Biosynthesis, Processing & Secretion Of Neuropeptides

生物合成、加工

• 批准号: 7198229
• 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/7198229
• 关键词: Golgi apparatus; PC12 cells; binding sites; biological signal transduction; brain derived neurotrophic factor; carboxypeptidase; chromogranins; diabetes mellitus; enkephalins; gene mutation; genetically modified animals; hormone regulation /control mechanism; hyperinsulinism; intracellular transport; laboratory mouse; neuropeptides; obesity; 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 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, 12-15? 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 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 sorting signal/receptor mediated mechanism for targeting prohormones, neuropeptides and the neurotrophin, BDNF, to the regulated secretory pathway in endocrine cells and neurons. 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. Interestingly, CPE knockout mice exhibit impaired glucose clearance and were insulin resistant. High levels of leptin in plasma were present in the KO mice, however, they showed no circulating fully-processed cocaine-amphetamine regulated transcript (CART), a peptide that is responsive to leptin-induced feedback inhibition of feeding. Aside from obesity and diabetes phenotypes, the KO mice were subfertile and showed deficits in GnRH processing in hypothalamus. They also showed abnormal retinograms and behavioral analyses revealed that the KO animals have diminished reactivity to stimuli, and reduced muscle strength, coordination and visual placing and toe-pinch reflexes. Thus CPE KO mice display a wide-range of neural and endocrine abnormalities and will be a useful model for understanding diseases such diabetes, obesity and deficits in learning and memory. 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 showed that all members of the family bearing the (Phe34Leu) mutation had only pro - and intermediate CART in their circulation, but no mature CART. In contrast , normal humans and the unaffected sibling have significant amounts of circulating CART. Cell biological studies demonstrated that mutant pro-CART was partially missorted, poorly processed to bioactive CART and was secreted via the constitutive pathway, in contrast to wild type proCART which was properly sorted, processed and secreted in the regulated secrtetory pathway. The reduction in levels of bioactive CART in humans is likely due to cellular missorting to the constitutive pathway which does not have the appropriate enzymes to process proCART. Thus the hyperphagia and obesity observed in humans bearing the CART (Leu34Phe) mutation is likely attributed to a putative defficiency of bioactive CART in the brain of these patients. Our recent studies showed that prohormone and neuropeptide processing enzymes, carboxypeptidase E (CPE) and prohormone convertases 1 and 2 (PC1 and PC2), 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 transmembrane association of their C-terminal 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 prohormones and their processing enzymes to the TGN. The importance of cholesterol in secretory granule biogenesis and in packaging of cargo into granules was analysed in vivo, in the pancreas of cholesterol-deficient mouse models of Smith-Lemli Opitz Syndrome (SLOS) and lathosterolosis (Sc5d-/-). SLOS and lathosterolosis are human disorders, respectively defective in 7-dehydrocholesterol reductase and lathosterol 5-desaturase, enzymes necessary for the final steps of cholesterol synthesis. Morphological analysis by light and electron microscopy of neonatal pancreas zymogen granules showed a marked decrease in the number of granules, as well as aberant granule morphology in both SLOS and Sc5d-/- versus control mice. Furthermore, in primary cultures of cholesterol deficient secretory cells in the exocrine pancreas, protein synthesis and regulated secretion were impaired as compared to control cells. Thus cholesterol defficiency in SLOS and Sc5d-/- impairs regulated secretory pathway granule biogenesis and maturation, leading to deficits in the secretory function in the exocrine pancreas, and possibly also in the endocrine and nervous systems. In another project we have studied the protein 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 demonstrate that chromogranin A (CgA) controls the formation of LDCG in neuroendocrine cells ex vivo and in vivo. We showed that an antisense mRNA transgenic mouse model deficient in CgA had severe aberrant granule formation quantitatively and qualitatively in the adrenal medulla.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, there was a deficit in LDCGs, regulated hormone secretion and degradation of granule proteins. We recently identified the Golgi as the site of degradation of the secretory granule proteins in these cells. Transfection of CgA into the 6T3 cells rescued the protein degradation and promoted granule biogenesis. Thus we propose that regulation of the stability of granule proteins at the Golgi by CgA is a mechanism f

