Autophagy mediates the degradation of harmful material within lysosomes. In aggrephagy, the pathway mediating the degradation of aggregated, ubiquitinated proteins, this cargo material is collected in larger condensates prior to its sequestration by autophagosomes. In this process, the autophagic cargo receptors SQSTM1/p62 and NBR1 drive cargo condensation, while TAX1BP1, which binds to NBR1, recruits the autophagy machinery to facilitate autophagosome biogenesis at the condensates. The mechanistic basis for the TAX1BP1-mediated switch from cargo collection to its sequestration is unclear. Here we show that TAX1BP1 is not a constitutive component of the condensates. Its recruitment correlates with the induction of autophagosome biogenesis. TAX1BP1 is sufficient to recruit the TBK1 kinase via the SINTBAD adapter. We define the NBR1-TAX1BP1-binding site, which is adjacent to the GABARAP/LC3 interaction site, and demonstrate that the recruitment of TAX1BP1 to cargo mimetics can be enhanced by an increased ubiquitin load. Our study suggests that autophagosome biogenesis is initiated once sufficient cargo is collected in the condensates.
{"title":"Recruitment of autophagy initiator TAX1BP1 advances aggrephagy from cargo collection to sequestration.","authors":"Bernd Bauer,Jonas Idinger,Martina Schuschnig,Luca Ferrari,Sascha Martens","doi":"10.1038/s44318-024-00280-5","DOIUrl":"https://doi.org/10.1038/s44318-024-00280-5","url":null,"abstract":"Autophagy mediates the degradation of harmful material within lysosomes. In aggrephagy, the pathway mediating the degradation of aggregated, ubiquitinated proteins, this cargo material is collected in larger condensates prior to its sequestration by autophagosomes. In this process, the autophagic cargo receptors SQSTM1/p62 and NBR1 drive cargo condensation, while TAX1BP1, which binds to NBR1, recruits the autophagy machinery to facilitate autophagosome biogenesis at the condensates. The mechanistic basis for the TAX1BP1-mediated switch from cargo collection to its sequestration is unclear. Here we show that TAX1BP1 is not a constitutive component of the condensates. Its recruitment correlates with the induction of autophagosome biogenesis. TAX1BP1 is sufficient to recruit the TBK1 kinase via the SINTBAD adapter. We define the NBR1-TAX1BP1-binding site, which is adjacent to the GABARAP/LC3 interaction site, and demonstrate that the recruitment of TAX1BP1 to cargo mimetics can be enhanced by an increased ubiquitin load. Our study suggests that autophagosome biogenesis is initiated once sufficient cargo is collected in the condensates.","PeriodicalId":501009,"journal":{"name":"The EMBO Journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-10-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142490374","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}
Senescent cells play a causative role in many diseases, and their elimination is a promising therapeutic strategy. Here, through a genome-wide CRISPR/Cas9 screen, we identify the gene PPIF, encoding the mitochondrial protein cyclophilin D (CypD), as a novel senolytic target. Cyclophilin D promotes the transient opening of the mitochondrial permeability transition pore (mPTP), which serves as a failsafe mechanism for calcium efflux. We show that senescent cells exhibit a high frequency of transient CypD/mPTP opening events, known as 'flickering'. Inhibition of CypD using genetic or pharmacologic tools, including cyclosporin A, leads to the toxic accumulation of mitochondrial Ca2+ and the death of senescent cells. Genetic or pharmacological inhibition of NCLX, another mitochondrial calcium efflux channel, also leads to senolysis, while inhibition of the main Ca2+ influx channel, MCU, prevents senolysis induced by CypD inhibition. We conclude that senescent cells are highly vulnerable to elevated mitochondrial Ca2+ ions, and that transient CypD/mPTP opening is a critical adaptation mechanism for the survival of senescent cells.
