Transmembrane Regulation of Ectodomain Shedding

项目总结 胞外结构域脱落，是一种完整的膜蛋白的蛋白水解性裂解，以释放 来自宿主细胞的胞外区，影响包括生长在内的各种生物重要蛋白质 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。长的- 本项目的学期目标是阐明脱落调节的分子和结构基础。 穿过细胞膜。细胞内的蛋白质可以通过直接与细胞内蛋白质相互作用来调节脱落。 脱落蛋白底物的细胞质结构域。最典型的例子就是钙调素 钙调素(CaM)与L-选择素联合抑制L-选择素的脱落。监管机制的一个线索是 有充分记录但无法解释的观察表明，膜-近端之间的距离 在L-选择素和细胞膜中脱落的裂解位点，而不是裂解位点的序列， 是减少活动的关键。缩短距离可取消掉绒。膜-近端区域 部分L-选择素胞质结构域与CaM相互作用，但只有12个残基长，远短于 典型的CaM结合序列。钙调素与L衍生的选择素相互作用的最新研究 多肽提示L-选择素的CaM结合区也可能包括L-选择素的一部分 跨膜结构域。我们推测CaM与L-选择素的相互作用影响其跨膜 膜中的结构域，进而改变脱落的构象和/或可获得性 膜另一侧的裂解部位。L-选择素跨膜结构域的一部分可以， 在Cam关联时，划分为Cam，从而移动整个TM结构域并剥离解理 并缩短脱落裂解部位与细胞质之间的距离。 能有效抑制L-选择素的脱落。在具体目标1中，能量和结构 CaM与L-选择素水溶性多肽相互作用的基础将进一步 特色化的。在特定目标2中，CaM与L-选择素片段在仿膜中的相互作用 环境将用核磁共振和荧光光谱进行表征。该片段包含 L-选择素的裂解部位、跨膜区和胞浆区。重点将放在 检测L-选择素跨膜结构域的构象和/或位置变化 凸轮协会。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联合引起的，包括脱落裂解位点之间的距离的任何变化 和膜双层，将被表征。周密考证卡门与L的情结 选择素肽有助于阐明钙调素调节L-选择素脱落的机制。 并提供了对总体上的脱落监管机制的见解。

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.