Pub Date : 2025-12-11DOI: 10.1038/s44318-025-00660-5
David S Fay,Boopathi Balasubramaniam,Sean M Harrington,Philip T Edeen
Proximity labeling has emerged as a powerful approach for identifying protein-protein interaction networks within living systems, particularly those involving weak or transient associations. Here, we present a comprehensive revised proximity labeling workflow, integrating TurboID labeling of endogenously expressed fusion proteins and data-independent acquisition (DIA) mass spectrometry (MS). We benchmark this pipeline with a study of five conserved Caenorhabditis elegans proteins-NEKL-2, NEKL-3, MLT-2, MLT-3, and MLT-4- that form two NEKL-MLT kinase-scaffold subcomplexes involved in membrane trafficking and actin regulation. Profiling of NEKL-MLT interactomes across 23 experiments validated our approach through the identification of known NEKL-MLT binding partners and conserved nekl-mlt genetic interactors, including the discovery of several novel functional interactors. Importantly, inclusion of methodological variations, stringent controls, and filtering strategies enhanced sensitivity and reproducibility, defining a set of intuitive quantitative metrics for routine assessment of experimental quality. We show that DIA-based interactome workflows produce physiologically relevant findings, even in the presence of experimental noise and variability across biological replicates. Our study underscores the utility of DIA mass spectrometry in proximity labeling applications and highlights the value of incorporating internal controls, quantitative metrics, and biological validation to enhance confidence in candidate interactors.
{"title":"Integrating endogenous TurboID and data-independent acquisition mass spectrometry for in vivo proximity labeling.","authors":"David S Fay,Boopathi Balasubramaniam,Sean M Harrington,Philip T Edeen","doi":"10.1038/s44318-025-00660-5","DOIUrl":"https://doi.org/10.1038/s44318-025-00660-5","url":null,"abstract":"Proximity labeling has emerged as a powerful approach for identifying protein-protein interaction networks within living systems, particularly those involving weak or transient associations. Here, we present a comprehensive revised proximity labeling workflow, integrating TurboID labeling of endogenously expressed fusion proteins and data-independent acquisition (DIA) mass spectrometry (MS). We benchmark this pipeline with a study of five conserved Caenorhabditis elegans proteins-NEKL-2, NEKL-3, MLT-2, MLT-3, and MLT-4- that form two NEKL-MLT kinase-scaffold subcomplexes involved in membrane trafficking and actin regulation. Profiling of NEKL-MLT interactomes across 23 experiments validated our approach through the identification of known NEKL-MLT binding partners and conserved nekl-mlt genetic interactors, including the discovery of several novel functional interactors. Importantly, inclusion of methodological variations, stringent controls, and filtering strategies enhanced sensitivity and reproducibility, defining a set of intuitive quantitative metrics for routine assessment of experimental quality. We show that DIA-based interactome workflows produce physiologically relevant findings, even in the presence of experimental noise and variability across biological replicates. Our study underscores the utility of DIA mass spectrometry in proximity labeling applications and highlights the value of incorporating internal controls, quantitative metrics, and biological validation to enhance confidence in candidate interactors.","PeriodicalId":501009,"journal":{"name":"The EMBO Journal","volume":"20 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-12-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145728508","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Microbial translation arrest peptides monitor intracellular environments and feedback-regulate downstream gene expression. Previous studies have identified a class of bacterial arrest peptides with C-terminal RAPP-like sequences, encoded upstream of genes involved in protein localization. In this study, we found that among RAPP-like sequences, RAPP (Arg-Ala-Pro-Pro) and RGPP (Arg-Gly-Pro-Pro) could more readily evolve into translation-impeding sequences with a particularly robust arrest that is refractory to EF-P. RAPP-like motifs were found to be strongly excluded from bacterial proteomes, likely reflecting the risk of disrupting the cellular translation system. Meanwhile, these motifs tended to occur near the C-terminus of relatively small secretory and membrane proteins. Notably, they were encoded upstream of genes with diverse functions beyond protein localization. Indeed, we identified seven RAPP/RGPP-containing arrest peptides from Streptomyces lividans encoded upstream of genes with diverse functions. These findings illustrate the bidirectional evolution of RAPP-containing proteins: their elimination from bacterial proteomes and their adaptation into arrest peptides with various regulatory roles.
