Activation of apoptosis signal-regulating kinase 1 by reactive oxygen species through dephosphorylation at serine 967 and 14-3-3 dissociation

Oxidative stress has been indicated in a variety of pathological processes such as atherosclerosis, diabetes, and neurodegenerative diseases. Understanding how intracellular signaling pathways respond to oxidative insults such as hydrogen peroxide (H(2)O(2)) would have significant therapeutic implications. Recent genetic studies have placed apoptosis signal-regulating kinase 1 (ASK1) in a pivotal position in transmitting H(2)O(2)-initiated signals. How ASK1 is activated by H(2)O(2), though, remains a subject of intense investigation. Here we report a mechanism by which H(2)O(2) induces ASK1 activation through dynamic control of its phosphorylation at serine 967. We found that treatment of COS7 cells with H(2)O(2) triggers dephosphorylation of Ser-967 through an okadaic acid-sensitive phosphatase, resulting in dissociation of the ASK1.14-3-3 complex with concomitant increase of ASK1 catalytic activity and ASK1-mediated activation of JNK and p38 pathways.     

Kinetic study of the antiport mechanism of an Escherichia coli zinc transporter, ZitB

ZitB is a member of the cation diffusion facilitator (CDF) family that mediates efflux of zinc across the plasma membrane of Escherichia coli. We describe the first kinetic study of the purified and reconstituted ZitB by stopped-flow measurements of transmembrane fluxes of metal ions using a metal-sensitive fluorescent indicator encapsulated in proteoliposomes. Metal ion filling experiments showed that the initial rate of Zn2+ influx was a linear function of the molar ratio of ZitB to lipid and was related to the concentration of Zn2+ or Cd2+ by a hyperbola with a Michaelis-Menten constant (K(m)) of 104.9 +/- 5.4 microm and 90.1 +/- 3.7 microm, respectively. Depletion of proton stalled Cd2+ transport down its diffusion gradient, whereas tetraethylammonium ion substitution for K+ did not affect Cd2+ transport, indicating that Cd2+ transport is coupled to H+ rather than to K+. H+ transport was inferred by the H+ dependence of Cd2+ transport, showing a hyperbolic relationship with a Km of 19.9 nm for H+. Applying H+ diffusion gradients across the membrane caused Cd2+ fluxes both into and out of proteoliposomes against the imposed H(+) gradients. Likewise, applying outwardly oriented membrane electrical potential resulted in Cd2+ efflux, demonstrating the electrogenic effect of ZitB transport. Taken together, these results indicate that ZitB is an antiporter catalyzing the obligatory exchange of Zn2+ or Cd2+ for H+. The exchange stoichiometry of metal ion for proton is likely to be 1:1.     

Crystal structure of quinone reductase 2 in complex with resveratrol

Resveratrol has been shown to have chemopreventive, cardioprotective, and antiaging properties. Here, we report that resveratrol is a potent inhibitor of quinone reductase 2 (QR2) activity in vitro with a dissociation constant of 35 nM and show that it specifically binds to the deep active-site cleft of QR2 using high-resolution structural analysis. All three resveratrol hydroxyl groups form hydrogen bonds with amino acids from QR2, anchoring a flat resveratrol molecule in parallel with the isoalloxazine ring of FAD. The unique active-site pocket in QR2 could potentially bind other natural polyphenols such as flavonoids, as proven by the high affinity exhibited by quercetin toward QR2. K562 cells with QR2 expression suppressed by RNAi showed similar properties as resveratrol-treated cells in their resistance to quinone toxicity. Furthermore, the QR2 knockdown K562 cells exhibit increased antioxidant and detoxification enzyme expression and reduced proliferation rates. These observations could imply that the chemopreventive and cardioprotective properties of resveratrol are possibly the results of QR2 activity inhibition, which in turn, up-regulates the expression of cellular antioxidant enzymes and cellular resistance to oxidative stress.     

The Pseudomonas syringae HopPtoV protein is secreted in culture and translocated into plant cells via the type III protein secretion system in a manner dependent on the ShcV type III chaperone

The bacterial plant pathogen Pseudomonas syringae depends on a type III protein secretion system and the effector proteins that it translocates into plant cells to cause disease and to elicit the defense-associated hypersensitive response on resistant plants. The availability of the P. syringae pv. tomato DC3000 genome sequence has resulted in the identification of many novel effectors. We identified the hopPtoV effector gene on the basis of its location next to a candidate type III chaperone (TTC) gene, shcV, and within a pathogenicity island in the DC3000 chromosome. A DC3000 mutant lacking ShcV was unable to secrete detectable amounts of HopPtoV into culture supernatants or translocate HopPtoV into plant cells, based on an assay that tested whether HopPtoV-AvrRpt2 fusions were delivered into plant cells. Coimmunoprecipitation and Saccharomyces cerevisiae two-hybrid experiments showed that ShcV and HopPtoV interact directly with each other. The ShcV binding site was delimited to an N-terminal region of HopPtoV between amino acids 76 and 125 of the 391-residue full-length protein. Our results demonstrate that ShcV is a TTC for the HopPtoV effector. DC3000 overexpressing ShcV and HopPtoV and DC3000 mutants lacking either HopPtoV or both ShcV and HopPtoV were not significantly impaired in disease symptoms or bacterial multiplication in planta, suggesting that HopPtoV plays a subtle role in pathogenesis or that other effectors effectively mask the contribution of HopPtoV in plant pathogenesis.     

