Biosynthesis, Processing And Secretion Of Neuropeptides

神经肽的生物合成、加工和分泌

• 批准号: 6990977
• 负责人: Yoke p Loh
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6990977
• 关键词: Golgi apparatus; PC12 cells; binding sites; biological signal transduction; brain derived neurotrophic factor; carboxypeptidase; chromogranins; enkephalins; gene mutation; genetically modified animals; hormone regulation /control mechanism; hyperinsulinism; intracellular transport; laboratory mouse; neuropeptides; peptide hormone biosynthesis; pituitary hormones; prohormone convertase; proinsulin; proopiomelanocortin; protein biosynthesis; protein transport; secretion; site directed mutagenesis

The intracellular sorting of pro-neuropeptides, prohormones 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-insulin, pro- cocaine-amphetamine regulated transcript (CART) and brain derived neurotrophic factor (BDNF) to the RSP was investigated. Such studies have led to the better understanding of diseases related to defects in hormone and neuropeptide targeting, obesity, diabetes, memory and learning.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, 12-15Ang apart from each other, exposed on the surface of these molecules, and two hydrophobic residues, 5-7Ang away from the acidic residues which are necessary for sorting these pro-proteins to the RSP. A RSP sorting receptor that was specific for the sorting signal of POMC, pro-insulin 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, CPE, to effect sorting to the RSP. Using a CPE knockout (KO) mouse model, we showed missorting of endogenous POMC and proinsulin in pituitary and pancreatic islet cells, respectively, in these animals. Furthermore BDNF which modulates synaptic plasticity was missorted in cortical and hippocampal neurons, which could account for the memory deficits observed in CPE KO mice. These studies provide evidence for a CPE-dependent, sorting signal/receptor mediated mechanism for targeting prohormones, neuropeptides and the neurotrophin, BDNF, to the regulated secretory pathway in endocrine cells and neurons. 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 shown to be missorted to the constitutive pathway and secreted in an unregulated manner when transfected into a cell line. Molecular modeling 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. The high levels of secreted mutant proinsulin in the plasma of these patients are therefore due to defects in sorting of these molecules, resulting from their genetic structural alterations. In another study, the sorting and processing of a mutant form of CART (CART Leu34Phe), found in a family of obese patients was investigated. CART, found in brain, is an anorexigenic peptide that has several physiological effects such as inhibiting feeding, regulating energy expenditure, and stress. CART acts downstream of leptin in the obesity controlling signaling pathway. We show that while pro-CART was substantially processed to active CART, mutant pro-CART was only minimally processed to yield an intermediate form. Furthermore, mutant pro-CART was partially missorted and secreted via the constitutive pathway, providing a possible molecular basis for the obese phenotype in these patients. We have also used our knowledge of the sorting motif of hormones to engineer biologically active mutant hormones that are redirected to the constitutive pathway. Such mutant hormones are currently being expressed in salivary glands for systemic secretion, with the ultimate aim of applying such technology to gene therapeutics. Recently we have also investigated the sorting of prohormone processing enzymes, 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 novel physical interaction of its cytoplasmic domain with ARF 6, a GTPase ADP-ribosylation factor. 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 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. The importance of CgA in large dense-core granule biogenesis in vivo was recently demonstrated using an antisense mRNA transgenic mouse model deficient in CgA.These mice showed severe aberrant granule formation quantitatively and qualitatively in the adrenal medulla.

