pcsk9基因突变与胆固醇血症

  常染色体显性高胆固醇血症（ADH）是家族早发性动脉粥样硬化的最主要的危险因素.在与ADH有关的基因突变中,LDL-R、apoB100基因突变导致ADH的机制已经比较明确,而pcsk9基因突变与ADH相关是最近发现的.pcsk9基因编码神经凋亡调节转化酶即NARC-1, 它通过在蛋白水平降低肝细胞上LDL受体的数量,使血液中LDL不能被清除,从而与高胆固醇血症相关联.研究pcsk9与LDL之间的关系,探索pcsk9在高胆固醇血症以及动脉粥样硬化发生中的作用及机制,将有助于高胆固醇血症和动脉粥样硬化发病机制的研究,也能为防治高胆固醇血症和动脉粥样硬化提供新思路.     

神经细胞凋亡调节转化酶-1在LDL胆固醇代谢中的作用

人类枯草溶菌素转化酶9（PCSK9）编码神经细胞凋亡调节转化酶-1,是一个属于丝氨酸蛋白酶K亚类的独特前蛋白转化酶。它能切割非碱性氨基酸,其底物特异性不同于许多其它前蛋白转化酶。它的唯一已知底物为前体NARC-1。PCSK9基因有多种序列变异,如错义突变S127R和F216L可引起常染色体显性高胆固醇血症;无义突变Y142X、C679X及错义变异R46L均可降低LDL胆固醇和冠心病发病率。通过研究这些变异和野生型PCSK9,发现PCSK9参与肝再生和神经分化,调节胆固醇代谢、含apoB脂蛋白代谢,并在动脉粥样硬化、冠心病等心血管疾病的发生发展和防治中发挥重要作用。     

PCSK9的药理学筛选靶点

前蛋白转化酶枯草溶菌素9（PCSK9）基因编码神经凋亡调节转化酶即NARC1,通过影响肝LDLR水平,在胆固醇代谢中发挥了重要的作用.其功能获得型突变使血浆胆固醇水平增高,而功能缺失型突变降低胆固醇水平.流行病学调查显示,高胆固醇血症是动脉粥样硬化和心脏病的主要危险因素.一些患者运用当前的降胆固醇药物治疗仍不能达到推荐的目标LDL水平,PCSK9作为新的降脂靶点引起了广泛的关注.本文将对PCSK9的结构、功能和药理学靶点进行综述.     

前蛋白转化酶枯草溶菌素9的生物学功能

前蛋白转化酶枯草溶菌素9(proprotein convertase subtilisin/kexin type 9,PCSK9)基因属于前蛋白转化酶(PC)家族,是一个新发现不久的与胆固醇代谢相关基因.近年来,PCSK9在其生物学效应及疾病中的作用越来越受到重视.大量的研究表明,除通过调节低密度脂蛋白受体(LDLR)影响胆固醇代谢外,PCSK9还参与细胞凋亡,促进肝发育、再生,促进神经系统发育,影响神经系统分化并且与炎症过程以及糖尿病相关.本文对PCSK9功能方面最新研究进展进行了综述。     

pcsk9 siRNA对oxLDL诱导的THP-1源性巨噬细胞凋亡的影响

为研究pcsk9基因沉默后对氧化型低密度脂蛋(oxLDL)诱导THP-1源性巨噬细胞凋亡的影响,用不同浓度oxLDL处理THP-1源性巨噬细胞48 h,Hoechst33258染色检测细胞凋亡,RT-PCR、Western blot分别检测pcsk9 mRNA、NARC-1蛋白的表达.应用Lipofectamine2000转染3对pcsk9 siRNAs进THP-1源性巨噬细胞中,筛选出最有效的siRNA再转染入THP-1源性巨噬细胞,24 h后加入oxLDL处理48 h,Hoechst33258染色观察细胞评价细胞凋亡,流式细胞术计数检测细胞凋亡率.结果发现,75 mg/L oxLDL处理THP-1源性巨噬细胞48 h后,Hoechst33258染色可见大量凋亡细胞.同时RT-PCR、Western blot检测发现,pcsk9 mRNA和NARC-1蛋白质表达量均随oxLDL浓度的增加而增加,75 mg/LoxLDL组增加最明显.不同浓度siRNA转染THP-1源性巨噬细胞后,RT-PCR筛选出3对siRNAs的终浓度为80 nmol/L均可出现明显的沉默效应.选取此浓度在蛋白质水平检测基因抑制情况,筛选出最有效的一对siRNA.将筛选出来的siRNA转染细胞24 h后,再用oxLDL处理48 h,Hoechst33258染色及流式细胞计数结果显示,转染siRNA组凋亡明显被抑制.结果表明,在本研究的浓度范围内,随着oxLDL浓度增加pcsk9的表达随之增加,同时,THP-1源性巨噬细胞凋亡也明显增加,75 mg/L oxLDL最明显,pcsk9 mRNA和蛋白质的表达也在该浓度最高.提示pcsk9 siRNA能有效抑制pcsk9基因的表达,从而有效抑制由oxLDL诱导的THP-1源性巨噬细胞的凋亡.     

