Molecular Genetic Analysis Of Lymphocyte Function

淋巴细胞功能的分子遗传学分析

• 批准号: 9354702
• 负责人: David Margulies
• 金额: $ 69.52万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9354702
• 关键词: Acute; Address; Affinity; Alleles; Amino Acid Sequence; Animal Model; Anti-Retroviral Agents; Antibodies; Antigens; Area; Autoantigens; Autoimmune Diseases; Autoimmune Process; Autoimmunity; Bacterial Infections; Binding; Binding Proteins; Biochemical; Biological Assay; CD8B1 gene; Cancerous; Cell surface; Communicable Diseases; Complex; Computer Simulation; Docking; ERp57; Engineering; Epitopes; Equilibrium; Fluorescence Polarization; Gastritis; Generations; Glutamic Acid; Goals; HLA-B Antigens; Histocompatibility; Hypersensitivity; Immediate hypersensitivity; Immune response; Immune system; Individual; Laboratories; Lead; Ligands; Lymphocyte Function; MHC Interaction; MHC binding peptide; Malignant Neoplasms; Measurement; Mediating; Modeling; Molecular; Molecular Biology; Molecular Chaperones; Molecular Genetics; Movement; Nature; Nuclear Magnetic Resonance; Parasitic infection; Pathway interactions; Peptide/MHC Complex; Peptides; Pharmaceutical Preparations; Play; Positioning Attribute; Predisposition; Protein Sequence Analysis; Proteins; Reaction; Regulation; Resolution; Roentgen Rays; Role; Shapes; Signal Transduction; Stress; Structure; Surface Plasmon Resonance; T cell response; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; Transgenic Animals; Transgenic Mice; Viral; Viral Antigens; Virus Diseases; Work; X-Ray Crystallography; abacavir; autoimmune gastritis; base; biophysical techniques; fly; genetic analysis; insight; interest; member; molecular dynamics; mouse model; neoplastic cell; novel; peptide B; peptide I; preference; response; simulation; tapasin; three dimensional structure; trafficking; two-dimensional

Parts (1) and (2) as listed above, deal with the MHC-I aspects of this project, and in general are directed to understand the molecular details of the loading of MHC-I molecules with self or antigenic peptides. Although hundreds of MHC-I and MHC-I-like three-dimensional structures have been determined, none of these is of a peptide-receptive (PR) form of the molecule. In previous studies, we determined the three-dimensional structure of a peptide epitope representative of a portion of the MHC-I molecule H2-Ld that is exposed only on partially unfolded, peptide receptive (PR) MHC-I molecules. This then defined one aspect of the PR form of the molecule, which was used as the input for molecular dynamics simulations of a fully hydrated model. This dynamics simulation has been examined extensively and provides a structural understanding of the way that MHC-I molecules change their shape from the metastable PR form to their stable PL form. Analysis of the two structures and of the transition from PR to PL suggests a detailed mechanism of how the MHC-I molecule works. To extend our understanding of the nature of peptide-loading, we have engineered the main chaperones involved in MHC-I loading, tapasin and Erp57. In addition, we have engineered a tapasin like molecule, known as TAPBPR, which is about 20% identical in amino acid sequence to tapasin and have undertaken studies examining the nature of its binding to the PR form of MHC-I. These studies suggest that TAPBPR interacts with a peptide-free, peptide-receptive form of MHC-I, and that this interaction is relaxed upon peptide binding. Additional studies of the TAPBPR/MHC-I interaction reveal direct interaction of antigenic peptides with the MHC-I and not the TAPBPR component of the complex. Furthermore, the interaction of peptide with the MHC-I molecule is quantitatively related to the strength of binding (the affinity) of the peptide for the MHC-I molecule. In additional studies of MHC-I/peptide interactions, we have explored the role that the anti-retroviral drug, abacavir, plays in binding to MHC-I and distorting the self-peptide repertoire bound by susceptible MHC-I alleles. In particular, we have shown, by peptide sequence analysis of the self peptides bound to the susceptible MHC-I allele, HLA-B*57:01, in the presence and absence of abacavir, that this drug can change the peptides that B*57:01 binds. This provides an explanation for the severe hypersensitivity reactions that are observed in a high proportion of HLA-B*57:01 individuals who receive the drug. We have developed transgenic mouse lines expressing various forms of HLA-B*57:01 as animal models for the effects of drugs in causing acute hypersensitivity reactions. These transgenic animals can be immunized to generate HLA-B*57:01-specific CD8-T cell responses. The third part of this project is focused on structural and functional studies of T cell receptor recognition of antigens, how this leads to T cell signaling, and how this leads to autoimmune disease. To provide a baseline for understanding antigen-specific structural changes in the TCR, we have determined the X-ray structure of a viral specific, MHC-I-restricted TCR, as well as its complex with its MHC-I/viral antigen ligand. Remarkably, although the MHC/peptide complex has a relatively rigid structure, the TCR shows great movement of its CDR3 alpha and beta loops, indicative of a fly-casting mechanism for ligand engagement. Further characterization of this fly-casting mechanism is reflected in other projects from this laboratory. Additional studies are underway to explore TCR/MHC-I interactions by novel biophysical techniques. In particular we have collaborated in studies that explore measurements of two-dimensional affinities of TCR/MHC interactions. In addition, we have collaboratively explored the use of NMR to study conformational changes in the TCR that accompany binding of a high affinity peptide/MHC complex. These studies offer new insight into possible allosteric mechanisms that contribute to T cell activation. Other approaches to understanding the TCR mediated aspects of autoimmunity include: 1) the characterization of antigenic peptides recognized by the autoimmune T cells in transgenic mouse models of autoimmune gastritis; and 2) the structural determination of MHC-II molecules bound to their antigenic peptides. We have determined the high resolution X-ray structure of IAd in complex with the Th2 peptide known as PLL, as well as the structures of two other IAd/peptide complexes in which the peptides are related to PLL, but are of higher intrinsic affinity. These structures, determined at high resolution reveal a previously unrecognized binding motif (exploiting residues 1,4,6,7, and 9 of the peptide) for IAd, particularly with respect to the preference of glutamic acid at position 9 of the peptide. This provides a framework for understanding interactions of autoimmune TCR with self MHC-II/peptide complexes. These experimental structural studies permitted the modeling of another gastritis-inducing peptide, known as PIT, bound to IAd, and provide further insight into the molecular basis of autoimmune gastritis. Similarities in this theme of the relevant anchors and the topology of the peptide bound to the MHC are consistent with other MHC-II/autoantigen complexes and suggest some common features that may be specifically relevant to autoimmune antigens.

