Abstract 706: HDL and ApoA-I Inhibit Palmitate-induced Lipid Raft Trafficking of TLR4 and Inflammatory Cytokines Expression in Adipocytes

Background: Adipose tissue inflammation induces inflammatory molecules production leading to insulin resistance and increased cardiovascular disease risk. Recently it has become apparent that innate immune system is activated by saturated fatty acids (SFAs) by a process involved in TLR4 signaling. HDL and apoA-I has anti-inflammatory effects, however the mechanisms are not well understood in adipocytes. Aims: We examined to elucidate the effect of HDL and/or ApoA-I mediated cholesterol efflux on induction of inflammatory gene expression by palmitate in adipocytes via TLR4 dependent immune system. Methods: 3T3-L1 adipocytes are exposure to 250uM palmitate for periods of up to 24 hours with or without HDL or Apo A-I. Antisense directed to ABCG-1 or ABCA-1 is added 24 hours prior to each reagent challenge. Cells were harvested and estimated for expression of Saa 3 gene using real-time PCR or detection of TLR4 in raft or non raft by western blotting. Lipid rafts were purified by ultracentrifugation on discontinuous sucrose gradient, and identified by the presence of caveolin-1. Results: TLR4 was translocated into lipid raft from non raft within 15 minutes and dislocated by 60 minutes after palmitate exposure. Saa 3 gene expression was significantly up-regulated by 24 hours palmitate exposure in adipocytes. TLR4 translocation to raft and Saa 3 gene up- regulation induced by palmitate was significantly inhibited by pretreatment of HDL or Apo A-I for 6 hours. Antisense directed to ABCG-1 or SRB1 abrogated these inhibitory effects by HDL, but not ApoA-I whereas antisense to ABCA-1 did by Apo A-I but not HDL. Conclusions: These findings suggest that TLR4 mediates palmitate-induced Saa 3 gene expression in adipocytes. A reduction in membrane cholesterol mediated by ABCG-1, SRB1 or ABCA-1 can account for the ability of HDL or ApoA-I to disrupt stimulation of inflammatory cytokines by palmitate

Abstract 583: Characterization of Cell Membrane Microdomains Facilitating HDL Biogenesis

High-density lipoprotein (HDL) particles, generated in the process of removing excess cellular cholesterol, play crucial roles in maintaining cholesterol homeostasis in arterial cells and in protecting the cardiovascular system from the development of atherosclerosis. Cholesterol-loaded cells increase their binding capacity to the HDL scaffolding protein, apolipoprotein A-I (ApoA-I), however, cell surface factors necessary for ApoA-I binding remains to be elucidated. To characterize cell membrane microdomains interacting with ApoA-I, primary human skin fibroblasts were incubated with ApoA-I for 1h at 4°C. After linking protein-protein interactions with a membrane-impermeable crosslinker, DTSSP, cells were subjected to homogenization. The cell homogenate was separated by a discontinuous sucrose gradient centrifugation and ten fractions were collected. ApoA-I-associated cell membrane fraction was located by immunoblotting for ApoA-I and organelle markers. Membrane-containing fractions were fragmented using sonication prior to immunoprecipitation of ApoA-I-associated microdomains using an anti-ApoA-I antibody. Major lipid classes present in the microdomains are phosphatidylcholine, phosphatidylserine, sphingomyelin and cholesterol. Two cell membrane proteins, caveolin and ABCA1, were excluded from the microdomains. These data suggest that ApoA-I bind to cholesterol-rich cell surface microdomains that are different from ABCA1 and caveolae domains. LC-MS/MS analysis identified the presence of 26 proteins in the microdomains. Among these, several desmosomal proteins, lipid binding proteins and protease inhibitors were identified. Overall, our results suggest that the initial binding of ApoA-I to cell surface occurs on the lateral sides of cell membranes where desmosomal proteins provide a binding site for ApoA-I, and that lipid binding proteins facilitate lipidation of ApoA-I while protease inhibitors protect ApoA-I and related proteins from degradation. In conclusion, we established a new method to isolate cell membrane microdomains interacting with ApoA-I. Using this method, we found that ApoA-I associates with desmosomal proteins for the formation of HDL.