PCSK9结构与功能

前蛋白转化酶枯草溶菌素9（PCSK9）基因属于前蛋白转化酶（PC）家族,由信号肽、前结构域、催化结构域和羧基末端结构域组成.大量研究发现,PCSK9能介导低密度脂蛋白受体（LDLR）降解,调节血浆LDL胆固醇（LDL-C）水平;而PCSK9的两类主要突变,功能获得型、功能缺失型可分别导致高胆固醇血症和低胆固醇血症. 因而研究PCSK9对相关心血管疾病的防治有重要意义. PCSK9结构特性与其生化功能密切相关,突变致使其调节胆固醇代谢的机制更为复杂.本文旨在总结PCSK9结构与功能的分子生物学特性,并指出目前研究中存在的问题,以利对PCSK9的进一步探索.     

pcsk9 siRNA对oxLDL诱导的THP-1源性巨噬细胞凋亡的影响

为研究pcsk9基因沉默后对氧化型低密度脂蛋白(oxLDL)诱导THP-1源性巨噬细胞凋亡的影响,用不同浓度oxLDL 处理THP-1源性巨噬细胞48 h,Hoechst33258染色检测细胞凋亡,RT-PCR、Western blot分别检测pcsk9 mRNA、NARC-1蛋白的表达．应用Lipofectamine2000转染3对pcsk9 siRNAs进THP-1源性巨噬细胞中,筛选出最有效的siRNA再转染入THP-1源性巨噬细胞,24 h后加入oxLDL处理 48 h,Hoechst33258染色观察细胞评价细胞凋亡,流式细胞术计数检测细胞凋亡率．结果发现,75 mg/L oxLDL处理THP-1源性巨噬细胞48 h后,Hoechst33258染色可见大量凋亡细胞．同时RT-PCR、Western blot检测发现,pcsk9 mRNA和NARC-1蛋白质表达量均随oxLDL浓度的增加而增加,75 mg/L oxLDL组增加最明显．不同浓度siRNA转染THP-1源性巨噬细胞后,RT-PCR筛选出3对siRNAs的终浓度为80 nmol/L均可出现明显的沉默效应．选取此浓度在蛋白质水平检测基因抑制情况,筛选出最有效的一对siRNA．将筛选出来的siRNA转染细胞24 h后,再用oxLDL处理48 h,Hoechst33258染色及流式细胞计数结果显示,转染siRNA组凋亡明显被抑制．结果表明,在本研究的浓度范围内,随着oxLDL浓度增加pcsk9的表达随之增加,同时,THP-1源性巨噬细胞凋亡也明显增加,75 mg/L oxLDL最明显,pcsk9 mRNA和蛋白质的表达也在该浓度最高．提示pcsk9 siRNA能有效抑制pcsk9基因的表达,从而有效抑制由oxLDL诱导的THP-1源性巨噬细胞的凋亡．     

BAG-1蛋白及其对神经系统疾病调控研究进展

B细胞CLL/淋巴瘤2关联凋亡基因1（B-cell CLL/lymphoma 2-associated athanogene-1,BAG-1）编码的蛋白是一种多功能结合蛋白,包含不同功能的结构域,可与抗凋亡蛋白（Bcl-2）、热激蛋白70（heat shock protein 70,Hsp70/Hsc70）、细胞转化分裂介质（Raf-1）、类固醇激素受体和DNA等结合,对于调节细胞凋亡、信号传导、基因转录、细胞增殖与分化等具有重要作用。BAG-1参与多种神经系统疾病（如阿尔茨海默病、帕金森氏病、亨廷顿舞蹈病、脑中风、脊髓损伤以及神经精神障碍疾病）的发生发展过程。主要就BAG-1的结构、功能及其对神经系统疾病影响的研究进展进行了综述,以期为神经系统疾病的研究和治疗提供参考。     