{"title":"Cyclophilin D plays a critical role in the survival of senescent cells.","authors":"Margherita Protasoni,Vanessa López-Polo,Camille Stephan-Otto Attolini,Julian Brandariz,Nicolas Herranz,Joaquin Mateo,Sergio Ruiz,Oscar Fernandez-Capetillo,Marta Kovatcheva,Manuel Serrano","doi":"10.1038/s44318-024-00259-2","DOIUrl":"https://doi.org/10.1038/s44318-024-00259-2","url":null,"abstract":"Senescent cells play a causative role in many diseases, and their elimination is a promising therapeutic strategy. Here, through a genome-wide CRISPR/Cas9 screen, we identify the gene PPIF, encoding the mitochondrial protein cyclophilin D (CypD), as a novel senolytic target. Cyclophilin D promotes the transient opening of the mitochondrial permeability transition pore (mPTP), which serves as a failsafe mechanism for calcium efflux. We show that senescent cells exhibit a high frequency of transient CypD/mPTP opening events, known as 'flickering'. Inhibition of CypD using genetic or pharmacologic tools, including cyclosporin A, leads to the toxic accumulation of mitochondrial Ca2+ and the death of senescent cells. Genetic or pharmacological inhibition of NCLX, another mitochondrial calcium efflux channel, also leads to senolysis, while inhibition of the main Ca2+ influx channel, MCU, prevents senolysis induced by CypD inhibition. We conclude that senescent cells are highly vulnerable to elevated mitochondrial Ca2+ ions, and that transient CypD/mPTP opening is a critical adaptation mechanism for the survival of senescent cells.","PeriodicalId":501009,"journal":{"name":"The EMBO Journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-10-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142490375","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 : 2024-10-24DOI: 10.1038/s44318-024-00278-z
Shuting Ding,Shuxian Feng,Shibo Zhou,Zhengran Zhao,Xiao Liang,Jiao Wang,Ruishuang Fu,Rui Deng,Tao Zhang,Shujun Shao,Jingquan Yu,Christine H Foyer,Kai Shi
Plants face constant threats from pathogens, leading to growth retardation and crop failure. Cell-surface leucine-rich repeat receptor-like kinases (LRR-RLKs) are crucial for plant growth and defense, but their specific functions, especially to necrotrophic fungal pathogens, are largely unknown. Here, we identified an LRR-RLK (Solyc06g069650) in tomato (Solanum lycopersicum) induced by the economically important necrotrophic pathogen Botrytis cinerea. Knocking out this LRR-RLK reduced plant growth and increased sensitivity to B. cinerea, while its overexpression led to enhanced growth, yield, and resistance. We named this LRR-RLK as BRAK (B. cinerea resistance-associated kinase). Yeast two-hybrid screen revealed BRAK interacted with phytosulfokine (PSK) receptor PSKR1. PSK-induced growth and defense responses were impaired in pskr1, brak single and double mutants, as well as in PSKR1-overexpressing plants with silenced BRAK. Moreover, BRAK and PSKR1 phosphorylated each other, promoting their interaction as detected by microscale thermophoresis. This reciprocal phosphorylation was crucial for growth and resistance. In summary, we identified BRAK as a novel regulator of seedling growth, fruit yield and defense, offering new possibilities for developing fungal disease-tolerant plants without compromising yield.
