Cell Biology of Bioactive Peptide Secretion

生物活性肽分泌的细胞生物学

基本信息

批准号：
8034505
负责人：
RICHARD E MAINS
金额：
$ 10万
依托单位：
UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-03-15 至 2011-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8034505
关键词：
ARHGEF1 gene Actins Address Affect Alzheimer&apos s Disease Anabolism Binding Biogenesis Biological Assay CRH gene Cell Culture Techniques Cell Nucleus Cell physiology Cells Cellular biology Complex Coronary Arteriosclerosis Corticotropin Cytoplasmic Granules Cytoskeleton DH Domain Data Endocrine Enzymes Epigenetic Process Essential Genes Exons Face Family Fluorescence G Protein-Coupled Receptor Signaling Gene Expression Genes Genetically Engineered Mouse Growth Guanine Nucleotide Exchange Factors Guanosine Triphosphate Health Hippocampus (Brain)Hormones Human In Vitro Individual Injection of therapeutic agent Integral Membrane Protein Knock-out Knockout Mice Knowledge Link Lipids Mediating Membrane Membrane Lipids Membrane Proteins Mixed Function Oxygenases Mus Mutation Neurophysiology - biologic function Nuclear Partner in relationship Pathway interactions Patients Peptides Phosphotransferases Physiological Pituitary Gland Pituitary Hormones Play Pro-Opiomelanocortin Production Protein Isoforms Proteins Proteomics RNA Splicing Recycling Regulation Role SH3 Domains Schizophrenia Secretory Vesicles Signal Transduction Site Somatotropin Spectrin Structure Subfamily lentivirinae Swelling Testing Variant Vasopressins Vesicle cell type early onset gel electrophoresis genetic analysis genetic linkage analysis improved in vitro testing in vivo inhibitor/antagonist mutant neoplastic cell neurotransmission peptide hormone peptidylglycine alpha-amidating monooxygenase promoter protein complex protein protein interaction public health relevance recombinase restraint stress rho rho GTP-Binding Proteins secretion process tool trafficking tumor two-dimensional vacuolar H+-ATPase

项目摘要

DESCRIPTION (provided by applicant): Proper control of the synthesis, storage and secretion of bioactive peptides is crucial to normal endocrine and neural function. Over the past several decades, we delineated the cell-type specific enzymatic steps leading from proopiomelanocortin (POMC) to its many product peptides. We selected one of these enzymes, peptidylglycine 1-amidating monooxygenase (PAM), as a focus of our studies because it is an integral membrane protein that can communicate information about the lumen of the regulated secretory pathway to cytosolic machinery and to the nucleus. Our focus on PAM led to the discovery of Kalirin, a GDP/GTP exchange factor (GEF) for small GTP binding proteins of the Rho family, that interacts with the cytosolic domain of PAM. Linkage studies associating Kalirin with coronary artery disease, the decrease in Kalirin expression associated with elevated iNOS in Alzheimer disease hippocampus, and the identification of Kalirin as one of the proteins essential for Ras-mediated epigenetic silencing of gene expression, motivate our studies of this complex, multidomain protein. By flanking an exon common to the major splice variants of Kalirin with loxp sites, Kalirin conditional knockout mice (KalCKO/CKO) and mice lacking the major isoforms of Kalirin (KalKO/KO) were created. While not yet well characterized, it is clear that normal storage and secretion of pituitary hormones require pituitary Kalirin. Using lentiviruses or mating with mice in which expression of Cre recombinase is driven by the POMC or growth hormone promoter, Kalirin expression in corticotropes or somatotropes will be eliminated. POMC and GH synthesis, processing and secretion will be evaluated in vivo and in cell culture. The hypothesis that G1q-mediated activation of the second GEF domain of Kalirin plays a key role in the ability of corticotropes to respond to specific secretagogues will be tested in vitro and in vivo. Endocytic trafficking of PAM will be evaluated to determine the role of Kalirin in recycling granule membrane proteins and PAM-mediated nuclear signaling. Proteomic analysis will be carried out on isolated immature granules, whose formation is regulated by Kalirin, and on isolated pituitary granules formed in the absence of Kalirin. Assays to detect activation of Rho GEFs and their effectors will be used to understand their physiological role in pituitary hormone secretion. Finally, we will use knowledge of its individual domains to test the hypothesis that Kalirin functions as a modular machine, coordinating multiple aspects of granule biogenesis and release. In particular, the physiological importance of the alternate N-termini of Kalirin, the ability of Kalirin to inhibit iNOS, and the ability of Kalirin to integrate signals from multiple pathways will be assessed in vivo and explored mechanistically in cell culture. PUBLIC HEALTH RELEVANCE: Precise control of the biosynthesis, storage and secretion of bioactive peptides requires the coordinate control of many different cellular processes. Genetically engineered mice were used to determine that Kalirin, a large protein with domains that allow it to interact with immature secretory granules, regulate the actin cytoskeleton, bind to lipid membranes and respond to multiple protein/protein interactions, plays an essential role in peptide hormone release. Mutations in the Kalirin gene or changes in Kalirin expression have been correlated with early-onset coronary artery disease, schizophrenia and Alzheimer Disease, making a better understanding of the functions of this complex protein relevant to human health.

