Transmembrane Regulation of Ectodomain Shedding

胞外域脱落的跨膜调控

基本信息

批准号：
8207999
负责人：
Renhao Li
金额：
$ 30.07万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-01-01 至 2013-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8207999
关键词：
Address Affect Amyloid beta-Protein Precursor Binding C-terminal Calcium Calmodulin Cell Adhesion Cell membrane Cell surface Cells Cleaved cell Complex Cytokine Receptors Cytoplasm Cytoplasmic Tail Disease Dissociation Environment Extracellular Domain Fluorescence Fluorescence Spectroscopy Growth Factor Inflammatory Integral Membrane Protein L-Selectin Lead Leukocytes Lipids Mediating Membrane Membrane Proteins Methods Molecular Molecular Conformation Mutagenesis Mutation NMR Spectroscopy Peptides Positioning Attribute Process Proteins Proteoglycan Public Health Regulation Relative (related person)Role Sequence Analysis Side Signal Transduction Site Solutions Surface Testing Thermodynamics Transmembrane Domain Vesicle Water adhesion receptor aqueous base extracellular inhibitor/antagonist insight novel therapeutics polypeptide C stoichiometry

项目摘要

PROJECT SUMMARY Ectodomain shedding, the proteolytic cleavage of an integral membrane protein to release the extracellular domain from the host cell, affects a variety of biologically important proteins including growth factor precursors, cytokine receptors, amyloid precursor proteins and cell adhesion receptors, and proteoglycans. Therefore malfunction of ectodomain shedding often leads to various diseases. The long- term objective of this project is to elucidate the molecular and structural basis for shedding regulation across the cell membrane. Intracellular proteins can regulate shedding by interacting directly with the cytoplasmic domain of the shedding protein substrate. The best characterized example is the calmodulin (CaM) association with L-selectin to inhibit shedding of L-selectin. One clue to the regulation mechanism is the well documented but unexplained observation that the distance between the membrane-proximal shedding cleavage site in L-selectin and the cell membrane, rather than the sequence at the cleavage site, is critical to shedding activity. Shortening the distance abolishes shedding. The membrane-proximal region of the L-selectin cytoplasmic domain interacts with CaM, but it is only 12 residues long, much shorter than a typical CaM-binding sequence. Our recent study on the interaction of CaM with L-selectin-derived peptides suggested that the CaM-binding region in L-selectin may also include a portion of the L-selectin transmembrane domain. We hypothesize that CaM interaction with L-selectin affects its transmembrane domain in the membrane, which in turn changes the conformation and/or accessibility of the shedding cleavage site on the other side of the membrane. A portion of the L-selectin transmembrane domain may, upon CaM association, partition into CaM, thereby moving the entire TM domain and shedding cleavage site toward the cytoplasm and shortening the distance between the shedding cleavage site and the membrane, and effectively inhibit shedding of L-selectin. In Specific Aim 1, the energetic and structural basis for the interaction of CaM with water-soluble peptides derived from L-selectin will be further characterized. In Specific Aim 2, the interaction of CaM with a L-selectin fragment in membrane-mimicking environments will be characterized with NMR and fluorescence spectroscopy. The fragment contains the shedding cleavage site, the transmembrane and cytoplasmic domains of L-selectin. The focus will be to detect any conformational and/or positional changes in the L-selectin transmembrane domain induced by CaM association. In Specific Aim 3, changes at the shedding cleavage site in the L-selectin fragment induced by CaM association, including any changes in the distance between the shedding cleavage site and the membrane bilayer, will be characterized. A careful study of the complexes of CaM with the L- selectin peptides will help to elucidate the mechanism underlying CaM regulation of L-selectin shedding, and provide insights into shedding regulation mechanisms in general.

项目概要胞外域脱落，整合膜蛋白的蛋白水解裂解释放来自宿主细胞的胞外结构域，影响多种生物学重要的蛋白质，包括生长因子前体、细胞因子受体、淀粉样前体蛋白和细胞粘附受体，以及蛋白多糖。因此，胞外域脱落功能障碍常常导致各种疾病。长- 该项目的长期目标是阐明脱落调节的分子和结构基础穿过细胞膜。细胞内蛋白质可以通过直接与细胞相互作用来调节脱落脱落蛋白底物的细胞质结构域。最具特征的例子是钙调蛋白 (CaM) 与 L-选择素结合抑制 L-选择素脱落。调控机制的线索之一是有据可查但无法解释的观察结果是，膜近端之间的距离脱落L-选择素和细胞膜中的切割位点，而不是切割位点的序列，对于脱落活动至关重要。缩短距离可以消除脱落。近膜区 L-选择素胞质结构域的一部分与CaM相互作用，但只有12个残基长，比典型的 CaM 结合序列。我们最近关于 CaM 与 L-选择素衍生的相互作用的研究肽表明 L-选择素中的 CaM 结合区也可能包括 L-选择素的一部分跨膜结构域。我们假设 CaM 与 L-选择素的相互作用影响其跨膜膜中的结构域，这反过来又改变了脱落的构象和/或可及性切割位点位于膜的另一侧。 L-选择素跨膜结构域的一部分可以， CaM 结合后，分裂成 CaM，从而移动整个 TM 结构域并摆脱裂解位点朝向细胞质并缩短脱落裂解位点和膜，并有效抑制L-选择素的脱落。在具体目标 1 中，能量和结构 CaM 与 L-选择素衍生的水溶性肽相互作用的基础将进一步得到证实特点。在具体目标 2 中，CaM 与 L-选择素片段在膜模拟中的相互作用环境将通过核磁共振和荧光光谱进行表征。该片段包含脱落裂解位点、L-选择素的跨膜结构域和细胞质结构域。重点将是检测 L-选择素跨膜结构域中任何构象和/或位置的变化 CaM 协会。在特定目标 3 中，L-选择素片段中脱落切割位点的变化由 CaM 关联诱导，包括脱落裂解位点之间距离的任何变化和膜双层，将被表征。仔细研究 CaM 与 L- 的复合物选择素肽将有助于阐明 CaM 调节 L-选择素脱落的机制，并提供对一般脱落调节机制的见解。