Pub Date : 2024-09-19DOI: 10.1038/s41594-024-01402-7
Vivek Singh, J. Conor Moran, Yuzuru Itoh, Iliana C. Soto, Flavia Fontanesi, Mary Couvillion, Martijn A. Huynen, L. Stirling Churchman, Antoni Barrientos, Alexey Amunts
{"title":"Publisher Correction: Structural basis of LRPPRC–SLIRP-dependent translation by the mitoribosome","authors":"Vivek Singh, J. Conor Moran, Yuzuru Itoh, Iliana C. Soto, Flavia Fontanesi, Mary Couvillion, Martijn A. Huynen, L. Stirling Churchman, Antoni Barrientos, Alexey Amunts","doi":"10.1038/s41594-024-01402-7","DOIUrl":"10.1038/s41594-024-01402-7","url":null,"abstract":"","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"31 11","pages":"1809-1809"},"PeriodicalIF":12.5,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.nature.com/articles/s41594-024-01402-7.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142273580","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 : 2024-09-17DOI: 10.1038/s41594-024-01378-4
Valentyn Petrychenko, Sung-Hui Yi, David Liedtke, Bee-Zen Peng, Marina V. Rodnina, Niels Fischer
The selection of an open reading frame (ORF) for translation of eukaryotic mRNA relies on remodeling of the scanning 48S initiation complex into an elongation-ready 80S ribosome. Using cryo-electron microscopy, we visualize the key commitment steps orchestrating 48S remodeling in humans. The mRNA Kozak sequence facilitates mRNA scanning in the 48S open state and stabilizes the 48S closed state by organizing the contacts of eukaryotic initiation factors (eIFs) and ribosomal proteins and by reconfiguring mRNA structure. GTPase-triggered large-scale fluctuations of 48S-bound eIF2 facilitate eIF5B recruitment, transfer of initiator tRNA from eIF2 to eIF5B and the release of eIF5 and eIF2. The 48S-bound multisubunit eIF3 complex controls ribosomal subunit joining by coupling eIF exchange to gradual displacement of the eIF3c N-terminal domain from the intersubunit interface. These findings reveal the structural mechanism of ORF selection in human cells and explain how eIF3 could function in the context of the 80S ribosome. Cryo-electron microscopy reveals the mechanism of human translation initiation from codon scanning to subunit joining. The structures show the roles of the Kozak sequence, GTP hydrolysis by eukaryotic initiation factor 2 (eIF2) and eIF5B in 48S remodeling, as well as that of eIF3 in the control of 60S docking.
{"title":"Structural basis for translational control by the human 48S initiation complex","authors":"Valentyn Petrychenko, Sung-Hui Yi, David Liedtke, Bee-Zen Peng, Marina V. Rodnina, Niels Fischer","doi":"10.1038/s41594-024-01378-4","DOIUrl":"10.1038/s41594-024-01378-4","url":null,"abstract":"The selection of an open reading frame (ORF) for translation of eukaryotic mRNA relies on remodeling of the scanning 48S initiation complex into an elongation-ready 80S ribosome. Using cryo-electron microscopy, we visualize the key commitment steps orchestrating 48S remodeling in humans. The mRNA Kozak sequence facilitates mRNA scanning in the 48S open state and stabilizes the 48S closed state by organizing the contacts of eukaryotic initiation factors (eIFs) and ribosomal proteins and by reconfiguring mRNA structure. GTPase-triggered large-scale fluctuations of 48S-bound eIF2 facilitate eIF5B recruitment, transfer of initiator tRNA from eIF2 to eIF5B and the release of eIF5 and eIF2. The 48S-bound multisubunit eIF3 complex controls ribosomal subunit joining by coupling eIF exchange to gradual displacement of the eIF3c N-terminal domain from the intersubunit interface. These findings reveal the structural mechanism of ORF selection in human cells and explain how eIF3 could function in the context of the 80S ribosome. Cryo-electron microscopy reveals the mechanism of human translation initiation from codon scanning to subunit joining. The structures show the roles of the Kozak sequence, GTP hydrolysis by eukaryotic initiation factor 2 (eIF2) and eIF5B in 48S remodeling, as well as that of eIF3 in the control of 60S docking.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"32 1","pages":"62-72"},"PeriodicalIF":12.5,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.nature.com/articles/s41594-024-01378-4.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142235054","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 : 2024-09-09DOI: 10.1038/s41594-024-01398-0
Sarah S. Henrikus, Marta H. Gross, Oliver Willhoft, Thomas Pühringer, Jacob S. Lewis, Allison W. McClure, Julia F. Greiwe, Giacomo Palm, Andrea Nans, John F. X. Diffley, Alessandro Costa
{"title":"Author Correction: Unwinding of a eukaryotic origin of replication visualized by cryo-EM","authors":"Sarah S. Henrikus, Marta H. Gross, Oliver Willhoft, Thomas Pühringer, Jacob S. Lewis, Allison W. McClure, Julia F. Greiwe, Giacomo Palm, Andrea Nans, John F. X. Diffley, Alessandro Costa","doi":"10.1038/s41594-024-01398-0","DOIUrl":"10.1038/s41594-024-01398-0","url":null,"abstract":"","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"31 11","pages":"1808-1808"},"PeriodicalIF":12.5,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.nature.com/articles/s41594-024-01398-0.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142165965","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 : 2024-09-09DOI: 10.1038/s41594-024-01384-6
