INTERACTIONS & MECHANISMS OF FUNCTION OF THE TAP COMPLEX

与抗原处理相关的转运蛋白(TAP)是至关重要的 I类主要组织相容性复合体(MHC)抗原的组成 呈现路径。TAP是两个亚单位TAP1和TAP2的复合体， 它的功能是将多肽从胞浆输送到内质 网状结构(ER)，用于呈递I类MHC抗原。TAP1和TAP2都是 包括跨膜结构域和ATP结合结构域(NBD)。这个 这里提出的研究将导致对TAP更好的理解 功能和对免疫的分子机制的洞察 该系统可以对抗病毒和癌症。在建议的第一条中， 研究表明，ATP与人类TAP亚基的相互作用将是 由ATP引起的分析和结构变化 结合将通过蛋白水解法、圆二色谱和表面法检测。 基于等离子体共振的分析。特别是，提出了实验方案。 为了确定核苷酸结合是否改变了一个 我们在TAP1和TAP2的NBD之间观察到的复杂性。在第二个 在拟议的研究中，ATP结合和 多肽需要TAP1和TAP2亚基的水解 通过抽头进行的易位是由世代和 预测会改变ATPase的突变体的特征 其中一个TAP亚基的活动。这些研究的基础是 人和啮齿动物TAP2序列自然突变的观察 在高度保守和已被证明是 对ATPase活性和其他成员的运输功能至关重要 三磷酸腺苷结合盒(ABC)家族的跨膜转运蛋白 哪个TAP是成员)。在最后一项拟议的研究中， TAP复合体与人类第I类MHC分子的相互作用 -B27和新发现的TAP相关蛋白Tapasin将是 检查过了。其他实验室的研究表明，新的 合成的I类MHC分子与内质网中的TAP形成络合物，并且 这种相互作用是由Tapasin桥接或稳定的。通过 表达TAP1、TAP2、TAPIN、 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 得到澄清，并将深入了解是否有其他未知 组件对于这些交互以及类I都是必需的 MHC抗原呈递。最后，分析TAP与 提出的人类白细胞抗原B27将提供对分子的洞察 B27与自身免疫相关的机制 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.