BIOSYNTHESIS, PROCESSING & SECRETION OF NEUROPEPTIDES & PITUITARY PEPTIDE HORMONE

靶向阿片黑素皮质素原(POMC， 促肾上腺皮质激素原/内啡肽)对调节的分泌途径 已确认身份。截短型POMC/CAT载体导入AtT-20细胞的研究 表明POMC的N-端26个氨基酸形成了一个 由两个二硫键稳定的发夹环路就足够了 将POMC靶向调节分泌途径所必需的。 POMC突变体具有完整的24个氨基酸发夹环或同时具有 二硫键缺失，不能靶向分泌颗粒。 然而，一个突变体被截断为11个氨基酸，带有一个二硫键 形成一个迷你发夹环的方法是正确的。这些发现 指示分选信号由特定构象编码。 POMC加工过程中的垂体分泌囊泡酶 ，并对其进行了研究。钙离子激活牛源天冬氨酸蛋白酶的研究 中叶分泌颗粒，POMC转换酶(PCE；EC 3.4.23.17)在成对的碱性残基上裂解POMC 裂解鳗鱼前生长抑素II的单基残留物 (APSSII)产生生长抑素28。一种酵母天冬氨酸蛋白酶(YAP3) 已经被提纯，并被证明具有类似的抑制物轮廓，大小 POMC和单碱基配对碱基的特异性 APSSII残留物为PCE。一种抗YAP3的抗体与 在Western印迹和放射免疫分析中检测PCE。免疫细胞化学 用这种抗体显示中间叶的细胞染色。 和发现PCE的前叶。PCE似乎是 在结构和催化活性上与YAP3同源。二 激素原加工酶候选者，PC1和PC2属于 枯草杆菌毒素相关的丝氨酸蛋白酶家族，被证明处理 POMC，炭疽保护性抗原(PA)和前TRH在 配对的碱性残基在体外。使用突变的PA底物 四元裂解位点揭示了次级和次级的重要性 三级结构决定了加工的特殊性。PC1和 PC2mRNA与TRH免疫阳性神经元共定位 原位杂交和免疫细胞化学，支持这些作用 体内加工促TRH原的酶。

The signal involved in the targeting of pro-opiomelanocortin (POMC, pro-ACTH/ endorphin) to the regulated secretory pathway was identified. Truncated POMC/CAT constructs transfected into AtT-20 cells indicated that the N-terminal 26 amino acids of POMC which form a hairpin loop stabilized by two disulfide bridges are sufficient and necessary for targeting POMC to the regulated secretory pathway. POMC mutants with the entire 24 amino acid hairpin loop or both the disulfide bridges deleted, failed to be targeted to secretory granules. However, a mutant truncated to 11 amino acids with one disulfide bridge to form a mini-hairpin loop was correctly sorted. These findings indicate that the sorting signal is encoded by a specific conformation. Pituitary secretory vesicle enzymes involved in the processing of POMC were studied. The Ca++ activated aspartic protease from bovine intermediate lobe secretory granules, POMC converting enzyme (PCE; EC 3.4.23.17) which cleaves POMC at paired basic residues has been shown to cleave at a monobasic residue of anglerfish pro-somatostatin II (aPSSII) to generate somatostatin 28. A yeast aspartic protease (YAP3) has been purified and shown to have a similar inhibitor profile, size and specificity for the paired basic residues of POMC and the monobasic residue of aPSSII as PCE. An antibody against YAP3 cross-reacted with PCE on Western blot and in a radioimmunoassay. Immunocytochemistry using this antibody showed staining in cells of the intermediate lobe and anterior lobe where PCE has been found. PCE appears to be homologous to YAP3 structurally and in catalytic activity. Two prohormone processing enzyme candidates, PC1 and PC2 belonging to the subtilisin-related family of serine proteases, were shown to process POMC, protective antigen (PA) of anthrax and pro-TRH correctly at paired basic residues in vitro. Use of PA substrates mutated at the tetrabasic cleavage site revealed the importance of the secondary and tertiary structure in dictating the specificity of processing. PC1 and PC2 mRNA have been colocalized with TRH immunopositive neurons by in situ hybridization and immunocytochemistry, supporting a role of these enzymes in processing pro-TRH in vivo.