对受调节的分泌途径（RSP）的促尿肽，促炎和神经营养蛋白的细胞内分选对于在神经内分泌细胞中的活性蛋白和肽的加工，储存和释放至关重要。研究对RSP的促蛋白酶素（POMC，促胰岛素/内啡肽），促胰岛素，促胰岛素 - 抑制转录本（CART）和脑衍生的神经营养因子（BDNF）的分类。此类研究导致对与激素和神经肽靶向，肥胖，糖尿病，记忆和学习的缺陷有关的疾病有了更好的了解。我们表明，这些前蛋白经历同型寡聚为浓度的步骤，因为它们会从内质网络中分类到conticiles netroce netroce contressement contressect（the）的网络（the）网络（the）网络（the）的网络（to）网络（the）的网站（the）网络（TO）。受监管的分泌途径的颗粒进行处理和分泌。定位的诱变研究确定了一个共有的分类基序，该基序由两个酸性残基组成，彼此之间相距12-15，暴露在这些分子的表面上，而两个疏水残基，与将这些促蛋白质分类为RSP所需的酸性残基5-7 g。针对POMC，前胰岛素和BDNF的分选信号的RSP排序受体被鉴定为膜羧肽酶E（CPE）。激素/pro-BDNF排序基序中的两个酸性残基特异性与两个基本残基R255和K260相互作用，分别受体CPE与RSP进行分类。使用CPE敲除（KO）小鼠模型，我们在这些动物中分别显示了垂体和胰岛细胞中内源性POMC和促胰岛素的错过。此外，在皮质和海马神经元中错过了调节突触可塑性的BDNF，这可以解释CPE KO小鼠中观察到的记忆缺陷。这些研究为CPE依赖性的信号/受体介导的机制提供了证据，用于靶向型肌动蛋白，神经肽和神经营养蛋白BDNF，以靶向内分泌细胞和神经元中调节的分泌途径。 研究了在异常血浆血浆蛋白异常高的血浆血症患者中发现的遗传突变蛋白的细胞内分选，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蛋白的一种形式，无法进行六聚体但形成二聚体的Hisb10ASP被证明被遗失在本构途径上，并在转染细胞系时以不受管制的方式分泌。该突变蛋白的二聚体的分子模型预测，RSP分类信号基序的两个酸性残基的分子距离太大，无法与排序受体CPE的结合位点中的基本残基相互作用。实际上，体外结合研究表明，该突变体没有与CPE结合，因此无法将其分类到RSP以促进依赖性的方式进行处理以处理胰岛素和分泌。因此，这些患者血浆中的高水平分泌突变蛋白是由于它们的遗传结构改变而导致的这些分子的分类缺陷。 在另一项研究中，研究了在肥胖患者家族中发现的一种突变形式的推车形式的分类和处理。在大脑中发现的购物车是一种厌食肽，具有多种生理作用，例如抑制喂养，调节能量消耗和压力。手推车在控制信号通路的肥胖症中作用于瘦素的下游。我们表明，虽然亲妻子基本上经过了活跃的手推车处理，但突变体pro-cart仅经过最小处理以产生中间形式。此外，突变亲 - 通过本构途径被部分遗漏并分泌，为这些患者提供了肥胖表型的分子基础。 我们还利用了对激素的排序基序的知识，用于重定向到本构途径的生物活性突变激素。目前，这种突变激素以用于系统性分泌的唾液腺中表达，最终目的是将这种技术应用于基因疗法。 最近，我们还研究了激素加工酶，CPE和激素转化酶1和2（PC1和PC2）的分类。我们已经表明，这些酶是跨膜蛋白，其在C末端具有非典型膜跨性别结构域。通过涉及将其C-末端跨膜结构域插入胆固醇 - 糖果氧化脂蛋白富含微域的脂质微域称为脂质筏的新机制，它们被分类为神经内分泌细胞中RSP的颗粒。从分泌颗粒膜中除去胆固醇，导致CPE（RSP分类受体结合货物）无能为力。通过治疗洛伐他汀的细胞治疗胆固醇的耗竭导致对RSP缺乏对CPE的排序。因此，与富含胆固醇的脂质筏的膜缔合对于将激素加工酶分类为TGN和分泌颗粒至关重要。我们还表明，在颗粒胞外增生后，将CPE从质膜回收为TGN，并且该酶的内在化取决于其细胞质结构域与ARF 6，GTPase ADP-核糖基化因子的新型物理相互作用。 在另一个项目中，我们研究了控制TGN大型致密颗粒（LDCG）的因素，这对于调节神经内分泌细胞的激素和神经肽的分泌至关重要。我们的研究发现了一个ON/OFF开关，即Chomogranin A（CGA），该开关控制了神经内分泌细胞中LDCG的形成。使用反义技术在大鼠PC12细胞中耗尽CGA导致了LDCG，调节分泌和包括CGB和SynaptoTagagmin在内的颗粒蛋白的降解。这些细胞中牛CGA的过表达营救了野生型表型。在突变的内分泌细胞系中，缺乏CGA，LDCG和受调节的激素分泌的6T3，CGA的转染恢复了这些细胞中的野生型表型。在没有颗粒生物发生的情况下，我们最近将高尔基体确定为分泌颗粒蛋白降解的部位。因此，我们建议通过CGA对高尔基体在高尔基体上的稳定性调节可能是神经内分泌细胞中颗粒生物发生的控制点。最近，使用反义mRNA转基因小鼠模型在CGA中证明了CGA在大型核心颗粒生物发生中的重要性。这些小鼠在肾上腺中表现出严重的异常颗粒形成。