the human MAPK1, which has been identified previously as being S-sulfenylated. We show that, by replacing Cys181 of recombinant AtMAPK4 by a redox-insensitive serine residue, the kinase activity decreased, indicating the importance of this noncatalytic cysteine for the kinase mechanism. Altogether, we quantitatively mapped the S-sulfenylated cysteines in Arabidopsis cells under H2O2 stress and thereby generated a comprehensive view on the S-sulfenylation landscape that will facilitate downstream plant redox studies.

Discovery of CAPE derivatives as dual EGFR and CSK inhibitors with anticancer activity in a murine model of hepatocellular carcinoma.

Caffeic acid phenethyl ester (CAPE), a bioactive component extracted from propolis of honeybee hives, can inhibit hepatocellular carcinoma (HCC). In order to explore more stable CAPE derivatives, 25 compounds were designed, synthesized, and pharmacologically assessed in vitro and in vivo as anti-tumor agents in HCC. Compounds 8d, 8f, 8l, 8j, and 8k showed favorable antiproliferative activity than other compounds including CAPE in the HCC cell lines. Based on the result of QTRP (Quantitative Thiol Reactivity Profiling), epidermal growth factor receptor (EGFR) and C-terminal Src kinase (CSK) were supposed to the targets of 8f, which was confirmed by binding mode analysis. Furthermore, compounds 8f, 8l, 8j, 8k, 8g, and 8h showed potent inhibitory effects against both CSK and EGFR than other derivatives in an ADP-Glo™ kinase assay. The representative compound, 8f, potently inhibited various tumor growth in murine model including murine hepatocellular carcinoma H22, meanwhile downregulating the EGFR/AKT pathway and enhancing T cell proliferation through inhibition of CSK. Metabolic stability in vitro suggested 8f and 8k were more stable in mouse plasma than CAPE and susceptible to metabolism in liver microsomes. The overall excellent profile of compound 8f makes it a potential candidate for further preclinical investigation.

Dynamic redox balance directs the oocyte-to-embryo transition via developmentally controlled reactive cysteine changes.

The metabolic and redox state changes during the transition from an arrested oocyte to a totipotent embryo remain uncharacterized. Here, we applied state-of-the-art, integrated methodologies to dissect these changes in Drosophila We demonstrate that early embryos have a more oxidized state than mature oocytes. We identified specific alterations in reactive cysteines at a proteome-wide scale as a result of this metabolic and developmental transition. Consistent with a requirement for redox change, we demonstrate a role for the ovary-specific thioredoxin Deadhead (DHD). dhd-mutant oocytes are prematurely oxidized and exhibit meiotic defects. Epistatic analyses with redox regulators link dhd function to the distinctive redox-state balance set at the oocyte-to-embryo transition. Crucially, global thiol-redox profiling identified proteins whose cysteines became differentially modified in the absence of DHD. We validated these potential DHD substrates by recovering DHD-interaction partners using multiple approaches. One such target, NO66, is a conserved protein that genetically interacts with DHD, revealing parallel functions. As redox changes also have been observed in mammalian oocytes, we hypothesize a link between developmental control of this cell-cycle transition and regulation by metabolic cues. This link likely operates both by general redox state and by changes in the redox state of specific proteins. The redox proteome defined here is a valuable resource for future investigation of the mechanisms of redox-modulated control at the oocyte-to-embryo transition.

Chemoproteomics Reveals Chemical Diversity and Dynamics of 4-Oxo-2-nonenal Modifications in Cells.

