IDENTIFICATION OF PROTEINS THAT STRUCTURE THE ENDOPLASMIC RETICULUM

描述(申请人提供)：内质网(ER)是由平板和弯曲的小管组成的连续的膜网络。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，从脂肪和蛋白质的合成和运输，到钙稳态。某些内质网过程被分成粗糙和光滑的亚域，以及特殊细胞类型内质网膜在形态和组织上的显著差异，这表明细胞器形式和功能之间存在联系。然而，对内质网亚室管膜和池膜网络的产生和维持机制缺乏明确的了解。我们的长期目标是确定哺乳动物内质网不同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识别支配内质网形态的蛋白质。虽然已经确定了一些可能参与内质网结构的候选者，但很少有人被证明是活细胞所需的。为了便于寻找所需的蛋白质，已在培养细胞中启动了基于候选的形态RNAi筛选。最近发表的由1430种成分组成的早期分泌途径蛋白质组(Gilchrist等人，2006，Cell 127：1265-1281)已经被削减到117个蛋白质候选库，其中大部分以前没有特征。这一筛选被证明是有效的，因为它已经产生了两次点击，对第一次的初步分析证实了它在调节内质网结构方面的作用。这项探索性建议的主要目标是继续筛选，以确定新的内质网结构调节器。从屏幕上出现的每一次点击都将通过测试编码目标蛋白的RNAi免疫结构的表达是否拯救RNAi诱导的ER形态异常来确认。一旦HITS得到验证，定点突变、蛋白质相互作用分析和高分辨率成像辅助的表型分析将使鉴定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伙伴成为可能。对第一个从屏幕上出现的确认HIT的初步研究已经确定了ER结构调节器中的一个关键氨基酸残基和一个可能的结合伙伴，这是调节其功能所需的。完成拟议的筛选和对这一探索性奖励下确定的蛋白质的初步分析有望对产生ER网络结构的机制产生新的见解。最终，了解RNAi诱导的结构缺陷或通过筛查确定的重排的功能后果可能会导致治疗由内质网功能障碍引起的疾病的新疗法。 与公共健康相关：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、脂肪和蛋白质生物合成和运输、蛋白质折叠和质量控制以及钙信号传递的场所。细胞器在所有组织中的功能重要性是显而易见的。此外，在特殊细胞类型中，内质网膜网络的结构和组织结构的显着差异导致了长期以来的观念，即细胞器的结构和亚区划对其功能至关重要。然而，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仍然知之甚少。这项建议的目的是确定控制内质网结构的新的哺乳动物蛋白。这些结果有望使人们更全面地了解细胞器的结构与功能之间的关系。最终，希望所获得的见解将有助于开发治疗由内质网功能障碍引起的疾病的疗法。

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.