Expression of caveolin-1 enhances cholesterol efflux in hepatic cells

HepG2 cells were stably transfected with human caveolin-1 (HepG2/cav cells). Transfection resulted in expression of caveolin-1 mRNA, a high abundance of caveolin-1 protein, and the formation of caveolae on the plasma membrane. Cholesterol efflux from HepG2/cav cells was 280 and 45% higher than that from parent HepG2 cells when human plasma and human apoA-I, respectively, were used as acceptors. The difference in efflux was eliminated by treatment of cells with progesterone. There was no difference in cholesterol efflux to cyclodextrin. Cholesterol efflux from plasma membrane vesicles was similar for the two cell types. Transfection led to a 40% increase in the amount of plasma membrane cholesterol in cholesterol-rich domains (caveolae and/or rafts) and a 67% increase in the rate of cholesterol trafficking from intracellular compartments to these domains. Cholesterol biosynthesis in HepG2/cav cells was increased by 2-fold, and cholesterol esterification was reduced by 50% compared with parent HepG2 cells. The proliferation rate of transfected cells was significantly lower than that of non-transfected cells. Transfection did not affect expression of ABCA1 or the abundance of ABCA1 protein, but decreased secretion of apoA-I. We conclude that overexpression of caveolin-1 in hepatic cells stimulates cholesterol efflux by enhancing transfer of cholesterol to cholesterol-rich domains in the plasma membrane.     

Apolipoprotein A-I-stimulated apolipoprotein E secretion from human macrophages is independent of cholesterol efflux

Apolipoprotein A-I (apoA-I)-mediated cholesterol efflux involves the binding of apoA-I to the plasma membrane via its C terminus and requires cellular ATP-binding cassette transporter (ABCA1) activity. ApoA-I also stimulates secretion of apolipoprotein E (apoE) from macrophage foam cells, although the mechanism of this process is not understood. In this study, we demonstrate that apoA-I stimulates secretion of apoE independently of both ABCA1-mediated cholesterol efflux and of lipid binding by its C terminus. Pulse-chase experiments using (35)S-labeled cellular apoE demonstrate that macrophage apoE exists in both relatively mobile (E(m)) and stable (E(s)) pools, that apoA-I diverts apoE from degradation to secretion, and that only a small proportion of apoA-I-mobilized apoE is derived from the cell surface. The structural requirements for induction of apoE secretion and cholesterol efflux are clearly dissociated, as C-terminal deletions in recombinant apoA-I reduce cholesterol efflux but increase apoE secretion, and deletion of central helices 5 and 6 decreases apoE secretion without perturbing cholesterol efflux. Moreover, a range of 11- and 22-mer alpha-helical peptides representing amphipathic alpha-helical segments of apoA-I stimulate apoE secretion whereas only the C-terminal alpha-helix (domains 220-241) stimulates cholesterol efflux. Other alpha-helix-containing apolipoproteins (apoA-II, apoA-IV, apoE2, apoE3, apoE4) also stimulate apoE secretion, implying a positive feedback autocrine loop for apoE secretion, although apoE4 is less effective. Finally, apoA-I stimulates apoE secretion normally from macrophages of two unrelated subjects with genetically confirmed Tangier Disease (mutations C733R and c.5220-5222delTCT; and mutations A1046D and c.4629-4630insA), despite severely inhibited cholesterol efflux. We conclude that apoA-I stimulates secretion of apoE independently of cholesterol efflux, and that this represents a novel, ABCA-1-independent, positive feedback pathway for stimulation of potentially anti-atherogenic apoE secretion by alpha-helix-containing molecules including apoA-I and apoE.     

The Ca2+ homeostasis defects in a pgm2Delta strain of Saccharomyces cerevisiae are caused by excessive vacuolar Ca2+ uptake mediated by the Ca2+-ATPase Pmc1p

Loss of the major isoform of phosphoglucomutase (PGM) causes an accumulation of glucose 1-phosphate when yeast cells are grown with galactose as the carbon and energy source. Remarkably, the pgm2Delta strain also exhibits a severe imbalance in intracellular Ca(2+) homeostasis when grown under these conditions. In the present study, we examined how the pgm2Delta mutation alters yeast Ca(2+) homeostasis in greater detail. We found that a shift from glucose to galactose as the carbon source resulted in a 2-fold increase in the rate of cellular Ca(2+) uptake in wild-type cells, whereas Ca(2+) uptake increased 8-fold in the pgm2Delta mutant. Disruption of the PMC1 gene, which encodes the vacuolar Ca(2+)-ATPase Pmc1p, suppressed the Ca(2+)-related phenotypes observed in the pgm2Delta strain. This suggests that excessive vacuolar Ca(2+) uptake is tightly coupled to these defects in Ca(2+) homeostasis. An in vitro assay designed to measure Ca(2+) sequestration into intracellular compartments confirmed that the pgm2Delta mutant contained a higher level of Pmc1p-dependent Ca(2+) transport activity than the wild-type strain. We found that this increased rate of vacuolar Ca(2+) uptake also coincided with a large induction of the unfolded protein response in the pgm2Delta mutant, suggesting that Ca(2+) uptake into the endoplasmic reticulum compartment was reduced. These results indicate that the excessive Ca(2+) uptake and accumulation previously shown to be associated with the pgm2Delta mutation are due to a severe imbalance in the distribution of cellular Ca(2+) into different intracellular compartments.