PROTEIN-PHOSPHOINOSITIDE BINDING RELATED TO EXOCYTOSIS

描述（改编自摘要）：胞吐作用是一种通用机制， 真核细胞分泌分泌物的能力。它涉及到 分泌囊泡与质膜由一个层次系列的蛋白质 相互作用和相互作用磷脂双层的重组。 聚磷酸肌醇似乎是胞吐作用的调节剂， 通过结合控制融合过程的蛋白质起作用。长期 本提案的目标是描述以下方面之间的相互作用： 多磷酸肌醇和分泌囊泡的膜蛋白 称为SCAMP，并确定这些相互作用如何有助于 胞吐作用初步研究表明，一种独特的肽对应 SCAMP家族一级结构中高度保守的片段 成员抑制大鼠腹腔肥大细胞的胞吐作用。所述肽还 与含有磷脂酰的磷脂囊泡紧密结合 肌醇-4，5-二磷酸和肌醇-1，4，5-三磷酸。进一步研究 计划确定绑定所需的结构要素， 比较相关聚磷酸肌醇之间的结合亲和力， 肌醇，然后将这些结果与抑制 肥大细胞胞吐作用。本研究的目的是：1）分析 通过核磁共振光谱法分析肽/磷酸肌醇复合物的结构，2） 使用位点特异性自旋标记确定肽如何结合膜， EPR光谱，3）设计突变体SCAMP，其将被表达和测试为 胞吐抑制剂和4）使用纯化的SCAMP重建， 含有聚磷酸肌醇的囊泡，以确认肌醇的结合 脂质与全长多肽的结合并测试结合引起的预测 一种可能反映膜融合催化作用的结构变化。

DESCRIPTION (Adapted from abstract): Exocytosis is a universal mechanism used by eukaryotic cells to export secretory products. It involves fusion of secretory vesicles with the plasma membrane by a hierarchical series of protein interactions and restructuring of the interaction phospholipid bilayers. Polyphosphoinositides appear to be regulators of exocytosis and are thought to act by binding proteins that control the fusion process. The long term objectives of this proposal are to characterize the interactions between polyphosphoinositides and integral membrane proteins of the secretory vesicles known as SCAMPs and to determine how these interactions contribute to exocytosis. Preliminary studies have shown that a unique peptide corresponding to a highly conserved segment within the primary structures of SCAMP family members inhibits exocytosis in rat peritoneal mast cells. The peptide also binds avidly to phospholipid vesicles containing phosphatidyl inositol-4,5-bisphosphate and to inositol-1,4,5-trisphosphate. Further studies are planned to identify the structural elements required for binding and to compare the binding affinities among related polyphosphoinositols and inositides and then correlate these results with potency in inhibiting exocytosis in mast cell. The aims of this study are to 1) analyze the molecular structure of the peptide/inositol phosphate complexes by NMR spectroscopy, 2) determine how the peptide binds membranes using site-specific spin labeling and EPR spectroscopy, 3) design mutant SCAMPs that will be expressed and tested as inhibitors of exocytosis and 4) use purified SCAMPs reconstituted in polyphosphoinositide-containing vesicles to confirm the binding of inositol lipid to full-length polypeptide and to test the prediction that binding causes a structural change that may reflect a role in catalysis of membrane fusion.