IDENTIFYING AND EXPLOITING POST-TRANSLATIONAL MODIFICATIONS IN ANTIGEN PRESENTATION USING NEXT GENERATION PROTEOMICS

在之前由bbsrc资助的工作中，通过达勒姆，利物浦和Scancell之间的新合作，我们已经确定了MHCII类分子（MHCII）向免疫系统呈递抗原的新途径。MHCII显示抗原以帮助T细胞控制细菌和其他病原体的感染，但也显示修饰的自身抗原（如新抗原）。Benham小组发现，一些MHCII类型，如HLA-DP4，独立于CD74组装，CD74是一种通常将MHCII引导到内体系统的蛋白质。这些HLA-DP4分子可以稳定地结合其他区室中的自肽，例如内质网和自噬囊泡。Scancell/Durrant等人已经证明，翻译后修饰（如瓜氨酸化）肽可以进入这些MHCII，产生新的修饰肽，可以破坏自我耐受性并引起免疫反应。这一机制对人类和牲畜传染病的控制、抗癌疫苗的开发以及自身免疫的定制疗法都有影响。目的、假设和实验方法我们假设HLA-DP4可以在非常规的细胞内运输过程中获得新抗原。该项目的首要目标是表征这些新型抗原，并建立驱动它们在细胞表面展示的细胞生物学。为了支持这一目标，我们将采用基础质谱技术，通过天然质谱来定义MHC组合修饰，用定量质谱绘制免疫蛋白质组图，并在两种系统（重组抗原提呈细胞和由CASE合作伙伴开发的人源化小鼠黑色素瘤模型）中鉴定翻译后修饰的MHC肽。学生还将接受广泛的实验室方法培训，包括共聚焦显微镜、组织培养、SDS-PAGE/western blotting、生化/细胞分析、分子生物学、生物信息学和统计计算(R)。学生将接受演讲技巧和报告写作方面的培训，鼓励他们在国际会议上展示他们的工作，并将参加定期的期刊俱乐部。与BBSRC战略的相关性该项目既以好奇心为导向，又具有创造性，受到技术能力新发展的推动。该项目广泛地位于世界级基础生物科学的范围内，并直接涉及生物科学的系统方法和生物科学的技术开发，通过强烈关注推进定量和机械蛋白质组学技术。

BackgroundIn previous BBSRC-funded work being advanced through this new collaboration between Durham, Liverpool and Scancell, we have identified novel routes for the presentation of antigens by MHC class II molecules (MHCII) to the immune system. MHCII display antigens to helper T cells to control infections by bacteria and other pathogens, but also present modified self-antigens (e.g. neoantigens). The Benham group has found that some MHCII types e.g. HLA-DP4, assemble independently of CD74, a protein that normally directs the MHCII to the endosomal system. These HLA-DP4 molecules can stably bind self- peptides in other compartments e.g. the Endoplasmic Reticulum and autophagic vesicles. Scancell/Durrant (amongst others) have shown that post-translationally modified (e.g. citrullinated) peptides can access these MHCII, generating novel modified peptides that can break self-tolerance and cause an immune response. This mechanism has implications for the control of infectious disease in humans and livestock, for the development of anti-cancer vaccines and for bespoke therapies for autoimmunity. Aims, Hypothesis and Experimental ApproachWe hypothesise that neoantigens can be accessed by HLA-DP4 during unconventional intracellular trafficking. The overarching aim of this project is to characterise these novel antigens and establish the cell biology that drives their display at the cell surface. Supporting this aim, we will employ underpinning mass spectrometry capability to define MHC combinatorial modifications by native ms, map the immuno-proteome with quantitative ms and identify post-translationally modified MHC peptides in two systems (reconstituted antigen presenting cells and a humanised mouse melanoma model developed by the CASE partner). The student will also be trained in a wide range of laboratory methods including confocal microscopy, tissue culture, SDS-PAGE/western blotting, biochemical/cellular assays, molecular biology, bioinformatics and statistical computing (R). The student will be trained in presentation skills and report writing, encouraged to present their work at international meetings, and will participate in a regular journal club. Relevance to BBSRC strategyThe project is both curiosity-led and creative, being catalysed by new developments in technological capability. The project sits broadly within the remit for world class underpinning Biosciences and relates directly to a systems approaches to the Biosciences and to technology development for the Biosciences, through the strong focus on advancing quantitative and mechanistic proteomic technologies.