Cellular protein maturation and degradation

描述(由申请人提供)：本项目的主要目标是确定哺乳动物内质网(ER)中蛋白质质量控制的机制。这项拟议的研究将利用定义明确的哺乳动物细胞系统来确定异常分泌货物是如何被选择和靶向销毁的，以及内质网内的质量控制机制如何协调蛋白质的转运和成熟。质量控制过程由一系列相互关联的步骤组成，这些步骤涉及对是否运输基质或将其改道以保留或降解内质网做出关键选择。绝大多数人 穿越哺乳动物分泌途径的蛋白质会得到N-连接的多糖，在一致的N-糖基化位点进入内质网管腔后不久就会加入。N-葡聚糖的组成是动态的，因为它们充当信号来招募有助于蛋白质成熟和质量控制过程的因子。N-葡聚糖支持结合单糖化侧链的碳水化合物结合伴侣钙粘蛋白和钙网状蛋白的募集。UDP-葡萄糖：糖蛋白葡萄糖基转移酶(UGT1)指导凝集素伴侣的持续结合。虽然UGT1已经在重组系统中进行了研究，但这个关键的ER因子在细胞中的作用还不是很清楚。甘露糖修剪被认为参与了将异常蛋白质分选到内质网相关降解(ERAD)途径中，因为抑制甘露糖苷酶活性稳定了ERAD底物。然而，许多问题仍然存在，关于甘露糖修剪在质量控制中的作用。虽然ER甘露糖苷酶样蛋白EDEM1的过表达加速了糖基化ERAD底物的周转，但它在质量控制和分选过程中的作用尚不清楚。我们的结果表明，EDEM1通过具有两部分结合特性，成为将缺陷货物运送到ERAD位错复合体的中心环节。最后，内质网精细的空间组织有助于提高内质网在时间上协调蛋白质成熟、质量控制和ERAD等过程的效率。四肽重复序列(TPR)结构域是一个通用的接头基序，它支持蛋白质之间的相互作用，参与翻译后易位和细胞内伴侣复合体的形成。我们最近发现了一个内质网驻留的富含TPR的蛋白家族，我们建议在内质网组织中发挥作用。这项研究计划有三个目的：(1)确定UGT1如何在细胞环境中参与内质网质量控制；(2)阐明EDEM1在ERAD过程中的作用和作用机制；(3)确定新的含TPR的蛋白质如何在内质网组织和动态平衡中发挥作用。深入了解早期分泌途径中的过程对于理解基本细胞生物学和一系列人类疾病的病因学具有广泛的意义，因为越来越多的疾病状态是由蛋白质成熟和内质网稳态缺陷引起的。 公共卫生相关性：对蛋白质细胞生命的洞察将有助于阐明蛋白质成熟和质量控制方面的缺陷所导致的病理。与成熟和质量控制缺陷相关的疾病包括白化病、肝硬变、肺气肿、囊性纤维化、Krabbe、Gaucher和一些形式的心脏病。此外，错误折叠的蛋白质的积累会导致与肥胖和糖尿病相关的应激反应的激活。了解穿越分泌途径的蛋白质的质量控制过程将有助于开发方法学或治疗剂，作为蛋白质折叠校正器，控制质量控制过程的严密性，或调节错误折叠蛋白质积累引起的应激反应。1

DESCRIPTION (provided by applicant): The overarching goals of this project are to define mechanisms of protein quality control in the mammalian endoplasmic reticulum (ER). The proposed research will utilize well-defined mammalian cell systems to determine how aberrant secretory cargo is selected and targeted for destruction and how organization of the quality control machinery within the ER coordinates protein transit and maturation. The quality control process is comprised of a series of interlocking steps that involve making critical choices on whether to transport a substrate or divert it for ER retention or degradation. The vast majority of proteins that traverse the mammalian secretory pathway receive N-linked glycans that are added shortly after a consensus N- glycosylation site emerges into the ER lumen. The composition of N-glycans is dynamic because they act as signals to recruit factors that assist the protein maturation and quality control processes. N-glycans support recruitment of the carbohydrate binding chaperones calnexin and calreticulin, which bind to monoglucosylated side chains. Persistent lectin chaperone binding is directed by UDP-glucose: glycoprotein glucosyltransferase (UGT1). Although UGT1 has been studied in reconstituted systems, the cellular role of this critical ER factor is poorly defined. Mannose trimming is proposed to be involved in sorting aberrant proteins to the ER-associated degradation (ERAD) pathway as inhibition of mannosidase activity stabilizes ERAD substrates. However, many questions still remain about the role of mannose trimming in quality control. While the over expression of the ER mannosidase-like protein EDEM1 accelerates the turnover of glycosylated ERAD substrates, its role in the quality control and sorting processes is unclear. Our results indicate that EDEM1 serves as a central link to deliver defective cargo to an ERAD dislocation complex by possessing bipartite binding properties. Finally, the exquisite spatial organization of the ER contributes to the efficiencies by which the ER temporally coordinates processes including protein maturation, quality control and ERAD. Tetratricopeptide repeat (TPR) domains are universal adaptor motifs that support protein-protein interactions involved in post-translational translocation and the formation of chaperone complexes within the cell. We have recently discovered a family of ER resident TPR-rich proteins that we propose to play a role in ER organization. This research proposal has three aims: (1) to define how UGT1 participates in ER quality control in a cellular context; (2) to elucidate the role and mechanism of action for EDEM1 in the ERAD process; and (3) to determine how novel TPR-containing proteins function in ER organization and homeostasis. A deeper knowledge of the processes in the early secretory pathway has widespread implications for understanding basic cell biology and the etiology of a range of human maladies as a growing number of diseases states are caused by defects in protein maturation and ER homeostasis. PUBLIC HEALTH RELEVANCE: Insights gained into the cellular life of a protein will shed light on pathologies caused by deficiencies in protein maturation and quality control. Diseases associated with maturation and quality control defects include albinism, liver cirrhosis, emphysema, cystic fibrosis, Krabbe, Gaucher, and some forms of heart disease to name a few. Furthermore, the accumulation of misfolded proteins results in the activation of stress responses that is associated with obesity and diabetes. Understanding the quality control process for proteins that traverse the secretory pathway will aid in the development of methodologies or therapeutic agents that serve as protein folding correctors control the stringency of the quality control process or modulate the stress response caused by the accumulation of misfolded proteins. 1