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Mark B Hampton - One of the best experts on this subject based on the ideXlab platform.
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Quaternary structure influences the peroxidase activity of Peroxiredoxin 3.
Biochemical and biophysical research communications, 2018Co-Authors: N. Amy Yewdall, Alexander V. Peskin, Mark B Hampton, David C. Goldstone, F. Grant Pearce, Juliet A. GerrardAbstract:Peroxiredoxins are abundant peroxidase enzymes that are key regulators of the cellular redox environment. A major subgroup of these proteins, the typical 2-Cys Peroxiredoxins, can switch between dimers and decameric or dodecameric rings, during the catalytic cycle. The necessity of this change in quaternary structure for function as a peroxidase is not fully understood. In order to explore this, human Peroxiredoxin 3 (Prx3) protein was engineered to form both obligate dimers (S75E Prx3) and stabilised dodecameric rings (S78C Prx3), uncoupling structural transformations from the catalytic cycle. The obligate dimer, S75E Prx3, retained catalytic activity towards hydrogen peroxide, albeit significantly lower than the wildtype and S78C proteins, suggesting an evolutionary advantage of having higher order self-assemblies.
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Structures of Human Peroxiredoxin 3 Suggest Self-Chaperoning Assembly that Maintains Catalytic State.
Structure (London England : 1993), 2016Co-Authors: N. Amy Yewdall, Mark B Hampton, Alok K. Mitra, Juliet A. Gerrard, David C. Goldstone, Hariprasad Venugopal, Ambroise Desfosses, Vahid Abrishami, Yuliana Yosaatmadja, Mazdak RadjainiaAbstract:Peroxiredoxins are antioxidant proteins primarily responsible for detoxification of hydroperoxides in cells. On exposure to various cellular stresses, Peroxiredoxins can acquire chaperone activity, manifested as quaternary reorganization into a high molecular weight (HMW) form. Acidification, for example, causes dodecameric rings of human Peroxiredoxin 3 (HsPrx3) to stack into long helical filaments. In this work, a 4.1-A resolution structure of low-pH-instigated helical filaments was elucidated, showing a locally unfolded active site and partially folded C terminus. A 2.8-A crystal structure of HsPrx3 was determined at pH 8.5 under reducing conditions, wherein dodecameric rings are arranged as a short stack, with symmetry similar to low-pH filaments. In contrast to previous observations, the crystal structure displays both a fully folded active site and ordered C terminus, suggesting that the HsPrx3 HMW form maintains catalytic activity. We propose a new role for the HMW form as a self-chaperoning assembly maintaining HsPrx3 function under stress.
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Cryo-Electron Microscopy Structure of Human Peroxiredoxin-3 Filament Reveals the Assembly of a Putative Chaperone
Structure (London England : 1993), 2015Co-Authors: Mazdak Radjainia, Mark B Hampton, Juliet A. Gerrard, N. Amy Yewdall, Hariprasad Venugopal, Ambroise Desfosses, Amy J. Phillips, Alok K. MitraAbstract:Peroxiredoxins (Prxs) are a ubiquitous class of thiol-dependent peroxidases that play an important role in the protection and response of cells to oxidative stress. The catalytic unit of typical 2-Cys Prxs are homodimers, which can self-associate to form complex assemblies that are hypothesized to have signaling and chaperone activity. Mitochondrial Prx3 forms dodecameric toroids, which can further stack to form filaments, the so-called high-molecular-weight (HMW) form that has putative holdase activity. We used single-particle analysis and helical processing of electron cryomicroscopy images of human Prx3 filaments induced by low pH to generate a ∼7-A resolution 3D structure of the HMW form, the first such structure for a 2-Cys Prx. The pseudo-atomic model reveals interactions that promote the stacking of the toroids and shows that unlike previously reported data, the structure can accommodate a partially folded C terminus. The HMW filament lumen displays hydrophobic patches, which we hypothesize bestow holdase activity.
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Mitochondrial respiratory chain involvement in Peroxiredoxin 3 oxidation by phenethyl isothiocyanate and auranofin
FEBS letters, 2010Co-Authors: Kristin K Brown, Andrew G Cox, Mark B HamptonAbstract:Mitochondrial Peroxiredoxin 3 (Prx 3) is rapidly oxidized in cells exposed to phenethyl isothiocyanate (PEITC) and auranofin (AFN), but the mechanism of oxidation is unclear. Using HL-60 cells deplete of mitochondrial DNA we show that Peroxiredoxin 3 oxidation and cytotoxicity requires a functional respiratory chain. Thioredoxin reductase (TrxR) could be inhibited by up to 90% by auranofin without direct oxidation of Peroxiredoxin 3. However, inhibition of thioredoxin reductase promoted Peroxiredoxin 3 oxidation and cytotoxicity in combination with phenethyl isothiocyanate or antimycin A. We conclude that rapid Peroxiredoxin 3 oxidation occurs as a consequence of increased oxidant production from the mitochondrial respiratory chain.
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redox potential and peroxide reactivity of human Peroxiredoxin 3
Biochemistry, 2009Co-Authors: Alexander V. Peskin, Louise N Paton, Christine C Winterbourn, Mark B HamptonAbstract:Peroxiredoxins (Prxs) are a ubiquitous family of thiol peroxidases that protect cells from peroxides and have a putative role in redox signaling. In this study, we investigated the redox properties of human Prx 3, a typical 2-Cys Prx that is localized to the mitochondrial matrix. We found that Prx 3 displayed strong reactivity with H2O2, with a competitive kinetic approach generating a second order rate constant of 2 × 107 M−1 s−1. This is considerably higher than typical thiols and similar to values for other mammalian 2-Cys Prxs. In contrast, Prx 3 reacted very slowly with the thiol alkylating agents iodoacetamide and N-ethylmaleimide. Using dithiothreitol redox buffers, we measured the redox potential of Prx 3 of −290 mV. This is similar to the redox potential of mitochondrial thioredoxin 2 and is consistent with optimal operation of Prx 3 in the mitochondrial matrix.
Andrew G Cox - One of the best experts on this subject based on the ideXlab platform.
