Biomedical subjects

Richard B Weinberg

Publications and source records attributed to Richard B Weinberg.

11 recordsLinked to original sources

PubMed2006

Common variants of apolipoprotein A-IV differ in their ability to inhibit low density lipoprotein oxidation.

Apolipoprotein A-IV (apoA-IV) inhibits lipid peroxidation, thus demonstrating potential anti-atherogenic properties. The aim of this study was to investigate how the inhibition of low density lipoprotein (LDL) oxidation was influenced by common apoA-IV isoforms. Recombinant wild type apoA-IV (100 microg/ml) significantly inhibited the oxidation of LDL (50 microg protein/ml) by 5 microM CuSO(4) (P<0.005), but not by 100 microM CuSO(4), suggesting that it may act by binding copper ions. ApoA-IV also inhibited the oxidation of LDL by the water-soluble free-radical generator 2,2'-azobis(amidinopropane) dihydrochloride (AAPH; 1 mM), as shown by the two-fold increase in the time for half maximal conjugated diene formation (T(1/2); P<0.05) suggesting it can also scavenge free radicals in the aqueous phase. Compared to wild type apoA-IV, apoA-IV-S347 decreased T(1/2) by 15% (P=0.036) and apoA-IV-H360 increased T(1/2) by 18% (P=0.046). All apoA-IV isoforms increased the relative electrophoretic mobility of native LDL, suggesting apoA-IV can bind to LDL and acts as a site-specific antioxidant. The reduced inhibition of LDL oxidation by apoA-IV-S347 compared to wild type apoA-IV may account for the previous association of the APOA4 S347 variant with increased CHD risk and oxidative stress.

Amidines↗

PubMed2006

Self-association and lipid binding properties of the lipoprotein initiating domain of apolipoprotein B.

The amino-terminal 20.1% of apolipoprotein B (apoB20.1; residues 1-912) is sufficient to initiate and direct the formation of nascent apoB-containing lipoprotein particles. To investigate the mechanism of initial lipid acquisition by apoB, we examined the lipid binding and interfacial properties of a carboxyl-terminal His6-tagged form of apoB20.1 (apoB20.1H). ApoB20.1H was expressed in Sf9 cells and purified by nickel affinity chromatography. ApoB20.1H was produced in a folded state as characterized by formation of intramolecular disulfide bonds and resistance to chemical reduction. Dynamic light scattering in physiological buffer indicated that purified apoB20.1H formed multimers, which were readily dissociable upon the addition of nonionic detergent (0.1% Triton X-100). ApoB20.1H was incapable of binding dimyristoylphosphatidylcholine multilamellar vesicles, unless its multimeric structure was first disrupted by guanidine hydrochloride. However, apoB20.1H multimers spontaneously dissociated and bound to the interface of naked and phospholipid-coated triolein droplets. These data reveal that the initiating domain of apoB contains solvent-accessible hydrophobic sequences, which, in the absence of a hydrophobic lipid interface or detergent, engage in self-association. The high affinity of apoB20.1H for neutral lipid is consistent with the membrane binding and desorption model of apoB-containing lipoprotein assembly.

Amino Acids↗

PubMed2005

Overexpression of apolipoprotein A-IV enhances lipid secretion in IPEC-1 cells by increasing chylomicron size.

