Pub Date : 2025-03-20DOI: 10.1016/j.chembiol.2025.01.009
Tian Qiu , Saara-Anne Azizi , Shubhashree Pani , Bryan C. Dickinson
Peroxiredoxins (PRDXs) are a highly conserved family of peroxidases that serve as the primary scavengers of peroxides. Post-translational modifications play crucial roles modulating PRDX activities, tuning the balance between reactive oxygen species (ROS) signaling and stress. We previously reported that S-acylation occurs at the “peroxidatic” cysteine (Cp) site of PRDX5 and that it inhibits PRDX5 activity. Here, we show that the PRDX family more broadly is subject to S-acylation at the Cp site of all PRDXs and that PRDX S-acylation dynamically responds to cellular ROS levels. Using activity-based fluorescent imaging with DPP-Red, a red-shifted fluorescent indicator for acyl-protein thioesterase (APT) activity, we also discover that the instigation of ROS-stress via exogenous H2O2 activates both the cytosolic and mitochondrial APTs, whereas epidermal growth factor (EGF)-stimulated endogenous H2O2 deactivates the cytosolic APTs. These results indicate that APTs help tune H2O2 signal transduction and ROS protection through PRDX S-deacylation.
{"title":"Dynamic PRDX S-acylation modulates ROS stress and signaling","authors":"Tian Qiu , Saara-Anne Azizi , Shubhashree Pani , Bryan C. Dickinson","doi":"10.1016/j.chembiol.2025.01.009","DOIUrl":"10.1016/j.chembiol.2025.01.009","url":null,"abstract":"<div><div>Peroxiredoxins (PRDXs) are a highly conserved family of peroxidases that serve as the primary scavengers of peroxides. Post-translational modifications play crucial roles modulating PRDX activities, tuning the balance between reactive oxygen species (ROS) signaling and stress. We previously reported that <em>S</em>-acylation occurs at the “peroxidatic” cysteine (Cp) site of PRDX5 and that it inhibits PRDX5 activity. Here, we show that the PRDX family more broadly is subject to <em>S</em>-acylation at the Cp site of all PRDXs and that PRDX <em>S</em>-acylation dynamically responds to cellular ROS levels. Using activity-based fluorescent imaging with DPP-Red, a red-shifted fluorescent indicator for acyl-protein thioesterase (APT) activity, we also discover that the instigation of ROS-stress via exogenous H<sub>2</sub>O<sub>2</sub> activates both the cytosolic and mitochondrial APTs, whereas epidermal growth factor (EGF)-stimulated endogenous H<sub>2</sub>O<sub>2</sub> deactivates the cytosolic APTs. These results indicate that APTs help tune H<sub>2</sub>O<sub>2</sub> signal transduction and ROS protection through PRDX <em>S</em>-deacylation.</div></div>","PeriodicalId":265,"journal":{"name":"Cell Chemical Biology","volume":"32 3","pages":"Pages 511-519.e5"},"PeriodicalIF":6.6,"publicationDate":"2025-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143486527","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-03-20DOI: 10.1016/j.chembiol.2025.02.003
Michael Ebner , Florian Fröhlich , Volker Haucke
Lysosomes are the central degradative organelle of mammalian cells and have emerged as major intersections of cellular metabolite flux. Macromolecules derived from dietary and intracellular sources are delivered to the acidic lysosomal lumen where they are subjected to degradation by acid hydrolases. Lipids derived from lipoproteins, autophagy cargo, or autophagosomal membranes themselves constitute major lysosomal substrates. Dysregulation of lysosomal lipid processing, defective export of lipid catabolites, and lysosomal membrane permeabilization underly diseases ranging from neurodegeneration to metabolic syndromes and lysosomal storage disorders. Mammalian cells are equipped with sophisticated homeostatic control mechanisms that protect the lysosomal limiting membrane from excessive damage, prevent the spillage of luminal hydrolases into the cytoplasm, and preserve the lysosomal membrane composition in the face of constant fusion with heterotypic organelles such as endosomes and autophagosomes. In this review we discuss the molecular mechanisms that govern lysosomal lipid homeostasis and, thereby, lysosome function in health and disease.
