Biosynthesis, Processing And Secretion Of Neuropeptides

在神经内分泌细胞中，前神经肽和神经营养因子在调节分泌通路（RSP）上的细胞内分选对于活性蛋白和肽的加工、储存和释放至关重要。研究了促肾上腺皮质激素（POMC）、促acth /内啡肽（acth /endorphin）、促脑啡肽（enk）、胰岛素原和脑源性神经营养因子（BDNF）在调节分泌通路（RSP）中的分选。我们发现这些前蛋白经历了同型寡聚化，作为一个浓缩步骤，它们从内质网的合成位点穿过细胞到达反式高尔基网络（TGN），在那里它们被分类成受调节的分泌途径的致密核颗粒进行加工和分泌。定点诱变研究确定了一个一致的排序基序，由两个酸性残基组成，12-15？彼此分开，暴露在这些分子表面，还有两个疏水残基，5-7？远离对RSP进行分类所必需的酸性残留物。对POMC、前脑啡肽胰岛素原和BDNF的分选信号具有特异性的RSP分选受体为膜羧基肽酶E （CPE）。激素原/ bdnf原分选基序中的两个酸性残基特异性地与分选受体羧基肽酶E （CPE）的两个碱性残基R255和K260相互作用，从而对RSP进行分选。在神经2a细胞的CPE零克隆中转染R255和K260突变为a的CPE突变体，以及在胰腺β细胞系INS细胞中转染显性CPE阴性突变体，分别导致POMC和胰岛素原分选错误，这表明CPE分选域的碱性残基在体内与POMC和胰岛素原分选信号中的酸性残基相互作用，从而对RSP进行分选。利用小鼠模型合成了在垂体中降解的突变CPE，我们能够在这些细胞中显示内源性POMC的错误分类。这些研究为在神经内分泌细胞中将前激素定向到受调节的分泌通路的分选信号/受体介导机制提供了证据。研究了在血浆胰岛素原水平异常高的高胰岛素原血症患者中发现的基因突变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突变胰岛素原HisB10Asp不能六聚体化，而是形成二聚体，当转染到细胞系中时，发现它在构成途径上被错分类并以不受调节的方式分泌。该突变体胰岛素原二聚体的分子模型预测，RSP分选信号基序的两个酸性残基的分子距离太大，无法与分选受体CPE结合位点的碱性残基相互作用。事实上，体外结合研究表明，该突变体不与CPE结合，从而导致其无法被分类到RSP以分泌依赖的方式加工成胰岛素和分泌。其他高胰岛素原血症胰岛素原突变体Arg65Pro和Arg65Leu也被发现是组成性分泌的，而不是储存的。结合研究表明，突变体Arg65Pro和Arg65Leu胰岛素原蛋白与CPE结合较差，导致未成熟分泌颗粒中缺乏分选和保留。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分类缺陷造成的，这是由它们的遗传结构改变造成的。最近，我们还研究了激素原和神经肽加工酶、羧肽酶E （CPE）和激素原转化酶1和2 （PC1和PC2）在调节分泌途径上的分类。我们已经证明这些酶是跨膜蛋白，在c端具有非典型的跨膜结构域。在神经内分泌细胞中，它们通过一种新机制被分类为RSP颗粒，这种新机制涉及它们的c端跨膜结构域插入到TGN富含胆固醇-糖鞘脂的微结构域（称为脂筏）中。从分泌颗粒膜上去除胆固醇导致RSP分选受体CPE无法结合货物；和用洛伐他汀治疗细胞的胆固醇消耗导致CPE对RSP缺乏分选。因此，与富含胆固醇的脂筏的膜关联对于将激素原加工酶分选到TGN和分泌颗粒是必不可少的。我们还发现，CPE在颗粒胞吐后从质膜再循环到TGN，并且该酶的内化依赖于其细胞质结构域与arf6的相互作用。

The intracellular sorting of pro-neuropeptides and neurotrophins to the regulated secretory pathway (RSP) is essential for processing, storage and release of active proteins and peptides in the neuroendocrine cell. The sorting of pro-opiomelanocortin (POMC, pro-ACTH/endorphin), pro-enkephalin (pro-ENK), proinsulin and brain derived neurotrophic factor (BDNF) to the regulated secretory pathway (RSP) was investigated. We show that these pro-proteins 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 consensus sorting motif consisting of two acidic residues, 12-15? apart from each other, exposed on the surface of these molecules, and two hydrophobic residues, 5-7? away from the acidic residues which are necessary for sorting to the RSP. A RSP sorting receptor that was specific for the sorting signal of POMC, pro-enkephalin proinsulin and BDNF was identified as membrane carboxypeptidase E (CPE). The two acidic residues in the prohormone/pro-BDNF sorting motif specifically interact with two basic residues, R255 and K260, of the sorting receptor, carboxypeptidase E (CPE), to effect sorting to the RSP. Transfection of a mutant CPE with R255 and K260 mutated to A in a CPE null clone of Neuro2a cells, and transfection of a dominant negative CPE mutant into INS cells, a pancreatic beta cell line, caused missorting of POMC and proinsulin to the constitutive pathway respectively, indicating that the basic residues in the sorting domain of CPE interacts with the acidic residues in the POMC and proinsulin sorting signal in vivo to effect sorting to the RSP. Using a mouse model which synthesizes a mutant CPE that is degraded in the pituitary, we were able to show missorting of endogenous POMC in these cells. 