ACS Chemical Biology最新文献

{"title":"Reconstitution and Characterization of Biosynthetic Machinery for Parageocin I, a Novel Thiazole-Rich Peptide from the Thermophilic Bacterium Parageobacillus caldoxylosilyticus","authors":"Ayane Yano,&nbsp;Hiroya Tomita*,&nbsp;Kentaro Miyazaki and Kohsuke Honda,&nbsp;","doi":"10.1021/acschembio.4c0075810.1021/acschembio.4c00758","DOIUrl":"https://doi.org/10.1021/acschembio.4c00758https://doi.org/10.1021/acschembio.4c00758","url":null,"abstract":"<p >Ribosomally synthesized and post-translationally modified peptides (RiPPs) are the representative microbial peptidyl secondary metabolites including the class of linear azol(in)e-containing peptides (LAPs). A substantial proportion of LAPs have been identified in mesophilic microorganisms, including actinomycetes. In this study, we report the biosynthetic reconstitution and characterization of parageocin I, a novel thiazole-rich LAP derived from the thermophilic bacterium <i>Parageobacillus caldoxylosilyticus</i> KH1-5 which exhibits optimal growth around 60 °C. The biosynthetic gene cluster (<i>pgc</i>) consists of four genes: <i>pgcA</i>, <i>pgcB</i>, <i>pgcC</i>, and <i>pgcD</i>, encoding the precursor peptide, dehydrogenase, YcaO family cyclodehydratase, and biosynthetic scaffold protein, respectively. The precursor peptide PgcA possesses 13 Cys and 2 Ser residues, with regularly repeated sequences interspaced between Cys residues. We first reconstituted the biosynthesis heterologously in <i>Escherichia coli</i>. Mass spectrometry analysis of the synthesized peptide, coupled with mutational analyses of the modified PgcA, revealed that the final product, designated as parageocin I, harbors 13 thiazole rings derived from the cyclization of Cys residues, while Ser residues remain intact. Furthermore, mutational studies of PgcA revealed three key principles governing heterocyclization by PgcC: (i) Cys is acceptable, but Ser and Thr are not; (ii) the presence of an acidic amino acid preceding Cys is not permissible; and (iii) a minimum of two amino acids must separate Cys residues. In addition, we successfully reconstituted the biosynthesis in vitro using the purified recombinant enzymes. This is the first report of LAP biosynthesis in thermophilic Bacillaceae, thereby expanding our understanding of not only LAPs but also secondary metabolism in thermophiles.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":"20 4","pages":"815–822 815–822"},"PeriodicalIF":3.5,"publicationDate":"2025-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143842272","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":"Amber Codon Mutational Scanning and Bioorthogonal PEGylation for Epitope Mapping of Antibody Binding Sites on Human Arginase-1","authors":"Jaime Fernández de Santaella,&nbsp;Nikolaj G. Koch,&nbsp;Lorenz Widmer and Michael A. Nash*,&nbsp;","doi":"10.1021/acschembio.4c0069210.1021/acschembio.4c00692","DOIUrl":"https://doi.org/10.1021/acschembio.4c00692https://doi.org/10.1021/acschembio.4c00692","url":null,"abstract":"<p >Epitope mapping is crucial for understanding immunological responses to protein therapeutics. Here, we combined genetic code expansion and bacterial surface display to incorporate S-allylcysteine (SAC) into human arginase-1 (hArg1) via <i>Methanococcoides burtonii</i> pyrrolysyl-tRNA synthetase. Using an amber codon deep mutational scanning and sequencing workflow, we mapped SAC incorporation efficiency across the hArg1 sequence, providing insights into structural and sequence dependencies of noncanonical amino acid incorporation. We used mutually bioorthogonal allyl/tetrazine and azide/DBCO chemistries to achieve site-specific PEGylation and fluorescent labeling of hArg1, revealing insights into SAC side chain reactivity and solvent accessibility of residues in hArg1. This system was further applied to determine the binding epitope of a monoclonal antibody on the surface of hArg1, providing high-resolution data on the impact of PEGylation residue position on antibody binding. Our method produces high dimensional data of noncanonical amino acid incorporation efficiency, site-specific functionalization enabled by mutually bioorthogonal chemistries, and epitope mapping of therapeutic proteins.