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

描述(由申请人提供)：尽管存在许多可从细胞外(例如治疗性抗体)或细胞质内(例如小分子抑制剂)访问的分子靶点的治疗策略，但它们不适用于位于膜双层内的分子靶点。疏水磷脂双层对水溶性的极性治疗药物施加了一道无法穿透的屏障。殷实验室最近开发了一种计算方法，计算螺旋抗膜蛋白(CHAMP)，以合理设计识别蛋白质跨膜结构域的多肽探针，具有高亲和力和高选择性。本研究利用这一前沿技术研究EB病毒致癌潜伏膜蛋白1(LMP-1)的激活机制。EBV是一种人类肿瘤病毒，与许多恶性肿瘤和淋巴增生性综合征有关。EB病毒感染B淋巴细胞并使其永生化的能力是其对人类疾病的贡献。EBV的转化活性依赖于LMP-1的表达和活性，LMP-1是一种病毒癌蛋白，在许多EBV依赖的淋巴瘤和淋巴增生性综合征中表达。LMP-1是一种具有结构性活性的肿瘤坏死因子受体(TNFR)，其活性需要其疏水跨膜区的功能。LMP-1在信号转导方面与TNFR CD40最为相似。与CD40活性需要配体激活不同，LMP-1的S活性是结构性的，不依赖于配体。LMP-1‘S跨膜区的结构性同源齐聚和脂筏结合在下游信号的激活中起着关键作用。由于LMP-1‘S在EB病毒依赖的B细胞转化中起重要作用，LMP-1’S在EB病毒依赖的淋巴瘤和淋巴增殖性综合征中起重要作用，以及EB病毒依赖LMP-1‘S疏水跨膜区的活性，因此本建议将LMP-1作为设计多肽抑制剂的模型膜蛋白靶点。本研究旨在开发一种新的靶向LMP-1‘S跨膜区的方法，以CHAMP设计的抗肽拮抗剂为探针，研究寡聚和RAFT结合在LMP-1激活中的作用，目的是抑制下游信号转导。本研究结果将有助于深入了解膜环境中分子间的相互作用和构成/致癌受体跨膜信号转导的机制，将揭示LMP-1‘S激活信号的机制，并将适用于未来针对依赖关键跨膜蛋白的疾病的新型治疗药物的开发。1)能否开发出与LMP-1‘S TMD-1具有高亲和力和特异性的抗TMD-1多肽探针？2)鉴定的多肽在体外能与LMP-1’S TMD-1特异结合并具有亲和力吗？3)靶向TMD-1的多肽(在AIMS 1和2中鉴定)能否干扰完整细胞中的LMP-1同源寡聚、RAFT结合和构成信号？

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?