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。研究了对RSP的促蛋白酶素（POMC，促胰岛素/内啡肽），促胰岛素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。定点诱变研究确定了由两个酸性残基组成的共识分类基序，12-15？除了彼此之间，暴露在这些分子的表面上，两个疏水残基5-7？远离将这些蛋白质分类为RSP所需的酸性残基。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。这些研究为靶向型促荷抗激素，神经肽和神经营养蛋白BDNF的分类信号/受体介导的机制提供了证据。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。 有趣的是，CPE敲除小鼠表现出受损的葡萄糖清除率，并且具有胰岛素耐药性。但是，在KO小鼠中存在高水平的血浆瘦素，但是，它们没有循环全面加工的可卡因 - 原苯丙胺调节的转录本（CART），这是一种对瘦素诱导的反馈抑制喂养的反应的肽。除了肥胖和糖尿病表型外，KO小鼠是子系数，并且在下丘脑的GNRH加工中显示出缺陷。他们还显示出异常的视网膜图和行为分析表明，KO动物对刺激的反应性降低，肌肉强度，协调和视觉放置以及脚趾刺激的反射。因此，CPE KO小鼠表现出广泛的神经和内分泌异常，并将成为理解此类糖尿病，肥胖和学习和记忆缺陷的疾病的有用模型。 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，带有（Phe34Leu）突变的家族成员只有pro pro-循环中的中间手推车，但没有成熟的购物车。相比之下，正常的人和未受影响的兄弟姐妹具有大量的循环车。细胞生物学研究表明，与野生型Procart相比，突变体pro-cart被部分遗漏，对生物活性推车的处理不佳，并通过本构途径分泌，该途径在受调节的保密途径中进行了适当的分类，处理和分泌。人类生物活性推车水平的降低可能是由于细胞遗漏到本构途径上，该途径没有适当的酶来处理Procart。因此，在携带手推车（LEU34PHE）突变的人类中观察到的心脏和肥胖可能归因于这些患者大脑中的生物活性推车的假定脱水。 我们最近的研究表明，激太激素和神经肽加工酶，羧肽酶E（CPE）和蛋白激素转化酶1和2（PC1和PC2）是跨膜蛋白，其在C-末端具有相同的膜跨度域。它们通过涉及将其C-末端结构域跨膜缔合到TGN的新机制中，该机制涉及将其C-末端结构域跨膜缔合到胆固醇 - 糖脂磷脂中的胆固醇 - 糖果脂域中，称为脂质筏。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对蛋白激素的分类及其加工酶对TGN至关重要。在体内分析了胆固醇在分泌颗粒生物发生和将货物包装到颗粒中的重要性，在体内，在史密斯 - 莱姆利OPITZ综合征（SLOS）和lathostropis（SC5D-/ - / - / - ）的胆固醇缺陷小鼠模型中。 SLOS和Lathosterolisois是人类疾病，分别在7-甲基胆固醇还原酶和lathosterol 5-尿素酶中分别有缺陷，这是胆固醇合成的最终步骤所必需的酶。新生儿胰腺酶颗粒的光和电子显微镜的形态学分析显示，颗粒的数量显着减少，而SLOS和SC5D - / - 与对照小鼠相比，颗粒的数量显着减少，以及Aberant颗粒的形态。此外，与对照细胞相比，在外分泌胰腺中胆固醇缺乏分泌细胞的原发性培养物中，蛋白质合成和调节分泌受损。因此，SLOS和SC5D - / - 的胆固醇脱酸会损害受调节的分泌途径颗粒生物发生和成熟，从而导致外分泌胰腺分泌功能缺陷，也可能在内分泌和神经系统中。 在另一个项目中，我们研究了蛋白质因子的蛋白质因子，该因子是在TGN上形成大型核颗粒（LDCG）的蛋白质因子，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。我们的研究表明，染色体蛋白A（CGA）控制神经内分泌细胞在体内和体内的形成。我们表明，在肾上腺髓质中，使用反义的固定形成了CGA缺乏的反义mRNA转基因小鼠模型。使用反义技术在大鼠PC12细胞中进行了定量和质量的形成。cGA的消耗导致LDCG丧失，调节的分泌物，调节的分泌物，包括颗粒蛋白包括CGB和cgb蛋白。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的6T3，并非颗粒蛋白的激素分泌和降解。我们最近将高尔基体确定为这些细胞中分泌颗粒蛋白降解的部位。将CGA转染到6T3细胞中挽救了蛋白质降解并促进了颗粒生物发生。因此，我们提出，CGA对高尔基颗粒蛋白在高尔基体上的稳定性的调节是一种机制F