{"title":"Mechanisms and functions of lysosomal lipid homeostasis","authors":"Michael Ebner , Florian Fröhlich , Volker Haucke","doi":"10.1016/j.chembiol.2025.02.003","DOIUrl":"10.1016/j.chembiol.2025.02.003","url":null,"abstract":"<div><div>Lysosomes are the central degradative organelle of mammalian cells and have emerged as major intersections of cellular metabolite flux. Macromolecules derived from dietary and intracellular sources are delivered to the acidic lysosomal lumen where they are subjected to degradation by acid hydrolases. Lipids derived from lipoproteins, autophagy cargo, or autophagosomal membranes themselves constitute major lysosomal substrates. Dysregulation of lysosomal lipid processing, defective export of lipid catabolites, and lysosomal membrane permeabilization underly diseases ranging from neurodegeneration to metabolic syndromes and lysosomal storage disorders. Mammalian cells are equipped with sophisticated homeostatic control mechanisms that protect the lysosomal limiting membrane from excessive damage, prevent the spillage of luminal hydrolases into the cytoplasm, and preserve the lysosomal membrane composition in the face of constant fusion with heterotypic organelles such as endosomes and autophagosomes. In this review we discuss the molecular mechanisms that govern lysosomal lipid homeostasis and, thereby, lysosome function in health and disease.</div></div>","PeriodicalId":265,"journal":{"name":"Cell Chemical Biology","volume":"32 3","pages":"Pages 392-407"},"PeriodicalIF":6.6,"publicationDate":"2025-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143560763","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-03-20DOI: 10.1016/j.chembiol.2024.09.008
Kelly H. Sokol , Cameron J. Lee , Thomas J. Rogers , Althea Waldhart , Abigail E. Ellis , Sahithi Madireddy , Samuel R. Daniels , Rachel (Rae) J. House , Xinyu Ye , Mary Olesnavich , Amy Johnson , Benjamin R. Furness , Ryan D. Sheldon , Evan C. Lien
Ferroptosis is a form of cell death caused by lipid peroxidation that is emerging as a target for cancer therapy, highlighting the need to identify factors that govern ferroptosis susceptibility. Lipid peroxidation occurs primarily on phospholipids containing polyunsaturated fatty acids (PUFAs). Here, we show that even though extracellular lipid limitation reduces cellular PUFA levels, lipid-starved cancer cells are paradoxically more sensitive to ferroptosis. Using mass spectrometry-based lipidomics with stable isotope fatty acid labeling, we show that lipid limitation induces a fatty acid trafficking pathway in which PUFAs are liberated from triglycerides to synthesize highly unsaturated PUFAs such as arachidonic and adrenic acid. These PUFAs then accumulate in phospholipids, including ether phospholipids, to promote ferroptosis sensitivity. Therefore, PUFA levels within cancer cells do not necessarily correlate with ferroptosis susceptibility. Rather, how cancer cells respond to extracellular lipid levels by trafficking PUFAs into proper phospholipid pools contributes to their sensitivity to ferroptosis.
