Adiponectin inhibits the binding of low-density lipoprotein to biglycan, a vascular proteoglycan

The aim of this study was to test the possibility that adiponectin has an antiatherogenic effect through the inhibition of LDL binding to proteoglycans, an initial event in atherogenesis. Both full-length and globular adiponectin inhibited LDL binding in a dose-dependent manner. Both types of adiponectin bound to biglycan in a dose-dependent manner. Immunoprecipitation and immunoblotting analysis showed interaction of full-length adiponectin with LDL. Pretreatment of biglycan with globular adiponectin prior to LDL addition diminished the inhibitory effect, while pretreatment with full-length adiponectin retained the effect. This is a new antiatherogenic property that appears independent of the receptor-mediated hormonal action of adiponectin.     

A novel cyanobacterial SmtB/ArsR family repressor regulates the expression of a CPx-ATPase and a metallothionein in response to both Cu(I)/Ag(I) and Zn(II)/Cd(II)

A novel SmtB/ArsR family metalloregulator, denoted BxmR, has been identified and characterized from the cyanobacterium Oscillatoria brevis. Genetic and biochemical evidence reveals that BxmR represses the expression of both bxa1, encoding a CPx-ATPase metal transporter, as well as a divergently transcribed operon encoding bxmR and bmtA, a heavy metal sequestering metallothionein. Derepression of the expression of all three genes is mediated by both monovalent (Ag(I) and Cu(I)) and divalent (Zn(II) and Cd(II)) heavy metal ions, a novel property among SmtB/ArsR metal sensors. Electrophoretic gel mobility shift experiments reveal that apoBxmR forms multiple resolvable complexes with oligonucleotides containing a single 12-2-12 inverted repeat derived from one of the two operator/promoter regions with similar apparent affinities. Preincubation with either monovalent or divalent metal ions induces disassembly of both the BxmR-bxa1 and BxmR-bxmR/bmtA operator/promoter complexes. Interestingly, the temporal regulation of expression of bxa1 and bmtA mRNAs is different in O. brevis with bxa1 induced first upon heavy metal treatment, followed by bmtA/bxmR. A dynamic interplay among Bxa1, BmtA, and BxmR is proposed that maintains metal homeostasis in O. brevis by balancing the relative rates of metal storage and efflux of multiple heavy metal ions.     

Intracellular uptake and inhibition of gene expression by PNAs and PNA-peptide conjugates

Peptide nucleic acids (PNAs) offer a distinct option for silencing gene expression in mammalian cells. However, the full value of PNAs has not been realized, and the rules governing the recognition of cellular targets by PNAs remain obscure. Here we examine the uptake of PNAs and PNA-peptide conjugates by immortal and primary human cells and compare peptide-mediated and DNA/lipid-mediated delivery strategies. We find that both peptide-mediated and lipid-mediated delivery strategies promote entry of PNA and PNA-peptide conjugates into cells. Confocal microscopy reveals a punctate distribution of PNA and PNA-peptide conjugates regardless of the delivery strategy used. Peptide D(AAKK)(4) and a peptide containing a nuclear localization sequence (NLS) promote the spontaneous delivery of antisense PNAs into cultured cells. The PNA-D(AAKK)(4) conjugate inhibits expression of human caveolin 1 (hCav-1) in both HeLa and primary endothelial cells. DNA/lipid-mediated delivery requires less PNA, while peptide-mediated delivery is simpler and is less toxic to primary cells. The ability of PNA-peptide conjugates to enter primary and immortal human cells and inhibit gene expression supports the use of PNAs as antisense agents for investigating the roles of proteins in cells. Both DNA/lipid-mediated and peptide-mediated delivery strategies are efficient, but the compartmentalized localization of PNAs suggests that improving the cellular distribution may lead to increased efficacy.     

Induction of antioxidant enzymes by FAK in a human leukemic cell line, HL-60

We have established several focal adhesion kinase (FAK) cDNA-transfected HL-60 (HL-60/FAK) cells which were highly resistant to oxidative stress-induced apoptosis. To identify target genes that are involved in HL-60/FAK cells, we performed cDNA microarray screening using apoptosis-chip. There, we identified the decrease of glutathione peroxidase (GPx). This result prompted us to investigate the changes of antioxidant enzymes. Here, we demonstrate that lipid peroxidation was suppressed after treatment with hydrogen peroxide in HL-60/FAK cells but not vector-transfected HL-60 (HL-60/Vect) cells. Furthermore, we demonstrate that HL-60/FAK cells have higher basal reactive oxygen species (ROS) levels than the parental HL-60 or HL-60/Vect cells, while ROS accumulation by hydrogen peroxide treatment was almost the same in these cells. Basal activity and mRNA expression of antioxidant enzymes, particularly of GSH reductase (GRe), phospholipid hydroperoxide glutathione peroxidase (PHGPx) were markedly elevated in HL-60/FAK cells. In contrast, GPx and catalase levels were decreased in HL-60/FAK cells. Further, a Src family kinases inhibitor, PP2, suppressed GRe and PHGPx mRNA by inactivation of FAK and c-Src in HL-60/FAK cells. These results suggest that FAK upregulates antioxidant enzymes and suppresses lipid peroxidation, resulting in the anti-apoptotic state for oxidative stress.     

