ACS Chemical Biology最新文献

{"title":"Bioorthogonal Cyclopropenones for Investigating RNA Structure.","authors":"Sharon Chen, Christopher D Sibley, Brandon Latifi, Sumirtha Balaratnam, Robert S Dorn, Andrej Lupták, John S Schneekloth, Jennifer A Prescher","doi":"10.1021/acschembio.4c00633","DOIUrl":"10.1021/acschembio.4c00633","url":null,"abstract":"<p><p>RNA sequences encode structures that impact protein production and other cellular processes. Misfolded RNAs can also potentiate disease, but a complete picture is lacking. To establish more comprehensive and accurate RNA structure-function relationships, new methods are needed to interrogate RNA in native environments. Existing tools rely primarily on electrophiles that are constitutively \"on\" or triggered by UV light, often resulting in high background. Here we describe an alternative, chemically triggered approach to cross-link RNAs using bioorthogonal cyclopropenones (CpOs). These reagents selectively react with phosphines to provide ketenes─electrophiles that can trap neighboring nucleophiles to forge covalent cross-links. As a proof-of-concept, we conjugated a CpO motif to thiazole orange (TO-1). TO-1-CpO bound selectively to a model RNA aptamer (Mango) with nanomolar affinity, as confirmed by fluorescence turn-on. After phosphine administration, covalent cross-links were formed between the CpO and RNA. Cross-linking was both time and dose dependent. We further applied the chemically triggered tools to model RNAs under biologically relevant conditions. Collectively, this work expands the toolkit of probes for studying RNA and its native conformations.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":"2406-2411"},"PeriodicalIF":3.5,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142783334","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Molecular Targeted Engagement of DPP9 in Rat Tissue Using CETSA, SP3 Processing, and Absolute Quantitation Mass Spectrometry.","authors":"Matthew T Mazur, Baojen Shyong, Qian Huang, Stacey L Polsky-Fisher, Carl J Balibar, Weixun Wang","doi":"10.1021/acschembio.4c00563","DOIUrl":"10.1021/acschembio.4c00563","url":null,"abstract":"<p><p>The cellular thermal shift assay (CETSA) provides a means of understanding the extent to which a small molecule ligand associates with a protein target of therapeutic interest, thereby inferring target engagement. Better analytical detection methods, including mass spectrometry, are being implemented to improve quantitation within these assays, providing both absolute quantitation and a very high analyte specificity. To understand the target engagement, and hence inhibition, of the protein dipeptidyl peptidase 9 (DPP9) in rat tissue, CETSA experiments, coupled with single-pot, solid-phase-enhanced sample preparation (\"SP3\") and absolute quantitation by high-resolution mass spectrometry, demonstrated a temperature-dependent \"melting curve\" by ex vivo incubation of compound with rat tissue and further demonstrated in vivo engagement by a dose-dependent response to treatment. These experiments illustrate the ability to extend the CETSA to in vivo dosed-animal samples using absolute quantitation of DPP9 by mass spectrometry and demonstrate a viable path for interrogating therapeutic molecules for drug discovery.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":"2477-2486"},"PeriodicalIF":3.5,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142789410","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Discovery of a Compound That Inhibits IRE1α <i>S</i>-Nitrosylation and Preserves the Endoplasmic Reticulum Stress Response under Nitrosative Stress.","authors":"Haruna Kurogi, Nobumasa Takasugi, Sho Kubota, Ashutosh Kumar, Takehiro Suzuki, Naoshi Dohmae, Daisuke Sawada, Kam Y J Zhang, Takashi Uehara","doi":"10.1021/acschembio.4c00403","DOIUrl":"10.1021/acschembio.4c00403","url":null,"abstract":"<p><p>Inositol-requiring enzyme 1α (IRE1α) is a sensor of endoplasmic reticulum (ER) stress and drives ER stress response pathways. Activated IRE1α exhibits RNase activity and cleaves mRNA encoding X-box binding protein 1, a transcription factor that induces the expression of genes that maintain ER proteostasis for cell survival. Previously, we showed that IRE1α undergoes <i>S</i>-nitrosylation, a post-translational modification induced by nitric oxide (NO), resulting in reduced RNase activity. Therefore, <i>S</i>-nitrosylation of IRE1α compromises the response to ER stress, making cells more vulnerable. We conducted virtual screening and cell-based validation experiments to identify compounds that inhibit the <i>S</i>-nitrosylation of IRE1α by targeting nitrosylated cysteine residues. We ultimately identified a compound (1ACTA) that selectively inhibits the <i>S</i>-nitrosylation of IRE1α and prevents the NO-induced reduction of RNase activity. Furthermore, 1ACTA reduces the rate of NO-induced cell death. Our research identified <i>S</i>-nitrosylation as a novel target for drug development for IRE1α and provides a suitable screening strategy.