Structure and Function of Novel Neuron-specific Calcium-binding Proteins

(1)对神经元特异性钙结合蛋白家族新成员进行了分子克隆：获得了蛙视杆感受器S-调制素、蛙视锥感受器S26、大鼠海马区HLP3和HLP4的克隆。(2)部分NCAP已在大肠杆菌中获得表达系统。分离得到了Hippocalin基因组基因，发现该基因位于人类第2条染色体上。对大鼠海马素基因的转录机制进行了研究，并确定了增强子和沉默子的位置。(3)找到了S-调制素和神经钙素的靶蛋白。在S-调制素的情况下，视紫红质激酶被发现是目标蛋白。这是通过功能分析和使用与S-调制素连接的类似物进行结构分析来完成的。神经降钙素的可能靶点是S100B和/或微管蛋白b。(4)恢复蛋白的N端与肉豆蔻酸发生酰化反应。对这种脂质修饰的作用进行了研究。研究发现，肉豆蔻酰化有助于恢复素与视紫红质激酶的结合。研究还表明，脂质修饰可促进视紫红质激酶靶向视盘细胞膜的形成。S26的膜结合力弱于S-调制素。发现这种差异的部分原因是S26在靠近C-末端的氨基酸残基上缺少正电荷。(5)研究了钙调蛋白在M受体磷酸化过程中的可能作用。钙结合形式的钙调蛋白抑制了GRK2对受体的磷酸化。(6)利用嵌合体钙调素研究了钙调蛋白各结构域的作用。为了发挥钙调蛋白的作用，需要配位的钙结合到一对钙结合部位。(7)研究了钙调蛋白的可能作用。研究发现，钙调神经蛋白可能会抑制长时程增强，这可能是一种记忆机制。

(1) Molecular cloning of new family members of neuron-specific calcium-binding proteins (NCaP) were performed : we obtained clones of frog rod photoreceptor S-modulin ; frog cone photoreceptor s26 ; rat hippocampal HLP3 and HLP4.(2) Expression systems in E.Coli are now available in some of the NCaPs. They included Hippocalcim, S-modulin and s26.(3) Hippocalcin genome gene was isolated and it was found that this gene is located on the 2nd human chromosome. The transcription mechansism of rat hippocalcin gene was investigated and the sites of enhancer and silencer were identified.(3) The target proteins of S-modulin and neurocalcin were found. In the case of S-modulin, rhodopsin kinase was found to be the target protein. This was done by functional analysis and also structural analysis using a liker conjugated with S-modulin. The possible target of neurocalcin was found to be S100b and/or tublin b.(4) The N-terminus of recoverin is acylated with myristic acid. The role of this lipid modification was examined. It was found that myristoylation facilitates the binding of recoverin to rhodopsin kinase. It was also demonstrated that the lipid modification induces facilitation of the targetting of rhodopsin kinase to disk membranes. The membrane binding is weaker in s26 than S-modulin. It was found that part of this difference is due to a lack of positive charges in s26 at amino acid residues close to C-terminus.(5) Possible role of calmodulin on the phosphorylation of muscarinic acetylcholine receptor was investigated. The calcium-bound form of calmodulin inhibited the receptor phosphorylation by GRK2.(6) The role of each domain of calmodulin was investigated using chimera calmodulin. To exert calmodulin function, co-ordinated calcium binding to a pair of calcium-binding site were required.(7) Possible role of calcineulin were examined. It was found that calcineulin may inhibit long term potentiation, a possible machinery of memory.