Biosynthesis, Processing And Secretion Of Neuropeptides And Pituitary Hormones

神经肽和垂体激素的生物合成、加工和分泌

• 批准号: 7734662
• 负责人: Yoke p Loh
• 金额: $ 117.9万
• 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/7734662
• 关键词: AQP1 gene; Adult; Amino Acids; Anabolism; Animals; Anterior Pituitary Gland; Antisense RNA; Apoptosis; Behavioral Assay; Binding; Binding Proteins; Biogenesis; Biological Assay; Brain; Brain-Derived Neurotrophic Factor; C-terminal; Cell Line; Cell Nucleus; Cell membrane; Cells; Cellular Neurobiology; Cellular biology; Chromogranin A; Chromosome Pairing; Corticotropin; Cytoplasm; Cytoplasmic Granules; Cytoplasmic Tail; Cytosol; Defect; Dense Core Vesicle; Diabetes Mellitus; Disease; Docking; Dynein ATPase; Eating; Electrons; Endocrine; Endopeptidases; Enzymes; Exocytosis; Fluorescence; Generations; Genetic Transcription; Glutamates; Glutathione S-Transferase; Golgi Apparatus; Hippocampus (Brain); Hormones; Hydrogen Peroxide; Hypothalamic structure; In Vitro; Knock-out; Knockout Mice; Lead; Learning; Length; Life; Link; Long-Term Potentiation; Measurement; Mediating; Memory; Messenger RNA; Microscopic; Microtubules; Molecular; Motor; Movement; Mus; Names; Nerve Degeneration; Nervous system structure; Neuroendocrine Cell; Neurons; Neuropeptides; Numbers; Obesity; Organelles; Oxidative Stress; PC12 Cells; POMC gene; Pathway interactions; Peptide Hydrolases; Peptides; Physiologic pulse; Physiological; Pituitary Gland; Pituitary Hormones; Play; Precipitation; Primary Cell Cultures; Pro-Opiomelanocortin; Process; Proinsulin; Proprotein Convertase 2; Protease Inhibitor; Protein Overexpression; Proteins; Pulse taking; Recruitment Activity; Regulation; Relative (related person); Research; Residual state; Role; Role playing therapy; Secretory Vesicles; Signal Transduction; Slice; Sorting - Cell Movement; Synapses; Synaptic Transmission; Synaptic Vesicles; Synaptic plasticity; Tail; Testing; Time; Tissues; Vesicle; Week; Work; base; brain cell; carboxypeptidase H; cellular imaging; density; dynactin; in vivo; inhibitor/antagonist; intracellular protein transport; knockout animal; membrane activity; morris water maze; mouse model; neurotrophic factor; nexin; novel; peptide hormone; preference; presynaptic; prevent; prohormone; protein degradation; protein transport; receptor; response; secretion process; secretogranin III; trafficking; water channel