Sicong Zhang, Robert G. Roeder
The bromodomain and extraterminal domain (BET) family of proteins are critical chromatin readers that bind to acetylated histones through their bromodomains to activate transcription. Here, we reveal that bromodomain inhibition fails to repress oncogenic targets of estrogen receptor because of an intrinsic transcriptional mechanism. While bromodomains are necessary for the transcription of many genes, bromodomain-containing protein 4 (BRD4) binds to estrogen receptor binding sites and activates transcription of critical oncogenes such as MYC, independently of its bromodomains. BRD4 associates with the Mediator complex and disruption of Mediator reduces BRD4’s enhancer occupancy. Profiling changes of the post-initiation RNA polymerase II (Pol II)-associated factors revealed that BET proteins regulate interactions between Pol II and elongation factors SPT5, SPT6 and the polymerase-associated factor 1 complex, which associate with BET proteins independently of their bromodomains and mediate their transcription elongation effect. Our findings highlight the importance of bromodomain-independent functions and interactions of BET proteins in the development of future therapeutic strategies. Here, the authors show that bromodomain-containing protein 4 is recruited to the MYC enhancer by Mediator and activates transcription through elongation factors independently of bromodomains. This mechanism contributes to bromodomain and extraterminal domain inhibitor resistance in estrogen receptor-positive breast cancer.
{"title":"Resistance of estrogen receptor function to BET bromodomain inhibition is mediated by transcriptional coactivator cooperativity","authors":"Sicong Zhang, Robert G. Roeder","doi":"10.1038/s41594-024-01384-6","DOIUrl":"10.1038/s41594-024-01384-6","url":null,"abstract":"The bromodomain and extraterminal domain (BET) family of proteins are critical chromatin readers that bind to acetylated histones through their bromodomains to activate transcription. Here, we reveal that bromodomain inhibition fails to repress oncogenic targets of estrogen receptor because of an intrinsic transcriptional mechanism. While bromodomains are necessary for the transcription of many genes, bromodomain-containing protein 4 (BRD4) binds to estrogen receptor binding sites and activates transcription of critical oncogenes such as MYC, independently of its bromodomains. BRD4 associates with the Mediator complex and disruption of Mediator reduces BRD4’s enhancer occupancy. Profiling changes of the post-initiation RNA polymerase II (Pol II)-associated factors revealed that BET proteins regulate interactions between Pol II and elongation factors SPT5, SPT6 and the polymerase-associated factor 1 complex, which associate with BET proteins independently of their bromodomains and mediate their transcription elongation effect. Our findings highlight the importance of bromodomain-independent functions and interactions of BET proteins in the development of future therapeutic strategies. Here, the authors show that bromodomain-containing protein 4 is recruited to the MYC enhancer by Mediator and activates transcription through elongation factors independently of bromodomains. This mechanism contributes to bromodomain and extraterminal domain inhibitor resistance in estrogen receptor-positive breast cancer.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"32 1","pages":"98-112"},"PeriodicalIF":12.5,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142158996","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 : 2024-09-06DOI: 10.1038/s41594-024-01388-2
Chanwoo Lee, Dante Lepore, Seung-Hak Lee, Tae Gyun Kim, Natasha Buwa, Jongchan Lee, Mary Munson, Tae-Young Yoon
Exocyst is a large multisubunit tethering complex essential for targeting and fusion of secretory vesicles in eukaryotic cells. Although the assembled exocyst complex has been proposed to tether vesicles to the plasma membrane and activate soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) for membrane fusion, the key biochemical steps that exocyst stimulates in SNARE-mediated fusion are undetermined. Here we use a combination of single-molecule and bulk fluorescence assays to investigate the roles of purified octameric yeast exocyst complexes in a reconstituted yeast exocytic SNARE assembly and vesicle fusion system. Exocyst had stimulatory roles in multiple distinct steps ranging from SNARE protein activation to binary and ternary complex assembly. Importantly, exocyst had a downstream role in driving membrane fusion and full content mixing of vesicle lumens. Our data suggest that exocyst provides extensive chaperoning functions across the entire process of SNARE complex assembly and fusion, thereby governing exocytosis at multiple steps. Exocytosis of secretory vesicles is required for cellular growth, cellular division and cell–cell communication. Lee et al. reveal that the exocyst tethering complex has stimulatory roles in exocytic soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly and SNARE-mediated vesicle fusion.
