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Michael P Murphy

Publications and source records attributed to Michael P Murphy.

At least 19 recordsLinked to original sources
PubMed2026

Tumor-derived antioxidants suppress immunity by depriving T cells of reactive oxygen species.

Reactive oxygen species (ROS) promote genomic instability and fuel oncogenic signaling in cancer, but antioxidant therapies have so far failed to improve, or worsen, cancer outcomes. Emerging data suggest that T cells depend on ROS for signal transduction. In this study, we show that tumors exploit this dependency, releasing antioxidant enzymes into the tumor environment to suppress T cell-mediated antitumor immunity. The interstitial fluid of tumors possesses potent antioxidant activity, associated with enrichment of the antioxidant enzyme peroxiredoxin 1 (PRDX1). Extracellular PRDX1 deprives T cells of ROS, preventing oxidative inactivation of phosphatases required for T cell receptor-driven kinase signaling and effector function. Prdx1 is up-regulated upon cancer immunoediting, and loss of PRDX1 within tumors enhances antitumor immunity and immunotherapy responses. These findings define a redox-dependent mechanism of tumor immunosuppression that is potentially amenable to therapeutic intervention.

Animals↗
PubMed2022

Immunodeficiency, autoimmunity, and increased risk of B cell malignancy in humans with TRAF3 mutations.

Tumor necrosis factor receptor-associated factor 3 (TRAF3) is a central regulator of immunity. TRAF3 is often somatically mutated in B cell malignancies, but its role in human immunity is not defined. Here, in five unrelated families, we describe an immune dysregulation syndrome of recurrent bacterial infections, autoimmunity, systemic inflammation, B cell lymphoproliferation, and hypergammaglobulinemia. Affected individuals each had monoallelic mutations in TRAF3 that reduced TRAF3 expression. Immunophenotyping showed that patients' B cells were dysregulated, exhibiting increased nuclear factor-κB 2 activation, elevated mitochondrial respiration, and heightened inflammatory responses. Patients had mild CD4+ T cell lymphopenia, with a reduced proportion of naïve T cells but increased regulatory T cells and circulating T follicular helper cells. Guided by this clinical phenotype, targeted analyses demonstrated that common genetic variants, which also reduce TRAF3 expression, are associated with an increased risk of B cell malignancies, systemic lupus erythematosus, higher immunoglobulin levels, and bacterial infections in the wider population. Reduced TRAF3 conveys disease risks by driving B cell hyperactivity via intrinsic activation of multiple intracellular proinflammatory pathways and increased mitochondrial respiration, with a likely contribution from dysregulated T cell help. Thus, we define monogenic TRAF3 haploinsufficiency syndrome and demonstrate how common TRAF3 variants affect a range of human diseases.

Autoimmunity↗
PubMed2007

Targeting antioxidants to mitochondria by conjugation to lipophilic cations.

Mitochondrial oxidative damage contributes to a range of degenerative diseases. Consequently, the selective inhibition of mitochondrial oxidative damage is a promising therapeutic strategy. One way to do this is to invent antioxidants that are selectively accumulated into mitochondria within patients. Such mitochondria-targeted antioxidants have been developed by conjugating the lipophilic triphenylphosphonium cation to an antioxidant moiety, such as ubiquinol or alpha-tocopherol. These compounds pass easily through all biological membranes, including the blood-brain barrier, and into muscle cells and thus reach those tissues most affected by mitochondrial oxidative damage. Furthermore, because of their positive charge they are accumulated several-hundredfold within mitochondria driven by the membrane potential, enhancing the protection of mitochondria from oxidative damage. These compounds protect mitochondria from damage following oral delivery and may therefore form the basis for mitochondria-protective therapies. Here we review the background and work to date on this class of mitochondria-targeted antioxidants.

Antioxidants↗
PubMed2006

Sequence-specific modification of mitochondrial DNA using a chimeric zinc finger methylase.

We used engineered zinc finger peptides (ZFPs) to bind selectively to predetermined sequences in human mtDNA. Surprisingly, we found that engineered ZFPs cannot be reliably routed to mitochondria by using only conventional mitochondrial targeting sequences. We here show that addition of a nuclear export signal allows zinc finger chimeric enzymes to be imported into human mitochondria. The selective binding of mitochondria-specific ZFPs to mtDNA was exemplified by targeting the T8993G mutation, which causes two mitochondrial diseases, neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP) and also maternally inherited Leigh's syndrome. To develop a system that allows the monitoring of site-specific alteration of mtDNA we combined a ZFP with the easily assayed DNA-modifying activity of hDNMT3a methylase. Expression of the mutation-specific chimeric methylase resulted in the selective methylation of cytosines adjacent to the mutation site. This is a proof of principle that it is possible to target and alter mtDNA in a sequence-specific manner by using zinc finger technology.

