C型尼曼-匹克蛋白1和2在细胞内脂质平衡中的作用

在正常细胞,细胞外的脂质与细胞膜成分采用小泡运输的方式从细胞膜通过早期内体和细胞内吞再循环小泡(endocytic recycling compartment,ERC)转运到晚期内体(late endosome,LE)和溶酶体(lyso-some,LY),在LE/LY中的脂质和细胞膜成分被消化并排出,然后被细胞的生物合成器再利用。     

ABC proteins: key molecules for lipid homeostasis

Forty-nine ABC protein genes exist on human chromosomes. Eukaryotic ABC proteins were originally recognized as drug efflux pumps involved in the multidrug resistance of cancer cells. However, it is now realized that one of their major physiological roles is cellular lipid transport and homeostasis, and their dysfunction is often associated with human diseases. ABCA1 and ABCA7 mediate the apolipoprotein-dependent formation of a high-density lipoprotein–cholesterol complex. ABCA3 is indispensable for pulmonary surfactant secretion. ABCG5 and ABCG8 are involved in the secretion of plant sterols and cholesterol into bile. However, the primary substrates and mechanism of action of these ABC proteins have not been precisely defined. In this review article, we first describe the general structure and functions of eukaryotic ABC proteins. The current model of ABCA1 functionality is then explained based on studies on a topological model, subcellular localization, apoA-I dependence of HDL formation, functional defects of Tangier disease mutants, and ATP hydrolysis of purified ABCA1. ABCA1 is supposed to function as a transporter of lipids as well as a receptor for apoA-I. ABCA3 is likely involved in accumulating phospholipids and cholesterol in lamellar bodies and in generating multivesicular structures.     

Apolipoprotein A-I but not high-density lipoproteins are internalised by RAW macrophages: roles of ATP-binding cassette transporter A1 and scavenger receptor BI

Accumulation of lipid-loaded macrophages (foam cells) within the vessel wall is an early hallmark of atherosclerosis. High-density lipoproteins (HDL) and apolipoprotein A-I (apoA-I) can efficiently promote cholesterol efflux from macrophages. Therefore, the interaction of HDL and apoA-I with macrophages appears to be important in the initial steps of reverse cholesterol transport, i.e. the transport of excess cholesterol from foam cells to the liver. However, although several cellular apoA-I and HDL receptors and transporters have been identified, it is as yet controversial how these interactions lead to cholesterol efflux from foam cells. In this study, we show that RAW264.7 macrophages bind HDL and apoA-I in a compatible manner. Furthermore, cell surface biotinylation experiments revealed that apoA-I but not HDL is specifically internalised. Binding of HDL to macrophages is decreased by reducing the expression of scavenger receptor BI (SR-BI) with cyclic adenosine monophosphate (cAMP), acetylated low-density lipoprotein (acLDL) or RNA interference. In contrast, apoA-I cell association and internalisation is modulated in parallel with ATP-binding cassette transporter A1 (ABCA1) expression which is altered by stimulating cells with cAMP and acLDL or expressing short hairpin RNA (shRNA) against ABCA1. Consistent with this, cell surface trapping of ABCA1 with cyclosporin A (CsA) results in increased apoA-I binding but reduced internalisation. Furthermore, blocking apoA-I uptake inhibits cholesterol efflux to apoA-I but not to HDL. Taken together, these data suggest that apoA-I- but not HDL-mediated cholesterol efflux may involve retroendocytosis.     

Impairment of the ABCA1 and SR-BI-mediated cholesterol efflux pathways and HDL anti-inflammatory activity in Alzheimer's disease

