Although it was recently recognized that sterol carrier protein‐2 (SCP‐2) interacts with fatty acids, little is known regarding the specificity of SCP‐2 for long‐chain fatty acids or branched‐chain ...fatty‐acid‐like molecules. Likewise the location of the fatty‐acid binding site within SCP‐2 is unresolved. A fluorescent cis‐parinaric acid displacement assay was used to show that SCP‐2 optimally interacted with 14–22 carbon chain lipidic molecules: polyunsaturated fatty acids > monounsaturated, saturated > branched‐chain isoprenoids > branched‐chain phytol‐derived fatty acids. In contrast, the other major fatty‐acid binding protein in liver, fatty‐acid binding protein (L‐FABP), displayed a much narrower carbon chain preference in general: polyunsaturated fatty acids > branched‐chain phytol‐derived fatty acids > 14‐ and 16‐carbon saturated > branched‐chain isoprenoids. However, both SCP‐2 and l‐FABP displayed a very similar unsaturated fatty‐acid specificity profile. The presence and location of the SCP‐2 lipid binding site were investigated by fluorescence energy transfer. The distance between the SCP‐2 Trp50 and bound cis‐parinaric acid was determined to be 40 Å. Thus, the SCP‐2 fatty‐acid binding site appeared to be located on the opposite side of the SCP‐2 Trp50. These findings not only contribute to our understanding of the SCP‐2 ligand binding site but also provide evidence suggesting a potential role for SCP‐2 and/or L‐FABP in metabolism of branched‐chain fatty acids and isoprenoids.
The human sterol carrier protein x (SCPx)/sterol carrier protein 2 (SCP2) gene gives rise to two mRNAs: a 2.8 kb mRNA encoding SCPx, a peroxisome-associated thiolase, and a 1.5 kb mRNA encoding SCP2, ...which is thought to be an intracellular lipid transfer protein. The SCPx/SCP2 gene is highly expressed in organs involved in lipid metabolism, but the relative abundance of SCPx and SCP2 mRNAs varies. Here we report that the two transcripts are produced under the direction of two independent promoters. We determined the DNA sequence of 3.4 kb of the proximal promoter governing the transcription of SCPx sequences. The promoter governing the transcription of SCP2 sequences was identified 45 kb downstream from the SCPx promoter in intron XI. This promoter initiates transcription within exon XII. Both the SCPx and SCP2 promoters lack TATA boxes and initiate transcription at multiple sites. They share features that are found in the promoters of genes encoding other peroxisomal proteins. The basal activities of the two promoters were tested as fusion gene constructs in selected host cells, including BeWo choriocarcinoma cells, HepG2 hepatoblastoma cells, murine Y1 adrenocortical tumor cells, and Balb 3T3 fibroblasts. Cell host-specific patterns of promoter activity were observed. In addition, 8-Br-cAMP and phorbol myristate acetate were found to increase SCPx promoter activity in a host cell-specific manner. The SCP2 promoter was not significantly influenced by these agents.
A greater frequency of vasomotor symptoms (VMSs) has been associated with higher low-density lipoprotein cholesterol (LDL-C), but the association with high-density lipoprotein cholesterol (HDL-C) ...remains unclear. Endogenous estradiol (E2) levels are associated with both VMS and lipid levels and thus may confound such associations.
To assess the relationship of VMS frequency with HDL-C, LDL-C, and lipoprotein concentrations (HDL and LDL particles HDL-P; LDL-P) and lipoprotein sizes in midlife women and to evaluate whether these associations are explained by E2.
Participants were from the Study of Women's Health Across the Nation (SWAN) HDL ancillary study who had both nuclear magnetic resonance (NMR) spectroscopy lipoprotein subclass metrics and self-reported frequency of VMS measured 2–5 times over the menopause transition. VMS frequency was categorized into none, 1–5 days (infrequent), or ≥6 days (frequent) within the past 2 weeks.
We evaluated 522 women at baseline: mean age 50.3 (SD: 2.8) years; infrequent VMS: 29.8%, frequent VMS: 16.5%. Adjusting for potential confounders except E2, frequent VMS was associated with smaller HDL size β(SE): −0.06 (0.03); P = .04 and higher concentrations of LDL-C β(SE): 3.58 (1.77); P = .04 and intermediate LDL-P β(SE): 0.09 (0.05); P = .04 than no VMS. These associations were largely explained by E2, all P's > .05.
