RSC Chemical Biology最新文献

{"title":"The mycobacterium lipid transporter MmpL3 is dimeric in detergent solution, SMALPs and reconstituted nanodiscs†","authors":"Sara Cioccolo, Joseph D. Barritt, Naomi Pollock, Zoe Hall, Julia Babuta, Pooja Sridhar, Alicia Just, Nina Morgner, Tim Dafforn, Ian Gould and Bernadette Byrne","doi":"10.1039/D4CB00110A","DOIUrl":"10.1039/D4CB00110A","url":null,"abstract":"<p >The mycobacterial membrane protein large 3 (MmpL3) transports key precursor lipids to the outer membrane of Mycobacterium species. Multiple structures of MmpL3 from both <em>M. tuberculosis</em> and <em>M. smegmatis</em> in various conformational states indicate that the protein is both structurally and functionally monomeric. However, most other resistance, nodulation and cell division (RND) transporters structurally characterised to date are either dimeric or trimeric. Here we present an in depth biophysical and computational analysis revealing that MmpL3 from <em>M. smegmatis</em> exists as a dimer in a variety of membrane mimetic systems (SMALPs, detergent-based solution and nanodiscs). Sucrose gradient separation of MmpL3 populations from <em>M. smegmatis</em>, reconstituted into nanodiscs, identified monomeric and dimeric populations of the protein using laser induced liquid bead ion desorption (LILBID), a native mass spectrometry technique. Preliminary cryo-EM analysis confirmed that MmpL3 forms physiological dimers. Untargeted lipidomics experiments on membrane protein co-purified lipids revealed PE and PG lipid classes were predominant. Molecular dynamics (MD) simulations, in the presence of physiologically-relevant lipid compositions revealed the likely dimer interface.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.2,"publicationDate":"2024-07-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.rsc.org/en/content/articlepdf/2024/cb/d4cb00110a?page=search","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141869244","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":"Enhancement of tryptophan 2-monooxygenase thermostability by semi-rational enzyme engineering: a strategic design to minimize experimental investigation†","authors":"Sirus Kongjaroon, Narin Lawan, Duangthip Trisrivirat and Pimchai Chaiyen","doi":"10.1039/D4CB00102H","DOIUrl":"10.1039/D4CB00102H","url":null,"abstract":"<p >Tryptophan 2-monooxygenase (TMO) is an FAD-bound flavoenzyme which catalyzes the oxidative decarboxylation of <small>L</small>-tryptophan to produce indole-3-acetamide (IAM) and carbon dioxide. The reaction of TMO is the first step of indole-3-acetic acid (IAA) biosynthesis. Although TMO is of interest for mechanistic studies and synthetic biology applications, the enzyme has low thermostability and soluble expression yield. Herein, we employed a combined approach of rational design using computational tools with site-saturation mutagenesis to screen for TMO variants with significantly improved thermostability properties and soluble protein expression. The engineered TMO variants, TMO-PWS and TMO-PWSNR, possess melting temperatures (<em>T</em><small>_m</small>) of 65 °C, 17 °C higher than that of the wild-type enzyme (TMO-WT). At 50 °C, the stabilities (<em>t</em><small>_1/2</small>) of TMO-PWS and TMO-PWSNR were 85-fold and 92.4-fold higher, while their soluble expression yields were 1.4-fold and 2.1-fold greater than TMO-WT, respectively. Remarkably, the kinetic parameters of these variants were similar to those of the wild-type enzymes, illustrating that they are promising candidates for future studies. Molecular dynamic simulations of the wild-type and thermostable TMO variants identified key interactions for enhancing these improvements in the biophysical properties of the TMO variants. The introduced mutations contributed to hydrogen bond formation and an increase in the regional hydrophobicity, thereby, strengthening the TMO structure.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.2,"publicationDate":"2024-07-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.rsc.org/en/content/articlepdf/2024/cb/d4cb00102h?page=search","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141772765","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 a new inhibitor for YTH domain-containing m6A RNA readers†","authors":"Chuan-Hui Wang and Huiqing Zhou","doi":"10.1039/D4CB00105B","DOIUrl":"10.1039/D4CB00105B","url":null,"abstract":"<p > <em>N</em> <small>⁶</small>-methyladenosine (m<small>⁶</small>A) is an abundant modification in mammalian mRNAs and plays important regulatory roles in gene expression, primarily mediated through specific recognition by “reader” proteins. YTH family proteins are one major family of known m<small>⁶</small>A readers, which specifically recognize m<small>⁶</small>A-modified transcripts <em>via</em> the YTH domains. Despite the significant relevance of YTH-m<small>⁶</small>A recognition in biology and diseases, few small molecule inhibitors are available for specifically perturbing this interaction. Here we report the discovery of a new inhibitor (“<strong>N-7</strong>”) for YTH-m<small>⁶</small>A RNA recognition, from the screening of a nucleoside analogue library against the YTH domain of the YTHDF1 protein. <strong>N-7</strong> is characterized to be a <em>pan</em>-inhibitor <em>in vitro</em> against five YTH domains from human YTHDF1, YTHDF2, YTHDF3, YTHDC1, and YTHDC2 proteins, with IC<small>₅₀</small> values in the range of 30–48 μM measured using a fluorescence polarization competition assay. We demonstrated that <strong>N-7</strong> directly interacts with the YTH domain proteins <em>via</em> a thermal shift assay. <strong>N-7</strong> expands the chemical structure landscape of the m<small>⁶</small>A YTH domain-containing reader inhibitors and potentiates future inhibitor development for reader functional studies and therapeutic efforts in targeting the epitranscriptome.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.2,"publicationDate":"2024-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.rsc.org/en/content/articlepdf/2024/cb/d4cb00105b?page=search","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141772766","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":"Metabolically activated proteostasis regulators that protect against erastin-induced ferroptosis†","authors":"Gabriel M. Kline, Nicole Madrazo, Christian M. Cole, Meera Pannikkat, Michael J. Bollong, Jessica D. Rosarda, Jeffery W. Kelly and R. Luke Wiseman","doi":"10.1039/D4CB00027G","DOIUrl":"10.1039/D4CB00027G","url":null,"abstract":"<p >We previously showed that the proteostasis regulator compound AA147 (<em>N</em>-(2-hydroxy-5-methylphenyl)benzenepropanamide) potently protects against neurotoxic insults, such as glutamate-induced oxytosis. Though AA147 is a selective activator of the ATF6 arm of the unfolded protein response in non-neuronal cells, AA147-dependent protection against glutamate toxicity in cells of neuronal origin is primarily mediated through activation of the NRF2 oxidative stress response. AA147 activates NRF2 through a mechanism involving metabolic activation of AA147 by endoplasmic reticulum (ER) oxidases, affording an AA147-based quinone methide that covalently targets the NRF2 repressor protein KEAP1. Previous results show that the 2-amino-<em>p</em>-cresol A-ring of AA147 is required for NRF2 activation, while the phenyl B-ring of AA147 is amenable to modification. Here we explore whether the protease-sensitive amide linker between the A- and B-rings of this molecule can be modified to retain NRF2 activation. We show that replacement of the amide linker of AA147 with a carbamate linker retains NRF2 activation in neuronal cells and improves protection against neurotoxic insults, including glutamate-induced oxytosis and erastin-induced ferroptosis. Moreover, we demonstrate that inclusion of this carbamate linker facilitates identification of next-generation AA147 analogs with improved cellular tolerance and activity in disease-relevant assays.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.2,"publicationDate":"2024-07-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.rsc.org/en/content/articlepdf/2024/cb/d4cb00027g?page=search","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141718946","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":"Identification of small molecules that are synthetically lethal upon knockout of the RNA ligase Rlig1 in human cells†","authors":"Florian M. Stumpf, Silke Müller and Andreas Marx","doi":"10.1039/D4CB00125G","DOIUrl":"10.1039/D4CB00125G","url":null,"abstract":"<p >Rlig1 is the first RNA ligase identified in humans utilising a classical 5′–3′ ligation mechanism. It is a conserved enzyme in all vertebrates and is mutated in various cancers. During our initial research on Rlig1, we observed that Rlig1-knockout (KO) HEK293 cells are more sensitive to the stress induced by menadione than their WT counterpart, representing a type of chemical synthetic lethality. To gain further insight into the biological pathways in which Rlig1 may be involved, we aimed at identifying new synthetically lethal small molecules. To this end, we conducted a high-throughput screening with a compound library comprising over 13 000 bioactive small molecules. This approach led to the identification of compounds that exhibited synthetic lethality in combination with Rlig1-KO. In addition to the aforementioned novel compounds that diverge structurally from menadione, we also tested multiple small molecules containing a naphthoquinone scaffold.