ENDOGENOUS MHC CLASS I ANTIGEN PROCESSING PATHWAY

描述：（改编自研究者摘要）： 该建议是通过以下方式来理解分子抗原处理机制： 内源性合成的蛋白质产生精确切割的肽/MHC 细胞表面的I类复合物。 这些肽/MHC的表达 复合物对于CD 8+细胞毒性T细胞免疫是必需的， 肿瘤、细胞内病原体、同种异体组织移植物， 自身免疫 肽/MHC复合物表达失败是一个主要机制 肿瘤细胞和病毒如单纯疱疹病毒，通过这种途径逃避免疫， 监视 阐明了内源性地 将被片段化并展示为肽/MHC的合成蛋白质也将 导致理解决定表位优势的关键因素， 从而改进疫苗的设计， 肿瘤和细胞内病原体。 新型的基于T细胞的“lacZ”测定具有 已经开发出允许定量和识别处理过的 阿托摩尔范围内的肽。 与表达DNA的方法一起 编码确定的抗原前体的构建体和用于表征所述抗原前体的构建体。 在模型细胞系中处理的肽前体和产物 问题将得到解决。 (1)无论是天然加工的肽 由于细胞蛋白质周转和/或来自新生蛋白质 将通过选择性改变抗原的稳定性来建立 前体蛋白通过N-末端规则。 (2)侧翼的影响 将通过以下方法测定产生加工肽的残留物： 在模型前体和全长蛋白质中引入特定突变。 改变免疫系统的定量或定性 抗原处理的结果将通过测量T细胞 对不同的显性和亚显性肽的反应， 前体 (3)在制备过程中加工的肽中间体的鉴别 前和后TAP步骤和他们的伴侣，最终产生最终的 精确切割的肽产物将通过HPLC分析来确定 分级细胞提取物和新的测定。 最后，（4）我们将生成 并分析抗原处理途径中的新突变。 这些遗传 突变体很可能揭示了在加工过程中发生的步骤， 活细胞中内源性合成的前体，但可能无法进入 传统的生化分析。

DESCRIPTION: (Adapted from Investigator's abstract): The long term goal of this proposal is to understand the molecular antigen processing mechanism by which endogenously synthesized proteins yield precisely cleaved peptide/MHC class I complexes on the cell surface. The expression of these peptide/MHC complexes is essential for CD8+ cytotoxic T-cell immunity specific for tumors, intracellular pathogens, allogeneic tissue grafts and for autoimmunity. Failure to express peptide/MHC complexes is a major mechanism by which tumor cells and viruses such as herpes simplex virus, escape immune surveillance. Elucidating the molecular mechanism that allows endogenously synthesized proteins to be fragmented and displayed as peptide/MHC will also lead to understanding a key factor in determining epitope dominance, and thus improve design of vaccines for eliciting immunity against established tumors and intracellular pathogens. Novel T-cell based "lacZ" assays have been developed that permit quantitation and identification of processed peptides in the attomole range. Together with methods to express DNA constructs encoding defined antigenic precursors and for characterizing the processed peptide precursors and products in model cell lines the following problems will be addressed. (1) Whether naturally processed peptides arise as a consequence of cellular protein turn-over and/or from nascent proteins will be established by selective alteration of stability of antigenic precursor proteins via the N-end rule. (2) The influence of flanking residues on the generation of processed peptides will be determined by introducing specific mutations in model precursors and full-length proteins. The immunological consequences of altering the quantitative or qualitative outcome of antigen processing will be evaluated by measurement of T-cell responses to different dominant and sub-dominant peptides in these precursors. (3) The identity of processed peptide intermediates during the pre- and post-TAP step and their chaperones, that eventually yield the final precisely cleaved peptide products will be determined by analysis of HPLC fractionated cell extracts and novel assays. Finally, (4) we will generate and analyze new mutations in the antigen processing pathway. These genetic mutants are likely to reveal steps that occur during processing of endogenously synthesized precursors in living cells but may be inaccessible to conventional biochemical analysis.