Interactions and mechanisms of function of the TAP complex

描述（由申请人提供）：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类分子被招募到与抗原加工相关的转运蛋白（TAP）、tapasin、ERp57、蛋白二硫异构酶、calnexin和calreticulin相互作用中。这个复杂的辅助蛋白质可以被认为是一个分子机器的工作就是(我)注入肽产品的蛋白质降解的MHC类我组装(ii)招募未装配的MHC类(3)促进MHC类I-peptide组装和(iv)确保监管释放最优加载类MHC i多有待理解这个复杂的分子机器的运作,这是我们研究的重点在过去的十一年。基于我们之前对TAP转运体的研究，我们能够提出ATP结合和水解如何与肽结合和转运结合的详细模型。在拟议的研究中，我们将研究TAP底物对核苷酸结合和交换的影响，以及核苷酸结合域（NBD）之间的相互作用。我们还将研究TAP的肽结合位点模型。这些研究将有助于更好地了解TAP如何被操纵来增强或抑制免疫反应，也将有助于更好地预测免疫显性细胞毒性T淋巴细胞（CTL）表位。基于对不同MHC I类同种异体在tapasin缺失细胞中的组装特征的分析，我们假设tapasin对于具有缓慢内在肽装载动力学的MHC I类同种异体的肽装载至关重要。将比较tapasin依赖性和非依赖性MHC I类同种异体在不同条件下的肽结合特性。我们的数据表明，tapasin负责将钙调蛋白和ERp57招募到肽装载复合物中。此外，不同构象状态的tapasin-ERp57复合物在增强MHC I类分子肽负载方面具有不同的活性。我们试图更好地理解这些差异的本质。我们还试图了解careticulin在tapasin辅助MHC I类组装中的作用。尽管所有MHC I类分子似乎在内质网内遵循相同的组装路线，但密切相关的HLA-B同种异体在其内在组装率和内质网退出率方面存在显着差异。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转运，并检查快速或缓慢转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，这将进一步研究。总之，这些研究将使我们更好地理解MHC I类组装路线的不同步骤，并将有助于开发更有效的策略来增强感染和癌症中的CTL反应。公共卫生相关性：了解TAP的底物相互作用位点和TAP的潜在静息状态构象（无活性构象）对未来设计TAP抑制剂非常重要，这些抑制剂可用于移植和自身免疫，此外还可用于设计其他ABC转运蛋白抑制剂以克服药物/抗生素耐药性。更好地了解tapasin的功能机制可能会导致新的策略来增强感染和癌症中特异性免疫原肽与MHC I类分子的组装。最后，了解HLA- b同种异体之间的转运差异如何影响其抗原递呈能力，对于更好地阐明不同HLA抗原对疾病易感性、缓解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.