Biosynthesis /Processing /Secretion of Neuropeptides And

研究了激素原、阿黑皮素原（POMC，pro-ACTH/endorphin）、脑啡肽原（pro-enkephalin，pro-ENK）和胰岛素原（pro-insulin，pro-insulin）在调节分泌通路（RSP）中的细胞内分选机制。这些激素原经历同型寡聚化，作为浓缩步骤，因为它们从内质网中的合成位点穿过细胞到达反式高尔基体网络（trans-Golgi network，TGN），在那里它们被分选成受调节的分泌途径的致密核心颗粒用于加工和分泌。定点诱变研究鉴定了由暴露在这些分子表面上的两个酸性残基（彼此相距12-15 Angs）和两个疏水残基（距离酸性残基5-7 Angs）组成的共有分选基序，所述两个疏水残基是分选RSP所必需的。对于POMC，残基是位于N-末端的D10、L11; E14、L18。在脑啡肽原的N端发现了一个类似的由残基D18、I19、E29、L32组成的分选基序。在单体胰岛素原中，分选信号基序由位于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被掩埋，并且基序由来自六聚体中两个相邻胰岛素原二聚体的A链中的两个残基贡献。RSP分选受体被鉴定为膜羧肽酶E（CPE），其对POMC、胰岛素原和脑啡肽原的分选信号具有特异性。激素原分选基序中的两个酸性残基与分选受体羧肽酶E（CPE）的两个碱性残基R255和K260特异性相互作用，以实现对RSP的分选。我们发现，CPE是一种跨膜蛋白，通过其C-末端两亲性尾锚定到独特的胆固醇-鞘糖脂丰富的微结构域称为筏，在TGN。通过甲基β-环糊精从分泌颗粒膜中去除胆固醇导致CPE无法结合货物;通过用洛伐他汀处理细胞来消耗胆固醇导致CPE和POMC缺乏对RSP的分选。因此，膜缔合对于TGN处CPE的激素原分选受体功能以及其自身对RSP的分选至关重要。在Neuro 2a细胞中通过反义RNA消耗CPE引起激素原向组成性途径的错误分选，表明CPE在体内起分选受体的作用。使用小鼠模型，合成的突变CPE，是差异降解垂体和胰腺，我们能够显示降低CPE水平和这些组织的细胞中的内源性激素原的分选错误的程度之间的相关性。这些研究为分选信号/受体介导的机制提供了证据，该机制用于将激素原靶向神经内分泌细胞中的调节分泌途径。研究了在具有异常高水平血浆胰岛素原的高胰岛素原血症患者中发现的遗传突变的胰岛素原的细胞内分选，以了解这些形式的糖尿病的分子基础。在这些患者中发现的一种形式的突变胰岛素原，HisB 10Asp，不能六聚体化，但形成二聚体，被发现被误分选到组成型途径，并在转染到细胞系中时以不受调控的方式分泌。该突变胰岛素原的二聚体的分子建模预测，RSP分选信号基序的两个酸性残基的分子距离将太大而不允许与分选受体CPE的结合位点中的碱性残基相互作用。实际上，体外结合研究表明，该突变体不与CPE结合，从而导致其不能被分选到RSP以加工成胰岛素并以促分泌素依赖性方式分泌。其他高胰岛素原血症胰岛素原突变体，Arg 65 Pro和Arg 65 Leu也被发现是组成性分泌的，不储存。因此，这些患者血浆中分泌的高水平突变胰岛素原是由于这些分子的遗传结构改变导致的分选、加工、储存和分泌缺陷。 在TGN的大致密核心颗粒（LDCG）的形成是必不可少的调节分泌的激素和神经内分泌细胞的神经肽。我们最近的研究发现了一个主开关，嗜铬粒蛋白A（CgA），它控制神经内分泌细胞中LDCG的形成。利用反义技术去除大鼠PC 12细胞中的CgA，导致LDCG的丢失，调节颗粒蛋白包括CgB和突触结合蛋白的分泌和降解。牛CgA在这些细胞中的过表达拯救了野生型表型。在缺乏CgA，LDCGs和调节激素分泌的突变内分泌细胞系中，CgA的转染恢复了这些细胞中的野生型表型。因此，CgA通过调节内分泌细胞和神经内分泌细胞中的分泌颗粒生物发生在控制激素分泌中起重要的高阶生理作用。

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.