Biosynthesis, Processing And Secretion Of Neuropeptides

神经肽的生物合成、加工和分泌

• 批准号: 7333363
• 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/7333363
• 关键词: Biosynthesis; Processing; Secretion; Neuropeptides

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 RSP for processing and secretion. Site-directed mutagenesis studies identified consensus sorting motifs 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). 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. BDNF which modulates synaptic plasticity was missorted in cortical and hippocampal neurons, and that may 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 BDNF, to the regulated secretory pathway in endocrine cells and neurons. We have used our knowledge of the sorting motif of hormones to engineer biologically active mutant hormones, e.g. mutant proinsulin that we have been able to express in salivary glands and redirect to the constitutive pathway for systemic secretion. The ultimate aim is to apply such technology to gene therapy in the treatment of diseases such as diabetes. 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 secretory 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 deficiency of bioactive CART in the brain of these patients. Recently, the role of CART in bone remodeling was investigated. We show that the CPE KO mouse lack plasma and hypothalamic CART, unlike WT mice. Bone density scans revealed that CPE KO mice had lower bone density compared to WT mice in both males and females. Furthermore, the osteoblast numbers were significantly reduced, while the osteoclast numbers were significantly increased in the CPE KO animals. This finding is consistent with the literature showing that CART-/- mice have increased osteoclast numbers and further supports a role of CART in bone remodeling. However, the CART-/- mice did not show a decrease in osteoblast number suggesting the exciting possibility that othher neuropeptides or peptide hormones known to be missing in the CPE KO mouse may play a role in osteoblast formation or survival. The role of CPE in the nervous system was investigated. Behavioral analyses revealed that CPE KO animals had diminished reactivity to stimuli, delayed learning in the water-maze test and abnormal neurotransmission from the photoreceptors to the inner retina, showing a loss of the b wave in their retinogram. Electron microscopic studies indicated that there were significantly decreased numbers of synaptic vesicles in the synaptic butons of the photo receptors of CPE KO mice, suggesting a defect in transport of synaptic vesicles. Indeed our recent studies indicate that CPE plays a role in transport and movement of peptidergic vesicles to the release site in endocrine cells and neurons. Absence of CPE in the KO mice could lead to failure in neurotransmission, deficits in learning and memory, and abnormal behavior. The importance of cholesterol in secretory granule biogenesis was investigated in vivo, in 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 of neonatal pancreas zymogen granules showed a marked decrease in the number of granules in both SLOS and Sc5d-/- versus control mice. Of the granules present in SLOS and Sc5d-/-animals, most were of an immature phenotype as compared to control animals, appearing as partially formed spheres. Furthermore, in primary cultures of cholesterol deficient secretory cells in the exocrine pancreas, granule biogenesis and regulated secretion of amylase was impaired. Addition of exogenous cholesterol to these cells rescued the phenotype. Granule biogenesis was also impaired in endocrine tissues of Sc5d-/- mice. We show that the defect in granule biogenesis and maturation is due to different physical contributions of sterols to membrane curvature. Lathosterol and 7-dehydro-cholesterol has a lower binding rigidity than cholesterol and exhibit poorer ability to form curvature. Thus genetic inhibition of cholesterol synthesis in SLOS and Sc5d-/- mice impairs regulated secretory pathway granule biogenesis and maturation, leading to deficits in the secretory function in the exocrine and endocrine systems. We have also investigated the protein factors governing the formation of large dense-core granules (LDCG)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. 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 resulted in the loss of LDCG, regulated secretion, and degradation of granule proteins. 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 found a protease inhibitor, protease nexin-1 (PN-1) in the Golgi in 6T3 cells that is transcriptionally activated by CgA. Transfection of PN-1 into 6T3 cells lacking CgA prevented LDCG protein degradation and rescued granule biogenesis. Furthermore, 6T3 cells incubated with conditioned medium from 6T3-bCgA cells expressing CgA, increased PN-1 mRNA and granule biogenesis. Thus CgA/CgA derived peptides secreted into the medium may bind to a receptor and transcriptionally activate expression of a protease inhibitor, PN-1, which then stabilizes granule proteins at the TGN to promote LDCG biogenesis in endocrine cells.

