SummaryWe have carried out a cell-based screen aimed at discovering small molecules that activate p53 and have the potential to decrease tumor growth. Here, we describe one of our hit compounds, tenovin-1, along with a more water-soluble analog, tenovin-6. Via a yeast genetic screen, biochemical assays, and target validation studies in mammalian cells, we show that tenovins act through inhibition of the protein-deacetylating activities of SirT1 and SirT2, two important members of the sirtuin family. Tenovins are active on mammalian cells at one-digit micromolar concentrations and decrease tumor growth in vivo as single agents. This underscores the utility of these compounds as biological tools for the study of sirtuin function as well as their potential therapeutic interest.

In Escherichia coli, a subset of periplasmic proteins is exported via the twin-arginine translocation (Tat) pathway. In the present study, we have purified the Tat complex from E. coli, and we show that it contains only TatA, TatB, and TatC. Within the purified complex, TatB and TatC are present in a strict 1:1 ratio, suggesting a functional association. This has been confirmed by expression of a translational fusion between TatB and TatC. This Tat(BC) chimera supports efficient Tat-dependent export, indicating that TatB and TatC act as a unit in both structural and functional terms. The purified Tat complex contains varying levels of TatA, suggesting a gradual loss during isolation and a looser association. The molecular mass of the complex is ϳ600 kDa, demonstrating the presence of multiple copies of TatA, B, and C. Co-immunoprecipitation experiments show that TatC is required for the interaction of TatA with TatB, suggesting that TatA may interact with the complex via binding to TatC.

Pyst1 is an inducible antagonist of FGF signaling in embryos and acts in a negative feedback loop to regulate the activity of MAPK. Our results demonstrate both the importance of MAPK signaling in neural induction and limb bud outgrowth and the critical role played by dual specificity MAP kinase phosphatases in regulating developmental outcomes in vertebrates.

MAP kinase phosphatase (MKP)-3 is a cytoplasmic dual specificity protein phosphatase that specifically binds to and inactivates the ERK1/2 MAP kinases in mammalian cells. However, the molecular basis of the cytoplasmic localization of MKP-3 or its physiological significance is unknown. We have used MKP-3-green fluorescent protein fusions in conjunction with leptomycin B to show that the cytoplasmic localization of MKP-3 is mediated by a chromosome region maintenance-1 (CRM1)-dependent nuclear export pathway. Furthermore, the nuclear translocation of MKP-3 seen in the presence of leptomycin B is mediated by an active process, indicating that MKP-3 shuttles between the nucleus and cytoplasm. The aminoterminal noncatalytic domain of MKP-3 is both necessary and sufficient for nuclear export of the phosphatase and contains a single functional leucine-rich nuclear export signal (NES). Even though this domain of the protein also mediates the binding of MKP-3 to MAP kinase, we show that mutations of the kinase interaction motif which abrogate ERK2 binding do not affect MKP-3 localization. Conversely, mutation of the NES does not affect either the binding or phosphatase activity of MKP-3 toward ERK2, indicating that the kinase interaction motif and NES function independently. Finally, we demonstrate that the ability of MKP-3 to cause the cytoplasmic retention of ERK2 requires both a functional kinase interaction motif and NES. We conclude that in addition to its established function in the regulated dephosphorylation and inactivation of MAP kinase, MKP-3 may also play a role in determining the subcellular localization of its substrate. Our results reinforce the idea that regulatory proteins such as MKP-3 may play a key role in the spatio-temporal regulation of MAP kinase activity.Dual specificity (Thr/Tyr) protein phosphatases play an important role in the regulation of signaling by mitogen-activated protein (MAP) 1 kinases in eukaryotic cells (1-3). These MAP kinase phosphatases (MKPs) act in direct opposition to the MAP kinase kinases (MKKs or MEKs) to dephosphorylate and inactivate the MAP kinase, thus regulating the duration and magnitude of activation and hence the biological outcome of signaling. Studies in Saccharomyces cerevisiae, Drosophila, and chicken embryos have also demonstrated that the expression of certain MKPs is induced in response to MAP kinase activation, indicating that they are components of negative feedback loops (4 -6). Furthermore, mathematical modeling of MAP kinase pathway dynamics emphasizes the role of MKPs in determining the timing and duration of MAP kinase activation in terms of bistable (switch-like) or monostable (proportional) response to agonists (7).There are 10 distinct MKPs in mammalian cells (3), and these enzymes feature a common structure comprising a carboxyl-terminal catalytic domain with sequence similarity to the prototypic VH1 dual specificity phosphatase of vaccinia virus (8) and an amino-terminal noncatalytic domain containing two short regions of homology with the cell ...

