Probing EBV-LMP-1's Transmembrane Activation Domain with Synthetic Peptide Antago

描述（由申请人提供）：尽管存在许多用于从细胞外部（例如治疗性抗体）或细胞质内（例如小分子抑制剂）可接近的分子靶标的治疗策略，但它们不适用于位于膜双层内的分子靶标。疏水磷脂双层对水溶性极性治疗剂施加了不可穿透的屏障。Yin实验室最近开发了一种计算方法，计算螺旋抗膜蛋白（CHAMP），以合理设计肽探针，以高亲和力和选择性识别蛋白质跨膜结构域。本研究利用这一尖端技术来研究EB病毒（EBV）致癌潜伏膜蛋白1（LMP-1）的激活机制。EBV是一种与多种恶性肿瘤和淋巴组织增生综合征相关的人类肿瘤病毒。EBV感染和永生化B淋巴细胞的能力是其对人类疾病的贡献的基础。EBV的转化活性取决于LMP-1的表达和活性，LMP-1是在许多EBV依赖性淋巴瘤和淋巴增生综合征中表达的病毒癌蛋白。LMP-1作为组成型活性肿瘤坏死因子受体（TNFR）发挥作用，其活性需要其疏水跨膜结构域的功能。LMP-1在其信号传导方面最类似于TNFR CD 40。与其活性需要通过配体活化的CD 40不同，LMP-1的活性是组成型的且不依赖于配体。LMP-1跨膜结构域的组成性同源寡聚化和脂筏缔合在下游信号转导的激活中起关键作用。由于LMP-1在EBV依赖性B细胞转化中的重要作用、LMP-1对EBV依赖性淋巴瘤和淋巴增生综合征的贡献以及EBV对LMP-1的疏水跨膜结构域的活性依赖性，该提议集中于LMP-1作为用于肽抑制剂设计的模型膜蛋白靶标。本研究旨在开发一种创新的方法来靶向LMP-1的跨膜结构域，使用CHAMP设计的抗肽拮抗剂作为探针来研究寡聚化和筏缔合对LMP-1活化的贡献，目的是抑制下游信号传导。本研究的结果将提供深入了解膜环境内的分子相互作用和跨膜的组成性/致癌受体信号转导的机制，将揭示LMP-1的组成性激活信号传导的机制，并将适用于未来开发新的治疗方法，靶向依赖于关键跨膜蛋白的疾病。具体地说，该提议提出了以下目的：1）能否开发对LMP-1的TMD-1具有高亲和力和特异性的抗TMD-1肽探针？2)鉴定的肽在体外是否特异性结合LMP-1的TMD-1并具有亲和力？和3）靶向TMD-1的肽（在目的1和2中鉴定的）能干扰完整细胞中的LMP-1同源寡聚化、筏缔合和组成性信号传导吗？

DESCRIPTION (provided by applicant): Although many therapeutic strategies exist for molecular targets accessible from the outside of the cell (e.g. therapeutic antibodies) or within the cytoplasm (e.g. small molecule inhibitors), they are not applicable to molecular targets that lie within the membrane bilayer. The hydrophobic phospholipid bilayer imposes an impenetrable barrier to water-soluble polar therapeutic agents. The Yin lab recently developed a computational method, Computed Helical Anti-Membrane Protein (CHAMP), to rationally design peptide probes that recognize protein transmembrane domains with high affinity and selectivity. This study utilizes this cutting edge technology to study the activation mechanism of oncogenic Latent Membrane Protein 1 (LMP-1) of Epstein-Barr virus (EBV). EBV is a human tumor virus associated with a number of malignancies and lymphoproliferative syndromes. EBV's ability to infect and immortalize B lymphocytes underlies its contribution to human disease. EBV's transforming activity depends on the expression and activity of LMP-1, the viral oncoprotein expressed in many EBV-dependent lymphomas and lymphoproliferative syndromes. LMP-1 functions as a constitutively active Tumor Necrosis Factor Receptor (TNFR) whose activity requires the function of its hydrophobic transmembrane domain. LMP-1 most resembles the TNFR CD40 in its signaling. Unlike CD40, whose activity requires activation by ligand, LMP-1's activity is constitutive and ligand-independent. Constitutive homo-oligomerization and lipid raft association, activities of LMP-1's transmembrane domain, play a key role in activation of downstream signaling. This proposal focuses on LMP-1 as a model membrane protein target for the design of peptide inhibitors because of LMP-1's essential role in EBV-dependent B cell transformation, LMP-1's contribution to EBV-dependent lymphoma and lymphoproliferative syndromes, and EBV's dependence on LMP-1's hydrophobic transmembrane domain for activity. This study aims to develop an innovative approach to target LMP-1's transmembrane domain, using CHAMP-designed anti-peptide antagonists as probes to study the contribution of oligomerization and raft association to LMP-1 activation, with the goal of inhibiting downstream signaling. Results of this research will provide insight into the molecular interactions within the membrane environment and the mechanisms underlying constitutive/oncogenic receptor signal transduction across membranes, will reveal the mechanism of LMP-1's constitutive activation of signaling, and will be applicable to the future development of novel therapeutics targeting diseases dependent on critical transmembrane proteins. Specifically, this proposal addresses the following Aims: 1) Can anti-TMD-1 peptides probes be developed that have high affinity and specificity for LMP-1's TMD-1? 2) Do identified peptides bind specifically and with affinity to LMP-1's TMD-1 in vitro? and 3) Can peptides that target TMD-1 (identified in Aims 1 and 2) interfere with LMP-1 homo-oligomerization, raft association, and constitutive signaling in intact cells?