Biomedical subjects

Victoria J Findlay

Publications and source records attributed to Victoria J Findlay.

8 recordsLinked to original sources

PubMed2006

A novel role for human sulfiredoxin in the reversal of glutathionylation.

Modification of protein cysteine residues by disulfide formation with glutathione (glutathionylation) is a reversible posttranslational modification of critical importance in controlling cell signaling events following oxidative and/or nitrosative stress. Here, we show that human sulfiredoxin, a small redox protein conserved in eukaryotes, can act as a novel regulator of the redox-activated thiol switch in cells by catalyzing deglutathionylation of a number of distinct proteins in response to oxidative and/or nitrosative stress. Actin and protein tyrosine phosphatase 1B were identified in vitro as targets of sulfiredoxin 1 (Srx1)-dependent deglutathionylation and confirmed in vivo by two-dimensional gel electrophoresis analysis. In addition, we show that Srx1-dependent deglutathionylation is functionally relevant through restoration of phosphatase activity. Human sulfiredoxin contains one cysteine residue (Cys(99)) that is conserved in all family members. Mutation of the cysteine residue inhibits deglutathionylation but did not affect its capacity to bind intracellular proteins. Furthermore, sulfiredoxin is not an acceptor molecule for the GS(-) moiety during the reaction process. Using two-dimensional gel electrophoresis, we identified multiple protein targets in vivo that are deglutathionylated by sulfiredoxin following oxidative and/or nitrosative stress. This novel deglutathionylation function of sulfiredoxin suggests it has a central role in redox control with potential implications in cell signaling.

4-Aminobenzoic Acid↗

PubMed2006

PABA/NO as an anticancer lead: analogue synthesis, structure revision, solution chemistry, reactivity toward glutathione, and in vitro activity.

PABA/NO is a diazeniumdiolate of structure Me(2)NN(O)=NOAr (where Ar is a 5-substituted-2,4-dinitrophenyl ring whose 5-substituent is N-methyl-p-aminobenzoic acid). It has shown activity against human ovarian cancer xenografts in mice rivaling that of cisplatin, but it is poorly soluble and relatively unstable in water. Here we report structure-based optimization efforts resulting in three analogues with improved solubility and stability in aqueous solution. We sought to explain PABA/NO's physicochemical uniqueness among these four compounds, whose aminobenzoic acid precursors differ structurally only in the presence or absence of the N-methyl group and/or the position of the carboxyl moiety (meta or para). Studies revealed that PABA/NO's N-methyl-p-aminobenzoic acid substituent is bound to the dinitrobenzene ring via its carboxyl oxygen while the other three are linked through the aniline nitrogen. This constitutes a revision of the previously published PABA/NO structure. All four analogues reacted with GSH to produce bioactive nitric oxide (NO), but PABA/NO was the most reactive. Consistent with PABA/NO's potent suppression of A2780 human ovarian cancer xenograft growth in mice, it was the most potent of the four in the OVCAR-3 cell line.

4-Aminobenzoic Acid↗

PubMed2005

A glutathione S-transferase pi-activated prodrug causes kinase activation concurrent with S-glutathionylation of proteins.

Nitric oxide (NO) is an endogenous, diffusible, transcellular messenger shown to affect regulatory and signaling pathways with impact on cell survival. Exposure to NO can impart direct post-translational modifications on target proteins such as nitration and/or nitrosylation. As an alternative, after interaction with oxygen, superoxide, glutathione, or certain metals, NO can lead to S-glutathionylation, a post-translational modification potentially critical to signaling pathways. A novel glutathione S-transferase pi (GSTpi)-activated pro-drug, O(2)-[2,4-dinitro-5-[4-(N-methylamino)benzoyloxy]phenyl]1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate (PABA/NO), liberates NO and elicits toxicity in vitro and in vivo. We now show that PABA/NO induces nitrosative stress, resulting in undetectable nitrosylation, limited nitration, and high levels of S-glutathionylation. After a single pharmacologically relevant dose of PABA/NO, S-glutathionylation occurs rapidly (<5 min) and is sustained for approximately 7 h, implying a half-life for the deglutathionylation process of approximately 3 h. Two-dimensional SDS-polyacrylamide gel electrophoresis and immunoblotting with a monoclonal antibody to S-glutathionylated residues indicated that numerous proteins were S-glutathionylated. Subsequent matrix-assisted laser desorption ionization/time of flight analysis identified 10 proteins, including beta-lactate dehydrogenase, Rho GDP dissociation inhibitor beta, ATP synthase, elongation factor 2, protein disulfide isomerase, nucleophosmin-1, chaperonin, actin, protein tyrosine phosphatase 1B (PTP1B), and glucosidase II. In addition, we showed that sustained S-glutathionylation was temporally concurrent with drug-induced activation of the stress kinases, known to be linked with cell death pathways. This is consistent with the fact that PABA/NO induces S-glutathionylation and inactivation of PTP1B, one phosphatase that can participate in deactivation of kinases. These effects were consistent with the presence of intracellular PABA/NO or metabolites, because cells overexpressing MRP1 were less sensitive to the drug and had reduced levels of S-glutathionylated proteins.