Amino Acid Sequence↗
PubMed2006

Accumulation of lipophilic dications by mitochondria and cells.

Lipophilic monocations can pass through phospholipid bilayers and accumulate in negatively-charged compartments such as the mitochondrial matrix, driven by the membrane potential. This property is used to visualize mitochondria, to deliver therapeutic molecules to mitochondria and to measure the membrane potential. In theory, lipophilic dications have a number of advantages over monocations for these tasks, as the double charge should lead to a far greater and more selective uptake by mitochondria, increasing their therapeutic potential. However, the double charge might also limit the movement of lipophilic dications through phospholipid bilayers and little is known about their interaction with mitochondria. To see whether lipophilic dications could be taken up by mitochondria and cells, we made a series of bistriphenylphosphonium cations comprising two triphenylphosphonium moieties linked by a 2-, 4-, 5-, 6- or 10-carbon methylene bridge. The 5-, 6- and 10-carbon dications were taken up by energized mitochondria, whereas the 2- and 4-carbon dications were not. The accumulation of the dication was greater than that of the monocation methyltriphenylphosphonium. However, the uptake of dications was only described by the Nernst equation at low levels of accumulation, and beyond a threshold membrane potential of 90-100 mV there was negligible increase in dication uptake. Interestingly, the 5- and 6-carbon dications were not accumulated by cells, due to lack of permeation through the plasma membrane. These findings indicate that conjugating compounds to dications offers only a minor increase over monocations in delivery to mitochondria. Instead, this suggests that it may be possible to form dications within mitochondria that then remain within the cell.

Adenosine Triphosphate↗
PubMed2006

Selective fluorescent imaging of superoxide in vivo using ethidium-based probes.

The putative oxidation of hydroethidine (HE) has become a widely used fluorescent assay for the detection of superoxide in cultured cells. By covalently joining HE to a hexyl triphenylphosphonium cation (Mito-HE), the HE moiety can be targeted to mitochondria. However, the specificity of HE and Mito-HE for superoxide in vivo is limited by autooxidation as well as by nonsuperoxide-dependent cellular processes that can oxidize HE probes to ethidium (Etd). Recently, superoxide was shown to react with HE to generate 2-hydroxyethidium [Zhao, H., Kalivendi, S., Zhang, H., Joseph, J., Nithipatikom, K., Vasquez-Vivar, J. & Kalyanaraman, B. (2003) Free Radic. Biol. Med. 34, 1359-1368]. However, 2-hydroxyethidium is difficult to distinguish from Etd by conventional fluorescence techniques exciting at 510 nm. While investigating the oxidation of Mito-HE by superoxide, we found that the superoxide product of both HE and Mito-HE could be selectively excited at 396 nm with minimal interference from other nonspecific oxidation products. The oxidation of Mito-HE monitored at 396 nm by antimycin-stimulated mitochondria was 30% slower than at 510 nm, indicating that superoxide production may be overestimated at 510 nm by even a traditional superoxide-stimulating mitochondrial inhibitor. The rate-limiting step for oxidation by superoxide was 4x10(6) M-1.s-1, which is proposed to involve the formation of a radical from Mito-HE. The rapid reaction with a second superoxide anion through radical-radical coupling may explain how Mito-HE and HE can compete for superoxide in vivo with intracellular superoxide dismutases. Monitoring oxidation at both 396 and 510 nm of excitation wavelengths can facilitate the more selective detection of superoxide in vivo.

Animals↗
PubMed2006

Effect of the mitochondrial antioxidant, Mito Vitamin E, on hypoxic-ischemic striatal injury in neonatal rats: a dose-response and stereological study.

A mitochondria-targeted antioxidant, Mito Vitamin E (MitoVit E), has previously been shown to prevent mitochondrial oxidative damage. The aim of this study was to investigate the effect of MitoVit E on neuronal survival in the rat striatum after acute perinatal hypoxia-ischemia. Continuous striatal infusion with 4.35 microM, 43.5 microM, or 148 microM of MitoVit E before, during, and after hypoxia-ischemia was not neuroprotective for striatal medium-spiny neurons. Pre- or posttreatment with 435 microM MitoVit E was neurotoxic. These results suggest that MitoVit E is not significantly neuroprotective for striatal medium-spiny neurons after acute perinatal hypoxic-ischemic brain injury. The results also suggest that mitochondrial oxidative damage does not contribute significantly to the death of striatal medium-spiny neurons after perinatal hypoxia-ischemia.