The aim of our study was to investigate the effect of Alzheimer's disease (AD) on the cholesterol efflux capacity and anti-inflammatory activity of HDL. HDL and apoA-I were isolated from 20 healthy subjects and from 39 AD patients. Our results showed that serum- and HDL-mediated cholesterol efflux is significantly impaired in AD patients. This impairment of serum and HDL cholesterol efflux capacity was significantly inversely correlated to the AD severity as evaluated by MMSE scores. Results obtained from SR-BI-enriched Fu5AH and ABCA1-enriched J774 cells revealed that AD impaired the interaction of HDL and apoA-I with both the ABCA1 transporter and SR-BI receptor. Purified apoA-I from AD patients also failed to remove free excess cholesterol from ABCA1-enriched J774 macrophages. Interestingly, the decrease in plasma α-tocopherol content and the increase in MDA formation and HDL relative electrophoretic mobility indicated that AD patients had higher levels of oxidative stress. The anti-inflammatory activity of HDL was also significantly lower in AD patients as measured by the level of ICAM-1 expression. In conclusion, our study provides evidence for the first time that the functionality of HDL is impaired in AD and that this alteration might be caused by AD-associated oxidative stress and inflammation.     

Role of apolipoproteins,ABCA1 and LCAT in the biogenesis of normal and aberrant high density lipoproteins

In this review, we focus on the pathway of biogenesis of HDL, the essential role of apoA-I, ATP binding cassette transporter A1 (ABCA1), and lecithin: cholesterol acyltransferase (LCAT) in the formation of plasma HDL; the generation of aberrant forms of HDL containing mutant apoA-I forms and the role of apoA-IV and apoE in the formation of distinct HDL subpopulations. The biogenesis of HDL requires functional interactions of the ABCA1 with apoA-I (and to a lesser extent with apoE and apoA-IV) and subsequent interactions of the nascent HDL species thus formed with LCAT. Mutations in apoA-I, ABCA1 and LCAT either prevent or impair the formation of HDL and may also affect the functionality of the HDL species formed. Emphasis is placed on three categories of apoA-I mutations. The first category describes a unique bio-engineered apoA-I mutation that disrupts interactions between apoA-I and ABCA1 and generates aberrant preβ HDL subpopulations that cannot be converted efficiently to α subpopulations by LCAT. The second category describes natural and bio-engineered apoA-I mutations that generate preβ and small size α4 HDL subpopulations, and are associated with low plasma HDL levels. These phenotypes can be corrected by excess LCAT. The third category describes bio-engineered apoA-I mutations that induce hypertriglyceridemia that can be corrected by excess lipoprotein lipase and also have defective maturation of HDL. The HDL phenotypes described here may serve in the future for diagnosis, prognoses and potential treatment of abnormalities that affect the biogenesis and functionality of HDL.     

Molecular defects in the ABCA1 pathway affect platelet function

Platelet function is sensitive to alterations in cholesterol metabolism, and hypercholesterolemia is associated with enhanced platelet reagibility. Atherogenic low-density lipoproteins (LDL), in particular oxidized LDL, activate src-kinase-family-dependent signalling. In contrast, antiatherogenic high-density lipoproteins(HDL) inhibit platelet aggregation and target the phosphatidylinositol phospholipase C (PI-PLC) pathway. Sphingosine 1-phosphate is a major HDL component and may be crucial for downstream reactions of collagen-induced glycoprotein VI signalling and phosphoinositide 3-kinase. The ATP-binding cassette transporter A1 (ABCA1) regulates cell membrane phospholipid and cholesterol homeostasis and their release to lipid-poor apolipoprotein AI to generate prebeta-HDL precursor particles. ABCA1 also interacts with modulators of vesicular trafficking and number and impaired release of dense bodies from platelets.The ABCA1-NH2-terminus-associated Syntaxin-13, a SNARE complex protein, interacts with syntaxin 13-interacting protein (pallidin) whose deficiency leads to impaired platelet granule release from the dense granule Adapter Protein-3 (AP-3)-related pathway. Interestingly, the cholesterol transporter ABCG1 in addition to ABCA1 is another constituent of the AP-3 pathway, and disorders of lysosome-related organelles such as the Hermansky-Pudlack syndrome complex, Chediak-Higashi syndrome and the ceroid lipofuscinoses provide new opportunities to understand AP-3 pathway-related disorders and the irrelation to membrane phospholipid processing. ABCA1 mutations are involved in dysregulated vesicular trafficking from the trans golgi compartment to the plasma membrane, and ABCA1 R1925Q was shown to contribute to Scott syndrome, a phospholipid-processing disorder of missing surface exposure of phosphatidlyserine. The P2Y12 receptor triggers dense granule secretion by downstream effectors including the G-protein-coupled inward rectifier K+ channel-4 (GIRK-4), and we found the sister geneGIRK-3 associated with the ABCA1 protein in macrophages. It is concluded that the presence of ABCA1 and ABCG1 in the AP-3 pathway will have major impact for membrane phospholipid processing and HDL metabolism and their relation to disorders of lysosome-related organelles.     