Crystal structure of human factor VIIa/tissue factor in complex with peptide mimetic inhibitor

The 3D structure of human factor VIIa/soluble tissue factor in complex with a peptide mimetic inhibitor, propylsulfonamide-D-Thr-Met-p-aminobenzamidine, is determined by X-ray crystallography. As compared with the interactions between thrombin and thrombin inhibitors, the interactions at S2 and S3 sites characteristic of factor VIIa and factor VIIa inhibitors are revealed. The S2 site has a small pocket, which is filled by the hydrophobic methionine side chain in P2. The small S3 site fits the small size residue, D-threonine in P3. The structural data and SAR data of the peptide mimetic inhibitor show that these interactions in the S2 and S3 sites play an important role for the improvement of selectivity versus thrombin. The results will provide valuable information for the structure-based drug design of specific inhibitors for FVIIa/TF.     

Acid denaturation and refolding of green fluorescent protein

Green fluorescent protein from the jellyfish Aequorea victoria can serve as a good model protein to understand protein folding in a complex environment with molecular chaperones and other macromolecules such as those in biological cells, but little is known about the detailed mechanisms of the in vitro folding of green fluorescent protein itself. We therefore investigated the kinetic refolding of a mutant (F99S/M153T/V163A) of green fluorescent protein, which is known to mature more efficiently than the wild-type protein, from the acid-denatured state; refolding was observed by chromophore fluorescence, tryptophan fluorescence, and far-UV CD, using a stopped-flow technique. In this study, we demonstrated that the kinetics of the refolding of the mutant have at least five kinetic phases and involve nonspecific collapse within the dead time of a stopped-flow apparatus and the subsequent formation of an on-pathway intermediate with the characteristics of the molten globule state. We also demonstrated that the slowest phase and a major portion of the second slowest phase were rate-limited by slow prolyl isomerization in the intermediate state, and this rate limitation accounts for a major portion of the observed kinetics in the folding of green fluorescent protein.     

Shp2, an SH2-containing protein-tyrosine phosphatase, positively regulates receptor tyrosine kinase signaling by dephosphorylating and inactivating the inhibitor Sprouty

Src homology 2-containing phosphotyrosine phosphatase (Shp2) functions as a positive effector in receptor tyrosine kinase (RTK) signaling immediately proximal to activated receptors. However, neither its physiological substrate(s) nor its mechanism of action in RTK signaling has been defined. In this study, we demonstrate that Sprouty (Spry) is a possible target of Shp2. Spry acts as a conserved inhibitor of RTK signaling, and tyrosine phosphorylation of Spry is indispensable for its inhibitory activity. Shp2 was able to dephosphorylate fibroblast growth factor receptor-induced phosphotyrosines on Spry both in vivo and in vitro. Shp2-mediated dephosphorylation of Spry resulted in dissociation of Spry from Grb2. Furthermore, Shp2 could reverse the inhibitory effect of Spry on FGF-induced neurite outgrowth and MAP kinase activation. These findings suggest that Shp2 acts as a positive regulator in RTK signaling by dephosphorylating and inactivating Spry.     

Oligomeric Hsp33 with enhanced chaperone activity: gel filtration, cross-linking, and small angle x-ray scattering (SAXS) analysis

Hsp33, an Escherichia coli cytosolic chaperone, is inactive under normal conditions but becomes active upon oxidative stress. It was previously shown to dimerize upon activation in a concentration- and temperature-dependent manner. This dimer was thought to bind to aggregation-prone target proteins, preventing their aggregation. In the present study, we report small angle x-ray scattering (SAXS), steady state and time-resolved fluorescence, gel filtration, and glutaraldehyde cross-linking analysis of full-length Hsp33. Our circular dichroism and fluorescence results show that there are significant structural changes in oxidized Hsp33 at different temperatures. SAXS, gel filtration, and glutaraldehyde cross-linking results indicate, in addition to the dimers, the presence of oligomeric species. Oxidation in the presence of physiological salt concentration leads to significant increases in the oligomer population. Our results further show that under conditions that mimic the crowded milieu of the cytosol, oxidized Hsp33 exists predominantly as an oligomeric species. Interestingly, chaperone activity studies show that the oligomeric species is much more efficient compared with the dimers in preventing aggregation of target proteins. Taken together, these results indicate that in the cell, Hsp33 undergoes conformational and quaternary structural changes leading to the formation of oligomeric species in response to oxidative stress. Oligomeric Hsp33 thus might be physiologically relevant under oxidative stress.