载脂蛋白A-I结构及其功能

载脂蛋白A-I（apolipoprotein A-1,ApoA-I）是高密度脂蛋白的重要组成成分,高密度脂蛋白对于冠心病有重要的保护作用。盘状高密度脂蛋白或者ApoA-I是体内胆固醇逆向转运过程的关键因子,而游离的ApoA-I是胆固醇和磷脂的受体,因此,了解其结构对于研究其功能显得尤其重要。本文对ApoA-I单体的结构、未结合脂质,以及结合磷脂后的构象及其相关功能作了综述。     

Sensing Domain Dynamics in Protein Kinase A-I�� Complexes by Solution X-ray Scattering

The catalytic (C) and regulatory (R) subunits of protein kinase A are exceptionally dynamic proteins. Interactions between the R- and C-subunits are regulated by cAMP binding to the two cyclic nucleotide-binding domains in the R-subunit. Mammalian cells express four different isoforms of the R-subunit (RIα, RIβ, RIIα, and RIIβ) that all interact with the C-subunit in different ways. Here, we investigate the dynamic behavior of protein complexes between RIα and C-subunits using small angle x-ray scattering. We show that a single point mutation in RIα, R333K (which alters the cAMP-binding properties of Domain B) results in a compact shape compared with the extended shape of the wild-type R·C complex. A double mutant complex that disrupts the interaction site between the C-subunit and Domain B in RIα, RIα_ABR333K·C(K285P), results in a broader P(r) curve that more closely resembles the P(r) profiles of wild-type complexes. These results together suggest that interactions between RIα Domain B and the C-subunit in the RIα·C complex involve large scale dynamics that can be disrupted by single point mutations in both proteins. In contrast to RIα·C complexes. Domain B in the RIIβ·C heterodimer is not dynamic and is critical for both inhibition and complex formation. Our study highlights the functional differences of domain dynamics between protein kinase A isoforms, providing a framework for elucidating the global organization of each holoenzyme and the cross-talk between the R- and C-subunits.     

牛血清载脂蛋白A-I的研究

本文采用区带密度梯度超离心,国内首次分离得到牛的纯化高密度脂蛋白(HDL)。然后用sephadex G-200凝胶过滤,将其中的载脂蛋白分为三个组分。通过聚丙烯酰胺凝胶电泳分析,确认组分Ⅱ为载脂蛋白A-Ⅰ(APO A-Ⅰ),占HDL的80—90％,测定其分子量为27040道尔顿,全部氨基酸组成中不含半胱氨酸。其N端是天冬氨酸。本文确认牛是含APO A-I较丰富的动物之一,同时也为提纯和分离HDL及APOA-I提供了可靠的方法。     

载脂蛋白A-I抗动脉粥样硬化的研究进展

载脂蛋白A—I是HDL最主要的结构成分,是卵磷脂胆固醇酰基转移酶的主要激活剂。它决定了HDL的代谢和在血浆中的浓度。实验已经证明,载脂蛋白A—I具有抗动脉粥样硬化症的功能。在这里阐述了载脂蛋白A—I的结构和制备;载脂蛋白A—I和HDL的关系以及它在抗动脉粥样硬化方面的作用和可能的机理：胆固醇的逆向转运、抗氧化作用和调节炎症反应。     

人载脂蛋白A-I（Apo A-I）的高级结构模型和主要功能域的确定

载脂蛋白A-I(Apo A—I)是高密度脂蛋白(HDL)的主要蛋白质组分,在HDL介导的胆固醇逆向转运中发挥重要作用。Apo A—I三级结构仍不清。在新生盘状HDL分子中,Apo A—I主要存在两种结构模型：栅栏模型和带状模型。Apo A-I可通过其N末端及C末端结构域与磷脂结合,引发HDL形成。Apo A—I作为细胞胆固醇的受体,促进HDL对外周组织胆固醇的摄取。Apo A-I氨基酸残基144～186为卵磷脂胆固醇酰基转移酶(LCAT)主要激活域,通过激活LCAT,促进胆固醇逆向转运。     

Probing the Structure–Function relationship and amyloidogenic propensities in natural variants of apolipoprotein A-I

