Targeting PCSK9 Transcription to Combat Hypercholesterolemia by Berberine

小檗碱靶向 PCSK9 转录对抗高胆固醇血症

基本信息

批准号：
8726287
负责人：
JINGWEN LIU
金额：
$ 34.19万
依托单位：
PALO ALTO VETERANS INSTIT FOR RESEARCH
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-30 至 2017-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8726287
关键词：
Affect Affinity Animal Model Animals Atherosclerosis Berberine Binding Binding Proteins Binding Sites Biological Assay Cell Line Cells Cholesterol Cholesterol Homeostasis Clinical Co-Immunoprecipitations Control Animal Coupled DNA Data Dissociation Down-Regulation Enzymes Event Gene Expression Gene Targeting Genes Genetic Transcription Goals Hamsters Heart Diseases Hepatic Hepatocyte Human Hydroxymethylglutaryl-CoA reductase Individual Knowledge LDL Cholesterol Lipoproteins Laboratories Liver Low Density Lipoprotein Receptor Low-Density Lipoproteins Mass Spectrum Analysis Mediating Methodology Molecular Mutation Nuclear Nuclear Extract Nucleotides Oligonucleotide Probes Pathway interactions Patients Pharmaceutical Preparations Plasma Play Poly(ADP-ribose) Polymerases Post-Translational Protein Processing Post-Translational Regulation Promoter Regions Proprotein Convertases Proteins Receptor Gene Regulation Regulatory Element Resolution Role Site Small Interfering RNA Sterols Subtilisins Surface Techniques Time Trans-Activators Transcriptional Regulation Treatment Efficacy Ubiquitin Ubiquitination Up-Regulation adenovirus mediated delivery attenuation base cholesterol biosynthesis cis acting element cofactor combat gene repression hepatic nuclear factor 1 hepatocyte nuclear factor hypercholesterolemia improved insight kexin mRNA Expression multicatalytic endopeptidase complex novel novel therapeutic intervention prevent promoter protein complex protein degradation protein profiling public health relevance receptor expression response small hairpin RNA success

项目摘要

DESCRIPTION (provided by applicant): PCSK9 (proprotein convertase subtilisin/kexin type 9) plays an important role in the control of circulating LDL- cholesterol (LDL-C) levels via modulation of rates of degradation of hepatic LDL receptor (LDLR). One important aspect of the PCSK9-LDLR pathway in mediating LDL-C clearance is that their transcription is coordinately regulated by sterols through a common sterol regulatory element (SRE) motif embedded in their gene promoters and is co-induced by current cholesterol lowering drugs such as statins through activation of SRE binding proteins. Statin treatment increases the transcription of both LDLR and PCSK9. The increased PCSK9 transcription leads to elevated plasma PCSK9 levels, which, in turn, reduces the number of LDLR on the surface of hepatocytes. The undesirable stimulatory action of statins on PCSK9 transcription is increasingly recognized as a major limitation to the therapeutic efficacy of statins in lowering plasma LDL-C. Our laboratory has made a novel discovery that PCSK9 gene transcription is regulated by the hepatocyte nuclear factor 1? (HNF1?) through a highly conserved HNF1 binding site of the PCSK9 gene promoter, whereas HNF1? binding site is absent on the LDLR gene promoter. Furthermore, we have demonstrated that HNF1 site and its trans-activator HNF1? are prominently involved in the inhibition of PCSK9 transcription by the natural cholesterol-lowering compound berberine (BBR) via a mechanism that reduces the intracellular protein level of HNF1?. Recently, we have obtained new evidence indicating the potential involvement of ubiquitin-proteasome pathway in BBR induced down regulation of HNF1? protein. In addition, by applying a unique technique that combines the DNA affinity pull-down (biotinylated HNF1-PCSK9 oligonucleotide probe) with high resolution mass spectrometry, we have identified the nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) as a novel HNF1? interacting protein, implicating its potential role in the regulation of PCSK9 transcription and repression by BBR. Altogether, our new findings have demonstrated for the first time that PCSK9 and LDLR transcription can be separately regulated and also provided a molecular basis for developing novel therapeutic interventions to selectively repress PCSK9 expression without diminishing LDLR transcription for the treatment of hypercholesterolemia and heart disease. The overall goal of this revised proposal is to elucidate the molecular and cellular mechanisms by which BBR inhibits PCSK9 transcription through its actions on HNF1? protein stability and its association with coactivators in hepatic cell lines and animal models. The three specific aims are: 1) to characterize HNF1? coactivators which are specifically involved in PCSK9 transcription and regulation by BBR; 2) to elucidate the molecular mechanisms by which BBR inhibits PCSK9 transcription through the ubiquitin-proteasome pathway mediated reduction of HNF1? protein levels; and 3) to examine the effects of liver-specific depletion of HNF1? on PCSK9, LDLR, plasma LDL-C metabolism in response to treatment of hyperlipidemic animals with either statin or BBR.