{"title":"Exocyst stimulates multiple steps of exocytic SNARE complex assembly and vesicle fusion","authors":"Chanwoo Lee, Dante Lepore, Seung-Hak Lee, Tae Gyun Kim, Natasha Buwa, Jongchan Lee, Mary Munson, Tae-Young Yoon","doi":"10.1038/s41594-024-01388-2","DOIUrl":"10.1038/s41594-024-01388-2","url":null,"abstract":"Exocyst is a large multisubunit tethering complex essential for targeting and fusion of secretory vesicles in eukaryotic cells. Although the assembled exocyst complex has been proposed to tether vesicles to the plasma membrane and activate soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) for membrane fusion, the key biochemical steps that exocyst stimulates in SNARE-mediated fusion are undetermined. Here we use a combination of single-molecule and bulk fluorescence assays to investigate the roles of purified octameric yeast exocyst complexes in a reconstituted yeast exocytic SNARE assembly and vesicle fusion system. Exocyst had stimulatory roles in multiple distinct steps ranging from SNARE protein activation to binary and ternary complex assembly. Importantly, exocyst had a downstream role in driving membrane fusion and full content mixing of vesicle lumens. Our data suggest that exocyst provides extensive chaperoning functions across the entire process of SNARE complex assembly and fusion, thereby governing exocytosis at multiple steps. Exocytosis of secretory vesicles is required for cellular growth, cellular division and cell–cell communication. Lee et al. reveal that the exocyst tethering complex has stimulatory roles in exocytic soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly and SNARE-mediated vesicle fusion.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"32 1","pages":"150-160"},"PeriodicalIF":12.5,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142142420","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 : 2024-09-03DOI: 10.1038/s41594-024-01386-4
Kevin Michalski, Taha Abdulla, Sam Kleeman, Lars Schmidl, Ricardo Gómez, Noriko Simorowski, Francesca Vallese, Harald Prüss, Manfred Heckmann, Christian Geis, Hiro Furukawa
Autoantibodies against neuronal membrane proteins can manifest in autoimmune encephalitis, inducing seizures, cognitive dysfunction and psychosis. Anti-N-methyl-d-aspartate receptor (NMDAR) encephalitis is the most dominant autoimmune encephalitis; however, insights into how autoantibodies recognize and alter receptor functions remain limited. Here we determined structures of human and rat NMDARs bound to three distinct patient-derived antibodies using single-particle electron cryo-microscopy. These antibodies bind different regions within the amino-terminal domain of the GluN1 subunit. Through electrophysiology, we show that all three autoantibodies acutely and directly reduced NMDAR channel functions in primary neurons. Antibodies show different stoichiometry of binding and antibody–receptor complex formation, which in one antibody, 003-102, also results in reduced synaptic localization of NMDARs. These studies demonstrate mechanisms of diverse epitope recognition and direct channel regulation of anti-NMDAR autoantibodies underlying autoimmune encephalitis. Anti-NMDA receptor encephalitis is the most common autoimmune encephalitis. Michalski et al. reveal epitope diversity, conformational changes and functional impacts of the autoantibodies using cryo-EM and electrophysiology.
