BIOSYNTHESIS, PROCESSING AND SECRETION OF NEUROPEPTIDES AND PITUITARY HORMONES

靶向阿黑皮素原（POMC， 促肾上腺皮质激素原（pro-ACTH/内啡肽）和脑啡肽原（pro-ENK） 分泌途径（RSP）。 定点诱变 研究在Cys 2和Cys 3之间的N-末端鉴定了两亲环。 Cys 8作为POMC分选信号。 两对酸性/疏水性 残基（D10 L11; E14 L18）暴露在环的表面上， 参与与分选受体的结合。 类似的分类主题 在脑啡肽原的N-末端鉴定出由两个 暴露在膜上的酸性/疏水性残基对（D18，I19; E29，L32） 由两个二硫键Cys 2-Cys 24稳定的双环表面 和Cys 6-Cys 28。 一种RSP分选受体，其特异性用于分选 POMC、胰岛素原、脑啡肽原和生长激素信号 鉴定为膜羧肽E. (CPE)CPE闭塞 在neuro 2a细胞和携带反义RNA的Cpefat/Cpefat小鼠中， CPE基因的突变导致POMC的错误分选， 脑啡肽原和胰岛素原的组成途径，表明 CPE在体内起到分选受体的作用。 我们的研究结果支持 分选信号/受体机制，用于将激素原分选到 调节神经内分泌细胞的分泌途径。 生物合成和分子基础的特异性的成员， 研究了一类新的天冬氨酸蛋白酶Yapsin 1（YAP 3 p）。 Yapsin 1是一种酵母激素前体加工酶， 作为一种无活性的酶原合成，并通过去除 前区通过两步，自催化过程。 酶也是 分裂成两个亚基，α和β，它们通过一个 二硫化物形成异二聚体。 使用野生型和 Sec 18酵母突变体显示，原Yapsin 1首先被切割成 内质网中的α和β亚基， 自催化去除高尔基体中的前区域。 分子建模 Yapsin 1的研究表明，它具有一个开放的，高度电负性的活性， S1亚位点中的位点口袋，有利于具有碱性残基的底物 在P1位置。 该模型还表明，S6、S2 '和S3' 亚位点是电负性的，底物中含有碱性残基， P6、P2'和P3'能增强结合和催化效率。 使用CCK 13 -33类似物的特异性研究表明，Yapsin 1切割 底物与碱性残基在P1优先和催化 在含有额外碱性残基的底物中， P6、P2 '、P3'和P6'的位置，与来自 该模型 Yapsin 1哺乳动物同源基因的筛选克隆 用Yapsin 1抗体构建垂体cDNA表达文库， 胃蛋白酶抑制剂的酶活性产生了一些积极的 目前正在测序的克隆。

The sorting signal motif for targeting pro-opiomelanocortin (POMC, pro-ACTH/endorphin) and pro-enkephalin (pro-ENK) to the regulated secretory pathway (RSP) was identified. Site directed mutagenesis studies identified an amphipathic loop at the N-terminus between Cys2 and Cys8 as the POMC sorting signal. Two pairs of acidic/hydrophobic residues (D10L11; E14L18) are exposed on the surface of the loop which are involved in binding to the sorting receptor. A similar sorting motif was identified in the N-terminus of pro-enkephalin consisting of two pairs of acidic/hydrophobic residues (D18, I19; E29,L32) exposed on the surface of a double loop stabilized by two disulfide bridges, Cys2-Cys24 and Cys6-Cys28. A RSP sorting receptor that is specific for the sorting signal of POMC, pro-insulin, pro-enkephalin and growth hormone was identified as membrane carboxypeptidese E. (CPE) Obliteration of CPE by antisense RNA in neuro2a cells and in the Cpefat/Cpefat mouse carrying a mutation in the CPE gene resulted in the missorting of POMC, pro-enkephalin and pro-insulin to the constitutive pathway, indicating that CPE functions as a sorting receptor in vivo. Our findings support a sorting signal/receptor mechanism for sorting prohormones to the regulated secretory pathway in neuro-endocrine cells. The biosynthesis and molecular basis for the specificity of a member of the novel class of aspartic protease, Yapsin1 (YAP3p) was studied. Yapsin1, a yeast pro-hormone processing enzyme was shown to be synthesized as an inactive proenzyme and activated by removal of the pro-region via a two step, autocatalytic process. The enzyme was also cleaved into two subunits, alpha and beta, which were linked by a disulfide to form a heterodimer. Pube-chase studies using wild type and the Sec 18 yeast mutant showed that pro-Yapsin1 was first cleaved into the alpha and beta subunits in the endoplasmic reticulum, followed by autocatalytic removal of the pro-region in the Golgi. Molecular modeling of Yapsin1 revealed that it has an open, highly electronegative active site pocket in the S1 subsite, favoring substrates with a basic residue in the P1 position. The model also showed that the S6, S2', and S3' subsites were electronegative and substrates with basic residues in the P6, P2' and P3' would enhance binding and the catalylic efficiency. Specificity studies using CCK13-33 analogues showed that Yapsin1 cleaved substrates with a basic residue in P1 preferentially and the catalytic efficiency was enhanced in substrates with additional basic residues in the P6, P2', P3', and P6' positions, consistent with the predictions from the model. Cloning of mammalian homologues of Yapsin1 by screening pituitary cDNA expression librabries with Yapsin1 antibodies and pepstatin inhibitable enzymatic activity have yielded several positive clones that are currently being sequenced.