MOLECULAR MECHANISM OF GRP94 FUNCTION

PI建议确定职能、结构和监管 GRP94的性质将其定义为(多)肽结合 蛋白。GRP94，hsp90的内质网(ER)同源物， 是内质网管腔内丰富的驻留分子伴侣。在……里面 除了它的伴侣作用，在动物致癌模型中的研究 已经确定GRP94可以作为肿瘤特异性疫苗发挥作用。 已知疫苗的活性需要摄取GRP94-肽 抗原提呈细胞的复合体，以及GRP94- 与内质网中新生的MHC I类分子结合的多肽。这个 控制GRP94释放的分子信号 相关的多肽目前尚不清楚，但肯定会证明 GRP94的分子机制研究具有重要意义 GRP94的功能及其作为免疫疗法的研究进展 探员。 为了实现这一目标，PI建议1)定义动力学和 与GRP94结合的多肽的调节。为此， GRP94与多肽结合的假说是由 将对核糖核苷酸进行测试，并将开发检测方法以识别 常驻内质网积分和管腔的潜在调节作用 蛋白质与GRP94多肽的结合和释放反应。2)至 对GRP94结合肽进行结构分析并鉴定 多肽结合模体。已制定程序，用于 天然GRP94的提纯，基于大规模的质谱学， 将启动结合多肽测序研究以检验这一假设 GRP94识别特定的多肽结构基序。3)至 用技术鉴定GRP94肽结合位点(S) 包括化学和光交联、蛋白水解区结构 研究，以及与纯化表达构建体的体外结合研究 和孤立的结构域。4)确定GRP94的作用 二聚化在多肽结合活性和体内调节中的作用 功能。为了测试功能是否需要二聚化， 阻止二聚化的组装结构域突变将是 通过多肽结合的体外研究和体内研究进行评估 蛋白质的组装和分泌。

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.