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":"2429-2437"},"PeriodicalIF":3.5,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142612630","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Single-Cell Multiomics Identifies Glycan Epitope LacNAc as a Potential Cell-Surface Effector Marker of Peripheral T Cells in Bladder Cancer Patients.","authors":"Xiangyu Wu, Zihan Zhao, Wenhao Yu, Siyang Liu, Meng Zhou, Ning Jiang, Xiang Du, Xin Yang, Jinbang Chen, Hongqian Guo, Rong Yang","doi":"10.1021/acschembio.4c00635","DOIUrl":"10.1021/acschembio.4c00635","url":null,"abstract":"<p><p>Cancer is a systemic disease continuously monitored and responded to by the human global immune system. Peripheral blood immune cells, integral to this surveillance, exhibit variable phenotypes during tumor progression. Glycosylation, as one of the most prevalent and significant post-translational modifications of proteins, plays a crucial role in immune system recognition and response. Glycan analysis has become a key method for biomarker discovery. LacNAc, a prominent glycosylation modification, regulates immune cell activity and function. Therefore, we applied our previously developed single-cell glycomic multiomics to analyze peripheral blood in cancer patients. This platform utilizes chemoenzymatic labeling with DNA barcodes for detecting and quantifying LacNAc levels at single-cell resolution without altering the transcriptional status of immune cells. For the first time, we systematically integrated single-cell transcriptome, T cell receptor (TCR) repertoire, and glycan epitope LacNAc analyses in tumor-patient-derived peripheral blood. Our integrated analysis reveals that lower-stage bladder cancer patients showed significantly higher levels of LacNAc in peripheral T cells, and peripheral T cells with high levels of cell-surface LacNAc exhibit higher cytotoxicity and TCR clonal expansion. In summary, we identified LacNAc as a potential cell-surface effector marker for peripheral T cells in bladder cancer patients, which enhances our understanding of peripheral immune cells and offers potential advancements in liquid biopsy.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":"2535-2547"},"PeriodicalIF":3.5,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142708486","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"<i>Mycobacterium tuberculosis</i> Mce3R TetR-like Repressor Forms an Asymmetric Four-Helix Bundle and Binds a Nonpalindrome Sequence†.","authors":"Navanjalee T Panagoda, Gábor Balázsi, Nicole S Sampson","doi":"10.1021/acschembio.4c00687","DOIUrl":"10.1021/acschembio.4c00687","url":null,"abstract":"<p><p><i>Mycobacterium tuberculosis</i> (<i>Mtb</i>), the causative agent of tuberculosis, is a major global health concern. TetR family repressors (TFRs) are important for <i>Mtb</i>'s adaptation to the human host environment. Our study focuses on one notable <i>Mtb</i> repressor, Mce3R, composed of an unusual double TFR motif. Mce3R-regulated genes encode enzymes implicated in cholesterol metabolism, resistance against reactive oxygen species, and lipid transport activities important for <i>Mtb</i> survival and persistence in the host and for the cellular activity of a 6-azasteroid derivative. Here, we present the structure of Mce3R bound to its DNA operator, unveiling a unique asymmetric assembly previously unreported. We obtained a candidate DNA-binding motif through MEME motif analysis, comparing intergenic regions of <i>mce3R</i> orthologues and identifying nonpalindromic regions conserved between orthologues. Using an electrophoretic mobility shift assay (EMSA), we confirmed that Mce3R binds to a 123-bp sequence that includes the predicted motif. Using scrambled DNA and DNA oligonucleotides of varying lengths with sequences from the upstream region of the <i>yrbE3A</i> (<i>mce3</i>) operon, we elucidated the operator region to be composed of two Mce3R binding sites, each a 25-bp asymmetric sequence separated by 53 bp. Mce3R binds with a higher affinity to the downstream site with a <i>K</i>_d of 2.4 ± 0.7 nM. The cryo-EM structure of Mce3R bound to the 123-bp sequence was refined to a resolution of 2.51 Å. Each Mce3R monomer comprises 21 α-helices (α1-α21) folded into an asymmetric TFR-like structure with a core asymmetric four-helix bundle. This complex has two nonidentical HTH motifs and a single ligand-binding domain. The two nonidentical HTHs from each TFR bind within the high-affinity, nonpalindromic operator motif, with Arg53 and Lys262 inserted into the major groove. Site-directed mutagenesis of Arg53 to alanine abrogated DNA binding, validating the Mce3R/DNA structure obtained. Among 811,645 particles, 63% were Mce3R homodimer bound to two duplex oligonucleotides. Mce3R homodimerizes primarily through α15, and each monomer binds to an identical site in the DNA duplex oligonucleotide.