The effects of NS5A inhibitors on NS5A phosphorylation, polyprotein processing and localization

Hepatitis C virus (HCV) non-structural protein 5A (NS5A) is a multi-functional protein that is expressed in basally phosphorylated (p56) and in hyperphosphorylated (p58) forms. NS5A phosphorylation has been implicated in regulating multiple aspects of HCV replication. We recently reported the identification of a class of compounds that potently inhibit HCV RNA replication by targeting NS5A. Although the precise mechanism of inhibition of these compounds is not well understood, one activity that has been described is their ability to block expression of the hyperphosphorylated form of NS5A. Here, we report that an NS5A inhibitor impaired hyperphosphorylation without affecting basal phosphorylation at the C-terminal region of NS5A. This inhibitor activity did not require NS5A domains II and III and was distinct from that of a cellular kinase inhibitor that also blocked NS5A hyperphosphorylation, results that are consistent with an inhibitor-binding site within the N-terminal region of NS5A. In addition, we observed that an NS5A inhibitor promoted the accumulation of an HCV polyprotein intermediate, suggesting that inhibitor binding to NS5A may occur prior to the completion of polyprotein processing. Finally, we observed that NS5A p56 and p58 separated into different membrane fractions during discontinuous sucrose gradient centrifugation, consistent with these NS5A phosphoforms performing distinct replication functions. The p58 localization pattern was disrupted by an NS5A inhibitor. Collectively, our results suggest that NS5A inhibitors probably impact several aspects of HCV expression and regulation. These findings may help to explain the exceptional potency of this class of HCV replication complex inhibitors.

Role of Caveolin-3 and Glucose Transporter-4 in Isoflurane-induced Delayed Cardiac Protection

Background Caveolae are small, flask-like invaginations of the plasma membrane. Caveolins are structural proteins found in caveolae that have scaffolding properties to allow organization of signaling. The authors tested the hypothesis that delayed cardiac protection induced by volatile anesthetics is caveolae or caveolin dependent. Methods An in vivo mouse model of ischemia-reperfusion injury with delayed anesthetic preconditioning (APC) was tested in wild-type, caveolin-1 knockout, and caveolin-3 knockout mice. Mice were exposed to 30 min of oxygen or isoflurane and allowed to recover for 24 h. After 24 h recovery, mice underwent 30-min coronary artery occlusion followed by 2 h of reperfusion at which time infarct size was determined. Biochemical assays were also performed in excised hearts. Results Infarct size as a percent of the area at risk was reduced by isoflurane in wild-type (24.0 +/- 8.8% vs. 45.1 +/- 10.1%) and caveolin-1 knockout mice (27.2 +/- 12.5%). Caveolin-3 knockout mice did not show delayed APC (41.5 +/- 5.0%). Microscopically distinct caveolae were observed in wild-type and caveolin-1 knockout mice but not in caveolin-3 knockout mice. Delayed APC increased the amount of caveolin-3 protein but not caveolin-1 protein in discontinuous sucrose-gradient buoyant fractions. In addition, glucose transporter-4 was increased in buoyant fractions, and caveolin-3/glucose transporter-4 colocalization was observed in wild-type and caveolin-1 knockout mice after APC. Conclusions These results show that delayed APC involves translocation of caveolin-3 and glucose transporter-4 to caveolae, resulting in delayed protection in the myocardium.

Raft Localization and Nucleotidase Activity of CD39 Depends on Internal Proteolytic Cleavage