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.2,"publicationDate":"2024-07-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.rsc.org/en/content/articlepdf/2024/cb/d4cb00125g?page=search","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141718945","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":"Advanced piperazine-containing inhibitors target microbial β-glucuronidases linked to gut toxicity†","authors":"Amanda L. Graboski, Joshua B. Simpson, Samuel J. Pellock, Naimee Mehta, Benjamin C. Creekmore, Yamuna Ariyarathna, Aadra P. Bhatt, Parth B. Jariwala, Josh J. Sekela, Mark E. Kowalewski, Natalie K. Barker, Angie L. Mordant, Valentina B. Borlandelli, Hermen Overkleeft, Laura E. Herring, Jian Jin, Lindsey I. James and Matthew R. Redinbo","doi":"10.1039/D4CB00058G","DOIUrl":"10.1039/D4CB00058G","url":null,"abstract":"<p >The gut microbiome plays critical roles in human homeostasis, disease progression, and pharmacological efficacy through diverse metabolic pathways. Gut bacterial β-glucuronidase (GUS) enzymes reverse host phase 2 metabolism, in turn releasing active hormones and drugs that can be reabsorbed into systemic circulation to affect homeostasis and promote toxic side effects. The FMN-binding and loop 1 gut microbial GUS proteins have been shown to drive drug and toxin reactivation. Here we report the structure–activity relationships of two selective piperazine-containing bacterial GUS inhibitors. We explore the potency and mechanism of action of novel compounds using purified GUS enzymes and co-crystal structures. Our results establish the importance of the piperazine nitrogen placement and nucleophilicity as well as the presence of a cyclohexyl moiety appended to the aromatic core. Using these insights, we synthesized an improved microbial GUS inhibitor, UNC10206581, that potently inhibits both the FMN-binding and loop 1 GUS enzymes in the human gut microbiome, does not inhibit bovine GUS, and is non-toxic within a relevant dosing range. Kinetic analyses demonstrate that UNC10206581 undergoes a slow-binding and substrate-dependent mechanism of inhibition similar to that of the parent scaffolds. Finally, we show that UNC10206581 displays potent activity within the physiologically relevant systems of microbial cultures and human fecal protein lysates examined by metagenomic and metaproteomic methods. Together, these results highlight the discovery of more effective bacterial GUS inhibitors for the alleviation of microbe-mediated homeostatic dysregulation and drug toxicities and potential therapeutic development.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.2,"publicationDate":"2024-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.rsc.org/en/content/articlepdf/2024/cb/d4cb00058g?page=search","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141722177","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":"Caged aminoluciferin probe for bioluminescent immunoproteasome activity analysis†","authors":"Cody A. Loy and Darci J. Trader","doi":"10.1039/D4CB00148F","DOIUrl":"10.1039/D4CB00148F","url":null,"abstract":"<p >The immunoproteasome (iCP) can be expressed under inflammatory conditions, such as exposure to interferon-gamma (IFN-γ), that alerts the cell to begin generating iCP preferentially over the standard proteasome (sCP). With the iCP becoming a widely targeted isoform in a variety of diseases, there is a need to understand its activity and expression in cells and <em>in vivo.</em> Activity-based probes for the iCP have been developed but their application has been limited due to their difficult synthesis and cannot be used in tissues or whole animals. Our lab has previously demonstrated we can monitor iCP activity using a 4-mer peptide linked to a fluorophore and a peptoid. This was utilized in the development of the first cell-permeable iCP activity-based probe that did not include a covalent reactive moiety. Here, we demonstrate that this same peptide recognition sequence can be appended to aminoluciferin, caging it, until its interaction with the iCP. This probe should be applicable to monitor iCP activity in animal models where tumor or other tissue has been engineered to produce luciferase. We anticipate it could also be applied to observe iCP activity as tumors are formed <em>in vivo</em>.