Mitochondrial inheritance and quality control

蛋白质稳态，或蛋白质稳态，依赖于对蛋白质合成、折叠和降解的精确控制。 前列腺癌错误会导致蛋白质聚集，这种聚集是有毒的，并与神经退行性疾病、心血管疾病、 肌肉和新陈代谢障碍，以及过早衰老。内质网和线粒体是蛋白质的主要部位 折叠，并由质量控制机制支持，以纠正蛋白质折叠或消除蛋白质或 细胞器受损，无法修复。内质网相关降解(ERAD)与线粒体相关 降解(MAD)是与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 鉴定，泛素化，从细胞器中提取，并被蛋白酶体降解。然而，这两条路径 都有局限性。以往的研究表明，MAD蛋白抑制作用仅限于线粒体外膜-- 膜(OM)蛋白，占线粒体蛋白的10%。此外，MAD和ERAD本身就是低吞吐量的 因为它们作用于单个蛋白质。我们由R35资助的研究表明，1)MAD在 线粒体和细胞在衰老模型中的适合性，2)MAD功能的丧失导致过早衰老，以及 3)MAD不仅在线粒体外膜中起蛋白抑制作用，而且在基质和 细胞器的内膜。在互补性研究中，我们确定了一条保守的内质网蛋白稳定途径 (ER-PERM)与ERAD功能重叠，但具有更高的吞吐量并导致ER压力 酵母、哺乳动物细胞和细胞模型对一种新发现的先天性肌营养不良症的反应 (CHKB CMD)。在ER-perm中，在ER膜上形成的细胞器--脂滴(LDs)起到逃逸的作用 用于大规模去除未折叠的内质网蛋白，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，这是一种保守但未被研究的自噬形式，不依赖于 将货物运送到液泡的自噬小体或核心ATG基因(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)生理方面的结果。 MAD介导的线粒体蛋白平衡的序列，以及3)确定成分和功能后果 急诊室烫发。我们请求资金更换已损坏、无法修复、已有15年历史的微板阅读器(Tecan NanoQuant)，它被大量用于完成我们R-35资助的研究所必需的分析 包括酵母生长曲线，蛋白质和核酸测定，酶分析和筛选 荧光标记在目的蛋白上的表达。虽然在其他实验室或设备中有平板阅读器 这些仪器不能用于长期、不间断的使用，如分析酵母生长曲线或 任何基于增长的屏幕(350小时/月)。因此，我们申请资金购买新的平板阅读器(Tecan 无限基板、M200和F200 PRO微板阅读器)和计算机硬件和软件来驱动 车牌阅读器。

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.