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Noninvasive Bioluminescence Imaging of Serum Albumins in Living Mice.
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-04-02 DOI: 10.1021/acschembio.4c00740
Sung-Bae Kim, Genta Kamiya, Tadaomi Furuta, Nobuo Kitada, Suresh Thangudu, Arutselvan Natarajan, Shojiro A Maki, Ramasamy Paulmurugan

Bioluminescence (BL) is an emerging optical readout that has been extensively used in various bioassays and molecular imaging systems. In this study, we present the bioanalytical application of marine luciferins as an excellent optical indicator for noninvasive imaging of serum albumins. We synthesized 30 kinds of regioisomeric coelenterazine (CTZ) analogs and investigated their specificities for major serum proteins from various species. The results found that some of the CTZ analogs exhibited surprisingly specific optical signals upon binding with the serum albumins. These CTZ analogs showed diverse emission spectra ranging from 495 to 558 nm according to the albumin species used acting as pseudoluciferases. The selective albumin indicators, TS1 and TS2, exhibited long and linear dose-response curves and were sensitive enough to determine clinically normal and abnormal (microalbuminuria) ranges of albumins in saliva and urine. The sensitivity of this assay is superior to that of the conventional Bromocresol purple (BCP) method. We further demonstrated the advantages of the albumin indicators through noninvasive imaging of liver-albumin in vivo in living mice. The in vivo and ex vivo imaging results confirmed that the CTZ analog TS2 can sensitively image the liver-albumin in vivo with high signal-to-background ratio. This study paves a new way to make use of CTZ analogs for noninvasive albumin imaging and conceptualizes the pseudoluciferase-based imaging. The distinct in vivo imaging of serum albumins can potentially aid clinicians in providing insight into patients' liver function and other vital factors needed for whole-body homeostasis.

{"title":"Noninvasive Bioluminescence Imaging of Serum Albumins in Living Mice.","authors":"Sung-Bae Kim, Genta Kamiya, Tadaomi Furuta, Nobuo Kitada, Suresh Thangudu, Arutselvan Natarajan, Shojiro A Maki, Ramasamy Paulmurugan","doi":"10.1021/acschembio.4c00740","DOIUrl":"https://doi.org/10.1021/acschembio.4c00740","url":null,"abstract":"<p><p>Bioluminescence (BL) is an emerging optical readout that has been extensively used in various bioassays and molecular imaging systems. In this study, we present the bioanalytical application of marine luciferins as an excellent optical indicator for noninvasive imaging of serum albumins. We synthesized 30 kinds of regioisomeric coelenterazine (CTZ) analogs and investigated their specificities for major serum proteins from various species. The results found that some of the CTZ analogs exhibited surprisingly specific optical signals upon binding with the serum albumins. These CTZ analogs showed diverse emission spectra ranging from 495 to 558 nm according to the albumin species used acting as pseudoluciferases. The selective albumin indicators, <b>TS1</b> and <b>TS2</b>, exhibited long and linear dose-response curves and were sensitive enough to determine clinically normal and abnormal (microalbuminuria) ranges of albumins in saliva and urine. The sensitivity of this assay is superior to that of the conventional Bromocresol purple (BCP) method. We further demonstrated the advantages of the albumin indicators through noninvasive imaging of liver-albumin <i>in vivo</i> in living mice. The <i>in vivo</i> and <i>ex vivo</i> imaging results confirmed that the CTZ analog <b>TS2</b> can sensitively image the liver-albumin <i>in vivo</i> with high signal-to-background ratio. This study paves a new way to make use of CTZ analogs for noninvasive albumin imaging and conceptualizes the pseudoluciferase-based imaging. The distinct <i>in vivo</i> imaging of serum albumins can potentially aid clinicians in providing insight into patients' liver function and other vital factors needed for whole-body homeostasis.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2025-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143762587","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}
引用次数: 0
Amber Codon Mutational Scanning and Bioorthogonal PEGylation for Epitope Mapping of Antibody Binding Sites on Human Arginase-1.
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-04-01 DOI: 10.1021/acschembio.4c00692
Jaime Fernández de Santaella, Nikolaj G Koch, Lorenz Widmer, Michael A Nash

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 Methanococcoides burtonii 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.