Frequent VMSs were associated with smaller HDL size and higher concentrations of LDL-C and intermediate LDL-P. These associations were explained by endogenous E2. Whether treating frequent VMS with exogenous E2 could simultaneously improve lipids/lipoproteins profile should be assessed in future studies.
•Frequent vasomotor symptom (VMS) is linked to smaller HDL size and higher LDL-C and intermediate LDL-P.•Endogenous E2 could be a pathway linking frequent VMS with lipoprotein subclasses.•Studies should assess effect of exogenous E2 on lipoproteins in women with VMS.
Relatively little is known regarding intracellular cholesterol trafficking pathways. To resolve some of these potential pathways, spontaneous and protein-mediated sterol transfer was examined between ...different donor-acceptor membrane pairs in vitro using L-cell fibroblast plasma membrane (PM) and microsomal (MICRO) and mitochondrial (MITO) membranes. Several new exciting insights were provided. First, the initial rate of spontaneous molecular sterol transfer was more dependent on the type of acceptor than donor membrane, i.e. spontaneous intracellular sterol trafficking was vectorial. Therefore, the rate of sterol desorption from the donor membrane was not necessarily the rate-limiting step in molecular sterol transfer. Second, the rate of molecular sterol transfer was not obligatorily correlated with the direction of the cholesterol gradient. For example, although PM had a 3.2-fold higher cholesterol/phospholipid ratio than MITO, spontaneous sterol transfer was 4–5-fold faster up (MITO to PM) rather than down (PM to MITO) the concentration gradient. Third, sterol carrier protein-2 differentially stimulated the initial rate of sterol transfer for all donor-acceptor combinations, being most effective with PM donors: PM-MICRO, 27-fold; and PM-MITO, 12-fold. Sterol carrier protein-2 was less effective in enhancing sterol transfer in the reverse direction, i.e. MICRO-PM and MITO-PM (5- and 4-fold, respectively). Fourth, liver fatty acid-binding protein was limited in stimulating the initial rate of sterol transfer from PM to PM (1.5-fold), from PM to MITO (3-fold), and from MICRO to MITO (3-fold). In summary, these observations present important insights into potential sterol trafficking pathways between the major membrane components of the cell.
The recent discovery that sterol carrier protein-2 (SCP-2) binds long chain fatty acyl-CoA (LCFA-CoA) with high affinity (A. Frolov et al., J. Biol. Chem. 271 (1997) 31878–31884) suggests new ...possible functions of this protein in LCFA-CoA metabolism. The purpose of the present investigation was to determine whether SCP-2 differentially modulated microsomal LCFA-CoA transacylation to cholesteryl esters, triacylglycerols, and phospholipids in vitro. Microsomal acyl-CoA:cholesterol acyltransferase (ACAT) activity measured with liposomal membrane cholesterol donors depended on substrate LCFA-CoA level, mol% cholesterol in the liposomal membrane, and total amount of liposomal cholesterol. As compared to basal activity without liposomes, microsomal ACAT was inhibited 30–50% in the presence of cholesterol poor (1.4 mol%) liposomes. In contrast, cholesterol rich (>25 mol%) liposomes stimulated ACAT up to 6.4-fold compared to basal activity without liposomes and nearly 10-fold as compared to cholesterol poor (1.4 mol%) liposomes. Increasing oleoyl-CoA reversed the inhibition of microsomal ACAT by cholesterol poor (1.4 mol%) liposomes, but did not further stimulate ACAT in the presence of cholesterol rich (35 mol%) liposomes. In contrast, high (100 μM) oleoyl-CoA inhibited ACAT nearly 3-fold. This inhibition was reversed by LCFA-CoA binding proteins, bovine serum albumin (BSA) and SCP-2. SCP-2 was 10-fold more effective (mole for mole) than BSA in reversing LCFA-CoA inhibited microsomal ACAT. Concomitantly, under conditions in which SCP-2 stimulated ACAT it equally enhanced transacylation of oleoyl-CoA into phospholipids, and 5.2-fold enhanced oleoyl-CoA transacylation to triacylglycerols. In summary, SCP-2 appeared to exert its greatest effects on microsomal transacylation in vitro by reversing LCFA-CoA inhibition of ACAT and by differentially targeting LCFA-CoA to triacylglycerols. These data suggest that the high affinity interaction of SCP-2s with LCFA-CoA may be physiologically important in microsomal transacylation reactions.
