BIOSYNTHESIS, PROCESSING AND SECRETION OF NEUROPEPTIDES AND PITUITARY HORMONES

确定了针对前阿片黑素皮质素(POMC)、前ACTH/内啡肽、前脑啡肽(PRO-ENK)和前胰岛素到调节分泌途径(RSP)的分选信号基序。定点突变研究发现，在这三个分子的表面，含有两对酸性/疏水残基的融合排序基序。对于POMC，残基为D10、L11；位于N-末端的E14、L18。在脑啡肽原的N-末端发现了类似的由残基D18，I19，E29，L32组成的排序基序。在单体胰岛素原中，分选信号基序由位于B链的残基E13和L17和位于A链的残基L16和E17组成。在六聚体胰岛素原中，B链上的E13残基被埋葬，该基序由六聚体中两个相邻的胰岛素原二聚体在A链上的两个残基贡献。膜羧肽酶E(CPE)是一种能特异性识别POMC、胰岛素原和脑啡肽原分选信号的RSP分选受体。CPE是一种跨膜蛋白，定位于跨高尔基体网络(TGN)中富含胆固醇-鞘糖脂的微区。前激素分选信号基序中的酸性残基与CPE上的两个碱性残基R255和K260特异性结合，从而在TGN上实现分选。在Neuro2a细胞中，CPE被反义RNA耗尽，导致POMC、脑啡肽原和胰岛素原错误地排列在构成通路上，表明CPE在体内起着分选受体的作用。使用一个小鼠模型合成了一种突变的CPE，这种突变的CPE在垂体和胰腺中被不同地降解，我们能够证明CPE水平降低与这些组织细胞中内源性前激素错选的程度之间的相关性。这些研究为神经内分泌细胞中前激素分选信号/受体调节分泌途径的机制提供了证据。本论文研究了新的一类Yapsin天冬氨酸蛋白酶成员Yapsin 1的生物合成、加工和分子基础。Yapsin 1是一种酵母激素前体加工酶，它是作为一种失活的酶原合成的，通过去除该酶原区域而被激活。该酶首先在前区被自动催化内部裂解，形成假蛋白1。然后假蛋白1在Asp45上游经过10-20个残基的自催化裂解，产生两个亚基a和b，这两个亚基由二硫键连接。用野生型和SEC 18酵母突变体进行的脉冲追逐研究表明，这些加工步骤发生在内质网中。在分泌之前，剩余的前区域被切割形成成熟的yapsin 1。对yapsin 1的分子模拟表明，它在S1亚基上有一个开放的、高度电负性的活性部位口袋，有利于在P1位置有碱性残基的底物。该模型还表明，S6、S2和S3亚位是电负性的，这解释了我们观察到在P6、P2和P3位置有碱性残基的底物由于与这些活性中心口袋的结合增强而具有更高的催化效率。另一个成员Yapsin 3被克隆，并被证明优先切割多肽前体的单个Lys。这与yapsin 1不同，yapsin 1更喜欢成对的碱性残基。对Yapsin 2的特异性分析表明，它更喜欢在一对Arg或Lys之后切割，不同于Yapsin 1在Lys之间切割。因此，Yapsin家族的不同成员在特异性上表现出细微的差异，并可能在体内切割不同的底物。

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.