Cell Biology of Bioactive Peptide Secretion

描述（由申请人提供）：正确控制生物活性肽的合成、储存和分泌对正常内分泌和神经功能至关重要。在过去的几十年里，我们描述了从proproiomelanocortin （POMC）到其许多产物肽的细胞类型特异性酶促步骤。我们选择其中一种酶，肽酰甘氨酸1-酰胺化单加氧酶（PAM）作为我们研究的重点，因为它是一种完整的膜蛋白，可以将有关调节分泌途径的管腔的信息传递给细胞质机制和细胞核。我们对PAM的关注导致了Kalirin的发现，Kalirin是一种GDP/GTP交换因子（GEF），用于Rho家族的小GTP结合蛋白，它与PAM的细胞质结构域相互作用。Kalirin与冠状动脉疾病相关的连锁研究，Kalirin表达降低与阿尔茨海默病海马iNOS升高相关的研究，以及Kalirin作为ras介导的基因表达表观遗传沉默所必需的蛋白质之一的鉴定，激发了我们对这种复杂的多结构域蛋白的研究。通过将Kalirin的主要剪接变体所共有的外显子与loxp位点连接在一起，创建了Kalirin条件敲除小鼠（KalCKO/CKO）和缺乏Kalirin主要同工型的小鼠（KalKO/KO）。虽然尚未很好地表征，但很明显，垂体激素的正常储存和分泌需要垂体加里林。使用慢病毒或与由POMC或生长激素启动子驱动Cre重组酶表达的小鼠交配，将消除促糖物或促生长物中的Kalirin表达。POMC和GH的合成、加工和分泌将在体内和细胞培养中进行评估。g1q介导的Kalirin第二GEF结构域的激活在促肾上腺皮质激素对特定分泌剂的反应能力中起关键作用，这一假设将在体外和体内进行验证。将评估PAM的内吞运输，以确定加里林在回收颗粒膜蛋白和PAM介导的核信号传导中的作用。蛋白质组学分析将对分离的未成熟颗粒（其形成受卡利林调节）和分离的垂体颗粒（在没有卡利林的情况下形成）进行。检测Rho gef及其效应物的激活将用于了解它们在垂体激素分泌中的生理作用。最后，我们将利用其各个领域的知识来测试Kalirin作为模块化机器的假设，协调颗粒生物发生和释放的多个方面。特别是，Kalirin的交替n末端的生理重要性，Kalirin抑制iNOS的能力，以及Kalirin整合多种途径信号的能力将在体内进行评估，并在细胞培养中进行机制探索。公共卫生相关性：精确控制生物活性肽的生物合成、储存和分泌需要协调控制许多不同的细胞过程。基因工程小鼠被用来确定Kalirin，一种大结构域的蛋白质，允许它与未成熟的分泌颗粒相互作用，调节肌动蛋白细胞骨架，结合脂质膜并响应多种蛋白质/蛋白质相互作用，在肽激素释放中起重要作用。Kalirin基因突变或表达变化与早发性冠状动脉疾病、精神分裂症和阿尔茨海默病相关，使人们更好地了解这种与人类健康相关的复杂蛋白的功能。

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.