4-Oxo-2-nonenal (ONE) derived from lipid peroxidation modifies nucleophiles and transduces redox signaling by its reactions with proteins. However, the molecular interactions between ONE and complex proteomes and their dynamics in situ remain largely unknown. Here we describe a quantitative chemoproteomic analysis of protein adduction by ONE in cells, in which the cellular target profile of ONE is mimicked by its alkynyl surrogate. The analyses reveal four types of ONE-derived modifications in cells, including ketoamide and Schiff-base adducts to lysine, Michael adducts to cysteine, and a novel pyrrole adduct to cysteine. ONE-derived adducts co-localize and exhibit crosstalk with many histone marks and redox sensitive sites. All four types of modifications derived from ONE can be reversed site-specifically in cells. Taken together, our study provides much-needed mechanistic insights into the cellular signaling and potential toxicities associated with this important lipid derived electrophile.

Systematic and Quantitative Assessment of Hydrogen Peroxide Reactivity With Cysteines Across Human Proteomes.

Protein cysteinyl residues are the mediators of hydrogen peroxide (H2O2)-dependent redox signaling. However, site-specific mapping of the selectivity and dynamics of these redox reactions in cells poses a major analytical challenge. Here we describe a chemoproteomic platform to systematically and quantitatively analyze the reactivity of thousands of cysteines toward H2O2 in human cells. We identified >900 H2O2-sensitive cysteines, which are defined as the H2O2-dependent redoxome. Although redox sites associated with antioxidative and metabolic functions are consistent, most of the H2O2-dependent redoxome varies dramatically between different cells. Structural analyses reveal that H2O2-sensitive cysteines are less conserved than their redox-insensitive counterparts and display distinct sequence motifs, structural features, and potential for crosstalk with lysine modifications. Notably, our chemoproteomic platform also provides an opportunity to predict oxidation-triggered protein conformational changes. The data are freely accessible as a resource at http://redox.ncpsb.org/OXID/.

Chemoproteomics Reveals Unexpected Lysine/Arginine-Specific Cleavage of Peptide Chains as a Potential Protein Degradation Machinery.

Proteins can undergo oxidative cleavage by in vitro metal-catalyzed oxidation (MCO) in either the α-amidation or the diamide pathway. However, whether oxidative cleavage of polypeptide-chain occurs in biological systems remains unexplored. We describe a chemoproteomic approach to globally and site-specifically profile electrophilic protein degradants formed from peptide backbone cleavages in human proteomes, including the known N-terminal α-ketoacyl products and >1000 unexpected N-terminal formyl products. Strikingly, such cleavages predominantly occur at the carboxyl side of lysine (K) and arginine (R) residues across native proteomes in situ, while MCO-induced oxidative cleavages randomly distribute on peptide/protein sequences in vitro. Furthermore, ionizing radiation-induced reactive oxygen species (ROS) also generate random oxidative cleavages in situ. These findings suggest that the endogenous formation of N-formyl and N-α-ketoacyl degradants in biological systems is more likely regulated by a previously unknown mechanism with a trypsin-like specificity, rather than the random oxidative damage as previously thought. More generally, our study highlights the utility of quantitative chemoproteomics in combination with unrestricted search tools as a viable strategy to discover unexpected chemical modifications of proteins labeled with active-based probes.

[Screening of nucleic acid aptamer of lung cancer cells based on cell exponential enrichment ligand system evolution and its application in tumor diagnosis and treatment].

Nucleic acid aptamer is an oligonucleotide sequence screened by the exponential enrichment ligand system evolution technology (SELEX). Previous studies have shown that nucleic acid aptamer has a good application prospect in tumor diagnosis and treatment. Therefore, we reviewed the selection and identification of nucleic acid aptamer of lung cancer cells in recent years, and discussed the effect of aptamer as targeting drugs and targeting vectors on the diagnosis of tumors, which provide a new idea for early diagnosis and treatment of tumor.

A Chemoproteomic Platform To Assess Bioactivation Potential of Drugs.

