Interactions and mechanisms of function of the TAP complex

描述（由申请方提供）：主要组织相容性复合体（MHC）I类分子的组装发生在细胞的内质网（ER）内。新合成的MHC I类分子被募集到与抗原加工相关的转运蛋白（TAP）、tapasin、ERp 57、蛋白质二硫键异构酶、钙连接蛋白和钙网蛋白的相互作用中。这种辅助蛋白的复合物可以被认为是一种分子机器，其工作是（i）将蛋白降解的肽产物泵入MHC I类组装区域，（ii）募集未组装的MHC I类，（iii）促进MHC I类-肽组装，以及（iv）确保最佳装载的MHC I类的调节释放。关于这台复杂的分子机器的工作原理，还有很多东西有待了解，这是我们过去11年来研究的重点。基于我们以前的工作与TAP转运蛋白，我们能够提出一个详细的模型，如何ATP结合和水解耦合肽结合和运输。在拟议的研究中，我们将检查TAP底物对TAP的核苷酸结合和交换的影响，以及对核苷酸结合结构域（NBD）之间的相互作用。还将检查TAP的肽结合位点的模型。这些调查将允许更好地了解如何TAP可以被操纵，以增强或抑制免疫反应，也将允许更好地预测免疫显性细胞毒性T淋巴细胞（CTL）表位。基于对tapasin缺陷细胞中各种MHC I类同种异型的组装特征的分析，我们假设tapasin对于具有缓慢的内在肽加载动力学的MHC I类同种异型的肽加载是必不可少的。将在不同条件下比较tapasin依赖性和独立性MHC I类同种异型的肽结合特性。我们的数据表明，tapasin负责招募钙网蛋白和ERp 57进入肽加载复合物。此外，不同构象状态的Tapasin-ERp 57复合物在增强MHC I类分子的肽负载方面具有不同的活性。我们寻求更好地理解差异的性质。我们还试图了解careticulin在塔帕辛辅助的MHC I类组装中的作用。虽然所有的MHC I类分子似乎遵循相同的装配路线内的ER，密切相关的HLA-B同种异型显着不同的内在率的组装和ER退出。在这里提出的研究中，我们试图将高频HLA-B等位基因分类为快速或缓慢贩运，并检查快速或缓慢贩运对抗原呈递和疾病进展的功能后果。我们的假设是，运输表型可以影响CTL反应和NK细胞反应，这将进一步检查。总之，这些研究将允许更好地了解MHC I类组装途径的不同步骤，并将有助于开发更有效的策略来增强感染和癌症中的CTL应答。 公共卫生相关性：对TAP的底物相互作用位点和TAP的潜在静息状态构象（非活性构象）的理解对于未来设计可用于移植和自身免疫的设置的TAP抑制剂以及另外设计其它ABC转运蛋白的抑制剂以克服药物/抗生素抗性将是重要的。更好地理解tapasin功能的机制可能会导致新的策略，用于增强感染和癌症中特定免疫原性肽与MHC I类分子的组装。最后，了解HLA-B同种异型之间的运输差异如何影响其抗原呈递能力对于更好地阐明不同HLA抗原对疾病易感性、消退和进展的影响将是重要的。

DESCRIPTION (provided by applicant): Assembly of major histocompatibility complex (MHC) class I molecules occurs within the endoplasmic reticulum (ER) of cells. Newly synthesized MHC class I molecules are recruited into interactions with the transporter associated with antigen processing (TAP), tapasin, ERp57, protein disulfide isomerase, calnexin and calreticulin. This complex of accessory proteins can be considered a molecular machine whose job it is to (i) pump the peptide products of protein degradation into the region of MHC class I assembly (ii) recruit unassembled MHC class I (iii) facilitate MHC class I-peptide assembly and (iv) ensure regulated release of optimally loaded MHC class I. Much remains to be understood about the workings of this intricate molecular machine, which has been the focus of our research for the past eleven years. Based on our previous work with the TAP transporter, we are able to propose a detailed model for how ATP binding and hydrolysis couple to peptide binding and transport. In the proposed studies we will examine effects of TAP substrates on nucleotide binding and exchange by TAP, and on interactions between the nucleotide binding domains (NBD). A model for the peptide-binding site of TAP will also be examined. These investigations will allow for better understanding of how TAP can be manipulated to enhance or suppress immune responses, and will also allow for better predictions of immunodominant cytotoxic T lymphocyte (CTL) epitopes. Based on analyses of the assembly characteristics of various MHC class I allotypes in tapasin-deficient cells, it is our hypothesis that tapasin is essential for peptide loading of MHC class I allotypes that have slow intrinsic peptide loading kinetics. Peptide binding properties of tapasin dependent and independent MHC class I allotypes will be compared under different conditions. Our data suggest that tapasin is responsible for recruiting calreticulin and ERp57 into the peptide loading complex. Furthermore different conformational states of tapasin-ERp57 complexes had different activities in enhancing peptide loading of MHC class I molecules. We seek to better understand the nature of the differences. We also seek to understand the role of careticulin in tapasin-assisted MHC class I assembly. Although all MHC class I molecules appear to follow the same assembly route within the ER, closely related HLA-B allotypes differ dramatically in their intrinsic rates of assembly and ER exit. In the studies proposed here, we seek to classify high frequency HLA-B alleles as rapid or slow trafficking, and to also examine the functional consequences of rapid or slow trafficking upon antigen presentation and disease progression. It is our hypothesis that the trafficking phenotypes can impact both the CTL response and the NK cell response, which will be further examined. Together, these studies will allow for a better understanding of the different steps of the MHC class I assembly route, and will contribute to the development of more effective strategies to enhance CTL responses in infection and cancer. PUBLIC HEALTH RELEVANCE: An understanding of the substrate interaction site of TAP, and of potential resting state conformations of TAP (inactive conformations) will be important for future designs of TAP inhibitors that could be of use in settings of transplantation and autoimmunity, and additionally in the design of inhibitors of other ABC transporters to overcome drug/antibiotic resistance. A better understanding of the mechanism of tapasin function could lead to new strategies for enhancing assembly of specific immunogenic peptides with MHC Class I molecules in infection and cancer. Finally, an understanding of how trafficking differences between HLA-B allotypes impact their antigen presenting ability will be important for better elucidating the effects of different HLA antigens on disease susceptibility, resolution, and progression.