The intracellular sorting and packaging of pro-neuropeptides, prohormones and neurotrophins to the granules of the regulated secretory pathway (RSP) is essential for processing, storage and release of active proteins and peptides in the neuroendocrine cell. Processing of these proproteins takes place in the secretory granules and therefore the proprotein converting enzymes (PC1/3, PC2 and carboxypeptidase E) must also be sorted into these organelles. We have investigated the sorting of pro-opiomelanocortin (POMC, pro-ACTH/endorphin), proinsulin and brain derived neurotrophic factor (BDNF) to the RSP. Such studies will lead to the better understanding of diseases related to defects in hormone and neuropeptide targeting such as; obesity, diabetes, memory and learning. Previously we have shown that carboxypeptidase E (CPE) has a non-enzymatic role, acting as a binding protein to facilitate the sorting of prohormones such as POMC into the granules of the RSP. Using a CPE knockout (KO) mouse model, we showed that 50% of newly synthesized POMC in primary cultures of the pituitary anterior lobe cells was degraded and suggests that in the absence of efficient sorting to the granules of the RSP due to the lack of CPE, POMC was targeted for degradation. However, some of the remaining POMC was sorted into the RSP. A candidate for a compensatory sorting receptor is Secretogranin III (SgIII) which has been shown to bind POMC in precipitation assays. Analysis of pituitary extracts show that the full length 65 kDa form, which was the form shown to bind POMC, is increased in the KO pituitary suggesting that this increase may facilitate sorting of the residual POMC into the RSP. The mechanism regulating the formation of large dense-core secretory granules (LDCGs) into which the prohomones are packaged is another major focus of our research. We previously showed that chromogranin A (CgA) plays a critical role in the control of LDCG formation and that the mechanism was through the control of granule protein degradation within the Golgi by regulating the level of a protease inhibitor. We identified the inhibitor as protease nexin-1 (PN-1) which expression was up-regulated transcriptionally by CgA (or a fragment of CgA). PN-1 prevented granule protein degradation and increased LDCG formation when up-regulated, but when reduced in its expression by PN-1 antisense-RNA, the proteins were degraded and LDCGs were not made. We hypothesized that CgA might be signaling the cell to increase the levels of PN-1 in response to reduced LDCGs after stimulated secretion. To this end we have identified a 26 amino acid C-terminal fragment of CgA in secretion medium, which we named serpinin, that was able to enhance PN-1 transcription and granule biogenesis in 6T3 cells, an endocrine cell line that normally lacks LDCGs. Serpinin was elevated in the medium after high K+ stimulation of AtT-20 cells. We propose that serpinin binds to a cognate receptor on the plasma membrane to cause signaling to the nucleus to enhance PN-1 mRNA transcription. Thus we have discovered a new CgA-derived peptide, serpinin, which is co-secreted with POMC-derived hormones upon stimulation of pituitary corticotrophs that signals replenishment of LDCGs by transcriptionally up-regulating the expression of a protease inhibitor, PN-1. PN-1 then stabilizes granule proteins resulting in increased LDCG biogenesis. Interestingly, in addition to PN-1, we found that the water channel protein, Aquaporin 1 (AQP1) was also up-regulated in its expression by CgA. We have now demonstrated that AQP1 is present in LDCGs of endocrine tissues and appears to play an integral role in the formation and/or function of the RSP. When down-regulated in its expression in AtT20 cells by antisense-RNA, the number of LDCGs decreased by 66% and in pulse-chase studies there was a defect in regulated secretion of the endogenous ACTH hormone, as well as increased degradation of newly synthesized granule proteins such as POMC and CPE at the Golgi apparatus. This indicated that AQP1 is necessary for maintaining hormone secretion and granule biogenesis in corticotrophs. Indeed, AQP1 knockout mice showed decreased circulating ACTH relative to control mice, thereby establishing for the first time an important intracellular role of this water channel in endocrine function in vivo. We have also investigated the mechanism of post-Golgi transport and delivery of hormone and BDNF vesicles to the plasma membrane for activity-dependent secretion which is critical for endocrine function and synaptic plasticity. We showed that the CPE cytoplasmic tail on these secretory granules plays an important role in their transport. Overexpression of the CPE cytoplasmic tail in the cytoplasm to compete with the endogenous tail diminished localization of endogenous POMC, BDNF and fluorescence-tagged CPE in the processes of an endocrine cell line, AtT20; and hippocampal neurons. In live hippocampal neurons, primary pituitary and AtT20 cell images, overexpression of the CPE tail inhibited the movement of BDNF- and POMC/CPE-containing vesicles to the processes, respectively. S-tagged CPE tail pulled down endogenous microtubule-based motors, dynactin (p150), dynein and KIF1A/KIF3A from cytosol of AtT20 and brain cells. Finally, overexpression of the CPE tail inhibited the regulated secretion of ACTH from AtT20 cells. Thus this study has uncovered a novel mechanism whereby the vesicular CPE cytoplasmic tail plays an important role in anchoring regulated secretory pathway POMC/ACTH and BDNF vesicles to microtubule-based motors for transport and activity-dependent secretion in endocrine cells and neurons. We recently showed that transmembrane CPE is not only associated with large dense core vesicles, but also with glutamate-containing synaptic vesicles in mouse hypothalamus. High K+ stimulated release of glutamate from cultured hippocampal neurons was absent in CPE-KO mice. Furthermore, electron microscopic analysis of 100 hypothalamic synaptic densities revealed that 40% of the synapses had no docked synaptic vesicles at the presynaptic density in CPE-KO mice in contrast to 15% for the WT mice, implicating that in some neurons, CPE may be involved in synaptic vesicle docking, possibly mediated by its cytoplasmic tail. In vitro GST pulldown assays using both brain and PC12 cell cytosol, GST tagged CPEC10, the CPE cytoplasmic tail, bound Rab27A and Rim1, molecules necessary for synaptic vesicle docking to the plasma membrane. These results suggest that the vesicular CPE cytoplasmic tail plays role in recruiting Rab27A and Rim1 necessary for docking and subsequent exocytosis of a subpopulation of synaptic vesicles in the hypothalamus. We have studied the role of CPE in the nervous system in vivo. We showed that CPE KO mice that are very obese lacked the anorexigenic neuropeptide, CART, in the hypothalamus. These animals over-eat, thus providing further evidence linking decrease of this neuropeptide to the cause of obesity. We demonstrated deficiencies in several behavioral assays including the Morris water maze and object preference tests indicating a problem with learning and memory in CPE KO mice. Indeed electrophysiological measurements have showed a defect in the generation of long term potentiation (LTP) in hippocampal slices of these mice. The major cause for this defect is due to the total degeneration of neurons in the CA3 region of the hippocampus. This was evident only in 4 week and older CPE-KO mice. Three week old KO animals were normal, suggesting that CPE is important in maintaining survival of CA3 neurons which are enriched in this enzyme. When CPE was overexpressed in hippocampal neurons, they were protected from apoptosis after induced oxidative stress using hydrogen peroxide. Thus, CPE has a novel neuroprotective role in adult hippocampal neurons.