Reactive metabolites (RM) formed from bioactivation of drugs can covalently modify liver proteins and cause mechanism-based inactivation of major cytochrome P450 (CYP450) enzymes. Risk of bioactivation of a test compound is routinely examined as part of lead optimization efforts in drug discovery. Here we described a chemoproteomic platform to assess in vitro and in vivo bioactivation potential of drugs. This platform enabled us to determine reactivity of thousands of proteomic cysteines toward RMs of diclofenac formed in human liver microsomes and living animals. We pinpointed numerous reactive cysteines as the targets of RMs of diclofenac, including the active (heme-binding) sites on several key CYP450 isoforms (1A2, 2E1 and 3A4 for human, 2C39 and 3A11 for mouse). This general platform should be applied to other drugs, drug candidates, and xenobiotics with potential hepatoxicity, including environmental organic substances, bioactive natural products, and traditional Chinese medicine.

A combination of 2D similarity search, pharmacophore, and molecular docking techniques for the identification of vascular endothelial growth factor receptor-2 inhibitors.

The human vascular endothelial growth factor receptor-2 (VEGFR-2) has been an attractive target for the inhibition of angiogenesis. In the current study, we used a hybrid protocol of virtual screening methods to retrieve new VEGFR-2 inhibitors from the Zinc-Specs Database (441 574 compounds). The hybrid protocol included the initial screening of candidates by comparing the 2D similarity to five reported top active inhibitors of 13 VEGFR-2 X-ray crystallography structures, followed by the pharmacophore modeling of virtual screening on the basis of receptor-ligand interactions and further narrowing by LibDOCK to obtain the final hits. Two compounds (AN-919/41439526 and AK-968/40939851) with a high libscore were selected as the final hits for a subsequent cell cytotoxicity study. The two compounds screened exerted significant inhibitory effects on the proliferation of cancer cells (U87 and MCF-7). The results indicated that the hybrid procedure is an effective approach for screening specific receptor inhibitors.

Tubulin inhibitors: pharmacophore modeling, virtual screening and molecular docking.

AIM: To construct a quantitative pharmacophore model of tubulin inhibitors and to discovery new leads with potent antitumor activities. METHODS: Ligand-based pharmacophore modeling was used to identify the chemical features responsible for inhibiting tubulin polymerization. A set of 26 training compounds was used to generate hypothetical pharmacophores using the HypoGen algorithm. The structures were further validated using the test set, Fischer randomization method, leave-one-out method and a decoy set, and the best model was chosen to screen the Specs database. Hit compounds were subjected to molecular docking study using a Molecular Operating Environment (MOE) software and to biological evaluation in vitro. RESULTS: Hypo1 was demonstrated to be the best pharmacophore model that exhibited the highest correlation coefficient (0.9582), largest cost difference (70.905) and lowest RMSD value (0.6977). Hypo1 consisted of one hydrogen-bond acceptor, a hydrogen-bond donor, a hydrophobic feature, a ring aromatic feature and three excluded volumes. Hypo1 was validated with four different methods and had a goodness-of-hit score of 0.81. When Hypo1 was used in virtual screening of the Specs database, 952 drug-like compounds were revealed. After docking into the colchicine-binding site of tubulin, 5 drug-like compounds with the required interaction with the critical amino acid residues and the binding free energies < -4 kcal/mol were selected as representative leads. Compounds 1 and 3 exhibited inhibitory activity against MCF-7 human breast cancer cells in vitro. CONCLUSION: Hypo1 is a quantitative pharmacophore model for tubulin inhibitors, which not only provides a better understanding of their interaction with tubulin, but also assists in discovering new potential leads with antitumor activities.

Global profiling of distinct cysteine redox forms reveals wide-ranging redox regulation in C. elegans.

Post-translational changes in the redox state of cysteine residues can rapidly and reversibly alter protein functions, thereby modulating biological processes. The nematode C. elegans is an ideal model organism for studying cysteine-mediated redox signaling at a network level. Here we present a comprehensive, quantitative, and site-specific profile of the intrinsic reactivity of the cysteinome in wild-type C. elegans. We also describe a global characterization of the C. elegans redoxome in which we measured changes in three major cysteine redox forms after H2O2 treatment. Our data revealed redox-sensitive events in translation, growth signaling, and stress response pathways, and identified redox-regulated cysteines that are important for signaling through the p38 MAP kinase (MAPK) pathway. Our in-depth proteomic dataset provides a molecular basis for understanding redox signaling in vivo, and will serve as a valuable and rich resource for the field of redox biology.