Intestinal apolipoprotein A-IV expression is highly regulated by dietary lipid in newborn swine, suggesting a role in lipid absorption. Constitutive overexpression of apoA-IV in newborn swine enterocytes enhances basolateral secretion of triacylglycerol (TG) in TG-rich lipoproteins 4.9-fold (Lu, S., Yao, Y., Meng, S., Cheng, X., and Black, D. D. (2002) J. Biol. Chem. 277, 31929-31937). To investigate the mechanism of this enhancement, IPEC-1 cells were transfected with a tetracycline-regulatable expression system (Tet-On). In cells incubated with oleic acid, a dose response relationship was observed between medium doxycycline concentration and basolateral apoA-IV and TG secretion. Similarly regulated expression of apoA-I did not enhance lipid secretion. The mean diameter of TG-rich lipoproteins secreted from doxycycline-treated cells was larger than from untreated cells (87.0 nm versus 53.4 nm). Basolateral apoB secretion decreased. Using the same expression system, full-length human apoA-IV (376 amino acids); a "pig-like" human apoA-IV, lacking the C-terminal EQQQ repeats (361 amino acids); and a "chicken-like" apoA-IV, further truncated to 343 amino acids, were expressed in IPEC-1 cells. With increasing protein secretion, cells expressing the full-length human apoA-IV displayed a 2-fold increase in TG secretion; in sharp contrast, cells expressing the pig-like human apoA-IV displayed a 25-fold increase in TG secretion and a 27-fold increase in lipoprotein diameter. When human apoA-IV was further truncated to yield a chicken-like protein, TG secretion was inhibited. We conclude that overexpression of swine apoA-IV enhances basolateral TG secretion in a dose-dependent manner by increasing the size of secreted lipoproteins. These data suggest that the region in the human apoA-IV protein from residues 344 to 354 is critical to its ability to enhance lipid secretion, perhaps by enabling the packaging of additional core TG into chylomicron particles. The EQQQ-rich region may play an inhibitory or modulatory role in chylomicron packaging in humans.

Amino Acid Sequence↗

PubMed2005

Specific sequences in the N and C termini of apolipoprotein A-IV modulate its conformation and lipid association.

Apolipoprotein (apoA-IV) is a 376-residue exchangeable apolipoprotein that may play a number of important roles in lipid metabolism, including chylomicron assembly, reverse cholesterol transport, and appetite regulation. In vivo, apoA-IV exists in both lipid-poor and lipid-associated forms, and the balance between these states may determine its function. We examined the structural elements that modulate apoA-IV lipid binding by producing a series of deletion mutants and determining their ability to interact with phospholipid liposomes. We found that the deletion of residues 333-343 strongly increased the lipid association rate versus native apoA-IV. Additional mutagenesis revealed that two phenylalanine residues at positions 334 and 335 mediated this lipid binding inhibitory effect. We also observed that residues 11-20 in the N terminus were required for the enhanced lipid affinity induced by deletion of the C-terminal sequence. We propose a structural model in which these sequences can modulate the conformation and lipid affinity of apoA-IV.

Amino Acid Sequence↗

PubMed2005

Apolipoprotein A-I helix 6 negatively charged residues attenuate lecithin-cholesterol acyltransferase (LCAT) reactivity.

Apolipoprotein A-I (apoA-I), the major protein in high density lipoprotein (HDL) regulates cholesterol homeostasis and is protective against atherosclerosis. An examination of the amino acid sequence of apoA-I among 21 species shows a high conservation of positively and negatively charged residues within helix 6, a domain responsible for regulating the rate of cholesterol esterification in plasma. These observations prompted an investigation to determine if charged residues in helix 6 maintain a structural conformation for protein-protein interaction with lecithin-cholesterol acyltransferase (LCAT) the enzyme for which apoA-I acts as a cofactor. Three apoA-I mutants were engineered; the first, (3)/(4) no negative apoA-I, eliminated 3 of the 4 negatively charged residues in helix 6, no negative apoA-I (NN apoA-I) eliminated all four negative charges, while all negative (AN apoA-I) doubled the negative charge. Reconstituted phospholipid-containing HDL (rHDL) of two discrete sizes and compositions were prepared and tested. Results showed that LCAT activation was largely influenced by both rHDL particle size and the net negative charge on helix 6. The 80 A diameter rHDL showed a 12-fold lower LCAT catalytic efficiency when compared to 96 A diameter rHDL, apparently resulting from an increased protein-protein interaction, at the expense of lipid-protein association on the 80 A rHDL. When mutant apoproteins were compared bound to the two different sized rHDL, a strong inverse correlation (r = 0.85) was found between LCAT catalytic efficiency and apoA-I helix 6 net negative charge. These results support the concept that highly conserved negatively charged residues in apoA-I helix 6 interact directly and attenuate LCAT activation, independent of the overall particle charge.