{"title":"Lipid availability influences ferroptosis sensitivity in cancer cells by regulating polyunsaturated fatty acid trafficking","authors":"Kelly H. Sokol , Cameron J. Lee , Thomas J. Rogers , Althea Waldhart , Abigail E. Ellis , Sahithi Madireddy , Samuel R. Daniels , Rachel (Rae) J. House , Xinyu Ye , Mary Olesnavich , Amy Johnson , Benjamin R. Furness , Ryan D. Sheldon , Evan C. Lien","doi":"10.1016/j.chembiol.2024.09.008","DOIUrl":"10.1016/j.chembiol.2024.09.008","url":null,"abstract":"<div><div>Ferroptosis is a form of cell death caused by lipid peroxidation that is emerging as a target for cancer therapy, highlighting the need to identify factors that govern ferroptosis susceptibility. Lipid peroxidation occurs primarily on phospholipids containing polyunsaturated fatty acids (PUFAs). Here, we show that even though extracellular lipid limitation reduces cellular PUFA levels, lipid-starved cancer cells are paradoxically more sensitive to ferroptosis. Using mass spectrometry-based lipidomics with stable isotope fatty acid labeling, we show that lipid limitation induces a fatty acid trafficking pathway in which PUFAs are liberated from triglycerides to synthesize highly unsaturated PUFAs such as arachidonic and adrenic acid. These PUFAs then accumulate in phospholipids, including ether phospholipids, to promote ferroptosis sensitivity. Therefore, PUFA levels within cancer cells do not necessarily correlate with ferroptosis susceptibility. Rather, how cancer cells respond to extracellular lipid levels by trafficking PUFAs into proper phospholipid pools contributes to their sensitivity to ferroptosis.</div></div>","PeriodicalId":265,"journal":{"name":"Cell Chemical Biology","volume":"32 3","pages":"Pages 408-422.e6"},"PeriodicalIF":6.6,"publicationDate":"2025-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142487024","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-03-20DOI: 10.1016/j.chembiol.2025.02.005
Yevheniia Bushman , Duhita A. Mirikar , Andrew.W. Truman
Molecular chaperones like Hsp70s are key players in protein quality control (PQC), capable of eliminating toxic intracellular condensates. In this issue of Cell Chemical Biology, Zhang et al.1 present a computational approach to design novel J-domain protein (JDP) constructs that bind to Hsp70 and enhance its chaperone activity.
{"title":"Targeting biomolecular condensates: The rise of engineered chaperones","authors":"Yevheniia Bushman , Duhita A. Mirikar , Andrew.W. Truman","doi":"10.1016/j.chembiol.2025.02.005","DOIUrl":"10.1016/j.chembiol.2025.02.005","url":null,"abstract":"<div><div>Molecular chaperones like Hsp70s are key players in protein quality control (PQC), capable of eliminating toxic intracellular condensates. In this issue of <em>Cell Chemical Biology</em>, Zhang et al.<span><span><sup>1</sup></span></span> present a computational approach to design novel J-domain protein (JDP) constructs that bind to Hsp70 and enhance its chaperone activity.</div></div>","PeriodicalId":265,"journal":{"name":"Cell Chemical Biology","volume":"32 3","pages":"Pages 381-383"},"PeriodicalIF":6.6,"publicationDate":"2025-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143660456","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-20DOI: 10.1016/j.chembiol.2024.12.006
Jian Tang , Ramkumar Moorthy , Laura E. Hirsch , Özlem Demir , Zachary D. Baker , Jordan A. Naumann , Katherine F.M. Jones , Michael J. Grillo , Ella S. Haefner , Ke Shi , Michaella J. Levy , Harshita B. Gupta , Hideki Aihara , Reuben S. Harris , Rommie E. Amaro , Nicholas M. Levinson , Daniel A. Harki
The N-Myc transcription factor, encoded by MYCN, is a mechanistically validated, yet challenging, target for neuroblastoma (NB) therapy development. In normal neuronal progenitors, N-Myc undergoes rapid degradation, while, in MYCN-amplified NB cells, Aurora kinase A (Aurora-A) binds to and stabilizes N-Myc, resulting in elevated protein levels. Here, we demonstrate that targeted protein degradation of Aurora-A decreases N-Myc levels. A potent Aurora-A degrader, HLB-0532259 (compound 4), was developed from an Aurora-A-binding ligand that engages the Aurora-A/N-Myc complex. HLB-0532259 promotes the degradation of Aurora-A, which elicits concomitant N-Myc degradation, with nanomolar potency and excellent selectivity. HLB-0532259 surpasses the cellular efficacy of established allosteric Aurora-A inhibitors, exhibits favorable pharmacokinetic properties, and elicits tumor reduction in a murine xenograft NB model. This study broadly delineates a strategy for targeting “undruggable” proteins that are reliant on accessory proteins for cellular stabilization.
