Genetic and Biochemical Analysis of INSIG Proteins

描述(申请人提供)：INSIG蛋白是哺乳动物体内固醇稳态的关键调节器，它们与具有SSD结构域(SSD)的蛋白质的跨膜结构域相互作用，并影响SSD客户的位置或稳定性的变化。我们最近发现，酿酒酵母含有INSIGs的同源物，称为Nsglp和Nsg2p，它们类似地调节酵母HMG-CoA还原酶同工酶Hmg2p的稳定性。酵母Hmg2p的跨膜区有一个SSD，这是酵母INSIGs相互作用和影响Hmg2p稳定性所必需的。因此，INSIG-SSD客户相互作用是广泛保守的，允许通过使用简易酵母方法来分析INSIG作用的未知特征，以研究Nsglp-Hmg2p相互作用，作为所有INSIG-SSD客户相互作用的模型。哺乳动物中的INSIG-SSD相互作用需要甾醇，我们的初步证据表明，Nsg1p-Hmg2p相互作用的这一方面类似地保守。 综上所述，这些结果表明INSIG-SSD客户相互作用在生物学中已经存在了超过10亿年，它在酵母中的研究将使我们能够在所有情况下了解INSIG蛋白的机制和作用。具体地说，我们计划1)测试晚期固醇途径产物调节Nsg1p-Hmg2p对相互作用的假设。我们将使用各种方法来发现这个调控因子的身份，并使用一些NSG功能的体外分析来确认其功能。2)检查Nsg1p-Hmg2p相互作用的序列要求。我们将测试Hmg2p SSD在Nsglp-Hmg2p相互作用中的作用，并类似地探索保守残基在NSG蛋白中的作用。我们还将使用酵母技术对这些蛋白质部分进行无偏筛选，以补充对保守基序的直接测试。通过这种方式，将首次描述INSIG-SSD域交互的细节。最后，我们将使用蛋白质组学技术和基于表型的遗传筛选相结合的方法来探索Nsglp蛋白的其他可能功能。由于INSIG蛋白在固醇调节中非常重要，任何将其用作潜在的临床靶点都需要详细了解它们的机制以及是否可能参与细胞生物学的未知方面。

DESCRIPTION (provided by applicant): The INSIG proteins are key regulators of sterol homeostasis in mammals, where they interact with the transmembrane domains of proteins with a Sterol Sensing Domain (SSD) and effect changes in the location or stability of the SSD clients. We recently discovered that the yeast S. cerevisiae has homologues of INSIGs, called Nsglp and Nsg2p, that similarly regulate the stability of yeast HMG-CoA reductase isozyme Hmg2p. Yeast Hmg2p has an SSD in its transmembrane region, which is required for the interaction and effects of yeast INSIGs on Hmg2p stability. Thus, the INSIG-SSD client interaction is broadly conserved allowing analysis of unknown features of INSIG action by use of facile yeast approaches to study the Nsglp- Hmg2p interaction as a model of all INSIG-SSD client interactions. The INSIG-SSD interaction in mammals requires sterols, and our preliminary evidence indicates that this aspect of the Nsg1p-Hmg2p interaction is similarly conserved. Taken together, these results indicate that the INSIG-SSD client interaction has been extant in biology for over 1 billion years, and its study in yeast will allow understanding of the mechanism and actions of the INSIG proteins in all cases. Specifically we plan to 1) Test the hypothesis that a late sterol pathway product regulates the interaction of the Nsg1p-Hmg2p pair. We will discover the identity of this regulator using a variety of approaches, and confirm its function using a number of in vitro assays of Nsg function. 2) Examine the sequence requirements for Nsg1p-Hmg2p interaction. We will test the role of the Hmg2p SSD in Nsglp- Hmg2p interaction, and similarly explore the role of conserved residues in the Nsg protein. We will also use yeast techniques to perform unbiased screens of these portions of the proteins to complement the directed tests of conserved motifs. In this way the details of an INSIG-SSD domain interaction will be delineated for the first time. Finally, 3) we will use a combination of proteomic techniques and phenotype-based genetic screens to explore other possible functions for the Nsglp protein. Because the INSIG proteins are so important in sterol regulation, any use of them as potential clinical targets will require a detailed understanding of they mechanisms and any possible involvement in unknown aspects of cell biology.