Apolipoprotein A-I↗

PubMed2004

apoA-IV tagged with the ER retention signal KDEL perturbs the intracellular trafficking and secretion of apoB.

To examine the role of apolipoprotein A-IV (apoA-IV) in the intracellular trafficking and secretion of apoB, COS cells were cotransfected with microsomal triglyceride transfer protein (MTP), apoB-41 (amino terminal 41% of apoB), and either native apoA-IV or apoA-IV modified with the carboxy-terminal endoplasmic reticulum (ER) retention signal, KDEL (apoA-IV-KDEL). As expected, apoA-IV-KDEL was inefficiently secreted relative to native apoA-IV. Coexpression of apoB-41 with apoA-IV-KDEL reduced the secretion of apoB-41 by approximately 80%. The apoA-IV-KDEL effect was specific, as neither KDEL-modified forms of human serum albumin or apoA-I affected apoB-41 secretion. Similar results were observed in McA-RH7777 rat hepatoma cells, which express endogenous MTP. The full inhibitory effect of apoA-IV-KDEL on apoB secretion was observed only for forms of apoB containing a minimum of the amino-terminal 25% of the protein (apoB-25). However, apoA-IV-KDEL inhibited the secretion of both lipid-associated and lipid-poor forms of apoB-25. Dual-label immunofluorescence microscopy of cells transfected with native apoA-IV and apoB-25 revealed that both apolipoproteins were localized to the ER and Golgi, as expected. However, when apoA-IV-KDEL was cotransfected with apoB-25, both proteins localized primarily to the ER. These data suggest that apoA-IV may physically interact with apoB in the secretory pathway, perhaps reflecting a role in modulating the process of triglyceride-rich lipoprotein assembly and secretion.

Animals↗

PubMed2003

Identification of the lipoprotein initiating domain of apolipoprotein B.

We have explored the minimum sequence requirement for the initiation of apolipoprotein B (apoB)-mediated triglyceride-rich lipoprotein assembly. A series of apoB COOH-terminal truncation mutants, spanning a range from apoB34 (amino acid residues 1-1544 of apoB100) to apoB19 (residues 1-862) were transfected into COS cells with and without coexpression of the microsomal triglyceride transfer protein (MTP). ApoB34, -25, -23, -21, -20.5, and -20.1 underwent efficient conversion to buoyant lipoproteins when coexpressed with MTP. ApoB19.5 (amino acids 1-884) also directed MTP-dependent particle assembly, although at reduced efficiency. When apoB19.5 was truncated by another 22 amino acids to form apoB19, MTP-dependent lipoprotein assembly was abolished. Analysis of the lipid stoichiometry of secreted lipoproteins revealed that all apoB truncation mutants formed spherical particles containing a hydrophobic core. Even highly truncated assembly-competent forms of apoB, such as apoB19.5 and 20.1, formed lipoproteins with surface:core lipid ratios of <1. We conclude that the translation of the first approximately 884 amino acids of apoB completes a domain capable of initiating nascent lipoprotein assembly. The composition of lipids recruited into lipoproteins by this initiating domain is consistent with formation of small emulsion particles, perhaps by simultaneous desorption of both polar and neutral lipids from a saturated bilayer.

Animals↗

PubMed2003

Structure and interfacial properties of human apolipoprotein A-V.

