RSC Chemical Biology最新文献

{"title":"A Lyt at the end of the tunnel? Unraveling the complex interactions of the <i>N</i>-acetylglucosaminidase LytG in cell wall metabolism.","authors":"Jazmeen Hernandez, Jett Duval, Taryn Rauff, Ethan Hall, Mika Gallati, Brad A Haubrich, Monica Thoma, Elimelec Aponte, Amit Basu, Joseph A DeGiorgis, Christopher W Reid","doi":"10.1039/d5cb00151j","DOIUrl":"10.1039/d5cb00151j","url":null,"abstract":"<p><p>The growth and division of the Gram-positive cell requires the coordinated action of enzymes involved in the synthesis and degradation of the heteropolymer peptidoglycan. Herein, we present the use of the diamide masarimycin, an inhibitor of the <i>exo-N</i>-acetylglucosaminidase (GlcNAcase) LytG from <i>Bacillus subtilis</i>, as a chemical biology probe to elucidate the biological role of this cell wall degrading enzyme. Using a combination of chemical biology and genetic approaches we provide the first evidence that LytG activity influences the elongation and division complexes in <i>B. subtilis</i>. Chemical inhibition of LytG resulted in dysregulated cell elongation and localization of the division plane and the induction of the cell wall stress response. In the presence of masarimycin, cells show asymmetrical thickening of the cell wall and dysregulation of division plane localization. The use of genetic and synergy/antagonism screens established connections to late-stage peptidoglycan synthesis, particularly related to cross-linking function. These results stand in stark contrast to those observed for the Δ<i>lytG</i> knockout, which does not exhibit these phenotypes. Cell-wall labelling with a fluorescent d-amino acid and muropeptide analysis has highlighted a functional connection between LytG, the carboxypeptidase DacA, and d,d-endopeptidases. These results highlight the use of chemical probes such as masarimycin to inform on the biological function of autolysins by providing insight into the role LytG plays in cell growth and division.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" ","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12505155/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145259501","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Discovery of microbial glycoside hydrolases <i>via</i> enrichment and metaproteomics.","authors":"Jitske M van Ede, Suzanne van der Steen, Geert M van der Kraan, Mark C M van Loosdrecht, Martin Pabst","doi":"10.1039/d5cb00049a","DOIUrl":"10.1039/d5cb00049a","url":null,"abstract":"<p><p>The immense microbial diversity on Earth represents a vast genomic resource, yet discovering novel enzymes from complex environments remains challenging. Here, we combine a microbial enrichment with metagenomics and metaproteomics to facilitate the identification of microbial glycoside hydrolases that operate under defined conditions. We enriched microbial communities on the carbohydrate polymer pullulan at elevated temperatures under acidic conditions. Pullulan is a natural polysaccharide composed of maltotriose units linked by α-1,6-glycosidic bonds. Pullulan, along with its hydrolyzing enzymes, has broad applications across various industries. The enrichment inocula were sampled from thermophilic compost and from soil from the bank of a pond. In both cases, <i>Alicyclobacillus</i> was identified as the dominant microorganism. Metaproteomic analysis of the enriched biomass and secretome enabled the identification of several pullulan-degrading enzyme candidates from this organism. These enzymes were absent in the metagenomic analysis of the initial inoculum, which is highly complex with a wide diversity of species. This underscores the effectiveness of combining microbial enrichment with multi-omics for uncovering novel enzymes and sequence variants that operate under defined conditions from complex microbial environments.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" ","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12510369/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145281472","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Sweet dicamba: a carbohydrate pro-herbicide strategy.","authors":"Karen J Deane, Joel Haywood, Michael D Wallace, Kalia Bernath-Levin, Mark T Waters, Joshua S Mylne, Keith A Stubbs","doi":"10.1039/d5cb00208g","DOIUrl":"10.1039/d5cb00208g","url":null,"abstract":"<p><p>Dicamba, although a potent and useful herbicide in weed management, is notorious for its off-target movement due to volatility. Here, we describe carbohydrate esters of dicamba as an unexplored pro-herbicide approach that addresses the volatility of dicamba, while maintaining its herbicidal qualities. Varying the carbohydrate and dicamba attachment point led to changes in potency and hydrolysis, potentially allowing for reactivity tuning of molecules for future weed management practices.