{"title":"A novel LRR receptor-like kinase BRAK reciprocally phosphorylates PSKR1 to enhance growth and defense in tomato.","authors":"Shuting Ding,Shuxian Feng,Shibo Zhou,Zhengran Zhao,Xiao Liang,Jiao Wang,Ruishuang Fu,Rui Deng,Tao Zhang,Shujun Shao,Jingquan Yu,Christine H Foyer,Kai Shi","doi":"10.1038/s44318-024-00278-z","DOIUrl":"https://doi.org/10.1038/s44318-024-00278-z","url":null,"abstract":"Plants face constant threats from pathogens, leading to growth retardation and crop failure. Cell-surface leucine-rich repeat receptor-like kinases (LRR-RLKs) are crucial for plant growth and defense, but their specific functions, especially to necrotrophic fungal pathogens, are largely unknown. Here, we identified an LRR-RLK (Solyc06g069650) in tomato (Solanum lycopersicum) induced by the economically important necrotrophic pathogen Botrytis cinerea. Knocking out this LRR-RLK reduced plant growth and increased sensitivity to B. cinerea, while its overexpression led to enhanced growth, yield, and resistance. We named this LRR-RLK as BRAK (B. cinerea resistance-associated kinase). Yeast two-hybrid screen revealed BRAK interacted with phytosulfokine (PSK) receptor PSKR1. PSK-induced growth and defense responses were impaired in pskr1, brak single and double mutants, as well as in PSKR1-overexpressing plants with silenced BRAK. Moreover, BRAK and PSKR1 phosphorylated each other, promoting their interaction as detected by microscale thermophoresis. This reciprocal phosphorylation was crucial for growth and resistance. In summary, we identified BRAK as a novel regulator of seedling growth, fruit yield and defense, offering new possibilities for developing fungal disease-tolerant plants without compromising yield.","PeriodicalId":501009,"journal":{"name":"The EMBO Journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-10-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142490376","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}
The identification of host factors with antiviral potential is important for developing effective prevention and therapeutic strategies against SARS-CoV-2 infection. Here, by using immortalized cell lines, intestinal organoids, ex vivo intestinal tissues and humanized ACE2 mouse model as proof-of-principle systems, we have identified lipolysis-stimulated lipoprotein receptor (LSR) as a crucial host defense factor against SARS-CoV-2 infection in the small intestine. Loss of endogenous LSR enhances ACE2-dependent infection by SARS-CoV-2 Spike (S) protein-pseudotyped virus and authentic SARS-CoV-2 virus, and exogenous administration of LSR protects against viral infection. Mechanistically, LSR interacts with ACE2 both in cis and in trans, preventing its binding to S protein, and thus inhibiting viral entry and S protein-mediated cell-cell fusion. Finally, a small LSR-derived peptide blocks S protein binding to the ACE2 receptor in vitro. These results identify both a previously unknown function for LSR in antiviral host defense against SARS-CoV-2, with potential implications for peptide-based pan-variant therapeutic interventions.
确定具有抗病毒潜能的宿主因子对于制定有效的预防和治疗策略来对抗 SARS-CoV-2 感染非常重要。在这里，我们利用永生化细胞系、肠道器官组织、体外肠道组织和人源化 ACE2 小鼠模型作为原理验证系统，鉴定出脂肪分解刺激脂蛋白受体（LSR）是小肠中抵抗 SARS-CoV-2 感染的关键宿主防御因子。内源性 LSR 的缺失会增强 SARS-CoV-2 Spike (S) 蛋白伪型病毒和真 SARS-CoV-2 病毒对 ACE2 的依赖性感染，而外源性给予 LSR 则可防止病毒感染。从机理上讲，LSR 与 ACE2 存在顺式和反式相互作用，阻止其与 S 蛋白结合，从而抑制病毒进入和 S 蛋白介导的细胞-细胞融合。最后，LSR 衍生的一种小肽在体外阻断了 S 蛋白与 ACE2 受体的结合。这些结果确定了 LSR 在抗 SARS-CoV-2 病毒宿主防御中的一种以前未知的功能，对基于肽的泛变异治疗干预具有潜在的意义。
