Dissecting Cell Signaling Mediated by Protein-Protein Interactions in Membranes

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），以合理设计具有高亲和力和选择性识别蛋白质跨膜结构域（TMDs）的肽探针。尽管如此，以前的所有工作都局限于单通道tmd。在占人类蛋白质组约25%至30%的膜相关蛋白中，多跨膜蛋白的研究尤其具有挑战性，因为这些跨膜蛋白的制备、表征和分析具有很高的难度。通过提出的研究，我们的目标是开发一种普遍适用的方法来研究以前无法获得的多跨膜蛋白关联。我们选择潜伏膜蛋白1 （Latent Membrane Protein 1, LMP-1）作为模型系统来测试这项技术，因为它的多跨越TMD在信号激活中的重要作用及其临床相关性。

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 (TMDs) with high affinity and selectivity. Nonetheless, all previous work has been limited to single-pass TMDs. Among the membrane-associating proteins that account for approximately 25% to 30% of the human proteome, multi-spanning membrane proteins are particularly challenging to study due to the high degree of difficulty involved in preparing, characterizing, and analyzing these transmembrane proteins. With the proposed studies, we aim to develop a generally applicable method to study previously inaccessible multi-spanning membrane protein associations. Latent Membrane Protein 1 (LMP-1) was chosen as a model system to test this technology because of the essential role of its multi-spanning TMD in activation of signaling and its clinical relevance, particularly to lymphoid malignancies and lymphoproliferative syndromes associated to human Epstein-Barr virus (EBV). EBV's ability to infect and immortalize B lymphocytes depends on the expression and activity of LMP-1, the multi-spanning, viral oncoprotein expressed in many EBV-dependent lymphomas and lymphoproliferative syndromes. LMP-1 most resembles the Tumor Necrosis Factor Receptor (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 of LMP-1's TMD plays a key role in activation of downstream signaling. This study aims to develop an innovative approach to target LMP-1's TMD, using CHAMP-designed anti-TMD peptide antagonists as probes to study the contribution of oligomerization to LMP-1 activation, with the goal of inhibiting downstream signaling. To the CHAMP method, we will introduce a novel screening dimension as well as a next generation of algorithm. 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 multi-spanning transmembrane proteins. Specifically, this proposal addresses the following Aims: 1) Develop specific peptide probes targeting individual TMDs of LMP-1; and 2) Determine the role of TMD-mediated oligomerization in LMP-1 activation.