Apolipoprotein M is required for prebeta-HDL formation and cholesterol efflux to HDL and protects against atherosclerosis

High-density lipoproteins (HDLs) are considered antiatherogenic because they mediate reverse cholesterol transport from the periphery to the liver for excretion and degradation. Here we show that mice deficient in apolipoprotein M (apoM), a component of the HDL particle, accumulated cholesterol in large HDL particles (HDL(1)) while the conversion of HDL to prebeta-HDL was impaired. Accordingly, apoM-deficient mice lacked prebeta-HDL, a subclass of lipid-poor apolipoproteins that serves as a key acceptor of peripheral cellular cholesterol. This deficiency led to a markedly reduced cholesterol efflux from macrophages to apoM-deficient HDL compared to normal HDL in vitro. Overexpression of apoM in Ldlr(-/-) mice protected against atherosclerosis when the mice were challenged with a cholesterol-enriched diet, showing that apoM is important for the formation of prebeta-HDL and cholesterol efflux to HDL, and thereby inhibits formation of atherosclerotic lesions.     

The carboxy-terminal region of apoA-I is required for the ABCA1-dependent formation of alpha-HDL but not prebeta-HDL particles in vivo

ATP-binding cassette transporter A-1 (ABCA1)-mediated lipid efflux to lipid-poor apolipoprotein A-I (apoA-I) results in the gradual lipidation of apoA-I. This leads to the formation of discoidal high-density lipoproteins (HDL), which are subsequently converted to spherical HDL by the action of lecithin:cholesterol acyltransferase (LCAT). We have investigated the effect of point mutations and deletions in the carboxy-terminal region of apoA-I on the biogenesis of HDL using adenovirus-mediated gene transfer in apoA-I-deficient mice. It was found that the plasma HDL levels were greatly reduced in mice expressing the carboxy-terminal deletion mutants apoA-I[Delta(185-243)] and apoA-I[Delta(220-243)], shown previously to diminish the ABCA1-mediated lipid efflux. The HDL levels were normal in mice expressing the WT apoA-I, the apoA-I[Delta(232-243)] deletion mutant, or the apoA-I[E191A/H193A/K195A] point mutant, which promote normal ABCA1-mediated lipid efflux. Electron microscopy and two-dimensional gel electrophoresis showed that the apoA-I[Delta(185-243)] and apoA-I[Delta(220-243)] mutants formed mainly prebeta-HDL particles and few spherical particles enriched in apoE, while WT apoA-I, apoA-I[Delta(232-243)], and apoA-I[E191A/H193A/K195A] formed spherical alpha-HDL particles. The findings establish that (a) deletions that eliminate the 220-231 region of apoA-I prevent the synthesis of alpha-HDL but allow the synthesis of prebeta-HDL particles in vivo, (b) the amino-terminal segment 1-184 of apoA-I can promote synthesis of prebeta-HDL-type particles in an ABCA1-independent process, and (c) the charged residues in the 191-195 region of apoA-I do not influence the biogenesis of HDL.     

Hypertriglyceridemia is associated with prebeta-HDL concentrations in subjects with familial low HDL

Prebeta-HDL particles act as the primary acceptors of cellular cholesterol in reverse cholesterol transport (RCT). An impairment of RCT may be the reason for the increased risk of coronary heart disease (CHD) in subjects with familial low HDL. We studied the levels of serum prebeta-HDL and the major regulating factors of HDL metabolism in 67 subjects with familial low HDL and in 64 normolipidemic subjects. We report that the subjects with familial low HDL had markedly reduced prebeta-HDL concentrations compared with the normolipidemic subjects (17.4 +/- 7.2 vs. 23.4 +/- 7.8 mg apolipoprotein A-I/dl; P < 0.001). A positive correlation was observed between prebeta-HDL concentration and serum triglyceride (TG) level (r = 0.334, P = 0.006). In addition, serum TG level was found to be the strongest predictor of prebeta-HDL concentration in subjects with familial low HDL. The activities of cholesteryl ester transfer protein and hepatic lipase were markedly increased in subjects with familial low HDL without a significant correlation to prebeta-HDL concentration. Our results support the hypothesis that impaired RCT is one mechanism behind the increased risk for CHD in subjects with familial low HDL.     

Mechanism of rhodopsin kinase activation

The role of the cytoplasmic loops and C-terminal region of bovine rhodopsin (Rho) in binding and activating rhodopsin kinase was investigated. The ability of various enzymatically truncated forms of photolyzed rhodopsin (Rho*) to stimulate rhodopsin kinase activity was quantified. Following endopeptidase Asp-N cleavage of all phosphorylation sites on the C-terminal, the resulting truncated Rho* (329G-Rho*) was not phosphorylated by rhodopsin kinase. This suggests that rhodopsin kinase only phosphorylates C-terminal sites of Rho*. However 329G-Rho* could bind rhodopsin kinase and stimulate phosphorylation of exogenous peptide. Kinase stimulation was investigated for other truncated forms of Rho* in which the C-terminal region was either partially or completely eliminated, and the V-VI loop was either cleaved or left intact (339K-Rho*, 341E239E-Rho*, 329G239E-Rho*, 327P240S-Rho*). Results suggest that the V-VI loop is crucial for kinase binding (similar to the binding of GT). Mastoparan, a model peptide for G-protein-coupled receptors, was found to stimulate rhodopsin kinase in a mechanism similar to that of truncated Rho*. We conclude that rhodopsin kinase binds to the cytoplasmic loops of Rho* to cause a stimulation of its catalytic activity.     

