Cell Biology of Bioactive Peptide Secretion

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，我们描绘了细胞类型的特定酶促步骤，导致阿黑皮素原（POMC）的许多产品肽。我们选择了这些酶之一，肽基甘氨酸1-酰胺化单加氧酶（PAM），作为我们的研究重点，因为它是一个完整的膜蛋白，可以沟通的信息调节分泌途径的细胞质机制和细胞核的管腔。我们对PAM的关注导致了Kalirin的发现，Kalirin是Rho家族的小GTP结合蛋白的GDP/GTP交换因子（GEF），其与PAM的胞质结构域相互作用。将Kalirin与冠状动脉疾病关联起来的连锁研究，与阿尔茨海默病海马中iNOS升高相关的Kalirin表达减少，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质的研究。通过将Kalirin的主要剪接变体共有的外显子与loxp位点侧接，产生Kalirin条件性敲除小鼠（KalCKO/CKO）和缺乏Kalirin的主要同种型的小鼠（KalKO/KO）。虽然尚未得到很好的表征，但很明显，垂体激素的正常储存和分泌需要垂体Kalirin。使用慢病毒或与Cre重组酶的表达由POMC或生长激素启动子驱动的小鼠交配，将消除促肾上腺皮质激素或促生长激素中的Kalirin表达。将在体内和细胞培养物中评价POMC和GH的合成、加工和分泌。G1q介导的Kalirin的第二GEF结构域的激活在促肾上腺皮质激素响应特定促分泌素的能力中起关键作用的假设将在体外和体内进行测试。将评价PAM的内吞运输以确定Kalirin在再循环颗粒膜蛋白和PAM介导的核信号传导中的作用。将对分离的未成熟颗粒（其形成受Kalirin调节）和在不存在Kalirin的情况下形成的分离的垂体颗粒进行蛋白质组学分析。检测Rho GEF及其效应物的活化的测定将用于了解它们在垂体激素分泌中的生理作用。最后，我们将使用其各个领域的知识来检验Kalirin作为模块化机器发挥作用的假设，协调颗粒生物发生和释放的多个方面。特别地，Kalirin的交替N-末端的生理重要性、Kalirin抑制iNOS的能力和Kalirin整合来自多个途径的信号的能力将在体内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，一个大的蛋白质与域，使其能够与未成熟的分泌颗粒相互作用，调节肌动蛋白细胞骨架，结合到脂质膜和响应多个蛋白质/蛋白质的相互作用，在肽激素释放中起着至关重要的作用。Kalirin基因的突变或Kalirin表达的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质的功能。

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.