{"title":"Targeting N-Myc in neuroblastoma with selective Aurora kinase A degraders","authors":"Jian Tang , Ramkumar Moorthy , Laura E. Hirsch , Özlem Demir , Zachary D. Baker , Jordan A. Naumann , Katherine F.M. Jones , Michael J. Grillo , Ella S. Haefner , Ke Shi , Michaella J. Levy , Harshita B. Gupta , Hideki Aihara , Reuben S. Harris , Rommie E. Amaro , Nicholas M. Levinson , Daniel A. Harki","doi":"10.1016/j.chembiol.2024.12.006","DOIUrl":"10.1016/j.chembiol.2024.12.006","url":null,"abstract":"<div><div>The N-Myc transcription factor, encoded by <em>MYCN</em>, is a mechanistically validated, yet challenging, target for neuroblastoma (NB) therapy development. In normal neuronal progenitors, N-Myc undergoes rapid degradation, while, in <em>MYCN</em>-amplified NB cells, Aurora kinase A (Aurora-A) binds to and stabilizes N-Myc, resulting in elevated protein levels. Here, we demonstrate that targeted protein degradation of Aurora-A decreases N-Myc levels. A potent Aurora-A degrader, HLB-0532259 (compound <strong>4</strong>), was developed from an Aurora-A-binding ligand that engages the Aurora-A/N-Myc complex. HLB-0532259 promotes the degradation of Aurora-A, which elicits concomitant N-Myc degradation, with nanomolar potency and excellent selectivity. HLB-0532259 surpasses the cellular efficacy of established allosteric Aurora-A inhibitors, exhibits favorable pharmacokinetic properties, and elicits tumor reduction in a murine xenograft NB model. This study broadly delineates a strategy for targeting “undruggable” proteins that are reliant on accessory proteins for cellular stabilization.</div></div>","PeriodicalId":265,"journal":{"name":"Cell Chemical Biology","volume":"32 2","pages":"Pages 352-362.e10"},"PeriodicalIF":6.6,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142935250","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-20DOI: 10.1016/j.chembiol.2024.10.001
Viviane S. De Paula , Abhinav Dubey , Haribabu Arthanari , Nikolaos G. Sgourakis
CRISPR-Cas9 has revolutionized genome engineering applications by programming its single-guide RNA, where high specificity is required. However, the precise molecular mechanism underscoring discrimination between on/off-target DNA sequences, relative to the guide RNA template, remains elusive. Here, using methyl-based NMR to study multiple holoenzymes assembled in vitro, we elucidate a discrete protein conformational state which enables recognition of DNA mismatches at the protospacer adjacent motif (PAM)-distal end. Our results delineate an allosteric pathway connecting a dynamic conformational switch at the REC3 domain, with the sampling of a catalytically competent state by the HNH domain. Our NMR data show that HiFi Cas9 (R691A) increases the fidelity of DNA recognition by stabilizing this "surveillance state" for mismatched substrates, shifting the Cas9 conformational equilibrium away from the active state. These results establish a paradigm of substrate recognition through an allosteric protein-based switch, providing unique insights into the molecular mechanism which governs Cas9 selectivity.