对受调节分泌途径（RSP）的颗粒的促尿肽，促肽和神经营养蛋白的细胞内分类和包装对于神经内分泌细胞中活性蛋白和肽的加工和释放至关重要。这些普洛蛋白的加工发生在分泌颗粒中，因此也必须将这些细胞器中的原则转化酶（PC1/3，PC2和羧肽酶E）进行分类。我们已经调查了促蛋白酶皮质素（POMC，Pro-ACTH/内啡肽），促胰岛素和脑衍生的神经营养因子（BDNF）的分类。这样的研究将使人们更好地了解与激素和神经肽靶向缺陷有关的疾病。肥胖，糖尿病，记忆和学习。以前，我们已经表明羧肽酶E（CPE）具有非酶作用，充当一种结合蛋白，可促进促ho酮（例如POMC）排序RSP的颗粒中。使用CPE敲除（KO）小鼠模型，我们表明50％的新合成POMC在垂体前叶的原代培养物中被降低，并表明由于缺乏CPE而缺乏RSP颗粒的有效分类，POMC缺乏pOMC的针对性，因此是降级的。但是，将其余的POMC分类到RSP中。补偿性分类受体的候选者是秘密素蛋白III（SGIII），已显示在降水测定中结合POMC。垂体提取物的分析表明，在KO垂体中增加了全长65 kDa形式，这是显示pOMC的形式，这表明这种增加可能有助于将残留的POMC分类到RSP中。 调节大型核心分泌颗粒（LDCG）的形成的机制是我们研究的另一个主要重点。我们先前表明，染色体蛋白A（CGA）在控制LDCG形成中起关键作用，并且该机制是通过控制Golgi内颗粒蛋白降解的机制，通过调节蛋白酶抑制剂的水平。我们将抑制剂确定为蛋白酶nexin-1（PN-1），表达在CGA（或CGA的片段）上被上调。 PN-1可防止颗粒蛋白降解并在上调时形成LDCG，但是当PN-1抗义RNA降低其表达时，蛋白质会降解并未制成LDCG。我们假设CGA可能在刺激分泌后响应LDCG的响应，以提高细胞以增加PN-1的水平。为此，我们在分泌培养基中确定了CGA的26个氨基酸C末端片段，我们将其命名为Serpinin，能够增强6T3细胞中的PN-1转录和颗粒生物发生，这是一种通常缺乏LDCG的内分泌细胞系。在高K+刺激ATT-20细胞之后，在培养基中升高了SERPININ。我们提出，Serpinin与质膜上的同源受体结合，以引起对细胞核的信号以增强PN-1 mRNA转录。因此，我们发现了一种新的CGA衍生的肽Serpinin，该肽在刺激垂体皮质营养物的刺激后与POMC衍生的激素共归类，该激素通过转录上调蛋白酶抑制剂PN-1的表达来表达信号为LDCG补充。然后，PN-1稳定颗粒蛋白，从而导致LDCG生物发生增加。有趣的是，除了PN-1外，我们还发现水通道蛋白Aquaporin 1（AQP1）在其表达中也被CGA上调。现在，我们已经证明了内分泌组织的LDCG中存在AQP1，并且似乎在RSP的形成和/或功能中起着不可或缺的作用。当通过反义RNA下调其在ATT20细胞中的表达时，LDCG的数量减少了66％，在脉冲练习研究中，内源性ACTH激素的调节分泌存在缺陷，以及增加了新合成的颗粒蛋白（例如POMC和CPE）的降解，例如，诸如POMC和CPE AT golgi Att golgi Aptus。这表明AQP1对于维持皮质营养的激素分泌和颗粒生物发生是必要的。实际上，AQP1敲除小鼠相对于对照小鼠的循环ACTH降低，从而首次确定该水通道在体内内分泌功能中的重要细胞内作用。 我们还研究了高尔基后传输以及激素和BDNF囊泡向质膜传递的机制，以进行活性依赖性分泌，这对于内分泌功能和突触可塑性至关重要。我们表明，这些分泌颗粒上的CPE细胞质尾巴在其运输中起着重要作用。在细胞质中的CPE细胞质尾巴的过表达与内源性尾巴竞争，在内分泌细胞系ATT20的过程中，内源性POMC，BDNF和荧光标记的CPE的定位减少了。和海马神经元。在活的海马神经元，原发性垂体和ATT20细胞图像中，CPE尾部的过表达抑制了含BDNF-和POMC/CPE的囊泡的运动，分别抑制了该过程。 S标记的CPE尾巴从ATT20和脑细胞的胞质醇中拉下了内源微管的电动机（P150），Dynein和kif1a/kif3a。最后，CPE尾部的过表达抑制了ACTH从ATT20细胞中的调节分泌。因此，这项研究发现了一种新的机制，即囊泡CPE细胞质尾巴在锚定调节的分泌途径POMC/ACTH和BDNF囊泡中起着至关重要的作用。 我们最近表明，跨膜CPE不仅与小鼠下丘脑中含有谷氨酸的突触小泡有关，而且与大型核心囊泡有关。 CPE-KO小鼠中不存在高K+刺激从培养的海马神经元释放的谷氨酸。 Furthermore, electron microscopic analysis of 100 hypothalamic synaptic densities revealed that 40% of the synapses had no docked synaptic vesicles at the presynaptic density in CPE-KO mice in contrast to 15% for the WT mice, implicating that in some neurons, CPE may be involved in synaptic vesicle docking, possibly mediated by its cytoplasmic tail.使用脑和PC12细胞胞质溶胶，GST标记CPEC10，CPE细胞质尾巴，结合的RAB27A和RIM1的体外GST下拉分析，用于突触囊泡对质膜的突触囊泡所需的分子。这些结果表明，囊泡CPE细胞质尾巴在募集RAB27A和RIM1中起作用，对于对接和随后的胞吐作用所必需的RIM1在下丘脑中突触囊泡的亚群。 我们已经研究了CPE在体内神经系统中的作用。我们表明，非常肥胖的CPE KO小鼠缺乏下丘脑中的厌食性神经肽CART。这些动物过度食用，因此提供了进一步的证据，将这种神经肽的降低与肥胖症的原因联系起来。我们证明了几种行为分析中的缺陷，包括莫里斯水迷宫和对象偏好测试，表明CPE KO小鼠的学习和记忆存在问题。实际上，电生理测量结果显示出这些小鼠海马切片中长期增强（LTP）的产生缺陷。该缺陷的主要原因是由于海马CA3区域的神经元的总变性所致。这仅在4周及以上的CPE-KO小鼠中很明显。三周大的KO动物是正常的，这表明CPE对于维持富含该酶的Ca3神经元的存活很重要。当CPE在海马神经元中过表达时，使用过氧化氢诱导氧化应激后，它们免受凋亡。因此，CPE在成人海马神经元中具有新颖的神经保护作用。