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" ","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-09-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12461606/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145186508","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Profiling polyamine-protein interactions in live cells through photoaffinity labeling.","authors":"Maciej Zakrzewski, Zuzanna Sas, Benjamin Cocom-Chan, Moh Egy Rahman Firdaus, Marcin Kałek, Karolina Szczepanowska, Piotr Gerlach, Anna Marusiak, Remigiusz A Serwa","doi":"10.1039/d5cb00103j","DOIUrl":"10.1039/d5cb00103j","url":null,"abstract":"<p><p>Polyamines are essential metabolites that play a crucial role in regulating key cellular processes. While previous studies have shown that polyamines modulate protein function through non-covalent interactions, the lack of robust analytical methods has limited the systematic identification of these interactions in living cells. To address this challenge, we synthesized a series of novel photoaffinity probes and applied them to a model cell line, identifying over 400 putative protein interactors with remarkable polyamine analog structure-dependent specificity. Analysis of probe-modified peptides revealed photocrosslinking sites for dozens of protein binders and demonstrated that all but one of the probes, the spermine analog, were intracellularly stable. The interaction profiles of these probes were visualized through in-gel fluorescence scanning, and their subcellular localization was examined using fluorescence microscopy. Spermidine analogs interacted with proteins in the nucleoplasm, colocalizing with nucleolar and nuclear-speckle proteins, as well as in the cytoplasm. By contrast, diamine analogs localized to vesicle-like structures near the Golgi apparatus, implying that different polyamine types exhibit a proclivity for specific cellular compartments. Notably, spermidine analogs bound preferentially to proteins containing acidic stretches, often located within intrinsically disordered regions. Focusing on one such case, we provide <i>in-cellulo</i> evidence of direct interactions between G3BP1/2 and spermidine analogs and advance the hypothesis that such interactions influence stress-granule dynamics. Overall, this study provides a comprehensive profile of polyamine analogs-protein interactions in live cells, offering valuable insights into their roles in cellular physiology.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" ","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12461607/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145187187","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Dynamics of single enzymes confined inside a nanopore.","authors":"Nicole Stéphanie Galenkamp, Marco van den Noort, Giovanni Maglia","doi":"10.1039/d5cb00149h","DOIUrl":"10.1039/d5cb00149h","url":null,"abstract":"<p><p>Enzymes are powerful catalysts that perform chemical reactions with remarkable speed and specificity. Their intrinsic dynamics often play a crucial role in determining their catalytic properties. To achieve a comprehensive understanding of enzymes, a diverse and sophisticated experimental toolbox capable of studying enzyme dynamics at the single-molecule level is necessary. In this review, we discuss nanopore technology as an emerging and powerful platform in single-molecule enzymology. We demonstrate how nanopores can be employed to probe enzyme dynamics in real-time, and we highlight how these studies have contributed to fundamentally and quantitatively elucidating enzymological concepts, such as allostery and hysteresis. Finally, we explore the potentials and limitations of nanopores in advancing single-molecule enzymology. By presenting the unique possibilities offered by nanopores, we aim to inspire the integration of this technology into future enzymology research.