CRISPR-Cas9 通过对需要高特异性的单导 RNA 进行编程，彻底改变了基因组工程应用。然而，相对于引导 RNA 模板而言，区分目标 DNA 序列的精确分子机制仍未确定。在这里，我们利用基于甲基的核磁共振技术研究了体外组装的多个全酶，阐明了一种离散的蛋白质构象状态，它能识别原间隔邻接基序（PAM）远端的 DNA 错配。我们的研究结果勾勒出了一条异构途径，它将 REC3 结构域的动态构象转换与 HNH 结构域的催化状态取样连接起来。我们的核磁共振数据显示，HiFi Cas9 (R691A)通过稳定这种针对不匹配底物的 "监视状态"，使 Cas9 的构象平衡偏离活性状态，从而提高了 DNA 识别的保真度。这些结果建立了一种通过基于异构蛋白的开关来识别底物的范例，为研究支配 Cas9 选择性的分子机制提供了独特的见解。
{"title":"Dynamic sampling of a surveillance state enables DNA proofreading by Cas9","authors":"Viviane S. De Paula , Abhinav Dubey , Haribabu Arthanari , Nikolaos G. Sgourakis","doi":"10.1016/j.chembiol.2024.10.001","DOIUrl":"10.1016/j.chembiol.2024.10.001","url":null,"abstract":"<div><div>CRISPR-Cas9 has revolutionized genome engineering applications by programming its single-guide RNA, where high specificity is required. However, the precise molecular mechanism underscoring discrimination between on/off-target DNA sequences, relative to the guide RNA template, remains elusive. Here, using methyl-based NMR to study multiple holoenzymes assembled <em>in vitro</em>, we elucidate a discrete protein conformational state which enables recognition of DNA mismatches at the protospacer adjacent motif (PAM)-distal end. Our results delineate an allosteric pathway connecting a dynamic conformational switch at the REC3 domain, with the sampling of a catalytically competent state by the HNH domain. Our NMR data show that HiFi Cas9 (R691A) increases the fidelity of DNA recognition by stabilizing this \"surveillance state\" for mismatched substrates, shifting the Cas9 conformational equilibrium away from the active state. These results establish a paradigm of substrate recognition through an allosteric protein-based switch, providing unique insights into the molecular mechanism which governs Cas9 selectivity.</div></div>","PeriodicalId":265,"journal":{"name":"Cell Chemical Biology","volume":"32 2","pages":"Pages 267-279.e5"},"PeriodicalIF":6.6,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142519849","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-20DOI: 10.1016/j.chembiol.2024.12.001
Bradley E. Poulsen , Thulasi Warrier , Sulyman Barkho , Josephine Bagnall , Keith P. Romano , Tiantian White , Xiao Yu , Tomohiko Kawate , Phuong H. Nguyen , Kyra Raines , Kristina Ferrara , A. Lorelei Golas , Michael FitzGerald , Andras Boeszoermenyi , Virendar Kaushik , Michael Serrano-Wu , Noam Shoresh , Deborah T. Hung
The surge of antimicrobial resistance threatens efficacy of current antibiotics, particularly against Pseudomonas aeruginosa, a highly resistant gram-negative pathogen. The asymmetric outer membrane (OM) of P. aeruginosa combined with its array of efflux pumps provide a barrier to xenobiotic accumulation, thus making antibiotic discovery challenging. We adapted PROSPECT, a target-based, whole-cell screening strategy, to discover small molecule probes that kill P. aeruginosa mutants depleted for essential proteins localized at the OM. We identified BRD1401, a small molecule that has specific activity against a P. aeruginosa mutant depleted for the essential lipoprotein, OprL. Genetic and chemical biological studies identified that BRD1401 acts by targeting the OM β-barrel protein OprH to disrupt its interaction with LPS and increase membrane fluidity. Studies with BRD1401 also revealed an interaction between OprL and OprH, directly linking the OM with peptidoglycan. Thus, a whole-cell, multiplexed screen can identify species-specific chemical probes to reveal pathogen biology.
