INTERACTIONS & MECHANISMS OF FUNCTION OF THE TAP COMPLEX

与抗原加工相关的转运蛋白（TAP）至关重要 I类主要组织相容性复合体（MHC）抗原的组分 呈递途径 TAP是由两个亚基TAP 1和TAP 2组成的复合物， 其功能是将肽从细胞质转运到内质 用于I类MHC抗原呈递的内质网（ER）。 TAP 1和TAP 2都是 包括跨膜结构域和ATP结合结构域（NBD）。 的 这里提出的研究将导致更好地了解TAP 功能和深入了解免疫系统的分子机制， 系统对抗病毒和癌症。 在第一个提议中， 研究中，ATP与人类TAP亚基的相互作用将是 分析和结构变化，诱导作为ATP的结果， 结合将使用蛋白水解，圆二色性和表面 基于等离子体共振的测定。特别是，提出了实验 为了确定核苷酸结合是否改变了 我们在TAP 1和TAP 2的NBD之间观察到的复合物。 在第二 在拟议的研究中，ATP结合和 肽需要TAP 1和TAP 2亚基水解 通过TAP的易位被解决，通过生成和 预测改变ATP酶的突变体的表征 技术咨询方案一个分单位的活动。 这些研究的基础是 人类和啮齿类动物TAP 2序列中天然突变的观察 在高度保守的位点上， 对ATP酶活性和其他成员的运输功能至关重要 跨膜转运蛋白的ATP结合盒（ABC）家族（例如， 其中一个成员）。 在最后一项研究中， TAP复合物与人I类MHC分子HLA的相互作用 -B27和新发现的TAP相关蛋白，tapasin将被 考察 其他实验室的研究表明， 合成的I类MHC分子在ER中与TAP形成复合物， 这种相互作用是由tapasin桥接或稳定的。 通过 表达蛋白质TAP 1、TAP 2、tapasin 以及昆虫细胞中的I类MHC重链和轻链， TAP/tapasin/I类MHC复合物的分子性质将是 “明”，“明”，“明”。 这些交互以及I类交互都需要组件 MHC抗原呈递。最后，分析TAP与 提出的HLA-B27将提供对分子生物学的深入了解。 B27与自身免疫性疾病相关的机制 关节炎疾病。

The transporters associated with antigen processing (TAP) are crucial components of the class I major histocompatibility complex (MHC) antigen presentation pathway. TAP is a complex of two subunits, TAP1 and TAP2, that functions to transport peptides from the cytosol to the endoplasmic reticulum (ER) for class I MHC antigen presentation. TAP1 and TAP2 both comprise a transmembrane domain and an ATP binding domain (NBD). The studies proposed here will result in a better understanding of TAP function and insights into molecular mechanisms by which the immune system combats viruses and cancers. In the first of the proposed studies, the interactions of ATP with human TAP subunits will be analyzed and structural changes that are induced as a consequence of ATP binding will be probed using proteolysis, circular dichroism and surface plasmon resonance-based assays. In particular, experiments are proposed to determine whether nucleotide binding alters the stability of a complex that we observe between the NBD of TAP1 and TAP2. In the second of the proposed studies, the question of whether ATP binding and hydrolysis by both TAP1 and TAP2 subunits is required for peptide translocation by TAP is addressed, by the generation and characterization of mutants that are predicted to alter the ATPase activities of one of the TAP subunits. The basis for these studies is the observation of natural mutations in human and rodent TAP2 sequences at sites that are highly conserved and that have been shown to be crucial for ATPase activity and transport function of other members of the ATP binding cassette (ABC) family of transmembrane transporters (of which TAP is a member). In the last of proposed studies, the interactions of the TAP complex with the human class I MHC molecule HLA -B27 and the newly discovered TAP-associated protein, tapasin will be examined. Studies in other laboratories have indicated that newly synthesized class I MHC molecules form complexes with TAP in the ER, and that this interaction is bridged or stabilized by tapasin. By expressing different combinations of the proteins TAP1 TAP2, tapasin, and the class I MHC heavy and light chains in insect cells, the molecular nature of the TAP/tapasin/class I MHC complexes will be elucidated, and insights will be obtained into whether other unknown components are required for these interactions, as well as for class I MHC antigen presentation. Finally, analysis of TAP interactions with HLA-B27 that are proposed will provide insight into the molecular mechanisms that underlie the association of B27 with autoimmune arthritic diseases.