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：
8907907
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 2016-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8907907
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 Health Heart Diseases HepG2 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 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（前蛋白转化酶枯草杆菌蛋白酶/kexin 9型）通过调节肝LDL受体（LDLR）的降解速率在控制循环LDL-胆固醇（LDL-C）水平中发挥重要作用。 PCSK9-LDLR 通路介导 LDL-C 清除的一个重要方面是，它们的转录通过嵌入其基因启动子中的通用甾醇调节元件 (SRE) 基序由甾醇协调调节，并由当前的降胆固醇药物共同诱导，例如通过激活 SRE 结合蛋白作为他汀类药物。他汀类药物治疗会增加 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 LDLR 的数量。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降低血浆 LDL-C 治疗效果的主要限制。本实验室新发现PCSK9基因转录受肝细胞核因子1？ (HNF1?) 通过 PCSK9 基因启动子的高度保守的 HNF1 结合位点，而 HNF1? LDLR 基因启动子上不存在结合位点。此外，我们还证明了HNF1位点及其反式激活因子HNF1？主要参与天然降胆固醇化合物小檗碱 (BBR) 通过降低细胞内 HNF1 蛋白水平的机制抑制 PCSK9 转录。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下调？蛋白质。此外，通过应用将DNA亲和力下拉（生物素化HNF1-PCSK9寡核苷酸探针）与高分辨率质谱相结合的独特技术，我们鉴定了核酶聚（ADP-核糖）聚合酶-1（PARP-1）作为小说 HNF1？相互作用蛋白，暗示其在调节 PCSK9 转录和 BBR 抑制中的潜在作用。总而言之，我们的新发现首次证明 PCSK9 和 LDLR 转录可以单独调节，并为开发新的治疗干预措施提供了分子基础，以选择性抑制 PCSK9 表达而不减少 LDLR 转录，从而治疗高胆固醇血症和心脏病。该修订提案的总体目标是阐明 BBR 通过对 HNF1 的作用来抑制 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剂的关联。这三个具体目标是：1）表征 HNF1？专门参与 PCSK9 转录和 BBR 调节的共激活因子； 2) 阐明BBR通过泛素-蛋白酶体途径介导的HNF1减少抑制PCSK9转录的分子机制？蛋白质水平； 3) 检查肝脏特异性去除 HNF1 的影响？对用他汀类药物或 BBR 治疗高脂血症动物的 PCSK9、LDLR、血浆 LDL-C 代谢的影响。