Apolipoprotein A-V (apoA-V), the newest member of the plasma apolipoprotein family, was recently discovered by comparison of the mouse and human genomes. Studies in rodents and population surveys of human apoA-V polymorphisms have noted a strong effect of apoA-V on plasma triglyceride levels. Toward the elucidation of the biologic function of apoA-V, we used spectroscopic and surface chemistry techniques to probe its structure and interfacial activity. Computer-assisted sequence analysis of apoA-V predicts that it is very hydrophobic, contains a significant amount of alpha-helical secondary structure, and probably is composed of discrete structural regions with varying degrees of lipid affinity. Fluorescence spectroscopy of recombinant human apoA-V provided evidence of tertiary folding, and light scattering studies indicated that apoA-V transforms dimyristoylphosphatidylcholine vesicles into discoidal complexes with an efficiency similar to that of apoA-I. Surface chemistry techniques revealed that apoA-V displays high affinity, low elasticity, and slow binding kinetics at hydrophobic interfaces, properties we propose may retard triglyceride-rich particle assembly. Metabolic labeling and immunofluorescence studies of COS-1 cells transfected with human apoA-V demonstrated that apoA-V is poorly secreted, remains associated with the endoplasmic reticulum, and does not traffic to the Golgi. Given that overexpression of the apoA-V gene lowers plasma triglycerides in mice, these data together suggest that apoA-V may function intracellularly to modulate hepatic VLDL synthesis and/or secretion.

Air↗

PubMed2002

Bryant.

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Employment↗

PubMed2002

Interfacial exclusion pressure determines the ability of apolipoprotein A-IV truncation mutants to activate cholesterol ester transfer protein.

We used a panel of recombinant human apolipoprotein (apo) A-IV truncation mutants, in which pairs of 22-mer alpha-helices were sequentially deleted along the primary sequence, to examine the impact of protein structure and interfacial activity on the ability of apoA-IV to activate cholesterol ester transfer protein. Circular dichroism and fluorescence spectroscopy revealed that the secondary structure, conformation, and molecular stability of recombinant human apoA-IV were identical to the native protein. However, deletion of any of the alpha-helical domains in apoA-IV disrupted its tertiary structure and impaired its molecular stability. Surprisingly, determination of the water/phospholipid interfacial exclusion pressure of the apoA-IV truncation mutants revealed that, for most, deletion of amphipathic alpha-helical domains increased their affinity for phospholipid monolayers. All of the truncation mutants activated the transfer of fluorescent-labeled cholesterol esters between high and low density lipoproteins at a rate higher than native apoA-IV. There was a strong positive correlation (r = 0.790, p = 0.002) between the rate constant for cholesterol ester transfer and interfacial exclusion pressure. We conclude that molecular interfacial exclusion pressure, rather than specific helical domains, determines the degree to which apoA-IV, and likely other apolipoproteins, facilitate cholesterol ester transfer protein-mediated lipid exchange.

Apolipoproteins A↗

PubMed2002

Apolipoprotein A-IV polymorphisms and diet-gene interactions.

Apolipoprotein A-IV is a 46kDa glycoprotein that is synthesized by intestinal enterocytes and is incorporated into the surface of nascent chylomicrons. Considerable evidence suggests that apolipoprotein A-IV plays a role in intestinal lipid absorption and chylomicron assembly. We have proposed that polymorphisms that alter the interfacial behavior of apolipoprotein A-IV may modulate the physical properties and metabolic fate of plasma chylomicrons. Of the reported genetic polymorphisms of apolipoprotein A-IV, two, Q360H and T347S, are known to occur at high frequencies among the world populations. Biophysical studies have established that the Q360H isoprotein displays higher lipid affinity; conversely the T347S isoprotein is predicted to be less lipid avid. Recent studies have shown that the Q360H polymorphism is associated with increased postprandial hypertriglyceridemia, a reduced low-density lipoprotein response to dietary cholesterol in the setting of a moderate fat intake, an increased high-density lipoprotein response to changes in total dietary fat content, and lower body mass and adiposity; the T347S polymorphism appears to confer the opposite effects. Studies on the diet-gene interactions of other apolipoprotein A-IV alleles are needed, as are studies on the interactions between apolipoprotein A-IV alleles and other apolipoprotein polymorphisms.

Apolipoproteins A↗