人肾上腺基因表达谱的建立及其功能的新认识

为深入理解人类肾上腺（AD）的功能,构建了正常人肾上腺cDNA文库,并利用大规模表达序列标签（ESTs）测序和生物信息学技术,研究显示参与基因/蛋白表达的基因类型表达最多,其次为能量代谢类.肾上腺中表达丰度最高的3个基因均为参与类固醇合成的酶类和蛋白.一些重要的基因首次显示在肾上腺表达,包括神经激素和神经肽,如促肾上腺皮质激素释放激素（CRH）,黑色素浓激素（MCH）,urocortin,可卡因和安非他明调节肽（CART）和垂体腺苷酸环化酶激活肽（PACAP）;许多重要介质的受体,如细胞因子、神经肽及神经递质受体;参与胆固醇代谢的基因,如LDL受体、HDL结合蛋白和胆固醇合成酶.研究结果表明在肾上腺表达丰度最高的基因与该器官的功能特异性有关,除类固醇激素外,许多神经肽、细胞因子在肾上腺产生,肾上腺与体内其他重要的系统间存在广泛的应答,而且在人肾上腺局部可能存在一个CRH-ACTH-皮质醇调节网络.     

单基因遗传性高胆固醇血症与非低密度脂蛋白受体致病基因的关系

家族性高胆固醇血症（FH）是一类异质性很强的单基因常染色体遗传性疾病。最近发现,除低密度脂蛋白受体和载脂蛋白B-100以外．前蛋白转化酶一枯草溶菌素9、衔接子蛋白、三磷酸腺苷结合盒转运蛋白G5和G8、胆固醇-7-α-羟化酶等多种基因的变异都能导致FH样表型。该文对利用基因剔除和过表达、RNA干扰、反义技术对单基因遗传性高胆固醇血症非低密度脂蛋白受体致病基因的功能和调控机制进行综述,有助于深入认识这些基因．从而为临床家族性高胆固醇血症的诊断和治疗提供新的思路。     

PCSK9 inhibition fails to alter hepatic LDLR,circulating cholesterol,and atherosclerosis in the absence of ApoE

LDL cholesterol (LDL-C) contributes to coronary heart disease. Proprotein convertase subtilisin/kexin type 9 (PCSK9) increases LDL-C by inhibiting LDL-C clearance. The therapeutic potential for PCSK9 inhibitors is highlighted by the fact that PCSK9 loss-of-function carriers exhibit 15–30% lower circulating LDL-C and a disproportionately lower risk (47–88%) of experiencing a cardiovascular event. Here, we utilized pcsk9^−/− mice and an anti-PCSK9 antibody to study the role of the LDL receptor (LDLR) and ApoE in PCSK9-mediated regulation of plasma cholesterol and atherosclerotic lesion development. We found that circulating cholesterol and atherosclerotic lesions were minimally modified in pcsk9^−/− mice on either an LDLR- or ApoE-deficient background. Acute administration of an anti-PCSK9 antibody did not reduce circulating cholesterol in an ApoE-deficient background, but did reduce circulating cholesterol (−45%) and TGs (−36%) in APOE*3Leiden.cholesteryl ester transfer protein (CETP) mice, which contain mouse ApoE, human mutant APOE3*Leiden, and a functional LDLR. Chronic anti-PCSK9 antibody treatment in APOE*3Leiden.CETP mice resulted in a significant reduction in atherosclerotic lesion area (−91%) and reduced lesion complexity. Taken together, these results indicate that both LDLR and ApoE are required for PCSK9 inhibitor-mediated reductions in atherosclerosis, as both are needed to increase hepatic LDLR expression.     

