权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

IDENTIFICATION OF PROTEINS THAT STRUCTURE THE ENDOPLASMIC RETICULUM

构建内质网的蛋白质的鉴定

基本信息

批准号：
8049736
负责人：
Christina H Lee
金额：
$ 22.97万
依托单位：
CARNEGIE-MELLON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-06-01 至 2013-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8049736
关键词：
Aftercare Am 80 Amino Acids Architecture Award Binding Biochemical Biological Assay Calcium Calcium Signaling Caliber Capsid Proteins Cells Code Computer Architectures Cultured Cells DNA Sequence Rearrangement Disease Drug Metabolic Detoxication Electrons Employee Strikes Endoplasmic Reticulum Epitopes Functional disorder Future Genetic Goals Golgi Apparatus Health Homeostasis Human Image Immune In Vitro Knowledge Lead Life Light Link Lipids Mammalian Cell Mediating Membrane Microscopic Molecular Morphology Organelles Pathway interactions Performance Pharmaceutical Preparations Phenotype Play Process Protein Analysis Protein Biosynthesis Proteins Proteome Proteomics Publishing Quality Control RNA Interference Regulation Resolution Resources Role Site Site-Directed Mutagenesis Small Interfering RNA Specificity Staging Structure Structure-Activity Relationship Testing Time Tissues Tubular formation Vesicle Yeasts base cell type insight lipid biosynthesis member mutant novel novel therapeutics protein folding protein function protein structure protein transport public health relevance segregation stem therapeutic development trafficking

项目摘要

DESCRIPTION (provided by applicant): The endoplasmic reticulum (ER) is a continuous membrane network of flat sheets and curved tubules. The network plays a central role in a variety of processes critical for human health, ranging from lipid and protein synthesis and transport, to calcium homeostasis. Segregation of certain ER processes into rough and smooth sub-domains as well as the striking differences in the morphology and organization of ER membranes in specialized cell types are suggestive of a link between organelle form and function. Yet a clear understanding of the mechanisms generating and maintaining the sub-compartmentalized tubular and cisternal membrane network of the ER is lacking. Our long-term objective is to define the molecular underpinnings and functional roles for the distinct structural features of the mammalian ER. The first step in achieving this goal is identification of the proteins that govern ER morphology. Although a number of candidates likely to participate in ER structuring have been identified, few have been shown to be required in living cells. To facilitate the search for the required proteins, a candidate-based morphological RNAi screen in cultured cells has been initiated. A recently published early secretory pathway proteome of 1430 constituents (Gilchrist et al, 2006, Cell 127:1265-1281) has been pared down to a candidate pool of 117 proteins, the majority of which are previously uncharacterized. The screen is proving effective, as it has already yielded two hits and preliminary analysis of the first is confirming its role in regulating ER structure. The primary objective of this exploratory proposal is to continue the screen to identify new ER structure regulators. Each hit to emerge from the screen will be confirmed by testing whether expression of an RNAi-immune construct coding for the targeted protein rescues the RNAi-induced ER morphological abnormalities. Once hits are validated, site-directed mutagenesis, protein interaction assays, and phenotypic analysis aided by high-resolution imaging will enable identification of the structural determinants and binding partners required for the ability of each newly identified protein to perform its organelle structuring function. Preliminary studies on the first confirmed hit to emerge from the screen have identified both a critical amino acid residue in the ER structure regulator and a likely binding partner required to mediate its function. The completion of the proposed screen and preliminary analysis of the proteins identified under this exploratory award promises to yield novel insights into the mechanisms that generate ER network architecture. Ultimately, an understanding of the functional consequences of the RNAi-induced structural deficits or rearrangements identified through the screen may lead to novel therapeutics in the treatment of diseases that stem from ER dysfunction. PUBLIC HEALTH RELEVANCE: The endoplasmic reticulum (ER) carries out a myriad of processes critical to human health and disease. It is the site of drug detoxification; lipid and protein biosynthesis and trafficking; protein folding and quality control; and calcium signaling. The functional importance of the organelle in all tissues is clear. Moreover the striking differences in the architecture and organization of the ER membrane network in specialized cells types have led to the long-standing notion that the structure and sub-compartmentalization of the organelle are critical for its functions. Yet the mechanisms that structure the ER and the exact relationship between organelle structure and function are only poorly understood. The objective of this proposal is to identify novel mammalian proteins that govern ER structure. The results are expected to lead to a fuller understanding of the structure-function relationship of the organelle. Ultimately, it is hoped that the insights gained will aid in the development of therapeutics for the treatment of diseases that stem from ER dysfunction.

描述（由申请人提供）：内质网（ER）是由平板和弯曲小管组成的连续膜网络。该网络在对人类健康至关重要的各种过程中发挥着核心作用，从脂质和蛋白质的合成和运输到钙稳态。某些内质网过程分为粗糙和光滑的子域，以及特殊细胞类型中内质网膜的形态和组织的显着差异，暗示了细胞器形式和功能之间的联系。然而，对于内质网亚室管状和脑池膜网络的生成和维持机制尚缺乏清晰的了解。我们的长期目标是确定哺乳动物内质网独特结构特征的分子基础和功能作用。实现这一目标的第一步是鉴定控制内质网形态的蛋白质。尽管已经确定了许多可能参与内质网构建的候选物，但很少有人被证明是活细胞中所需要的。为了促进寻找所需蛋白质，在培养细胞中启动了基于候选的形态学 RNAi 筛选。最近发表的包含 1430 种成分的早期分泌途径蛋白质组（Gilchrist 等人，2006，Cell 127：1265-1281）已被削减为 117 种蛋白质的候选池，其中大部分以前未被表征。该筛选被证明是有效的，因为它已经产生了两次结果，并且对第一次的初步分析证实了其在调节 ER 结构中的作用。该探索性提案的主要目标是继续筛选以确定新的 ER 结构调节剂。通过测试编码目标蛋白的 RNAi 免疫构建体的表达是否可以挽救 RNAi 诱导的 ER 形态异常，可以确认从屏幕中出现的每个命中。一旦命中得到验证，定点诱变、蛋白质相互作用测定和高分辨率成像辅助的表型分析将能够识别每个新鉴定的蛋白质执行其细胞器结构功能所需的结构决定因素和结合配偶体。对屏幕上出现的第一个已确认的命中的初步研究已经确定了 ER 结构调节剂中的关键氨基酸残基以及介导其功能所需的可能结合伴侣。该探索性奖项所确定的蛋白质筛选和初步分析的完成有望对生成 ER 网络架构的机制产生新的见解。最终，了解通过筛选确定的 RNAi 诱导的结构缺陷或重排的功能后果可能会导致治疗由 ER 功能障碍引起的疾病的新疗法。公共卫生相关性：内质网 (ER) 执行对人类健康和疾病至关重要的无数过程。它是药物解毒的场所；脂质和蛋白质的生物合成和运输；蛋白质折叠和质量控制；和钙信号传导。细胞器在所有组织中的功能重要性是显而易见的。此外，特殊细胞类型中内质网膜网络的结构和组织的显着差异导致了一个长期存在的观念，即细胞器的结构和亚区室化对其功能至关重要。然而，人们对内质网的结构机制以及细胞器结构和功能之间的确切关系知之甚少。该提案的目的是鉴定控制 ER 结构的新型哺乳动物蛋白。预计结果将有助于更全面地了解细胞器的结构与功能关系。最终，希望所获得的见解将有助于开发治疗内质网功能障碍引起的疾病的疗法。