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类分子参与与抗原处理相关的转运蛋白(TAP)、Tapasin、ERp57、蛋白质二硫键异构酶、钙粘蛋白和钙网蛋白的相互作用。这种辅助蛋白的复合体可以被认为是一台分子机器，它的工作是(I)将蛋白质降解的多肽产物泵入MHC I类组装区域(Ii)招募未组装的MHC I类多肽组装(Iii)促进MHC I类多肽组装和(Iv)确保最佳负载的MHC I类多肽的调节释放。关于这台复杂的分子机器的工作原理仍有许多有待了解的地方，这一直是我们过去11年来研究的重点。基于我们以前对TAP转运体的工作，我们能够提出一个详细的模型来解释ATP结合和水解偶联到肽的结合和运输。在拟议的研究中，我们将检查TAP底物对TAP与核苷酸结合和交换的影响，以及对核苷酸结合域(NBD)之间相互作用的影响。还将研究TAP的多肽结合部位的模型。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，也将使更好地预测免疫优势细胞毒性T淋巴细胞(CTL)表位。基于对不同MHC-I类异型在Tapasin缺乏细胞中的组装特征的分析，我们的假设是Tapasin对内源性多肽负载动力学缓慢的MHC-I类异型的多肽负载是必不可少的。在不同的条件下，将比较Tapasin依赖的和独立的MHC I类同种异型的多肽结合特性。我们的数据表明，Tapasin负责将钙网蛋白和ERp57重新招募到多肽负载复合体中。此外，不同构象的Tapasin-ERp57复合体在增强MHC I类分子的多肽负载量方面具有不同的活性。我们寻求更好地理解差异的本质。我们还试图了解Careticlin在Tapasin辅助的MHC I类组装中的作用。尽管所有MHC I类分子在内质网中似乎遵循相同的组装路线，但密切相关的人类白细胞抗原-B同种异型在其内在组装和退出内质网的速度上存在显著差异。在这里提出的研究中，我们试图将高频的HLA-B等位基因分类为快速或缓慢运输，并检查快速或缓慢运输对抗原呈递和疾病进展的功能后果。我们的假设是，贩运表型可以影响CTL反应和NK细胞反应，这一点将进一步研究。总之，这些研究将使我们能够更好地了解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，以增强感染和癌症中的CTL反应。公共卫生相关性：了解TAP的底物相互作用部位，以及TAP的潜在静止状态构象(非活性构象)，对于未来设计可用于移植和自身免疫环境的TAP抑制剂，以及设计其他ABC转运体的抑制剂以克服药物/抗生素耐药性将是重要的。更好地了解Tapasin的作用机制可能会导致在感染和癌症中加强特定免疫原肽与MHC-I类分子的组装的新策略。最后，了解人类白细胞抗原B同种异型之间的转运差异如何影响其抗原提呈能力，对于更好地阐明不同的人类白细胞抗原对疾病易感性、分辨率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.