The molecular chaperone, alpha-crystallin, inhibits amyloid formation by apolipoprotein C-II.
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Under lipid-free conditions, human apolipoprotein C-II (apoC-II) exists in an unfolded conformation that over several days forms amyloid ribbons. We examined the influence of the molecular chaperone, alpha-crystallin, on amyloid formation by apoC-II. Time-dependent changes in apoC-II turbidity (at 0.3 mg/ml) were suppressed potently by substoichiometric subunit concentrations of alpha-crystallin (1-10 microg/ml). alpha-Crystallin also inhibits time-dependent changes in the CD spectra, thioflavin T binding, and sedimentation coefficient of apoC-II. This contrasts with stoichiometric concentrations of alpha-crystallin required to suppress the amorphous aggregation of stressed proteins such as reduced alpha-lactalbumin. Two pieces of evidence suggest that alpha-crystallin directly interacts with amyloidogenic intermediates. First, sedimentation equilibrium and velocity experiments exclude high affinity interactions between alpha-crystallin and unstructured monomeric apoC-II. Second, the addition of alpha-crystallin does not lead to the accumulation of intermediate sized apoC-II species between monomer and large aggregates as indicated by gel filtration and sedimentation velocity experiments, suggesting that alpha-crystallin does not inhibit the relatively rapid fibril elongation upon nucleation. We propose that alpha-crystallin interacts stoichiometrically with partly structured amyloidogenic precursors, inhibiting amyloid formation at nucleation rather than the elongation phase. In doing so, alpha-crystallin forms transient complexes with apoC-II, in contrast to its chaperone behavior with stressed proteins. (+info)
Functional analyses of human apolipoprotein CII by site-directed mutagenesis: identification of residues important for activation of lipoprotein lipase.
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Apolipoprotein CII (apoCII) activates lipoprotein lipase (LPL). Seven residues, located on one face of a model alpha-helix spanning residues 59-75, are fully conserved in apoCII from ten different animal species. We have mutated these residues one by one. Substitution of Ala(59) by glycine, or Thr(62) and Gly(65) by alanine did not change the activation, indicating that these residues are outside the LPL-binding site. Replacement of Tyr(63), Ile(66), Asp(69), or Gln(70) by alanine lowered the affinity for LPL and the catalytic activity of the LPL-apoCII complex. For each residue several additional replacements were made. Most mutants retained some activating ability, but replacement of Tyr(63) by phenylalanine or tryptophan and Gln(70) by glutamate caused almost complete loss of activity. All mutants bound to liposomes with similar affinity as wild-type apoCII, and they also bound with similar affinity to LPL in the absence of hydrolyzable lipids. However, the inactive mutants did not compete with wild-type apoCII in the activation assay. Therefore, we conclude that the productive apoCII-LPL interaction may be dependent on substrate molecules. In summary, our data demonstrate that residues 63, 66, 69, and 70 are of special importance for the function of apoCII, but no single amino acid residue is absolutely crucial. (+info)
Macromolecular crowding accelerates amyloid formation by human apolipoprotein C-II.
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Human apolipoprotein C-II (apoC-II) slowly forms amyloid fibers in lipid-free solutions at physiological pH and salt concentrations (Hatters, D. M., MacPhee, C. E., Lawrence, L. J., Sawyer, W. H., and Howlett, G. J. (2000) Biochemistry 39, 8276--8283). Measurements of the time dependence of solution turbidity, thioflavin T reactivity, and the amount of sedimentable aggregate reveal that the rate and extent of amyloid formation are significantly increased by the addition of an inert polymer, dextran T10, at concentrations exceeding 20 g/liter. High dextran concentrations do not alter the secondary structure of the protein, fiber morphology, or the thioflavin T and Congo Red binding capacity of apoC-II amyloid. Analytical ultracentrifugation studies show that monomeric apoC-II does not associate significantly with dextran. The observed dependence of the overall rate of amyloid formation on dextran concentration may be accounted for quantitatively by a simple model for nonspecific volume exclusion. The model predicts that an increase in the fractional volume occupancy of macromolecules in a physiological fluid can nonspecifically accelerate the formation of amyloid fibers by any amyloidogenic protein. (+info)
Regulated expression of the apolipoprotein E/C-I/C-IV/C-II gene cluster in murine and human macrophages. A critical role for nuclear liver X receptors alpha and beta.
