Phenethylamine in chlorella alleviates high-fat diet-induced mouse liver damage by regulating generation of methylglyoxal

This study examined the effects of oral administration of water extract of chlorella (WEC) (100 mg/kg bodyweight) and phenethylamine (10 μg/kg bodyweight) on high-fat diet (HFD)-induced liver damage in mice. Phenethylamine significantly mitigated HFD-induced lipid oxidation (generation of malondialdehyde) and liver damage without markedly decreasing hepatic lipid accumulation. WEC exerted similar effects although with decreased efficacy. In addition, WEC and phenethylamine decreased the methylglyoxal levels and increased the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein levels in the liver. Methylglyoxal is generated from substrates of GAPDH, dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. These facts indicate that methylglyoxal triggers oxidation of accumulated lipid, which generates malondialdehyde and consequently induces liver damage. Suppression of generation of toxic aldehydes by WEC and phenethylamine was also confirmed by maintaining hepatic cysteine, highly reactive to aldehydes. Thus, trace amounts of phenethylamine alleviate HFD-induced liver damage by regulating methylglyoxal via increase of GAPDH.

Abstract 15762: Gapdh Interaction With Ape1 Endonuclease Protects Vascular Smooth Muscle Cells Against Apoptosis: Potential Role of These Enzymes in Prevention of Atherosclerotic Plaque Destabilization

We have shown that oxidized lipids (OxLDL) co-localized with apoptotic vascular smooth muscle cells (VSMC) in atherosclerotic plaque and that OxLDL downregulated the major glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in cultured VSMC via H2O2-dependent mechanism. We hypothesized that maintenance of sufficient GAPDH levels is critical for cell survival under oxidative stress. H2O2 (220 uM, 12h) decreased GAPDH protein (55±5% decrease) and induced apoptosis (TUNEL assay) in human VSMC. Human VSMC transfection with pCMV-GAPDH increased GAPDH protein (3.5-fold) and prevented H2O2-induced apoptosis (74±4% decrease) vs. pCMV-GFP. We generated rat VSMC with constitutive 2-fold overexpression GAPDH (R3-VSMC). R3-VSMC had increased glycolysis (2.5 folds increase, pyruvate levels), ATP levels (38±2% increase) and reduced oxidative DNA damage (40±2% reduction in number of apurinic/apyrimidinic (AP) sites) and these effects correlated with suppressed H2O2-induced cell apoptosis (>90% reduction, TUNEL assay; 45.7±3.6% reduction, Cell Death ELISA). GAPDH-targeted siRNA reduced GAPDH protein (for 55%) and potentiated H2O2-induced apoptosis (9.4±0.3 folds increase, TUNEL; 8.6±0.4 folds increase, ELISA) in WT-VSMC and blocked anti-apoptotic effect seen in R3-VSMC. GAPDH known to bind apurinic/apyrimidinic (AP) endonuclease 1 (APE1), a major DNA repair enzyme. We found that GAPDH was co-immunoprecipitated and co-localized with APE1 in cytosolic/nuclear fraction and H2O2 induced further GAPDH-Ape1 co-localization and migration toward nuclei. APE1 specific activity was increased in H2O2-treated R3-VSMC vs. H2O2-treated WT VSMC, suggesting that GAPDH preserved APE1 activity under oxidative stress. APE1-targeted siRNA abrogated the protection of GAPDH overexpression against H2O2-induced apoptosis in VMSC. In summary, GAPDH downregulation mediates oxidant-induced VSMC apoptosis and forced expression of GAPDH protects VSMC from H2O2-induced cell death potentially via activation of APE1-dependent DNA repair. Our data suggest that preservation of GAPDH and APE1 activity in plaque VSMC could be a potential strategy to stabilize plaque and prevent acute coronary events.