{"title":"Evolutionary adaptation of bacterial proteomes to translation-impeding sequences.","authors":"Keigo Fujiwara,Naoko Tsuji,Karen Sakiyama,Hironori Niki,Shinobu Chiba","doi":"10.1038/s44318-025-00651-6","DOIUrl":"https://doi.org/10.1038/s44318-025-00651-6","url":null,"abstract":"Microbial translation arrest peptides monitor intracellular environments and feedback-regulate downstream gene expression. Previous studies have identified a class of bacterial arrest peptides with C-terminal RAPP-like sequences, encoded upstream of genes involved in protein localization. In this study, we found that among RAPP-like sequences, RAPP (Arg-Ala-Pro-Pro) and RGPP (Arg-Gly-Pro-Pro) could more readily evolve into translation-impeding sequences with a particularly robust arrest that is refractory to EF-P. RAPP-like motifs were found to be strongly excluded from bacterial proteomes, likely reflecting the risk of disrupting the cellular translation system. Meanwhile, these motifs tended to occur near the C-terminus of relatively small secretory and membrane proteins. Notably, they were encoded upstream of genes with diverse functions beyond protein localization. Indeed, we identified seven RAPP/RGPP-containing arrest peptides from Streptomyces lividans encoded upstream of genes with diverse functions. These findings illustrate the bidirectional evolution of RAPP-containing proteins: their elimination from bacterial proteomes and their adaptation into arrest peptides with various regulatory roles.","PeriodicalId":501009,"journal":{"name":"The EMBO Journal","volume":"44 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-12-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145710858","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Post-meiotic development of spermatids is under the control of a sophisticated RNA metabolic network, wherein the N6-methyladenosine (m6A) modification of mRNA, and proteins that bind to it, exert crucial functions in regulating sperm development from spermatogonia to spermatocytes. However, which m6A recognition proteins are involved in male post-meiotic spermiogenesis, and via which regulatory mechanisms, remains largely unknown. Here, we uncover the involvement of the m6A reader protein IGF2BP3 in the regulation of post-meiotic spermatid development. Genetic ablation of Igf2bp3 results in spermatogenesis defects, leading to male sub-fertility or even infertility. Mechanistically, IGF2BP3 loss-of-function leads to the excessive translation of its target RNAs associated with histone-to-protamine replacement, particularly Dot1l and Hdac11. IGF2BP3 translationally represses these targets through its m6A-binding property and through its interaction with its binding partner YBX2. Sperm developmental defects of IGF2BP3 knockout mouse can be rescued by siRNAs targeting Dot1l and Hdac11. Collectively, our findings define the essential role of IGF2BP3-dependent regulation of protein biosynthesis in histone-to-protamine replacement during spermiogenesis, helping to understand the functions of m6A RNA modification in sperm development and male fertility.
{"title":"IGF2BP3 recognizes m6A to regulate histone-to-protamine replacement during mouse sperm development.","authors":"Dazhuang Wang,Zhenyi Huang,Yichun Zhou,Peiyan Chen,Gang Chang,Liwei Ke,Congying Jing,Haojie Yang,Jiexiang Zhao,Shaofang Ren,Yi Zheng,Yuhan Chen,Yunfan Xiang,Jun Liu,Mei Wang","doi":"10.1038/s44318-025-00659-y","DOIUrl":"https://doi.org/10.1038/s44318-025-00659-y","url":null,"abstract":"Post-meiotic development of spermatids is under the control of a sophisticated RNA metabolic network, wherein the N6-methyladenosine (m6A) modification of mRNA, and proteins that bind to it, exert crucial functions in regulating sperm development from spermatogonia to spermatocytes. However, which m6A recognition proteins are involved in male post-meiotic spermiogenesis, and via which regulatory mechanisms, remains largely unknown. Here, we uncover the involvement of the m6A reader protein IGF2BP3 in the regulation of post-meiotic spermatid development. Genetic ablation of Igf2bp3 results in spermatogenesis defects, leading to male sub-fertility or even infertility. Mechanistically, IGF2BP3 loss-of-function leads to the excessive translation of its target RNAs associated with histone-to-protamine replacement, particularly Dot1l and Hdac11. IGF2BP3 translationally represses these targets through its m6A-binding property and through its interaction with its binding partner YBX2. Sperm developmental defects of IGF2BP3 knockout mouse can be rescued by siRNAs targeting Dot1l and Hdac11. Collectively, our findings define the essential role of IGF2BP3-dependent regulation of protein biosynthesis in histone-to-protamine replacement during spermiogenesis, helping to understand the functions of m6A RNA modification in sperm development and male fertility.","PeriodicalId":501009,"journal":{"name":"The EMBO Journal","volume":"54 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145680711","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-05DOI: 10.1038/s44318-025-00647-2