{"title":"Tight junction protein LSR is a host defense factor against SARS-CoV-2 infection in the small intestine.","authors":"Yanan An,Chao Wang,Ziqi Wang,Feng Kong,Hao Liu,Min Jiang,Ti Liu,Shu Zhang,Kaige Du,Liang Yin,Peng Jiao,Ying Li,Baozhen Fan,Chengjun Zhou,Mingxia Wang,Hui Sun,Jie Lei,Shengtian Zhao,Yongfeng Gong","doi":"10.1038/s44318-024-00281-4","DOIUrl":"https://doi.org/10.1038/s44318-024-00281-4","url":null,"abstract":"The identification of host factors with antiviral potential is important for developing effective prevention and therapeutic strategies against SARS-CoV-2 infection. Here, by using immortalized cell lines, intestinal organoids, ex vivo intestinal tissues and humanized ACE2 mouse model as proof-of-principle systems, we have identified lipolysis-stimulated lipoprotein receptor (LSR) as a crucial host defense factor against SARS-CoV-2 infection in the small intestine. Loss of endogenous LSR enhances ACE2-dependent infection by SARS-CoV-2 Spike (S) protein-pseudotyped virus and authentic SARS-CoV-2 virus, and exogenous administration of LSR protects against viral infection. Mechanistically, LSR interacts with ACE2 both in cis and in trans, preventing its binding to S protein, and thus inhibiting viral entry and S protein-mediated cell-cell fusion. Finally, a small LSR-derived peptide blocks S protein binding to the ACE2 receptor in vitro. These results identify both a previously unknown function for LSR in antiviral host defense against SARS-CoV-2, with potential implications for peptide-based pan-variant therapeutic interventions.","PeriodicalId":501009,"journal":{"name":"The EMBO Journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142489624","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}
Maintaining mitochondrial homeostasis is crucial for cell survival and organismal health, as evidenced by the links between mitochondrial dysfunction and various diseases, including Alzheimer's disease (AD). Here, we report that lncMtDloop, a non-coding RNA of unknown function encoded within the D-loop region of the mitochondrial genome, maintains mitochondrial RNA levels and function with age. lncMtDloop expression is decreased in the brains of both human AD patients and 3xTg AD mouse models. Furthermore, lncMtDloop binds to mitochondrial transcription factor A (TFAM), facilitates TFAM recruitment to mtDNA promoters, and increases mitochondrial transcription. To allow lncMtDloop transport into mitochondria via the PNPASE-dependent trafficking pathway, we fused the 3'UTR localization sequence of mitochondrial ribosomal protein S12 (MRPS12) to its terminal end, generating a specified stem-loop structure. Introducing this allotropic lncMtDloop into AD model mice significantly improved mitochondrial function and morphology, and ameliorated AD-like pathology and behavioral deficits of AD model mice. Taken together, these data provide insights into lncMtDloop as a regulator of mitochondrial transcription and its contribution to Alzheimer's pathogenesis.
{"title":"The mitochondrial long non-coding RNA lncMtloop regulates mitochondrial transcription and suppresses Alzheimer's disease.","authors":"Wandi Xiong,Kaiyu Xu,Jacquelyne Ka-Li Sun,Siling Liu,Baizhen Zhao,Jie Shi,Karl Herrup,Hei-Man Chow,Lin Lu,Jiali Li","doi":"10.1038/s44318-024-00270-7","DOIUrl":"https://doi.org/10.1038/s44318-024-00270-7","url":null,"abstract":"Maintaining mitochondrial homeostasis is crucial for cell survival and organismal health, as evidenced by the links between mitochondrial dysfunction and various diseases, including Alzheimer's disease (AD). Here, we report that lncMtDloop, a non-coding RNA of unknown function encoded within the D-loop region of the mitochondrial genome, maintains mitochondrial RNA levels and function with age. lncMtDloop expression is decreased in the brains of both human AD patients and 3xTg AD mouse models. Furthermore, lncMtDloop binds to mitochondrial transcription factor A (TFAM), facilitates TFAM recruitment to mtDNA promoters, and increases mitochondrial transcription. To allow lncMtDloop transport into mitochondria via the PNPASE-dependent trafficking pathway, we fused the 3'UTR localization sequence of mitochondrial ribosomal protein S12 (MRPS12) to its terminal end, generating a specified stem-loop structure. Introducing this allotropic lncMtDloop into AD model mice significantly improved mitochondrial function and morphology, and ameliorated AD-like pathology and behavioral deficits of AD model mice. Taken together, these data provide insights into lncMtDloop as a regulator of mitochondrial transcription and its contribution to Alzheimer's pathogenesis.","PeriodicalId":501009,"journal":{"name":"The EMBO Journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142451434","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}