{"title":"Amber Codon Mutational Scanning and Bioorthogonal PEGylation for Epitope Mapping of Antibody Binding Sites on Human Arginase-1.","authors":"Jaime Fernández de Santaella, Nikolaj G Koch, Lorenz Widmer, Michael A Nash","doi":"10.1021/acschembio.4c00692","DOIUrl":"https://doi.org/10.1021/acschembio.4c00692","url":null,"abstract":"<p><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":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143762580","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}
引用次数: 0
Structural and Biochemical Insights into Lignin-Oxidizing Activity of Bacterial Peroxidases against Soluble Substrates and Kraft Lignin.
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-03-27 DOI: 10.1021/acschembio.4c00788
Zahra Choolaei, Anna N Khusnutdinova, Tatiana Skarina, Peter Stogios, Patrick Diep, Sofia Lemak, Elizabeth A Edwards, Alexei Savchenko, Alexander F Yakunin

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 Frankia casuarinae, PF3257 from Pseudomonas fluorescens, and PR9465 from Pseudomonas rhizosphaerae. 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.

{"title":"Structural and Biochemical Insights into Lignin-Oxidizing Activity of Bacterial Peroxidases against Soluble Substrates and Kraft Lignin.","authors":"Zahra Choolaei, Anna N Khusnutdinova, Tatiana Skarina, Peter Stogios, Patrick Diep, Sofia Lemak, Elizabeth A Edwards, Alexei Savchenko, Alexander F Yakunin","doi":"10.1021/acschembio.4c00788","DOIUrl":"https://doi.org/10.1021/acschembio.4c00788","url":null,"abstract":"<p><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":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143717570","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}
引用次数: 0
MYC-Targeting PROTACs Lead to Bimodal Degradation and N-Terminal Truncation.
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-03-27 DOI: 10.1021/acschembio.4c00864
Shelton R Boyd, Srinivas Chamakuri, Alexander J Trostle, Hu Chen, Zhandong Liu, Antrix Jian, Jian Wang, Anna Malovannaya, Damian W Young

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.

{"title":"MYC-Targeting PROTACs Lead to Bimodal Degradation and N-Terminal Truncation.","authors":"Shelton R Boyd, Srinivas Chamakuri, Alexander J Trostle, Hu Chen, Zhandong Liu, Antrix Jian, Jian Wang, Anna Malovannaya, Damian W Young","doi":"10.1021/acschembio.4c00864","DOIUrl":"https://doi.org/10.1021/acschembio.4c00864","url":null,"abstract":"<p><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":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143726998","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}
引用次数: 0
Chitinivorax: The New Kid on the Block of Bacterial 2-Alkyl-4(1H)-quinolone Producers.
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-03-27 DOI: 10.1021/acschembio.5c00046
Viktoriia Savchenko, Xiaoqian Annie Yu, Martin F Polz, Thomas Böttcher

2-Alkyl-4(1H)-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 Pseudomonas aeruginosa various species of Burkholderia and Pseudoalteromonas are known to produce 2-alkyl-4(1H)-quinolones. However, the evolutionary relationships of their biosynthetic gene clusters remain largely unexplored. To address this, we investigated the phylogeny of 2-alkyl-4(1H)-quinolone biosynthetic gene clusters, leading to the discovery of Chitinivorax as a fourth genus capable of producing 2-alkyl-4(1H)-quinolones, expanding our knowledge of the diversity of bacteria involved in quinolone-biosynthesis.