Leticia Lemus,Hadar Meyer,Ana I Rodríguez-Rosado,Maya Schuldiner,Veit Goder
Protein quality control (PQC) in the secretory pathway, a process critically linked to numerous human diseases, begins in the endoplasmic reticulum (ER) and involves ER-associated degradation (ERAD) of terminally misfolded proteins. In this study, we conducted genome-wide screens in baker's yeast (Saccharomyces cerevisiae) to investigate the degradation of Gas1*, a misfolded version of an O-mannosylated, glycosylphosphatidylinositol (GPI)-anchored protein. In combination with detailed biochemical and genetic analyses, these screens revealed an unexpected bifunctionality of the evolutionarily conserved heteromeric enzyme complex Pbn1-Gpi14: while it has been previously recognized as a GPI-mannosyltransferase, we here find that it catalyzes the O-mannosylation of misfolded proteins, thereby promoting their ERAD. This process is particularly relevant for misfolded proteins that lack N-glycans. Our results suggest that protein O-mannosylation constitutes a distinct type of glycan-dependent mechanism for promoting ERAD.
{"title":"O-mannosylation of misfolded ER proteins promotes ERAD.","authors":"Leticia Lemus,Hadar Meyer,Ana I Rodríguez-Rosado,Maya Schuldiner,Veit Goder","doi":"10.1038/s44318-025-00647-2","DOIUrl":"https://doi.org/10.1038/s44318-025-00647-2","url":null,"abstract":"Protein quality control (PQC) in the secretory pathway, a process critically linked to numerous human diseases, begins in the endoplasmic reticulum (ER) and involves ER-associated degradation (ERAD) of terminally misfolded proteins. In this study, we conducted genome-wide screens in baker's yeast (Saccharomyces cerevisiae) to investigate the degradation of Gas1*, a misfolded version of an O-mannosylated, glycosylphosphatidylinositol (GPI)-anchored protein. In combination with detailed biochemical and genetic analyses, these screens revealed an unexpected bifunctionality of the evolutionarily conserved heteromeric enzyme complex Pbn1-Gpi14: while it has been previously recognized as a GPI-mannosyltransferase, we here find that it catalyzes the O-mannosylation of misfolded proteins, thereby promoting their ERAD. This process is particularly relevant for misfolded proteins that lack N-glycans. Our results suggest that protein O-mannosylation constitutes a distinct type of glycan-dependent mechanism for promoting ERAD.","PeriodicalId":501009,"journal":{"name":"The EMBO Journal","volume":"162 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145680710","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Localization of lysosomes influences their properties, e.g., perinuclear lysosomes are more acidic but less mobile compared with the peripheral ones. Furthermore, the endoplasmic reticulum (ER) can actively regulate the dynamics and functions of lysosomes via membrane contact sites. In this study, we find that ER-resident apoptosis-resistant E3 ubiquitin protein ligase 1 (AREL1) establishes membrane contacts with lysosomes by directly interacting with the Voa subunit of V-ATPase. AREL1 also catalyzes K33-linked polyubiquitylation of V-ATPase V1B2 subunit, inducing its binding to UBAC2 localized in the perinuclear ER. Depletion of AREL1 or UBAC2 increases the number of peripheral lysosomes that possess partially assembled V-ATPase, elevated luminal pH, and attenuated degradative capacity. Knockdown of ZRANB1, the deubiquitylating enzyme that antagonizes AREL1-mediated V1B2 ubiquitylation, promotes perinuclear clustering of lysosomes and increases lysosomal acidity and degradation. Mice lacking Arel1 exhibit age-dependent Purkinje cell loss, an ataxic phenotype, and motor impairment. Lipofuscin accumulation in the residual Purkinje cells of Arel1-/- mice indicates lysosomal dysfunction. Orchestration of lysosomal positioning and function by the AREL1-UBAC2-V-ATPase axis underscores the physiological significance of ER-regulated perinuclear lysosomal positioning in neurons.
