Redox regulation of RAD51 Cys319 and homologous recombination by peroxiredoxin 1.

RAD51 is a critical recombinase that functions in concert with auxiliary mediator proteins to direct the homologous recombination (HR) DNA repair pathway. We show that Cys319 RAD51 possesses nucleophilic characteristics and is important for irradiation-induced RAD51 foci formation and resistance to inhibitors of poly (ADP-ribose) polymerase (PARP). We have previously identified that cysteine (Cys) oxidation of proteins can be important for activity and modulated via binding to peroxiredoxin 1 (PRDX1).

S-Glutathionylated Serine Proteinase Inhibitors as Biomarkers for Radiation Exposure in Prostate Cancer Patients.

In biological tissues, radiation causes the formation of reactive oxygen species (ROS), some of which lead to sequential oxidation of certain protein cysteine residues. Resultant cysteinyl radicals are subject to post-translational modification through S-glutathionylation. The present clinical trial was designed to determine if S-glutathionylated serine protease inhibitors (serpins) in blood could be used as biomarkers of exposure to radiation.

Isoflavone ME-344 Disrupts Redox Homeostasis and Mitochondrial Function by Targeting Heme Oxygenase 1.

ME-344 is a second-generation isoflavone with unusual cytotoxic properties that is in clinical testing in cancer. To identify targets that contribute to its anticancer activity and therapeutic index, we used lung cancer cell lines that are naturally sensitive or resistant to ME-344. Drug-induced apoptosis was linked with enhanced levels of reactive oxygen species and this initiated a nuclear erythroid factor 2-like 2 signaling response, downstream of which, heme oxygenase 1 (HO-1) was also found to be time-dependently inhibited by ME-344.

Inhibitors of the protein disulfide isomerase family for the treatment of multiple myeloma.

Multiple Myeloma (MM) is highly sensitive to disruptions in cellular protein homeostasis. Proteasome inhibitors (PIs) are initially effective in the treatment of MM, although cures are not achievable and the emergence of resistance limits the durability of responses. New therapies are needed for refractory patients, and those that combat resistance to standard of care agents would be particularly valuable.

-Glutathionylation of estrogen receptor α affects dendritic cell function.

Glutathione -transferase Pi (GSTP) is a thiolase that catalyzes the addition of glutathione (GSH) to receptive cysteines in target proteins, producing an -glutathionylated residue. Accordingly, previous studies have reported that -glutathionylation is constitutively decreased in cells from mice lacking GSTP (/). Here, we found that bone marrow-derived dendritic cells (BMDDCs) from / mice have proliferation rates that are greater than those in their WT counterparts (/).

Glutaminase inhibitor CB-839 synergizes with carfilzomib in resistant multiple myeloma cells.

Curative responses in the treatment of multiple myeloma (MM) are limited by the emergence of therapeutic resistance. To address this problem, we set out to identify druggable mechanisms that convey resistance to proteasome inhibitors (PIs; e.g.

Redox Signaling and Bioenergetics Influence Lung Cancer Cell Line Sensitivity to the Isoflavone ME-344.

ME-344 [(3R,4S)-3,4-bis(4-hydroxyphenyl)-8-methyl-3,4-dihydro-2H-chromen-7-ol] is a second-generation derivative natural product isoflavone presently under clinical development. ME-344 effects were compared in lung cancer cell lines that are either intrinsically sensitive or resistant to the drug and in primary immortalized human lung embryonic fibroblasts (IHLEF). Cytotoxicity at low micromolar concentrations occurred only in sensitive cell lines, causing redox stress, decreased mitochondrial ATP production, and subsequent disruption of mitochondrial function.

Glutathione S-Transferase P-Mediated Protein S-Glutathionylation of Resident Endoplasmic Reticulum Proteins Influences Sensitivity to Drug-Induced Unfolded Protein Response.

Aims: S-glutathionylation of cysteine residues, catalyzed by glutathione S-transferase Pi (GSTP), alters structure/function characteristics of certain targeted proteins. Our goal is to characterize how S-glutathionylation of proteins within the endoplasmic reticulum (ER) impact cell sensitivity to ER-stress inducing drugs.

Results: We identify GSTP to be an ER-resident protein where it demonstrates both chaperone and catalytic functions.

N-acetyl-L-cysteine sensitizes pancreatic cancers to gemcitabine by targeting the NFκB pathway.