Some DNA helicases play central and specific roles in genome maintenance and plasticity through their branch migration activity in different pathways of homologous recombination. RadA is a highly conserved bacterial helicase involved in DNA repair throughout all bacterial species. In Gram-positive Firmicutes, it also has a role in natural transformation, while in Gram-negative bacteria, ComM is the canonical transformation-specific helicase. Both RadA and ComM helicases form hexameric rings and use ATP hydrolysis as an energy source to propel themselves along DNA. In this study, we present the cryoEM structures of RadA and ComM interacting with DNA and ATP analogs. These structures reveal important molecular interactions that couple ATP hydrolysis and DNA binding in RadA, as well as the role of the Lon protease-like domain, shared by RadA and ComM, in this process. Taken together, these results provide new molecular insights into the mechanisms of DNA branch migration in different pathways of homologous recombination.
一些 DNA 螺旋酶通过在同源重组的不同途径中的分支迁移活动，在基因组的维护和可塑性方面发挥着核心和特殊的作用。RadA 是一种高度保守的细菌螺旋酶，参与所有细菌物种的 DNA 修复。在革兰氏阳性真菌中，它还在自然转化中发挥作用，而在革兰氏阴性细菌中，ComM 是典型的转化特异性螺旋酶。RadA 和 ComM 螺旋酶都形成六聚体环，并利用 ATP 水解作为能量来源，推动自身沿着 DNA 运行。在这项研究中，我们展示了 RadA 和 ComM 与 DNA 和 ATP 类似物相互作用的冷冻电镜结构。这些结构揭示了 RadA 中 ATP 水解与 DNA 结合之间的重要分子相互作用，以及 RadA 和 ComM 共享的 Lon 蛋白酶样结构域在这一过程中的作用。总之，这些结果为了解同源重组不同途径中 DNA 分支迁移的机制提供了新的分子见解。
{"title":"Structural insights into the mechanism of DNA branch migration during homologous recombination in bacteria.","authors":"Leonardo Talachia Rosa,Émeline Vernhes,Anne-Lise Soulet,Patrice Polard,Rémi Fronzes","doi":"10.1038/s44318-024-00264-5","DOIUrl":"https://doi.org/10.1038/s44318-024-00264-5","url":null,"abstract":"Some DNA helicases play central and specific roles in genome maintenance and plasticity through their branch migration activity in different pathways of homologous recombination. RadA is a highly conserved bacterial helicase involved in DNA repair throughout all bacterial species. In Gram-positive Firmicutes, it also has a role in natural transformation, while in Gram-negative bacteria, ComM is the canonical transformation-specific helicase. Both RadA and ComM helicases form hexameric rings and use ATP hydrolysis as an energy source to propel themselves along DNA. In this study, we present the cryoEM structures of RadA and ComM interacting with DNA and ATP analogs. These structures reveal important molecular interactions that couple ATP hydrolysis and DNA binding in RadA, as well as the role of the Lon protease-like domain, shared by RadA and ComM, in this process. Taken together, these results provide new molecular insights into the mechanisms of DNA branch migration in different pathways of homologous recombination.","PeriodicalId":501009,"journal":{"name":"The EMBO Journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142451435","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 : 2024-10-17DOI: 10.1038/s44318-024-00273-4
Nan Zhao,Antonio Michelucci,Laura Pietrangelo,Sundeep Malik,Linda Groom,Jennifer Leigh,Thomas N O'Connor,Takahiro Takano,Paul D Kingsley,James Palis,Simona Boncompagni,Feliciano Protasi,Robert T Dirksen