{"title":"E3 ligase AREL1 controls perinuclear localization of lysosomes and supports Purkinje cell survival.","authors":"Luyi Jiang,Jiangfen Tang,Ya-Fen Zhang,Wen-Xuan Zou,Gang Deng,Na Tian,Xiaolu Zhao,Lei Han,Kai Liu,Bao-Liang Song,Jie Luo","doi":"10.1038/s44318-025-00654-3","DOIUrl":"https://doi.org/10.1038/s44318-025-00654-3","url":null,"abstract":"Localization of lysosomes influences their properties, e.g., perinuclear lysosomes are more acidic but less mobile compared with the peripheral ones. Furthermore, the endoplasmic reticulum (ER) can actively regulate the dynamics and functions of lysosomes via membrane contact sites. In this study, we find that ER-resident apoptosis-resistant E3 ubiquitin protein ligase 1 (AREL1) establishes membrane contacts with lysosomes by directly interacting with the Voa subunit of V-ATPase. AREL1 also catalyzes K33-linked polyubiquitylation of V-ATPase V1B2 subunit, inducing its binding to UBAC2 localized in the perinuclear ER. Depletion of AREL1 or UBAC2 increases the number of peripheral lysosomes that possess partially assembled V-ATPase, elevated luminal pH, and attenuated degradative capacity. Knockdown of ZRANB1, the deubiquitylating enzyme that antagonizes AREL1-mediated V1B2 ubiquitylation, promotes perinuclear clustering of lysosomes and increases lysosomal acidity and degradation. Mice lacking Arel1 exhibit age-dependent Purkinje cell loss, an ataxic phenotype, and motor impairment. Lipofuscin accumulation in the residual Purkinje cells of Arel1-/- mice indicates lysosomal dysfunction. Orchestration of lysosomal positioning and function by the AREL1-UBAC2-V-ATPase axis underscores the physiological significance of ER-regulated perinuclear lysosomal positioning in neurons.","PeriodicalId":501009,"journal":{"name":"The EMBO Journal","volume":"19 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145656855","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-28DOI: 10.1038/s44318-025-00650-7
Zhibin Zhang,Ying Zhang,Judy Lieberman
The successes of cancer immunotherapy have inspired research aiming to increase the number of immune-responsive cancers. The first effective immunotherapeutic strategies-immune checkpoint blockade (ICB) and CAR T cells-were designed to overcome limitations in CD8+ T cell recognition and killing of tumor cells. However, most solid tumors still do not respond to these measures and new treatment approaches are needed. Tumors evolve many strategies to avoid immune control. One way to identify immunotherapy strategies is to study what distinguishes immunotherapy-responsive and -unresponsive tumors. Another way is to identify the differences in tumors that emerge after carcinogen exposure in immunocompetent versus immunodeficient hosts. Still another way is to identify changes in gene expression in emerging tumors that enable them to escape immunosurveillance (known as tumor immunoediting). Evolving tumors suppress antigen processing and presentation to avoid triggering tumor-specific T cells but also repress key innate immune genes that transmit danger signals to immune cells. In this perspective, we discuss the roles of innate immunity in anti-tumor responses and consider how innate immunity could be harnessed to make tumors more immune-responsive.
