Involvement of the p53 and p73 transcription factors in neuroAIDS

HIV-associated dementia (HAD) is the most common AIDS-associated neurological disorder and is characterized by the development of synaptodendritic injury to neurons. To advance HAD therapy, it is crucial to identify the mechanisms and factors involved. The viral protein HIV-1 Tat is among those factors and is released by HIV-1-infected cells and can be taken up by adjacent neuronal cells leading to neurotoxic effects. Multiple cellular host proteins have been identified as Tat cofactors in causing neuronal injury. Interestingly, most of these factors function through activation of the p53 pathway. We have now examined the ability of Tat to activate the p53 pathway leading to the induction of endogenous p53 and p73 in neuronal cells. We found that Tat induced p53 and p73 levels in SH-SY5Y cells and that this induction caused retraction of neurites. In the absence of either p53 or p73, Tat failed to induce dendritic retraction or to activate the proapoptotic proteins, such as Bax. Further, we found that p53-accumulation in Tat-treated cells depends on the presence of p73. Therefore, we conclude that Tat contributes to neuronal degeneration through activation of a pathway involving p53 and p73. This information will be valuable for the development of therapeutic agents that affect these pathways to protect CNS neurons and prevent HAD.     

Interactions of nitroaromatic compounds with the mammalian selenoprotein thioredoxin reductase and the relation to induction of apoptosis in human cancer cells

Here we described novel interactions of the mammalian selenoprotein thioredoxin reductase (TrxR) with nitroaromatic environmental pollutants and drugs. We found that TrxR could catalyze nitroreductase reactions with either one- or two-electron reduction, using its selenocysteine-containing active site and another redox active center, presumably the FAD. Tetryl and p-dinitrobenzene were the most efficient nitroaromatic substrates with a k(cat) of 1.8 and 2.8 s(-1), respectively, at pH 7.0 and 25 degrees C using 50 muM NADPH. As a nitroreductase, TrxR cycled between four- and two-electron-reduced states. The one-electron reactions led to superoxide formation as detected by cytochrome c reduction and, interestingly, reductive N-denitration of tetryl or 2,4-dinitrophenyl-N-methylnitramine, resulting in the release of nitrite. Most nitroaromatics were uncompetitive and noncompetitive inhibitors with regard to NADPH and the disulfide substrate 5,5'-dithiobis(2-nitrobenzoic acid), respectively. Tetryl and 4,6-dinitrobenzofuroxan were, however, competitive inhibitors with respect to 5,5'-dithiobis(2-nitrobenzoic acid) and were clearly substrates for the selenolthiol motif of the enzyme. Furthermore, tetryl and 4,6-dinitrobenzofuroxan efficiently inactivated TrxR, likely by alkylation of the selenolthiol motif as in the inhibition of TrxR by 1-chloro-2,4-dinitrobenzene/dinitrochlorobenzene (DNCB) or juglone. The latter compounds were the most efficient inhibitors of TrxR activity in a cellular context. DNCB, juglone, and tetryl were highly cytotoxic and induced caspase-3/7 activation in HeLa cells. Furthermore, DNCB and juglone were potent inducers of apoptosis also in Bcl2 overexpressing HeLa cells or in A549 cells. Based on these findings, we suggested that targeting of intracellular TrxR by alkylating nitroaromatic or quinone compounds may contribute to the induction of apoptosis in exposed human cancer cells.     

Regeneration of the antioxidant ubiquinol by lipoamide dehydrogenase, thioredoxin reductase and glutathione reductase

Ubiquinol is a powerful antioxidant, which is oxidized in action and needs to be replaced or regenerated to be capable of a sustained effort. This article summarises current knowledge of extramitochondrial reduction of ubiquinone by three flavoenzymes, i.e. lipoamide dehydrogenase, glutathione reductase and thioredoxin reductase, belonging to the same pyridine nucleotide-disulfide oxidoreductase family. These three enzymes are the most efficient extramitochondrial ubiquinone reductases so far described. The reduction of ubiquinone by lipoamide dehydrogenase and glutathione reductase is potently stimulated by zinc and the highest rate of reduction is achieved at acidic pH and the rates are equal with either NADPH or NADH as co-factors. The most efficient ubiquinone reductases are mammalian cytosolic thioredoxin reductases, which are selenoenzymes with a number of biological functions. Reduction of ubiquinone by thioredoxin reductase is in contrast to the other two enzymes investigated, inhibited by zinc and shows a sharp physiological pH optimum at pH 7.5. Furthermore, the reaction is selenium dependent as revealed from experiments using truncated and mutant forms of the enzyme and also in a cellular context by selenium treatment of transfected thioredoxin reductase overexpressing stable cell lines. The reduction of ubiquinone by the three enzymes offers a multifunctional system for extramitochondrial regeneration of an important antioxidant.     

Mutational analysis of human thioredoxin reductase 1. Effects on p53-mediated gene expression and interferon and retinoic acid-induced cell death

The interferon (IFN)-beta and all-trans-retinoic acid combination suppresses tumor growth by inducing apoptosis in several tumor cell lines. A genetic technique permitted the isolation of human thioredoxin reductase (TR) as a critical regulator of IFN/all-trans-retinoic acid-induced cell death. Our recent studies have shown that TR1:thioredoxin 1-regulated cell death is effected in part through the activation of p53-dependent responses. To understand its death regulatory function, we have performed a mutational analysis of TR. Human TR1 has three major structural domains, the FAD binding domain, the NADPH binding domain, and an interface domain (ID). Here, we show that the deletion of the C-terminal interface domain results in a constitutive activation of TR-dependent death responses and promotes p53-dependent gene expression. TR mutant without the ID still retains its dependence on thioredoxin for promoting these responses. Thus, our data suggest that TR-ID acts as a regulatory domain.     

Interaction of regulators Mdm2 and Mdmx with transcription factors p53, p63 and p73

The negative regulation of p53, a major human tumor suppressor, by Mdm2 and Mdmx is crucial for the survival of a cell, whereas its aberrant function is a common feature of cancer. Both Mdm proteins act through the spatial occlusion of the p53 transactivation (TA) domain and by the ubiquitination of p53, resulting in its degradation. Two p53 homologues, p63 and p73, have been described in humans. Unlike p53, these proteins regulate developmental processes rather than genome stability. Both p63 and p73 contain TA domains homologous to that of p53, but relatively little is known about their regulation by Mdm2 or Mdmx. Here, we present a detailed characterization of the interaction of Mdm2 and Mdmx with the TA domains of p63 and p73. Earlier reports of Mdm2 and Mdmx interactions with p73 are substantiated by the detailed quantitative characterization reported in this study. Most importantly, earlier contradictions concerning the presumed interaction of the Mdm proteins with p63 are convincingly resolved and for the first time, the affinities of these interactions are determined. Finally, the contribution of these findings to our understanding of the physiological role of these interactions is discussed.