We have previously shown that CD39 undergoes limited cleavage and that inhibition of proteolysis results in a decrease in ATPase activity. The reduction in enzymatic activity correlated with a decrease in the fraction of full-length CD39 present in active membrane raft-localized oligomeric complexes. We exploited N-and C-terminal VP16-and V5-tagged CD39, both transiently and stably expressed in 293 cells, to further elucidate the role of cleavage in the regulation of CD39 processing and activity. To characterize the complexes generated by cross-linking, N-terminal VP16-tagged and C-terminal V5-tagged CD39 were co-expressed in 293 cells. Following crosslinking of membranes with DTSSP and immunoprecipitation with anti-V5, DTT-cleaved species were visualized by Western Blot using VP16 antibody. Interestingly, both VP16-tagged full-length and N-terminal fragments (30 kDa) were immunoprecipitated by anti-V5. This indicates that both full-length CD39 and the N-terminal cleavage fragment are present in raft-localized complexes. The composition of raft-localized CD39 complexes was studied by separating membrane fractions on a discontinuous sucrose gradient using a non-detergent method. When overexpressed, CD39 and its C-terminal fragment distribute across the gradient as visualized by Western with anti-VP16. Importantly, specific activity (expressed as ATPase activity divided by total CD39 content) was 8 times greater in low-density raft-enriched fractions than in high density raft-free fractions. In addition, relative ADPase activity was higher in fractions containing a higher proportion of C-terminal CD39 relative to full-length CD39. Thus, CD39 forms oligomeric complexes and possesses optimal enzyme activity in lipid rafts. The relationship between CD39 cleavage, ATPase activity and raft localization was further studied in 293 cells transfected with C-or N-terminal VP16-tagged CD39. Subcellular fractionation on a discontinuous sucrose gradient yielded membrane fractions enriched in endoplasmic reticulum (ER), early endosomes (EE) and plasma membrane/Golgi (PM-Golgi). Importantly, the EE fraction contained both full-length and C-terminal (or N-terminal) CD39 at the same level as seen in the PM-Golgi fraction, suggesting that near 50% of CD39 resides in the EE compartment. Furthermore, EE-expressed CD39 exhibited an ATPase and ADPase activity equivalent to that seen in Golgi-PM fractions. This led us to examine effects of NH4Cl and bafilomycin (which block acidification of EE), and chloroquine (blocks EE maturation) on CD39 cleavage, activity and raft localization. Each treatment inhibited CD39 cleavage and correspondingly decreased ATPase activity. A shift of ~50% of full-length CD39 from raft fractions to high density membrane fractions was observed upon sucrose gradient fractionation following chloroquine treatment of cells transfected with N-terminal VP16 tagged CD39. This redistribution of CD39 in the membrane correlated with a 40% decrease in ATPase activity and a striking inhibition of CD39 cleavage. Here, at a lower level of expression than cited above, ATPase activity in low-density raft fractions was ~100-fold greater than in high density fractions. Thus, cleavage of a portion of CD39 molecules is required for both raft localization of full-length CD39 and optimal enzyme activity. Regulated proteolytic cleavage of CD39 would allow for rapid upregulation of CD39 activity in response to alterations in cell environment. This would occur via cycling of CD39 between plasma membrane and endosomal compartments, the proposed site of CD39 cleavage and assembly of fully active oligomeric complexes.

Abstract 1616: The Role For Caveolin And Caveolae In Delayed Protection From Ischemia-reperfusion Injury

Introduction: Caveolae are small, flask-like invaginations of the plasma membrane. Caveolins are structural proteins found in caveolae that have scaffolding properties to allow organization of signaling. We have recently shown that both caveolin-1 and caveolin-3 knockout (Cav-1 KO and Cav-3 KO, respectively) mice are unable to be protected from myocardial ischemia-reperfusion injury by acute treatment with volatile anesthetics. Therefore, we tested the hypothesis that delayed cardiac protection induced by volatile anesthetics is caveolin-dependent. Methods: Biochemical assays were performed in excised hearts. Electron microscopy was used to assess caveolae formation. An in vivo mouse model of ischemia-reperfusion injury with delayed anesthetic preconditioning (delayed APC) was tested in wild-type (WT), Cav-1 KO, and Cav-3 KO mice. Mice were exposed to 30 min isoflurane or oxygen and allowed to recover for 24 h. After 24 h recovery, mice underwent 30 min left anterior descending coronary artery occlusion, followed by 2 h of reperfusion at which time infarct size was determined. Results: To elucidate a role for caveolins in delayed APC, wild-type mice were exposed to delayed APC and hearts were fractionated on a discontinuous sucrose gradient to isolate buoyant caveolar membranes. Delayed APC increased the amount of Cav-3 protein but not Cav-1 protein in buoyant fractions. Glucose transporter-4 (GLUT-4), known to interact with Cav-3 and affect cardiac protection, was also increased in buoyant fractions after APC. Microscopically distinct caveolae were observed in WT and Cav-1 KO mice but not Cav-3 KO mice. We assessed the impact of caveolae formation in induction of delayed APC. Infarct size as a percent of the area at risk was reduced by isoflurane in WT (24.0 ± 2.5% vs. 45.1 ± 2.9%, p < 0.05) and Cav-1 KO mice (27.2 ± 4.4%). Cav-3 KO mice did not show delayed APC (41.5 ± 2.2%). Conclusions: These results demonstrate that isoflurane-induced delayed preconditioning involves translocation of Cav-3 and GLUT-4 to caveolae and the presence of microscopically distinct caveolae (dependent on Cav-3 expression) are a requisite for induction of delayed protection in the myocardium. This research has received full or partial funding support from the American Heart Association, AHA Western States Affiliate (California, Nevada & Utah).