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":"2580-2592"},"PeriodicalIF":3.5,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142638004","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Understanding Polysulfide-Mediated Papain Inhibition and Differentiating between Disulfide vs Persulfide Formation.","authors":"Meg Shieh, Anna Y Chung, Stephen Lindahl, Melany Veliz, Charlotte A Bain, Ming Xian","doi":"10.1021/acschembio.4c00573","DOIUrl":"10.1021/acschembio.4c00573","url":null,"abstract":"<p><p>Protein cysteine residues are sensitive to redox-regulating molecules, including reactive sulfur species (RSS). As an important member of the RSS family, polysulfides are known to react with protein cysteines to form persulfides and disulfides, both affecting protein functions. In this work, we studied how polysulfides could impact cysteine proteases through careful mechanistic and kinetic studies. The model protein papain was treated with different polysulfides to elucidate the efficacy of polysulfides as inhibitors for this protein. We also explored the effects of different reductants that could regenerate papain activity after polysulfide-mediated inhibition. A triarylphosphine reagent, TXPTS, was found to be efficient in differentiating between papain persulfidation and disulfide formation.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":"2487-2493"},"PeriodicalIF":3.5,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142638005","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of a Versatile Plant-Derived Mitochondrial Targeting Sequence Based on a Reporter Protein Sorting Analysis and Biological Information.","authors":"Naoya Abe, Masaki Odahara, Shamitha Rao Morey, Keiji Numata","doi":"10.1021/acschembio.4c00625","DOIUrl":"10.1021/acschembio.4c00625","url":null,"abstract":"<p><p>Methods for the delivery of exogenous substances to specific organelles are important because each organelle functions according to its own role. Specifically, mitochondria play an important role in energy production. Recently, plant mitochondrial transformation via delivery methods to mitochondria has been actively researched. Mitochondrial targeting sequences (MTSs) are essential for transporting bioactive molecules, such as nucleic acids, to mitochondria. However, the selectivity and efficacy of MTSs as carrier molecules in plants are not yet sufficient. In this study, we developed an effective MTS in plants via a quantitative comparison of the targeting functions of several MTSs. The presequence of HSP60 from <i>Nicotiana tabacum</i>, which is highly similar to that of several other model plants, showed high mitochondrial-targeting ability among the MTSs tested. This result suggests the applicability of the HSP60 presequence for MTSs in various plants. We further investigated this HSP60 presequence through stepwise shortening on the basis of secondary structure prediction, aiming to simplify synthesis and increase the solubility of the peptides. As shown by assessment of the mitochondrial targeting ability, the 15 residues from the N-terminus of the HSP60 presequence for the MTS, which is particularly conserved among various model plants, retained a targeting efficacy comparable to that of the full-length HSP60 presequence. This developed sequence from the HSP60 sequence is a promising MTS for transfection into plant mitochondria.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":"2515-2524"},"PeriodicalIF":3.5,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142764589","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mapping the FGF2 Interactome Identifies a Functional Proteoglycan Coreceptor.","authors":"Meg Critcher, Jia Meng Pang, Mia L Huang","doi":"10.1021/acschembio.4c00475","DOIUrl":"https://doi.org/10.1021/acschembio.4c00475","url":null,"abstract":"<p><p>Fibroblast growth factor 2 (FGF2) is a multipotent growth factor and signaling protein that exhibits broad functions across multiple cell types. These functions are often initiated by binding to growth factor receptors and fine-tuned by glycosaminoglycan (GAG)-modified proteins called proteoglycans. The various outputs of FGF2 signaling and functions arise from a dynamic and cell type-specific set of binding partners. However, the interactome of FGF2 has yet to be comprehensively determined. Moreover, the identity of the proteoglycan proteins carrying GAG chains is often overlooked and remains unknown in most cell contexts. Here, we perform peroxidase-catalyzed live cell proximity labeling using an engineered APEX2-FGF2 fusion protein to map the interactome of FGF2. Across two cell lines with established and distinct FGF2-driven functions, we greatly expand upon the known FGF2 interactome, identifying >600 new putative FGF2 interactors. Notably, our results demonstrate a key role for the GAG binding capacity of FGF2 in modulating its interactome.