Animals↗
PubMed2006

Persistent S-nitrosation of complex I and other mitochondrial membrane proteins by S-nitrosothiols but not nitric oxide or peroxynitrite: implications for the interaction of nitric oxide with mitochondria.

S-nitrosation of mitochondrial proteins has been proposed to contribute to the pathophysiological interactions of nitric oxide (NO) and its derivatives with mitochondria but has not been shown directly. Furthermore, little is known about the mechanism of formation or the fate of these putative S-nitrosothiols. Here we have determined whether mitochondrial membrane protein thiols can be S-nitrosated on exposure to free NO from 3,3-bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene (DETA-NONOate) by interaction with S-nitrosoglutathione or S-nitroso-N-acetylpenicillamine (SNAP) and by the NO derivative peroxynitrite. S-Nitrosation of protein thiols was measured directly by chemiluminescence detection. S-Nitrosoglutathione and S-nitroso-N-acetylpenicillamine led to extensive protein thiol oxidation, with about 30% of the modified protein thiols persistently S-nitrosated. In contrast, there was no protein thiol oxidation or S-nitrosation on exposure to 3,3-bis (aminoethyl)-1-hydroxy-2-oxo-1-triazene. Peroxynitrite extensively oxidized protein thiols but produced negligible amounts of S-nitrosothiols. Therefore, mitochondrial membrane protein thiols are S-nitrosated by preformed S-nitrosothiols but not by NO or by peroxynitrite. These S-nitrosated protein thiols were readily reduced by glutathione, so S-nitrosation will only persist when the mitochondrial glutathione pool is oxidized. Respiratory chain complex I was S-nitrosated by S-nitrosothiols, consistent with it being an important target for S-nitrosation during nitrosative stress. The S-nitrosation of complex I correlated with a significant loss of activity that was reversed by thiol reductants. S-Nitrosation was also associated with increased superoxide production from complex I. These findings point to a significant role for complex I S-nitrosation and consequent dysfunction during nitrosative stress in disorders such as Parkinson disease and sepsis.

Animals↗
PubMed2006

The effects of exogenous antioxidants on lifespan and oxidative stress resistance in Drosophila melanogaster.

We used the fruit fly Drosophila melanogaster to test the effects of feeding the superoxide dismutase (SOD) mimetic drugs Euk-8 and -134 and the mitochondria-targeted mitoquinone (MitoQ) on lifespan and oxidative stress resistance of wild type and SOD-deficient flies. Our results reaffirm the findings by other workers that exogenous antioxidant can rescue pathology associated with compromised defences to oxidative stress, but fail to extend the lifespan of normal, wild type animals. All three drugs showed a dose-dependent increase in toxicity in wild type flies, an effect that was exacerbated in the presence of the redox-cycling drug paraquat. However, important findings from this study were that in SOD-deficient flies, where the antioxidant drugs increased lifespan, the effects were sex-specific and, for either sex, the effects were also variable depending on (1) the stage of development from which the drugs were given, and (2) the magnitude of the dose. These findings place significant constraints on the role of oxidative stress in normal ageing.

Aging↗
PubMed2006

Mitochondrial production of oxidants is necessary for physiological calcium oscillations.

Mitochondrial involvement in Ca2+ signaling is thought to be due to the effect of mitochondrial Ca2+ removal from and Ca2+ release to cytosolic domains close to ryanodine and IP3 Ca2+ channels. However, mitochondria are a source of low levels of endogenous reactive oxygen species, and Ca2+ release channels are known to be redox-sensitive. In the present work, we studied the role of mitochondrial production of oxygen species in Ca2+ oscillations during physiological stimulation. Mitochondria-targeted antioxidants and mitochondrial inhibitors quickly inhibited calcium oscillations in pancreatic acinar cells stimulated by postprandial levels of the gut hormone cholecystokinin. Confocal microscopy using different redox-sensitive dyes showed that cholecystokinin-induced oscillations are associated with mitochondrial production of reactive oxygen species. This production is inhibited by application of mitochondria-targeted antioxidants and mitochondrial inhibitors. In addition, we found no correlation between inhibition of oscillations and mitochondrial depolarization. We conclude that low level production of reactive oxygen species by mitochondria is a necessary element in the development of Ca2+ oscillations during physiological stimulation. This study unveils a new and unexplored aspect of the participation of mitochondria in calcium signals.