{"title":"Chitinivorax: The New Kid on the Block of Bacterial 2-Alkyl-4(1<i>H</i>)-quinolone Producers.","authors":"Viktoriia Savchenko, Xiaoqian Annie Yu, Martin F Polz, Thomas Böttcher","doi":"10.1021/acschembio.5c00046","DOIUrl":"https://doi.org/10.1021/acschembio.5c00046","url":null,"abstract":"<p><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":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143717568","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}
引用次数: 0
Formation of the i-motif Structures by Human Telomeric c-Rich Sequences d(CCCTAA)n and Its Recognition by Bisbenzylisoquinoline Alkaloids.
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-03-27 DOI: 10.1021/acschembio.4c00844
Junliu Huang, Zexuan Lin, Jishun Yang, Huining Tang, Yang Yang, Yi Tang, Feixian Luo, Wenshu Wang, Xiaojie Cui

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.

{"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, Zexuan Lin, Jishun Yang, Huining Tang, Yang Yang, Yi Tang, Feixian Luo, Wenshu Wang, Xiaojie Cui","doi":"10.1021/acschembio.4c00844","DOIUrl":"https://doi.org/10.1021/acschembio.4c00844","url":null,"abstract":"<p><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)<sub>4</sub> (hTeloC-24mer) and d(CCCTAA)<sub>8</sub> (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":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143717569","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}
引用次数: 0
The Hunt for the Putative Epoxyeicosatrienoic Acid Receptor.
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-03-24 DOI: 10.1021/acschembio.5c00047
William R Arnold, Sona Jain, Vidya Sinha, Aditi Das

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.

{"title":"The Hunt for the Putative Epoxyeicosatrienoic Acid Receptor.","authors":"William R Arnold, Sona Jain, Vidya Sinha, Aditi Das","doi":"10.1021/acschembio.5c00047","DOIUrl":"https://doi.org/10.1021/acschembio.5c00047","url":null,"abstract":"<p><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":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2025-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143699060","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}
引用次数: 0
Synthesis of Differentially Halogenated Lissoclimide Analogues To Probe Ribosome E-Site Binding. 合成不同卤代的利索克利酰胺类似物以探查核糖体 E 位点结合情况
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-03-22 DOI: 10.1021/acschembio.4c00825
Salvatore Terrosu, Liliia Nurullina, Nantamon Supantanapong, Bonnie S Pak, Sierra Nguyen, Mikael Holm, Cheng Wu, Min Lin, David Horne, Matthew S Sachs, Scott C Blanchard, Marat Yusupov, Christopher D Vanderwal

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.

{"title":"Synthesis of Differentially Halogenated Lissoclimide Analogues To Probe Ribosome E-Site Binding.","authors":"Salvatore Terrosu, Liliia Nurullina, Nantamon Supantanapong, Bonnie S Pak, Sierra Nguyen, Mikael Holm, Cheng Wu, Min Lin, David Horne, Matthew S Sachs, Scott C Blanchard, Marat Yusupov, Christopher D Vanderwal","doi":"10.1021/acschembio.4c00825","DOIUrl":"https://doi.org/10.1021/acschembio.4c00825","url":null,"abstract":"<p><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":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2025-03-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143676572","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}
引用次数: 0
Inhibitors of Lysinoalanine Cross-Linking in the Flagella Hook as Antimicrobials against Spirochetes.
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-03-21 Epub Date: 2025-02-25 DOI: 10.1021/acschembio.4c00749
Michael J Lynch, Kurni Kurniyati, Maithili Deshpande, Nyles W Charon, Chunhao Li, Brian R Crane

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 Treponema denticola (Td) and Borreliella burgdorferi (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.

{"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}
引用次数: 0
Characterization of the Iron-Sulfur Cluster in the NCOA4 Fragment (383-522) and Its Interaction with Ferritin.
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-03-21 Epub Date: 2025-02-27 DOI: 10.1021/acschembio.4c00877
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

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.

{"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}
引用次数: 0
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