4-Aminobenzoic Acid↗

PubMed2005

Oxidation of a eukaryotic 2-Cys peroxiredoxin is a molecular switch controlling the transcriptional response to increasing levels of hydrogen peroxide.

Although activation of the AP-1-like transcription factor Pap1 in Schizosaccharomyces pombe is important for oxidative stress-induced gene expression, this activation is delayed at higher concentrations of peroxide. Here, we reveal that the 2-Cys peroxiredoxin (2-Cys Prx) Tpx1 is required for the peroxide-induced activation of Pap1. Tpx1, like other eukaryotic 2-Cys Prxs, is highly sensitive to oxidation, which inactivates its thioredoxin peroxidase activity. Our data suggest that the reduced thioredoxin peroxidase-active form of Tpx1 is required for the peroxide-induced oxidation and nuclear accumulation of Pap1. Indeed, in contrast to the previously described role for Tpx1 in the activation of the Sty1 stress-activated protein kinase by peroxide, we find that both catalytic cysteines of Tpx1 are required for Pap1 activation. Moreover, overexpression of the conserved sulfiredoxin Srx1, which interacts with and reduces Tpx1, allows rapid activation of Pap1 at higher concentrations of H(2)O(2). Conversely, loss of Srx1 prevents the reduction of oxidized Tpx1 and prolongs the inhibition of Pap1 activation. Collectively, these data suggest that redox regulation of the thioredoxin peroxidase activity of Tpx1 acts as a molecular switch controlling the transcriptional response to H(2)O(2). Furthermore, they reveal that a single eukaryotic 2-Cys Prx regulates peroxide signaling by multiple independent mechanisms.

Active Transport, Cell Nucleus↗

PubMed2005

Sulfiredoxin: a potential therapeutic agent?

The importance of antioxidants in maintaining homeostasis has long been accepted and includes antioxidant proteins such as, peroxiredoxin (Prx), superoxide dismutase and glutathione S transferases. Sulfiredoxin (Srx) is a recently identified antioxidant protein with a role in signaling through catalytic reduction of oxidative modifications. It was first characterized for its regulation of Prx(s) through reduction of the conserved cysteine from sulfinic to sulfenic acid, thereby impacting the role of Prx in regulation of downstream transcription factors and kinase signaling pathways. Furthermore, the reduction of sulfinic to sulfenic acid prevents further oxidation of the conserved cysteine residue to sulfonic acid, the end result of which is degradation. Srx also has a role in the reduction of glutathionylation a post-translational, oxidative modification that occurs on numerous proteins and has been implicated in a wide variety of pathologies, including Parkinson's disease. The regulation of glutathionylation/deglutathionylation (or thiol switch) has been likened to phosphorylation/dephosphorylation, another post-translational modification involved in the regulation of signaling pathways. Unlike, the reduction of Prx over-oxidation, Srx-dependent deglutathionylation appears to be non-specific. Deglutathionylation of multiple proteins has been observed both in vitro and in vivo in response to oxidative and/or nitrosative stress. This review discusses Srx as a novel antioxidant, and focuses on its potential role in the regulation of glutathionylation/deglutathionylation pathways, that have been implicated in a growing number of disease states.

Animals↗

PubMed2004

A 2-Cys peroxiredoxin regulates peroxide-induced oxidation and activation of a stress-activated MAP kinase.

