mRNA Template-free Protein Elongation: a New Paradigm for Quality Control at the Ribosome

mRNA 无模板蛋白质延伸：核糖体质量控制的新范式

基本信息

批准号：
9325337
负责人：
Onn Brandman
金额：
$ 30.28万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-08-01 至 2020-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9325337
关键词：
Address Affect Alanine Amyloid Area Automobile Driving Basic Science Binding Biochemical Biochemistry Biological Biological Assay Biology C-terminal CDC48 protein Cells Characteristics Collaborations Complex Data Disease Eukaryotic Cell Event Failure Foundations Fractionation Future Genetic Genetic Translation Goals Heat shock proteins Homeostasis Image Inclusion Bodies Induced Mutation Investigation Knowledge Label Libraries Life Link Measurement Measures Mediating Messenger RNA Methods Microscopy Molecular Molecular Chaperones Mus Mutation Names Nerve Degeneration Neurocognitive Neurodegenerative Disorders Orthologous Gene Pathologic Pathology Pathway interactions Peptides Phenotype Physiologic pulse Process Property Proteins Proteomics Quality Control Reaction Recruitment Activity Research Research Proposals Ribosomes Role Stress System Tail Testing Therapeutic Therapeutic Intervention Threonine Time Transfer RNA Aminoacylation Translating Translations Typha Visit Work Yeasts base experimental study human disease in vivo in vivo imaging multicatalytic endopeptidase complex neurodegenerative phenotype novel novel strategies polyalanine polypeptide professor protein aggregate proteotoxicity public health relevance response ribosome profiling role model stress protein synthetic peptide

Address Affect Alanine Amyloid Area Automobile Driving Basic Science Binding Biochemical Biochemistry Biological Biological Assay Biology C-terminal CDC48 protein Cells Characteristics Collaborations Complex Data Disease Eukaryotic Cell Event Failure Foundations Fractionation Future Genetic Genetic Translation Goals Heat shock proteins Homeostasis Image Inclusion Bodies Induced Mutation Investigation Knowledge Label Libraries Life Link Measurement Measures Mediating Messenger RNA Methods Microscopy Molecular Molecular Chaperones Mus Mutation Names Nerve Degeneration Neurocognitive Neurodegenerative Disorders Orthologous Gene Pathologic Pathology Pathway interactions Peptides Phenotype Physiologic pulse Process Property Proteins Proteomics Quality Control Reaction Recruitment Activity Research Research Proposals Ribosomes Role Stress System Tail Testing Therapeutic Therapeutic Intervention Threonine Time Transfer RNA Aminoacylation Translating Translations Typha Visit Work Yeasts base experimental study human disease in vivo in vivo imaging multicatalytic endopeptidase complex neurodegenerative phenotype novel novel strategies polyalanine polypeptide professor protein aggregate proteotoxicity public health relevance response ribosome profiling role model stress protein synthetic peptide

