Transmembrane Regulation of Ectodomain Shedding

描述（由申请人提供）：胞外域脱落，即完整膜蛋白的蛋白水解裂解，从宿主细胞释放胞外结构域，影响多种生物学上重要的蛋白质，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聚糖。因此，胞外域脱落功能障碍常常导致各种疾病。该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。细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蛋白 (CaM) 与 L-选择素结合以抑制 L-选择素的脱落。调节机制的一条线索是有据可查但无法解释的观察结果，即 L-选择素中近膜脱落裂解位点与细胞膜之间的距离，而不是裂解位点的序列，对脱落活性至关重要。缩短距离可以消除脱落。 L-选择素胞质结构域的近膜区域与 CaM 相互作用，但它只有 12 个残基长，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-选择素中的 CaM 结合区也可能包括 L-选择素跨膜结构域的一部分。我们假设 CaM 与 L-选择素的相互作用影响其在膜中的跨膜结构域，这反过来又改变了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结合后分裂成CaM，从而将整个TM结构域和脱落裂解位点移向细胞质，缩短脱落裂解位点与膜之间的距离，有效抑制L-选择素脱落。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肽相互作用的能量和结构基础。在具体目标 2 中，将通过 NMR 和荧光光谱来表征模拟膜环境中 CaM 与 L-选择素片段的相互作用。该片段包含脱落切割位点、L-选择素的跨膜结构域和细胞质结构域。重点是检测 CaM 关联诱导的 L-选择素跨膜结构域中的任何构象和/或位置变化。在具体目标 3 中，将表征由 CaM 关联诱导的 L-选择素片段中脱落裂解位点的变化，包括脱落裂解位点与膜双层之间距离的任何变化。仔细研究 CaM 与 L-选择素肽的复合物将有助于阐明 CaM 调节 L-选择素脱落的机制，并为一般脱落调节机制提供见解。公共健康相关性：与公共健康的相关性：胞外域脱落会影响多种具有生物学重要意义的蛋白质。它的故障常常会导致疾病。阐明钙调蛋白调节 L-选择素脱落的机制将有助于更好地理解脱落调节。我们的发现可能会带来新的治疗策略。

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.