Mitochondrial inheritance and quality control

线粒体遗传和质量控制

基本信息

批准号：
10799088
负责人：
Liza A Pon
金额：
$ 2.76万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-04-01 至 2027-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10799088
关键词：
15 year old Aging Autophagocytosis Autophagosome Biological Assay Cardiovascular Diseases Cells Computer Hardware Computer software Enzymes Equipment Excision Fluorescence Functional disorder Funding Future Gene Delivery Goals Growth Individual Lead Link Lipids Lysosomes Mammalian Cell Mediating Membrane Membrane Proteins Metabolic Diseases Mitochondria Mitochondrial Inheritance Mitochondrial Proteins Modeling Myopathy Neurodegenerative Disorders Nucleic Acids Organelles Outer Mitochondrial Membrane Pathway interactions Physiological Play Premature aging syndrome Protein Biosynthesis Proteins Quality Control Reader Research Role Site United States National Institutes of Health Vacuole Yeasts base biological adaptation to stress congenital muscular dystrophy endoplasmic reticulum stress fitness hatching instrument interest misfolded protein multicatalytic endopeptidase complex protein aggregation protein folding proteostasis repaired screening

项目摘要

Protein homeostasis, or proteostasis, relies on precise control of protein synthesis, folding and degradation. Proteostatic errors lead to protein aggregates, which are toxic and linked to neurodegenerative, cardiovascular, muscular and metabolic disorders, and to premature aging. The ER and mitochondria are major sites for protein folding and are supported by quality control mechanisms that correct protein folding or eliminate proteins or organelles that are damaged beyond repair. ER-associated degradation (ERAD) and mitochondria-associated degradation (MAD) are functionally and mechanistically related mechanisms. In both, misfolded proteins are identified, ubiquitinated, extracted from organelles and degraded by the proteasome. However, both pathways have limitations. Previous studies suggested that MAD proteostasis was restricted to mitochondrial outer mem- brane (OM) proteins, <10% of mitochondrial proteins. Moreover, MAD and ERAD are inherently low-throughput because they act on individual proteins. Our R35-funded research revealed that 1) MAD plays a major role in mitochondrial and cellular fitness in a model for aging, 2) loss of MAD function results in premature aging, and 3) MAD functions in proteostatic control not just in the mitochondrial outer membrane, but also in the matrix and inner membrane of the organelle. In complementary studies, we identified a conserved ER proteostasis pathway (ER-PERM) that has overlapping function with ERAD, but has higher throughput and contributes to the ER stress response in yeast, mammalian cells and cellu models for a newly identified congenital muscular dystrophy (CHKB CMD). In ER-PERM, lipid droplets (LDs), organelles that form at ER membranes, act as escape hatches for large-scale removal of unfolded ER proteins and degradation of those proteins and their LD carriers. Here, degradation occurs by microautophagy, a conserved but understudied form of autophagy that does not rely on autophagosomes or core ATG genes for delivery of cargoes to the vacuole (yeast lysosome). Important future goals are to 1) understand the mechanism of MAD function within mitochondria, 2) the physiological conse- quences of MAD-mediated mitochondrial proteostasis, and 3) identify components and functional consequences of ER-PERM. We request funds to replace a broken, unrepairable, >15 year-old microplate reader (Tecan NanoQuant) that was heavily used for assays that are essential to the completion of our R-35-funded research including yeast growth curves, protein and nucleic acid determination, enzyme assays, and screening for expression of fluorescence tags on proteins of interest. While there are plate readers in other labs or equipment cores, those instruments are not available for long-term, uninterrupted use like analysis of yeast growth curve or any growth-based screens (350 hrs/month). Thus, we request funds to purchase a new plate reader (Tecan Infinite Base Unit, M200 and F200 PRO Microplate Reader) and the computer hardware and software to drive the plate reader.

蛋白质稳态或蛋白抑制性依赖于蛋白质合成，折叠和降解的精确控制。蛋白抑制导致蛋白质聚集体，这些蛋白质骨料有毒，与神经退行性，心血管相关，肌肉和代谢性疾病，并过早衰老。 ER和线粒体是蛋白质的主要部位折叠并受到质量控制机制的支持，这些机制纠正蛋白质折叠或消除蛋白质或损坏的细胞器无法修复。 ER相关的降解（ERAD）和线粒体相关降解（MAD）在功能和机械上相关的机制上。在这两者中，错误折叠的蛋白质是从细胞器中鉴定，泛素化，从细胞器中提取并被蛋白酶体降解。但是，这两种途径有局限性。先前的研究表明，疯狂的蛋白抑制剂仅限于线粒体外膜 Brane（OM）蛋白，<线粒体蛋白的10％。而且，疯狂和埃拉德本质上是低通量的因为它们对单个蛋白质作用。我们的R35资助研究表明，1）MAD在衰老模型中的线粒体和细胞适应性，2）MAD功能的丧失导致过早衰老，并且 3）MAD不仅在线粒体外膜中，而且在矩阵和矩阵和细胞器的内膜。在互补研究中，我们确定了一个保守的ER蛋白毒素途径（ER-Perm）与Erad重叠的功能，但具有更高的吞吐量，并有助于ER应力在酵母中的反应，哺乳动物细胞和Cellu模型对新近鉴定的先天性肌肉营养不良（CHKB CMD）。在Er-Perm，脂质液滴（LDS），在ER膜上形成的细胞器，充当逃生舱口用于大规模去除展开的ER蛋白质以及这些蛋白质及其LD载体的降解。这里，降解是由微自噬发生的，一种保守而研究的自噬形式，不依赖于自噬小体或核心ATG基因将货物传递到液泡（酵母溶酶体）。重要的未来目标是1）了解线粒体中疯狂功能的机制，2）疯狂介导的线粒体蛋白抑制剂的问题，以及3）识别组件和功能后果 Er-Perm。我们要求资金代替破碎的，不可修复的，> 15岁的微板读取器（Tecan）纳米量）大量用于完成R-35资助的研究至关重要的测定包括酵母生长曲线，蛋白质和核酸测定，酶测定和筛查荧光标签在感兴趣的蛋白质上的表达。虽然其他实验室或设备中有板板读取器核心，这些仪器不可用于长期，不间断的使用，例如对酵母生长曲线的分析或任何基于增长的屏幕（每月350小时）。因此，我们要求资金购买新的板块读取器（Tecan 无限基础单元，M200和F200 Pro Microplate读取器）以及计算机硬件和软件驱动器板读取器。