Phosphorylation-dependent Feed-forward Regulation of SREBP-1c

描述(由申请人提供)： 与肥胖相关的2型糖尿病及其并存，动脉粥样硬化和中风，在我们退伍军人老龄化的人口中都是太常见的疾病。这些疾病总是与异常的脂平衡有关，表现为过度合成和循环中极低密度脂蛋白和甘油三酯的高水平。我们研究的长期目标是从分子水平深入了解肝脂平衡和血脂异常及其心血管并发症的发病机制。固醇调节元件结合蛋白-1c(SREBP-1c)作为一种转录因子，调节肝脏中参与脂肪合成的关键基因，在这一过程中发挥着重要作用。新生pSREBP-1c位于内质网(ER)，必须运输到高尔基体，在那里它经历受调控的膜内蛋白分解(RIP)以释放其转录活性核区nSREBP-1c，激活其自身的启动子。胰岛素对肝脏SREBP-1c的前馈调节是选择性胰岛素抵抗和高脂血症及其临床后果的关键机制。根据已发表的数据和我们正在进行的研究，我们假设通过胰岛素诱导的激酶PI3K-Akt/PKB及其下游的mTOR和p70S6K的磷酸化，调节RIP介导的成熟，以及核内nSREBP-1c的反式激活效力和稳定性。此外，在慢性肥胖的啮齿动物和人类中，通过与胰岛素信号通路的串扰，MAP通路也会影响SREBP-1c的前馈调节。为了在实验上验证这一假说，我们根据已发表的观察和我们的理论分析，选择了17个位点特异性突变，AKT/mTOR/S6K和MAPK特定的磷酸化位点。基于对McArdle肝癌细胞系和原代大鼠肝细胞中WT和突变的SREBP-1c表型的严格评估，我们选择了四个位点[mTOR(S57和S140)，p70S6K(S956)和MAP激酶(S39)]来详细研究它们在SREBP-1c前馈调节中的作用。我们建议的短期目标是：(I)鉴定SREBP-1c的序列基序，参与胰岛素增强的、依赖于磷酸化的RIP调节和体内外nSREBP-1c的反式激活能力；(Ii)破译磷酸化如何改变SREBP-1c在ER到高尔基转移以及S1P和S2P蛋白酶降解过程中的分子相互作用；以及(Iii)阐明磷酸化如何改变nSREBP-1c在细胞核、体外和体内的稳定性和转录功能。为了实现这些目标，我们将使用我们独特的定制设计的腺病毒和质粒载体，在培养的大鼠肝细胞以及正常、肥胖和高胰岛素血症MC4R基因敲除大鼠的肝脏中表达标记的pSREBP-1c和nSREBP-1c蛋白的野生型和磷酸化获得或丢失突变体。我们将比较RIP介导的WT和突变的nSREBP-1c蛋白的成熟率，以及它们在胰岛素存在和不存在的情况下的反式激活潜力。我们将确定磷酸化是否/如何改变全长SREBP-1c与其他参与内质网到高尔基体转运和蛋白降解的分子之间的分子相互作用。此外，我们还将阐明磷酸化如何改变nSREBP-1c的反式激活潜力，从而激活其自身的启动子和调节肝脏脂肪合成的下游靶基因的启动子。我们的具体目标是阐明SREBP-1c在体外和体内对脂质稳态的磷酸化依赖作用的机制。这些研究的成功完成有可能揭开治疗慢性肥胖症及其对心血管系统的病理后果的新治疗靶点的潜力。

DESCRIPTION (provided by applicant): Obesity-associated type-2 diabetes and its comorbidities, atherosclerosis and stroke, are all too common diseases in the aging population of our Veterans. These illnesses are invariably linked to aberrant lipid homeostasis as manifested by excessive synthesis and high circulating levels of very low-density lipoproteins and triglycerides. The long-term objective of our studies is to gain molecular insights into the mechanisms of hepatic lipid homeostasis and dyslipidemia and their cardiovascular complications. As a transcription factor that regulates key genes involved de novo lipid synthesis in the liver, sterol regulatory element binding protein-1c (SREBP-1c) is a central player in these processes. Nascent pSREBP-1c resides in the endoplasmic reticulum (ER) and must be transported to the Golgi where it undergoes regulated intra- membrane proteolysis (RIP) to release its transcriptionally active nuclear domain, nSREBP-1c that activates its own promoter. This feed-forward regulation SREBP-1c in the liver by insulin is a key mechanism of selective insulin resistance and hyperlipidemia and their clinical consequences. Based on compelling published data and our ongoing studies we hypothesize that phosphorylation via insulin-induced kinase PI3K-Akt/PKB, and its downstream kinases mTOR and p70S6K, regulate the RIP-mediated maturation, and trans-activation potency and stability of nSREBP-1c in the nucleus. Additionally, via crosstalk with insulin signaling kinases, MAP kinases also impinge on the feed-forward regulation of SREBP-1c in chronically obese rodents and man. With a goal to experimentally test this hypothesis, we have site-specifically mutated seventeen, AKT/mTOR/S6K and MAPK specific putative phosphorylation sites, selected on the basis of published observations and our theoretical analyses. Based on a rigorous assessment of the phenotypes of WT and mutated SREBP-1c in a McArdle hepatoma cell line and primary rat hepatocytes, we have selected FOUR sites [mTOR (S57 and S140), p70S6K (S956) and MAP kinase (S39)] for detailed examination of their role in the feed-forward regulation of SREBP-1c. The short-term goals of our proposal are (i) to identify sequence motifs of SREBP-1c involved in insulin-enhanced, phosphorylation- dependent regulation of RIP and trans-activation ability of nSREBP-1c in vitro and in vivo and (ii) to decipher how phosphorylation alters the molecular interactions of SREBP-1c during its ER to Golgi transit and proteolysis by S1P and S2P proteases, and (iii) to elucidate how phosphorylation alters the stability and transcriptional function of nSREBP-1c in the nucleus, in vitro and in vivo. To accomplish these goals, we will express wild type, and gain- or loss-of-phosphorylation mutants of tagged pSREBP-1c and nSREBP-1c proteins in rat hepatocytes in culture, and in livers of normal and obese and hyper-insulinemic MC4R knockout, rats, using our unique custom-designed adenovirus and plasmid vectors. We will compare the rates of RIP-mediated maturation of WT and mutated nSREBP-1c proteins and their trans-activation potential in the presence and absence of insulin. We will determine if/how phosphorylation alters the molecular interactions between full-length SREBP-1c and other molecules that are involved in ER-to-Golgi transit and proteolysis. Additionally, we will elucidate how phosphorylation alters the trans-activation potential of nSREBP-1c to activate its own promoter and the promoters of downstream target genes that regulate hepatic lipid synthesis. Our Specific Aims are designed to shed mechanistic light on phosphorylation-dependent actions of SREBP-1c on lipid homeostasis in vitro and in vivo. A successful completion of these studies has the potential to unravel novel therapeutic targets to treat chronic obesity and its pathological consequences for the cardiovascular system.