Genetic and Biochemical Analysis of INSIG Proteins

描述（由申请人提供）：INSIG蛋白是哺乳动物中固醇稳态的关键调节剂，其中它们与具有固醇传感结构域（SSD）的蛋白质的跨膜结构域相互作用，并影响SSD客户端的位置或稳定性的变化。我们最近发现，酵母S。酿酒酵母具有INSIG的同系物，称为Nsglp和Nsg 2 p，其类似地调节酵母HMG-CoA还原酶同工酶Hmg 2 p的稳定性。酵母Hmg 2 p在其跨膜区具有SSD，这是酵母INSIG对Hmg 2 p稳定性的相互作用和影响所必需的。因此，INSIG-SSD客户端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，以研究作为所有INSIG-SSD客户端相互作用模型的Nsglp-Hmg 2 p相互作用。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，这方面的Nsg 1 p-Hmg 2 p相互作用是类似的保守。 综上所述，这些结果表明，INSIG-SSD客户端相互作用在生物学中已经存在了超过10亿年，其在酵母中的研究将有助于了解INSIG蛋白在所有情况下的机制和作用。具体来说，我们计划1）测试的假设，一个晚甾醇途径的产物调节Nsg 1 p-Hmg 2 p对的相互作用。我们将使用各种方法来发现这种调节剂的身份，并使用一些NSG功能的体外测定来确认其功能。2)检查Nsg 1 p-Hmg 2 p相互作用的序列要求。我们将测试Hmg 2 p SSD在Nsglp-Hmg 2 p相互作用中的作用，并类似地探索Nsg蛋白中保守残基的作用。我们还将使用酵母技术对蛋白质的这些部分进行无偏筛选，以补充保守基序的定向测试。通过这种方式，将首次描述INSIG-SSD域相互作用的细节。最后，3）我们将使用蛋白质组学技术和基于表型的遗传筛选的组合来探索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，因此将其用作潜在的临床靶标将需要详细了解其机制以及任何可能涉及细胞生物学未知方面的内容。

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.