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项目摘要

DESCRIPTION (provided by applicant): Translating mRNA into protein is a process that is essential to all life. Recently, mutations that cause stalls in translation or to an associated response pathway, the Ribosome Quality control Complex (RQC), were shown to cause neurodegeneration, although the mechanisms driving pathology are unknown. This research proposal is focused on understanding the mechanism of this quality control pathway and its role in disease. In yeast, the RQC pathway includes four highly conserved proteins, Ltn1p, Rqc1p, Tae2p, and Cdc48p that bind to 60S ribosome-nascent chain complexes that have stalled mid-translation of an mRNA and split off from the 40S ribosomal subunit and mRNA. We previously found that disrupting the RQC components Ltn1p or Rqc1p causes proteotoxic stress, perhaps relating to the neurodegenerative phenotype caused by a hypomorph of the mouse ortholog of Ltn1p. However, we also found RQC-mediated proteotoxic stress is dependent upon the Tae2p. Our new preliminary data reveals a surprising role for the protein Tae2p in the response to translation failure: Tae2p binds to the dissociated 60S-nascent chain complex, engages the stalled nascent chain, and recruits tRNAs charged with alanine and threonine so that the 60S catalyzes the C terminal addition of the nascent chain with Alanine and Threonine tails ("CAT tails"). This protein elongation reaction happens without an mRNA-template or the 40S ribosomal subunit, and is therefore a novel form of elongation never before observed in vivo. RQC-mediated proteotoxic stress is dependent on synthesis of CAT tail synthesis, and therefore CAT tails may be toxic. Consistent with this hypothesis, CAT tailed proteins form inclusions and this may be caused by alanine, which form amyloid and are the cause of a at least nine diseases, many of which include neurocognitive phenotypes. The discovery of CAT tails raises a number of immediate questions about the quality control mechanisms they take part in and their role in disease. What substrates are CAT tailed? How do CAT tails cause proteotoxic stress? What happens to ribosomes targeted by the RQC? In order to explore these areas the following specific aims will be carried out (1) identify the characteristics and identity of CAT tailed substrates. (2) Identify the fate of CAT tailed proteins. (3) Identify the fate of ribosomes targeted by Tae2p and the RQC. To address achieve these goals, a variety of experiments will be performed, including biochemical analysis of purified RQC-nascent chain complexes, genetic perturbations to intracellular protein quality control systems, in vivo imaging, biochemical studie of synthetic RQC substrates, assays measuring translation (ribosome profiling), and covalent labeling of stalled ribosomes targeted by the RQC. Relevance: The proposed investigations will lay the foundation for future investigations into the basic biology and therapeutic applications of a new paradigm of protein elongation that is linked to disease: mRNA template- free protein elongation. This work will likely increase our knowledge of fundamental biology, protein quality control, and the cause of neurodegenerative diseases.

描述（由适用提供）：将mRNA转化为蛋白质是对所有生命必不可少的过程。最近，尽管未知的机制尚不清楚，但显示导致核糖体质量控制复合物（RQC）的突变，即核糖体质量控制复合物（RQC）引起神经变性。该研究建议的重点是了解该质量控制途径的机制及其在疾病中的作用。在酵母中，RQC途径包括四种高度组成的蛋白，LTN1P，RQC1P，TAE2P和Cdc48p，它们与60S核糖体 - 脱粒链复合物结合，这些链链链络合物已停滞了mRNA的中期，并从40s的核糖体亚基和mRNA中脱离。我们以前发现，破坏RQC成分LTN1P或RQC1P会导致蛋白质毒性应激，这可能与由LTN1P小鼠直立性的低变形物引起的神经退行性表型有关。但是，我们还发现RQC介导的蛋白毒性应激取决于TAE2P。 Our new preliminary data reveals a surprising role for the protein Tae2p in the response to translation failure: Tae2p binds to the dissociated 60S-nascent chain complex, engages the stalled nascent chain, and recruits tRNAs charged with alanine and threonine so that the 60S catalyzes the C terminal addition of the nascent chain with Alanine and Threonine tails ("CAT tails").这种蛋白质伸长反应在没有mRNA-template或40S核糖体亚基的情况下发生，因此是一种新颖的伸长形式，从未在体内观察到。 RQC介导的蛋白毒性应激取决于猫尾合成的合成，因此猫尾蛋白形成夹杂物，这可能是由丙氨酸引起的，丙氨酸会形成淀粉样蛋白，并且至少是九种疾病的原因，其中许多疾病包括神经认知表型。猫尾巴的发现提出了许多有关其参与质量控制机制及其在疾病中的作用的直接问题。猫尾巴的底物是什么？猫的尾巴如何引起蛋白质毒性应激？ RQC靶向的核糖体会发生什么？为了探索这些领域，将执行以下特定目标（1）确定猫尾底物的特征和身份。（2）确定猫尾蛋白的命运。（3）确定核糖体的命运由TAE2P和RQC靶向。为了解决实现这些目标，将进行各种实验，包括对纯化的RQC-NASENCENT链络合物的生化分析，对细胞内蛋白质质量控制系统的遗传扰动，在体内成像，生化室内的生化室，Synthetic RQC的生物化学工作室，Synthetic RQC底物，测量型（核核Cobosome thristect transe）（核核）的瞄准（核核）和cos的靶向效果。相关性：拟议的调查将为未来对基本生物学和治疗应用的投资奠定基础与疾病有关的新蛋白质伸长范围：mRNA模板 - 无蛋白质伸长。这项工作可能会增加我们对基本生物学，蛋白质质量控制以及神经退行性疾病的原因的了解。