氧化修饰高密度脂蛋白的研究进展

血浆高密度脂蛋白(HDL)与低密度脂蛋白(LDL)一样可以在体内外发生氧化修饰,引起其理化性质发生一系列的改变,如多不饱和脂肪酸过氧化,卵磷脂水解,蛋白质发生聚合或分解等.活体内HDL可能在动脉壁巨噬细胞、内皮细胞及中性粒细胞、单核细胞的作用下发生氧化修饰.氧化修饰HDL可能通过清道夫受体途径代谢.氧化修饰HDL产生多种致动脉粥样硬化作用.维生素E、C的摄入可能有助于防止脂蛋白的氧化.     

A Novel Anti-Inflammatory Effect for High Density Lipoprotein

High density lipoprotein has anti-inflammatory effects in addition to mediating reverse cholesterol transport. While many of the chronic anti-inflammatory effects of high density lipoprotein (HDL) are attributed to changes in cell adhesion molecules, little is known about acute signal transduction events elicited by HDL in endothelial cells. We now show that high density lipoprotein decreases endothelial cell exocytosis, the first step in leukocyte trafficking. ApoA-I, a major apolipoprotein of HDL, mediates inhibition of endothelial cell exocytosis by interacting with endothelial scavenger receptor-BI which triggers an intracellular protective signaling cascade involving protein kinase C (PKC). Other apolipoproteins within the HDL particle have only modest effects upon endothelial exocytosis. Using a human primary culture of endothelial cells and murine apo-AI knockout mice, we show that apo-AI prevents endothelial cell exocytosis which limits leukocyte recruitment. These data suggest that high density lipoprotein may inhibit diseases associated with vascular inflammation in part by blocking endothelial exocytosis.     

Superhelix Density Heterogeneity of Intracellular Simian Virus 40 Deoxyribonucleic Acid

Covalently closed intracellular and viral simian virus 40 (SV40) deoxyribonucleic acid (DNA) were separately isolated from infected African green monkey cells (BSC-1) grown in culture. The two DNA species form overlapping bands centered at different positions in a propidium di-iodide-cesium chloride (PDI-CsCl) buoyant density gradient capable of separating closed DNA species with different superhelix densities. When the dense side of a (32)P-labeled intracellular DNA band was mixed with the light side of a (3)H-labeled intracellular DNA band and again centrifuged in a PDI-CsCl density gradient, two overlapping bands formed with modes displaced from each other. Similar band-splitting experiments performed with viral DNA always gave superimposable bands. The foregoing experiments demonstrate that the intracellular DNA is heterogeneous in superhelix density, whereas, by the same criteria, the viral DNA is homogeneous. The mean superhelix density of the intracellular closed DNA is approximately three-fourths as large as the superhelix density of the viral DNA. These results rule out the possibility that closed SV40 DNA is drawn randomly from the intracellular pool and assembled without a further nicking-closing step into virions. When the cells were grown and infected in the presence of ethidium bromide (EB), the intracellular closed DNA was found to be homogeneous in superhelix density and to have the same superhelix density as the viral DNA which, in turn, was unaffected by the presence of the drug. The foregoing results were explained by postulating that the intracellular DNA is formed with a homogeneous superhelix density and becomes heterogeneous in the absence of EB as a result of a nicking-closing cycle that occurs in a spacially or temporally heterogeneous environment. The drug EB would inhibit this action by inhibiting the nicking enzyme(s).     

