MOLECULAR MECHANISM OF GRP94 FUNCTION

PI建议确定功能、结构和监管 GRP 94的性质，将其定义为（多）肽结合 蛋白 GRP 94，hsp 90的内质网（ER）同源物， 是内质网腔丰富的常驻分子伴侣。 在 除了它的伴侣作用，在动物致癌模型中的研究 已经确定GRP 94可以作为肿瘤特异性疫苗发挥作用。 已知疫苗活性需要摄取GRP 94-肽 复合物的抗原呈递细胞，和转移的GRP 94- 结合肽与ER中新生的MHC I类分子。 的 控制发现GRP 94释放的分子信号 相关的肽目前是未知的，但肯定会证明 这对阐明GRP 94的分子机制具有重要意义 功能和GRP 94作为免疫疗法的发展 剂 为了实现这一目标，PI建议1）定义动力学， 调节（多）肽与GRP 94的结合。 为此中央 假设GRP 94与肽的结合受 将对核糖核苷酸进行检测，并开发检测方法， 常驻ER整体和内腔的潜在调节作用 蛋白质与GRP 94肽的结合和释放反应。2)到 进行GRP 94结合肽的结构分析并鉴定 肽结合基序。 在制定了程序， 天然GRP 94的纯化，大规模的，基于质谱的， 将启动结合肽测序研究以检验假设 GRP 94识别特定的肽结构基序。 3)到 通过技术鉴定GRP 94肽结合位点 包括化学和光交联、蛋白水解结构域结构 研究和纯化表达构建体的体外结合研究 和孤立的结构域。 4)确定GRP 94的作用 二聚化在肽结合活性调节和体内 功能 为了测试二聚化是否是功能所必需的， 阻断二聚化的组装结构域中的突变将被 通过肽结合的体外研究和 蛋白质组装和分泌。

The PI proposes to identify the functional, structural and regulatory properties of GRP94 which define it as a (poly) peptide binding protein. GRP94, the endoplasmic reticulum (ER) homolog of hsp90, is an abundant resident molecular chaperone of the ER lumen. In addition to its chaperone role, studies in animal carcinogenesis models have established that GRP94 can function as a tumor-specific vaccine. Vaccine activity is know to require the uptake of GRP94-peptide complexes by antigen-presenting cells, and the transfer of GRP94- bound peptides to nascent MHC class I molecules in the ER. The molecular signals governing the finding the release of the GRP94 associated peptides are currently unknown, but are certain to prove significant to the identification of the molecular mechanism of GRP94 function and to the development of GRP94 as an immunotherapy agent. To achieve this goal, the PI proposes 1) to define the kinetics and regulation of (poly) peptide binding to GRP94. To this end, the hypothesis that peptide binding by GRP94 is regulated by ribonucleotides will be tested and assays will be developed to identify potential regulatory contributions of resident ER integral and lumenal proteins to the GRP94 peptide binding and release reactions. 2) To perform structural analyses of GRP94 bound peptides and identify peptide binding motifs. Having developed procedures for the purification of native GRP94, a large scale, mass spectrometry based, bound peptide sequencing study will be initiated to test the hypothesis that GRP94 recognizes specific peptide structural motifs. 3) To identify the GRP94 peptide binding site(s) through techniques including chemical and photo-crosslinking, proteolytic domain structure studies, and in vitro binding studies with purified expression constructs and isolated structural domains. 4) To determine the role of GRP94 dimerization in the regulation of peptide binding activity and in vivo function. To test whether dimerization is necessary for function, mutations in the assembly domain that block dimerization will be assessed by in vitro studies of peptide binding and in vivo studies of protein assembly and secretion.