{"title":"Structural and functional mechanisms of anti-NMDAR autoimmune encephalitis","authors":"Kevin Michalski, Taha Abdulla, Sam Kleeman, Lars Schmidl, Ricardo Gómez, Noriko Simorowski, Francesca Vallese, Harald Prüss, Manfred Heckmann, Christian Geis, Hiro Furukawa","doi":"10.1038/s41594-024-01386-4","DOIUrl":"10.1038/s41594-024-01386-4","url":null,"abstract":"Autoantibodies against neuronal membrane proteins can manifest in autoimmune encephalitis, inducing seizures, cognitive dysfunction and psychosis. Anti-N-methyl-d-aspartate receptor (NMDAR) encephalitis is the most dominant autoimmune encephalitis; however, insights into how autoantibodies recognize and alter receptor functions remain limited. Here we determined structures of human and rat NMDARs bound to three distinct patient-derived antibodies using single-particle electron cryo-microscopy. These antibodies bind different regions within the amino-terminal domain of the GluN1 subunit. Through electrophysiology, we show that all three autoantibodies acutely and directly reduced NMDAR channel functions in primary neurons. Antibodies show different stoichiometry of binding and antibody–receptor complex formation, which in one antibody, 003-102, also results in reduced synaptic localization of NMDARs. These studies demonstrate mechanisms of diverse epitope recognition and direct channel regulation of anti-NMDAR autoantibodies underlying autoimmune encephalitis. Anti-NMDA receptor encephalitis is the most common autoimmune encephalitis. Michalski et al. reveal epitope diversity, conformational changes and functional impacts of the autoantibodies using cryo-EM and electrophysiology.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"31 12","pages":"1975-1986"},"PeriodicalIF":12.5,"publicationDate":"2024-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142123517","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 : 2024-09-03DOI: 10.1038/s41594-024-01387-3
Han Wang, Chun Xie, Bo Deng, Jinjun Ding, Na Li, Zengwei Kou, Mengmeng Jin, Jie He, Qinrui Wang, Han Wen, Jinbao Zhang, Qinming Zhou, Sheng Chen, Xiangjun Chen, Ti-Fei Yuan, Shujia Zhu
Antibodies against N-methyl-d-aspartate receptors (NMDARs) are most frequently detected in persons with autoimmune encephalitis (AE) and used as diagnostic biomarkers. Elucidating the structural basis of monoclonal antibody (mAb) binding to NMDARs would facilitate the development of targeted therapy for AE. Here, we reconstructed nanodiscs containing green fluorescent protein-fused NMDARs to label and sort individual immune B cells from persons with AE and further cloned and identified mAbs against NMDARs. This allowed cryo-electron microscopy analysis of NMDAR–Fab complexes, revealing that autoantibodies bind to the R1 lobe of the N-terminal domain of the GluN1 subunit. Small-angle X-ray scattering studies demonstrated NMDAR–mAb stoichiometry of 2:1 or 1:2, structurally suitable for mAb-induced clustering and endocytosis of NMDARs. Importantly, these mAbs reduced the surface NMDARs and NMDAR-mediated currents, without tonically affecting NMDAR channel gating. These structural and functional findings imply that the design of neutralizing antibody binding to the R1 lobe of NMDARs represents a potential therapy for AE treatment. The authors cloned anti-NMDAR (N-methyl-d-aspartate receptor) monoclonal antibodies from the immune B cells of persons with autoimmune encephalitis and revealed their precise binding epitopes on NMDARs and the pathological mechanism underlying the downregulation of synaptic function.