{"title":"Discovery of a Pseudomonas aeruginosa-specific small molecule targeting outer membrane protein OprH-LPS interaction by a multiplexed screen","authors":"Bradley E. Poulsen , Thulasi Warrier , Sulyman Barkho , Josephine Bagnall , Keith P. Romano , Tiantian White , Xiao Yu , Tomohiko Kawate , Phuong H. Nguyen , Kyra Raines , Kristina Ferrara , A. Lorelei Golas , Michael FitzGerald , Andras Boeszoermenyi , Virendar Kaushik , Michael Serrano-Wu , Noam Shoresh , Deborah T. Hung","doi":"10.1016/j.chembiol.2024.12.001","DOIUrl":"10.1016/j.chembiol.2024.12.001","url":null,"abstract":"<div><div>The surge of antimicrobial resistance threatens efficacy of current antibiotics, particularly against <em>Pseudomonas aeruginosa</em>, a highly resistant gram-negative pathogen. The asymmetric outer membrane (OM) of <em>P. aeruginosa</em> combined with its array of efflux pumps provide a barrier to xenobiotic accumulation, thus making antibiotic discovery challenging. We adapted PROSPECT, a target-based, whole-cell screening strategy, to discover small molecule probes that kill <em>P. aeruginosa</em> mutants depleted for essential proteins localized at the OM. We identified BRD1401, a small molecule that has specific activity against a <em>P. aeruginosa</em> mutant depleted for the essential lipoprotein, OprL. Genetic and chemical biological studies identified that BRD1401 acts by targeting the OM β-barrel protein OprH to disrupt its interaction with LPS and increase membrane fluidity. Studies with BRD1401 also revealed an interaction between OprL and OprH, directly linking the OM with peptidoglycan. Thus, a whole-cell, multiplexed screen can identify species-specific chemical probes to reveal pathogen biology.</div></div>","PeriodicalId":265,"journal":{"name":"Cell Chemical Biology","volume":"32 2","pages":"Pages 307-324.e15"},"PeriodicalIF":6.6,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142888682","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-20DOI: 10.1016/j.chembiol.2024.12.013
Cong Shen , Aldo I. Salazar-Morales , Wonhyeuk Jung , Joey Erwin , Yangqi Gu , Anthony Coelho , Kallol Gupta , Sibel Ebru Yalcin , Fadel A. Samatey , Nikhil S. Malvankar
Microbial extracellular electron transfer (EET) drives various globally important environmental phenomena and has biotechnology applications. Diverse prokaryotes have been proposed to perform EET via surface-displayed “nanowires” composed of multi-heme cytochromes. However, the mechanism that enables only a few cytochromes to polymerize into nanowires is unclear. Here, we identify a highly conserved omcS-companion (osc) cluster that drives the formation of cytochrome OmcS nanowires in Geobacter sulfurreducens. Through a combination of genetic, biochemical, and biophysical methods, we establish that prolyl isomerase-containing chaperon OscH, channel-like OscEFG, and β-propeller-like OscD are involved in the folding, secretion, and morphology maintenance of OmcS nanowires, respectively. OscH and OscG can interact with OmcS. Furthermore, overexpression of oscG accelerates EET by overproducing nanowires in an ATP-dependent manner. Heme loading splits OscD; ΔoscD accelerates cell growth, bundles nanowires into cables. Our findings establish the mechanism and prevalence of a specialized and modular assembly system for nanowires across phylogenetically diverse species and environments
{"title":"A widespread and ancient bacterial machinery assembles cytochrome OmcS nanowires essential for extracellular electron transfer","authors":"Cong Shen , Aldo I. Salazar-Morales , Wonhyeuk Jung , Joey Erwin , Yangqi Gu , Anthony Coelho , Kallol Gupta , Sibel Ebru Yalcin , Fadel A. Samatey , Nikhil S. Malvankar","doi":"10.1016/j.chembiol.2024.12.013","DOIUrl":"10.1016/j.chembiol.2024.12.013","url":null,"abstract":"<div><div>Microbial extracellular electron transfer (EET) drives various globally important environmental phenomena and has biotechnology applications. Diverse prokaryotes have been proposed to perform EET via surface-displayed “nanowires” composed of multi-heme cytochromes. However, the mechanism that enables only a few