Cell Biology of Bioactive Peptide Secretion

描述(申请人提供)：适当控制生物活性多肽的合成、储存和分泌对正常的内分泌和神经功能至关重要。在过去的几十年里，我们描述了从前阿片黑素皮质素(POMC)到它的许多产物肽的细胞型特异性酶步骤。我们选择了其中的一种酶，肽甘氨酸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，可以将调节的分泌途径的管腔信息传递到细胞质机械和细胞核。我们对PAM的关注导致了Kalirin的发现，Kalirin是一种针对Rho家族小GTP结合蛋白的GDP/GTP交换因子，它与PAM的胞浆结构域相互作用。将Kalirin与冠状动脉疾病联系起来的研究，Kalirin在阿尔茨海默病海马区iNOS升高导致的表达下降，以及Kalirin是RAS介导的表观遗传沉默基因表达所必需的蛋白质之一，推动了我们对这种复杂的多结构域蛋白的研究。通过在Kalirin主要剪接变异体的共同外显子两侧加上loxP位点，创建了Kalirin条件性基因敲除小鼠(KalCKO/CKO)和缺乏Kalirin主要异构体的小鼠(KalKO/KO)。虽然还没有得到很好的描述，但很明显，正常的脑下垂体激素的储存和分泌需要脑下垂体激动素。使用慢病毒或与Cre重组酶由POMC或生长激素启动子驱动表达的小鼠交配，将消除促肾上腺皮质细胞或生长激素细胞中Kalirin的表达。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第二个全球环境基金结构域的激活在促肾上腺皮质激素对特定促分泌剂的反应能力中起关键作用的假说将在体外和体内得到验证。将评估PAM的胞内转运，以确定Kalirin在回收颗粒膜蛋白和PAM介导的核信号中的作用。蛋白质组学分析将对分离的未成熟颗粒进行，其形成受到Kalirin的调节，以及在没有Kalirin的情况下形成的分离的垂体颗粒。检测Rho GEF及其效应器激活的实验将被用来了解它们在脑下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测试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。特别是，Kalirin的N末端交替的生理意义，Kalirin抑制iNOS的能力，以及Kalirin整合来自多个途径的信号的能力将在体内进行评估，并在细胞培养中进行机械探索。公共卫生相关性：生物活性多肽的生物合成、储存和分泌的精确控制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是一种大蛋白，其结构域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，与脂膜结合，并对多种蛋白质/蛋白质相互作用做出反应，在多肽激素释放中发挥重要作用。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.