N-甲基-d-天冬氨酸受体（NMDAR）抗体最常在自身免疫性脑炎（AE）患者体内检测到，并被用作诊断生物标志物。阐明单克隆抗体（mAb）与 NMDARs 结合的结构基础将有助于开发治疗自身免疫性脑炎的靶向疗法。在这里，我们重建了含有绿色荧光蛋白融合 NMDARs 的纳米圆盘，以标记和分拣来自 AE 患者的单个免疫 B 细胞，并进一步克隆和鉴定了针对 NMDARs 的 mAb。这样就可以对 NMDAR-Fab 复合物进行冷冻电镜分析，发现自身抗体与 GluN1 亚基 N 端结构域的 R1 叶结合。小角 X 射线散射研究表明，NMDAR-mAb 的配比为 2:1 或 1:2，在结构上适合 mAb 诱导的 NMDAR 聚集和内吞。重要的是，这些 mAb 减少了表面 NMDARs 和 NMDAR 介导的电流，而不会影响 NMDAR 通道门控。这些结构和功能研究结果表明，设计与 NMDARs R1 叶结合的中和抗体是一种潜在的 AE 治疗方法。
{"title":"Structural basis for antibody-mediated NMDA receptor clustering and endocytosis in autoimmune encephalitis","authors":"Han Wang, Chun Xie, Bo Deng, Jinjun Ding, Na Li, Zengwei Kou, Mengmeng Jin, Jie He, Qinrui Wang, Han Wen, Jinbao Zhang, Qinming Zhou, Sheng Chen, Xiangjun Chen, Ti-Fei Yuan, Shujia Zhu","doi":"10.1038/s41594-024-01387-3","DOIUrl":"10.1038/s41594-024-01387-3","url":null,"abstract":"Antibodies against N-methyl-d-aspartate receptors (NMDARs) are most frequently detected in persons with autoimmune encephalitis (AE) and used as diagnostic biomarkers. Elucidating the structural basis of monoclonal antibody (mAb) binding to NMDARs would facilitate the development of targeted therapy for AE. Here, we reconstructed nanodiscs containing green fluorescent protein-fused NMDARs to label and sort individual immune B cells from persons with AE and further cloned and identified mAbs against NMDARs. This allowed cryo-electron microscopy analysis of NMDAR–Fab complexes, revealing that autoantibodies bind to the R1 lobe of the N-terminal domain of the GluN1 subunit. Small-angle X-ray scattering studies demonstrated NMDAR–mAb stoichiometry of 2:1 or 1:2, structurally suitable for mAb-induced clustering and endocytosis of NMDARs. Importantly, these mAbs reduced the surface NMDARs and NMDAR-mediated currents, without tonically affecting NMDAR channel gating. These structural and functional findings imply that the design of neutralizing antibody binding to the R1 lobe of NMDARs represents a potential therapy for AE treatment. The authors cloned anti-NMDAR (N-methyl-d-aspartate receptor) monoclonal antibodies from the immune B cells of persons with autoimmune encephalitis and revealed their precise binding epitopes on NMDARs and the pathological mechanism underlying the downregulation of synaptic function.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"31 12","pages":"1987-1996"},"PeriodicalIF":12.5,"publicationDate":"2024-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.nature.com/articles/s41594-024-01387-3.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142123520","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 : 2024-09-03DOI: 10.1038/s41594-024-01381-9
Ashleigh King, Pia I. Reichl, Jean S. Metson, Robert Parker, Daniella Munro, Catarina Oliveira, Lucia Sommerova, Jordan R. Becker, Daniel Biggs, Chris Preece, Benjamin Davies, J. Ross Chapman
Tumor suppressor p53-binding protein 1 (53BP1) regulates DNA end joining in lymphocytes, diversifying immune antigen receptors. This involves nucleosome-bound 53BP1 at DNA double-stranded breaks (DSBs) recruiting Rap1-interacting factor 1 homolog (RIF1) and shieldin, a poorly understood DNA-binding complex. The 53BP1–RIF1–shieldin axis is pathological in BRCA1-mutated cancers, blocking homologous recombination (HR) and driving illegitimate nonhomologous end joining (NHEJ). However, how this axis regulates DNA end joining and HR suppression remains unresolved. We investigated shieldin and its interplay with the Ctc1–Stn1–Ten1 (CST) complex, which was recently implicated downstream of 53BP1. Immunophenotypically, mice lacking shieldin or CST are equivalent, with class-switch recombination coreliant on both complexes. Ataxia-telangiectasia mutated kinase-dependent DNA damage signaling underpins this cooperation, inducing physical interactions between these complexes that reveal shieldin as a DSB-responsive CST adaptor. Furthermore, DNA polymerase ζ functions downstream of shieldin, establishing DNA fill-in synthesis as the physiological function of shieldin–CST. Lastly, we demonstrate that 53BP1 suppresses HR and promotes NHEJ in BRCA1-deficient mice and cells independently of shieldin. These findings showcase the versatility of the 53BP1 pathway, achieved through the collaboration of chromatin-bound 53BP1 complexes and DNA end-processing effector proteins. Here, using mouse genetics, biochemistry and cell-based experiments, the authors reveal that shieldin primarily catalyzes DNA priming and polymerase-dependent fill-in synthesis at 5′ recessed DNA ends during the joining of activation-induced cytidine deaminase-dependent DNA breaks.