Pteridine formation during lectin-induced lymphocyte activation

After iodine oxidation, biopterin, 6-hydroxymethylpterin, and 6-formylpterin were identified in mouse spleen lymphocytes by means of reverse-phase HPLC, Crithidia assay, and oxidative degradation. Concanavalin A activation induces a 30-fold increase in the pteridine amounts; biopterin as well as the sum of the carbinol and the aldehyde attain levels of 6-8 X 10(-12) mol/10(6) cells. The most rapid increase occurs during the first 24 hr. Thus, pteridine accumulation precedes the period of lymphocyte proliferation; maximum DNA synthesis was found after 72 hr. Biopterin remains largely inside the cells, whereas 6-hydroxymethylpterin and 6-formylpterin were found in the supernatant if the stimulated cells were subsequently incubated in a phosphate buffered salt solution (PBS). Isoxanthopterin was found in the PBS supernatant of control cells that previously were kept in medium alone rather than subjected to lectin stimulation. Only minimal amounts were found inside these cells, and this pterin was absent from the stimulated lymphocytes. The early increase in cellular pteridines and their differential release may well provide the basis for their modulating effect on interleukin-2 activity (Ziegler I, et al: Lymphokine Research 3:284, 1984).     

Thermal activation of DNA frayed wire formation

Dynamic light scattering has been used to study the formation of stable multistranded DNA complexes called frayed wires. DNA frayed wires arise from the indefinite self-association of oligonucleotides with long terminal tracks of guanines, e.g. d(A15G15). The complexes are stabilized via guanine-guanine interactions resulting in the formation of a guanine stem. Non-guanine portions of the oligonucleotide are disposed away from the stem and form single stranded arms. The indefinite nature of the self-association of these oligonucleotides leads to a distribution of aggregate molecular weights. The distribution arises from aggregated species that differ from one another by the number of self-associated oligonucleotides. In light-scattering experiments, the autocorrelation functions collected for frayed wires are bimodal. The slow mode, often observed for samples containing DNA and other polyelectrolytes, has been ascribed to the formation of large unspecific aggregates due to electrostatic or other long-range interactions. We attribute the fast mode to the translational diffusion of the polydisperse population in the frayed wire sample. We use the mean of the fast mode to characterize the growth of the frayed wires. Consistent with the gel electrophoresis studies, the aggregation of frayed wires is promoted by the presence of magnesium ions and incubation at high temperature. The rate of aggregate formation increases with temperature, indicating the positive activation energy for the reaction. We propose an energy diagram for the formation/disruption of frayed wires revealing the catalytic-like role of the complementary strand in the denaturation of high molecular weight complexes.     

Mechanism of formation of chromatid exchanges

Chinese hamster cells with different patterns of distribution of 5-bromodeoxyuridine (BrdUrd) between chromosome subunits were subjected, during the G2 stage, to UV irradiation, which only produced breaks in BrdUrd substituted DNA. The frequency of chromatid and subchromatid interchanges as well as isochromatid aberrations was estimated. It was found that only BrdUrd containing chromatids were involved into aberrations; this result challenges the so called "molecular theory" for aberration production proposed by Leenhouts and Chadwick. A very small increase of the aberration yield in chromosomes without BrdUrd may be connected with the action of UV on the frequency of recombination. The observed frequency of interchanges was not proportional to the BrdUrd content in chromosomes and depended on the time of its incorporation: more exchanges were induced in the chromatids incorporating BrdUrd during the last round of replication. These regularities may be connected with some molecular peculiarities of chromosome structure and function.     

Mechanism of formation of actomyosin interface

Force generation in muscle results from binding of myosin to F-actin. ATP binding to myosin provides energy to dissociate actomyosin complex while the hydrolysis of ATP is needed for re-binding of myosin to F-actin. At the end of each cycle myosin and actin form a tight complex with a substantial interface area. We investigated the dynamics of formation of actomyosin interface in presence and absence of nucleotides by quenched flow cross-linking technique. We showed previously that myosin head (subfragment 1, S1) directly interacts with at least two monomers in the actin filament. The quenched flow cross-linking experiments revealed that the initial contact (in presence or absence of nucleotides) occurs between loop 635-647 of S1 and 1-12 N-terminal residues of one actin and, then, the second contact forms between loop 567-574 of S1 and the N terminus of the second actin. The distance between these two loops in S1 corresponds to the distance between N termini of two actins in the same strand (53 A) but is smaller than that between two actins from the different strands (102 A). The formation of the actomyosin complex proceeds in ordered sequence: S1 initially binds to one actin then binds with the second actin located in the same strand but probably closer to the barbed end of F-actin. The presence of nucleotides slows down the interaction of S1 with the second actin, which correlates with recently proposed cleft movement in a 50 kDa domain of S1. The sequential mechanism of formation of actomyosin interface starting from one end and developing towards the barbed end might be involved in force generation and directional movement in actin-myosin system.