Interactions and mechanisms of function of the TAP complex

TAP 复合体的相互作用和功能机制

• 批准号: 8006401
• 负责人: MALINI RAGHAVAN
• 金额: $ 37.85万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-15 至 2013-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8006401
• 关键词: ATP Hydrolysis; ATP-Binding Cassette Transporters; Acquired Immunodeficiency Syndrome; Affect; African American; Alleles; American; Antibiotic Resistance; Antigen Presentation; Antigens; Autoimmunity; Binding; Binding Sites; Calnexin; Cell surface; Cells; Cellular Membrane; Characteristics; Complex; Cytosol; Cytotoxic T-Lymphocytes; Data; Dependence; Development; Disease; Disease Progression; Disease susceptibility; Dissociation; ERp57; Endoplasmic Reticulum; Ensure; European; Family; Frequencies; Future; Goals; HLA A*0201 antigen; HLA Antigens; HLA-B Antigens; Health; Human; Hydrolysis; Immune response; Immunologic Surveillance; Infection; Integral Membrane Protein; Investigation; Kinetics; Lead; Lectin; Major Histocompatibility Complex; Malignant Neoplasms; Membrane Potentials; Modeling; Molecular Chaperones; Molecular Conformation; Molecular Machines; Natural Killer Cells; Nature; Nucleotides; Occupations; Oxidoreductase; Pathway interactions; Pattern; Peptide Transport; Peptides; Pharmaceutical Preparations; Phenotype; Population; Property; Protein Disulfide Isomerase; Proteins; Proteolysis; Pump; Recruitment Activity; Refractory; Research; Resolution; Role; Route; Site; Substrate Interaction; T-Cell Receptor; T-Lymphocyte; T-Lymphocyte Epitopes; T-Lymphocyte and Natural Killer Cell; Testing; Transplantation; Variant; Viral; Work; antigenic peptide transporter; base; calreticulin; design; immunogenic; inhibitor/antagonist; insight; mutant; peptide I; protein degradation; protein function; response; reticulum cell; tapasin; trafficking; tumor

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.

描述（由申请人提供）：主要组织相容性复合物（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类分子被募集到与与抗原加工（TAP），小吃，ERP57，蛋白质二硫化物异构酶，钙网络蛋白和钙网蛋白相关的转运蛋白相互作用中。这种辅助蛋白的复合物可以视为一种分子机器，它的工作是（i）将蛋白质降解的肽产物泵入MHC I类组装（II）招募未组装的MHC I（iii）的MHC I（iii）的MHC I型肽组合和（IV）的MOREC型MOREC的释放，以确保MHC的释放量很大，该型号均在内，该机构均置于最佳的MHC释放，以确保MHC的释放量很高，因此该机构均置入了最佳的MHC释放。在过去的11年中，这一直是我们研究的重点。根据我们以前与Tap Transporter的工作，我们能够提出一个详细的模型，以了解ATP结合和水解对夫妇如何与肽结合和运输。在拟议的研究中，我们将检查TAP底物对通过TAP的核苷酸结合和交换的影响，以及核苷酸结合结构域（NBD）之间的相互作用。还将检查TAP的肽结合位点的模型。这些研究将可以更好地理解如何操纵TAP以增强或抑制免疫反应，还可以更好地预测免疫主导性细胞毒性T淋巴细胞（CTL）表位。基于对小吃缺陷型细胞中各种MHC I类同种型的组装特性的分析，我们的假设是，木薯蛋白对于具有缓慢的内在肽载荷动力学的MHC I类同种型的肽负载至关重要。在不同条件下，将比较帕替辛和独立MHC I类同型的肽结合特性。我们的数据表明，木薯素负责将钙网蛋白和ERP57募集到肽载体中。此外，西班牙蛋白酶ERP57复合物的不同构象状态在增强MHC I类分子的肽负荷方面具有不同的活性。我们试图更好地理解差异的本质。我们还寻求了解载质蛋白在木薯蛋白辅助的MHC I类组装中的作用。尽管所有MHC I类分子似乎都遵循ER内的相同组装途径，但与HLA-B同种型的内在组装和ER出口的内在速率截然不同。在此处提出的研究中，我们试图将高频HLA-B等位基因分类为迅速或缓慢的运输，并检查抗原表现和疾病进展时快速或缓慢运输的功能后果。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，这些研究将可以更好地了解MHC I类组装途径的不同步骤，并有助于制定更有效的策略，以增强感染和癌症中的CTL反应。 公共卫生相关性：对TAP的底物互动站点的理解，以及潜在的TAP（不活动构象）的静止状态构象，对于未来的TAP抑制剂设计非常重要，这些设计可能在移植和自身免疫性的情况下使用，以及在ABC Transporters抑制剂抑制剂以克服抗毒药的抑制剂的设计中，以克服抗毒药/抗抗近比的抗近比伯伯物的抑制剂。更好地理解拿花功能的机制可能会导致新的策略，以增强特定的免疫原性肽在感染和癌症中的MHC I类分子中的组装。最后，了解HLA-B同种型之间的运输差异如何影响其抗原表现能力对于更好地阐明不同HLA抗原对疾病易感性，分辨率和进展的影响至关重要。