Genetic Requirements for Protein Degradation at the Endoplasmic Reticulum Translocon

内质网易位蛋白降解的遗传要求

• 批准号: 10512586
• 负责人: Eric Meyer Rubenstein
• 金额: $ 44.7万
• 依托单位: BALL STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10512586
• 关键词: ATP phosphohydrolase; Address; Apolipoproteins B; Area; Biological Models; Cell physiology; Cells; Cellular biology; Cholesterol; Communication; Data; Data Collection; Defect; Development; Diabetes Mellitus; Elements; Endoplasmic Reticulum; Environment; Enzymes; Eukaryota; Exhibits; Extracellular Space; Family; Functional disorder; Funding; Genetic; Genetic Determinism; Goals; Growth; Health; Homeostasis; Human; Impairment; Investigation; Low-Density Lipoproteins; Mammals; Mediating; Medical; Membrane; Metabolic; Metalloproteases; Microscopic; Mission; Modeling; Molecular; National Institute of General Medical Sciences; Nature; Neurologic; Pathway interactions; Peptide Hydrolases; Phenotype; Physiology; Play; Proteins; Publishing; Quality Control; Research; Role; Saccharomyces cerevisiae; Students; System; Testing; Therapeutic; Translations; Ubiquitin; Universities; Vacuole; Work; Yeasts; Zinc; cofactor; design; experimental study; fitness; healthy aging; human disease; hypercholesterolemia; improved; insight; islet amyloid polypeptide; multicatalytic endopeptidase complex; novel; prevent; protein degradation; protein function; recruit; rhomboid; student mentoring; therapeutic target; ubiquitin ligase; undergraduate student

PROJECT SUMMARY Most endomembrane system and secreted eukaryotic proteins traverse the endoplasmic reticulum (ER) translocon during or shortly after synthesis. Underscoring the critical nature of maintaining functional translocons, eukaryotes possess multiple conserved translocon quality control (TQC) mechanisms that promote degradation of channel-clogging proteins. TQC remains poorly understood relative to other ER protein quality control pathways. The highly conserved yeast zinc metalloprotease Ste24 (ZMPSte24 in mammals) catalyzes degradation of translocon-clogging proteins. Preliminary data indicate that ER rhomboid-family pseudoprotease Dfm1 (homologous to mammalian derlin proteins) contributes to TQC, likely via the Ste24 mechanism. Consistent with the mission of the National Institute of General Medical Sciences, the objective of the proposed work is an improved understanding of TQC, a medically important facet of cell biology, using yeast as a model system. Ste24 promotes healthy aging and, by virtue of its function in TQC, is likely to play a protective role against the progression of diabetes. Further, at least two proteins with profound significance for metabolic physiology persistently engage translocons. The proposed experiments will test the hypothesis that Dfm1 and its cofactor, the Cdc48 ATPase, partially extract ubiquitylated clogging proteins from the translocon to enable cleavage by Ste24, whereupon the cytosolic fragment is degraded by the proteasome and the luminal fragment is trafficked through the endomembrane system to the vacuole for degradation. The specific aims of this project are to (1) characterize STE24 function in TQC and (2) determine the role of Dfm1 and Cdc48 in TQC. To address these objectives, several mechanistic aspects of Ste24 and Dfm1/Cdc48 function in TQC will be explored. The breadth of Ste24 and Dfm1/Cdc48 TQC substrates will be determined. Experiments will address whether Ste24 TQC substrates are ubiquitylated prior to recognition, if TQC substrates are cleaved in a Ste24-dependent manner, and how Ste24 TQC substrates are ultimately degraded (i.e. by the proteasome or vacuole). Dfm1 association with the translocon and TQC substrates will be assessed, as will the contributions of Dfm1 and Cdc48 to Ste24-mediated and Ste24-independent TQC. The role of conserved Dfm1 elements in TQC will be determined. Finally, yeast lacking DFM1 expressing TQC substrates exhibit a profound fitness defect that is rapidly and stably suppressed. The genetic determinants of suppression will be identified. Undergraduate and master’s students will participate in all aspects of this project, including experiment design, data collection, and communication of results. These experiments will yield novel mechanistic insights into conserved, biomedically relevant mechanisms of TQC. Upon completion, the proposed work has the strong potential to inform the development of improved therapeutic strategies for multiple human conditions, including elevated cholesterol and diabetes.

项目概要 大多数内膜系统和分泌的真核蛋白穿过内质网 (ER) 在合成过程中或合成后不久易位。强调维持功能易位子的关键性质， 真核生物拥有多种保守的易位子质量控制（TQC）机制，可促进降解 通道堵塞蛋白。相对于其他 ER 蛋白质质量控​​制，TQC 仍然知之甚少 途径。高度保守的酵母锌金属蛋白酶 Ste24（哺乳动物中为 ZMPSte24）催化 易位子堵塞蛋白的降解。初步数据表明 ER 菱形家族假蛋白酶 Dfm1（与哺乳动物 derlin 蛋白同源）可能通过 Ste24 机制促进 TQC。 与国家普通医学科学研究所的使命相一致，拟议的目标 工作是使用酵母作为模型，加深对 TQC（细胞生物学的医学重要方面）的理解 系统。 Ste24 促进健康衰老，并凭借其在 TQC 中的功能，可能发挥保护作用 对抗糖尿病的进展。此外，至少有两种对代谢具有深远意义的蛋白质 生理学持续涉及易位子。所提出的实验将检验 Dfm1 和 它的辅助因子 Cdc48 ATPase 从易位子中部分提取泛素化的堵塞蛋白，以实现 被 Ste24 切割，随后胞质片段被蛋白酶体和管腔片段降解 通过内膜系统运输至液泡进行降解。该项目的具体目标 目的是 (1) 表征 STE24 在 TQC 中的功能，以及 (2) 确定 Dfm1 和 Cdc48 在 TQC 中的作用。致地址 为了实现这些目标，我们将探讨 Ste24 和 Dfm1/Cdc48 在 TQC 中功能的几个机制方面。这 Ste24 和 Dfm1/Cdc48 TQC 底物的宽度将被确定。实验将解决 Ste24 是否 如果 TQC 底物以 Ste24 依赖性方式裂解，则 TQC 底物在识别前被泛素化 方式，以及 Ste24 TQC 底物最终如何降解（即通过蛋白酶体或液泡）。 DFM1 将评估与易位子和 TQC 底物的关联，以及 Dfm1 和 Cdc48 到 Ste24 介导的和 Ste24 独立的 TQC。保守的 Dfm1 元素在 TQC 中的作用将是 决定。最后，缺乏表达 DFM1 的 TQC 底物的酵母表现出严重的适应性缺陷，即 迅速而稳定地压制。抑制的遗传决定因素将被确定。本科及 硕士生将参与该项目的各个方面，包括实验设计、数据收集和 结果的沟通。这些实验将对保守的生物医学产生新的机制见解 TQC的相关机制。完成后，拟议的工作有很大的潜力为人们提供信息 开发针对多种人类疾病（包括胆固醇升高）的改进治疗策略 和糖尿病。