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Mitochondrial respiratory chain involvement in Peroxiredoxin 3 oxidation by phenethyl isothiocyanate and auranofin
FEBS letters, 2010Co-Authors: Kristin K Brown, Andrew G Cox, Mark B HamptonAbstract:Mitochondrial Peroxiredoxin 3 (Prx 3) is rapidly oxidized in cells exposed to phenethyl isothiocyanate (PEITC) and auranofin (AFN), but the mechanism of oxidation is unclear. Using HL-60 cells deplete of mitochondrial DNA we show that Peroxiredoxin 3 oxidation and cytotoxicity requires a functional respiratory chain. Thioredoxin reductase (TrxR) could be inhibited by up to 90% by auranofin without direct oxidation of Peroxiredoxin 3. However, inhibition of thioredoxin reductase promoted Peroxiredoxin 3 oxidation and cytotoxicity in combination with phenethyl isothiocyanate or antimycin A. We conclude that rapid Peroxiredoxin 3 oxidation occurs as a consequence of increased oxidant production from the mitochondrial respiratory chain.
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Mitochondrial Peroxiredoxin 3 is more resilient to hyperoxidation than cytoplasmic Peroxiredoxins
The Biochemical journal, 2009Co-Authors: Andrew G Cox, Christine C Winterbourn, Juliet M. Pullar, W. Todd Lowther, Andree G. Pearson, Thomas J. Jönsson, Mark B HamptonAbstract:The Prxs (Peroxiredoxins) are a family of cysteine-dependent peroxidases that decompose hydrogen peroxide. Prxs become hyperoxidized when a sulfenic acid formed during the catalytic cycle reacts with hydrogen peroxide. In the present study, Western blot methodology was developed to quantify hyperoxidation of individual 2-Cys Prxs in cells. It revealed that Prx 1 and 2 were hyperoxidized at lower doses of hydrogen peroxide than would be predicted from in vitro data, suggesting intracellular factors that promote hyperoxidation. In contrast, mitochondrial Prx 3 was considerably more resistant to hyperoxidation. The concentration of Prx 3 was estimated at 125 microM in the mitochondrial matrix of Jurkat T-lymphoma cells. Although the local cellular environment could influence susceptibility, purified Prx 3 was also more resistant to hyperoxidation, suggesting that despite having C-terminal motifs similar to sensitive eukaryote Prxs, other structural features must contribute to the innate resilience of Prx 3 to hyperoxidation.
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the thioredoxin reductase inhibitor auranofin triggers apoptosis through a bax bak dependent process that involves Peroxiredoxin 3 oxidation
Biochemical Pharmacology, 2008Co-Authors: Andrew G Cox, Kristin K Brown, Elias S J Arner, Mark B HamptonAbstract:Abstract Thioredoxin reductase (TrxR) is a key selenoprotein antioxidant enzyme and a potential target for anti-cancer drugs. One potent inhibitor of TrxR is the gold (I) compound auranofin, which can trigger mitochondrial-dependent apoptosis pathways. The exact mechanism of apoptosis induction by auranofin is not yet clear, but there are indications that mitochondrial oxidative stress is a central event. We assessed the redox state of the Peroxiredoxins (Prxs) in Jurkat T-lymphoma cells treated with auranofin, and found that mitochondrial Prx3 was considerably more sensitive to oxidation than the cytosolic Prx1 and 2, indicating selective mitochondrial stress. Prx3 oxidation was detected at apoptotic doses of auranofin in several cell types, and occurred before other mitochondrial events including cytochrome c release and mitochondrial depolarisation. Auranofin was also able to sensitise U937 cells to TNF-α-mediated apoptosis. Auranofin-induced apoptosis was effectively blocked by the overexpression of Bcl-2, and Bax/Bak deficient mouse embryonic fibroblasts were also resistant to apoptosis, indicating a central role for the pro-apoptotic proteins of this family in auranofin-triggered apoptosis. Auranofin exposure inhibited the proliferation of apoptosis-resistant cells, and at higher doses of auranofin could cause cell death through necrosis. We conclude that auranofin induces apoptosis in cells through a Bax/Bak-dependent mechanism associated with selective disruption of mitochondrial redox homeostasis in conjunction with oxidation of Prx3.
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The thioredoxin reductase inhibitor auranofin triggers apoptosis through a Bax/Bak-dependent process that involves Peroxiredoxin 3 oxidation.
Biochemical pharmacology, 2008Co-Authors: Andrew G Cox, Kristin K Brown, Elias S J Arner, Mark B HamptonAbstract:Abstract Thioredoxin reductase (TrxR) is a key selenoprotein antioxidant enzyme and a potential target for anti-cancer drugs. One potent inhibitor of TrxR is the gold (I) compound auranofin, which can trigger mitochondrial-dependent apoptosis pathways. The exact mechanism of apoptosis induction by auranofin is not yet clear, but there are indications that mitochondrial oxidative stress is a central event. We assessed the redox state of the Peroxiredoxins (Prxs) in Jurkat T-lymphoma cells treated with auranofin, and found that mitochondrial Prx3 was considerably more sensitive to oxidation than the cytosolic Prx1 and 2, indicating selective mitochondrial stress. Prx3 oxidation was detected at apoptotic doses of auranofin in several cell types, and occurred before other mitochondrial events including cytochrome c release and mitochondrial depolarisation. Auranofin was also able to sensitise U937 cells to TNF-α-mediated apoptosis. Auranofin-induced apoptosis was effectively blocked by the overexpression of Bcl-2, and Bax/Bak deficient mouse embryonic fibroblasts were also resistant to apoptosis, indicating a central role for the pro-apoptotic proteins of this family in auranofin-triggered apoptosis. Auranofin exposure inhibited the proliferation of apoptosis-resistant cells, and at higher doses of auranofin could cause cell death through necrosis. We conclude that auranofin induces apoptosis in cells through a Bax/Bak-dependent mechanism associated with selective disruption of mitochondrial redox homeostasis in conjunction with oxidation of Prx3.
