4-O-Carboxymethylascochlorin Inhibits Expression Levels of on Inflammation-Related Cytokines and Matrix Metalloproteinase-9 Through NF–κB/MAPK/TLR4 Signaling Pathway in LPS-Activated RAW264.7 Cells

Toll-like receptor 4 (TLR4) and matrix metalloproteinase-9 (MMP-9) are known to play important roles in inflammatory diseases such as arteriosclerosis and plaque instability. The purpose of this study was to perform the effect of 4-O-carboxymethylascochlorin (AS-6) on MMP-9 expression in lipopolysaccharide (LPS)-induced murine macrophages and signaling pathway involved in its anti-inflammatory effect. Effect of AS-6 on MAPK/NF-κB/TLR4 signaling pathway in LPS-activated murine macrophages was examined using ELISA, Western blotting, reverse transcription polymerase chain reaction (RT-PCR) and fluorescence immunoassay. MMP-9 enzyme activity was examined by gelatin zymography. AS-6 significantly suppressed MMP-9 and MAPK/NF-κB expression levels in LPS-stimulated murine macrophages. Expression levels of inducible nitric oxide synthase (iNOS), COX2, MMP-9, JNK, ERK, p38 phosphorylation, and NF-κB stimulated by LPS were also decreased by AS-6. Moreover, AS-6 suppressed TLR4 expression and dysregulated LPS-induced activators of transcription signaling pathway. The results of this study showed that AS-6 can inhibit LPS-stimulated inflammatory response by suppressing TLR4/MAPK/NF-κB signals, suggesting that AS-6 can be used to induce the stability of atherosclerotic plaque and prevent inflammatory diseases in an in vitro model

The nucleotide sequence of yeast chromosome XVI

info:eu-repo/semantics/publishe

HslVU ATP-dependent Protease Utilizes Maximally Six among Twelve Threonine Active Sites during Proteolysis*

HslVU is a bacterial ATP-dependent protease distantly related to eukaryotic proteasomes consisting of hexameric HslU ATPase and dodecameric HslV protease. As a homolog of the 20 S proteasome β-subunits, HslV also uses the N-terminal threonine as the active site residue. However, unlike the proteasome that has only 6 active sites among the 14 β-subunits, HslV has 12 active sites that could potentially contribute to proteolytic activity. Here, by using a series of HslV dodecamers containing different numbers of active sites, we demonstrate that like the proteasome, HslV with only ∼6 active sites is sufficient to support full catalytic activity. However, a further reduction of the number of active sites leads to a proportional decrease in activity. Using proteasome inhibitors, we also demonstrate that substrate-mediated stabilization of the HslV-HslU interaction remains unchanged until the number of the active sites is decreased to ∼6 but is gradually compromised upon further reduction. These results with a mathematical model suggest HslVU utilizes no more than 6 active sites at any given time, presumably because of the action of HslU. These results also suggest that each ATP-bound HslU subunit activates one HslV subunit and that substrate bound to the HslV active site stimulates the HslU ATPase activity by stabilizing the HslV-HslU interaction. We propose this mechanism plays an important role in supporting complete degradation of substrates while preventing wasteful ATP hydrolysis in the resting state by controlling the interaction between HslV and HslU through the catalytic engagement of the proteolytic active sites