BackgroundApolipoprotein A-I (apoA-I) protects against atherosclerosis and participates in the removal of excess cellular cholesterol from peripheral organs. Several naturally occurring apoA-I mutations are associated with familial systemic amyloidosis, with deposition of amyloid aggregates in peripheral organs, resulting in multiple organ failure. Systematic studies on naturally occurring variants are needed to delineate their roles and involvement in pathogenesis.MethodsWe performed a comparative structure–function analysis of five naturally occurring apoA-I variants and the wild-type protein. Circular dichroism, Fourier-transform infrared spectroscopy, thioflavin T and congo red fluorescence assays, thermal, chemical, and proteolytic stability assays, and 1,2-Dimyristoyl-sn-glycero-3-phosphocholine clearance analyses were used to assess the effects of mutations on the structure, function, stability, aggregation, and proteolytic susceptibility of the proteins to explore the mechanisms underlying amyloidosis and hypercholesterolemia.ResultsWe observed structural changes in the mutants independent of fibril formation, suggesting the influence of the surrounding environment. The mutants were involved in aggregate formation to varying degree; L170P, R173P, and V156E showed an increased propensity to aggregate under different physiological conditions. β sheet formation indicates that L170P and R173P participate in amyloid formation. Compared to WT, V156E and L170P exhibited higher capacity for lipid clearance.ConclusionsThe selected point mutations, including those outside the hot spot regions of apoA-I structure, perturb the physiochemical and conformational behavior of the protein, influencing its function.General significanceThe study provides insights into the structure–function relationships of naturally occurring apoA-I variants outside the hot spot mutation sites.     

Can novel Apo A-I polymorphisms be responsible for low HDL in South Asian immigrants?

Coronary artery disease (CAD) is the leading cause of death in the world. Even though its rates have decreased worldwide over the past 30 years, event rates are still high in South Asians. South Asians are known to have low high-density lipoprotein (HDL) levels. The objective of this study was to identify Apolipoprotein A-I (Apo A-I) polymorphisms, the main protein component of HDL and explore its association with low HDL levels in South Asians. A pilot study on 30 South Asians was conducted and 12-h fasting samples for C-reactive protein, total cholesterol, HDL, low-density lipoprotein (LDL), triglycerides, Lipoprotein (a), Insulin, glucose levels, DNA extraction, and sequencing of Apo A-I gene were done. DNA sequencing revealed six novel Apo A-I single nucleotide polymorphisms (SNPs) in South Asians, one of which (rs 35293760, C938T) was significantly associated with low (<40 mg/dl) HDL levels (P = 0.004). The association was also seen with total cholesterol (P = 0.026) and LDL levels (P = 0.032). This pilot work has highlighted some of the gene-environment associations that could be responsible for low HDL and may be excess CAD in South Asians. Further larger studies are required to explore and uncover these associations that could be responsible for excess CAD risk in South Asians.     

Apolipoprotein A-I infiltration in rheumatoid arthritis synovial tissue: a control mechanism of cytokine production?

The production of tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β) by monocytes is strongly induced by direct contact with stimulated T lymphocytes, and this mechanism may be critical in the pathogenesis of rheumatoid arthritis (RA). Apolipoprotein A-I (apoA-I) blocks contact-mediated activation of monocytes, causing inhibition of TNF-α and IL-1β production. This study examined the hypothesis that apoA-I may have a regulatory role at sites of macrophage activation by T lymphocytes in inflamed RA synovial tissue. Synovial tissue samples were obtained after arthroscopy from patients with early untreated RA or treated RA and from normal subjects. As determined by immunohistochemistry, apoA-I was consistently present in inflamed synovial tissue that contained infiltrating T cells and macrophages, but it was absent from noninflamed tissue samples obtained from treated patients and from normal subjects. ApoA-I staining was abundant in the perivascular areas and extended in a halo-like pattern to the surrounding cellular infiltrate. C-reactive protein and serum amyloid A were not detected in the same perivascular areas of inflamed tissues. The abundant presence of apoA-I in the perivascular cellular infiltrates of inflamed RA synovial tissue extends the observations in vitro that showed that apoA-I can modify contact-mediated macrophage production of TNF-α and IL-1β. ApoA-I was not present in synovium from patients in apparent remission, suggesting that it has a specific role during phases of disease activity. These findings support the suggestion that the biologic properties of apoA-I, about which knowledge is newly emerging, include anti-inflammatory activities and therefore have important implications for the treatment of chronic inflammatory diseases.     