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.2,"publicationDate":"2024-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.rsc.org/en/content/articlepdf/2024/cb/d4cb00148f?page=search","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141718947","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":"Harnessing acetogenic bacteria for one-carbon valorization toward sustainable chemical production","authors":"Jiyun Bae, Chanho Park, Hyunwoo Jung, Sangrak Jin and Byung-Kwan Cho","doi":"10.1039/D4CB00099D","DOIUrl":"10.1039/D4CB00099D","url":null,"abstract":"<p >The pressing climate change issues have intensified the need for a rapid transition towards a bio-based circular carbon economy. Harnessing acetogenic bacteria as biocatalysts to convert C1 compounds such as CO<small>₂</small>, CO, formate, or methanol into value-added multicarbon chemicals is a promising solution for both carbon capture and utilization, enabling sustainable and green chemical production. Recent advances in the metabolic engineering of acetogens have expanded the range of commodity chemicals and biofuels produced from C1 compounds. However, producing energy-demanding high-value chemicals on an industrial scale from C1 substrates remains challenging because of the inherent energetic limitations of acetogenic bacteria. Therefore, overcoming this hurdle is necessary to scale up the acetogenic C1 conversion process and realize a circular carbon economy. This review overviews the acetogenic bacteria and their potential as sustainable and green chemical production platforms. Recent efforts to address these challenges have focused on enhancing the ATP and redox availability of acetogens to improve their energetics and conversion performances. Furthermore, promising technologies that leverage low-cost, sustainable energy sources such as electricity and light are discussed to improve the sustainability of the overall process. Finally, we review emerging technologies that accelerate the development of high-performance acetogenic bacteria suitable for industrial-scale production and address the economic sustainability of acetogenic C1 conversion. Overall, harnessing acetogenic bacteria for C1 valorization offers a promising route toward sustainable and green chemical production, aligning with the circular economy concept.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.2,"publicationDate":"2024-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.rsc.org/en/content/articlepdf/2024/cb/d4cb00099d?page=search","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141567824","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":"Correction: Virtual screening, identification and in vitro validation of small molecule GDP-mannose dehydrogenase inhibitors","authors":"Jonathan P. Dolan, Sanaz Ahmadipour, Alice J. C. Wahart, Aisling Ní Cheallaigh, Suat Sari, Chatchakorn Eurtivong, Marcelo A. Lima, Mark A. Skidmore, Konstantin P. Volcho, Jóhannes Reynisson, Robert A. Field and Gavin J. Miller","doi":"10.1039/D4CB90026J","DOIUrl":"10.1039/D4CB90026J","url":null,"abstract":"<p >Correction for ‘Virtual screening, identification and <em>in vitro</em> validation of small molecule GDP-mannose dehydrogenase inhibitors’ by Jonathan P. Dolan <em>et al.</em>, <em>RSC Chem. Biol.</em>, 2023, <strong>4</strong>, 865–870, https://doi.org/10.1039/D3CB00126A.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.2,"publicationDate":"2024-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.rsc.org/en/content/articlepdf/2024/cb/d4cb90026j?page=search","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141567823","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 toolbox for enzymatic modification of nucleic acids with photosensitizers for photodynamic therapy†","authors":"Germain Niogret, Camille Chériaux, Frédéric Bonhomme, Fabienne Levi-Acobas, Carlotta Figliola, Gilles Ulrich, Gilles Gasser and Marcel Hollenstein","doi":"10.1039/D4CB00103F","DOIUrl":"10.1039/D4CB00103F","url":null,"abstract":"<p >Photodynamic therapy (PDT) is an approved cancer treatment modality. Despite its high efficiency, PDT is limited in terms of specificity and by the poor solubility of the rather lipophilic photosensitizers (PSs). In order to alleviate these limitations, PSs can be conjugated to oligonucleotides. However, most conjugation methods often involve complex organic synthesis and result in the appendage of single modifications at the 3′/5′ termini of oligonucleotides. Here, we have investigated the possibility of bioconjugating a range of known PSs by polymerase-mediated synthesis. We have prepared a range of modified nucleoside triphosphates by different conjugation methods and investigated the substrate tolerance of these nucleotides for template-dependent and -independent DNA polymerases. This method represents a mild and versatile approach for the conjugation of single or multiple PSs onto oligonucleotides and can be useful to further improve the efficiency of the PDT treatment.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.2,"publicationDate":"2024-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.rsc.org/en/content/articlepdf/2024/cb/d4cb00103f?page=search","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141567822","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}