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。这样的研究使人们对与激素和神经肽靶向，肥胖，糖尿病，记忆和学习相关的疾病有了更好的了解。我们表明，这些前蛋白会作为浓度步骤进行同型寡聚化，因为它们从内质网中的合成部位横穿细胞到反式高尔基网络（TGN），并将它们分类为RSP的密集核颗粒进行处理和分泌。定点诱变研究确定了将这些前蛋白分类为RSP所必需的共识分类基序。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）。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。调节突触可塑性的BDNF在皮质和海马神经元中被错过，这可能是CPE KO小鼠中观察到的记忆缺陷。这些研究提供了一种证据，证明了靶向促炎性，神经肽和BDNF的分类信号/受体介导的机制，可用于内分泌细胞和神经元中受调节的分泌途径。我们已经利用了对激素的分类基序的知识来设计具有生物活性的突变激素，例如我们已经能够在唾液腺中表达并重定向到全身分泌的概要途径的突变蛋白。最终目的是将这种技术应用于基因治疗，以治疗糖尿病等疾病。 研究了在肥胖患者家族中发现的突变形式的推车形式的分类和加工。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，突变的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，该途径在受调节的分泌途径中进行了正确分类，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缺乏。最近，研究了卡车在骨骼重塑中的作用。我们表明，与WT小鼠不同，CPE KO小鼠缺乏血浆和下丘脑推车。骨密度扫描表明，与男性和女性的WT小鼠相比，CPE KO小鼠的骨密度较低。此外，成骨细胞数量显着降低，而CPE KO动物的破骨细胞数则显着增加。这一发现与文献一致，表明卡特 - / - 小鼠的破骨细胞数量增加，并进一步支持购物车在骨骼重塑中的作用。但是，卡车 - / - 小鼠没有显示成骨细胞数量的减少，这表明在CPE KO小鼠中已知的神经肽或肽激素可能在成骨细胞形成或存活中起作用。 研究了CPE在神经系统中的作用。行为分析表明，CPE KO动物对刺激的反应性降低，在水迷宫测试中延迟学习以及从光感受器到内部视网膜的神经传递异常，显示其视网膜图中B波的丧失。电子显微镜研究表明，在CPE KO小鼠的照片受体的突触受体中，突触囊泡数量明显减少，这表明突触囊泡的运输缺陷。实际上，我们最近的研究表明，CPE在肽基囊泡向内分泌细胞和神经元中释放位点的运输和运动中起作用。 KO小鼠中缺少CPE可能会导致神经传递，学习和记忆缺陷以及异常行为失败。 在体内研究了胆固醇在分泌颗粒生物发生中的重要性，在史密斯 - 莱姆利OPITZ综合征（SLOS）和Lathosterolosis（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形态学分析显示，SLOS和SC5D - / - 与对照小鼠的颗粒数显着减少。与对照动物相比，SLOS和SC5D - / - 动物中存在的颗粒中，大多数是未成熟的表型，它们是部分形成的球体。此外，在外分泌胰腺中胆固醇缺乏分泌细胞的原发性培养中，颗粒生物发生和淀粉酶的调节分泌受损。在这些细胞中添加外源胆固醇挽救了表型。 SC5D - / - 小鼠的内分泌组织也受损颗粒生物发生。我们表明，颗粒生物发生和成熟的缺陷是由于固醇对膜曲率的不同物理贡献所致。 laterosterol和7-脱氢 - 胆固醇的结合刚度低于胆固醇，并且表现出形成曲率的能力较差。因此，遗传抑制SLOS和SC5D - / - 小鼠中胆固醇合成的损害会损害受调节的分泌途径颗粒生物发生和成熟，从而导致外分泌和内分泌系统中分泌功能的缺陷。 我们还研究了控制大型核颗粒（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中的LDCG的形成。 CGA缺乏的反义mRNA转基因小鼠模型在肾上腺髓质中定量和质量地形成严重的异常颗粒。 CGA在大鼠PC12细胞中的耗竭导致LDCG的损失，调节分泌和颗粒蛋白的降解。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的6T3，并非颗粒蛋白的激素分泌和降解。我们在6T3细胞中的高尔基体中发现了一种蛋白酶抑制剂，蛋白酶Nexin-1（PN-1），该蛋白酶抑制剂是由CGA转录激活的。将PN-1转染到缺乏CGA的6T3细胞中，阻止了LDCG蛋白质降解并营救了颗粒生物发生。此外，从表达CGA的6T3-BCGA细胞中与条件培养基孵育的6T3细胞增加了PN-1 mRNA和颗粒生物发生。因此，分泌到培养基中的CGA/CGA衍生的肽可能与受体结合并转录激活蛋白酶抑制剂PN-1的表达，然后将颗粒蛋白稳定在TGN上，以促进内分泌细胞中的LDCG生物发生。