Dissecting Cell Signaling Mediated by Protein-Protein Interactions in Membranes

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(TMD)的多肽探针，具有高亲和力和高选择性。尽管如此，以前的所有工作都局限于单程TMD。在约占人类蛋白质组25%~30%的膜结合蛋白中，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、鉴定和分析涉及到很高的难度。通过拟议的研究，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。选择潜伏膜蛋白1(LMP-1)作为模型系统来测试这项技术，是因为它的多跨TMD在信号激活中的关键作用及其临床意义， 尤其涉及与人类EB病毒(EBV)相关的淋巴样恶性肿瘤和淋巴增殖性综合征。EB病毒感染和永生化B淋巴细胞的能力依赖于LMP-1的表达和活性，LMP-1是一种多跨病毒癌蛋白，在许多EBV依赖的淋巴瘤和淋巴增殖性综合征中表达。LMP-1在信号转导方面与肿瘤坏死因子受体(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 TMD的结构性同源齐聚在下游信号的激活中起着关键作用。本研究旨在开发一种新的靶向LMP-1‘S TMD的方法，以CHAMP设计的抗TMD多肽拮抗剂为探针，研究寡聚对LMP-1激活的贡献，目的是抑制下游信号转导。对于CHAMP方法，我们将引入一种新的筛选维度以及下一代算法。本研究结果将为深入了解膜环境中的分子相互作用和构成/致癌受体跨膜信号转导的机制提供依据，将揭示LMP-1的S信号的组成性激活机制，并将适用于未来针对依赖关键多跨膜蛋白的疾病的新疗法的开发。具体地说，该建议旨在解决以下目标：1)开发针对LMP-1单个TMD的特异性多肽探针；以及2)确定TMD介导的寡聚在LMP-1激活中的作用。

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.