A Clickable APEX Probe for Proximity-Dependent Proteomic Profiling in Yeast.

The engineered ascorbate peroxidase (APEX) is a powerful tool for the proximity-dependent labeling of proteins and RNAs in live cells. Although widely use in mammalian cells, APEX applications in microorganisms have been hampered by the poor labeling efficiency of its biotin-phenol (BP) substrate. In this study, we sought to address this challenge by designing and screening a panel of alkyne-functionalized substrates. Our best probe, Alk-Ph, substantially improves APEX-labeling efficiency in intact yeast cells, as it is more cell wall-permeant than BP. Through a combination of protein-centric and peptide-centric chemoproteomic experiments, we have identified 165 proteins with a specificity of 94% in the yeast mitochondrial matrix. In addition, we have demonstrated that Alk-Ph is useful for proximity-dependent RNA labeling in yeast, thus expanding the scope of APEX-seq. We envision that this improved APEX-labeling strategy would set the stage for the large-scale mapping of spatial proteome and transcriptome in yeast.

Direct Proteomic Mapping of Cysteine Persulfidation.

Aims: Cysteine persulfidation (also called sulfhydration or sulfuration) has emerged as a potential redox mechanism to regulate protein functions and diverse biological processes in hydrogen sulfide (H2S) signaling. Due to its intrinsically unstable nature, working with this modification has proven to be challenging. Although methodological progress has expanded the inventory of persulfidated proteins, there is a continued need to develop methods that can directly and unequivocally identify persulfidated cysteine residues in complex proteomes. Results: A quantitative chemoproteomic method termed as low-pH quantitative thiol reactivity profiling (QTRP) was developed to enable direct site-specific mapping and reactivity profiling of proteomic persulfides and thiols in parallel. The method was first applied to cell lysates treated with NaHS, resulting in the identification of overall 1547 persulfidated sites on 994 proteins. Structural analysis uncovered unique consensus motifs that might define this distinct type of modification. Moreover, the method was extended to profile endogenous protein persulfides in cells expressing H2S-generating enzyme, mouse tissues, and human serum, which led to additional insights into mechanistic, structural, and functional features of persulfidation events, particularly on human serum albumin. Innovation and Conclusion: Low-pH QTRP represents the first method that enables direct and unbiased proteomic mapping of cysteine persulfidation. Our method allows to generate the most comprehensive inventory of persulfidated targets of NaHS so far and to perform the first analysis of in vivo persulfidation events, providing a valuable tool to dissect the biological functions of this important modification.

Identification of Sulfenylated Cysteines in Arabidopsis thaliana Proteins Using a Disulfide-Linked Peptide Reporter.

In proteins, hydrogen peroxide (H2O2) reacts with redox-sensitive cysteines to form cysteine sulfenic acid, also known as S-sulfenylation. These cysteine oxidation events can steer diverse cellular processes by altering protein interactions, trafficking, conformation, and function. Previously, we had identified S-sulfenylated proteins by using a tagged proteinaceous probe based on the yeast AP-1-like (Yap1) transcription factor that specifically reacts with sulfenic acids and traps them through a mixed disulfide bond. However, the identity of the S-sulfenylated amino acid residues within a protein remained enigmatic. By using the same transgenic YAP1C probe, we present here a technological advancement to identify in situ sulfenylated cysteine sites in Arabidopsis thaliana cells under control condition and oxidative stress. Briefly, the total extract of transgenic YAP1C A. thaliana cells was initially purified on IgG-Sepharose beads, followed by a tryptic digest. Then, the mixed disulfide-linked peptides were further enriched at the peptide level on an anti-YAP1C-derived peptide (C598SEIWDR) antibody. Subsequent mass spectrometry analysis with pLink 2 identified 1,745 YAP1C cross-linked peptides, indicating sulfenylated cysteines in over 1,000 proteins. Approximately 55% of these YAP1C-linked cysteines had previously been reported as redox-sensitive cysteines (S-sulfenylation, S-nitrosylation, and reversibly oxidized cysteines). The presented methodology provides a noninvasive approach to identify sulfenylated cysteines in any species that can be genetically modified.