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":"20 4","pages":"791–801 791–801"},"PeriodicalIF":3.5,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143842217","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":"Structural and Biochemical Insights into Lignin-Oxidizing Activity of Bacterial Peroxidases against Soluble Substrates and Kraft Lignin","authors":"Zahra Choolaei,&nbsp;Anna N. Khusnutdinova,&nbsp;Tatiana Skarina,&nbsp;Peter Stogios,&nbsp;Patrick Diep,&nbsp;Sofia Lemak,&nbsp;Elizabeth A. Edwards,&nbsp;Alexei Savchenko and Alexander F. Yakunin*,&nbsp;","doi":"10.1021/acschembio.4c0078810.1021/acschembio.4c00788","DOIUrl":"https://doi.org/10.1021/acschembio.4c00788https://doi.org/10.1021/acschembio.4c00788","url":null,"abstract":"<p >Great interest has recently been drawn to the production of value-added products from lignin; however, its recalcitrance and high chemical complexity have made this challenging. Dye-decolorizing peroxidases and catalase-peroxidases are among the enzymes that are recognized to play important roles in environmental lignin oxidation. However, bacterial lignin-oxidizing enzymes remain less characterized compared to related proteins from fungi. In this study, screening of 18 purified bacterial peroxidases against the general chromogenic substrate 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) revealed the presence of peroxidase activity in all proteins. Agarose plate-based screens with kraft lignin identified detectable and high lignin oxidation activity in 15 purified proteins. Crystal structures were determined for the DyP-type peroxidases FC2591 from <i>Frankia casuarinae</i>, PF3257 from <i>Pseudomonas fluorescens</i>, and PR9465 from <i>Pseudomonas rhizosphaerae</i>. The structures revealed the presence of hemes with bound oxygens coordinated by conserved His, Arg, and Asp residues as well as three molecular tunnels connecting the heme with the protein surface. Structure-based site-directed mutagenesis of FC2591 identified at least five active site residues as essential for oxidase activity against both ABTS and lignin, whereas the S370A mutant protein showed a three- to 4-fold activity increase with both substrates. HPLC analysis of reaction products of the wild-type FC2591 and S370A mutant proteins with the model lignin dimer guaiacylglycerol-β-guaiacyl ether and kraft lignin revealed the formation of products consistent with the radical coupling of the reaction intermediates. Thus, this study identified novel bacterial heme peroxidases with lignin oxidation activity and provided further insights into our understanding of these enzymes.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":"20 4","pages":"830–844 830–844"},"PeriodicalIF":3.5,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143842298","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":"Chitinivorax: The New Kid on the Block of Bacterial 2-Alkyl-4(1H)-quinolone Producers","authors":"Viktoriia Savchenko,&nbsp;Xiaoqian Annie Yu,&nbsp;Martin F. Polz and Thomas Böttcher*,&nbsp;","doi":"10.1021/acschembio.5c0004610.1021/acschembio.5c00046","DOIUrl":"https://doi.org/10.1021/acschembio.5c00046https://doi.org/10.1021/acschembio.5c00046","url":null,"abstract":"<p >2-Alkyl-4(1<i>H</i>)-quinolones play a key role in bacterial communication, regulating biofilm formation, and virulence. Their antimicrobial properties also support bacterial survival and interspecies competition in microbial communities. In addition to the human pathogen <i>Pseudomonas aeruginosa</i> various species of <i>Burkholderia</i> and <i>Pseudoalteromonas</i> are known to produce 2-alkyl-4(1<i>H</i>)-quinolones. However, the evolutionary relationships of their biosynthetic gene clusters remain largely unexplored. To address this, we investigated the phylogeny of 2-alkyl-4(1<i>H</i>)-quinolone biosynthetic gene clusters, leading to the discovery of <i>Chitinivorax</i> as a fourth genus capable of producing 2-alkyl-4(1<i>H</i>)-quinolones, expanding our knowledge of the diversity of bacteria involved in quinolone-biosynthesis.