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Oxidation of mitochondrial Peroxiredoxin 3 during the initiation of receptor-mediated apoptosis
Free radical biology & medicine, 2007Co-Authors: Andrew G Cox, Juliet M. Pullar, Gillian Hughes, Elizabeth C. Ledgerwood, Mark B HamptonAbstract:Abstract It is hypothesized that activation of death receptors disrupts the redox homeostasis of cells and that this contributes to the induction of apoptosis. The redox status of the Peroxiredoxins, which are extremely sensitive to increases in H2O2 and disruption of the thioredoxin system, were monitored in Jurkat T lymphoma cells undergoing Fas-mediated apoptosis. The only detectable change during the early stages of apoptosis was oxidation of mitochondrial Peroxiredoxin 3. Increased H2O2 triggers Peroxiredoxin overoxidation to a sulphinic acid; however during apoptosis Peroxiredoxin 3 was captured as a disulfide, suggesting impairment of the thioredoxin system responsible for maintaining Peroxiredoxin 3 in its reduced form. Peroxiredoxin 3 oxidation was an early event, occurring within the same timeframe as increased mitochondrial oxidant production, caspase activation and cytochrome c release. It preceded other major apoptotic events including mitochondrial permeability transition and phosphatidylserine exposure, and glutathione depletion, global thiol protein oxidation and protein carbonylation. Peroxiredoxin 3 oxidation was also observed in U937 cells stimulated with TNF-α. We hypothesize that the selective oxidation of Peroxiredoxin 3 leads to an increase in mitochondrial H2O2 and that this may influence the progression of apoptosis.
Kristin K Brown - One of the best experts on this subject based on the ideXlab platform.
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Mitochondrial respiratory chain involvement in Peroxiredoxin 3 oxidation by phenethyl isothiocyanate and auranofin
FEBS letters, 2010Co-Authors: Kristin K Brown, Andrew G Cox, Mark B HamptonAbstract:Mitochondrial Peroxiredoxin 3 (Prx 3) is rapidly oxidized in cells exposed to phenethyl isothiocyanate (PEITC) and auranofin (AFN), but the mechanism of oxidation is unclear. Using HL-60 cells deplete of mitochondrial DNA we show that Peroxiredoxin 3 oxidation and cytotoxicity requires a functional respiratory chain. Thioredoxin reductase (TrxR) could be inhibited by up to 90% by auranofin without direct oxidation of Peroxiredoxin 3. However, inhibition of thioredoxin reductase promoted Peroxiredoxin 3 oxidation and cytotoxicity in combination with phenethyl isothiocyanate or antimycin A. We conclude that rapid Peroxiredoxin 3 oxidation occurs as a consequence of increased oxidant production from the mitochondrial respiratory chain.
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the thioredoxin reductase inhibitor auranofin triggers apoptosis through a bax bak dependent process that involves Peroxiredoxin 3 oxidation
Biochemical Pharmacology, 2008Co-Authors: Andrew G Cox, Kristin K Brown, Elias S J Arner, Mark B HamptonAbstract:Abstract Thioredoxin reductase (TrxR) is a key selenoprotein antioxidant enzyme and a potential target for anti-cancer drugs. One potent inhibitor of TrxR is the gold (I) compound auranofin, which can trigger mitochondrial-dependent apoptosis pathways. The exact mechanism of apoptosis induction by auranofin is not yet clear, but there are indications that mitochondrial oxidative stress is a central event. We assessed the redox state of the Peroxiredoxins (Prxs) in Jurkat T-lymphoma cells treated with auranofin, and found that mitochondrial Prx3 was considerably more sensitive to oxidation than the cytosolic Prx1 and 2, indicating selective mitochondrial stress. Prx3 oxidation was detected at apoptotic doses of auranofin in several cell types, and occurred before other mitochondrial events including cytochrome c release and mitochondrial depolarisation. Auranofin was also able to sensitise U937 cells to TNF-α-mediated apoptosis. Auranofin-induced apoptosis was effectively blocked by the overexpression of Bcl-2, and Bax/Bak deficient mouse embryonic fibroblasts were also resistant to apoptosis, indicating a central role for the pro-apoptotic proteins of this family in auranofin-triggered apoptosis. Auranofin exposure inhibited the proliferation of apoptosis-resistant cells, and at higher doses of auranofin could cause cell death through necrosis. We conclude that auranofin induces apoptosis in cells through a Bax/Bak-dependent mechanism associated with selective disruption of mitochondrial redox homeostasis in conjunction with oxidation of Prx3.
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The thioredoxin reductase inhibitor auranofin triggers apoptosis through a Bax/Bak-dependent process that involves Peroxiredoxin 3 oxidation.
Biochemical pharmacology, 2008Co-Authors: Andrew G Cox, Kristin K Brown, Elias S J Arner, Mark B HamptonAbstract:Abstract Thioredoxin reductase (TrxR) is a key selenoprotein antioxidant enzyme and a potential target for anti-cancer drugs. One potent inhibitor of TrxR is the gold (I) compound auranofin, which can trigger mitochondrial-dependent apoptosis pathways. The exact mechanism of apoptosis induction by auranofin is not yet clear, but there are indications that mitochondrial oxidative stress is a central event. We assessed the redox state of the Peroxiredoxins (Prxs) in Jurkat T-lymphoma cells treated with auranofin, and found that mitochondrial Prx3 was considerably more sensitive to oxidation than the cytosolic Prx1 and 2, indicating selective mitochondrial stress. Prx3 oxidation was detected at apoptotic doses of auranofin in several cell types, and occurred before other mitochondrial events including cytochrome c release and mitochondrial depolarisation. Auranofin was also able to sensitise U937 cells to TNF-α-mediated apoptosis. Auranofin-induced apoptosis was effectively blocked by the overexpression of Bcl-2, and Bax/Bak deficient mouse embryonic fibroblasts were also resistant to apoptosis, indicating a central role for the pro-apoptotic proteins of this family in auranofin-triggered apoptosis. Auranofin exposure inhibited the proliferation of apoptosis-resistant cells, and at higher doses of auranofin could cause cell death through necrosis. We conclude that auranofin induces apoptosis in cells through a Bax/Bak-dependent mechanism associated with selective disruption of mitochondrial redox homeostasis in conjunction with oxidation of Prx3.