Tubular aggregate myopathy (TAM) is a heritable myopathy primarily characterized by progressive muscle weakness, elevated levels of creatine kinase (CK), hypocalcemia, exercise intolerance, and the presence of tubular aggregates (TAs). Here, we generated a knock-in mouse model based on a human gain-of-function mutation which results in a severe, early-onset form of TAM, by inducing a glycine-to-serine point mutation in the ORAI1 pore (Orai1G100S/+ or GS mice). By 8 months of age, GS mice exhibited significant muscle weakness, exercise intolerance, elevated CK levels, hypocalcemia, and robust TA presence. Unexpectedly, constitutive Ca2+ entry in mutant mice was observed in muscle only during early development and was abolished in adult skeletal muscle, partly due to reduced ORAI1 expression. Consistent with proteomic results, significant mitochondrial damage and dysfunction was observed in skeletal muscle of GS mice. Thus, GS mice represent a powerful model for investigation of the pathophysiological mechanisms that underlie key TAM symptoms, as well as those compensatory responses that limit the damaging effects of uncontrolled ORAI1-mediated Ca2+ influx.
{"title":"An Orai1 gain-of-function tubular aggregate myopathy mouse model phenocopies key features of the human disease.","authors":"Nan Zhao,Antonio Michelucci,Laura Pietrangelo,Sundeep Malik,Linda Groom,Jennifer Leigh,Thomas N O'Connor,Takahiro Takano,Paul D Kingsley,James Palis,Simona Boncompagni,Feliciano Protasi,Robert T Dirksen","doi":"10.1038/s44318-024-00273-4","DOIUrl":"https://doi.org/10.1038/s44318-024-00273-4","url":null,"abstract":"Tubular aggregate myopathy (TAM) is a heritable myopathy primarily characterized by progressive muscle weakness, elevated levels of creatine kinase (CK), hypocalcemia, exercise intolerance, and the presence of tubular aggregates (TAs). Here, we generated a knock-in mouse model based on a human gain-of-function mutation which results in a severe, early-onset form of TAM, by inducing a glycine-to-serine point mutation in the ORAI1 pore (Orai1G100S/+ or GS mice). By 8 months of age, GS mice exhibited significant muscle weakness, exercise intolerance, elevated CK levels, hypocalcemia, and robust TA presence. Unexpectedly, constitutive Ca2+ entry in mutant mice was observed in muscle only during early development and was abolished in adult skeletal muscle, partly due to reduced ORAI1 expression. Consistent with proteomic results, significant mitochondrial damage and dysfunction was observed in skeletal muscle of GS mice. Thus, GS mice represent a powerful model for investigation of the pathophysiological mechanisms that underlie key TAM symptoms, as well as those compensatory responses that limit the damaging effects of uncontrolled ORAI1-mediated Ca2+ influx.","PeriodicalId":501009,"journal":{"name":"The EMBO Journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-10-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142449240","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 : 2024-10-16DOI: 10.1038/s44318-024-00274-3
Yichen Lu,Ke Liang,Xiechao Zhan
Pre-mRNA splicing, a fundamental step in eukaryotic gene expression, is executed by the spliceosomes. While there is extensive knowledge of the composition and structure of spliceosomes in yeasts and humans, the structural diversity of spliceosomes in non-canonical organisms remains unclear. Here, we present a cryo-EM structure of a step II catalytically activated spliceosome (C* complex) derived from the unicellular green alga Chlamydomonas reinhardtii at 2.6 Å resolution. This Chlamydomonas C* complex comprises 29 proteins and four RNA elements, creating a dynamic assembly that shares a similar overall architecture with yeast and human counterparts but also has unique features of its own. Distinctive structural characteristics include variations in protein compositions as well as some noteworthy RNA features. The splicing factor Prp17, with four fragments and a WD40 domain, is engaged in intricate interactions with multiple protein and RNA components. The structural elucidation of Chlamydomonas C* complex provides insights into the molecular mechanism of RNA splicing in plants and understanding splicing evolution in eukaryotes.