上面列出的第（1）和（2）部分涉及本项目的MHC-I方面，并且通常旨在理解MHC-I分子负载自身或抗原肽的分子细节。虽然已经确定了数百种MHC-I和MHC-I样三维结构，但这些都不是肽受体（PR）形式的分子。在以前的研究中，我们确定了代表MHC-I分子H2-Ld的一部分的肽表位的三维结构，所述肽表位仅暴露于部分未折叠的肽受体（PR）MHC-I分子上。然后，这定义了分子的PR形式的一个方面，其被用作完全水合模型的分子动力学模拟的输入。这种动力学模拟已被广泛研究，并提供了一个结构的理解，MHC-I分子改变其形状的方式，从亚稳态PR形式到其稳定的PL形式。对这两种结构以及从PR到PL的转变的分析表明了MHC-I分子如何工作的详细机制。为了扩展我们对肽加载性质的理解，我们已经设计了参与MHC-I加载的主要分子伴侣，tapasin和Erp 57。此外，我们还设计了一种Tapasin样分子，称为TAPBPR，其氨基酸序列与Tapasin约20%相同，并进行了研究，检查其与MHC-I的PR形式结合的性质。这些研究表明，TAPBPR与无肽、肽受体形式的MHC-I相互作用，并且这种相互作用在肽结合后是松弛的。 TAPBPR/MHC-I相互作用的其他研究揭示了抗原肽与MHC-I而不是复合物的TAPBPR组分的直接相互作用。 此外，肽与MHC-I分子的相互作用与肽与MHC-I分子的结合强度（亲和力）定量相关。 在MHC-I/肽相互作用的其他研究中，我们探索了抗逆转录病毒药物阿巴卡韦在与MHC-I结合和扭曲易感MHC-I等位基因结合的自身肽库中所起的作用。特别地，我们通过在存在和不存在阿巴卡韦的情况下与易感的MHC-I等位基因HLA-B *57：01结合的自身肽的肽序列分析，表明该药物可以改变与B*57：01结合的肽。这为在接受药物的高比例HLA-B*57：01个体中观察到的重度超敏反应提供了解释。 我们开发了表达各种形式HLA-B*57：01的转基因小鼠系，作为药物引起急性超敏反应的影响的动物模型。这些转基因动物可以被免疫以产生HLA-B*57：01特异性CD 8-T细胞应答。 该项目的第三部分重点是T细胞受体识别抗原的结构和功能研究，这如何导致T细胞信号传导，以及这如何导致自身免疫性疾病。为了提供理解TCR中抗原特异性结构变化的基线，我们已经确定了病毒特异性MHC-I限制性TCR的X射线结构，以及其与其MHC-I/病毒抗原配体的复合物。值得注意的是，尽管MHC/肽复合物具有相对刚性的结构，但TCR显示其CDR 3 α和β环的巨大运动，表明配体接合的飞投机制。该实验室的其他项目反映了这种飞抛机制的进一步表征。 其他研究正在进行中，以探索TCR/MHC-I的相互作用，通过新的生物物理技术。特别是，我们合作的研究，探索测量的二维亲和力的TCR/MHC相互作用。此外，我们还合作探索了使用核磁共振来研究伴随高亲和力肽/MHC复合物结合的TCR构象变化。这些研究提供了新的见解，可能的变构机制，有助于T细胞活化。 理解TCR介导的自身免疫方面的其他方法包括：1）在自身免疫性胃炎的转基因小鼠模型中由自身免疫性T细胞识别的抗原肽的表征;和2）与其抗原肽结合的MHC-II分子的结构测定。我们已经确定了高分辨率的X-射线结构的IAd与称为PLL的Th 2肽的复合物，以及其他两个IAd/肽复合物的结构，其中肽与PLL相关，但具有更高的内在亲和力。在高分辨率下测定的这些结构揭示了先前未识别的IAd结合基序（利用肽的残基1、4、6、7和9），特别是关于肽的9位谷氨酸的偏好。这为理解自身免疫TCR与自身MHC-II/肽复合物的相互作用提供了框架。这些实验性结构研究允许另一种胃炎诱导肽（称为PIT）与IAD结合的模型，并提供了对自身免疫性胃炎分子基础的进一步了解。在这个主题的相关锚和拓扑结构的肽结合到MHC的相似性是一致的，与其他MHC-II/自身抗原复合物，并建议一些共同的特点，可能是特别相关的自身免疫抗原。