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Mitochondrial Peroxiredoxin 3 is rapidly oxidized in cells treated with isothiocyanates.
Free radical biology & medicine, 2008Co-Authors: Kristin K Brown, Elias S J Arner, Sofi Eriksson, Mark B HamptonAbstract:Isothiocyanates are phytochemicals with anti-cancer properties that include the ability to trigger apoptosis. A substantial body of evidence suggests that reaction of the electrophilic isothiocyanate moiety with cysteine residues in cellular proteins and glutathione accounts for their biological activity. In this study we investigated the effect of several different isothiocyanates on the redox states of the cysteine-dependent Peroxiredoxins (Prx) in Jurkat T lymphoma cells, and compared this to known effects on the selenoprotein thioredoxin reductase, glutathione reductase and intracellular GSH levels. Interestingly, oxidation of mitochondrial Prx3 could be detected as early as 5 min after exposure of cells to phenethyl isothiocyanate, with complete oxidation occurring at doses that only had small inhibitory effects on total cellular thioredoxin reductase and glutathione reductase activities. Peroxiredoxin oxidation was specific to the mitochondrial isoform with cytoplasmic Prx1 and Prx2 maintained in their reduced forms at all analyzed time points and concentrations of isothiocyanate. Phenethyl isothiocyanate could react with purified Prx3 directly, but it did not oxidize Prx3 or promote its oxidation by hydrogen peroxide. A selection of aromatic and alkyl isothiocyanates were tested and while all lowered cellular GSH levels, only the isothiocyanates that caused Prx3 oxidation were able to trigger cell death. We propose that pro-apoptotic isothiocyanates selectively disrupt mitochondrial redox homeostasis, as indicated by Prx3 oxidation, and that this contributes to their pro-apoptotic activity.
Nicholas H. Heintz - One of the best experts on this subject based on the ideXlab platform.
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Unique Cellular and Biochemical Features of Human Mitochondrial Peroxiredoxin 3 Establish the Molecular Basis for Its Specific Reaction with Thiostrepton.
Antioxidants (Basel Switzerland), 2021Co-Authors: Kimberly J. Nelson, Nicholas H. Heintz, Stacie L. Beuschel, Terri L. Messier, Stephanie Milczarek, Alexis Saaman, Uma Gandhi, Terrence L. Smalley, W. Todd Lowther, Brian CunniffAbstract:A central hallmark of tumorigenesis is metabolic alterations that increase mitochondrial reactive oxygen species (mROS). In response, cancer cells upregulate their antioxidant capacity and redox-responsive signaling pathways. A promising chemotherapeutic approach is to increase ROS to levels incompatible with tumor cell survival. Mitochondrial Peroxiredoxin 3 (PRX3) plays a significant role in detoxifying hydrogen peroxide (H2O2). PRX3 is a molecular target of thiostrepton (TS), a natural product and FDA-approved antibiotic. TS inactivates PRX3 by covalently adducting its two catalytic cysteine residues and crosslinking the homodimer. Using cellular models of malignant mesothelioma, we show here that PRX3 expression and mROS levels in cells correlate with sensitivity to TS and that TS reacts selectively with PRX3 relative to other PRX isoforms. Using recombinant PRXs 1–5, we demonstrate that TS preferentially reacts with a reduced thiolate in the PRX3 dimer at mitochondrial pH. We also show that partially oxidized PRX3 fully dissociates to dimers, while partially oxidized PRX1 and PRX2 remain largely decameric. The ability of TS to react with engineered dimers of PRX1 and PRX2 at mitochondrial pH, but inefficiently with wild-type decameric protein at cytoplasmic pH, supports a novel mechanism of action and explains the specificity of TS for PRX3. Thus, the unique structure and propensity of PRX3 to form dimers contribute to its increased sensitivity to TS-mediated inactivation, making PRX3 a promising target for prooxidant cancer therapy.
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Peroxiredoxin 3 levels regulate a mitochondrial redox setpoint in malignant mesothelioma cells
Redox biology, 2014Co-Authors: Brian Cunniff, Alexandra N. Wozniak, Patrick Sweeney, Kendra Decosta, Nicholas H. HeintzAbstract:Peroxiredoxin 3 (PRX3), a typical 2-Cys Peroxiredoxin located exclusively in the mitochondrial matrix, is the principal peroxidase responsible for metabolizing mitochondrial hydrogen peroxide, a byproduct of cellular respiration originating from the mitochondrial electron transport chain. Mitochondrial oxidants are produced in excess in cancer cells due to oncogenic transformation and metabolic reorganization, and signals through FOXM1 and other redox-responsive factors to support a hyper-proliferative state. Over-expression of PRX3 in cancer cells has been shown to counteract oncogene-induced senescence and support tumor cell growth and survival making PRX3 a credible therapeutic target. Using malignant mesothelioma (MM) cells stably expressing shRNAs to PRX3 we show that decreased expression of PRX3 alters mitochondrial structure, function and cell cycle kinetics. As compared to control cells, knockdown of PRX3 expression increased mitochondrial membrane potential, basal ATP production, oxygen consumption and extracellular acidification rates. shPRX3 MM cells failed to progress through the cell cycle compared to wild type controls, with increased numbers of cells in G2/M phase. Diminished PRX3 expression also induced mitochondrial hyperfusion similar to the DRP1 inhibitor mdivi-1. Cell cycle progression and changes in mitochondrial networking were rescued by transient expression of either catalase or mitochondrial-targeted catalase, indicating high levels of hydrogen peroxide contribute to perturbations in mitochondrial structure and function in shPRX3 MM cells. Our results indicate that PRX3 levels establish a redox set point that permits MM cells to thrive in response to increased levels of mROS, and that perturbing the redox status governed by PRX3 impairs proliferation by altering cell cycle-dependent dynamics between mitochondrial networking and energy metabolism.