{"title":"Structure of a step II catalytically activated spliceosome from Chlamydomonas reinhardtii.","authors":"Yichen Lu,Ke Liang,Xiechao Zhan","doi":"10.1038/s44318-024-00274-3","DOIUrl":"https://doi.org/10.1038/s44318-024-00274-3","url":null,"abstract":"Pre-mRNA splicing, a fundamental step in eukaryotic gene expression, is executed by the spliceosomes. While there is extensive knowledge of the composition and structure of spliceosomes in yeasts and humans, the structural diversity of spliceosomes in non-canonical organisms remains unclear. Here, we present a cryo-EM structure of a step II catalytically activated spliceosome (C* complex) derived from the unicellular green alga Chlamydomonas reinhardtii at 2.6 Å resolution. This Chlamydomonas C* complex comprises 29 proteins and four RNA elements, creating a dynamic assembly that shares a similar overall architecture with yeast and human counterparts but also has unique features of its own. Distinctive structural characteristics include variations in protein compositions as well as some noteworthy RNA features. The splicing factor Prp17, with four fragments and a WD40 domain, is engaged in intricate interactions with multiple protein and RNA components. The structural elucidation of Chlamydomonas C* complex provides insights into the molecular mechanism of RNA splicing in plants and understanding splicing evolution in eukaryotes.","PeriodicalId":501009,"journal":{"name":"The EMBO Journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142447963","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}
The C2-WW-HECT domain ubiquitin ligase Nedd4L regulates membrane sorting during endocytosis through the ubiquitination of cargo molecules such as the epithelial sodium channel (ENaC). Nedd4L is catalytically autoinhibited by an intramolecular interaction between its C2 and HECT domains, but the protein's activation mechanism is poorly understood. Here, we show that Nedd4L activation is linked to membrane shape by FCHO2, a Bin-Amphiphysin-Rsv (BAR) domain protein that regulates endocytosis. FCHO2 was required for the Nedd4L-mediated ubiquitination and endocytosis of ENaC, with Nedd4L co-localizing with FCHO2 at clathrin-coated pits. In cells, Nedd4L was specifically recruited to, and activated by, the FCHO2 BAR domain. Furthermore, we reconstituted FCHO2-induced recruitment and activation of Nedd4L in vitro. Both the recruitment and activation were mediated by membrane curvature rather than protein-protein interactions. The Nedd4L C2 domain recognized a specific degree of membrane curvature that was generated by the FCHO2 BAR domain, with this curvature directly activating Nedd4L by relieving its autoinhibition. Thus, we show for the first time a specific function (i.e., recruitment and activation of an enzyme regulating cargo sorting) of membrane curvature by a BAR domain protein.
