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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

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.