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mitochondrial targeted nitroxides disrupt mitochondrial architecture and inhibit expression of Peroxiredoxin 3 and foxm1 in malignant mesothelioma cells
Journal of Cellular Physiology, 2013Co-Authors: Brian Cunniff, Kheng Newick, Paul Held, Kira Benson, Jason Stumpff, Douglas J Taatjes, Joy Joseph, B Kalyanaraman, Nicholas H. HeintzAbstract:Malignant mesothelioma (MM) is an intractable tumor of the peritoneal and pleural cavities primarily linked to exposure to asbestos. Recently, we described an interplay between mitochondrial-derived oxidants and expression of FOXM1, a redox-responsive transcription factor that has emerged as a promising therapeutic target in solid malignancies. Here we have investigated the effects of nitroxides targeted to mitochondria via triphenylphosphonium (TPP) moieties on mitochondrial oxidant production, expression of FOXM1 and Peroxiredoxin 3 (PRX3), and cell viability in MM cells in culture. Both Mito-carboxy-proxyl (MCP) and Mito-TEMPOL (MT) caused dose-dependent increases in mitochondrial oxidant production that was accompanied by inhibition of expression of FOXM1 and PRX3 and loss of cell viability. At equivalent concentrations TPP, CP, and TEMPOL had no effect on these endpoints. Live cell ratiometric imaging with a redox-responsive green fluorescent protein targeted to mitochondria (mito-roGFP) showed that MCP and MT, but not CP, TEMPOL, or TPP, rapidly induced mitochondrial fragmentation and swelling, morphological transitions that were associated with diminished ATP levels and increased production of mitochondrial oxidants. Mdivi-1, an inhibitor of mitochondrial fission, did not rescue mitochondria from fragmentation by MCP. Immunofluorescence microscopy experiments indicate a fraction of FOXM1 coexists in the cytoplasm with mitochondrial PRX3. Our results indicate that MCP and MT inhibit FOXM1 expression and MM tumor cell viability via perturbations in redox homeostasis caused by marked disruption of mitochondrial architecture, and suggest that both compounds, either alone or in combination with thiostrepton or other agents, may provide credible therapeutic options for the management of MM.
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Mitochondrial-Targeted Nitroxides Disrupt Mitochondrial Architecture and Inhibit Expression of Peroxiredoxin 3 and FOXM1 in Malignant Mesothelioma Cells
Journal of cellular physiology, 2012Co-Authors: Brian Cunniff, Kheng Newick, Paul Held, Kira Benson, Jason Stumpff, Douglas J Taatjes, Joy Joseph, B Kalyanaraman, Nicholas H. HeintzAbstract:Mitochondria are dynamic organelles, constantly adapting their structure and function in response to environmental cues and intracellular signals (Mitra et al., 2009; Hamanaka and Chandel, 2010; Antico Arciuch et al., 2012). Beyond their role as the primary source of ATP in the cell, mitochondria have emerged as signaling hubs that regulate normal and pathological cellular processes through redox-responsive signaling cascades, as reviewed in (Hamanaka and Chandel, 2010; Tait and Green, 2010). It has long been appreciated that cancer cells harbor mitochondria with altered energy production and structural aberrations (de Oliveira et al., 2012). The “Warburg effect” first described altered metabolism in malignant tissues that is characterized by increases in aerobic glycolysis, lactic acid production, and loss of oxidative phosphorylation (Diaz-Ruiz et al., 2011). Along with altered energy metabolism, the mitochondria of tumor cells produce increased amounts of oxidants (Fried and Arbiser, 2008; Klaunig et al., 2011), mainly through electron leakage to molecular oxygen in the electron transport chain (ETC) located in the inner mitochondrial membrane. Leakage of electrons from the ETC to molecular oxygen leads to the formation of superoxide radical which is spontaneously and enzymatically dismutated to hydrogen peroxide, the primary oxidant capable of freely crossing membranes (Jones, 2006; Rhee, 2006; Janssen-Heininger et al., 2008; Murphy, 2009). Through oxidation of reactive cysteine residues in signaling factors, hydrogen peroxide has been implicated in the modulation of regulatory pathways that control proliferation, apoptosis, metabolism, migration, and survival (Droge, 2002; Jones, 2010). It is important to note that the balance between oxidant production and metabolism, as well as the array of susceptible targets expressed in the cell, is critical in determining phenotypic responses. Moreover, redox-signaling by endogenous hydrogen peroxide involves significant spatial and temporal regulation, as either too little or too much hydrogen peroxide impairs cell cycle progression and viability (Burhans and Heintz, 2009). Activation of certain oncogenes, such as Ras, leads to increased production of cellular oxidants, a metabolic response that in most normal cells induces senescence (Lee et al., 1999). Tumor cells evade senescence and tolerate constitutive increases in the production of cellular oxidants, either through loss of checkpoint function or adaptive responses, including the up-regulation of anti-oxidant enzymes. Indeed, some tumor types appear to rely on enhanced production of oxidants for viability and other properties of malignancy (Fried and Arbiser, 2008; Gupta et al., 2012). FOXM1, a redox-responsive transcription factor that regulates genes involved in S phase and the G2/M transition, functions at the interface between oxidative stress, aging, and cancer (Laoukili et al., 2007; Myatt and Lam, 2007; Park et al., 2009). Because FOXM1 is up-regulated in all carcinomas examined to date, and is expressed only in proliferating cells (Laoukili et al., 2007), FOXM1 has emerged as a promising therapeutic target in cancer treatment (Wang et al., 2010). FOXM1 has also been shown to respond to changes in cellular redox status, with its expression increasing in response to exposure to low levels of exogenous hydrogen peroxide and decreasing following overnight treatment of cells with the free radical scavenger TEMPOL (Park et al., 2009). Through up-regulation of anti-oxidant enzymes that include mitochondrial superoxide dismutase (SOD2) and Peroxiredoxin 3 (PRX3), FOXM1 permits cells to escape senescence induced by activated Ras (Park et al., 2009). Previously, we showed that malignant mesothelioma (MM) cells in culture constitutively produce twofold to threefold more mitochondrial superoxide than non-transformed mesothelial cells, and that compounds that inactivate the major mitochondrial anti-oxidant network of thioredoxin reductase 2 (TR2)—thioredoxin 2 (TRX2)—PRX3 increase mitochondrial oxidative stress and block FOXM1 expression (Newick et al., 2012), albeit through an unknown pathway. Other small molecules that perturb mitochondrial redox status may therefore prove useful for inhibiting FOXM1 expression and the clinical management of MM. Due to its high negative membrane potential, compounds can be selectively targeted to mitochondria through the conjugation of a triphenylphosphonium (TPP) moiety, which provides a large, dispersed positive charge to the test agent (Murphy, 1997). The selective accumulation of TPP-containing compounds in mitochondria has allowed for targeted delivery of a large number of test agents, with levels that can be 100- to 500-fold higher than the bulk concentration (Murphy, 1997; Dhanasekaran et al., 2005). The higher negative membrane potential of tumor mitochondria also facilitates increased accumulation in tumor cell mitochondria versus normal cell mitochondria (Modica-Napolitano and Aprille, 2001; Millard et al., 2010). In this study we evaluated the activity of two TPP conjugated mitochondrial-targeted nitroxides, Mito-carboxy proxyl (MCP) (Dhanasekaran et al., 2005) and Mito-TEMPOL (MT) (Trnka et al., 2008) on MM tumor cell proliferation and survival (Supplementary Fig. S1). Our results indicate that MCP and MT inhibit FOXM1 expression by inducing marked mitochondrial fragmentation and increased production of mitochondrial oxidants, a phenotypic response that appears distinct from mitochondrial fission. In contrast, the parent compounds, carboxy proxyl (CP), TEMPOL, and TPP alone had no effect on FOXM1 expression, mitochondrial architecture, or cell viability at equivalent concentrations. These observations demonstrate that altered mitochondrial energy and oxidant metabolism in tumor cells is linked to FOXM1 expression, thereby providing a rationale for exploiting this relationship for cancer therapy.
