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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.

蛋白质稳态或蛋白质稳态依赖于蛋白质合成、折叠和降解的精确控制。蛋白质抑制错误会导致蛋白质聚集，这是有毒的，与神经退行性、心血管、肌肉和代谢紊乱，以及过早衰老。内质网和线粒体是蛋白质的主要场所折叠并得到质量控制机制的支持，纠正蛋白质折叠或消除蛋白质或受损无法修复的细胞器。 ER 相关降解 (ERAD) 和线粒体相关降解（MAD）是功能和机械相关的机制。在这两种情况下，错误折叠的蛋白质都是被鉴定、泛素化、从细胞器中提取并被蛋白酶体降解。然而，这两条途径有局限性。先前的研究表明，MAD 蛋白质稳态仅限于线粒体外膜。膜 (OM) 蛋白，<10% 的线粒体蛋白。此外，MAD 和 ERAD 本质上是低通量的因为它们作用于单个蛋白质。我们的 R35 资助的研究表明 1) MAD 在衰老模型中的线粒体和细胞适应性，2) MAD 功能丧失导致过早衰老，以及 3) MAD不仅在线粒体外膜中发挥蛋白质抑制控制作用，而且还在基质和基质中发挥作用。细胞器的内膜。在补充研究中，我们发现了一条保守的 ER 蛋白质稳态途径 (ER-PERM) 与 ERAD 具有重叠功能，但具有更高的吞吐量并有助于 ER 应力酵母、哺乳动物细胞和细胞模型对新发现的先天性肌营养不良症的反应（CHKB CMD）。在 ER-PERM 中，脂滴 (LD) 是在内质网膜上形成的细胞器，充当逃生口用于大规模去除未折叠的 ER 蛋白并降解这些蛋白及其 LD 载体。这里，降解是通过微自噬发生的，微自噬是一种保守但尚未得到充分研究的自噬形式，不依赖于自噬体或核心 ATG 基因，用于将货物递送至液泡（酵母溶酶体）。重要的未来目标是 1) 了解线粒体内 MAD 功能的机制，2) 生理学结果 MAD 介导的线粒体蛋白质稳态的序列，以及 3) 识别成分和功能后果 ER-PERM。我们请求资金来更换损坏的、无法修复的、使用超过 15 年的酶标仪 (Tecan NanoQuant）被大量用于对完成我们 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、酶测定以及筛选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读板器核心，这些仪器不能长期不间断地使用，例如分析酵母生长曲线或任何基于生长的筛选（350 小时/月）。因此，我们请求资金购买新的读板器（Tecan Infinite Base Unit、M200 和 F200 PRO 酶标仪）以及驱动的计算机硬件和软件读板器。