Dissecting Cell Signaling Mediated by Protein-Protein Interactions in Membranes

解析膜中蛋白质-蛋白质相互作用介导的细胞信号传导

• 批准号: 8797241
• 负责人: Hang Hubert Yin
• 金额: $ 6.71万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8797241
• 关键词: Accounting; Address; Affect; Affinity; Algorithms; Antibodies; Artificial Membranes; B Cell Proliferation; B-Lymphocytes; Binding; Biological Assay; Biological Models; Biophysics; Cell Survival; Cells; Cellular Membrane; Chemistry; Computing Methodologies; Cytoplasm; Dependence; Detergents; Development; Dimensions; Disease; Drug Targeting; Engineering; Environment; Extracellular Domain; Family member; Fluorescence Resonance Energy Transfer; Future; Generations; Goals; Herpesviridae; Homo; Human; Human Herpesvirus 4; Individual; Integral Membrane Protein; Lead; Length; Libraries; Ligands; Lymphoma; Lymphoproliferative Disorders; Malignant Neoplasms; Malignant lymphoid neoplasm; Measures; Mediating; Membrane; Membrane Proteins; Memory B-Lymphocyte; Methods; Micelles; Modeling; Molecular Probes; Molecular Target; Monitor; Oncogene Proteins; Oncogenic; Oncogenic Viruses; Peptides; Phospholipids; Play; Prevention; Process; Proteins; Proteome; Proteomics; Receptor Signaling; Reporter; Research; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Structure; Syndrome; System; TNFRSF5 gene; Technology; Tertiary Protein Structure; Testing; Therapeutic; Therapeutic Agents; Therapeutic antibodies; Transmembrane Domain; Tumor Necrosis Factor Receptor; Vesicle; Viral; Virus Activation; Water; Work; Yin; assay development; clinically relevant; design; human disease; improved; inhibitor/antagonist; innovation; insight; molecular recognition; new therapeutic target; next generation; novel; novel therapeutic intervention; oncology; prevent; protein aminoacid sequence; protein protein interaction; public health relevance; screening; small molecule; tool

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.

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（TMD）。尽管如此，所有以前的工作一直局限于单通TMD。在占人类蛋白质组约25%至30%的膜相关蛋白中，多跨膜蛋白由于涉及制备、表征和分析这些跨膜蛋白的高度困难而特别具有挑战性。与拟议的研究，我们的目标是开发一个普遍适用的方法来研究以前无法访问的多跨膜蛋白协会。选择潜伏膜蛋白1（LMP-1）作为模型系统来测试该技术，因为其多跨TMD在信号传导激活中的重要作用及其临床相关性， 特别是与人EB病毒（EBV）相关的淋巴恶性肿瘤和淋巴增生综合征。EBV感染和永生化B淋巴细胞的能力取决于LMP-1的表达和活性，LMP-1是在许多EBV依赖性淋巴瘤和淋巴组织增生综合征中表达的多跨越病毒癌蛋白。LMP-1在其信号传导方面最类似于肿瘤坏死因子受体（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的TMD的组成性同源寡聚化在下游信号传导的激活中起关键作用。本研究旨在开发一种创新的方法来靶向LMP-1的TMD，使用CHAMP设计的抗TMD肽拮抗剂作为探针来研究寡聚化对LMP-1激活的贡献，目的是抑制下游信号传导。对于CHAMP方法，我们将引入一个新的筛选维度以及下一代算法。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，靶向依赖于关键多跨膜蛋白的疾病。具体而言，该提议解决了以下目的：1）开发靶向LMP-1的各个TMD的特异性肽探针;和2）确定TMD介导的寡聚化在LMP-1活化中的作用。