Salicylic Acid Inhibits the Replication of Tomato bushy stunt virus by Directly Targeting a Host Component in the Replication Complex

Although the plant hormone salicylic acid (SA) plays a central role in signaling resistance to viral infection, the underlying mechanisms are only partially understood. Identification and characterization of SA’s direct targets have been shown to be an effective strategy for dissecting the complex SA-mediated defense signaling network. In search of additional SA targets, we previously developed two sensitive approaches that utilize SA analogs in conjunction with either a photoaffinity labeling technique or surface plasmon resonance-based technology to identify and evaluate candidate SA-binding proteins (SABPs) from Arabidopsis. Using these approaches, we have now identified several members of the Arabidopsis glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein family, including two chloroplast-localized and two cytosolic isoforms, as SABPs. Cytosolic GAPDH is a well-known glycolytic enzyme; it also is an important host factor involved in the replication of Tomato bushy stunt virus (TBSV), a single-stranded RNA virus. Using a yeast cell-free extract, an in vivo yeast replication system, and plant protoplasts, we demonstrate that SA inhibits TBSV replication. SA does so by inhibiting the binding of cytosolic GAPDH to the negative (−)RNA strand of TBSV. Thus, this study reveals a novel molecular mechanism through which SA regulates virus replication.

Effect of altering fibroblast integrin associated protein expression on the growth and protein expression of pancreas cancer cells.

251 Background: The lack of success of current treatments for pancreatic ductal adenocarcinoma (PDA) may be due in part to the presence of the dense surrounding stromal response and the interactions between the stroma and cancer cells. We have shown that integrin associated proteins, in particular PINCH, are expressed to a higher degree in the stroma adjacent to the tumor cells and PINCH expression is positively correlated with poorer PDA patient outcomes. We hypothesize that decreasing PINCH protein expression in the tumor associated stroma will decrease the growth and expression of growth promoting proteins in PDA cells. Methods: Red fluorescent protein (RFP) stably expressing MiaPaCa-2 cells were co-cultured with WI38 fibroblasts, or WI38 fibroblasts with shRNA knockdown of PINCH protein (PINCH KD). Cell growth of the PDA cells alone and upon exposure to WI38 and PINCH KD fibroblasts was determined using RFP at 48 hours. PINCH, Akt, and pAKt protein expression in cultured PDA cells, exposed to media, WI38, or WI38 PINCH KD cells via a transwell system, were determined by western blot with values normalized to GAPDH protein expression at 48 hours. Results: MiaPaCa-2 cells grown in co-culture with WI38 cells had a 70%±5% increase in RFP, while those grown in co-culture with WI38 PINCH KD cells had a 40%±3% increase relative to MiaPaCa2 cells grown alone. In terms of relative PINCH protein expression there was an increase in MiaPaCa-2 (20%±5%) cells grown in co-culture with WI38 cells compared to alone but not when grown with PINCH KD (6%±3%). MiaPaCa-2 cells had a greater increase in AKT (47%±6% compared with 19%±4%) and pAKT (36%±5 and 22%±4%) in the cells grown with WI38 cells compared to PDA cells grown with WI38 PINCH KD. Conclusions: Reductions in fibroblast PINCH protein expression are associated with reductions in the growth of adjacent PDA cells, as well as the expression of growth enhancing proteins (PINCH, AKT, and pAKT). Since greater PINCH expression within PDA stromal cells is associated with poorer patient outcomes, understanding the mechanisms associated with this tumor-stromal interaction may provide intervention opportunities.

Comparison of the Regulation, Metabolic Functions, and Roles in Virulence of the Glyceraldehyde-3-Phosphate Dehydrogenase Homologues gapA and gapB in Staphylococcus aureus

ABSTRACT The Gram-positive bacterium Staphylococcus aureus contains two glyceraldehyde-3-phosphate dehydrogenase (GAPDH) homologues known as GapA and GapB. GapA has been characterized as a functional GAPDH protein, but currently there is no biological evidence for the role of GapB in metabolism in S. aureus. In this study we show through a number of complementary methods that S. aureus GapA is essential for glycolysis while GapB is essential in gluconeogenesis. These proteins are reciprocally regulated in response to glucose concentrations, and both are influenced by the glycolysis regulator protein GapR, which is the first demonstration of the role of this regulator in S. aureus and the first indication that GapR homologues control genes other than those within the glycolytic operon. Furthermore, we show that both GapA and GapB are important in the pathogenesis of S. aureus in a Galleria mellonella model of infection, showing for the first time in any bacteria that both glycolysis and gluconeogenesis have important roles in virulence.