Transmembrane Regulation of Ectodomain Shedding

描述(申请人提供)：胞外结构域脱落，是一种完整的膜蛋白的蛋白水解性裂解，从宿主细胞释放胞外结构域，影响各种生物重要的蛋白质，包括生长因子前体、细胞因子受体、淀粉样前体蛋白和细胞黏附受体，以及蛋白多糖。因此，胞外区脱落的故障往往会导致各种疾病。这个项目的长期目标是阐明跨细胞膜脱落调节的分子和结构基础。胞内蛋白可以通过直接与脱落蛋白底物的细胞质结构域相互作用来调节脱落。最典型的例子是钙调素(CaM)与L-选择素联合抑制L-选择素的脱落。调控机制的一个线索是有充分证据但无法解释的观察，即L-选择素的膜-近端脱落切割位点与细胞膜之间的距离，而不是切割位点上的序列，是脱落活性的关键。缩短距离可取消掉绒。L-选择素胞质结构域的膜-近端区域与CaM相互作用，但它只有12个残基，比典型的CaM结合序列短得多。我们最近对CaM与L-选择素衍生肽相互作用的研究表明，L-选择素的CaM结合区也可能包括L-选择素跨膜区的一部分。我们假设，CaM与L-选择素的相互作用影响其在膜上的跨膜结构域，进而改变膜另一侧脱落裂解位点的构象和/或可及性。L-选择素跨膜结构域的一部分可在CaM结合后分割成CaM，从而使整个TM结构域和脱落的裂解部位向细胞质移动，缩短脱落的裂解部位与细胞膜的距离，从而有效地抑制L-选择素的脱落。在具体目标1中，将进一步表征CaM与L-选择素衍生的水溶性多肽相互作用的能量和结构基础。在特定目标2中，将用核磁共振和荧光光谱表征CaM与L-选择素片段在仿膜环境中的相互作用。该片段含有L-选择素的裂解位点、跨膜区和细胞质结构域。重点将是检测由CaM结合引起的L-选择素跨膜结构域的任何构象和/或位置变化。在具体目标3中，将表征由CaM结合引起的L-选择素片段中脱落裂解部位的变化，包括脱落裂解部位与膜双层之间的距离的任何变化。深入研究CaM与L-选择素多肽的复合体，有助于阐明CaM调控L-选择素脱落的机制，并有助于从整体上探讨其脱落调控机制。与公共卫生的相关性：与公共健康的相关性：胞外结构域的脱落会影响各种生物重要蛋白质。它的故障往往会导致疾病。阐明钙调蛋白调控L-选择素脱落的机制将有助于更好地理解其脱落调控。我们的发现可能会导致新的治疗策略。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Relevance to public health: Ectodomain shedding affects a variety of biologically important proteins. It malfunction often leads to diseases. Elucidating the mechanism underlying calmodulin regulation of L- selectin shedding will contribute to a better understanding of shedding regulation. Our findings may lead to novel therapeutic strategies.