描述（由申请人提供）：适当控制生物活性肽的合成，储存和分泌对于正常内分泌和神经功能至关重要。在过去的几十年中，我们描绘了从proopiomelanocortin（POMC）导致其许多产品肽的细胞类型特异性酶促步骤。我们选择了这些酶之一，即肽基甘氨酸1-增强单加氧酶（PAM），作为我们研究的重点，因为它是一种成符膜蛋白，可以传达有关细胞质机械和核和原子质的受调节分泌途径的信息。我们对PAM的关注导致发现Kalirin是Rho家族的小GTP结合蛋白的GDP/GTP交换系数（GEF），该蛋白与PAM的胞质结构域相互作用。将卡利林与冠状动脉疾病相关联的连锁研究，与阿尔茨海默氏病海马iNOS升高有关的卡利林表达的降低，以及将kalirin鉴定为Kalirin作为RAS介导的基因表达的表观遗传沉默所必需的蛋白质之一，激励我们对这种复杂的这种复杂的研究，多细胞质体蛋白质。通过在Kalirin的主要剪接变体中使用LOXP位点的主要剪接变体，Kalirin条件敲除小鼠（Kalcko/CKO）和缺乏Kalirin（Kalko/KO）的主要同工型的小鼠。虽然还没有很好的特征，但很明显，垂体激素的正常存储和分泌需要垂体卡利林。使用慢病毒或与小鼠交配，其中CRE重组酶的表达是由POMC或生长激素启动子驱动的，将消除皮质激素或体状中的Kalirin表达。 POMC和GH合成，加工和分泌将在体内和细胞培养中进行评估。 g1q介导的第二个GEF结构域的G1Q介导的激活在皮质体对特定促分泌物反应的能力中起关键作用的假设将在体外和体内进行测试。将评估PAM的内吞运输，以确定卡利林在回收颗粒膜蛋白和PAM介导的核信号传导中的作用。蛋白质组学分析将在分离的未成熟颗粒上进行，其形成由卡利林（Kalirin）调节，并在没有卡利林（Kalirin）的情况下形成的分离的垂体颗粒。检测Rho GEF及其效应子的分析将用于了解其在垂体激素分泌中的生理作用。最后，我们将使用其各个领域的知识来检验卡利林（Kalirin）作为模块化机器的假设，从而协调颗粒生物发生和释放的多个方面。特别是，Kalirin的替代N-末端，卡利林抑制iNOS的能力以及卡利林在体内评估了多个途径的信号的能力的生理重要性，并在细胞培养中进行了机械探索。公共卫生相关性：生物合成，存储和分泌生物活性肽的精确控制需要对许多不同细胞过程进行协调控制。基因工程的小鼠用于确定Kalirin是一种具有域的大蛋白，使其与未成熟的分泌颗粒相互作用，调节肌动蛋白细胞骨架，与脂质膜结合并响应多种蛋白质/蛋白质相互作用，在肽激素释放中起着重要的作用。卡利林基因的突变或卡利林表达的变化与早期发作的冠状动脉疾病，精神分裂症和阿尔茨海默氏病有关，从而更好地了解了这种与人类健康相关的复杂蛋白质的功能。