描述（由申请人提供）：PCSK9（普洛蛋白转化酶枯草蛋白/KEXIN 9型）通过调节肝LDL受体（LDLR）降解率来控制循环LDL-胆固醇（LDL-C）水平的重要作用。 PCSK9-LDLR途径的一个重要方面在介导LDL-C清除率时，它们的转录是通过固醇通过固醇的常见调节元素（SRE）基序协调调节的，该基元嵌入了其基因启动子中，并由当前胆固醇降低的药物（如诸如STATINS）通过STATINS（例如脂肪素）通过SROTICATICS蛋白质诱导的SRE蛋白来诱导。他汀类药物的治疗增加了LDLR和PCSK9的转录。 PCSK9转录增加导致血浆PCSK9水平升高，进而减少肝细胞表面的LDLR数量。他汀类药物对PCSK9转录的不良刺激作用越来越被认为是他汀类药物在降低血浆LDL-C中的治疗功效的主要限制。我们的实验室已经有一个新发现的发现，即PCSK9基因转录受肝细胞核因子1的调节？（HNF1？）通过PCSK9基因启动子的高度保守的HNF1结合位点，而HNF1？ LDLR基因启动子不存在结合位点。此外，我们已经证明了HNF1位点及其反式激活剂HNF1？通过降低HNF1的细胞内蛋白水平的机制，自然降解胆固醇的复合碱（BBR）抑制PCSK9转录的抑制。最近，我们获得了新的证据，表明泛素 - 蛋白酶体途径可能参与BBR诱导的HNF1的调节？蛋白质。此外，通过应用一种将DNA亲和力下拉（生物素化的HNF1-PCSK9寡核苷酸探针）与高分辨率质谱法结合的独特技术，我们已经将核酶聚（ADP-核糖）聚合酶1（Parp-1）确定为新型HNF1？相互作用的蛋白质，暗示其在BBR调节PCSK9转录和抑制中的潜在作用。总的来说，我们的新发现首次证明了PCSK9和LDLR转录可以分别调节，还为开发新型治疗干预措施提供了分子基础，以选择性地抑制PCSK9的表达，而无需减少LDLR转录，而无需减少高胆固醇血症和心脏病的治疗。该修订建议的总体目标是阐明BBR通过其对HNF1的作用抑制PCSK9转录的分子和细胞机制？蛋白质稳定性及其与肝细胞系和动物模型中的共激活因子的关联。三个具体目标是：1）表征HNF1？由BBR专门参与PCSK9转录和调节的共激活剂； 2）阐明BBR通过泛素 - 蛋白酶体途径抑制PCSK9转录的分子机制，介导了HNF1的还原？蛋白质水平； 3）检查HNF1的肝脏特异性耗竭的影响？在PCSK9上，LDLR，血浆LDL-C代谢，以响应他汀类药物或BBR治疗高脂动物。