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":"20 4","pages":"960–966 960–966"},"PeriodicalIF":3.5,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acschembio.5c00046","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143842313","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Formation of the i-motif Structures by Human Telomeric c-Rich Sequences d(CCCTAA)n and Its Recognition by Bisbenzylisoquinoline Alkaloids","authors":"Junliu Huang,&nbsp;Zexuan Lin,&nbsp;Jishun Yang,&nbsp;Huining Tang,&nbsp;Yang Yang,&nbsp;Yi Tang,&nbsp;Feixian Luo,&nbsp;Wenshu Wang and Xiaojie Cui*,&nbsp;","doi":"10.1021/acschembio.4c0084410.1021/acschembio.4c00844","DOIUrl":"https://doi.org/10.1021/acschembio.4c00844https://doi.org/10.1021/acschembio.4c00844","url":null,"abstract":"<p >The human telomeric repeat CCCTAA has been reported to form a higher-order structure called an intercalated motif (i-motif) that plays important roles in telomere function and telomerase activity regulation, and small molecule ligands targeting human telomeric i-motif (hTelo-iM) is a promising therapeutic strategy for cancer treatment, yet the i-motif folding pattern of long CCCTAA repeats and the hTelo-iM ligand screening have not been studied extensively. In this study, we systematically investigated the i-motif structures formed by four and eight telomeric C-rich repeats d(CCCTAA)₄ (hTeloC-24mer) and d(CCCTAA)₈ (hTeloC-48mer) under varied conditions and found that the long hTeloC-48mer probably forms unstacked tandem i-motif consisting of two hTeloC-24mer i-motif monomers under near physiological conditions. Moreover, natural bisbenzylisoquinoline (BBI) alkaloids, isofangchinoline, fangchinoline, cepharanthine, and tetrandrine, were screened from 33 natural small molecules to effectively disrupt and destabilize the hTelo-iM structures mainly through major groove hydrogen bonding and van der Waals interactions. Further, telomerase repeated amplification protocol (TRAP) assay suggested that the selected BBI alkaloids can inhibit the telomere extension by telomerase. These findings provide a theoretical basis for further telomere structure research as well as a novel class of natural small molecule compounds regulating the hTelo-iM structure and telomerase activity, which may contribute to the anticancer drug design and strategy development taking the hTelo-iM as a target.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":"20 4","pages":"870–879 870–879"},"PeriodicalIF":3.5,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143842312","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":"MYC-Targeting PROTACs Lead to Bimodal Degradation and N-Terminal Truncation","authors":"Shelton R. Boyd,&nbsp;Srinivas Chamakuri,&nbsp;Alexander J. Trostle,&nbsp;Hu Chen,&nbsp;Zhandong Liu,&nbsp;Antrix Jian,&nbsp;Jian Wang,&nbsp;Anna Malovannaya and Damian W. Young*,&nbsp;","doi":"10.1021/acschembio.4c0086410.1021/acschembio.4c00864","DOIUrl":"https://doi.org/10.1021/acschembio.4c00864https://doi.org/10.1021/acschembio.4c00864","url":null,"abstract":"<p >MYC is a master regulatory transcription factor whose sustained dysregulation promotes the initiation and maintenance of numerous cancers. While MYC is a regarded as a potenial therapeutic target in cancer, its intrinsically disordered structure has proven to be a formidable barrier toward the development of highly effective small molecule inhibitors. We rationalized that proteolysis targeting chimeras (PROTACs), which might accomplish the targeted degradation of MYC, would achieve more potent cell killing in MYC-driven cancer cells than reversible inhibitors. PROTACs are bifunctional small molecules designed to produce a ternary complex between a target protein and an E3 ligase leading the target’s ubiquitination and degradation by the 26S proteasome. We generated PROTAC MTP3 based on modifications of the previously reported MYC-targeting compound KJ-Pyr-9. We found that MTP3 depletes endogenous full-length MYC proteins and uniquely induces increasing levels of a functional, N-terminally truncated MYC species, tMYC. Furthermore, MTP3 perturbs cellular MYC levels in favor of a tMYC-dominated state whose gene regulatory landscape is not significantly altered compared to that of wild type MYC. Moreover, although it lacks ∼10 kDa of MYC’s N-terminal transactivation domain, tMYC is sufficient to maintain an oncogenic proliferative state. Our results highlight the complexities of proximity-inducing compounds against highly regulated and conformationally dynamic protein targets such as MYC and indicate that PROTACs can induce alternative outcomes beyond target protein degradation.