磷脂转运蛋白在毕赤酵母中的高效表达

为深入了解磷脂转运蛋白的结构和功能，从中国人胎肝组织总RNA成功克隆了磷脂转运蛋白成熟肽基因，所得磷脂转运蛋白基因通过同源重组整合至毕酵母细胞染色体的氧化酶AOX1基因中，甲醇诱导后经SDS聚丙烯酰胺凝胶电泳显示诱导表达蛋白分子量约为75kDa，薄层扫描显示表达蛋白中酵母蛋白总量的28%，为大量制备重组磷脂转运蛋白奠定了基础。     

胆固醇酯转运蛋白基因突变的研究进展

胆固醇酯转运蛋白促进脂蛋白中各种中性脂质转运和交换,在胆固醇逆转运中起转运蛋白基因突变和基因缺陷导致血浆胆固醇酯转运蛋白含量或活性降低,引起脂蛋白代谢发生显著变化,高密度脂蛋白水平升高,常伴有高α-脂蛋白血症.胆固醇酯转运蛋白基因变异和脂蛋白代谢异常及动脉粥样硬化的相关研究,已成为脂代谢和冠心病研究热点之一.本文综述了胆固醇酯转运蛋白基因突变及其限制片段长度多态性的研究进展.     

冠心病患者血清磷脂转运蛋白活性与冠状动脉病变严重程度的相关分析

目的分析冠心病患者血清磷脂转运蛋白活性水平与冠状动脉病变严重程度的相关性。方法 2015年6月至2016年6月期间,无锡市锡山人民医院心血管科顺序入选胸痛患者202例,行冠状动脉造影术后分为冠心病组(n=121)和非冠心病组(n=81),计算Gensini评分,测定血清磷脂转运蛋白活性水平并行常规实验室检查。结果冠心病组磷脂转运蛋白活性水平和Gensini评分显著高于非冠心病组[90.3(67~118)mg/dL比77.4(54.6~103.3)mg/dL,P0.05;29(8~48)比2(0~6),P0.001]。70~79岁与69岁及以下、男性与女性、糖尿病与非糖尿病患者相比,磷脂转运蛋白活性水平和Gensini评分明显升高(P0.05)。多重线性回归分析提示冠心病组患者磷脂转运蛋白与Gensini评分相关(Beta=0.356,P0.001)。ROC曲线分析显示磷脂转运蛋白曲线下面积为0.749(P0.001)。结论冠心病组磷脂转运蛋白活性水平与冠状动脉病变严重程度相关,磷脂转运蛋白活性水平可预测冠状动脉病变严重程度。     

胆固醇酯转运蛋白基因突变的研究进展

胆固醇酯转运蛋白促进脂蛋白中各种中性脂质转运和交换，在胆固醇逆转运中起关键作用。胆固醇酯转运蛋白基因突变和基因缺陷导致血浆胆固醇酯转运蛋白含量或活性降低，引起脂蛋白代谢发生显著变化，高密度脂蛋白水平升高，常伴有高α-脂蛋白血症。胆固醇酯转运蛋白基因变异和脂蛋白代谢异常及动脉粥样硬化的相关研究，已成为脂代谢和冠心病研究热点之一。本文综述了胆固醇酯转运蛋白基因突变及其限制片段长度多态性的研究进展。     

高密度脂蛋白的代谢相关基因表达产物与动脉粥样硬化

高密度脂蛋白胆固醇水平降低是动脉粥样硬化性心脏病独立和重要的危险因素。血浆高密度脂蛋白胆固醇水平不仅决定于它的生成速率,更重要的是取决于它的代谢水平。一系列的基因及其相关产物参与了高密度脂蛋白参与的胆固醇逆向转运过程,包括与升高血浆高密度脂蛋白胆固醇水平的基因及其产物如三磷酸腺苷结合盒转运A1、磷脂酰胆碱胆固醇酰基转移酶、磷脂转运蛋白和脂蛋白脂酶等,以及降低血浆高密度脂蛋白胆固醇水平的基因和产物如清道夫受体B1、胆固醇酯转运蛋白、肝脂肪酶和内皮细胞脂肪酶等。而高密度脂蛋白代谢与动脉粥样硬化的关系也是多方面的,不能仅由血浆高密度脂蛋白胆固醇水平来明确推断对动脉粥样硬化的影响,降低血浆高密度脂蛋白胆固醇水平与动脉粥样硬化有一定的关联但不是必然的联系。     