{"title":"Innate immunity in tumour immunoediting and immunosurveillance.","authors":"Zhibin Zhang,Ying Zhang,Judy Lieberman","doi":"10.1038/s44318-025-00650-7","DOIUrl":"https://doi.org/10.1038/s44318-025-00650-7","url":null,"abstract":"The successes of cancer immunotherapy have inspired research aiming to increase the number of immune-responsive cancers. The first effective immunotherapeutic strategies-immune checkpoint blockade (ICB) and CAR T cells-were designed to overcome limitations in CD8+ T cell recognition and killing of tumor cells. However, most solid tumors still do not respond to these measures and new treatment approaches are needed. Tumors evolve many strategies to avoid immune control. One way to identify immunotherapy strategies is to study what distinguishes immunotherapy-responsive and -unresponsive tumors. Another way is to identify the differences in tumors that emerge after carcinogen exposure in immunocompetent versus immunodeficient hosts. Still another way is to identify changes in gene expression in emerging tumors that enable them to escape immunosurveillance (known as tumor immunoediting). Evolving tumors suppress antigen processing and presentation to avoid triggering tumor-specific T cells but also repress key innate immune genes that transmit danger signals to immune cells. In this perspective, we discuss the roles of innate immunity in anti-tumor responses and consider how innate immunity could be harnessed to make tumors more immune-responsive.","PeriodicalId":501009,"journal":{"name":"The EMBO Journal","volume":"146 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-11-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145613274","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-27DOI: 10.1038/s44318-025-00628-5
Olivia Hunker,Franziska Bleichert
DNA replication initiation is a tightly regulated process that requires the coordinated assembly of replication machineries throughout the genome. During the first step of initiation, origin licensing, the MCM replicative helicase motor is loaded onto replication origins by the origin recognition complex (ORC) as a head-to-head double hexamer complex. Distinct mechanisms have been proposed to facilitate human MCM double hexamer loading, but the physiological relevance of each of them remains unclear. Here, we investigate the evolutionary conservation of these pathways using an AlphaFold-guided structural phylogenetics approach. Our analyses reveal that ORC6, a subunit of ORC previously thought to be essential for origin licensing in vivo, has been lost in multiple metazoan lineages. Despite this loss, many of these species retain an element in ORC3, the ORC3 tether, that can interact with MCM and facilitate an ORC6-independent MCM loading mechanism. AlphaFold2 Multimer predictions suggest that ORC3 tether interactions with MCM are broadly conserved across Metazoa. Our findings support the physiological relevance of ORC6-independent MCM loading, provide experimentally testable hypotheses on origin licensing mechanisms in diverse metazoan species, and highlight how AlphaFold can be leveraged to investigate protein evolution and function over large timescales.
{"title":"AlphaFold-guided phylogenetic analyses suggest surprising heterogeneity in metazoan replication origin licensing mechanisms.","authors":"Olivia Hunker,Franziska Bleichert","doi":"10.1038/s44318-025-00628-5","DOIUrl":"https://doi.org/10.1038/s44318-025-00628-5","url":null,"abstract":"DNA replication initiation is a tightly regulated process that requires the coordinated assembly of replication machineries throughout the genome. During the first step of initiation, origin licensing, the MCM replicative helicase motor is loaded onto replication origins by the origin recognition complex (ORC) as a head-to-head double hexamer complex. Distinct mechanisms have been proposed to facilitate human MCM double hexamer loading, but the physiological relevance of each of them remains unclear. Here, we investigate the evolutionary conservation of these pathways using an AlphaFold-guided structural phylogenetics approach. Our analyses reveal that ORC6, a subunit of ORC previously thought to be essential for origin licensing in vivo, has been lost in multiple metazoan lineages. Despite this loss, many of these species retain an element in ORC3, the ORC3 tether, that can interact with MCM and facilitate an ORC6-independent MCM loading mechanism. AlphaFold2 Multimer predictions suggest that ORC3 tether interactions with MCM are broadly conserved across Metazoa. Our findings support the physiological relevance of ORC6-independent MCM loading, provide experimentally testable hypotheses on origin licensing mechanisms in diverse metazoan species, and highlight how AlphaFold can be leveraged to investigate protein evolution and function over large timescales.","PeriodicalId":501009,"journal":{"name":"The EMBO Journal","volume":"1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-11-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145613338","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-26DOI: 10.1038/s44318-025-00644-5
Anna Huhn,Mikhail A Kutuzov,Keir Maclean,Lion F K Uhl,Jagdish M Mahale,Audrey Gérard,P Anton van der Merwe,Omer Dushek
T cells use their T-cell receptors (TCRs) to discriminate between higher-affinity foreign and lower-affinity self-peptide-MHC (pMHC) antigen complexes. The OT-I mouse TCR is widely used to study antigen discrimination between foreign and self-pMHC antigens, and previous work suggested it achieved near-perfect discrimination between higher- and lower-affinity antigens. However, other TCRs show imperfect discrimination. To resolve these discrepancies, we developed in this study a protocol for measuring ultra-low TCR-pMHC binding affinities to determine the 3D solution affinities of OT-I TCR for 19 pMHCs. These revised 3D affinities now strongly correlate with 2D membrane affinities and predict T-cell functional responses. Our results indicate that OT-I exhibits enhanced yet imperfect discrimination, similar to other TCRs, explaining how T cells can detect abnormally high levels of low-affinity self-antigens. We also show that OT-I discrimination is consistent with the kinetic proofreading model, which highlights that discrimination is most effective for low-affinity pMHC ligands. Our work underscores the ability of T cells to gauge proxies for 3D affinity within the 2D interface, with implications for the mechanisms underlying antigen discrimination.