载脂蛋白E在ABCA1和ABCG1介导的胆固醇流出中的作用

目的:探讨载脂蛋白E(ApoE)在ATP结合盒转运蛋白A1(ABCA1)和ATP结合盒转运蛋白G1(ABCG1)介导的胆固醇流出中的作用及可能机制。方法:将RAW264.7细胞随机分为3组:空白对照组:用单纯培养基培养;低密度脂蛋白受体基因敲除(LDLr^-/-)组:用20mg/LLDLr^-/-小鼠脂蛋白处理细胞;ApoE基因敲除(ApoE^-/-)组:用20mg/LApoE^-/-小鼠脂蛋白处理细胞。采用透射电镜和酶法检测细胞内脂质含量,用液闪仪技术检测胆固醇流出变化,用免疫印迹技术和实时定量PCR技术检测ABCA1和ABCG1的表达水平。结果:与对照组相比,LDLr^-/-小鼠脂蛋白处理后细胞内脂质含量并未明显改变,而ApoE^-/-小鼠脂蛋白处理48h后,细胞内胆固醇酯增加了60%;同时,与LDLr^-/-小鼠脂蛋白处理组相比,用ApoE^-/-小鼠脂蛋白处理后,巨噬细胞胆固醇流出至载脂蛋白A-I(ApoA-I)和高密度脂蛋白(HDL)的量均明显降低。与对照组相比,LDLr^-/-小鼠脂蛋白和ApoE^-/-小鼠脂蛋白处理显著增加ABCA1和ABCG1的蛋白和mRNA水平,但是ApoE^-/-小鼠脂蛋白对ABCA1和ABCG1蛋白和mRNA水平的诱导程度明显低于LDLr^-/-小鼠脂蛋白。结论:ApoE在介导巨噬细胞胆固醇流出中发挥重要作用,该作用与其调节ABCA1和ABCG1的表达水平有关。     

Lipoxin A4 promotes ABCA1 expression and cholesterol efflux through the LXRα signaling pathway in THP-1 macrophage-derived foam cells

Adenosine triphosphate-binding cassette transporter A1 (ABCA1) is a crucial cholesterol transporter and plays a central role in the high density lipoproteins (HDL) cholesterol metabolism and lipid clearance from the foam cell. Lipoxin A4 (LXA4) is an endogenous lipid mediator that requires cell-cell interaction or cell-platelet interaction for its synthesis. The roles of LXA4 on inflammatory responses are well described, while its effects on mediating ABCA1 and underlying mechanisms remain unclear. In this study, we showed that LXA4 significantly increases expression of ABCA1 and LXRα in a dose-dependent manner in THP-1 macrophage-derived foam cells. Cellular cholesterol content was decreased while cholesterol efflux was increased by LXA4 treatment. However, after short interfering RNA of LXRα, the effects of LXA4 on ABCA1 expression and cholesterol metabolism were significantly abolished. These results provide evidence that LXA4 increases ABCA1 expression and promotes cholesterol efflux through LXRα pathway in THP-1 macrophage-derived foam cells.     