磷脂转运蛋白与脂蛋白代谢和动脉粥样硬化

动脉粥样硬化性心脑血管疾病是困扰人类健康的首要慢性炎症性疾病。而脂蛋白代谢异常是公认的动脉粥样硬化主要危险因素。2001年哥伦比亚大学的研究人员发现磷脂转运蛋白可以通过影响脂蛋白代谢进而改变动脉粥样硬化的易感性，从而开启了长达十多年的磷脂转运蛋白热点研究。　从动物实验到人群调查使得人们对磷脂转运蛋白在脂蛋白代谢和动脉粥样硬化中作用的认识不断深入。     

三磷酸腺苷结合盒转运子A7在脂质转运过程中作用的研究

目的:探讨三磷酸腺苷结合盒转运子A7(ABCA7)在细胞内脂质流出过程中的作用。方法:以apoAI刺激转染ABCA7-或ABCA1-基因的HEK293细胞24h,利用蛋白印迹法以及酶分析法分别测定ABCA7的变化与细胞内胆固醇和磷脂的流出。结果:apoAI分别上调ABCA7或ABCA1蛋白量,ABCA7与ABCA1同样促进细胞内脂质的转运,其中磷脂的流出较胆固醇流出更为明显。结论:ABCA7与ABCA1同样具有促进细胞内脂质转运的功能。     

胆固醇酯转运蛋白在动脉粥样硬化中的作用

胆固醇酯转运蛋白介导血浆高密度脂蛋白、低密度脂蛋白和极低密度脂蛋白之间胆固醇酯、磷脂和甘油三酯的转运,直接关联各种脂蛋白分子的组成、大小和浓度,在胆固醇的逆向转运中起关键作用,与动脉粥样硬化的发生发展密切相关。胆固醇酯转运蛋白基因有多种突变,基因突变者胆固醇酯转运蛋白浓度和活性发生改变,并伴有高密度脂蛋白胆固醇水平升高等显著的脂代谢异常。雌激素对胆固醇酯转运蛋白水平也有调节作用。胆固醇酯转运蛋白在小颗粒致密的B型低密度脂蛋白形成中起重要作用,大而轻的A型低密度脂蛋白在高甘油三酯的条件下,经胆固醇酯转运蛋白介导极低密度脂蛋白中甘油三酯与低密度脂蛋白中胆固醇酯交换,导致典型的A型低密度脂蛋白变成强致病性的B型低密度脂蛋白。     

人的hAVcDNA的克隆、表达载体的构建、序列鉴定及诱导表达

构建含有与ＨＢｓＡｇ结合的人的钙磷脂结合蛋白Ｖ(ｈＡＶ)全部 960ｂｐ基因原核表达载体。采用逆转录 ＰＣＲ ,从 1名正常人的血清中 ,扩增出ｈＡＶ编码蛋白的全部基因片段 ,将该片段插入载体ｐＧＥＸ 2Ｔ中 ,在大肠杆菌中表达。结果表明 ,ＳＤＳ ＰＡＧＥ电泳后 ,表达产物的分子量与预计相同 ,表达量占菌体蛋白量的 2 0 .5% ,成功地构建了含有ｈＡＶ全部基因的原核表达载体。该载体可以大量表达 ,为下一步测定ｈＡＶ蛋白的活性打下了基础。人的hAVcDNA的克隆、表达载体的构建、序列鉴定及诱导表达@范振平!100039$北京解放军第三○二医…     

中国人胆固醇酯转运蛋白TaqIB基因多态性与冠心病关系的Meta分析

目的对中国人胆固醇酯转运蛋白TaqIB基因多态性与冠心病关系进行Meta分析。方法通过中国生物医学文献数据库、中国学术期刊全文数据库和Medline等文献检索途径,全面检索2005年12月31日以前发表的有关中国人胆固醇酯转运蛋白TaqIB基因多态性与冠心病关系的病例对照研究,由2名独立的研究者根据纳入标准评价筛选合格文献,漏斗图检验入选文献的发表偏倚,分析各研究的异质性并采用相应的数学模型进行数据合并,统计其总的比数比和95%可信区间。采用Meta分析专用软件Review Manager(4.2版)进行统计分析。结果7个研究共1 161名冠心病患者和1 149名对照者被纳入Meta分析,入选研究无明显发表偏倚,研究结果之间无明显异质性(χ2=3.53,P=0.74),按Peto固定模型进行数据合并,数据合并后胆固醇酯转运蛋白TaqIB中B1B1基因型与B1B2 B2B2基因型的比数比为1.34,95%可信区间为1.12~1.60(P=0.002)。结论胆固醇酯转运蛋白基因TaqIB酶切位点多态性与中国人冠心病易感性相关,B1等位基因可能是冠心病的遗传危险因素。     

