Taurine chloramine inhibits inducible nitric oxide synthase and TNF-alpha gene expression in activated alveolar macrophages: decreased NF-kappaB activation and IkappaB kinase activity

Taurine prevents tissue damage in a variety of models that involve inflammation, including oxidant-induced lung damage. The mechanism of protection is uncertain, but is postulated to involve the actions of taurine chloramine (Tau-Cl) derived via halide-dependent myeloperoxidase associated with neutrophils. Understanding the influence of Tau-Cl on the production of inflammatory mediators by alveolar macrophages provides an opportunity for determining the mechanism of Tau-Cl action. The effects of Tau-Cl were evaluated on the production of NO and TNF-alpha in NR8383, a cloned cell line derived from rat alveolar macrophages (RAM), and in primary cultures of RAM. Production of NO and TNF-alpha, and expression of inducible NO synthase was inhibited by Tau-Cl in activated NR8383 cells as well as in RAM. Temporal (2, 4, 8, 24 h) expression of inducible NO synthase and TNF-alpha mRNAs was reduced by Tau-Cl in NR8383 cells. Tau-Cl depressed NF-kappaB migration into the nucleus of activated NR8383 cells and caused a more sustained presence of IkappaB in the cytoplasm. Stabilization of cytoplasmic IkappaB-alpha in Tau-Cl-treated cells resulted from decreased phosphorylation of IkappaB-alpha serine-32 and a lower activity of IkappaB kinase (IKK). Additional experiments demonstrated that Tau-Cl does not directly inhibit IKK activity. These results suggest that Tau-Cl exerts its effects at some level upstream of IKK in the signaling pathway and inhibits production of inflammatory mediators through a mechanism that, at least in part, involves inhibition of NF-kappaB activation.     

NF-kappaB activation by double-stranded-RNA-activated protein kinase (PKR) is mediated through NF-kappaB-inducing kinase and IkappaB kinase

The interferon (IFN)-inducible double-stranded-RNA (dsRNA)-activated serine-threonine protein kinase (PKR) is a major mediator of the antiviral and antiproliferative activities of IFNs. PKR has been implicated in different stress-induced signaling pathways including dsRNA signaling to nuclear factor kappa B (NF-kappaB). The mechanism by which PKR mediates activation of NF-kappaB is unknown. Here we show that in response to poly(rI). poly(rC) (pIC), PKR activates IkappaB kinase (IKK), leading to the degradation of the inhibitors IkappaBalpha and IkappaBbeta and the concomitant release of NF-kappaB. The results of kinetic studies revealed that pIC induced a slow and prolonged activation of IKK, which was preceded by PKR activation. In PKR null cell lines, pIC failed to stimulate IKK activity compared to cells from an isogenic background wild type for PKR in accord with the inability of PKR null cells to induce NF-kappaB in response to pIC. Moreover, PKR was required to establish a sustained response to tumor necrosis factor alpha (TNF-alpha) and to potentiate activation of NF-kappaB by cotreatment with TNF-alpha and IFN-gamma. By coimmunoprecipitation, PKR was shown to be physically associated with the IKK complex. Transient expression of a dominant negative mutant of IKKbeta or the NF-kappaB-inducing kinase (NIK) inhibited pIC-induced gene expression from an NF-kappaB-dependent reporter construct. Taken together, these results demonstrate that PKR-dependent dsRNA induction of NF-kappaB is mediated by NIK and IKK activation.     

Chemical modification of tryptophanase by chloramine T: a possible involvement of the methionine residue in enzyme activity

Tryptophanase purified from Escherichia coli B/1t7-A was irreversibly inactivated by chloramine T (sodium N-chloro-p-toluenesulfonamide). The mode of inactivation was rather complex and did not follow pseudo-first-order kinetics. The inactivation of the apoenzyme was much faster than that of the holoenzyme. The Km value for the synthetic substrate S-o-nitrophenyl-L-cysteine (SOPC) increased concomitantly with the modification. In contrast, the Km value for the coenzyme, pyridoxal 5'-phosphate (PLP), was not altered. L-Serine, another substrate, and L-alanine, a competitive inhibitor, protected the enzyme from inactivation. Determination of SH groups in the enzyme protein with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) showed that modification of two SH groups per enzyme subunit resulted in a complete inactivation. When the enzyme was subjected to chloramine T-modification following the SH group modification with DTNB, further inactivation was still observed, even after the addition of dithiothreitol. The SH-blocked enzyme preparation thus obtained, however, exhibited less pH dependency of inactivation by chloramine T than that of the native enzyme. The amino acid analysis of the chloramine T-modified enzyme showed that modification of four or five methionine residues among the 16 residues per subunit proceeded concomitantly with the complete inactivation. Modification of the enzyme with chloramine T quenched the absorption peak near 500 nm, characteristic of a quinoidal structure formed by labilization of the alpha-proton. These results suggest the possibility that chloramine T modifies not only the SH groups, but also methionine residues important for the catalytic activity of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

IkappaB kinase-independent IkappaBalpha degradation pathway: functional NF-kappaB activity and implications for cancer therapy

