Physicochemical Properties of the Mammalian Molecular Chaperone HSP60

The E. coli GroEL/GroES chaperonin complex acts as a folding cage by producing a bullet-like asymmetric complex, and GroEL exists as double rings regardless of the presence of adenosine triphosphate (ATP). Its mammalian chaperonin homolog, heat shock protein, HSP60, and co-chaperonin, HSP10, play an essential role in protein folding by capturing unfolded proteins in the HSP60/HSP10 complex. However, the structural transition in ATPase-dependent reaction cycle has remained unclear. We found nucleotide-dependent association and dissociation of the HSP60/HSP10 complex using various analytical techniques under near physiological conditions. Our results showed that HSP60 exist as a significant number of double-ring complexes (football- and bullet-type complexes) and a small number of single-ring complexes in the presence of ATP and HSP10. HSP10 binds to HSP60 in the presence of ATP, which increased the HSP60 double-ring formation. After ATP is hydrolyzed to Adenosine diphosphate (ADP), HSP60 released the HSP10 and the dissociation of the double-ring to single-rings occurred. These results indicated that HSP60/HSP10 undergoes an ATP-dependent transition between the single- and double-rings in their system that is highly distinctive from the GroEL/GroES system particularly in the manner of complex formation and the roles of ATP binding and hydrolysis in the reaction cycle

Low Concentration Platinum Nanoparticles Effectively Scavenge Reactive Oxygen Species in Rat Skeletal L6 Cells

Prolonged exposure to excessive reactive oxygen species (ROS) increases risk factors for many diseases. Therefore, elimination of ROS as well as prevention of its production becomes critically important. In the present study, we evaluated the levels of cytotoxicity and ROS scavenging activity induced by synthetic platinum nanoparticles (PtNPs). Average size of synthesized PtNPs was 2.2 nm. Synthetic PtNPs were found to scavenge both induced and endogenous H2O2 significantly in L6 rat skeletal muscle cells at a very low concentration (10-2 mg/l). To investigate the mechanism of action, the hierarchical oxidative stress model was used as an experimental model. To evaluate this possibility, we assessed glutathione concentration and gene levels of several antioxidant enzymes in PtNPs-treated (10-3-10 mg/l) L6 cells. Reduced glutathione (GSH) was increased in the range of 10-3-1 mg/l, but not in the 10 mg/l PtNP-treated cells. The GSH/GSSG ratio increased significantly at 1 mg/l and decreased in the 10 mg/l PtNPtreated cells. Most of the gene transcripts for oxidative stress inducible heme oxygenase-1 (HO-1), glutathione reductase (GR), copper-zinc superoxide dismutase (CuZn-SOD), manganese superoxide dismutase (Mn-SOD), glutathione peroxidase (GPx), and catalase were increased significantly by PtNPs at 10-1-10 mg/l. Such upregulatory effects induced by synthetic PtNPs at high concentrations (1-10 mg/l) in L6 cells can be explained by the hierarchical oxidative stress model. However, the cellular responses induced by low levels (10-3-10-2 mg/l) of PtNPs could not be fully explained by this model

Suppressive effects of electrochemically reduced water on matrix metalloproteinase-2 activities and in vitro invasion of human fibrosarcoma HT1080 cells

It has been demonstrated that hydrogen peroxide (H2O2) is directly associated with elevated matrix metalloproteinase-2 (MMP-2) expression in several cell lines. Electrochemically reduced water (ERW), produced near the cathode during electrolysis, and scavenges intracellular H2O2 in human fibrosarcoma HT1080 cells. RT-PCR and zymography analyses revealed that when HT1080 cells were treated with ERW, the gene expression of MMP-2 and membrane type 1 MMP and activation of MMP-2 was repressed, resulting in decreased invasion of the cells into matrigel. ERW also inhibited H2O2-induced MMP-2 upregulation. To investigate signal transduction involved in MMP-2 downregulation, mitogen-activated protein kinase (MAPK)-specific inhibitors, SB203580 (p38 MAPK inhibitor), PD98059 (MAPK/extracellular regulated kinase kinase 1 inhibitor) and c-Jun NH2-terminal kinase inhibitor II, were used to block the MAPK signal cascade. MMP-2 gene expression was only inhibited by SB203580 treatment, suggesting a pivotal role of p38 MAPK in regulation of MMP-2 gene expression. Western blot analysis showed that ERW downregulated the phosphorylation of p38 both in H2O2-treated and untreated HT1080 cells. These results indicate that the inhibitory effect of ERW on tumor invasion is due to, at least in part, its antioxidative effect