Genetic variation in phospholipid transfer protein modulates lipoprotein profiles in hyperalphalipoproteinemia

We previously demonstrated the role of a phospholipid transfer protein (PLTP) gene variation (rs2294213) in determining levels of high-density lipoprotein cholesterol (HDL-C) in hypoalphalipoproteinemia (HypoA). We have now explored the role of PLTP in hyperalphalipoproteinemia (HyperA). The human PLTP gene was screened for sequence anomalies by DNA melting in 107 subjects with HyperA. The association with plasma lipoprotein levels was evaluated. We detected 7 sequence variations: 1 previously reported variation (rs2294213) and 5 novel mutations including 1 missense mutation (L106F). The PLTP activity was unchanged in the p.L106F mutation. The frequency of the rs2294213 minor allele was markedly increased in the HyperA group (7.0%) in comparison with a control group (4.3%) and the hypoalphalipoproteinemia group (2.2%). Moreover, rs2294213 was strongly associated with HDL-C levels. Linear regression models predict that possession of the rs2294213 minor allele increases HDL-C independent of triglycerides. These findings extend the association of rs2294213 with HDL-C levels into the extremes of the HDL distribution.     

Haplotypes in the phospholipid transfer protein gene are associated with obesity-related phenotypes: the Québec Family Study

BACKGROUND: The phospholipid transfer protein (PLTP) may play a role in body fat regulation. OBJECTIVE: To investigate the association between PLTP genetic variants and obesity-related phenotypes. METHODS: Two intronic variants, one in intron 1 (c.-87G>A) and the other in intron 12 (c.1175+68T>G), were genotyped in 811 participants of the Québec Family Study. Nine obesity-related phenotypes were investigated, including body mass index (BMI), obesity (BMI> or =30 kg/m(2)), and waist circumference, percentage of fat, fat mass and fat-free mass measured by hydrostatic weighing as well as total, visceral and subcutaneous abdominal adipose tissue areas assessed by computed tomography. Single markers and haplotypes were tested for associations in family-based designs using the FBAT program. RESULTS: The SNP located in intron 1 showed significant associations with obesity, BMI, waist circumference and fat-free mass (P<0.05). The low-frequency allele (A allele) was associated with higher trait values, suggesting that the transmission of this allele is associated with an increased risk of being obese. Significant associations were observed between haplotypes and obesity, waist circumference, percentage of fat and fat-free mass (P<0.05). The transmission of the AT haplotype (frequency=0.180) was positively associated with obesity-related phenotypes. After sequencing the promoter and the coding regions of the PLTP gene, we were unable to identify a mutation that could replicate these results. CONCLUSION: Intronic variants of the PLTP gene are significantly associated with obesity-related phenotypes. Considering the number and the relevance of candidate genes surrounding the PLTP locus and the absence of missense polymorphisms in the coding region, the associations could be mediated by a second gene allele in linkage disequilibrium with the marker locus.     

Serum phospholipid transfer protein activity and genetic variation of the PLTP gene.

The inverse relationship between serum levels of high density lipoproteins (HDL) and risk of coronary heart disease is well established. The phospholipid transfer protein (PLTP) promotes the transfer of phospholipids between lipoproteins and modulates HDL size and composition. It thus plays a central role in HDL metabolism. Serum PLTP activity was measured in 400 healthy Finnish individuals in order to determine normal PLTP serum values. PLTP activity increased with age (P<0.001), so that the PLTP activity was 3.81+/-0.84 micromol/ml per h (mean +/- S.D., n = 52) for men and 3.97+/-0.11 micromol/ml per h (n = 52) for women in the youngest age group (25-35 years), while it was 6.77+/-0.17 micromol/ml per h (n = 45) for men and 6.68+/-0.15 micromol/ml per h (n = 40) for women in the oldest age group (56-65 years). PLTP activity correlated significantly (P<0.001) with body mass index (r = 0.22), serum total cholesterol (r = 0.17), the ratio of HDL-cholesterol/total cholesterol (r = -0.20), triglycerides (r = 0.20), apo A-II (r = 0.20), and gamma glutamyl transferase (r = 0.22) values. Serum PLTP activity correlated negatively (r = -0.20, P<0.001) with levels of apolipoprotein A-I in HDL particles that contained only apo A-I [Lp(A-I) particles]. The allelic frequencies of six intragenic polymorphisms, -79G/T, -56G/A, -37T/C, -31A/G, Phe2Leu, Arg121Trp, and two neutral polymorphisms, located in the immediate vicinity of the PLTP gene were determined. There were no significant associations between these polymorphisms and serum PLTP activity.     