Antiapoptotic activity of NF-kappaB in tumors contributes to acquisition of resistance to chemotherapy. Degradation of IkappaB is a seminal step in activation of NF-kappaB. The IkappaB kinases, IKK1 and IKK2, have been implicated in both IkappaB degradation and subsequent modifications of NFkappaB. Using mouse embryo fibroblasts (MEFs) devoid of both IKK1 and IKK2 genes (IKK1/2(-/-)), we document a novel IkappaB degradation mechanism. We show that this degradation induced by a chemotherapeutic agent, doxorubicin (DoxR), does not require the classical serine 32 and 36 phosphorylation or the PEST domain of IkappaBalpha. Degradation of IkappaBalpha is partially blocked by phosphatidylinositol 3-kinase inhibitor LY294002 and is mediated by the proteasome. Free NF-kappaB generated by DoxR-induced IkappaB degradation in IKK1/2(-/-) cells is able to activate chromatin based NF-kappaB reporter gene and expression of the endogenous target gene, IkappaBalpha. These results also imply that modification of NF-kappaB by IKK1 or IKK2 either prior or subsequent to its release from IkappaB is not essential for NF-kappaB-mediated gene expression at least in response to DNA damage. In addition, DoxR-induced cell death in IKK1/2(-/-) MEFs is enhanced by simultaneous inhibition of NF-kappaB activation by blocking the proteasome activity. These results reveal an additional pathway of activating NF-kappaB during the course of anticancer therapy and provide a mechanistic basis for the observation that proteasome inhibitors could be used as adjuvants in chemotherapy.     

TAK1 is critical for IkappaB kinase-mediated activation of the NF-kappaB pathway

Cytokine treatment stimulates the IkappaB kinases, IKKalpha and IKKbeta, which phosphorylate the IkappaB proteins, leading to their degradation and activation of NF-kappaB regulated genes. A clear definition of the specific roles of IKKalpha and IKKbeta in activating the NF-kappaB pathway and the upstream kinases that regulate IKK activity remain to be elucidated. Here, we utilized small interfering RNAs (siRNAs) directed against IKKalpha, IKKbeta and the upstream regulatory kinase TAK1 in order to better define their roles in cytokine-induced activation of the NF-kappaB pathway. In contrast to previous results with mouse embryo fibroblasts lacking either IKKalpha or IKKbeta, which indicated that only IKKbeta is involved in cytokine-induced NF-kappaB activation, we found that both IKKalpha and IKKbeta were important in activating the NF-kappaB pathway. Furthermore, we found that the MAP3K TAK1, which has been implicated in IL-1-induced activation of the NF-kappaB pathway, was also critical for TNFalpha-induced activation of the NF-kappaB pathway. TNFalpha activation of the NF-kappaB pathway is associated with the inducible binding of TAK1 to TRAF2 and both IKKalpha and IKKbeta. This analysis further defines the distinct in vivo roles of IKKalpha, IKKbeta and TAK1 in cytokine-induced activation of the NF-kappaB pathway.     

Metal-mediated inhibition is a viable approach for inhibiting cellular methionine aminopeptidase

Methionine aminopeptidase (MetAP) plays an essential role for cell survival. Hence, MetAP is a promising target for developing broad spectrum antibacterial agents. MetAP can be activated in vitro by a number of divalent metals, and X-ray structures show that the active site can accommodate two cations. Herein, we demonstrate bacterial growth inhibition by a compound that targets MetAP by recruitment of a third auxiliary metal. Contrary to previous beliefs, this shows that metal-mediated inhibition is a viable approach for discovering MetAP inhibitors that are effective for therapeutic application.     

The heat-shock-induced suppression of the IkappaB/NF-kappaB cascade is due to inactivation of upstream regulators of IkappaBalpha through insolubilization

Heat shock (HS) was found to suppress the IkappaB/NF-kappaB cascade via the inhibition of IkappaB kinase (IKK) activity; however, the mechanism has not been clear. This study was undertaken to elucidate the detail of the mechanism involved. TNF-alpha-induced activation of IKK was suppressed by HS in human bronchial epithelial cells, and this was associated with the absence of IKK in the immunoprecipitates. It was not due to a degradation of IKK, but due to a conversion of IKK from a soluble to an insoluble form. IKK lost its activity rapidly upon exposure to HS in vitro. The time course of the insolubilization of IKK coincided with the decrease in IKK activity. However, inhibition of IKK insolubilization by the induction of thermotolerance did not reverse the HS-induced suppression of IKK activation and IkappaBalpha degradation. Upstream activators of IKK, such as NF-kappaB-inducing kinase (NIK) and IL-1R-associated kinase (IRAK) were also insolubilized by HS. The HS-induced insolubilization of NIK was not blocked by the induction of thermotolerance. Overexpression of NIK resumed TNF-alpha-induced activation of IKK in thermotolerant cells. These results indicate that the loss of activity of NIK, IRAK, and IKK through insolubilization is responsible for the HS-induced suppression of IkappaB/NF-kappaB pathway.     

Comparative aspects of aminooxyacetate inhibition of glycine oxidation and aminotransferase activity of pea leaf mitochondria

Aminooxyacetate (AOA) was found to inhibit glycine oxidation by pea leaf mitochondria at micromolar levels. The inhibition resulted from an inhibition of both glycine decarboxylase and serine hydroxymethyltransferase (SHMT) activity. Aspartate: 2-oxoglutarate aminotransferase (AsAT) and alanine: 2-oxoglutarate aminotransferase activities of pea leaf mitochondria were also very sensitive to AOA inhibition. Inhibition of both glycine oxidation and aminotransferase activity was likely competitive with respect to the amino group substrate, but also displayed a time-dependent increase in inhibition at constant AOA concentration. In the case of glycine oxidation, this time-dependent component may be related to the rate of penetration of AOA across the inner mitochondrial membrane. Furthermore, the AOA-inhibition of glycine oxidation could be reversed by pyridoxal 5-phosphate (PLP), whereas AOA-inhibited aminotransferase activity was not reversed. The results indicate that the pyridoxal 5-phosphate antagonist, AOA, results in varying types of inhibition depending on the type of enzyme involved.