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）： 肥胖相关的2型糖尿病及其合并症，动脉粥样硬化和中风，在我们的退伍军人老龄化人口中是非常常见的疾病。这些疾病总是与异常的脂质稳态有关，表现为极低密度脂蛋白和甘油三酯的过度合成和高循环水平。本研究的长期目标是从分子水平深入了解肝脏脂质稳态和血脂异常及其心血管并发症的机制。固醇调节元件结合蛋白-1c（sterol regulatory element binding protein-1c，SREBP-1c）作为调控肝脏脂质从头合成的关键基因的转录因子，在这些过程中起着重要作用。新生pSREBP-1c存在于内质网（ER）中，必须被转运到高尔基体，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nSREBP-1c，nSREBP-1c激活其自身启动子.胰岛素对肝脏中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。基于令人信服的已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及其下游激酶mTOR和p70 S6K的磷酸化调节RIP介导的成熟，以及nSREBP-1c在细胞核中的反式激活效力和稳定性。此外，通过与胰岛素信号激酶的串扰，MAP激酶也影响了SREBP-1c在慢性肥胖啮齿类动物和man.With实验测试这一假设的目标的前馈调节，我们有位点特异性突变的十七个，AKT/mTOR/S6K和MAPK特异性推定的磷酸化位点，选择的基础上发表的意见和我们的理论分析。基于对McArdle肝癌细胞系和原代大鼠肝细胞中WT和突变SREBP-1c表型的严格评估，我们选择了四个位点[mTOR（S57和S140），p70 S6K（S956）和MAP激酶（S39）]，以详细检查它们在SREBP-1c前馈调节中的作用。我们的建议的短期目标是（i）鉴定SREBP-1c的序列基序，所述序列基序参与RIP的胰岛素增强的磷酸化依赖性调节和nSREBP-1c的体外和体内反式激活能力，和（ii）破译磷酸化如何改变SREBP-1c在其ER到高尔基体转运和通过S1P和S2P蛋白酶的蛋白水解期间的分子相互作用，和（iii）阐明磷酸化如何改变nSREBP-1c在细胞核中的稳定性和转录功能，在体外和体内。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中以及在正常和肥胖和高胰岛素血症MC4R敲除大鼠的肝脏中表达标记的pSREBP-1c和nSREBP-1c蛋白的野生型和获得或失去磷酸化突变体。我们将比较RIP介导的WT和突变的nSREBP-1c蛋白成熟的速率，以及在存在和不存在胰岛素的情况下它们的反式激活潜力。我们将确定磷酸化是否/如何改变全长SREBP-1c和其他参与ER到高尔基体转运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的反式激活潜力，以激活其自身的启动子和调节肝脏脂质合成的下游靶基因的启动子。我们的特定目的旨在阐明SREBP-1c在体外和体内对脂质稳态的磷酸化依赖性作用的机制。这些研究的成功完成有可能揭示新的治疗靶点，以治疗慢性肥胖及其对心血管系统的病理后果。