High Density Lipoprotein Protects against Polymicrobe-induced Sepsis in Mice

HDL has been considered to be a protective factor in sepsis; however, most contributing studies were conducted using the endotoxic animal model, and evidence from clinically relevant septic animal models remains limited and controversial. Furthermore, little is known about the roles of HDL in sepsis other than LPS neutralization. In this study, we employed cecal ligation and puncture (CLP), a clinically relevant septic animal model, and utilized apoA-I knock-out (KO) and transgenic mice to elucidate the roles of HDL in sepsis. ApoA-I-KO mice were more susceptible to CLP-induced septic death as shown by the 47.1% survival of apoA-I-KO mice versus the 76.7% survival of C57BL/6J (B6) mice (p = 0.038). ApoA-I-KO mice had exacerbated inflammatory cytokine production during sepsis compared with B6 mice. Further study indicated that serum from apoA-I-KO mice displayed less capacity for LPS neutralization compared with serum from B6 mice. In addition, apoA-I-KO mice had less LPS clearance, reduced corticosterone generation, and impaired leukocyte recruitment in sepsis. In contrast to apoA-I-KO mice, apoA-I transgenic mice were moderately resistant to CLP-induced septic death compared with B6 mice. In conclusion, our findings reveal multiple protective roles of HDL in CLP-induced sepsis. In addition to its well established role in neutralization of LPS, HDL exerts its protection against sepsis through promoting LPS clearance and modulating corticosterone production and leukocyte recruitment. Our study supports efforts to raise HDL levels as a therapeutic approach for sepsis.     

Interfacial Tension and Surface Pressure of High Density Lipoprotein,Low Density Lipoprotein,and Related Lipid Droplets

Lipid droplets play a central role in energy storage and metabolism on a cellular scale. Their core is comprised of hydrophobic lipids covered by a surface region consisting of amphiphilic lipids and proteins. For example, high and low density lipoproteins (HDL and LDL, respectively) are essentially lipid droplets surrounded by specific proteins, their main function being to transport cholesterol. Interfacial tension and surface pressure of these particles are of great interest because they are related to the shape and the stability of the droplets and to protein adsorption at the interface. Here we use coarse-grained molecular-dynamics simulations to consider a number of related issues by calculating the interfacial tension in protein-free lipid droplets, and in HDL and LDL particles mimicking physiological conditions. First, our results suggest that the curvature dependence of interfacial tension becomes significant for particles with a radius of ～5 nm, when the area per molecule in the surface region is <1.4 nm². Further, interfacial tensions in the used HDL and LDL models are essentially unaffected by single apo-proteins at the surface. Finally, interfacial tensions of lipoproteins are higher than in thermodynamically stable droplets, suggesting that HDL and LDL are kinetically trapped into a metastable state.     

高脂饮食豚鼠高密度脂蛋白代谢的特点

目的研究豚鼠高脂饮食后高密度脂蛋白代谢的特点,并与大鼠进行比较。方法将豚鼠和大鼠分别随机分为正常组（NC）和高脂组（HF）,正常组均给予普通饲料,高脂组给予高脂饲料诱导10周后,测定血清LDL-C、HDL-C水平,HDL3/HDL2比值和LCAT、CETP的表达;采用real-time RT-PCR方法检测肝脏SR-BI表达的变化。结果与正常组相比,豚鼠高脂组血清HDL-C水平显著升高,高密度脂蛋白亚型HDL3/HDL2的比值升高,血清CETP表达均显著增加,血清LCAT表达下降,肝脏SR-BI mRNA表达水平是正常组的2.27倍。而相同高脂饲料条件下,大鼠的上述指标均无明显变化。结论豚鼠摄入高脂饮食后HDL代谢与大鼠有所不同,主要表现为血清HDL-C升高,肝脏SR-BI受体表达增加,高密度脂蛋白亚型组分发生变化,大颗粒HDL2含量相对减少,小颗粒HDL3堆积,其机制与血清CETP、LCAT的变化密切相关。     

HDL能抵抗内毒素引起得小鼠损伤

目的:观察HDL、LPS和(HDL+LPS)对小鼠血液SOD及MDA的影响,研究HDL抗LPS的作用.方法:(1)用不同浓度的PEG-6000离心人血浆脂蛋白,提取HDL并脱脂;(2)给小鼠注射HDL,LPS或HDL+LPS,对照组小鼠注射生理盐水;观察小鼠存活时间,测定其血液中SOD活性及MDA含量.结果:(1)相比对照组及HDL组,LPS组和HDL+LPS组小鼠的存活时间明显缩短,且后两者之间存在显著性差异;(2)LPS组小鼠血浆中SOD活性降低,MDA含量升高,均和其他三组有显著性差异.结论:LPS能使内毒素损伤小鼠血浆中SOD活性降低,MDA含量升高,HDL有抗内毒素损伤的作用.     