Plasma apolipoprotein E concentration is an important determinant of phospholipid transfer protein activity in type 2 diabetes mellitus

BACKGROUND: Phospholipid transfer protein (PLTP) transfers phospholipids between lipoproteins and plays an important role in HDL metabolism. PLTP exists as a high-activity and a low-activity form in the circulation. In vitro studies have shown that apolipoprotein (apo) E is involved in maintaining PLTP in the active form, while the low-activity form is associated with apo AI. We have therefore investigated whether plasma apo AI, B and E concentrations are important determinants of plasma PLTP activity in type 2 diabetes, a condition associated with increased plasma PLTP activity. METHODS: Plasma PLTP activity was assayed by measuring the transfer of radiolabelled phosphatidylcholine from liposomes to HDL; apo AI and B by rate nephelometry and apo E by a 2-point turbidimetric assay. RESULTS: Type 2 diabetic patients (n = 230) had higher PLTP activity than controls (n = 97) (2374 +/- 628 nmol/mL/h versus 1862 +/- 585 respectively, p < 0.01). They also had increased fasting triglyceride and low HDL. Plasma apo B (p < 0.01) and apo E (p < 0.05) were increased, whereas apo AI was reduced (p < 0.01). Univariate analysis showed that plasma PLTP activity correlated mainly with apolipoproteins AI and E. Stepwise regression analysis showed that apo E was the main determinant of plasma PLTP activity, accounting for 23% of its variability in the diabetic subjects and 8% in the controls respectively. CONCLUSIONS: The associations between plasma apo AI and E concentrations and PLTP activity suggest that these apolipoproteins are important regulators of PLTP activity in vivo. The increase in PLTP activity in type 2 diabetes is partly related to the changes in these apolipoproteins.     

Plasma phospholipid transfer protein activity and subclinical inflammation in type 2 diabetes mellitus

Phospholipid transfer protein (PLTP) transfers phospholipids between lipoproteins, and plays an essential role in HDL metabolism. The regulation of PLTP is poorly understood and recent evidence suggests that PLTP activity increases during acute-phase response. Since type 2 diabetes is associated with chronic subclinical inflammation, the objective is to determine whether inflammation modulates PLTP in diabetes. Plasma PLTP activity was assayed by measuring the transfer of radiolabeled phosphatidylcholine from liposomes to HDL and high-sensitivity C-reactive protein (CRP) by immunoturbidimetric assay in 280 type 2 diabetic patients and 105 controls. Plasma PLTP activity (2364+/-651 nmol/ml/h versus 1880+/-586 nmol/ml/h in control, mean +/- S.D., P <0.01) and CRP (1.64(0.89-3.23)mg/l versus 0.99(0.53-2.23 mg/l, median (interquartile range), P<0.01) were increased in diabetic subjects. PLTP activity correlated significantly with age, BMI, HbA1c, log(CRP) and apolipoprotein AI and B in diabetic subjects. General linear model analysis showed that only apolipoprotein AI, age, BMI, and log(CRP) were independent determinants of PLTP activity. In conclusion, PLTP activity is increased in diabetes and apolipoprotein AI is a major determinant of PLTP activity. There is also an independent association between CRP and PLTP activity, suggesting that subclinical inflammation may influence PLTP activity in diabetes.     

Liver phospholipid transfer protein (PLTP) expression with a PLTP-null background promotes very low-density lipoprotein production in mice

It is known that plasma phospholipid transfer protein (PLTP) activity influences lipoprotein metabolism. The liver is one of the major sites of lipoprotein production and degradation, as well as of PLTP expression. To address the impact of liver-expressed PLTP on lipoprotein metabolism, we created a mouse model that expresses PLTP in the liver acutely and specifically, with a PLTP-null background. This approach in mouse model preparations can also be used universally for evaluating the function of many other genes in the liver. We found that liver PLTP expression dramatically increases plasma levels of non-high-density lipoprotein (HDL) cholesterol (2.7-fold, P < 0.0001), non-HDL phospholipid (2.5-fold, P < 0.001), and triglyceride (51%, P < 0.01), but has no significant influence on plasma HDL lipids compared with controls. Plasma apolipoprotein (apo)B levels were also significantly increased in PLTP-expressing mice (2.2-fold, P < 0.001), but those of apoA-I were not. To explore the mechanism involved, we examined the lipidation and secretion of nascent very low-density lipoprotein (VLDL), finding that liver PLTP expression significantly increases VLDL lipidation in hepatocyte microsomal lumina, and also VLDL secretion into the plasma. CONCLUSION: It is possible to prepare a mouse model that expresses the gene of interest only in the liver, but not in other tissues. Our results suggest, for the first time, that the major function of liver PLTP is to drive VLDL production and makes a small contribution to plasma PLTP activity.     