NARC-1/PCSK9 and its natural mutants: zymogen cleavage and effects on the low density lipoprotein (LDL) receptor and LDL cholesterol

The discovery of autosomal dominant hypercholesterolemic patients with mutations in the PCSK9 gene, encoding the proprotein convertase NARC-1, resulting in the missense mutations suggested a role in low density lipoprotein (LDL) metabolism. We show that the endoplasmic reticulum-localized proNARC-1 to NARC-1 zymogen conversion is Ca2+-independent and that within the zymogen autocatalytic processing site SSVFAQ [downward arrow]SIP Val at P4 and Pro at P3' are critical. The S127R and D374Y mutations result in approximately 50-60% and > or =98% decrease in zymogen processing, respectively. In contrast, the double [D374Y + N157K], F216L, and R218S natural mutants resulted in normal zymogen processing. The cell surface LDL receptor (LDLR) levels are reduced by 35% in lymphoblasts of S127R patients. The LDLR levels are also reduced in stable HepG2 cells overexpressing NARC-1 or its natural mutant S127R, and this reduction is abrogated in the presence of 5 mm ammonium chloride, suggesting that overexpression of NARC-1 increases the turnover rate of the LDLR. Adenoviral expression of wild type human NARC-1 in mice resulted in a maximal approximately 9-fold increase in circulating LDL cholesterol, while in LDLR-/- mice a delayed approximately 2-fold increase in LDL cholesterol was observed. In conclusion, NARC-1 seems to affect both the level of LDLR and that of circulating apoB-containing lipoproteins in an LDLR-dependent and -independent fashion.     

Cholesterol-related genes in Alzheimer's disease

Experimental data show that cholesterol can modulate central processes in the pathogenesis of Alzheimer's disease (AD). The epidemiological link between elevated plasma cholesterol at midlife and increased risk for AD and the possibility that 3-hydroxy-3-methylglutaryl-coenzym A reductase inhibitors (statins) may be protective against AD support a role of cholesterol metabolism in AD and have rendered it a potential therapeutic target in the treatment and prevention of the disease. The strong association of AD and AD endophenotypes with the APOE gene provides a genetic link between AD and cholesterol metabolism, because the apolipoprotein E (ApoE) is the most prevalent cholesterol transport protein in the central nervous system. Against this background several other genes with a role in cholesterol metabolism have been investigated for association with AD. In this review a compilation of genes related to cholesterol based on the information of the AmiGo gene ontology database is matched with the AlzGene database of AD candidate genes. 56 out of 149 (37.6%) genes with a relation to cholesterol metabolism have been investigated for association with AD. Given that only 660 out of about 23,000 (2.9%) genes have been assessed in hypothesis-driven candidate gene studies on AD, the cholesterol metabolic pathway is strongly represented among these genes. Among 34 cholesterol-related genes for which association with AD has been described APOE, CH25H, CLU, LDLR, SORL1 outstand with positive meta-analyses. However, it is unclear, if their association with AD is mediated by cholesterol-related mechanisms or by more specific direct effects of the respective proteins on Aβ metabolism.     

Proprotein Convertase Subtilisin/Kexin Type 7 (PCSK7) Is Essential for the Zebrafish Development and Bioavailability of Transforming Growth Factor β1a (TGFβ1a)

Proprotein convertase subtilisin/kexin (PCSK) enzymes convert proproteins into bioactive end products. Although other PCSK enzymes are known to be essential for biological processes ranging from cholesterol metabolism to host defense, the in vivo importance of the evolutionarily ancient PCSK7 has remained enigmatic. Here, we quantified the expressions of all pcsk genes during the 1st week of fish development and in several tissues. pcsk7 expression was ubiquitous and evident already during the early development. To compare mammalian and zebrafish PCSK7, we prepared homology models, which demonstrated remarkable structural conservation. When the PCSK7 function in developing larvae was inhibited, we found that PCSK7-deficient fish have defects in various organs, including the brain, eye, and otic vesicle, and these result in mortality within 7 days postfertilization. A genome-wide analysis of PCSK7-dependent gene expression showed that, in addition to developmental processes, several immune system-related pathways are also regulated by PCSK7. Specifically, the PCSK7 contributed to the mRNA expression and proteolytic cleavage of the cytokine TGFβ1a. Consequently, tgfβ1a morphant fish displayed phenotypical similarities with pcsk7 morphants, underscoring the importance of this cytokine in the zebrafish development. Targeting PCSK activity has emerged as a strategy for treating human diseases. Our results suggest that inhibiting PCSK7 might interfere with normal vertebrate development.     