Role of apoA-I, ABCA1, LCAT, and SR-BI in the biogenesis of HDL

The concentration, composition, shape, and size of plasma high-density lipoprotein (HDL) are determined by numerous proteins that influence its biogenesis, remodeling, and catabolism. The discoveries of the HDL receptor (scavenger receptor class B type I, SR-BI) and the ABCA1 (ATP-binding cassette transporter A1) lipid transporter provided two missing links that were necessary to understand the biogenesis and some of the functions of HDL. Existing data indicate that functional interactions between apoA-I and ABCA1 are necessary for the initial lipidation of apoA-I. Through a series of intermediate steps, lipidated apoA-I proceeds to form discoidal HDL particles that can be converted to spherical particles by the action of lecithin:cholesterol acyltransferase (LCAT). Discoidal and spherical HDL can interact functionally with SR-BI and these interactions lead to selective lipid uptake and net efflux of cholesterol and thus remodel HDL. Defective apoA-I/ABCA1 interactions prevent lipidation of apoA-I that is necessary for the formation of HDL particles. In the same way, specific mutations in apoA-I or LCAT prevent the conversion of discoidal to spherical HDL particles. The interactions of lipid-bound apoA-I with SR-BI are affected in vitro by specific mutations in apoA-I or SR-BI. Furthermore, deficiency of SR-BI affects the lipid and apolipoprotein composition of HDL and is associated with increased susceptibility to atherosclerosis. Here we review the current status of the pathway of HDL biogenesis and mutations in apoA-I, ABCA1, and SR-BI that disrupt different steps of the pathway and may lead to dyslipidemia and atherosclerosis in mouse models. The phenotypes generated in experimental mouse models for apoA-I, ABCA1, LCAT, SR-BI, and other proteins of the HDL pathway may facilitate early diagnosis of similar phenotypes in the human population and provide guidance for proper treatment.     

冠心病患者来源HDL对小鼠腹腔巨噬细胞脂质沉积及凋亡的影响

目的:探讨冠心病(CAD)患者来源的高密度脂蛋白(HDL)对小鼠腹腔巨噬细胞脂质沉积及凋亡的影响。方法:分离提取健康人群、稳定型冠心病(SCAD)及急性心肌梗死(AMI)患者的HDL,并观察其对氧化型低密度脂蛋白(ox-LDL)诱导的泡沫细胞内脂质沉积及细胞凋亡的影响。采用油红O检测细胞内脂质沉积,用DCFH-DA检测活性氧簇(ROS)的水平,annexin V/PI检测巨噬细胞凋亡率,Western blot检测巨噬细胞ATP结合盒转运体(ABC)A1、ABCG1、Bcl-2和Bax的表达。结果:ox-LDL处理后巨噬细胞脂质沉积增加,ABCA1和ABCG1表达上调(P<0.05);与单纯ox-LDL处理组相比,联合健康人HDL(HDL healthy)处理后巨噬细胞脂质沉积减少,ABCA1和ABCG1表达上调(P<0.05),而联合SCAD患者HDL(HDL SCAD)或AMI患者HDL(HDL AMI)处理后巨噬细胞脂质沉积进一步增加,ABCA1和ABCG1表达下调(P<0.05);与HDL SCAD处理组比较,HDL AMI处理后巨噬细胞脂质沉积进一步增加,ABCA1和ABCG1表达进一步下调(P<0.05)。ox-LDL处理后巨噬细胞内的ROS水平及凋亡比例增加,凋亡相关蛋白Bcl-2表达下调,Bax表达上调(P<0.05);与ox-LDL单独处理相比,联合HDL healthy处理后巨噬细胞内的ROS生成减少,细胞凋亡比例下降,Bcl-2表达上调,Bax表达下调(P<0.05),而联合HDL SCAD和HDL AMI处理后巨噬细胞内的ROS水平及细胞凋亡比例增高,Bcl-2表达下调,Bax表达上调;与HDL SCAD组处理相比,HDL AMI组巨噬细胞内的ROS水平及细胞凋亡进一步增加,Bcl-2表达下调,Bax表达上调(P<0.05)。结论:HDL healthy可通过上调ABCA1和ABCG1表达减轻ox-LDL诱导的泡沫细胞脂质沉积及凋亡,而HDL CAD可通过下调ABCA1和ABCG1表达加重泡沫细胞内脂质沉积并促进细胞凋亡。     

ABCA1, from pathology to membrane function

The ABCA1 transporter is the prototype of the A class of mammalian adenosine triphosphate binding cassette transporters and one of the largest members of this family. ABCA1 has been originally identified as an engulfment receptor on macrophages and, more recently, it has been shown to play an essential role in the handling of cellular lipids. Indeed by promoting the effluxes of membrane phospholipids and cholesterol to lipid-poor apoprotein acceptors, ABCA1 controls the formation of high-density lipoproteins and thus the whole process of reverse cholesterol transport. A number of additional phenotypes have been found in the mouse model of invalidation of the ABCA1 gene. In spite of their clinical diversity, they all are extremely sensitive to variations in the physicochemical properties of the cell membrane, which ABCA1 controls as a lipid translocator.     