Animals↗
PubMed2006

Targeting dinitrophenol to mitochondria: limitations to the development of a self-limiting mitochondrial protonophore.

The protonmotive force (Deltap) across the mitochondrial inner membrane drives ATP synthesis. In addition, the energy stored in Deltap can be dissipated by proton leak through the inner membrane, contributing to basal metabolic rate and thermogenesis. Increasing mitochondrial proton leak pharmacologically should decrease the efficiency of oxidative phosphorylation and counteract obesity by enabling fatty acids to be oxidised with decreased ATP production. While protonophores such as 2,4-dinitrophenol (DNP) increase mitochondrial proton leak and have been used to treat obesity, a slight increase in DNP concentration above the therapeutically effective dose disrupts mitochondrial function and leads to toxicity. Therefore we set out to develop a less toxic protonophore that would increase proton leak significantly at high Deltap but not at low Deltap. Our design concept for a potential self-limiting protonophore was to couple the DNP moiety to the lipophilic triphenylphosphonium (TPP) cation and this was achieved by the preparation of 3-(3,5-dinitro-4-hydroxyphenyl)propyltriphenylphosphonium methanesulfonate (MitoDNP). TPP cations accumulate within mitochondria driven by the membrane potential (Deltapsi), the predominant component of Deltap. Our hypothesis was that MitoDNP would accumulate in mitochondria at high Deltapsi where it would act as a protonophore, but that at lower Deltapsi the accumulation and uncoupling would be far less. We found that MitoDNP was extensively taken into mitochondria driven by Deltapsi. However MitoDNP did not uncouple mitochondria as judged by its inability to either increase respiration rate or decrease Deltapsi. Therefore MitoDNP did not act as a protonophore, probably because the efflux of deprotonated MitoDNP was inhibited.

Animals↗
PubMed2006

Stem cell use in a patient with an ischemic foot ulcer: a case study.

Stem cell research has been discussed in both the political and popular arenas lately. Some types of stem cell research are controversial; however, not all stem cell research involves harvesting cells from an embryo or an aborted fetus. Another type of stem cell, the adult stem cell, resides within tissues and organs of the body and is responsible for repair after injury. Recent interest has focused on using adult stem cells isolated from a patient's bone marrow to stimulate the development of new blood vessels (a process called angiogenesis) in patients with peripheral vascular disease. These specialized stem cells are referred to as endothelial progenitor cells (EPCs). In laboratory and clinical studies it has been shown that these EPCs are involved in arterial repair and remodeling, as well as angiogenesis. The following case presentation will show how a patient with an ischemic toe ulcer and rest pain, who had no options for conventional revascularization, benefited from the injection of stem cells into her calf muscle. The process of angiogenesis using EPCs will be examined discussing the screening criteria and possible adverse events. The patient's history and progress throughout the recovery period will be reviewed.

Aged↗
PubMed2006

Activation of mitogen-activated protein kinases by lysophosphatidylcholine-induced mitochondrial reactive oxygen species generation in endothelial cells.

Lysophosphatidylcholine (lysoPC) evokes diverse biological responses in vascular cells including Ca(2+) mobilization, production of reactive oxygen species, and activation of the mitogen-activated protein kinases, but the mechanisms linking these events remain unclear. Here, we provide evidence that the response of mitochondria to the lysoPC-dependent increase in cytosolic Ca(2+) leads to activation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase through a redox signaling mechanism in human umbilical vein endothelial cells. ERK activation was attenuated by inhibitors of the electron transport chain proton pumps (rotenone and antimycin A) and an uncoupler (carbonyl cyanide p-trifluoromethoxyphenylhydrazone), suggesting that mitochondrial inner membrane potential plays a key role in the signaling pathway. ERK activation was also selectively attenuated by chain-breaking antioxidants and by vitamin E targeted to mitochondria, suggesting that transduction of the mitochondrial hydrogen peroxide signal is mediated by a lipid peroxidation product. Inhibition of ERK activation with MEK inhibitors (PD98059 or U0126) diminished induction of the antioxidant enzyme heme oxygenase-1. Taken together, these data suggest a role for mitochondrially generated reactive oxygen species and Ca(2+) in the redox cell signaling path-ways, leading to ERK activation and adaptation of the pathological stress mediated by oxidized lipids such as lysoPC.