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Lipid-loaded macrophage "foam cells" accumulate in the subendothelial space during the development of fatty streaks and atherosclerotic lesions. To better understand the consequences of such lipid loading, murine peritoneal macrophages were isolated and incubated with ligands for two nuclear receptors, liver X receptor (LXR) and retinoic acid receptor (RXR). Analysis of the expressed mRNAs using microarray technology led to the identification of four highly induced genes that encode apolipoproteins E, C-I, C-IV, and C-II. Northern blot analysis confirmed that the mRNA levels of these four genes were induced 2-14-fold in response to natural or synthetic ligands for LXR and/or RXR. The induction of all four mRNAs was greatly attenuated in peritoneal macrophages derived from LXRalpha/beta null mice. The two LXR response elements located within the multienhancers ME.1 and ME.2 were shown to be essential for the induction of apoC-II promoter-reporter genes by ligands for LXR and/or RXR. Finally, immunohistochemical studies demonstrate that apoC-II protein co-localizes with macrophages within murine arterial lesions. Taken together, these studies demonstrate that activated LXR induces the expression of the apoE/C-I/C-IV/C-II gene cluster in both human and murine macrophages. These results suggest an alternative mechanism by which lipids are removed from macrophage foam cells. (+info)
Characterization of the lipolytic activity of endothelial lipase.
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Endothelial lipase (EL) is a new member of the triglyceride lipase gene family previously reported to have phospholipase activity. Using radiolabeled lipid substrates, we characterized the lipolytic activity of this enzyme in comparison to lipoprotein lipase (LPL) and hepatic lipase (HL) using conditioned medium from cells infected with recombinant adenoviruses encoding each of the enzymes. In the absence of serum, EL had clearly detectable triglyceride lipase activity. Both the triglyceride lipase and phospholipase activities of EL were inhibited in a dose-dependent fashion by the addition of serum. The ratio of triglyceride lipase to phospholipase activity of EL was 0.65, compared with ratios of 24.1 for HL and 139.9 for LPL, placing EL at the opposite end of the lipolytic spectrum from LPL. Neither lipase activity of EL was influenced by the addition of apolipoprotein C-II (apoC-II), indicating that EL, like HL, does not require apoC-II for activation. Like LPL but not HL, both lipase activities of EL were inhibited by 1 M NaCl. The relative ability of EL, versus HL and LPL, to hydrolyze lipids in isolated lipoprotein fractions was also examined using generation of FFAs as an end point. As expected, based on the relative triglyceride lipase activities of the three enzymes, the triglyceride-rich lipoproteins, chylomicrons, VLDL, and IDL, were efficiently hydrolyzed by LPL and HL. EL hydrolyzed HDL more efficiently than the other lipoprotein fractions, and LDL was a poor substrate for all of the enzymes. (+info)
Suppression of apolipoprotein C-II amyloid formation by the extracellular chaperone, clusterin.
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The effect of the extracellular chaperone, clusterin, on amyloid fibril formation by lipid-free human apolipoprotein C-II (apoC-II) was investigated. Sub-stoichiometric levels of clusterin, derived from either plasma or semen, potently inhibit amyloid formation by apoC-II. Inhibition is dependent on apoC-II concentration, with more effective inhibition by clusterin observed at lower concentrations of apoC-II. The average sedimentation coefficient of apoC-II fibrils formed from apoC-II (0.3 mg.mL-1) is reduced by coincubation with clusterin (10 microg x mL(-1)). In contrast, addition of clusterin (0.1 mg x mL(-1)) to preformed apoC-II amyloid fibrils (0.3 mg x mL(-1)) does not affect the size distribution after 2 days. This sedimentation velocity data suggests that clusterin inhibits fibril growth but does not promote fibril dissociation. Electron micrographs indicate similar morphologies for amyloid fibrils formed in the presence or absence of clusterin. The substoichiometric nature of the inhibition suggests that clusterin interacts with transient amyloid nuclei leading to dissociation of the monomeric subunits. We propose a general role for clusterin in suppressing the growth of extracellular amyloid. (+info)
Therapeutic factor VIII levels and negligible toxicity in mouse and dog models of hemophilia A following gene therapy with high-capacity adenoviral vectors.