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Peroxiredoxin 3 is a redox-dependent target of thiostrepton in malignant mesothelioma cells.
PloS one, 2012Co-Authors: Kheng Newick, Brian Cunniff, Kelsey Preston, Paul Held, Jack L. Arbiser, Harvey I. Pass, Brooke T. Mossman, Arti Shukla, Nicholas H. HeintzAbstract:Thiostrepton (TS) is a thiazole antibiotic that inhibits expression of FOXM1, an oncogenic transcription factor required for cell cycle progression and resistance to oncogene-induced oxidative stress. The mechanism of action of TS is unclear and strategies that enhance TS activity will improve its therapeutic potential. Analysis of human tumor specimens showed FOXM1 is broadly expressed in malignant mesothelioma (MM), an intractable tumor associated with asbestos exposure. The mechanism of action of TS was investigated in a cell culture model of human MM. As for other tumor cell types, TS inhibited expression of FOXM1 in MM cells in a dose-dependent manner. Suppression of FOXM1 expression and coincidental activation of ERK1/2 by TS were abrogated by pre-incubation of cells with the antioxidant N-acetyl-L-cysteine (NAC), indicating its mechanism of action in MM cells is redox-dependent. Examination of the mitochondrial thioredoxin reductase 2 (TR2)-thioredoxin 2 (TRX2)-Peroxiredoxin 3 (PRX3) antioxidant network revealed that TS modifies the electrophoretic mobility of PRX3. Incubation of recombinant human PRX3 with TS in vitro also resulted in PRX3 with altered electrophoretic mobility. The cellular and recombinant species of modified PRX3 were resistant to dithiothreitol and SDS and suppressed by NAC, indicating that TS covalently adducts cysteine residues in PRX3. Reduction of endogenous mitochondrial TRX2 levels by the cationic triphenylmethane gentian violet (GV) promoted modification of PRX3 by TS and significantly enhanced its cytotoxic activity. Our results indicate TS covalently adducts PRX3, thereby disabling a major mitochondrial antioxidant network that counters chronic mitochondrial oxidative stress. Redox-active compounds like GV that modify the TR2/TRX2 network may significantly enhance the efficacy of TS, thereby providing a combinatorial approach for exploiting redox-dependent perturbations in mitochondrial function as a therapeutic approach in mesothelioma.
Brian Cunniff - One of the best experts on this subject based on the ideXlab platform.
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Unique Cellular and Biochemical Features of Human Mitochondrial Peroxiredoxin 3 Establish the Molecular Basis for Its Specific Reaction with Thiostrepton.
Antioxidants (Basel Switzerland), 2021Co-Authors: Kimberly J. Nelson, Nicholas H. Heintz, Stacie L. Beuschel, Terri L. Messier, Stephanie Milczarek, Alexis Saaman, Uma Gandhi, Terrence L. Smalley, W. Todd Lowther, Brian CunniffAbstract:A central hallmark of tumorigenesis is metabolic alterations that increase mitochondrial reactive oxygen species (mROS). In response, cancer cells upregulate their antioxidant capacity and redox-responsive signaling pathways. A promising chemotherapeutic approach is to increase ROS to levels incompatible with tumor cell survival. Mitochondrial Peroxiredoxin 3 (PRX3) plays a significant role in detoxifying hydrogen peroxide (H2O2). PRX3 is a molecular target of thiostrepton (TS), a natural product and FDA-approved antibiotic. TS inactivates PRX3 by covalently adducting its two catalytic cysteine residues and crosslinking the homodimer. Using cellular models of malignant mesothelioma, we show here that PRX3 expression and mROS levels in cells correlate with sensitivity to TS and that TS reacts selectively with PRX3 relative to other PRX isoforms. Using recombinant PRXs 1–5, we demonstrate that TS preferentially reacts with a reduced thiolate in the PRX3 dimer at mitochondrial pH. We also show that partially oxidized PRX3 fully dissociates to dimers, while partially oxidized PRX1 and PRX2 remain largely decameric. The ability of TS to react with engineered dimers of PRX1 and PRX2 at mitochondrial pH, but inefficiently with wild-type decameric protein at cytoplasmic pH, supports a novel mechanism of action and explains the specificity of TS for PRX3. Thus, the unique structure and propensity of PRX3 to form dimers contribute to its increased sensitivity to TS-mediated inactivation, making PRX3 a promising target for prooxidant cancer therapy.
