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病毒感染引起许多生理过程的改变，其病因学知之甚少。最初的步骤是合胞体的形成，目前被认为涉及HIV gp 120和gp 41，CD 4和趋化因子受体。后来的发展包括表达CD 4的T细胞和表达免疫球蛋白的B细胞的耗竭，以及细胞因子调节的变化。理解这些过程和制定与这些变化相关的治疗策略的关键是确定对变化中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用于表位测定的方法（保护测定和表面修饰反应结合质谱法）来探测与HIV感染相关的受体-配体对，包括：a）CD 4和gp 120; B）gp 120、CD 4和趋化因子受体CXCR 4之间的复合物。gp 120、CD 4和趋化因子受体之间的三元复合物现在被认为是HIV细胞感染中所涉及的关键相互作用。最近，已经报道了突变gp 120、突变CD 4和抗体的抗原结合片段之间的1：1：1复合物的晶体结构。然而，本研究中使用的gp 120不含可变环，也不完全糖基化。鉴于晶体结构中使用的gp 120的高度突变结构，关于溶液中全长、完全糖基化的gp 120中的复杂化学计量和相互作用位点的信息仍然不确定。甚至更重要的是，用于晶体结构测定的gp 120构建体中不存在的gp 120的V-3环（及其相关聚糖）与趋化因子受体和CD 4的结合有关。我们的方法来探测的gp 120/CD 4相互作用位点使用一种新的策略，基于使用一种新的可切割的荧光交联剂的蛋白质相互作用位点的特异性标记。非共价复合物在非变性条件下形成，然后与交联剂偶联。交联复合物已被分离并裂解成组分蛋白质，现在携带荧光标记。在一项类似的研究中，HIV-整合酶和两种抑制剂之间的相互作用位点正在与NCI小组合作，通过光亲和标记和MS肽图谱进行研究。- 艾滋病，受体-配体相互作用，质谱，SAED交联