项目成果

Oligomerization of pro-opiomelanocortin is independent of pH, calcium and the sorting signal for the regulated secretory pathway.

阿片黑皮质素原的寡聚化与 pH、钙和受调节分泌途径的分选信号无关。

• DOI: 10.1016/s0014-5793(00)01961-x
• 发表时间: 2000
• 期刊: FEBS letters
• 影响因子: 3.5
• 作者: Cawley,NX;Normant,E;Chen,A;Loh,YP
• 通讯作者: Loh,YP

Identification of a novel prohormone sorting signal-binding site on carboxypeptidase E, a regulated secretory pathway-sorting receptor.

• DOI: 10.1210/mend.13.4.0267
• 发表时间: 1999-04
• 期刊: Molecular endocrinology
• 作者: Chun-fa Zhang;Christopher R. Snell;Y. Loh

Altered biosynthesis and secretion of pro-opiomelanocortin in the intermediate and anterior pituitary of carboxypeptidase E-deficient, Cpe(fat)/ Cpe(fat)mice.

改变羧肽酶 E 缺陷 Cpe(fat)/Cpe(fat) 小鼠垂体中叶和垂体前叶阿片黑皮质素原的生物合成和分泌。

• DOI: 10.1054/npep.1999.0045
• 发表时间: 1999
• 期刊: Neuropeptides
• 影响因子: 2.9
• 作者: Shen,FS;Aguilera,G;Loh,YP
• 通讯作者: Loh,YP

Sorting of carboxypeptidase E to the regulated secretory pathway requires interaction of its transmembrane domain with lipid rafts.

• DOI: 10.1042/bj20020827
• 发表时间: 2003-02
• 期刊: The Biochemical journal
• 作者: Chun-fa Zhang;S. Dhanvantari;H. Lou;Y. Loh

Synthesis and characterization of the first potent inhibitor of yapsin 1. Implications for the study of yapsin-like enzymes.

第一个强效yapsin抑制剂的合成和表征1.对yapsin类酶研究的启示。

• DOI: 10.1074/jbc.m207230200
• 发表时间: 2003
• 期刊: The Journal of biological chemistry
• 作者: Cawley,NiamhX;Chino,Masao;Maldonado,Alex;Rodriguez,YazminM;Loh,YPeng;Ellman,JonathanA
• 通讯作者: Ellman,JonathanA