JCL roundtable: High-density lipoprotein function and reverse cholesterol transport

High-density lipoproteins (HDL) have been known since the 1960s to be associated with protection from atherosclerotic cardiovascular disease. However, the mechanisms of this protection are unclear. The extent to which HDL per se vs other correlated metabolic factors may mitigate atherosclerosis has been seriously questioned. In fact, new epidemiologic studies have found that in some clinical settings, very high HDL cholesterol levels correlate with increased atherosclerotic risk. Most importantly, over the past 2 decades, randomized clinical trials targeting HDL have failed to reproduce the usual epidemiologic inverse relation of HDL cholesterol to atherosclerotic events. In this roundtable discussion, we bring together 3 expert investigators working in the HDL field to elucidate questions of HDL function. One area of agreement is that reverse cholesterol transport remains a primary hypothesis for an anti-atherogenic role of HDL. Bioassays that measure cholesterol efflux capacity of HDL (or of apolipoprotein [apo] B-depleted plasma) have emerged as potentially accurate surrogates for reverse cholesterol transport. ApoA-I is the major functional apoprotein of HDL, but apoE- and apoC-III-containing subpopulations of HDL may have significant roles. Anti- and pro-inflammatory functions of various HDL particles, as well as the role of oxidative and other modifications, are gaining attention.     

Neurodegeneration in Niemann-Pick type C disease mice

Niemann-Pick disease type C (NP-C) is an inherited neurodegenerative disorder associated with intracellular cholesterol and glycolipid trafficking defects. Two separate genes, NPC1 and NPC2, have been linked to NP-C. NPC1 encodes a polytopic membrane-bound protein with a putative sterol-sensing domain. NPC2 has been recently identified as epididymal secretory glycoprotein 1. The NPC1 protein functions in the vesicular redistribution of endocytosed lysosomal cargo, but how its inactivation leads to neurodegeneration is not known. The neurological symptoms of NP-C typically appear after a period of normal early development and reflect progressive degeneration of widespread brain regions. Here we have delineated the pattern of neurodegeneration in NP-C mice, whose genetic defect has been shown to be an inactivating mutation of the mouse NPC1 gene. The results reveal a spatially and temporally specific pattern of degeneration of nerve fibers followed by degeneration of neuronal cell bodies beginning as early as day 9 and continuing throughout life. We have recently showed that in the primate brain, the NPC1 protein is localized predominantly within perisynaptic astrocytic processes. The present observations suggest that a functional disturbance in NPC1 could disrupt vesicular transport of cholesterol, glycolipids and possibly other endocytic cargo in glia, which is critical for maintaining the integrity of neurons.     

Role of the gut in lipid homeostasis

Intestinal lipid transport plays a central role in fat homeostasis. Here we review the pathways regulating intestinal absorption and delivery of dietary and biliary lipid substrates, principally long-chain fatty acid, cholesterol, and other sterols. We discuss the regulation and functions of CD36 in fatty acid absorption, NPC1L1 in cholesterol absorption, as well as other lipid transporters including FATP4 and SRB1. We discuss the pathways of intestinal sterol efflux via ABCG5/G8 and ABCA1 as well as the role of the small intestine in high-density lipoprotein (HDL) biogenesis and reverse cholesterol transport. We review the pathways and genetic regulation of chylomicron assembly, the role of dominant restriction points such as microsomal triglyceride transfer protein and apolipoprotein B, and the role of CD36, l-FABP, and other proteins in formation of the prechylomicron complex. We will summarize current concepts of regulated lipoprotein secretion (including HDL and chylomicron pathways) and include lessons learned from families with genetic mutations in dominant pathways (i.e., abetalipoproteinemia, chylomicron retention disease, and familial hypobetalipoproteinemia). Finally, we will provide an integrative view of intestinal lipid homeostasis through recent findings on the role of lipid flux and fatty acid signaling via diverse receptor pathways in regulating absorption and production of satiety factors.     