Abstract-Human essential hypertension is a classic example of a complex, multifactorial, polygenic disease with a substantial genetic influence in which the underlying genetic components remain unknown. The stroke-prone spontaneously hypertension rat (SHRSP) is a well-characterized experimental model for essential hypertension and endothelial dysfunction. Previous work, identified glutathione S-transferase type 1, a protein involved in detoxification of reactive oxygen species, as a positional and functional candidate gene. Quantitative real-time polymerase chain reaction showed a highly significant, 4-fold reduction of glutathione S-transferase type 1 mRNA expression in 5-and 16-week-old SHRSP compared with the congenic and normotensive Wistar Kyoto rats. This suggests that differential expression is not attributable to long-term changes in blood pressure. DNA sequencing identified one coding single nucleotide polymorphism (R202H) and multiple single nucleotide polymorphisms in the promoter region. mRNA expression changes were reflected at the protein level, with significant reductions in the SHRSP glutathione S-transferase type 1. Protein was colocalized with aquaporin 2 to the principle cells of the renal collecting ducts.Coupled to significant increases in nitrotyrosine levels in the kidney, this suggests a pathophysiological role of this protein in hypertension and oxidative stress. Similar processes may underlie oxidative stress in the vasculature. Key Words: rats, stroke-prone SHR Ⅲ hypertension, genetic Ⅲ gene expression Ⅲ oxidative stress H uman essential hypertension is a classic example of a complex, multifactorial, polygenic disease with a substantial genetic influence in which the underlying genetic components remain unknown. [1][2][3][4] The methodological difficulties in studying the genetic determinants of hypertension have given a major impetus for development of similar but inherently simpler paradigms in rodent models of genetic hypertension that remain under complex control. 5-7 Genetic heterogeneity can be reduced by the use of inbred strains with complete control over environmental influences. Moreover, the ability to produce genetic crosses and analyze large numbers of progeny facilitate genetic analysis, including genetic dissection of complex phenotypes, gene-gene and gene-environment interactions. 8 -10 The stroke-prone spontaneously hypertensive rat (SHRSP) is a well-characterized experimental model for essential hypertension and endothelial dysfunction. 11-13 Genome-wide scans performed on several rat crosses have identified quantitative trait loci (QTLs), which are involved in blood pressure regulation. 5 However, these represent large chromosomal regions and contain too many putative candidate genes to pursue classic positional cloning strategies. More recently, generation of congenic strains in which blood pressure QTLs from a normotensive strain have been introgressed into a hypertensive background has allowed genetic dissection of QTLs in hypertensive strains. [13][14][15][16] ...

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Aerobic cells produce reactive oxygen species as a consequence of normal cellular metabolism, and an array of antioxidant systems are in place to maintain the redox balance. When the redox equilibrium of the cell is upset by pro-oxidant environmental stimuli, adaptive responses to the redox stress take place, which can result in up-regulation of antioxidant proteins and detoxification enzymes. Over the past few years, it has become apparent that members of the CNC (cap 'n' collar)-basic leucine zipper family of transcription factors are principal mediators of defensive responses to redox stress. In mammals, the CNC family members nuclear factor-erythroid 2 p45-related factors 1 and 2 (Nrf1 and Nrf2) have been shown to be involved in the transcriptional up-regulation of cytoprotective genes including those encoding glutamate cysteine ligase, NAD(P)H:quinone oxidoreductase, glutathione S-transferases and aldo-keto reductases. An evolutionarily conserved system exists in Caenorhabditis elegans, and it is possible that Drosophila melanogaster may also utilize CNC transcription factors to induce antioxidant genes in response to pro-oxidant chemicals. The advent of microarray and proteomic technologies has advanced our understanding of the gene batteries regulated by oxidative insult, but has highlighted the complexity of gene regulation by environmental factors. This review focuses on the antioxidant response to environmental stress, and the impact that microarrays and proteomics have made in this field.

The twin-arginine translocation (Tat) system catalyzes the transport of folded proteins across the bacterial plasma membrane or the chloroplast thylakoid membrane. In Escherichia coli and most other species, three important tat genes have been identi¢ed but the structure and mechanism of this system are poorly understood; the role and location of TatA are particularly unclear. In this report we have used site-speci¢c mutagenesis to probe the signi¢cance of conserved features of the related TatA/B subunits. We ¢nd that an apparent 'hinge' region between the transmembrane (TM) span and an adjacent amphipathic region is important in both proteins, in that substitution of turn-inducing residues inhibits the export of a natural Tat substrate. Surprisingly, large-scale mutagenesis of the conserved amphipathic regions of TatA and TatB leads only to minor e¡ects on Tat-dependent export suggesting that this particular feature is not central to the translocation mechanism. This domain is, however, critical for the translocation process and we identify Gly/Pro residues in these regions of TatA/B that are essential for e⁄cient export. ß