First-line therapy for pancreatic cancer is gemcitabine. Although tumors may initially respond to the gemcitabine treatment, soon tumor resistance develops leading to treatment failure. Previously, we demonstrated in human MIA PaCa-2 pancreatic cancer cells that N-acetyl-l-cysteine (NAC), a glutathione (GSH) precursor, prevents NFκB activation via S-glutathionylation of p65-NFκB, thereby blunting expression of survival genes.

Glutathione S-transferase P influences redox and migration pathways in bone marrow.

To interrogate why redox homeostasis and glutathione S-transferase P (GSTP) are important in regulating bone marrow cell proliferation and migration, we isolated crude bone marrow, lineage negative and bone marrow derived-dendritic cells (BMDDCs) from both wild type (WT) and knockout (Gstp1/p2(-/-)) mice. Comparison of the two strains showed distinct thiol expression patterns. WT had higher baseline and reactive oxygen species-induced levels of S-glutathionylated proteins, some of which (sarco-endoplasmic reticulum Ca2(+)-ATPase) regulate Ca(2+) fluxes and subsequently influence proliferation and migration.

Pleiotropic functions of glutathione S-transferase P.

Glutathione S-transferase P (GSTP) is one member of the GST superfamily that is prevalently expressed in mammals. Known to possess catalytic activity through deprotonating glutathione allowing formation of thioether bonds with electrophilic substrates, more recent discoveries have broadened our understanding of the biological roles of this protein. In addition to catalytic detoxification, other properties so far ascribed to GSTP include chaperone functions, regulation of nitric oxide pathways, regulation of a variety of kinase signaling pathways, and participation in the forward reaction of protein S-glutathionylation.

Causes and consequences of cysteine S-glutathionylation.

Post-translational S-glutathionylation occurs through the reversible addition of a proximal donor of glutathione to thiolate anions of cysteines in target proteins, where the modification alters molecular mass, charge, and structure/function and/or prevents degradation from sulfhydryl overoxidation or proteolysis. Catalysis of both the forward (glutathione S-transferase P) and reverse (glutaredoxin) reactions creates a functional cycle that can also regulate certain protein functional clusters, including those involved in redox-dependent cell signaling events. For translational application, S-glutathionylated serum proteins may be useful as biomarkers in individuals (who may also have polymorphic expression of glutathione S-transferase P) exposed to agents that cause oxidative or nitrosative stress.

Voltage-dependent anion channels modulate mitochondrial metabolism in cancer cells: regulation by free tubulin and erastin.

Respiratory substrates and adenine nucleotides cross the mitochondrial outer membrane through the voltage-dependent anion channel (VDAC), comprising three isoforms--VDAC1, 2, and 3. We characterized the role of individual isoforms in mitochondrial metabolism by HepG2 human hepatoma cells using siRNA. With VDAC3 to the greatest extent, all VDAC isoforms contributed to the maintenance of mitochondrial membrane potential, but only VDAC3 knockdown decreased ATP, ADP, NAD(P)H, and mitochondrial redox state.

Sulfiredoxin redox-sensitive interaction with S100A4 and non-muscle myosin IIA regulates cancer cell motility.

Sulfiredoxin (Srx) is a redox active protein that participates in the reduction of oxidized cysteine residues. Here we identify a novel function of Srx through its specific binding to S-glutathionylated S100A4 affecting its interaction with non-muscle myosin (NMIIA), thereby modulating the effect of S100A4 on NMIIA function and impacting cell adhesion and migration. Srx forms a complex with S100A4 (and has stronger affinity for S-glutathionylated S100A4), regulates its activity, and mediates redox regulation of the interaction of S100A4 with NMIIA.

S-Glutathionylation of Protein Disulfide Isomerase Regulates Estrogen Receptor α Stability and Function.

S-Glutathionylation of cysteine residues within target proteins is a posttranslational modification that alters structure and function. We have shown that S-glutathionylation of protein disulfide isomerase (PDI) disrupts protein folding and leads to the activation of the unfolded protein response (UPR). PDI is a molecular chaperone for estrogen receptor alpha (ERα).

S-glutathionylated serine proteinase inhibitors as plasma biomarkers in assessing response to redox-modulating drugs.

Many cancer drugs impact cancer cell redox regulatory mechanisms and disrupt redox homeostasis. Pharmacodynamic biomarkers that measure therapeutic efficacy or toxicity could improve patient management. Using immunoblot analyses and mass spectrometry, we identified that serpins A1 and A3 were S-glutathionylated in a dose- and time-dependent manner following treatment of mice with drugs that alter reactive oxygen or nitrogen species.