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" ","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12445297/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145114405","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Convergent construction of N-terminally modified CCL5 chemokines for photoaffinity receptor pull-down using cross-aldol bioconjugations.","authors":"Afzaal Tufail, Matthew E Warnes, Nathalie Signoret, Martin A Fascione","doi":"10.1039/d5cb00162e","DOIUrl":"10.1039/d5cb00162e","url":null,"abstract":"<p><p>Chemokines such as CCL5 (RANTES) mediate immune responses <i>via</i> interaction with G-protein-coupled receptors like CCR5, which also serves as a co-receptor for HIV-1 entry into host cells. Modified CCL5 analogues have shown promise as CCR5 antagonists for anti-HIV strategies, but current approaches involve hydrolytically unstable linkages or laborious synthesis. Here, we demonstrate the use of an organocatalyst-mediated protein aldol ligation (OPAL) to construct N-terminally modified CCL5 analogues bearing hydrolytically stable carbon-carbon linkages. Using a recombinant CCL5 P2G mutant and selective oxidation to introduce an α-oxo aldehyde at the N-terminus, we achieved efficient OPAL bioconjugation with various aldehyde donors, including alkyl and aryl acetaldehydes. Notably, a 4-azido aryl acetaldehyde CCL5 OPAL product was utilised as a CCR5 photoaffinity probe. This modified chemokine successfully captured CCR5 from mammalian cells <i>via</i> photo-crosslinking, enabling receptor pull-down for biochemical analysis. Our work showcases cross-aldol bioconjugations as a versatile and convergent strategy for stable chemokine functionalisation, with potential applications in therapeutic development and mechanistic studies of chemokine-receptor interactions. This method offers a promising chemical biology platform for modulating or probing the CCL5-CCR5 axis with enhanced precision and synthetic accessibility.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" ","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12455665/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145138587","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A trifunctional probe for generation of fluorogenic glycan-photocrosslinker conjugates.","authors":"Brandon Vreulz, Daphnée De Crozals, Samy Cecioni","doi":"10.1039/d5cb00206k","DOIUrl":"10.1039/d5cb00206k","url":null,"abstract":"<p><p>Interactions between cell surface glycans and lectins mediate vital biological processes, yet their characterization is hindered by the low affinity of these binding events. While photoaffinity labeling can capture these interactions, traditional custom probes often demand tedious synthesis, are limited to simple glycans, and lack versatility. To overcome these limitations, we report a trifunctional scaffold enabling modular assembly of glycan probes. This scaffold integrates orthogonal sites for: (i) efficient late-stage ligation of native oligosaccharides <i>via</i> an <i>N</i>-alkoxy-amine, preserving glycan structure; (ii) flexible amide coupling of various photocrosslinkers, including a recently developed fluorogenic azidocoumarin for traceable labeling; and (iii) conjugation to reporter tags (<i>e.g.</i>, biotin) or multivalent carriers through a carboxylic acid motif. We demonstrate the scaffold's utility by synthesizing probes bearing various fucosylated glycans. Probes incorporating the fluorogenic photocrosslinker achieved specific, light-induced labeling of the model lectin BambL. The platform's adaptability was further confirmed by generating monovalent biotinylated probes displaying the photoactive glycan. This modular strategy offers a practical solution to rapidly construct advanced chemical probes, facilitating the investigation of complex glycan recognition events in diverse biological systems.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" ","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12459285/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145151474","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Novel terbium-sensitizing peptide substrates for cyclin-dependent kinase 5 (CDK5) and their demonstration in luminescence kinase assays.","authors":"Jason L Heier, Dylan J Boselli, Laurie L Parker","doi":"10.1039/d5cb00189g","DOIUrl":"10.1039/d5cb00189g","url":null,"abstract":"<p><p>Novel time-resolved terbium luminescence assays were developed for CDK5 and CDK2 by designing synthetic substrates which incorporate phospho-inducible terbium sensitizing motifs with kinase substrate consensus sequences. A substrate designed for CDK5 showed no phosphorylation by CDK2, opening the possibility for CDK5-specific assay development for selective drug discovery.