Diversities and similarities in pH dependency among bacterial NhaB-like Na+/H+ antiporters.

NhaB-like antiporters were the second described class of Na(+)/H(+) antiporters, identified in bacteria more than 20 years ago. While nhaB-like gene sequences have been found in a number of bacterial genomes, only a few of the NhaB-like antiporters have been functionally characterized to date. Although earlier studies have identified a few pH-sensitive and -insensitive NhaB-like antiporters, the mechanisms that determine their pH responses still remain elusive. In this study, we sought to investigate the diversities and similarities among bacterial NhaB-like antiporters, with particular emphasis on their pH responsiveness. Our phylogenetic analysis of NhaB-like antiporters, combined with pH profile analyses of activities for representative members of several phylogenetic groups, demonstrated that NhaB-like antiporters could be classified into three distinct types according to the degree of their pH dependencies. Interestingly, pH-insensitive NhaB-like antiporters were only found in a limited proportion of enterobacterial species, which constitute a subcluster that appears to have diverged relatively recently among enterobacterial NhaB-like antiporters. Furthermore, kinetic property analyses of NhaB-like antiporters at different pH values revealed that the degree of pH sensitivity of antiport activities was strongly correlated with the magnitude of pH-dependent change in apparent Km values, suggesting that the dramatic pH sensitivities observed for several NhaB-like antiporters might be mainly due to the significant increases of apparent Km at lower pH. These results strongly suggested the possibility that the loss of pH sensitivity of NhaB-like antiporters had occurred relatively recently, probably via accumulation of the mutations that impair pH-dependent change of Km in the course of molecular evolution.

Mechanism of inhibition of lipopolysaccharide-induced interferon-ß production by 2-aminopurine.

2-Aminopurine (2-AP) is widely used as an inhibitor for double stranded RNA-dependent protein kinase (PKR). Previously, we reported that 2-AP inhibits Toll-like receptor (TLR) ligand-induced nitric oxide production through the prevention of interferon (IFN)-ß production. In this study, we investigated the mechanisms for 2-AP inhibition of lipopolysaccharide (LPS)-induced IFN-ß production. A reporter gene assay showed that LPS-induced IFN-ß promoter, but not nuclear factor (NF)-κB, activation was significantly inhibited by 2-AP. IFN-ß promoter activation induced by the overexpression of Toll/interleukin-1 receptor domain-containing adaptor inducing IFN-ß (TRIF) was significantly inhibited by 2-AP in a dose-dependent manner, while TRIF- or myeloid differentiation primary response gene 88-dependent NF-κB activation was not inhibited. IFN-ß promoter activation induced by expression of the downstream signaling molecules, tumor necrosis factor receptor-associated factor family member-associated NF-κB activator-binding kinase 1, inhibitor of NF-κB kinase i and a constitutively active mutant of interferon regulatory factor (IRF)-3, was also inhibited by 2-AP. Another PKR inhibitor harboring the imidazolo-oxindole structure, however, did not affect TRIF signaling molecules-induced IFN-ß promoter activation, suggesting that the inhibition of IFN-ß transcription by 2-AP is independent of PKR inhibition. Further, we examined the effect of 2-AP on LPS-induced IRF-3 activation by immunoblotting. While 2-AP did not affect LPS-induced phosphorylation of IRF-3, nuclear translocation of IRF-3 was inhibited. Moreover, we revealed that LPS-induced phosphorylation of Akt, another key molecule involved in IRF-3 activation, was inhibited by 2-AP. These results suggest that 2-AP inhibits nuclear translocation of phosphorylated-IRF-3 by inhibiting Akt activation.