Generating Antigenic Peptide/MHC By Cryptic Translation

描述（由申请人提供）：本项目的长期目标是了解抗原加工途径如何通过隐蔽翻译产生肽/MHC I类复合物（pMHC I）。来自许多不同实验室的大量研究表明，肿瘤、病毒感染细胞、转染细胞或甚至正常细胞表面上的天然加工的pMHC I来自预期不被翻译的mRNA区域。相对于其他传统来源，允许细胞对这种抗原前体的神秘来源进行采样的机制尚不清楚。此外，隐藏肽在多大程度上来自正常mRNA，并有助于由MHC I类分子呈递的肽的多样性池是未知的。在这里，我们将测试的假设，（a）这些不寻常的，隐藏的肽不仅来自突变或外源引入的基因，而且从正常的基因和（B）他们的表达可能受到调节压力。此外，我们将测试的假设，（c）隐蔽翻译结果从一组新的核糖体，启动翻译不是在传统的AUG密码子，但在隐蔽的非AUG密码子。我们将使用引物延伸抑制（脚趾印）和直接分析它们的t-RNA，蛋白质和RNA成分来表征这些核糖体。我们预计，对隐藏的pMHC I是如何产生的更好的理解不仅将提供一种手段来挖掘抗原肽的新来源和疫苗设计的新方法，而且还将深入了解蛋白质翻译和免疫监视机制。

DESCRIPTION (provided by applicant): The long term goals of this project are to understand how the antigen processing pathway generates peptide/MHC class I complexes (pMHC I) by cryptic translation. Numerous studies, from many different laboratories, have shown that naturally processed pMHC I on the surface of tumors, virally infected cells, transfected or even normal cells arise from regions of mRNAs that were not expected to be translated. The mechanism that allows cells to sample this cryptic source of antigenic precursors versus other conventional sources is obscure. In addition, to what extent cryptic peptides arise from normal mRNAs and contribute to the diverse pool of peptides presented by MHC class I molecules is unknown. Here we will test the hypotheses that (a) these unusual, cryptic peptides arise not only from mutated or exogenously introduced genes but also from normal genes and (b) their expression may be regulated under stress. Furthermore, we will test the hypothesis that (c) cryptic translation results from a novel set of ribosomes that initiate translation not at conventional AUG codons, but at cryptic non-AUG codons. We will characterize these ribosomes using primer extension inhibition (toeprinting) and by direct analyses of their t-RNAs, proteins and RNA constituents. We anticipate that an improved understanding of how cryptic pMHC I are generated will not only provide a means to tap into a novel sources of antigenic peptides and new approaches to vaccine design, but will also yield insights into protein translation and immune surveillance mechanisms.