Oxidative stress-induced cell damage is an important component of many diseases and ageing. In eukaryotes, activation of JNK/p38 stress-activated protein kinase (SAPK) signaling pathways is critical for the cellular response to stress. 2-Cys peroxiredoxins (2-Cys Prx) are highly conserved, extremely abundant antioxidant enzymes that catalyze the breakdown of peroxides to protect cells from oxidative stress. Here we reveal that Tpx1, the single 2-Cys Prx in Schizosaccharomyces pombe, is required for the peroxide-induced activation of the p38/JNK homolog, Sty1. Tpx1 activates Sty1, downstream of previously identified redox sensors, by a mechanism that involves formation of a peroxide-induced disulphide complex between Tpx1 and Sty1. We have identified conserved cysteines in Tpx1 and Sty1 that are essential for normal peroxide-induced Tpx1-Sty1 disulphide formation and Tpx1-dependent regulation of peroxide-induced Sty1 activation. Thus we provide new insight into the response of SAPKs to diverse stimuli by revealing a mechanism for SAPK activation specifically by oxidative stress.

Amino Acid Sequence↗

PubMed2004

Tumor cell responses to a novel glutathione S-transferase-activated nitric oxide-releasing prodrug.

We have used structure-based design techniques to introduce the drug O(2)-[2,4-dinitro-5-(N-methyl-N-4-carboxyphenylamino) phenyl] 1-N,N-dimethylamino)diazen-1-ium-1,2-diolate (PABA/NO), which is efficiently metabolized to potentially cytolytic nitric oxide by the pi isoform of glutathione S-transferase, an enzyme expressed at high levels in many tumors. We have used mouse embryo fibroblasts (MEFs) null for GSTpi (GSTpi(-/-)) to show that the absence of GSTpi results in a decreased sensitivity to PABA/NO. Cytotoxicity of PABA/NO was also examined in a mouse skin fibroblast (NIH3T3) cell line that was stably transfected with GSTpi and/or various combinations of gamma-glutamyl cysteine synthetase and the ATP-binding cassette transporter MRP1. Overexpression of MRP1 conferred the most significant degree of resistance, and in vitro transport studies confirmed that a GSTpi-activated metabolite of PABA/NO was effluxed by MRP1 in a GSH-dependent manner. Additional studies showed that in the absence of MRP1, PABA/NO activated the extracellular-regulated and stress-activated protein kinases ERK, c-Jun NH(2)-terminal kinase (JNK), and p38. Selective inhibition studies showed that the activation of JNK and p38 were critical to the cytotoxic effects of PABA/NO. Finally, PABA/NO produced antitumor effects in a human ovarian cancer model grown in SCID mice.

3T3 Cells↗

PubMed2002

Distinct regulatory proteins control the graded transcriptional response to increasing H(2)O(2) levels in fission yeast Schizosaccharomyces pombe.

The signaling pathways that sense adverse stimuli and communicate with the nucleus to initiate appropriate changes in gene expression are central to the cellular stress response. Herein, we have characterized the role of the Sty1 (Spc1) stress-activated mitogen-activated protein kinase pathway, and the Pap1 and Atf1 transcription factors, in regulating the response to H(2)O(2) in the fission yeast Schizosaccharomyces pombe. We find that H(2)O(2) activates the Sty1 pathway in a dose-dependent manner via at least two sensing mechanisms. At relatively low levels of H(2)O(2), a two component-signaling pathway, which feeds into either of the two stress-activated mitogen-activated protein kinase kinase kinases Wak1 or Win1, regulates Sty1 phosphorylation. In contrast, at high levels of H(2)O(2), Sty1 activation is controlled predominantly by a two-component independent mechanism and requires the function of both Wak1 and Win1. Individual transcription factors were also found to function within a limited range of H(2)O(2) concentrations. Pap1 activates target genes primarily in response to low levels of H(2)O(2), whereas Atf1 primarily controls the transcriptional response to high concentrations of H(2)O(2). Our results demonstrate that S. pombe uses a combination of stress-responsive regulatory proteins to gauge and effect the appropriate transcriptional response to increasing concentrations of H(2)O(2).

Activating Transcription Factor 1↗