CYP46 T/C Polymorphism is not Associated with Alzheimer’s Dementia in a Population from Hungary

Multiple genetic and environmental factors regulate the susceptibility to Alzheimer’s disease (AD). Recently, several independent studies have reported that a locus on chromosome 14q32.1, where a gene encoding a cholesterol degrading enzyme of the brain, called 24-hydroxylase (CYP46A1) is located, has been linked with AD. The single nucleotide polymorphism (T/C) in intron 2 of CYP46 gene has been found to confer the risk for AD. The water soluble 24(S)-hydroxysterol is the product of the CYP46A1, and elevated plasma and cerebrospinal fluid hydroxysterol concentrations have been found in AD, reflecting increased brain cholesterol turnover or cellular degradation, due to the neurodegenerative process. A case–control study was performed on 125 AD and 102 age- and gender-matched control subjects (CNT) from Hungary, to test the association of CYP46 T/C and apolipoprotein E (ApoE) gene polymorphisms in AD. The frequency of the CYP46 C allele was similar (χ²=0.647, df=1, P=0.421, exact P=0.466, OR=0.845; 95% CI: 0.561–1.274) in both groups (CNT: 27%; 95% CI: 21.3–33.4; AD 30%; 95% CI: 25.0–36.3). The ApoE ɛ4 allele was significantly over-represented (χ²=11.029, df=2, P=0.004) in the AD population (23.2%; 95% CI: 18.2–29.0) when compared with the CNT (11.3%; 95% CI: 7.4–16.6). The presence or absence of one or two CYP46C alleles together with the ApoE ɛ4 allele did not increase the risk of AD (OR=3.492; 95% CI: 1.401–8.707; P<0.007 and OR=3.714; 95% CI: 1.549–8.908; P<0.003, respectively). Our results indicate that the intron 2 T/C polymorphism of CYP46 gene (neither alone, nor together with the ɛ4 allele) does not increase the susceptibility to late-onset sporadic AD in the Hungarian population.     

A liver X receptor and retinoid X receptor heterodimer mediates apolipoprotein E expression, secretion and cholesterol homeostasis in astrocytes

Apolipoprotein E (apoE) is an important protein involved in lipoprotein clearance and cholesterol redistribution. ApoE is abundantly expressed in astrocytes in the brain and is closely linked to the pathogenesis of Alzheimer's disease (AD). We report here that small molecule ligands that activate either liver X receptors (LXR) or retinoid X receptor (RXR) lead to a dramatic increase in apoE mRNA and protein expression as well as secretion of apoE in a human astrocytoma cell line (CCF-STTG1 cells). Examination of primary mouse astrocytes also revealed significant induction of apoE mRNA, and protein expression and secretion following incubation with LXR/RXR agonists. Moreover, treatment of mice with a specific synthetic LXR agonist T0901317 resulted in up-regulation of apoE mRNA and protein in both hippocampus and cerebral cortex, indicating that apoE expression in brain can be up-regulated by LXR agonists in vivo. Along with a dramatic induction of ABCA1 cholesterol transporter expression, these ligands effectively mediate cholesterol efflux in both CCF-STTG1 cells and mouse astrocytes in the presence or absence of apolipoprotein AI (apoAI). Our studies provide strong evidence that small molecule LXR/RXR agonists can effectively mediate apoE synthesis and secretion as well as cholesterol homeostasis in astrocytes. LXR/RXR agonists may have significant impact on the pathogenesis of multiple neurological diseases, including AD.     

江西地区畲族人群pcsk9基因变异的SNaPshot分析

目的 研究江西地区畲族人群pcsk9基因SNP位点的变异,为开展冠心病风险研究提供可借鉴的资料.方法 通过SNaPshot法对84例畲族人外周血DNA pcsk9基因外显子7个SNP位点进行分型,ABI 3730XL扫描分型结果.结果 7个SNP位点中,仅rs505151A/G位点检测到基因变异,其G基因的频率为0.036.结论 江西地区畲族人群pcsk9基因7个SNP位点的基因型频率和等位基因频率与国内外其他民族间均存在一定差异,提示畲族人群可能具有独特的冠心病风险标记位点及变异特征.本研究为开展畲族人群pcsk9基因的冠心病风险关联性研究提供了参考依据.