CHARACTERIZATION OF RECEPTOR LIGAND INTERACTIONS RELEVANT TO HIV INFECTION

与 HIV 感染相关的受体配体相互作用的表征

• 批准号: 6290025
• 负责人: Kenneth Tomer
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 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/6290025
• 关键词: B lymphocyte; CD4 molecule; CHO cells; HIV envelope protein gp120; HIV infections; MHC class II antigen; glycosylation; helper T lymphocyte; immunoglobulins; intermolecular interaction; ligands; monoclonal antibody; proteolysis; receptor; recombinant virus; tumor necrosis factor alpha

Summary of WorkInfection by the HIV virus causes a change in a number of physiological processes, the etiologies of which are poorly understood. The initial step is syncitium formation, currently accepted as involving HIV gp120 and gp41, CD4 and a chemokine receptor. Later developments include the depletion of T cells expressing CD4 and B cells expressing immunoglobulins, and changes in the regulation of cytokines. Crucial to an understanding of these processes and to developing therapeutic strategies related to these changes is the determination of the structural motifs critical to the physiological processes involved in the changes. We are employing the methodologies we have been using for epitope determination (protection assays and surface modification reactions combined with mass spectrometry) to probe receptor-ligand pairs relevant to HIV infection including: a) CD4 and gp120 and; b) the complex between gp120, CD4 and chemokine receptor CXCR4. The ternary complex between gp120, CD4 and a chemokine receptor is now accepted as the crucial interaction involved in cellular infection by the HIV. Recently, the crystal structure of a 1:1:1 complex between mutant gp120, mutant CD4 and an antigen-binding fragment of an antibody has been reported. The gp120 used in this study, however, did not contain the variable loops nor was it fully glycosylated. In view of the highly mutated structure of the gp120 used in the crystal structure, information about complex stoichiometry and sites of interaction in the full length, fully glycosylated gp120 in solution is still uncertain. Even more importantly, the V-3 loop (and its associated glycans) of gp120, which were not present in the gp120 construct used for the crystal structure determination, has been implicated in binding with chemokine receptors and CD4. Our approach to probing the gp120/CD4 interaction site uses a new strategy based on the specific labeling of the protein interaction site using a novel cleavable fluorescent crosslinker. The non-covalent complex is formed under non-denaturing conditions and then coupled with the crosslinker. The crosslinked complex has been isolated and cleaved into the component proteins, now carrying fluorescent tags. Characterization of the residues modified by the crosslinker is currently in progress.In a similar study, the sites of interaction between HIV-integrase and two inhibitors is being studied by photoaffinity labeling and MS peptide mapping in collaboration with the NCI group. - HIV, AIDS, Receptor- ligand interactions, mass spectrometry, SAED crosslinking

HIV病毒的工作感染摘要导致许多生理过程发生了变化，其病因的理解很少。最初的步骤是副形成，目前被接受为HIV GP120和GP41，CD4和趋化因子受体。后来的发展包括表达CD4和B细胞表达免疫球蛋白的T细胞的耗竭以及细胞因子调节的变化。对于对这些过程的理解和制定与这些变化相关的治疗策略至关重要的是确定对变化所涉及的生理过程至关重要的结构基序。我们正在使用我们一直使用的方法论来确定表位（保护分析和表面修饰反应与质谱法结合）来探测与HIV感染相关的受体配体，包括：a）CD4和GP120和； b）GP120，CD4和趋化因子受体CXCR4之间的复合物。 GP120，CD4和趋化因子受体之间的三元复合物现在被接受为与HIV有关细胞感染的关键相互作用。最近，已经报道了突变体GP120，突变体CD4和抗体的抗原结合片段之间的1：1：1的晶体结构。但是，本研究中使用的GP120不包含可变环，也不包含完全糖基化的循环。鉴于在晶体结构中使用的GP120的高度突变结构，有关复杂化学计量的信息以及全长的相互作用位点，溶液中完全糖基化的GP120仍不确定。更重要的是，用于晶体结构测定的GP120构建体中不存在的GP120的V-3环（及其相关的聚糖）与趋化因子受体和CD4的结合有关。我们探测GP120/CD4相互作用位点的方法采用了一种新策略，基于使用新型的可裂解荧光交联链链接蛋白相互作用位点的特定标记。非共价络合物在非判断性条件下形成，然后与交联链链接。交联的复合物已被分离并裂解到组件蛋白中，现在携带荧光标签。当前正在进行的交联链链接所修饰的残基的表征。在一项类似的研究中，通过与NCI组合作，正在研究HIV - 积极酶和两个抑制剂之间相互作用的位点。 - 艾滋病毒，艾滋病，受体相互作用，质谱法，交联成