Mechanisms Responsible for the Compositional Heterogeneity of Nascent High Density Lipoprotein

Apolipoprotein (apo) A-I-containing nascent HDL particles produced by the ATP binding cassette transporter A1 have different sizes and compositions. To understand the molecular basis for this heterogeneity, the HDL particles produced by apoA-I-mediated solubilization of phospholipid (PL)/free (unesterified) cholesterol (FC) bilayer membranes in cell and cell-free systems are compared. Incubation of apoA-I with ATP binding cassette transporter A1-expressing baby hamster kidney cells leads to formation of two populations of FC-containing discoidal nascent HDL particles. The larger 11-nm diameter particles are highly FC-enriched (FC/PL = 1.2/1 mol/mol) relative to the smaller 8 nm particles and the cell plasma membrane (FC/PL = 0.4/1). ApoA-I-mediated spontaneous solubilization of either multilamellar or unilamellar vesicles made of a membrane-PL mixture and FC yields discoidal HDL particles with diameters in the range 9–17 nm and, as found with the cell system, the larger particles are relatively enriched in FC despite the fact that all particles are created by solubilization of a common FC/PL membrane domain. The size-dependent distribution of FC among HDL particles is due to varying amounts of PL being sequestered in a boundary layer by interaction with apoA-I at the disc edge. The presence of a relatively large boundary layer in smaller discoidal HDL promotes preferential distribution of phosphatidylserine to such particles. However, phosphatidylcholine and sphingomyelin which are the primary PL constituents of nascent HDL do not exhibit selective incorporation into HDL discs of different sizes. This understanding of the mechanisms responsible for the heterogeneity in lipid composition of nascent HDL particles may provide a basis for selecting subspecies with preferred cardio-protective properties.     

Direct Determination of the Superhelix Density of Closed Circular DNA by Viscometric Titration

A method has been developed for determining directly the superhelix density of DNA; this is more sensitive and subject to fewer constraints than sedimentation analysis.     

Identification of Critical Paraoxonase 1 Residues Involved in High Density Lipoprotein Interaction

Paraoxonase 1 (PON1) is a high density lipoprotein (HDL)-associated protein with atherosclerosis-protective and systemic anti-oxidant functions. We recently showed that PON1, myeloperoxidase, and HDL bind to one another in vivo forming a functional ternary complex (Huang, Y., Wu, Z., Riwanto, M., Gao, S., Levison, B. S., Gu, X., Fu, X., Wagner, M. A., Besler, C., Gerstenecker, G., Zhang, R., Li, X. M., Didonato, A. J., Gogonea, V., Tang, W. H., et al. (2013) J. Clin. Invest. 123, 3815–3828). However, specific residues on PON1 involved in the HDL-PON1 interaction remain unclear. Unambiguous identification of protein residues involved in docking interactions to lipid surfaces poses considerable methodological challenges. Here we describe a new strategy that uses a novel synthetic photoactivatable and click chemistry-taggable phospholipid probe, which, when incorporated into HDL, was used to identify amino acid residues on PON1 that directly interact with the lipoprotein phospholipid surface. Several specific PON1 residues (Leu-9, Tyr-185, and Tyr-293) were identified through covalent cross-links with the lipid probes using affinity isolation coupled to liquid chromatography with on-line tandem mass spectrometry. Based upon the crystal structure for PON1, the identified residues are all localized in relatively close proximity on the surface of PON1, defining a domain that binds to the HDL lipid surface. Site-specific mutagenesis of the identified PON1 residues (Leu-9, Tyr-185, and Tyr-293), coupled with functional studies, reveals their importance in PON1 binding to HDL and both PON1 catalytic activity and stability. Specifically, the residues identified on PON1 provide important structural insights into the PON1-HDL interaction. More generally, the new photoactivatable and affinity-tagged lipid probe developed herein should prove to be a valuable tool for identifying contact sites supporting protein interactions with lipid interfaces such as found on cell membranes or lipoproteins.     

