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

描述（由申请人提供）：尽管许多治疗策略存在于可从细胞外（如治疗性抗体）或细胞质内（如小分子抑制剂）获得的分子靶点，但它们不适用于位于膜双分子层内的分子靶点。疏水磷脂双分子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（Computed Helical Anti-Membrane Protein， CHAMP），以合理设计肽探针，识别具有高亲和力和选择性的蛋白质跨膜结构域。本研究利用这一前沿技术研究eb病毒（EBV）致癌潜伏膜蛋白1 （LMP-1）的激活机制。EBV是一种与许多恶性肿瘤和淋巴增生性综合征相关的人类肿瘤病毒。EBV感染和使B淋巴细胞永生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是许多EBV依赖性淋巴瘤和淋巴增生性综合征中表达的病毒癌蛋白。LMP-1作为一种组成活性的肿瘤坏死因子受体（TNFR），其活性需要其疏水跨膜结构域的功能。LMP-1在信号传导方面与TNFR CD40最相似。与CD40不同，CD40的活性需要配体的激活，LMP-1的活性是组成型的，与配体无关。LMP-1跨膜结构域的活性在下游信号的激活中起关键作用。由于LMP-1在EBV依赖性B细胞转化中起重要作用，LMP-1在EBV依赖性淋巴瘤和淋巴增生性综合征中起重要作用，以及EBV依赖LMP-1的疏水跨膜结构域的活性，本提案将重点关注LMP-1作为肽抑制剂设计的模型膜蛋白靶点。本研究旨在开发一种针对LMP-1跨膜结构域的创新方法，使用champ设计的抗肽拮抗剂作为探针，研究寡聚化和筏关联对LMP-1激活的贡献，目的是抑制下游信号传导。这项研究的结果将为膜环境中的分子相互作用和跨膜组成/致癌受体信号转导的机制提供深入的见解，将揭示LMP-1的组成激活信号的机制，并将适用于未来开发针对依赖关键跨膜蛋白的疾病的新疗法。具体而言，本提案旨在解决以下目标：1)能否开发出对LMP-1的TMD-1具有高亲和力和特异性的抗TMD-1肽探针？2)鉴定出的肽是否能在体外特异性和亲和力地结合LMP-1的TMD-1 ？3)靶向TMD-1的肽（在Aims 1和2中鉴定）是否会干扰完整细胞中LMP-1的同质寡聚、raft association和组成信号？

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?