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142862499","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"γ-Secretase Cleaves Bifunctional Fatty Acid-Conjugated Small Molecules with Amide Bonds in Mammalian Cells.","authors":"Kai Tahara, Akinobu Nakamura, Xiaotong Wang, Keishi Mitamura, Yuki Ichihashi, Keiko Kano, Emi Mishiro-Sato, Kazuhiro Aoki, Yasuteru Urano, Toru Komatsu, Shinya Tsukiji","doi":"10.1021/acschembio.4c00432","DOIUrl":"10.1021/acschembio.4c00432","url":null,"abstract":"<p><p>Connecting two small molecules, such as ligands, fluorophores, or lipids, together via a linker with amide bonds is a widely used strategy to generate synthetic bifunctional molecules for various biological and biomedical applications. Such bifunctional molecules have been used in live-cell experiments under the assumption that they should be stable in cells. However, we recently found that a membrane-targeting bifunctional molecule, composed of a lipopeptide and the small-molecule ligand trimethoprim, referred to as mgcTMP, underwent amide-bond cleavage in mammalian cells. In this work, we first identified γ-secretase as the major protease degrading mgcTMP in cells. We next investigated the intracellular degradation of several different types of amide-linked bifunctional compounds and found that <i>N</i>-terminally fatty acid-conjugated small molecules are susceptible to γ-secretase-mediated amide-bond cleavage. In contrast, amide-linked bifunctional molecules composed of two small molecules, such as ligands and hydrophobic groups, which lack lipid modification, did not undergo intracellular degradation. These findings highlight a previously overlooked consideration for the development and application of lipid-based bifunctional molecules in chemical biology research.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":"2438-2450"},"PeriodicalIF":3.5,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142680231","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Expanding the Substrate Selectivity of the Fimsbactin Biosynthetic Adenylation Domain, FbsH.","authors":"Syed Fardin Ahmed, Adam Balutowski, Jinping Yang, Timothy A Wencewicz, Andrew M Gulick","doi":"10.1021/acschembio.4c00512","DOIUrl":"10.1021/acschembio.4c00512","url":null,"abstract":"<p><p>Nonribosomal peptide synthetases (NRPSs) produce diverse natural products including siderophores, chelating agents that many pathogenic bacteria produce to survive in low iron conditions. Engineering NRPSs to produce diverse siderophore analogs could lead to the generation of novel antibiotics and imaging agents that take advantage of this unique iron uptake system in bacteria. The highly pathogenic and antibiotic-resistant bacteria <i>Acinetobacter baumannii</i> produces fimsbactin, an unusual branched siderophore with iron-binding catechol groups bound to a serine or threonine side chain. To explore the substrate promiscuity of the assembly line enzymes, we report a structure-guided investigation of the stand-alone aryl adenylation enzyme FbsH. We report structures bound to its native substrate 2,3-dihydroxybenzoic acid (DHB) as well as an inhibitor that mimics the adenylate intermediate. We produced enzyme variants with an expanded binding pocket that are more tolerant for analogs containing a DHB C4 modification. Wild-type and mutant enzymes were then used in an in vitro reconstitution analysis to assess the production of analogs of the final product as well as several early stage intermediates. This analysis shows that some altered substrates progress down the fimsbactin assembly line to the downstream domains. However, analogs from alternate building blocks are produced at lower levels, indicating that selectivity exists in the downstream catalytic domains. These findings expand the substrate scope of producing condensation products between serine and aryl acids and identify the bottlenecks for chemoenzymatic production of fimsbactin analogs.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":"2451-2461"},"PeriodicalIF":3.5,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142602145","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}