载脂蛋白A-I基因的DNA多态性与高脂血症的关系

载脂蛋白A-I是高密度脂蛋白的主要成份。高密度脂蛋白在肪类代谢中起着极其重要的作用,A-I组分的异常或含量的减少都将影响正常的脂类代谢、与高血脂和动脉硬化的发生有相关性。本文利用apoA-I基因探针,在中国人中,对18例血脂正常的人和15例高甘油三酯病人进行了初步的RFLP分析,结果如下。 方法:分别取10ml正常人与高甘油三酯病人末稍血提取其基因组DNA;用限制性内切酶Sstl进行完全酶解;琼脂糖凝胶电泳;Southern印迹至硝酸纤维素膜上;用α-~32P dCTP通过缺口平移标记探针,然后进行分子杂交;放射性自显影;酶谱结果分析。     

Apolipoprotein A-I Deficiency Increases Cerebral Amyloid Angiopathy and Cognitive Deficits in APP/PS1ΔE9 Mice

A hallmark of Alzheimer disease (AD) is the deposition of amyloid β (Aβ) in brain parenchyma and cerebral blood vessels, accompanied by cognitive decline. Previously, we showed that human apolipoprotein A-I (apoA-I) decreases Aβ₄₀ aggregation and toxicity. Here we demonstrate that apoA-I in lipidated or non-lipidated form prevents the formation of high molecular weight aggregates of Aβ₄₂ and decreases Aβ₄₂ toxicity in primary brain cells. To determine the effects of apoA-I on AD phenotype in vivo, we crossed APP/PS1ΔE9 to apoA-I^KO mice. Using a Morris water maze, we demonstrate that the deletion of mouse Apoa-I exacerbates memory deficits in APP/PS1ΔE9 mice. Further characterization of APP/PS1ΔE9/apoA-I^KO mice showed that apoA-I deficiency did not affect amyloid precursor protein processing, soluble Aβ oligomer levels, Aβ plaque load, or levels of insoluble Aβ in brain parenchyma. To examine the effect of Apoa-I deletion on cerebral amyloid angiopathy, we measured insoluble Aβ isolated from cerebral blood vessels. Our data show that in APP/PS1ΔE9/apoA-I^KO mice, insoluble Aβ₄₀ is increased more than 10-fold, and Aβ₄₂ is increased 1.5-fold. The increased levels of deposited amyloid in the vessels of cortices and hippocampi of APP/PS1ΔE9/apoA-I^KO mice, measured by X-34 staining, confirmed the results. Finally, we demonstrate that lipidated and non-lipidated apoA-I significantly decreased Aβ toxicity against brain vascular smooth muscle cells. We conclude that lack of apoA-I aggravates the memory deficits in APP/PS1ΔE9 mice in parallel to significantly increased cerebral amyloid angiopathy.     

北京鸭血清LDL-apo A-I及其性质的初步研究

载脂蛋白(apo)A-I主要存在于血清高密度脂蛋白(HDL)组分中,少量存在于极低密度脂蛋白(VLDL)中。我们在研究北京鸭血清脂蛋白时偶然发现鸭低密度脂蛋白(LDL)     

Human apolipoprotein A-I polymorphism. Identification of amino acid substitutions in three electrophoretic variants of the Münster-3 type

Variant forms of apolipoprotein A-I (apo-A-I) have been shown to exist in the human population. One mutant form, referred to as apo-A-I-Münster-3, is one charge unit more basic than normal apo-A-I on isoelectric focusing gels. This variant has the same immunologic characteristics and molecular weight as normal apo-A-I. The apo-A-I-Münster-3 from subjects in three unrelated families (in two of which the trait has been shown to be transmitted as an autosomal co-dominant) has been analyzed by partial amino acid sequencing to define the cause of the electrophoretic abnormality. In the apo-A-I of family A, the abnormality was shown to occur in the smallest cyanogen bromide fragment, CB-2 (residues 87-112), and amino acid sequencing revealed asparagine instead of the usual aspartic acid at residue 103. Subjects with this mutant form have shown no signs of dyslipoproteinemia. The NH2-terminal cyanogen bromide fragment (CB-1, residues 1-86) from the apo-A-I of family B was shown to differ electrophoretically from normal CB-1, and amino acid sequencing revealed that a substitution of arginine for proline at residue 4 was responsible for this variant form. Analysis of the plasma lipids of one affected family B member demonstrated that the percentage of the total cholesterol that was esterified was somewhat lower than that normally observed. In a third family, family C, a variant having the same electrophoretic abnormality as the other two was determined to have an amino acid substitution at yet a different position. In this variant, histidine was found at residue 3 in the apo-A-I sequence, rather than the usual proline. In all three cases, the substitution could account for the electrophoretic abnormality. It is proposed that these three apo-A-I-Münster-3 variants be designated apo-A-I(Asp103----Asn), apo-A-I(Pro4----Arg), and apo-A-I(Pro3----His), respectively, to indicate the substitution that accounts for the abnormality in isoelectric focusing gels.     

