BIOSYNTHESIS, PROCESSING AND SECRETION OF NEUROPEPTIDES AND PITUITARY HORMONES

确定了将促肾上腺皮质激素（POMC）、促acth /内啡肽（pro- acth /endorphin）、促脑啡肽（pro- enk）和促胰岛素（pro- insulin）靶向调节分泌通路（RSP）的分选信号基序。位点定向诱变研究发现了一个一致的分类基序，包含两对暴露在这三个分子表面的酸性/疏水性残基。对于POMC，残基为D10， L11；E14 L18位于n端。类似的排序基序，由残基D18， I19组成；E29、L32在前脑啡肽的n端存在。在单体胰岛素原中，排序信号基序由位于B链上的残基E13和L17以及位于A链上的残基L16和E17组成。在六聚体胰岛素原中，B链上的残基E13被传递，基序由六聚体中两个相邻胰岛素原二聚体的A链上的两个残基提供。一个对POMC、胰岛素前体和脑啡肽前体的分选信号具有特异性的RSP分选受体被鉴定为膜羧基肽酶E （CPE）。CPE被证明是一种跨膜蛋白，它被锚定在反式高尔基网络（TGN）中富含胆固醇-糖鞘脂的微结构域。激素原分选信号基序中的酸性残基特异性地结合CPE上的两个基本残基R255和K260，从而在TGN上进行分选。在Neuro2a细胞中，反义RNA对CPE的损耗导致POMC、前脑啡肽和前胰岛素在组成通路上的错误分选，表明CPE在体内具有分选受体的功能。利用小鼠模型合成了在垂体和胰腺中不同降解的CPE突变体，我们能够显示CPE水平降低与这些组织细胞中内源性原激素的错分类程度之间的相关性。这些研究为神经内分泌细胞中存在一种信号/受体介导的前激素分选机制提供了证据。本文研究了yapsin 1的生物合成、加工及其特异性的分子基础。yapsin 1是一类新的yapsin天冬氨酸蛋白酶。Yapsin 1是一种酵母激素前加工酶，是一种无活性的前酶，通过去除前区而被激活。该酶首先在前区内部自催化裂解形成pseudoyapsin 1。然后，Pseudoyapsin 1在Asp45上游进行10-20个残基的自催化裂解，产生两个亚基，a和b，它们通过二硫桥连接。利用野生型和sec18酵母突变体进行的脉冲追踪研究表明，这些加工步骤发生在内质网中。在分泌之前，剩余的前区域被裂解形成成熟的yapsin 1。yapsin1的分子模型显示，它在S1亚位点上有一个开放的、高度电负性的活性位点口袋，有利于在P1位置上有碱性残基的底物。该模型还显示，S6、S2和S3亚位具有电负性，这说明我们观察到，由于与这些活性位点口袋的结合增强，在P6、P2和P3位置具有碱性残基的底物具有更高的催化效率。另一个成员yapsin 3被克隆，并被证明优先切割肽前体的单赖氨酸。这与yapsin 1不同，yapsin 1偏爱成对的碱基残基。对yapsin 2的特异性分析表明，它更倾向于在一对精氨酸或赖氨酸之后切割，而不同于在赖氨酸之间切割的yapsin 1。因此，yapsin家族的不同成员在特异性上表现出微妙的差异，并可能在体内被定制为切割不同的底物。

The sorting signal motif for targeting pro-opiomelanocortin (POMC, pro-ACTH/endorphin) pro-enkephalin (pro-ENK) and pro- insulin to the regulated secretory pathway (RSP) was identified. Site directed mutagenesis studies identified a concensus sorting motif containing two pairs of acidic/hydrophobic residues exposed on the surface of these three molecules. For POMC, the residues are D10, L11; E14, L18 located at the N-terminus. A similar sorting motif consisting of residues D18, I19; E29,L32 was found in the N-terminus of pro-enkephalin. In monomeric proinsulin, the sorting signal motif consists of residues E13 and L17 located on the B chain and L16 and E17 located on the A chain. In hexameric proinsulin, residue E13 on the B chain is burried and the motif is contributed by the two residues in the A chain from two adjacent proinsulin dimers in the hexamer. A RSP sorting receptor that is specific for the sorting signal of POMC, pro- insulin and pro-enkephalin was identified as membrane carboxypeptidase E (CPE). CPE was shown to be a transmembrane protein which is anchored in cholesterol-glycosphingolipid rich microdomains in the trans-golgi network (TGN). The acidic residues in the prohormone sorting signal motif specifically bind the two basic residues, R255 and K260, on CPE, to effect sorting at the TGN. Depletion of CPE by antisense RNA in Neuro2a cells 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. Using a mouse model which synthesizes a mutant CPE that is differentially degraded in pituitary and pancreas, we were able to show a correlation between lowered CPE levels and the degree of missorting of endogenous prohormones in the cells of these tissues. These studies provide evidence for a sorting signal/receptor mediated mechanism for sorting prohormones to the regulated secretory pathway in neuro-endocrine cells.The biosynthesis, processing and molecular basis for the specificity of yapsin 1, a member of the novel class of yapsin aspartic proteases was studied. Yapsin 1, a yeast prohormone processing enzyme is synthesized as an inactive proenzyme and is activated by removal of the pro-region. The enzyme is first cleaved internally in the proregion autocatalytically to form pseudoyapsin 1. Pseudoyapsin 1 then undergoes autocatalytic cleavage 10-20 residues upstream of Asp45 to yield two subunits, a and b, which are linked by a disulfide bridge. Pulse-chase studies using wild type and the Sec 18 yeast mutant showed that these processing steps occur in the endoplasmic reticulum. Prior to secretion, the remaining pro- region is cleaved to form mature yapsin 1. 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, accounting for our observations that substrates with basic residues in the P6, P2 and P3 positions are cleaved with higher catalytic efficiency due to enhanced binding to these active site pockets. Another member, yapsin 3 was cloned and shown to preferentially cleave single Lys of peptide precursors. This differs from yapsin 1 which prefers paired basic residues. Analysis of yapsin 2 specificity indicated that it prefers to cleave after a pair of Arg or Lys, differing from yapsin 1 which cleaves in between the Lys. Thus the various members of the yapsin family show subtle differences in specificity and are likely tailored to cleave different substrates in vivo.