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High-capacity adenoviral (HC-Ad) vectors expressing B-domain-deleted human or canine factor VIII from different liver-specific promoters were evaluated for gene therapy of hemophilia A. Intravenous administration of these vectors into hemophilic FVIII-deficient immunodeficient SCID mice (FVIIIKO-SCID) at a dose of 5 x 10(9) infectious units (IU) resulted in efficient hepatic gene delivery and long-term expression of supraphysiologic FVIII levels (exceeding 15 000 mU/mL), correcting the bleeding diathesis. Injection of only 5 x 10(7) IU still resulted in therapeutic FVIII levels. In immunocompetent hemophilic FVIII-deficient mice (FVIIIKO), FVIII expression levels peaked at 75 000 mU/mL but declined thereafter because of neutralizing anti-FVIII antibodies and a cellular immune response. Vector administration did not result in thrombocytopenia, anemia, or elevation of the proinflammatory cytokine interleukin-6 (IL-6) and caused no or only transient elevations in serum transaminases. Following transient in vivo depletion of macrophages before gene transfer, significantly higher and stable FVIII expression levels were observed. Injection of only 5 x 10(6) HC-Ad vectors after macrophage depletion resulted in long-term therapeutic FVIII levels in the FVIIIKO and FVIIIKO-SCID mice. Intravenous injection of an HC-Ad vector into a hemophilia A dog at a dose of 4.3 x 10(9) IU/kg led to transient therapeutic canine FVIII levels that partially corrected whole-blood clotting time. Inhibitory antibodies to canine FVIII could not be detected, and there were no signs of hepatotoxicity or of hematologic abnormalities. These results contribute to a better understanding of the safety and efficacy of HC-Ad vectors and suggest that the therapeutic window of HC-Ad vectors could be improved by minimizing the interaction between HC-Ad vectors and the innate immune system. (+info)
Apolipoprotein B in cholesterol-containing drusen and basal deposits of human eyes with age-related maculopathy.
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Lipids accumulate in Bruch's membrane (BrM), a specialized vascular intima of the eye, and in extracellular lesions associated with aging and age-related maculopathy (ARM). We tested the hypothesis that ARM and atherosclerotic cardiovascular disease share molecules and mechanisms pertaining to extracellular lipid accumulation by localizing cholesterol and apolipoprotein B (apo B) in BrM, basal deposits, and drusen. Human donor eyes were preserved <4 hours postmortem and cryosectioned. Sections were stained with traditional lipid stains and filipin for esterified and unesterified cholesterol or probed with antibodies to apo B, apo E, and apo C-III. Normal adult retinal pigment epithelium (RPE) was subjected to RT-PCR and Western blot analysis for apolipoprotein mRNA and protein. Esterified and unesterified cholesterol was present in all drusen and basal deposits of ARM and normal eyes. Both apo B and apo E but not apo C-III were found in BrM, drusen, and basal deposits. Fewer macular drusen were stained by traditional lipid stains and apolipoprotein antibodies than peripheral drusen. RPE contained apo B and apo E mRNA and protein. Finding cholesterol and apo B in sub-RPE deposits links ARM with important molecules and mechanisms in atherosclerosis initiation and progression. The combination of apo B mRNA and protein in RPE raises the possibility that intraocular assembly of apo B-containing lipoproteins is a pathway involved in forming cholesterol-enriched lesions in ARM. (+info)