The Effect of Tetrahydrocortisol–Apolipoprotein A-I Complex on the RNA Polymerase Interaction with Eukaryotic DNA and the Rate of Protein Biosynthesis in Hepatocytes

A mechanism of activation of protein biosynthesis in hepatocytes was proposed as effected by the conditioned medium of nonparenchymal liver cells incubated in the presence of high density lipoproteins, cortisol, and lipopolysaccharides. It was found that the increase in the biosynthesis rate was associated with the formation of the tetrahydrocortisol–apolipoprotein A-I (THC–apoA-I) complex in macrophages, which display 5- and 5-reductase activity and are constituents of nonparenchymal liver cell. Using the small-angle X-ray scattering technique, it was shown that the THC–apoA-I–eukaryotic DNA interaction may break hydrogen bonds between pairs of complementary nucleic bases and cause the formation of single-stranded DNA fragments capable of binding to DNA-dependent RNA polymerase. The interaction is highly cooperative and has a saturating mode, up to six enzyme molecules being bound per DNA molecule.     

Effect of TNFα on activities of different promoters of human apolipoprotein A-I gene

Human apolipoprotein A-I (ApoA-I) is a major structural and functional protein component of high-density lipoproteins. The expression of the apolipoprotein A-I gene (apoA-I) in hepatocytes is repressed by pro-inflammatory cytokines such as IL-1β and TNFα. Recently, two novel additional (alternative) promoters for human apoA-I gene have been identified. Nothing is known about the role of alternative promoters in TNFα-mediated downregulation of apoA-I gene. In this article we report for the first time about the different effects of TNFα on two alternative promoters of human apoA-I gene. Stimulation of HepG2 cells by TNFα leads to activation of the distal alternative apoA-I promoter and downregulation of the proximal alternative and the canonical apoA-I promoters. This effect is mediated by weakening of the promoter competition within human apoA-I 5′-regulatory region (apoA-I promoter switching) in the cells treated by TNFα. The MEK1/2-ERK1/2 cascade and nuclear receptors PPARα and LXRs are important for TNFα-mediated apoA-I promoter switching.     

Interaction of Tetrahydrocortisol–Apolipoprotein A-I Complex with Eukaryotic DNA and with Single-Stranded Oligonucleotides

The complex formed by tetrahydrocortisol (THC) and apolipoprotein A-I (ApoAI) specifically interacts with eukaryotic DNA from rat liver. Taken together, physical and chemical data and the results of small-angle X-ray scattering analysis show that interaction of the THC–ApoAI complex with eukaryotic DNA results in deformation of the DNA double helix. Single-stranded fragments were demonstrated to cause deformation of the double helix. In this state DNA forms complexes with DNA-dependent RNA polymerase. This interaction is cooperative and of saturating type; up to six enzyme molecules bind with one DNA molecule. The putative site of complex binding with DNA is the sequence CC(GCC)_n found in many genes including the human ApoAI gene. An oligonucleotide of this type was synthesized. Its association constant (K _a) was 1.66·10⁶ M^–1. Substitution of THC with cortisol considerably decreases the K _a. We suggest that THC interacting with GC pairs of the binding site forms hydrogen bonds with cytosine, inducing rupture of the bonds within the complementary nucleic base pair.     

