Biosynthesis /Processing /Secretion of Neuropeptides And

研究了前激素、前阿片黑素皮质素(POMC)、前ACTH/内啡肽、前脑啡肽(PRO-ENK)和胰岛素原对调节分泌途径(RSP)的细胞内分选机制。这些前激素经历同型齐聚，作为一个浓缩步骤，当它们从内质网中的合成部位穿过细胞到跨高尔基网络(TGN)时，在那里它们被分类为调节分泌途径的致密核心颗粒进行处理和分泌。定点突变研究确定了一个集中的排序基序，由两个相互间隔12-15个氨基酸的酸性残基和两个疏水残基组成，两个疏水残基远离酸性残基5-7个残基，这是分选到RSP所必需的。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残基被埋没，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。前激素分选基序中的两个酸性残基与分选受体羧基肽酶E(CPE)的两个碱性残基R255和K260特异地相互作用，影响对RSP的分选。我们发现CPE是一种跨膜蛋白，通过其C-末端两亲性尾巴锚定到TGN中独特的富含胆固醇-鞘糖脂的微域。甲基β-环糊精去除分泌颗粒膜上的胆固醇，导致CPE不能结合货物；洛伐他汀处理细胞使胆固醇耗尽，导致CPE和POMC不能与RSP分离。因此，膜结合对于CPE在TGN的激素前体分选受体功能以及对RSP本身的分选是至关重要的。在Neuro2a细胞中，CPE被反义RNA耗尽，导致前激素在构成途径上的错误分选，表明CPE在体内作为分选受体发挥作用。利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靶向调节分泌途径的分选信号/受体调节机制提供了证据。研究了在血浆胰岛素原水平异常高的高胰岛素血症患者中发现的基因突变的胰岛素原的细胞内分类，以了解这些形式的糖尿病的分子基础。在这些患者中发现了一种突变的胰岛素原，HisB10Asp，它不能六角化，但形成二聚体，当转染到细胞系中时，被发现错误地分类到构成途径并以不受调控的方式分泌。对这个突变的胰岛素原二聚体的分子模拟预测，RSP分选信号基序的两个酸性残基的分子距离太大，不能与分选受体CPE结合部位的碱性残基相互作用。事实上，体外结合研究表明，该突变体不能与CPE结合，从而导致它不能被RSP分类，以分泌依赖的方式加工成胰岛素和分泌。其他高胰岛素血症原突变体Arg65Pro和Arg65Leu也被发现是结构性分泌而不是储存的。因此，这些患者血浆中高水平分泌突变的胰岛素原是由于这些分子的分类、加工、储存和分泌方面的缺陷，这是由于它们的遗传结构变化造成的。在TGN形成大的致密核颗粒(LDCG)是调节神经内分泌细胞分泌激素和神经肽的关键。我们最近的研究发现了一种主要的开/关开关，嗜铬粒素A(CGA)，它控制神经内分泌细胞中LDCG的形成。应用反义技术去除大鼠PC12细胞中的CgA，导致LDCG的丢失，调节分泌和降解颗粒蛋白，包括Cgb和synaptopagmin。牛CGA在这些细胞中的过表达挽救了野生型的表型。在缺乏CGA、LDCGs和调节激素分泌的突变内分泌细胞系中，CGA的转基因恢复了这些细胞的野生型表型。因此，CGA通过调节内分泌和神经内分泌细胞分泌颗粒的生物发生，在控制激素分泌方面发挥着重要的高级生理作用。

The mechanism of intracellular sorting of prohormones, pro-opiomelanocortin (POMC, pro-ACTH/endorphin) pro-enkephalin (pro-ENK) and pro-insulin to the regulated secretory pathway (RSP) was investigated. These prohormones undergo homotypic oligomerization, as a concentration step as they traverse the cell from the site of synthesis in the endoplasmic reticulum to the trans-Golgi network(TGN)where they are sorted into dense-core granules of the regulated secretory pathway for processing and secretion. Site-directed mutagenesis studies identified a concensus sorting motif consisting of two acidic residues, 12-15 Angs apart from each other, exposed on the surface of these molecules, and two hydrophobic residues, 5-7 Angs away from the acidic residues which are necessary for sorting to the RSP. 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 buried 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 was specific for the sorting signal of POMC, pro-insulin and pro-enkephalin was identified as membrane carboxypeptidase E (CPE). The two acidic residues in the prohormone sorting motif specifically interact with two basic residues, R255 and K260, of the sorting receptor, carboxypeptidase E (CPE), to effect sorting to the RSP. We showed that CPE is a transmembrane protein which is anchored via its C-terminal amphipathic tail to unique cholesterol-glycosphingolipid rich microdomains known as rafts, in the TGN. Removal of cholesterol from secretory granule membranes by methyl beta-cyclodextrin resulted in the inability of CPE to bind cargo; and cholesterol depletion by treatment of cells with lovastatin resulted in lack of sorting of CPE and POMC to the RSP. Thus membrane association is essential for the prohormone sorting receptor function of CPE at the TGN, as well as its own sorting to the RSP. Depletion of CPE by antisense RNA in Neuro2a cells caused missorting of prohormones 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 targeting prohormones to the regulated secretory pathway in neuro-endocrine cells. The intracellular sorting of genetically mutated proinsulins found in hyperproinsulinemia patients who have abnormally high levels of plasma proinsulin was investigated to understand the molecular basis of these forms of diabetes. One form of mutant proinsulin found in these patients, HisB10Asp, which is unable to hexamerize but forms dimers, was found to be missorted to the constitutive pathway and secreted in an unregulated manner when transfected into a cell line. Molecular modelling of the dimer of this mutant proinsulin predicted that the molecular distance of the two acidic residues of the RSP sorting signal motif would be too large to allow interaction with the basic residues in the binding site of the sorting receptor, CPE. Indeed in vitro binding studies showed that this mutant did not bind to CPE, thus resulting in its inability to be sorted to the RSP for processing to insulin and secretion in a secretogogue-dependant manner. Other hyperproinsulinemia proinsulin mutants, Arg65Pro and Arg65Leu were also found to be secreted constitutively and not stored. The high levels of secreted mutant proinsulins in the plasma of these patients are therefore due to defects in sorting, processing, storage and secretion of these molecules resulting from their genetic structural alterations. Formation of large dense-core granules(LDCG) at the TGN is essential for regulated secretion of hormones and neuropeptides from neuroendocrine cells. Our recent studies uncovered a master on/off switch , chromogranin A (CgA), that controls the formation of LDCG in neuroendocrine cells. Depletion of CgA in rat PC12 cells using antisense technology resulted in the loss of LDCG, regulated secretion and degradation of granule proteins including CgB and synaptotagmin. Overexpression of bovine CgA in these cells rescued the wild type phenotype. In a mutant endocrine cell line lacking CgA, LDCGs and regulated hormone secretion, transfection of CgA restored the wild type phenotype in these cells. Thus CgA serves an important higher-order physiological role in controlling hormone secretion through regulating secretory granule biogenesis in endocrine and neuroendocrine cells.