Adiponectin upregulates ABCA1 expression through liver X receptor alpha signaling pathway in RAW 264.7 macrophages

ATP-binding cassette transporter A1 (ABCA1) plays a crucial role in reverse cholesterol transport and anti-atherosclerosis. Liver X receptor alpha (LXRα) can stimulate cholesterol efflux through ABCA1. It has been well known that adiponectin has cardiovascular protection. In this study, we attempted to clarify the effect of adiponectin on expression of ABCA1, and explored the role of LXRα in the regulation of ABCA1 in RAW 264.7 macrophages. Our results showed that adiponectin increased ABCA1 expression at both the mRNA and protein levels in a dose-dependent and time-dependent manner. Consequently, adiponectin promoted cholesterol efflux and decreased cholesterol content in RAW 264.7 macrophages. Moreover, adiponectin up-regulated the expression of LXRα in a dose-dependent and time-dependent manner in RAW 264.7 macrophages. LXRα small interfering RNA completely abolished the promotion effects of adiponectin. In summary, adiponectin up-regulates ABCA1 expression via the LXRα pathway in RAW 264.7 macrophages. This novel insight could prove useful for developing new treatment strategies for cardiovascular diseases.     

miRNA-33对Hcy干预RAW264.7巨噬细胞衍生的泡沫细胞表达ABCA1/ABCG1的影响

目的:研究同型半胱氨酸(Hcy)是否通过微小RNA-33 (miRNA-33)抑制三磷酸腺苷结合盒转运体A1(ABCA1)和三磷酸腺苷结合盒转运体G1(ABCG1)的表达,从而降低胆固醇逆转运(RCT)效率。方法:复制RAW264.7巨噬细胞经氧化型低密度脂蛋白(ox-LDL)诱导为泡沫细胞的模型,油红O染色确定模型是否复制成功,用LipofectamineTM2000将miRNA-33 mimics和miRNA-33 inhibitor分别转染入细胞内,再给予5 mmol/L的Hcy干预24 h后进行实验;用油红O染色观察细胞内脂滴情况;real-time PCR和Western blot检测ABCA1和ABCG1的mRNA和蛋白表达水平;液体闪烁计数法检测细胞内胆固醇流出率;高效液相色谱分析细胞内胆固醇含量。结果:(1)与空白对照组相比,miRNA-33 mimics组细胞内脂滴含量增加,ABCA1和ABCG1的mRNA和蛋白表达量降低(P 0.05),细胞内胆固醇含量逐渐增加(P 0.05),细胞内胆固醇流出率逐渐减少(P 0.05);(2)与空白对照组相比,miRNA-33 inhibitor组检测结果与miRNA-33 mimics组的结果相反,差异具有统计学意义(P 0.05);(3)空白对照组、miRNA-33 mimics-NC组和miRNA-33 inhibitor-NC组3组间相比,所有检测结果的差异均无统计学显著性。结论:Hcy可间接通过诱导miRNA-33表达而抑制ABCA1和ABCG1的蛋白表达,降低RCT效率。     

ATP结合盒A1在胆固醇流出和动脉粥样硬化中的作用

ABCA 1是ATP结合盒 (ABC)转运子超家族成员。ABCA 1基因的表达受到多种代谢物的严格调控。ABCA 1基因的突变引起Tangier病 (TD)。胆固醇逆向转运清除组织过量的胆固醇 ,防止动脉粥样硬化(AS)的产生。而细胞内胆固醇的流出是胆固醇逆向转运的限速步骤 ,ABCA 1促进胆固醇的流出而参与胆固醇的逆向转运过程。TD杂合子和转ABCA 1基因的研究证实 ,ABCA 1具有防止早期AS的作用。ABCA 1激活剂已成为临床上预防和治疗AS的新靶点。