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" ","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12441597/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145087654","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Sources of mismeasurement of RNA knockdown by DNAzymes and XNAzymes","authors":"Maria J. Donde, Alicia Montulet and Alexander I. Taylor","doi":"10.1039/D5CB00182J","DOIUrl":"10.1039/D5CB00182J","url":null,"abstract":"<p >RNA-cleaving oligonucleotide catalysts composed of DNA and/or nucleic acid analogues (DNAzymes, modified DNAzymes and XNAzymes) are promising agents for specific knockdown of disease-associated RNAs. However, we and others have identified discrepancies between their apparent activity <em>in vitro versus</em> when transfected into cells. Here, using examples of catalysts targeting the codon 12 region of <em>KRAS</em> RNA – an unmodified DNAzyme based on the classic “10–23” motif, a modified DNAzyme (“10–23_v46”) or an XNAzyme (“FR6_1_KRas12B”) – we examine confounding effects including unintended activity during standard RNA work-up steps, leading to mismeasurement of knockdown. We find that catalysts are not irreversibly denatured by typical cell lysis reagents, nor fully degraded by typical DNase treatments, exacerbated by nuclease resistant modification chemistries. In standard RT-qPCR workflows, DNAzymes and XNAzymes were found to be capable of cleaving their target RNAs during (1) DNase treatment and (2) reverse transcription (RT) reactions, in both instances with enhanced rates compared with under quasi-physiological conditions, producing cleavage-dependent false positives. Furthermore, catalysts were found to site-specifically inhibit cDNA synthesis (<em>i.e.</em> producing cleavage-independent false positives) and in the case of DNAzymes also had the capacity to act as primers during RT, leading to an enhancement of target site cDNA as judged by digital PCR, producing (cleavage-independent) false negatives. These effects could be broadly mitigated by purification to remove catalysts at the point of RNA extraction, under denaturing conditions. We recommend that studies of oligo catalysts in cells must include a 0 h timepoint after catalyst delivery or transfection to assess the collective impact of these mismeasurements on a case by case basis.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" 10","pages":" 1595-1606"},"PeriodicalIF":3.1,"publicationDate":"2025-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12426771/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145065981","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cell-based high-throughput screening using a target-NanoLuc fusion construct to identify molecular glue degraders of c-Myc oncoprotein.","authors":"Muyu Xu, Jinying Qiu, Lin Tan, Jiayu Xu, Yi Wang, Wenyue Kong, Hongda Liao, Anran Chen, Xiaolan Chen, Jiying Zhang, Cookson K C Chiu, Meiying Zhang, Yingying Tian, Caohui Li, Biao Ma, Leiming Wang, Jingpeng Fu, Seung H Choi, Jeffrey Hill, Weijun Shen","doi":"10.1039/d5cb00093a","DOIUrl":"10.1039/d5cb00093a","url":null,"abstract":"<p><p>Oncoprotein c-Myc (Myc) plays a critical role in regulating cellular gene expression. Although Myc dysregulation is found in more than 70% of cancers and can facilitate tumor initiation and progression, it is still considered to be an \"undruggable\" oncotarget years after its first discovery. Recent advances in the field of targeted protein degradation provide alternative Myc-targeting strategies. Here, we develop the first Myc-NanoLuc fusion plasmid transfected cell-based high-throughput screening assay to identify Myc-downregulating small molecules. We verified the effectiveness of our assay by demonstrating that previously known Myc-downregulating compounds (G9 and SY-1365) were successfully identified from a library of bioactive compounds with established biological function. Next, we screened another 108 800 compounds from the diverse ChemDiv library collection, and 14 novel Myc-downregulating compounds were identified after cherry-pick triplicate confirmation, counter-screening, dose-response and western blotting experiments. A cellular thermal shift assay further demonstrated that five out of the 14 Myc-downregulating compounds bound to endogenous Myc protein in crude 293T whole-cell lysate. Subsequently, compound C1 was shown to selectively degrade Myc protein at a DC₅₀ value of around 5 μM. Further characterization showed that C1 killed cancer cells with high Myc expression at a lower dose than it killed cancer cells with low Myc expression. Moreover, C1 selectively reduced the expression of various Myc-target genes. Intriguingly, co-immunoprecipitation showed that C1 functionally acted like a molecular glue to aggregate Myc proteins and block Myc/Max interaction. The self-aggregation of Myc and the dissociation of the Myc/Max dimer by C1 promoted Myc degradation. Using a target-NanoLuc fusion strategy in our novel cell-based high-throughput screening system, we identified a molecular glue-like small molecule degrader of Myc.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" ","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12447757/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145114433","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}