北京鸭Apo A-I溴化氰裂解片段的分离、纯化及其功能域定位

溴化氰可裂解北京鸭ａｐｏＡ－Ｉ为１１个肽段,通过测定纯化后各片段分子量和Ｎ末端氨基酸序列确定片段３～１０在ａｐｏＡ－Ｉ分子中的位置,分别为６４～２４０,７４～２４０,６４～２０６,１～１３６,１～６３,１７１～２０６,２０７～２４０和１３７～１７０．并对上述片段功能进行研究,结果为：（１）这些片段均可与脂质结合形成大小不同的脂质体,其大小与片段长短成正比。（２）ＡｐｏＡ－Ｉ溴化氰片段３～１０激活ＬＣＡＴ活性分别为完整ａｐｏＡ－Ｉ的６５％、５２％、６０％、３９％、８％、７％、０％和２％,说明激活ＬＣＡＴ的活性主要存在于６４～１３６之间。（３）只有片段３、４和９形成的脂质体可与肝ＨＤＬ受体结合,其他均无明显结合力,显示氨基酸２０７～２４０是ａｐｏＡ－Ｉ与ＨＤＬ受体结合片段。     

Influence of C-terminal α-helix hydrophobicity and aromatic amino acid content on apolipoprotein A-I functionality

The apoA-I molecule adopts a two-domain tertiary structure and the properties of these domains modulate the ability to form HDL particles. Thus, human apoA-I differs from mouse apoA-I in that it can form smaller HDL particles; the C-terminal α-helix is important in this process and human apoA-I is unusual in containing aromatic amino acids in the non-polar face of this amphipathic α-helix. To understand the influence of these aromatic amino acids and the associated high hydrophobicity, apoA-I variants were engineered in which aliphatic amino acids were substituted with or without causing a decrease in overall hydrophobicity. The variants human apoA-I (F225L/F229A/Y236A) and apoA-I (F225L/F229L/A232L/Y236L) were compared to wild-type (WT) apoA-I for their abilities to (1) solubilize phospholipid vesicles and form HDL particles of different sizes, and (2) mediate cellular cholesterol efflux and create nascent HDL particles via ABCA1. The loss of aromatic residues and concomitant decrease in hydrophobicity in apoA-I (F225L/F229A/Y236A) has no effect on protein stability, but reduces by a factor of about three the catalytic efficiencies (V_max/K_m) of vesicle solubilization and cholesterol efflux; also, relatively large HDL particles are formed. With apoA-I (F225L/F229L/A232L/Y236L) where the hydrophobicity is restored by the presence of only leucine residues in the helix non-polar face, the catalytic efficiencies of vesicle solubilization and cholesterol efflux are similar to those of WT apoA-I; this variant forms smaller HDL particles. Overall, the results show that the hydrophobicity of the non-polar face of the C-terminal amphipathic α-helix plays a critical role in determining apoA-I functionality but aromatic amino acids are not required. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

Effects of a lipid environment on the fibrillogenic pathway of the N-terminal polypeptide of human apolipoprotein A-I, responsible for in vivo amyloid fibril formation

In amyloidosis associated with apolipoprotein A-I (ApoA-I), heart amyloid deposits are mainly constituted by the 93-residue ApoA-I N-terminal region. A recombinant form of the amyloidogenic polypeptide, named [1-93]ApoA-I, shares conformational properties and aggregation propensity with its natural counterpart. The polypeptide, predominantly in a random coil state at pH 8.0, following acidification to pH 4.0 adopts a helical/molten globule transient state, which leads to formation of aggregates. Here we provide evidence that fibrillogenesis occurs also in physiologic-like conditions. At pH 6.4, [1-93]ApoA-I was found to assume predominantly an α-helical state, which undergoes aggregation at 37°C over time at a lower rate than at pH 4.0. After 7 days at pH 6.4, protofibrils were observed by atomic force microscopy (AFM). Using a multidisciplinary approach, including circular dichroism (CD), fluorescence, electrophoretic, and AFM analyses, we investigated the effects of a lipid environment on the conformational state and aggregation propensity of [1-93]ApoA-I. Following addition of the lipid-mimicking detergent Triton X-100, the polypeptide was found to be in a helical state at both pH 8.0 and 6.4, with no conformational transition occurring upon acidification. These helical conformers are stable and do not generate aggregated species, as observed by AFM after 21 days. Similarly, analyses of the effects of cholesterol demonstrated that this natural ApoA-I ligand induces formation of α-helix at physiological concentrations at both pH 8.0 and 6.4. Zwitterionic, positively charged, and negatively charged liposomes were found to affect [1-93]ApoA-I conformation, inducing helical species. Our data support the idea that lipids play a key role in [1-93]ApoA-I aggregation in vivo.