A quantitative thiol reactivity profiling platform to analyze redox and electrophile reactive cysteine proteomes.

Cysteine is unique among all protein-coding amino acids, owing to its intrinsically high nucleophilicity. The cysteinyl thiol group can be covalently modified by a broad range of redox mechanisms or by various electrophiles derived from exogenous or endogenous sources. Measuring the response of protein cysteines to redox perturbation or electrophiles is critical for understanding the underlying mechanisms involved. Activity-based protein profiling based on thiol-reactive probes has been the method of choice for such analyses. We therefore adapted this approach and developed a new chemoproteomic platform, termed 'QTRP' (quantitative thiol reactivity profiling), that relies on the ability of a commercially available thiol-reactive probe IPM (2-iodo-N-(prop-2-yn-1-yl)acetamide) to covalently label, enrich and quantify the reactive cysteinome in cells and tissues. Here, we provide a detailed and updated workflow of QTRP that includes procedures for (i) labeling of the reactive cysteinome from cell or tissue samples (e.g., control versus treatment) with IPM, (ii) processing the protein samples into tryptic peptides and tagging the probe-modified peptides with isotopically labeled azido-biotin reagents containing a photo-cleavable linker via click chemistry reaction, (iii) capturing biotin-conjugated peptides with streptavidin beads, (iv) identifying and quantifying the photo-released peptides by mass spectrometry (MS)-based shotgun proteomics and (v) interpreting MS data by a streamlined informatic pipeline using a proteomics software, pFind 3, and an automatic post-processing algorithm. We also exemplified here how to use QTRP for mining H2O2-sensitive cysteines and for determining the intrinsic reactivity of cysteines in a complex proteome. We anticipate that this protocol should find broad applications in redox biology, chemical biology and the pharmaceutical industry. The protocol for sample preparation takes 3 d, whereas MS measurements and data analyses require 75 min and <30 min, respectively, per sample.

MicroRNA-29b-3p enhances radiosensitivity through modulating WISP1-mediated mitochondrial apoptosis in prostate cancer cells.

Radiotherapy is frequently applied for clinically localized prostate cancer while its efficacy could be significantly hindered by radioresistance. MicroRNAs (miRNAs) are important regulators in mediating cellular responses to ionizing radiation (IR), and strongly associate with radiosensitivity in many cancers. In this study, enhancement of radiosensitivity by miR-29b-3p was demonstrated in prostate cancer cell line LNCaP in vitro. Results showed that miR-29b-3p expression was significantly upregulated in response to IR from both X-rays and carbon ion irradiations. Knockdown of miR-29b-3p resulted in radioresistance while overexpression of miR-29b-3p led to increased radiosensitivity (showing reduced cell viability, suppressed cell proliferation and decreased colony formation). In addition, miR-29b-3p was found to directly target Wnt1-inducible-signaling protein 1 (WISP1). Inhibition of WISP1 facilitated the mitochondrial apoptosis pathway through suppressing Bcl-XL expression while activating caspase-3 and poly (ADP-ribose) polymerase (PARP). The results indicated that miR-29b-3p was a radiosensitizing miRNAs and could enhance radiosensitivity of LNCaP cells by targeting WISP1. These findings suggested a novel treatment to overcome radioresistance in prostate cancer patients, especially those with higher levels of the WISP1 expression.