肿瘤抑制因子 p53 结合蛋白 1（53BP1）调节淋巴细胞中的 DNA 端接，使免疫抗原受体多样化。这涉及到核糖体结合的 53BP1 在 DNA 双链断裂（DSB）处招募 Rap1-interacting factor 1 homolog（RIF1）和盾牌蛋白，后者是一种鲜为人知的 DNA 结合复合物。在 BRCA1 基因突变的癌症中，53BP1-RIF1-shieldin 轴是病态的，它会阻碍同源重组（HR）并驱动非法的非同源末端连接（NHEJ）。然而，该轴如何调控DNA末端连接和HR抑制仍未解决。我们研究了屏蔽素及其与Ctc1-Stn1-Ten1（CST）复合物的相互作用，最近发现CST与53BP1下游有关。从免疫表型上看，缺乏屏蔽素或CST的小鼠是等同的，类开关重组核心依赖于这两种复合体。依赖于共济失调-特朗日病突变激酶的DNA损伤信号是这种合作的基础，它诱导了这些复合体之间的物理相互作用，从而揭示了屏蔽素是DSB反应性CST适配体。此外，DNA聚合酶ζ在屏蔽素的下游发挥作用，从而确定了DNA填充合成是屏蔽素-CST的生理功能。最后，我们证明在 BRCA1 缺失的小鼠和细胞中，53BP1 可独立于屏蔽素抑制 HR 并促进 NHEJ。这些发现展示了 53BP1 通路的多功能性，它是通过与染色质结合的 53BP1 复合物和 DNA 末端处理效应蛋白的协作实现的。
{"title":"Shieldin and CST co-orchestrate DNA polymerase-dependent tailed-end joining reactions independently of 53BP1-governed repair pathway choice","authors":"Ashleigh King, Pia I. Reichl, Jean S. Metson, Robert Parker, Daniella Munro, Catarina Oliveira, Lucia Sommerova, Jordan R. Becker, Daniel Biggs, Chris Preece, Benjamin Davies, J. Ross Chapman","doi":"10.1038/s41594-024-01381-9","DOIUrl":"10.1038/s41594-024-01381-9","url":null,"abstract":"Tumor suppressor p53-binding protein 1 (53BP1) regulates DNA end joining in lymphocytes, diversifying immune antigen receptors. This involves nucleosome-bound 53BP1 at DNA double-stranded breaks (DSBs) recruiting Rap1-interacting factor 1 homolog (RIF1) and shieldin, a poorly understood DNA-binding complex. The 53BP1–RIF1–shieldin axis is pathological in BRCA1-mutated cancers, blocking homologous recombination (HR) and driving illegitimate nonhomologous end joining (NHEJ). However, how this axis regulates DNA end joining and HR suppression remains unresolved. We investigated shieldin and its interplay with the Ctc1–Stn1–Ten1 (CST) complex, which was recently implicated downstream of 53BP1. Immunophenotypically, mice lacking shieldin or CST are equivalent, with class-switch recombination coreliant on both complexes. Ataxia-telangiectasia mutated kinase-dependent DNA damage signaling underpins this cooperation, inducing physical interactions between these complexes that reveal shieldin as a DSB-responsive CST adaptor. Furthermore, DNA polymerase ζ functions downstream of shieldin, establishing DNA fill-in synthesis as the physiological function of shieldin–CST. Lastly, we demonstrate that 53BP1 suppresses HR and promotes NHEJ in BRCA1-deficient mice and cells independently of shieldin. These findings showcase the versatility of the 53BP1 pathway, achieved through the collaboration of chromatin-bound 53BP1 complexes and DNA end-processing effector proteins. Here, using mouse genetics, biochemistry and cell-based experiments, the authors reveal that shieldin primarily catalyzes DNA priming and polymerase-dependent fill-in synthesis at 5′ recessed DNA ends during the joining of activation-induced cytidine deaminase-dependent DNA breaks.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"32 1","pages":"86-97"},"PeriodicalIF":12.5,"publicationDate":"2024-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.nature.com/articles/s41594-024-01381-9.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142123592","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 : 2024-09-02DOI: 10.1038/s41594-024-01379-3
Jiayue Su, Xuyang Tian, Hang Cheng, Desheng Liu, Ziyi Wang, Shan Sun, Hong-Wei Wang, Sen-Fang Sui