{"title":"The Nedd4L ubiquitin ligase is activated by FCHO2-generated membrane curvature.","authors":"Yasuhisa Sakamoto,Akiyoshi Uezu,Koji Kikuchi,Jangmi Kang,Eiko Fujii,Toshiro Moroishi,Shiro Suetsugu,Hiroyuki Nakanishi","doi":"10.1038/s44318-024-00268-1","DOIUrl":"https://doi.org/10.1038/s44318-024-00268-1","url":null,"abstract":"The C2-WW-HECT domain ubiquitin ligase Nedd4L regulates membrane sorting during endocytosis through the ubiquitination of cargo molecules such as the epithelial sodium channel (ENaC). Nedd4L is catalytically autoinhibited by an intramolecular interaction between its C2 and HECT domains, but the protein's activation mechanism is poorly understood. Here, we show that Nedd4L activation is linked to membrane shape by FCHO2, a Bin-Amphiphysin-Rsv (BAR) domain protein that regulates endocytosis. FCHO2 was required for the Nedd4L-mediated ubiquitination and endocytosis of ENaC, with Nedd4L co-localizing with FCHO2 at clathrin-coated pits. In cells, Nedd4L was specifically recruited to, and activated by, the FCHO2 BAR domain. Furthermore, we reconstituted FCHO2-induced recruitment and activation of Nedd4L in vitro. Both the recruitment and activation were mediated by membrane curvature rather than protein-protein interactions. The Nedd4L C2 domain recognized a specific degree of membrane curvature that was generated by the FCHO2 BAR domain, with this curvature directly activating Nedd4L by relieving its autoinhibition. Thus, we show for the first time a specific function (i.e., recruitment and activation of an enzyme regulating cargo sorting) of membrane curvature by a BAR domain protein.","PeriodicalId":501009,"journal":{"name":"The EMBO Journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-10-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142439545","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 : 2024-10-14DOI: 10.1038/s44318-024-00265-4
Madalena M Reimão-Pinto,Andrew Behrens,Sergio Forcelloni,Klemens Fröhlich,Selay Kaya,Danny D Nedialkova
Embryogenesis entails dramatic shifts in mRNA translation and turnover that reprogram gene expression during cellular proliferation and differentiation. Codon identity modulates mRNA stability during early vertebrate embryogenesis, but how the composition of tRNA pools is matched to translational demand is unknown. By quantitative profiling of tRNA repertoires in zebrafish embryos during the maternal-to-zygotic transition, we show that zygotic tRNA repertoires are established after the onset of gastrulation, succeeding the major wave of zygotic mRNA transcription. Maternal and zygotic tRNA pools are distinct, but their reprogramming does not result in a better match to the codon content of the zygotic transcriptome. Instead, we find that an increase in global translation at gastrulation sensitizes decoding rates to tRNA supply, thus destabilizing maternal mRNAs enriched in slowly translated codons. Translational activation and zygotic tRNA expression temporally coincide with an increase of TORC1 activity at gastrulation, which phosphorylates and inactivates the RNA polymerase III repressor Maf1a/b. Our data indicate that a switch in global translation, rather than tRNA reprogramming, determines the onset of codon-dependent maternal mRNA decay during zebrafish embryogenesis.
{"title":"The dynamics and functional impact of tRNA repertoires during early embryogenesis in zebrafish.","authors":"Madalena M Reimão-Pinto,Andrew Behrens,Sergio Forcelloni,Klemens Fröhlich,Selay Kaya,Danny D Nedialkova","doi":"10.1038/s44318-024-00265-4","DOIUrl":"https://doi.org/10.1038/s44318-024-00265-4","url":null,"abstract":"Embryogenesis entails dramatic shifts in mRNA translation and turnover that reprogram gene expression during cellular proliferation and differentiation. Codon identity modulates mRNA stability during early vertebrate embryogenesis, but how the composition of tRNA pools is matched to translational demand is unknown. By quantitative profiling of tRNA repertoires in zebrafish embryos during the maternal-to-zygotic transition, we show that zygotic tRNA repertoires are established after the onset of gastrulation, succeeding the major wave of zygotic mRNA transcription. Maternal and zygotic tRNA pools are distinct, but their reprogramming does not result in a better match to the codon content of the zygotic transcriptome. Instead, we find that an increase in global translation at gastrulation sensitizes decoding rates to tRNA supply, thus destabilizing maternal mRNAs enriched in slowly translated codons. Translational activation and zygotic tRNA expression temporally coincide with an increase of TORC1 activity at gastrulation, which phosphorylates and inactivates the RNA polymerase III repressor Maf1a/b. Our data indicate that a switch in global translation, rather than tRNA reprogramming, determines the onset of codon-dependent maternal mRNA decay during zebrafish embryogenesis.","PeriodicalId":501009,"journal":{"name":"The EMBO Journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-10-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142439546","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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