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. Binding studies showed that mutant Arg65Pro and Arg65Leu proinsulins bound poorly to CPE, accounting for the lack of sorting and retention in the immature secretory granule. The high levels of secreted mutant proinsulins in the plasma of these patients are therefore due to defects in sorting of these molecules, resulting from their genetic structural alterations.Recently we have also investigated the sorting of prohormone and neuropeptide processing enzymes, carboxypeptidase E (CPE) and prohormone convertases 1 and 2 (PC1 and PC2) to the regulated secretory pathway. We have shown that these enzymes are transmembrane proteins with an atypical membrane spanning domain at the C-terminus. They are sorted into granules of the RSP in neuroendocrine cells by a novel mechanism involving insertion of their C-terminal transmembrane domain into cholesterol-glycosphingolipid rich microdomains known as lipid rafts, at the TGN. Removal of cholesterol from secretory granule membranes resulted in the inability of CPE, the RSP sorting receptor to bind cargo; and cholesterol depletion by treatment of cells with lovastatin resulted in lack of sorting of CPE to the RSP. Thus membrane association with cholesterol-rich lipid rafts is essential for sorting of the prohormone processing enzymes to the TGN and secretory granules. We also showed that CPE is recycled back from the plasma membrane to the TGN after granule exocytosis, and the internalization of the enzyme is dependent on the interaction of its cytoplasmic domain with ARF 6. In another project we have studied the factors governing the formation of large dense-core granules (LDCG) at the TGN, which is essential for regulated secretion of hormones and neuropeptides from neuroendocrine cells. Our recent studies uncovered an 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, 6T3, lacking CgA, LDCGs and regulated hormone secretion, transfection of CgA restored the wild type phenotype in these cells. We have recently identified the Golgi as the site of degradation of the secretory granule proteins in the absence of granule biogenesis. Thus we propose that regulation of the stability of granule proteins at the Golgi by CgA may be a point of control of granule biogenesis in neuroendocrine cells. Recently, we compared gene expression in 6T3 cells lacking LDCGs and 6T3 cells stably transfected with CgA using microarrays. We found that aquaporin-1(AQP1, a water channel), a secretory granule protein was significantly up-regulated in 6T3 cells expressing CgA. These findings suggest that CgA may have a new regulatory role in secretory granule protein expression at the transcriptional level which in turn may regulate granule biogenesis.