Isolation of High Density Lipoprotein Subclasses by Electrofiltration and Their Chemical Components

Abstract

The exact role of high density lipoprotein in atheroprotection is not well understood yet due to its complex nature; it comprises more than ten subclasses that vary in size, composition, and function. Isolation and characterization of these subclasses is an important step for further studies addressing their functions in health and disease. In this work, we present a novel method that is relatively simple and efficient for isolation of high density lipoprotein subclasses. The method depends on fractional filtration of the subclasses through a preformed gel membrane system under the effect of an electric field, where the stepwise isolation of the subclasses depends on differences in their rates of migration in polyacrylamide gel. Using this design, we were able to isolate seven high density lipoprotein subclasses with relative molecular masses of 42,000–50,000; 71,000; 103,000; 124,000; 150,000; 182,000; and 219,000. All the subclasses contained apolipoprotein A-I, phosphatidylcholine, sphingomyelin, free cholesterol, esterified cholesterol, and triacylglycerols. Some fractions of some samples contained the apolipoproteins A-II, C-I, C-II, C-III, and E. A subclass of molecular mass of 106,000 was identified and isolated from a healthy young subject that contained albumin and apoA-I with some free and esterified cholesterol, but with no triacylglycerols. This electrofiltration technique offers a novel tool for isolating pure native high density lipoprotein subclasses in a concentrated form that can be used directly for detailed studies of their physicochemical and physiological properties.     

次氯酸与Cu^2＋氧化修饰高密度脂蛋白

为了进一步研究氧化修饰高密度脂蛋白（Ox－HDL）的性质,观察了次氯酸（HOCl )及Cu^2 氧化修饰,使其颗粒表面负电荷增加,TBARS值上升;另外,在HOCl氧化修饰下,HDL的载脂蛋白AI及AII发生降解,而在Cu^2 氧化下,HDLapoAI则聚合为二聚体;HOCl和Cu^2 诱导下Ox－HDL卵磷脂降解成溶血卵磷脂,同时其载脂蛋白还发生羰基修饰。然而HOCl和Cu^2 诱导下Ox-HDL卵磷脂降解成溶血卵磷脂,同时其载脂蛋白还发生羰基修饰。然而HOCl和Cu^2 介导的HDL氧化修饰在性质及动力学改变上各不相同。     

Measurement of Aldehydes in Low Density Lipoprotein by High Performance Liquid Chromatography

A modified procedure is presented for the HPLC determination of nanomolar concentrations of n-alkanals, hydroxyalkenals, malondialdehyde and furfural in biological fluid. The modifications allow aldehyde profile analysis of small samples of fresh, human, low density lipoprotein (LDL), enabling more detailed studies of LDL fatty acid peroxidation. Aldehydes are reacted with 1,3-cyclohexanedione to produce fluorescent derivatives which are separated by gradient, reversed phase, high performance liquid chromatography (HPLC). Analysis time has been reduced by shortening the sample preparation. Sensitivity has been increased by miniaturization of the derivatisation procedure, reducing required sample size. Recoveries of added aldehydes have been improved. In addition, the method presented allows determination of three further aldehydes, not measured previously by CHD methods: malondialdehyde, formaldehyde and furfural. Recovery and variability data and concentrations of aldehydes found in human LDL are given. The capacity of the method for further development, to enable determination of other aldehydes such as the trans, 2-alkenals, is also demonstrated.     

Agonistic Human Antibodies Binding to Lecithin-Cholesterol Acyltransferase Modulate High Density Lipoprotein Metabolism

Drug discovery opportunities where loss-of-function alleles of a target gene link to a disease-relevant phenotype often require an agonism approach to up-regulate or re-establish the activity of the target gene. Antibody therapy is increasingly recognized as a favored drug modality due to multiple desirable pharmacological properties. However, agonistic antibodies that enhance the activities of the target enzymes are rarely developed because the discovery of agonistic antibodies remains elusive. Here we report an innovative scheme of discovery and characterization of human antibodies capable of binding to and agonizing a circulating enzyme lecithin cholesterol acyltransferase (LCAT). Utilizing a modified human LCAT protein with enhanced enzymatic activity as an immunogen, we generated fully human monoclonal antibodies using the XenoMouse^TM platform. One of the resultant agonistic antibodies, 27C3, binds to and substantially enhances the activity of LCAT from humans and cynomolgus macaques. X-ray crystallographic analysis of the 2.45 Å LCAT-27C3 complex shows that 27C3 binding does not induce notable structural changes in LCAT. A single administration of 27C3 to cynomolgus monkeys led to a rapid increase of plasma LCAT enzymatic activity and a 35% increase of the high density lipoprotein cholesterol that was observed up to 32 days after 27C3 administration. Thus, this novel scheme of immunization in conjunction with high throughput screening may represent an effective strategy for discovering agonistic antibodies against other enzyme targets. 27C3 and other agonistic human anti-human LCAT monoclonal antibodies described herein hold potential for therapeutic development for the treatment of dyslipidemia and cardiovascular disease.     