{"title":"Murine T-cell receptor OT-I exhibits imperfect discrimination between foreign and self-antigens.","authors":"Anna Huhn,Mikhail A Kutuzov,Keir Maclean,Lion F K Uhl,Jagdish M Mahale,Audrey Gérard,P Anton van der Merwe,Omer Dushek","doi":"10.1038/s44318-025-00644-5","DOIUrl":"https://doi.org/10.1038/s44318-025-00644-5","url":null,"abstract":"T cells use their T-cell receptors (TCRs) to discriminate between higher-affinity foreign and lower-affinity self-peptide-MHC (pMHC) antigen complexes. The OT-I mouse TCR is widely used to study antigen discrimination between foreign and self-pMHC antigens, and previous work suggested it achieved near-perfect discrimination between higher- and lower-affinity antigens. However, other TCRs show imperfect discrimination. To resolve these discrepancies, we developed in this study a protocol for measuring ultra-low TCR-pMHC binding affinities to determine the 3D solution affinities of OT-I TCR for 19 pMHCs. These revised 3D affinities now strongly correlate with 2D membrane affinities and predict T-cell functional responses. Our results indicate that OT-I exhibits enhanced yet imperfect discrimination, similar to other TCRs, explaining how T cells can detect abnormally high levels of low-affinity self-antigens. We also show that OT-I discrimination is consistent with the kinetic proofreading model, which highlights that discrimination is most effective for low-affinity pMHC ligands. Our work underscores the ability of T cells to gauge proxies for 3D affinity within the 2D interface, with implications for the mechanisms underlying antigen discrimination.","PeriodicalId":501009,"journal":{"name":"The EMBO Journal","volume":"116 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-11-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145609974","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-26DOI: 10.1038/s44318-025-00648-1
Haoqiang K He,Shintaroh Kubo,Xuwei Chen,Qianru H Lv,Azusa Kage,Muneyoshi Ichikawa
Eukaryotic cilia and flagella are thin structures present on the surface of cells, playing vital roles in signaling and cellular motion. Cilia assembly depends on intraflagellar transport (IFT) along doublet microtubules (doublets). Unlike dynein-1, which works on cytoplasmic singlet microtubules, dynein-2 works on the doublets inside cilia. Previous studies have shown that retrograde IFT, driven by dynein-2, occurs on the A-tubule of the doublet, suggesting an elusive mechanism by which dynein-2 recruits retrograde IFT to the A-tubule. Here, we investigated the molecular basis of this mechanism using cryo-electron tomography (cryo-ET), molecular dynamics (MD) simulations, and biochemical analysis. Our biochemical assays revealed that the microtubule-binding domain of dynein-2 exhibits a higher affinity for the ciliary doublets than dynein-1. Cryo-ET further visualized the preferential binding of dynein-2 to the A-tubule of the doublet. MD simulations suggest that dynein-2 prefers the tyrosinated tubulin lattice as is present in the A-tubule. These findings reveal a recruitment mechanism of retrograde IFT by dynein-2, providing new insights into the spatial and functional specialization of ciliary doublets.