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Disabling Mitochondrial Peroxide Metabolism via Combinatorial Targeting of Peroxiredoxin 3 as an Effective Therapeutic Approach for Malignant Mesothelioma
PloS one, 2015Co-Authors: Brian Cunniff, Alexandra N. Wozniak, Kheng Newick, Kimberly J. Nelson, Stacie L. Beuschel, Bruce J. Leavitt, Anant D. Bhave, Kelly J. Butnor, Andreas Koenig, Edward T. ChouchaniAbstract:Dysregulation of signaling pathways and energy metabolism in cancer cells enhances production of mitochondrial hydrogen peroxide that supports tumorigenesis through multiple mechanisms. To counteract the adverse effects of mitochondrial peroxide many solid tumor types up-regulate the mitochondrial thioredoxin reductase 2 - thioredoxin 2 (TRX2) - Peroxiredoxin 3 (PRX3) antioxidant network. Using malignant mesothelioma cells as a model, we show that thiostrepton (TS) irreversibly disables PRX3 via covalent crosslinking of peroxidatic and resolving cysteine residues in homodimers, and that targeting the oxidoreductase TRX2 with the triphenylmethane gentian violet (GV) potentiates adduction by increasing levels of disulfide-bonded PRX3 dimers. Due to the fact that activity of the PRX3 catalytic cycle dictates the rate of adduction by TS, immortalized and primary human mesothelial cells are significantly less sensitive to both compounds. Moreover, stable knockdown of PRX3 reduces mesothelioma cell proliferation and sensitivity to TS. Expression of catalase in shPRX3 mesothelioma cells restores defects in cell proliferation but not sensitivity to TS. In a SCID mouse xenograft model of human mesothelioma, administration of TS and GV together reduced tumor burden more effectively than either agent alone. Because increased production of mitochondrial hydrogen peroxide is a common phenotype of malignant cells, and TS and GV are well tolerated in mammals, we propose that targeting PRX3 is a feasible redox-dependent strategy for managing mesothelioma and other intractable human malignancies.
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Peroxiredoxin 3 levels regulate a mitochondrial redox setpoint in malignant mesothelioma cells
Redox biology, 2014Co-Authors: Brian Cunniff, Alexandra N. Wozniak, Patrick Sweeney, Kendra Decosta, Nicholas H. HeintzAbstract:Peroxiredoxin 3 (PRX3), a typical 2-Cys Peroxiredoxin located exclusively in the mitochondrial matrix, is the principal peroxidase responsible for metabolizing mitochondrial hydrogen peroxide, a byproduct of cellular respiration originating from the mitochondrial electron transport chain. Mitochondrial oxidants are produced in excess in cancer cells due to oncogenic transformation and metabolic reorganization, and signals through FOXM1 and other redox-responsive factors to support a hyper-proliferative state. Over-expression of PRX3 in cancer cells has been shown to counteract oncogene-induced senescence and support tumor cell growth and survival making PRX3 a credible therapeutic target. Using malignant mesothelioma (MM) cells stably expressing shRNAs to PRX3 we show that decreased expression of PRX3 alters mitochondrial structure, function and cell cycle kinetics. As compared to control cells, knockdown of PRX3 expression increased mitochondrial membrane potential, basal ATP production, oxygen consumption and extracellular acidification rates. shPRX3 MM cells failed to progress through the cell cycle compared to wild type controls, with increased numbers of cells in G2/M phase. Diminished PRX3 expression also induced mitochondrial hyperfusion similar to the DRP1 inhibitor mdivi-1. Cell cycle progression and changes in mitochondrial networking were rescued by transient expression of either catalase or mitochondrial-targeted catalase, indicating high levels of hydrogen peroxide contribute to perturbations in mitochondrial structure and function in shPRX3 MM cells. Our results indicate that PRX3 levels establish a redox set point that permits MM cells to thrive in response to increased levels of mROS, and that perturbing the redox status governed by PRX3 impairs proliferation by altering cell cycle-dependent dynamics between mitochondrial networking and energy metabolism.
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mitochondrial targeted nitroxides disrupt mitochondrial architecture and inhibit expression of Peroxiredoxin 3 and foxm1 in malignant mesothelioma cells
Journal of Cellular Physiology, 2013Co-Authors: Brian Cunniff, Kheng Newick, Paul Held, Kira Benson, Jason Stumpff, Douglas J Taatjes, Joy Joseph, B Kalyanaraman, Nicholas H. HeintzAbstract:Malignant mesothelioma (MM) is an intractable tumor of the peritoneal and pleural cavities primarily linked to exposure to asbestos. Recently, we described an interplay between mitochondrial-derived oxidants and expression of FOXM1, a redox-responsive transcription factor that has emerged as a promising therapeutic target in solid malignancies. Here we have investigated the effects of nitroxides targeted to mitochondria via triphenylphosphonium (TPP) moieties on mitochondrial oxidant production, expression of FOXM1 and Peroxiredoxin 3 (PRX3), and cell viability in MM cells in culture. Both Mito-carboxy-proxyl (MCP) and Mito-TEMPOL (MT) caused dose-dependent increases in mitochondrial oxidant production that was accompanied by inhibition of expression of FOXM1 and PRX3 and loss of cell viability. At equivalent concentrations TPP, CP, and TEMPOL had no effect on these endpoints. Live cell ratiometric imaging with a redox-responsive green fluorescent protein targeted to mitochondria (mito-roGFP) showed that MCP and MT, but not CP, TEMPOL, or TPP, rapidly induced mitochondrial fragmentation and swelling, morphological transitions that were associated with diminished ATP levels and increased production of mitochondrial oxidants. Mdivi-1, an inhibitor of mitochondrial fission, did not rescue mitochondria from fragmentation by MCP. Immunofluorescence microscopy experiments indicate a fraction of FOXM1 coexists in the cytoplasm with mitochondrial PRX3. Our results indicate that MCP and MT inhibit FOXM1 expression and MM tumor cell viability via perturbations in redox homeostasis caused by marked disruption of mitochondrial architecture, and suggest that both compounds, either alone or in combination with thiostrepton or other agents, may provide credible therapeutic options for the management of MM.