双碳源单细胞蛋白高密度培养

单细胞蛋白(SCP)培养具有增殖速度快、原料来源广、蛋白质含量高等优点,正在成为重要的蛋白质来源之一。生产SCP的原料包括烷烃、低碳醇类及碳水化合物。由碳水化合物,尤其是由各种农副产品加工的废料、造纸工业废水及木质纤维素水解产物等,生产SCP属于废弃资源的综合利用,具有重要的经济和社会意义。这类原料中常含有多种碳源,例如,在木质纤维水解液中,有以木糖为主的五碳糖,也有以葡萄糖为主的六碳糖,木糖与葡萄糖之比约为1:2。这样,木糖的利用就成了一个关键问题。葡萄糖对木糖代谢的抑制作用随葡萄糖比例的降低而减轻;Slinger和Bothast研究了用混合糖培养Candida shehetane的过程,当木糖与葡萄糖的比例为3:1及1:1时,葡萄糖的利用速度分别比木糖高1.6倍及3.4倍;在低还原势时,酵母产酒精速率降低而细胞增长速率加快,可以达到较高细胞浓度。     

Mathematical Model for Low Density Lipoprotein (LDL) Endocytosis by Hepatocytes

Individuals with elevated levels of plasma low density lipoprotein (LDL) cholesterol (LDL-C) are considered to be at risk of developing coronary heart disease. LDL particles are removed from the blood by a process known as receptor-mediated endocytosis, which occurs mainly in the liver. A series of classical experiments delineated the major steps in the endocytotic process; apolipoprotein B-100 present on LDL particles binds to a specific receptor (LDL receptor, LDL-R) in specialized areas of the cell surface called clathrin-coated pits. The pit comprising the LDL–LDL-R complex is internalized forming a cytoplasmic endosome. Fusion of the endosome with a lysosome leads to degradation of the LDL into its constituent parts (that is, cholesterol, fatty acids, and amino acids), which are released for reuse by the cell, or are excreted. In this paper, we formulate a mathematical model of LDL endocytosis, consisting of a system of ordinary differential equations. We validate our model against existing in vitro experimental data, and we use it to explore differences in system behavior when a single bolus of extracellular LDL is supplied to cells, compared to when a continuous supply of LDL particles is available. Whereas the former situation is common to in vitro experimental systems, the latter better reflects the in vivo situation. We use asymptotic analysis and numerical simulations to study the longtime behavior of model solutions. The implications of model-derived insights for experimental design are discussed.     

人高密度脂蛋白受体胞外片段在甲醇酵母中的表达

高密度脂蛋白受体在胆固醇逆向运输中起重要作用,并且其可以做为动脉粥样硬化的新型治疗靶点。为了构建以竞争性受体-配体相互作用为基础的体外高通量药物筛选模型,在甲醇酵母中表达了高密度脂蛋白受体胞外结构域。应用RT-PCR方法从人肝癌Bel-7402总RNA中扩增了编码高密度脂蛋白受体胞外结构域的基因片段,此基因片段测序证实后亚克隆至甲醇酵母分泌表达质粒pPIC9K中。经测序验证读码框正确后将重组质粒电转至Pichia pastorisGS115中。应用PCR方法确定目的基因的整合和Mut^ 表型。重组菌株用1％甲醇诱导5天后取发酵上清进行SDS-PAGE和Western blot分析。结果表明,在分子量约64.5kDa处有sHDLR表达。随后应用Dil-AcLDL为配体证明了表达的sHDLR具有结合其配体AcLDL的生物学活性。