The enzymes 3-methylcrotonyl-coenzyme A (CoA) carboxylase (MCC), pyruvate carboxylase and propionyl-CoA carboxylase belong to the biotin-dependent carboxylase family located in mitochondria. They participate in various metabolic pathways in human such as amino acid metabolism and tricarboxylic acid cycle. Many human diseases are caused by mutations in those enzymes but their structures have not been fully resolved so far. Here we report an optimized purification strategy to obtain high-resolution structures of intact human endogenous MCC, propionyl-CoA carboxylase and pyruvate carboxylase in different conformational states. We also determine the structures of MCC bound to different substrates. Analysis of MCC structures in different states reveals the mechanism of the substrate-induced, multi-element synergistic activation of MCC. These results provide important insights into the catalytic mechanism of the biotin-dependent carboxylase family and are of great value for the development of new drugs for the treatment of related diseases. This work reveals structures of biotin-dependent carboxylases in different states, provides notable insight into their catalytic mechanism and may help the development of new drugs for the treatment of related diseases.
{"title":"Structural insight into synergistic activation of human 3-methylcrotonyl-CoA carboxylase","authors":"Jiayue Su, Xuyang Tian, Hang Cheng, Desheng Liu, Ziyi Wang, Shan Sun, Hong-Wei Wang, Sen-Fang Sui","doi":"10.1038/s41594-024-01379-3","DOIUrl":"10.1038/s41594-024-01379-3","url":null,"abstract":"The enzymes 3-methylcrotonyl-coenzyme A (CoA) carboxylase (MCC), pyruvate carboxylase and propionyl-CoA carboxylase belong to the biotin-dependent carboxylase family located in mitochondria. They participate in various metabolic pathways in human such as amino acid metabolism and tricarboxylic acid cycle. Many human diseases are caused by mutations in those enzymes but their structures have not been fully resolved so far. Here we report an optimized purification strategy to obtain high-resolution structures of intact human endogenous MCC, propionyl-CoA carboxylase and pyruvate carboxylase in different conformational states. We also determine the structures of MCC bound to different substrates. Analysis of MCC structures in different states reveals the mechanism of the substrate-induced, multi-element synergistic activation of MCC. These results provide important insights into the catalytic mechanism of the biotin-dependent carboxylase family and are of great value for the development of new drugs for the treatment of related diseases. This work reveals structures of biotin-dependent carboxylases in different states, provides notable insight into their catalytic mechanism and may help the development of new drugs for the treatment of related diseases.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"32 1","pages":"73-85"},"PeriodicalIF":12.5,"publicationDate":"2024-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142118124","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 : 2024-08-22DOI: 10.1038/s41594-024-01374-8
Methyl-CpG-binding protein 2 (MeCP2) is a master regulator of neuronal gene expression, and its genetic mutations cause the neurodevelopmental disorder Rett syndrome. Single-molecule experiments have enabled the direct visualization of the dynamics of MeCP2 on DNA, shedding light on how the specific chromatin context tunes MeCP2 function.
{"title":"Dynamics of an epigenetic regulator on chromatin observed at the single-molecule level","authors":"","doi":"10.1038/s41594-024-01374-8","DOIUrl":"10.1038/s41594-024-01374-8","url":null,"abstract":"Methyl-CpG-binding protein 2 (MeCP2) is a master regulator of neuronal gene expression, and its genetic mutations cause the neurodevelopmental disorder Rett syndrome. Single-molecule experiments have enabled the direct visualization of the dynamics of MeCP2 on DNA, shedding light on how the specific chromatin context tunes MeCP2 function.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"31 11","pages":"1648-1649"},"PeriodicalIF":12.5,"publicationDate":"2024-08-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142021862","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}
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