Serum phospholipid transfer protein mass as a possible protective factor for coronary heart diseases.

BACKGROUND: Phospholipid transfer protein (PLTP) can generate pre-beta high-density lipoprotein (HDL), an efficient acceptor of peripheral cholesterol, by mediating a process called HDL conversion. The transfer of phospholipids to immature HDL is also essential in maintaining reverse cholesterol transport. The phospholipid transfer activity of PLTP has been associated with various patho-physiological conditions; however, little information is available concerning the relationship between PLTP mass and disease. METHODS AND RESULTS: Using a sandwich enzyme-linked immunosorbent assay, PLTP concentration was measured and related to the risk of developing cardiovascular disease in a worksite-based cohort of Japanese men (n=2,567). Multiple linear regression analysis showed significant associations between PLTP and HDL cholesterol, triglycerides, low-density lipoprotein cholesterol, and body mass index (standardized beta=0.395, -0.191, -0.064, and -0.064, respectively; R(2)=0.31). During the follow-up period, there were 10 cases of coronary heart disease (CHD) and 7 of stroke. The multivariate adjusted relative risk of CHD was 0.46 (95% confidence interval, 0.20-1.07) for an increase of 1 standard deviation in the PLTP value (p=0.071). PLTP concentration was not related to the risk of stroke. CONCLUSIONS: The results of this prospective study indicate that the serum PLTP concentration would serve as a predictor of CHD, independent of HDL cholesterol, triglycerides and other established risk factors.     

Influence of leptin and insulin on lipid transfer proteins in human hepatoma cell line, HepG2.

AIM: Phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein (CETP) are key enzymes in lipoprotein metabolism facilitating the transfer and exchange of cholesteryl esters, triglycerides and phospholipids between lipoproteins. In the study presented here, we investigated the influence of two hormones-the adipocyte-derived hormone leptin as well as insulin on the hepatic secretion of both, PLTP and CETP. METHODS: PLTP activity and CETP concentration-measured by exogenous substrate assay and enzyme-linked immunosorbent assay-were determined in supernatant of human hepatoma cell line HepG2 after single or combined exposure to leptin and insulin at physiological and supraphysiological concentrations, respectively. Messenger-RNA of PLTP and CETP was quantified by Northern blot analysis. RESULTS: Leptin suppressed PLTP activity and CETP-concentration by up to 33% and 23%, respectively. Insulin also suppressed PLTP activity by up to 11% and CETP-concentration by up to 16%. In combination, the two hormones had additive suppressive effects for both, PLTP activity and CETP-concentration. Northern blot analysis showed no difference in m-RNA levels after exposure to leptin or insulin. CONCLUSIONS: Leptin and insulin, both known to increase with body fat mass, suppress production of PLTP and CETP in HepG2 cells. When extrapolated to the in vivo situation, this suppressive effect may constitute a mechanism counteracting the potentially harmful action of lipid transfer proteins, particularly reduction of HDL-cholesterol, in conditions frequently associated with increased plasma triglyceride levels such as obesity and insulin resistance.     

The impact of phospholipid transfer protein (PLTP) on HDL metabolism

High-density lipoproteins (HDL) play a major protective role against the development of coronary artery disease. Phospholipid transfer protein (PLTP) is a main factor regulating the size and composition of HDL in the circulation and plays an important role in controlling plasma HDL levels. This is achieved via both the phospholipid transfer activity of PLTP and its capability to cause HDL conversion. The present review focuses on the impact of PLTP on HDL metabolism. The basic characteristics and structure of the PLTP protein are described. The two main functions of PLTP, PLTP-mediated phospholipid transfer and HDL conversion are reviewed, and the mechanisms and control, as well as the physiological significance of these processes are discussed. The relationship between PLTP and the related cholesteryl ester transfer protein (CETP) is reviewed. Thereafter other functions of PLTP are recapitulated: the ability of PLTP to transfer cholesterol, alpha-tocopherol and lipopolysaccharide (LPS), and the suggested involvement of PLTP in cellular cholesterol traffic. The discussion on PLTP activity and mass in (patho)physiological settings includes new data on the presence of two forms of PLTP in the circulation, one catalytically active and the other inactive. Finally, future directions for PLTP research are outlined.