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":"20 4","pages":"896–906 896–906"},"PeriodicalIF":3.5,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143842299","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":"The Hunt for the Putative Epoxyeicosatrienoic Acid Receptor","authors":"William R. Arnold,&nbsp;Sona Jain,&nbsp;Vidya Sinha and Aditi Das*,&nbsp;","doi":"10.1021/acschembio.5c0004710.1021/acschembio.5c00047","DOIUrl":"https://doi.org/10.1021/acschembio.5c00047https://doi.org/10.1021/acschembio.5c00047","url":null,"abstract":"<p >Epoxyeicosatrienoic acids, or EETs, are signaling molecules formed by the metabolism of arachidonic acid by cytochrome P450 enzymes. They are well-known for their anti-inflammatory effects, their ability to lower blood pressure, and benefits to cardiovascular outcomes. Despite the wealth of data demonstrating their physiological benefits, the putative high-affinity receptor that mediates these effects is yet to be identified. The recent report that the sphingosine-1-phosphate receptor 1 (S1PR1) is a high-affinity receptor for a related epoxy lipid prompted us to ask, “Why has the putative EET receptor not been discovered yet? What information about the discoveries of lipid epoxide receptors can help us identify the putative EET receptor?” In this review, we summarize the evidence supporting that the putative EET receptor exists. We then review the data showing EETs binding to other, low-affinity receptors and the discovery of receptors for similar lipid metabolites that can serve as a model for identifying the putative EET receptor. We hope this review will revitalize the search for this important receptor, which can facilitate the development of anti-inflammatory and cardiovascular therapeutics.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":"20 4","pages":"762–777 762–777"},"PeriodicalIF":3.5,"publicationDate":"2025-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acschembio.5c00047","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143842207","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Synthesis of Differentially Halogenated Lissoclimide Analogues To Probe Ribosome E-Site Binding","authors":"Salvatore Terrosu,&nbsp;Liliia Nurullina,&nbsp;Nantamon Supantanapong,&nbsp;Bonnie S. Pak,&nbsp;Sierra Nguyen,&nbsp;Mikael Holm,&nbsp;Cheng Wu,&nbsp;Min Lin,&nbsp;David Horne,&nbsp;Matthew S. Sachs*,&nbsp;Scott C. Blanchard*,&nbsp;Marat Yusupov* and Christopher D. Vanderwal*,&nbsp;","doi":"10.1021/acschembio.4c0082510.1021/acschembio.4c00825","DOIUrl":"https://doi.org/10.1021/acschembio.4c00825https://doi.org/10.1021/acschembio.4c00825","url":null,"abstract":"<p >Halogenated natural products from marine sources often demonstrate potent activity against microorganisms and cancer cell lines. During the last three decades, the lissoclimide class of chlorinated labdane diterpenoids has been characterized with respect to structure and cytotoxic activity. Recently, our laboratories have developed different strategies to produce a broad range of naturally occurring lissoclimides and designed synthetic analogues. This work led to the discovery of a novel halogen−π dispersion interaction between the C2 chloride of chlorolissoclimide and guanine residues in the tRNA exit (E) site of the ribosome. In this study, we aimed to synthesize lissoclimide analogues bearing different substituents in place of the chloride to investigate the importance of the halogen identity for binding, translation inhibition, and cytotoxicity. With previous access to the protio and chloro compounds (haterumaimide Q and chlorolissoclimide), we synthesized two more halogenated variants, fluorolissoclimide and bromolissoclimide, as well as a methylated analogue, methyllissoclimide, to complete a panel of chemical probes for functional and structural studies. Using an integrative approach, we explored the effects of these analogues on the eukaryotic translational machinery in vivo and in vitro. X-ray cocrystal structures with the eukaryotic ribosome were solved for each probe molecule, and the effects on ribosomal thermal stability and FRET-derived ribosome binding constants were determined. Together, these data provide a detailed understanding of the different modes of binding of lissoclimides and insight into their relative activities, which vary according to the substitutions that interact with the eukaryote-specific ribosomal protein eL42. Ultimately, we learned that the presence of a lissoclimide C2-halogen atom─offering a potentially stabilizing halogen−π interaction─appears to facilitate or to synergize with a hydrogen-bonding interaction between the C7-hydroxyl group and the backbone of the ribosomal protein eL42, leading to stronger translation inhibition. We therefore conclude that the C2-halogen and C7-hydroxyl groups are critical contributors to potency, and this idea is borne out in the observations of reduced biological activities in the absence of either group.