Regulation of insulin-like growth factors by the ER chaperone GRP94

描述(由申请人提供)：胰岛素样生长因子(IGF)在胚胎发育、细胞生长信号和对代谢应激的反应中调节分化决定。我们发现，这些重要生长因子的产生依赖于内质网伴侣葡萄糖调节蛋白94(GRP94)的活性，GRP94是它们生物合成所必需的。GRP94与其他分子伴侣的不同之处在于，它只对少量的客户蛋白有选择性，对晚期折叠中间产物有偏好，并且没有任何已知的辅助因子。所有这些方面都为GRP94提出了一种独特的行动模式。这个项目将利用细胞的生长依赖于GRP94-IGF轴的优势来阐明GRP94神秘的作用周期。在我们的第一个目标中，我们将使用一种新的基于细胞的分析来筛选不支持IGF-II产生的GRP94突变，从而定义这种伴侣功能所必需的氨基酸。在目标2中，我们将确定这些突变体是否在一般机制方面存在缺陷，如ATP结合和水解或钙结合，或者这些突变是否定义了一个客户结合域。我们还将测试GRP94突变体支持IGF以外的客户的能力，以发现是否存在客户特定的突变体。我们的第三个目标是通过利用一组胰岛素样蛋白质之间的结构相似和差异来理解GRP94是如何识别IGF的。这些研究将提供有关GRP94客户选择性的信息，目前尚不清楚。最后，我们将利用IGF缺乏的临床后果来寻找具有重要功能的人类GRP94变体。根据我们的发现，至少有一个这样的变异会影响IGF的产生，我们将对IGF缺乏的患者群体进行基因分型和排序，以寻找可能解释低IGF产生的等位基因GRP94变异。综上所述，这些结果将导致对GRP94如何陪伴客户蛋白的新理解。 公共卫生相关性：该项目旨在破译代表HSP90伴侣蛋白家族的GRP94是如何选择其客户蛋白并影响其正常生产的。这项工作将使用一种新的基于细胞的分析方法，以胰岛素样生长因子为客户，确定GRP94的功能并从生化角度表征GRP94的自然和定点突变。

DESCRIPTION (provided by applicant): Insulin-like growth factors (IGFs) mediate differentiation decisions during embryonic development, cell growth signaling and responses to metabolic stress. We found that the production of these important growth factors is dependent on the activity of the endoplasmic reticulum chaperone glucose regulate protein 94 (GRP94), which is essential for their biosynthesis. GRP94 is different from other molecular chaperones in its selectivity towards only a small number of client proteins, preference for late folding intermediates and the absence of any known co-factors. All these aspects suggest a unique mode of action for GRP94. This project will elucidate the enigmatic action cycle of GRP94 by taking advantage of cells whose growth depends on the GRP94-IGF axis. In our first aim we will use a novel cell-based assay to screen for mutants of GRP94 that do not support IGF-II production and thus define amino acids that are essential for this chaperone function. In Aim 2, we will determine whether these mutants are defective in general mechanistic aspects, such as ATP binding and hydrolysis or calcium binding, or whether the mutations define a client binding domain. We will also test the ability of GRP94 mutants to support clients other than IGF, to discover whether there are client-specific mutants. Our third aim is to understand how GRP94 recognizes IGF, by exploiting the structural similarities and differences among a set of insulin-like proteins. These studies will inform on the client selectivity of GRP94, which is currently not understood. Finally, we will take advantage of the clinical consequences of IGF deficiency to search for functionally important human GRP94 variants. Following on our finding that at least one such variant affects IGF production, we will genotype and sequence IGF-deficient patient populations to seek allelic GRP94 variants that may explain low IGF production. Together, these results will lead to a new understanding of how GRP94 chaperones client proteins. PUBLIC HEALTH RELEVANCE: This project aims to decipher how GRP94, representing the family of HSP90 chaperones, selects its client proteins and affects their proper production. The work will use a novel cell-based assay, with insulin-like growth factor as the client, to determine the functionality and characterize biochemically both natural and site-directed mutations in GRP94.