cytochromes to polymerize into nanowires is unclear. Here, we identify a highly conserved <em><u>o</u>mc<u>S</u></em>-<u>c</u>ompanion (<em>osc</em>) cluster that drives the formation of cytochrome OmcS nanowires in <em>Geobacter sulfurreducens</em>. Through a combination of genetic, biochemical, and biophysical methods, we establish that prolyl isomerase-containing chaperon OscH, channel-like OscEFG, and β-propeller-like OscD are involved in the folding, secretion, and morphology maintenance of OmcS nanowires, respectively. OscH and OscG can interact with OmcS. Furthermore, overexpression of <em>oscG</em> accelerates EET by overproducing nanowires in an ATP-dependent manner. Heme loading splits OscD; Δ<em>oscD</em> accelerates cell growth, bundles nanowires into cables. Our findings establish the mechanism and prevalence of a specialized and modular assembly system for nanowires across phylogenetically diverse species and environments</div></div>","PeriodicalId":265,"journal":{"name":"Cell Chemical Biology","volume":"32 2","pages":"Pages 239-254.e7"},"PeriodicalIF":6.6,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142981366","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-20DOI: 10.1016/j.chembiol.2025.01.002
Joseph J. Smith , Taylor R. Valentino , Austin H. Ablicki , Riddhidev Banerjee , Adam R. Colligan , Debra M. Eckert , Gabrielle A. Desjardins , Katharine L. Diehl
Acetyl-coenzyme A is a central metabolite that participates in many cellular pathways. Evidence suggests that acetyl-CoA metabolism is highly compartmentalized in mammalian cells. Yet methods to measure acetyl-CoA in living cells are lacking. Herein, we engineered an acetyl-CoA biosensor from the bacterial protein PanZ and circularly permuted green fluorescent protein (cpGFP). The sensor, “PancACe,” has a maximum change of ∼2-fold and a response range of ∼10 μM–2 mM acetyl-CoA. We demonstrated that the sensor has a greater than 7-fold selectivity over coenzyme A, butyryl-CoA, malonyl-CoA, and succinyl-CoA, and a 2.3-fold selectivity over propionyl-CoA. We expressed the sensor in E. coli and showed that it enables detection of rapid changes in acetyl-CoA levels. By localizing the sensor to either the cytoplasm, nucleus, or mitochondria in human cells, we showed that it enables subcellular detection of changes in acetyl-CoA levels, the magnitudes of which agreed with an orthogonal PicoProbe assay.
{"title":"A genetically encoded fluorescent biosensor for visualization of acetyl-CoA in live cells","authors":"Joseph J. Smith , Taylor R. Valentino , Austin H. Ablicki , Riddhidev Banerjee , Adam R. Colligan , Debra M. Eckert , Gabrielle A. Desjardins , Katharine L. Diehl","doi":"10.1016/j.chembiol.2025.01.002","DOIUrl":"10.1016/j.chembiol.2025.01.002","url":null,"abstract":"<div><div>Acetyl-coenzyme A is a central metabolite that participates in many cellular pathways. Evidence suggests that acetyl-CoA metabolism is highly compartmentalized in mammalian cells. Yet methods to measure acetyl-CoA in living cells are lacking. Herein, we engineered an acetyl-CoA biosensor from the bacterial protein PanZ and circularly permuted green fluorescent protein (cpGFP). The sensor, “PancACe,” has a maximum change of ∼2-fold and a response range of ∼10 μM–2 mM acetyl-CoA. We demonstrated that the sensor has a greater than 7-fold selectivity over coenzyme A, butyryl-CoA, malonyl-CoA, and succinyl-CoA, and a 2.3-fold selectivity over propionyl-CoA. We expressed the sensor in <em>E. coli</em> and showed that it enables detection of rapid changes in acetyl-CoA levels. By localizing the sensor to either the cytoplasm, nucleus, or mitochondria in human cells, we showed that it enables subcellular detection of changes in acetyl-CoA levels, the magnitudes of which agreed with an orthogonal PicoProbe assay.</div></div>","PeriodicalId":265,"journal":{"name":"Cell Chemical Biology","volume":"32 2","pages":"Pages 325-337.e10"},"PeriodicalIF":6.6,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143044669","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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