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Mitochondrial-Targeted Nitroxides Disrupt Mitochondrial Architecture and Inhibit Expression of Peroxiredoxin 3 and FOXM1 in Malignant Mesothelioma Cells
Journal of cellular physiology, 2012Co-Authors: Brian Cunniff, Kheng Newick, Paul Held, Kira Benson, Jason Stumpff, Douglas J Taatjes, Joy Joseph, B Kalyanaraman, Nicholas H. HeintzAbstract:Mitochondria are dynamic organelles, constantly adapting their structure and function in response to environmental cues and intracellular signals (Mitra et al., 2009; Hamanaka and Chandel, 2010; Antico Arciuch et al., 2012). Beyond their role as the primary source of ATP in the cell, mitochondria have emerged as signaling hubs that regulate normal and pathological cellular processes through redox-responsive signaling cascades, as reviewed in (Hamanaka and Chandel, 2010; Tait and Green, 2010). It has long been appreciated that cancer cells harbor mitochondria with altered energy production and structural aberrations (de Oliveira et al., 2012). The “Warburg effect” first described altered metabolism in malignant tissues that is characterized by increases in aerobic glycolysis, lactic acid production, and loss of oxidative phosphorylation (Diaz-Ruiz et al., 2011). Along with altered energy metabolism, the mitochondria of tumor cells produce increased amounts of oxidants (Fried and Arbiser, 2008; Klaunig et al., 2011), mainly through electron leakage to molecular oxygen in the electron transport chain (ETC) located in the inner mitochondrial membrane. Leakage of electrons from the ETC to molecular oxygen leads to the formation of superoxide radical which is spontaneously and enzymatically dismutated to hydrogen peroxide, the primary oxidant capable of freely crossing membranes (Jones, 2006; Rhee, 2006; Janssen-Heininger et al., 2008; Murphy, 2009). Through oxidation of reactive cysteine residues in signaling factors, hydrogen peroxide has been implicated in the modulation of regulatory pathways that control proliferation, apoptosis, metabolism, migration, and survival (Droge, 2002; Jones, 2010). It is important to note that the balance between oxidant production and metabolism, as well as the array of susceptible targets expressed in the cell, is critical in determining phenotypic responses. Moreover, redox-signaling by endogenous hydrogen peroxide involves significant spatial and temporal regulation, as either too little or too much hydrogen peroxide impairs cell cycle progression and viability (Burhans and Heintz, 2009). Activation of certain oncogenes, such as Ras, leads to increased production of cellular oxidants, a metabolic response that in most normal cells induces senescence (Lee et al., 1999). Tumor cells evade senescence and tolerate constitutive increases in the production of cellular oxidants, either through loss of checkpoint function or adaptive responses, including the up-regulation of anti-oxidant enzymes. Indeed, some tumor types appear to rely on enhanced production of oxidants for viability and other properties of malignancy (Fried and Arbiser, 2008; Gupta et al., 2012). FOXM1, a redox-responsive transcription factor that regulates genes involved in S phase and the G2/M transition, functions at the interface between oxidative stress, aging, and cancer (Laoukili et al., 2007; Myatt and Lam, 2007; Park et al., 2009). Because FOXM1 is up-regulated in all carcinomas examined to date, and is expressed only in proliferating cells (Laoukili et al., 2007), FOXM1 has emerged as a promising therapeutic target in cancer treatment (Wang et al., 2010). FOXM1 has also been shown to respond to changes in cellular redox status, with its expression increasing in response to exposure to low levels of exogenous hydrogen peroxide and decreasing following overnight treatment of cells with the free radical scavenger TEMPOL (Park et al., 2009). Through up-regulation of anti-oxidant enzymes that include mitochondrial superoxide dismutase (SOD2) and Peroxiredoxin 3 (PRX3), FOXM1 permits cells to escape senescence induced by activated Ras (Park et al., 2009). Previously, we showed that malignant mesothelioma (MM) cells in culture constitutively produce twofold to threefold more mitochondrial superoxide than non-transformed mesothelial cells, and that compounds that inactivate the major mitochondrial anti-oxidant network of thioredoxin reductase 2 (TR2)—thioredoxin 2 (TRX2)—PRX3 increase mitochondrial oxidative stress and block FOXM1 expression (Newick et al., 2012), albeit through an unknown pathway. Other small molecules that perturb mitochondrial redox status may therefore prove useful for inhibiting FOXM1 expression and the clinical management of MM. Due to its high negative membrane potential, compounds can be selectively targeted to mitochondria through the conjugation of a triphenylphosphonium (TPP) moiety, which provides a large, dispersed positive charge to the test agent (Murphy, 1997). The selective accumulation of TPP-containing compounds in mitochondria has allowed for targeted delivery of a large number of test agents, with levels that can be 100- to 500-fold higher than the bulk concentration (Murphy, 1997; Dhanasekaran et al., 2005). The higher negative membrane potential of tumor mitochondria also facilitates increased accumulation in tumor cell mitochondria versus normal cell mitochondria (Modica-Napolitano and Aprille, 2001; Millard et al., 2010). In this study we evaluated the activity of two TPP conjugated mitochondrial-targeted nitroxides, Mito-carboxy proxyl (MCP) (Dhanasekaran et al., 2005) and Mito-TEMPOL (MT) (Trnka et al., 2008) on MM tumor cell proliferation and survival (Supplementary Fig. S1). Our results indicate that MCP and MT inhibit FOXM1 expression by inducing marked mitochondrial fragmentation and increased production of mitochondrial oxidants, a phenotypic response that appears distinct from mitochondrial fission. In contrast, the parent compounds, carboxy proxyl (CP), TEMPOL, and TPP alone had no effect on FOXM1 expression, mitochondrial architecture, or cell viability at equivalent concentrations. These observations demonstrate that altered mitochondrial energy and oxidant metabolism in tumor cells is linked to FOXM1 expression, thereby providing a rationale for exploiting this relationship for cancer therapy.