Cocaine-induced adaptations in cellular redox balance contributes to enduring behavioral plasticity.

Impaired glutamate homeostasis in the nucleus accumbens has been linked to cocaine relapse in animal models, and results in part from cocaine-induced downregulation of the cystine-glutamate exchanger. In addition to regulating extracellular glutamate, the uptake of cystine by the exchanger is a rate-limiting step in the synthesis of glutathione (GSH). GSH is critical for balancing cellular redox in response to oxidative stress.

The role of glutathione S-transferase P in signaling pathways and S-glutathionylation in cancer.

Glutathione S-transferase P is abundantly expressed in some mammalian tissues, particularly those associated with malignancies. While the enzyme can catalyze thioether bond formation between some electrophilic chemicals and GSH, novel nondetoxification functions are now ascribed to it. This review summarizes recent material that implicates GSTP in mediating S-glutathionylation of specific clusters of target proteins and in reactions that define a negative regulatory role in some kinase pathways through ligand or protein:protein interactions.

Diazeniumdiolate mediated nitrosative stress alters nitric oxide homeostasis through intracellular calcium and S-glutathionylation of nitric oxide synthetase.

Background: PABA/NO is a diazeniumdiolate that acts as a direct nitrogen monoxide (NO) donor and is in development as an anticancer drug. Its mechanism of action and effect on cells is not yet fully understood.

Methodology/principal Findings: We used HPLC and mass spectrometry to identify a primary nitroaromatic glutathione metabolite of PABA/NO and used fluorescent assays to characterize drug effects on calcium and NO homeostasis, relating these to endothelial nitric oxide synthase (eNOS) activity.

Maternal and fetal oxidative stress and intrapartum term fever.

Objective: The association between maternal chorioamnionitis and fetal oxidative stress has not been well established.

Study Design: A nested case control study was performed within a prospective cohort of term nulliparous women: 20 cases (intrapartum fever of >100.4 degrees F) and 20 afebrile controls.

Cellular resistance to a nitric oxide releasing glutathione S-transferase P-activated prodrug, PABA/NO.

PABA/NO is a diazeniumdiolate selectively activated by glutathione S-transferase P (GSTP) to release nitric oxide (NO) and is a potent inducer of protein S-glutathionylation, a redox-sensitive post-translational modification of cysteine residues. Using a procedure that incrementally increased exposure of cells to PABA/NO, an acquired drug resistant human promyelocytic leukemia HL60 cell line (HL60(PABA)) that exhibited 1.9-fold resistance to the drug (IC(50) 15 μM vs ~8 μM for wild-type) was created.

Peroxiredoxin 1 and its role in cell signaling.

Peroxiredoxins (Prdxs) are a family of small (22-27 kDa) nonseleno peroxidases currently known to possess six mammalian isoforms. Although their individual roles in cellular redox regulation and antioxidant protection are quite distinct, they all catalyze peroxide reduction of H(2)O(2), organic hydroperoxides and peroxynitrite. They are found to be expressed ubiquitously and in high levels, suggesting that they are both an ancient and important enzyme family.

Nitration of the mitochondrial complex I subunit NDUFB8 elicits RIP1- and RIP3-mediated necrosis.

Nitric oxide (NO) and other reactive nitrogen species target multiple sites in the mitochondria to influence cellular bioenergetics and survival. Kinetic imaging studies revealed that NO from either activated macrophages or donor compounds rapidly diffuses to the mitochondria, causing a dose-dependent progressive increase in NO-dependent DAF fluorescence, which corresponded to mitochondrial membrane potential loss and initiated alterations in cellular bioenergetics that ultimately led to necrotic cell death. Cellular dysfunction is mediated by an elevated 3-nitrotyrosine signature of the mitochondrial complex I subunit NDUFB8, which is vital for normal mitochondrial function as evidenced by selective knockdown via siRNA.

Nitrosative stress-induced s-glutathionylation of protein disulfide isomerase leads to activation of the unfolded protein response.

The rapid proliferation of cancer cells mandates a high protein turnover. The endoplasmic reticulum (ER) is intimately involved in protein processing. An accumulation of unfolded or misfolded proteins in the ER leads to a cascade of transcriptional and translational events collectively called the unfolded protein response (UPR).