{"title":"Cryo-ET and MD simulations reveal that dynein-2 is tuned for binding to the A-tubule of the ciliary doublet.","authors":"Haoqiang K He,Shintaroh Kubo,Xuwei Chen,Qianru H Lv,Azusa Kage,Muneyoshi Ichikawa","doi":"10.1038/s44318-025-00648-1","DOIUrl":"https://doi.org/10.1038/s44318-025-00648-1","url":null,"abstract":"Eukaryotic cilia and flagella are thin structures present on the surface of cells, playing vital roles in signaling and cellular motion. Cilia assembly depends on intraflagellar transport (IFT) along doublet microtubules (doublets). Unlike dynein-1, which works on cytoplasmic singlet microtubules, dynein-2 works on the doublets inside cilia. Previous studies have shown that retrograde IFT, driven by dynein-2, occurs on the A-tubule of the doublet, suggesting an elusive mechanism by which dynein-2 recruits retrograde IFT to the A-tubule. Here, we investigated the molecular basis of this mechanism using cryo-electron tomography (cryo-ET), molecular dynamics (MD) simulations, and biochemical analysis. Our biochemical assays revealed that the microtubule-binding domain of dynein-2 exhibits a higher affinity for the ciliary doublets than dynein-1. Cryo-ET further visualized the preferential binding of dynein-2 to the A-tubule of the doublet. MD simulations suggest that dynein-2 prefers the tyrosinated tubulin lattice as is present in the A-tubule. These findings reveal a recruitment mechanism of retrograde IFT by dynein-2, providing new insights into the spatial and functional specialization of ciliary doublets.","PeriodicalId":501009,"journal":{"name":"The EMBO Journal","volume":"94 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-11-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145609976","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Changes in host carbohydrate metabolism determine the outcome of host-parasite relationships, but the underlying mechanistic basis remains elusive. Here, we show that the parasitoid wasp Cotesia vestalis induces trehalose accumulation in its host, the moth Plutella xylostella, largely independently of insulin/adipokinetic hormone signalling and food intake. Instead, parasitoids rewire host carbohydrate metabolism via two pathways activated by the evolutionarily conserved short neuropeptide F (sNPF), a functional analogue of mammalian neuropeptide Y. Parasitoid-derived teratocytes secrete sNPF that interacts with the sNPF receptor (sNPFR) on host cells, and contributes to host hypertrehalosemia by promoting glycogenolysis in the fat body. We further find that a parasitoid-symbiotic virus induces expression of host-encoded sNPF, which stimulates glycolysis in the host midgut. Furthermore, we show that the host sNPF-sNPFR complex stimulates Gq/Ca2+ signalling, while the parasitoid sNPF, exhibiting higher receptor affinity, triggers Gi/cAMP signalling. Molecular docking analyses suggest that the observed distinct receptor activation properties may be attributed to structural variations in the sNPF-sNPFR binding pocket. Collectively, our findings uncover an unexpected role of peripheral sNPFs in the regulation of carbohydrate metabolism during host-parasite interactions.
{"title":"Dual interference with host neuropeptide signaling allows parasitoid wasp to hijack host sugar metabolism.","authors":"Zhi-Zhi Wang,Ruo-Fei Ma,Li-Cheng Gu,Li-Zhi Wang,Ting Chen,Pei Yang,Jia-Ni Zou,Jiang-Yan Zhu,Zhi-Wei Wu,Yue-Nan Zhou,Min Shi,Xing-Xing Shen,Jian-Hua Huang,Xue-Xin Chen","doi":"10.1038/s44318-025-00636-5","DOIUrl":"https://doi.org/10.1038/s44318-025-00636-5","url":null,"abstract":"Changes in host carbohydrate metabolism determine the outcome of host-parasite relationships, but the underlying mechanistic basis remains elusive. Here, we show that the parasitoid wasp Cotesia vestalis induces trehalose accumulation in its host, the moth Plutella xylostella, largely independently of insulin/adipokinetic hormone signalling and food intake. Instead, parasitoids rewire host carbohydrate metabolism via two pathways activated by the evolutionarily conserved short neuropeptide F (sNPF), a functional analogue of mammalian neuropeptide Y. Parasitoid-derived teratocytes secrete sNPF that interacts with the sNPF receptor (sNPFR) on host cells, and contributes to host hypertrehalosemia by promoting glycogenolysis in the fat body. We further find that a parasitoid-symbiotic virus induces expression of host-encoded sNPF, which stimulates glycolysis in the host midgut. Furthermore, we show that the host sNPF-sNPFR complex stimulates Gq/Ca2+ signalling, while the parasitoid sNPF, exhibiting higher receptor affinity, triggers Gi/cAMP signalling. Molecular docking analyses suggest that the observed distinct receptor activation properties may be attributed to structural variations in the sNPF-sNPFR binding pocket. Collectively, our findings uncover an unexpected role of peripheral sNPFs in the regulation of carbohydrate metabolism during host-parasite interactions.","PeriodicalId":501009,"journal":{"name":"The EMBO Journal","volume":"21 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-11-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145609975","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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