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

SREBP-1c 的磷酸化依赖性前馈调节

• 批准号: 9057856
• 负责人: RAJENDRA RAGHOW
• 金额: --
• 依托单位: MEMPHIS VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-10-01 至 2018-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9057856
• 关键词: 70-kDa Ribosomal Protein S6 Kinases; ADD-1 protein; Adenovirus Vector; Adenoviruses; Affect; Affinity; Alanine; Amino Acids; Anabolism; Antibodies; Aspartic Acid; Atherosclerosis; Binding; Cardiovascular system; Cell Line; Cell Nucleus; Cells; Chronic; Clinical; Comorbidity; Custom; Data; Disease; Dyslipidemias; Endoplasmic Reticulum; Epitopes; FASN gene; FRAP1 gene; Gene Targeting; Genes; Goals; Golgi Apparatus; Heart Diseases; Hepatic; Hepatocyte; Homeostasis; Hyperlipidemia; In Vitro; Insulin; Insulin Resistance; Knock-out; Length; Light; Link; Lipids; Liver; Luciferases; MAP Kinase Gene; MAPK14 gene; MAPK8 gene; Mediating; Melanocortin 4 Receptor; Membrane; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Molecular; Molecular Conformation; Mono-S; Mutate; N-terminal; Non-Insulin-Dependent Diabetes Mellitus; Nuclear; Nuclear Envelope; Obesity; Peptide Hydrolases; Phenotype; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Plasmid Cloning Vector; Process; Proteins; Proteolysis; Proto-Oncogene Proteins c-akt; Publishing; Rattus; Regulation; Reporter; Risk; Rodent; Role; Serine; Site; Starvation; Stimulus; Stroke; Testing; Threonine; Transactivation; Veterans; aging population; base; design; feeding; hepatoma cell; in vivo; insight; insulin signaling; lipoprotein triglyceride; man; migration; mimetics; mutant; new therapeutic target; promoter; public health relevance; recombinant adenovirus; response; tandem mass spectrometry; transcription factor; very low density lipoprotein triglyceride

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.

描述（由申请人提供）： 与肥胖相关的2型糖尿病及其合并症，动脉粥样硬化和中风，在我们退伍军人的老龄化中都是太常见的疾病。这些疾病总是与异常的脂质稳态有关，这表现为过度合成和高密度脂蛋白和甘油三酸酯的高循环水平。我们研究的长期目标是获得分子见解，以了解肝脂质稳态和血脂异常及其心血管并发症的机制。作为调节关键基因的转录因子，涉及肝脏中脂质合成的主要基因，固醇调节元件结合蛋白1C（SREBP-1C）是这些过程中的核心参与者。新生的PSREBP-1C驻留在内质网（ER）中，必须将其转移到高尔基体中，在那里它经历受调节的膜内蛋白水解（RIP）以释放其转录活性核结构域，NSREBPP-1C激活其自身的启动子。胰岛素中肝脏中的这种馈送调节SREBP-1C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。基于引人注目的发表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磷酸化以及其下游激酶MTOR和p70S6K，调节了RIP介导的成熟，以及NSREBP-1C的NSREBP-1C的透射激活和稳定性。此外，通过与胰岛素信号激酶的串扰，MAP激酶还构成了慢性肥胖啮齿动物和人类中SREBP-1C的前馈调节。为了通过实验检验这一假设的目标，我们根据已发表的观测值和我们的理论分析选择了站点特异性突变的17个，Akt/mTOR/S6K和MAPK特定的推定磷酸化位点。基于对Mcardle肝癌细胞系和原发性大鼠肝细胞中WT和SREBP-1C表型的严格评估，我们选择了四个位点[MTOR（S57和S140），P70S6K（S956）（S956）和MAP KINASE（S39）]，以详细研究SSC。我们提案的短期目标是（i）确定与胰岛素增强，磷酸化相关的SREBP-1C序列基序，依赖于RIP的磷酸化调节和NSREBP-1C在体外，体内和体内和（II）中磷酸化的磷酸化和（II）在Sreemartion中的磷酸化goL srebp-1c-1C的沟渠中的pro依赖的dcc-1C的交流能力和反式激活能力。 S1P和S2P蛋白酶，以及（III），以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，我们将在培养物中大鼠肝细胞中的标记为PSREBP-1C和NSREBP-1C蛋白的野生类型，以及使用我们独特的独特的adenenovirus and plastics and psrebp-1c和NSREBP-1C蛋白，以及正常和肥胖和高胰岛素MC 4R敲除大鼠的肝脏中的NSREBP-1C蛋白。我们将在存在和不存在胰岛素的情况下比较RIP介导的WT和突变的NSREBP-1C蛋白的成熟速率及其反式激活潜力。我们将确定/磷酸化如何改变全长SREBP-1C与其他参与ER-Golgi Transit和蛋白水解的分子之间的分子相互作用。此外，我们将阐明磷酸化如何改变NSREBP-1C激活其自身启动子和调节肝脂质合成的下游靶基因的启动子的反式激活潜力。我们的具体目的旨在使SREBP-1C在体外和体内对脂质稳态的磷酸化依赖性作用的机械介绍。这些研究的成功完成有可能揭示新的治疗靶标，以治疗慢性肥胖症及其对心血管系统的病理后果。