Binding of fibrinogen to platelet glycoprotein (GP) IIb/IIIa induces clot retraction. Significant differences among GP IIb/IIIa antagonists were previously noted to inhibit thromboelastography in ...whole blood specimens. The relationship between efficacy of these agents and inhibition of clot retraction is unclear. Here, we use a plasma-free clot retraction assay to evaluate potency of GP IIb/IIIa antagonists to inhibit clot retraction and modulate platelet signaling, and to address whether these effects are realized in the clinically relevant dose range. The potencies for inhibition of clot retraction and aggregation are similar for antagonists with high affinity for resting platelets and slow off-rates, whereas lower affinity and fast off-rate antagonists are disproportionately less effective in blocking clot retraction. A positive correlation is observed between inhibition of clot retraction and inhibition of tyrosine dephosphorylation across a number of GP IIb/IIIa antagonist pharmacophores. For lower affinity and fast off-rate antagonists, the concentrations required for inhibition of clot retraction clearly exceed the clinical dose range. Site occupancy studies combined with clot retraction experiments addressed whether high affinity and slow off-rate compounds can alter clot retraction during the dosing interval. Binding studies using
3H Roxifiban, a high affinity GP IIb/IIIa antagonist, indicate that occupancy of >95% of GP IIb/IIIa sites is required to inhibit clot retraction. This level of occupancy is not routinely achieved in the clinic and is not tolerated, at least for chronic therapy. These results suggest that inhibition of clot retraction is not necessary for efficacy of GP IIb/IIIa antagonists.
Abstract Objective We report a novel apolipoprotein (apo) A-I truncation (apoA-IMytilene ) due to a heterozygous nonsense mutation (c.718C > T, p.Gln216*) in a 68-year-old male proband with premature ...coronary heart disease (CHD), corneal arcus, and very low plasma concentrations of HDL cholesterol (HDL-C) and apoA-I. Two family members also had the same mutation. Our objectives were to characterize the kindred and to examine the kinetics of apoA-I, as well as cellular cholesterol efflux capacity in the proband. Methods We carried out the kinetic studies using a primed constant infusion of 5,5,5-D3 L-leucine and isotopic enrichment was determined by gas chromatography mass spectrometry in the proband and seven controls with low HDL-C. To assess cellular cholesterol efflux capacity, we used a validated ex vivo system that involved incubation of J774 macrophages with apoB-depleted serum from the proband, five controls with normal HDL-C, and two controls with low HDL-C. Results Stable isotope kinetic studies indicated that the proband had an apoA-I production rate (PR) that was 41% lower than the mean PR observed in low HDL-C controls ( n = 7). The cellular cholesterol efflux capacity assessment showed normalized cholesterol efflux capacity in the proband was decreased by 36% compared to the mean normalized cholesterol efflux capacity of normal controls ( n = 5). Conclusions Our data indicate that this novel heterozygous apoA-I truncation is associated with markedly decreased levels of HDL-C, plasma apoA-I, and apoA-I in large α-1 HDL particles, as well as decreased total cellular cholesterol efflux and decreased apoA-I production.
Although sterol carrier protein 2 (SCP-2) has long been regarded primarily as a sterol transfer protein, its actual physiological function is not known. The recent discovery that SCP-2 binds long ...chain fatty acyl-CoAs (LCFA-CoAs) with high affinity suggests additional roles for SCP-2 in cellular utilization of LCFA-CoAs for synthesis of glycerides and cholesterol esters. Concomitant to these anabolic pathways, LCFA-CoAs are also degraded by cellular hydrolases. The purpose of the work presented herein was to determine if SCP-2 altered the aqueous pool of LCFA-CoA by (i) extracting LCFA-CoA from microsomal membranes, and (ii) protecting LCFA-CoA from microsomal hydrolase activity. The data demonstrated for the first time that SCP-2 increases the aqueous pool of oleoyl-CoA by increasing the aqueous/membrane distribution oleoyl-CoA by 2.4-fold. In addition, SCP-2 inhibited the hydrolysis of oleoyl-CoA by microsomal acyl-CoA hydrolase 1.6-2.4 fold, depending on the concentration of oleoyl-CoA. By simultaneously extracting LCFA-CoA from membranes and inhibiting LCFA-CoA degradation SCP-2 may potentiate LCFA-CoA transacylation and modulate the role of LCFA-CoAs as intracellular signaling molecules.
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