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":"20 4","pages":"858–869 858–869"},"PeriodicalIF":3.5,"publicationDate":"2025-03-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acschembio.4c00825","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143842202","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Inhibitors of Lysinoalanine Cross-Linking in the Flagella Hook as Antimicrobials against Spirochetes.","authors":"Michael J Lynch, Kurni Kurniyati, Maithili Deshpande, Nyles W Charon, Chunhao Li, Brian R Crane","doi":"10.1021/acschembio.4c00749","DOIUrl":"10.1021/acschembio.4c00749","url":null,"abstract":"<p><p>Spirochetes are especially invasive bacteria that are responsible for several human diseases, including Lyme disease, periodontal disease, syphilis, and leptospirosis. Spirochetes rely on an unusual form of motility based on periplasmic flagella (PFs) to infect hosts and evade the immune system. The flexible hook of these PFs contains a post-translational modification in the form of a lysinoalanine (Lal) cross-link between adjacent subunits of FlgE, which primarily comprise the hook. Lal cross-linking has since been found in key species across the phylum and involves residues that are highly conserved. The requirement of the Lal cross-link for motility of the pathogens <i>Treponema denticola</i> (Td) and <i>Borreliella burgdorferi</i> (Bb) establish Lal as a potential therapeutic target for the development of antimicrobials. Herein, we present the design, development, and application of a NanoLuc-based high-throughput screen that was used to successfully identify two structurally related Lal cross-link inhibitors (hexachlorophene and triclosan) from a library of clinically approved small molecules. A structure-activity relationship study further expanded the inhibitor set to a third compound (dichlorophene), and each inhibitor was demonstrated to biochemically block autocatalytic cross-linking of FlgE from several pathogenic spirochetes with varied mechanisms and degrees of specificity. The most potent inhibitor, hexachlorophene, alters Lal cross-linking in cultured cells of Td and reduces bacterial motility in swimming plate assays. Overall, these results provide a proof-of-concept for the discovery and development of Lal-cross-link inhibitors to combat spirochete-derived illnesses.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":"620-631"},"PeriodicalIF":3.5,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143497470","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":"Characterization of the Iron-Sulfur Cluster in the NCOA4 Fragment (383-522) and Its Interaction with Ferritin.","authors":"Ayush Srivastava, Maximilian Beyer, Colby Hladun, Rebekah Tardif, Aneeta Arshad, Costel C Darie, Yeonni Zoo, Georgia C Papaefthymiou, Weijing Liu, Rosa Viner, Paolo Arosio, Fadi Bou-Abdallah","doi":"10.1021/acschembio.4c00877","DOIUrl":"10.1021/acschembio.4c00877","url":null,"abstract":"<p><p>Ferritin degradation pathways, particularly NCOA4-mediated ferritinophagy, are crucial for maintaining iron homeostasis. Here, we demonstrate the coexistence of two NCOA4 isoforms, one iron-sulfur cluster-free and one iron-sulfur cluster-bound, in oxygenated cell cultures. Using a combination of spectroscopic and analytical techniques, in vitro characterization of the NCOA4 fragment (383-522), denoted NCOA4-D, revealed a predominance of monomeric species with a relatively stable [2Fe-2S] cluster under normoxic conditions. The results demonstrate distinct interactions between NCOA4-D isoforms and ferritin, underscoring the influence of cellular oxygen and iron concentrations on NCOA4's regulatory functions, pathways, and ferritin's fate. Our findings suggest that different NCOA4-initiated degradation pathways may concurrently occur in cells and highlight the necessity of further exploring the role of the Fe-S cluster in NCOA4 as an iron-sensing mechanism for maintaining cellular iron homeostasis.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":"731-745"},"PeriodicalIF":3.5,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143513987","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}