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: 7881378
• 负责人: MALINI RAGHAVAN
• 金额: $ 2.29万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-14 至 2011-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7881378
• 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; 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; public health relevance; 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 类分子被招募来与抗原加工 (TAP)、塔帕辛、ERp57、蛋白质二硫键异构酶、钙联蛋白和钙网蛋白相关的转运蛋白相互作用。这种辅助蛋白复合物可以被认为是一个分子机器，其作用是（i）将蛋白质降解的肽产物泵入 MHC I 类组装区域（ii）招募未组装的 MHC I 类（iii）促进 MHC I 类- (iv) 确保最佳加载的 MHC I 类的调节释放。对于这种复杂的分子机器的工作原理，还有很多需要了解的地方，这一直是我们过去 11 年研究的重点。基于我们之前对 TAP 转运蛋白的研究，我们能够提出一个关于 ATP 结合和水解如何与肽结合和转运耦合的详细模型。在拟议的研究中，我们将检查 TAP 底物对 TAP 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。还将检查 TAP 肽结合位点的模型。这些研究将有助于更好地了解如何操纵 TAP 来增强或抑制免疫反应，并且还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 Tapasin 缺陷细胞中各种 MHC I 类同种异型组装特征的分析，我们假设 Tapasin 对于具有缓慢内在肽加载动力学的 MHC I 类同种异型的肽加载至关重要。将在不同条件下比较 Tapasin 依赖性和独立 MHC I 类同种异型的肽结合特性。我们的数据表明，tapasin 负责将钙网蛋白和 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本质。我们还试图了解 Careticulin 在 Tapasin 辅助的 MHC I 类组装中的作用。尽管所有 MHC I 类分子在 ER 内似乎都遵循相同的组装路线，但密切相关的 HLA-B 同种异型在其内在组装速率和 ER 退出方面存在显着差异。在此提出的研究中，我们试图将高频 HLA-B 等位基因分类为快速或慢速运输，并检查快速或慢速运输对抗原呈递和疾病进展的功能影响。我们假设贩运表型会影响 CTL 反应和 NK 细胞反应，这一点将得到进一步检验。总之，这些研究将有助于更好地了解 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 反应。 公共健康相关性：了解 TAP 的底物相互作用位点以及 TAP 的潜在静息态构象（非活性构象）对于 TAP 抑制剂的未来设计非常重要，这些抑制剂可用于移植和自身免疫环境，此外还可以用于设计其他 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 功能机制可能会带来新的策略，以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、消退和进展的影响非常重要。