Calcium↗
PubMed2006

Beta-amyloid mediated nitration of manganese superoxide dismutase: implication for oxidative stress in a APPNLH/NLH X PS-1P264L/P264L double knock-in mouse model of Alzheimer's disease.

Alzheimer's disease is a multifactorial, progressive, age-related neurodegenerative disease. In familial Alzheimer's disease, Abeta is excessively produced and deposited because of mutations in the amyloid precursor protein, presenilin-1, and presenilin-2 genes. Here, we generated a double homozygous knock-in mouse model that incorporates the Swedish familial Alzheimer's disease mutations and converts mouse Abeta to the human sequence in amyloid precursor protein and had the P264L familial Alzheimer's disease mutation in presenilin-1. We observed Abeta deposition in double knock-in mice beginning at 6 months as well as an increase in the levels of insoluble Abeta1-40/1-42. Brain homogenates from 3-, 6-, 9-, 12-, and 14-month-old mice showed that protein levels of manganese superoxide dismutase (MnSOD) were unchanged in the double knock-in mice compared to controls. Genotype-associated increases in nitrotyrosine levels were observed. Protein immunoprecipitation revealed MnSOD as a target of this nitration. Although the levels of MnSOD protein did not change, MnSOD activity and mitochondrial respiration decreased in knock-in mice, suggesting compromised mitochondrial function. The compromised activity of MnSOD, a primary antioxidant enzyme protecting mitochondria, may explain mitochondrial dysfunction and provide the missing link between Abeta-induced oxidative stress and Alzheimer's disease.

Aging↗
PubMed2005

Anti-Abeta42- and anti-Abeta40-specific mAbs attenuate amyloid deposition in an Alzheimer disease mouse model.

Accumulation and aggregation of amyloid beta peptide 1-42 (Abeta42) in the brain has been hypothesized as triggering a pathological cascade that causes Alzheimer disease (AD). To determine whether selective targeting of Abeta42 versus Abeta40 or total Abeta is an effective way to prevent or treat AD, we compared the effects of passive immunization with an anti-Abeta42 mAb, an anti-Abeta40 mAb, and multiple Abeta(1-16) mAbs. We established in vivo binding selectivity of the anti-Abeta42 and anti-Abeta40 mAbs using novel TgBRI-Abeta mice. We then conducted a prevention study in which the anti-Abeta mAbs were administered to young Tg2576 mice, which have no significant Abeta deposition, and therapeutic studies in which mAbs were administered to Tg2576 or CRND8 mice with modest levels of preexisting Abeta deposits. Anti-Abeta42, anti-Abeta40, and anti-Abeta(1-16) mAbs attenuated plaque deposition in the prevention study. In contrast, anti-Abeta42 and anti-Abeta40 mAbs were less effective in attenuating Abeta deposition in the therapeutic studies and were not effective in clearing diffuse plaques following direct injection into the cortex. These data suggest that selective targeting of Abeta42 or Abeta40 may be an effective strategy to prevent amyloid deposition, but may have limited benefit in a therapeutic setting.

Alzheimer Disease↗
PubMed2005

Mitochondrial reactive oxygen species regulate the temporal activation of nuclear factor kappaB to modulate tumour necrosis factor-induced apoptosis: evidence from mitochondria-targeted antioxidants.

ROS (reactive oxygen species) from mitochondrial and non-mitochondrial sources have been implicated in TNFalpha (tumour necrosis factor alpha)-mediated signalling. In the present study, a new class of specific mitochondria-targeted antioxidants were used to explore directly the role of mitochondrial ROS in TNF-induced apoptosis. MitoVit E {[2-(3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl)ethyl]triphenylphosphonium bromide} (vitamin E attached to a lipophilic cation that facilitates accumulation of the antioxidant in the mitochondrial matrix) enhanced TNF-induced apoptosis of U937 cells. In time course analyses, cleavage and activation of caspase 8 in response to TNF were not affected by MitoVit E, whereas the activation of caspase 3 was significantly increased. Furthermore, there was an increased cleavage of the proapoptotic Bcl-2 family member Bid and an increased release of cytochrome c from mitochondria, in cells treated with TNF in the presence of MitoVit E. We considered several mechanisms by which MitoVit E might accelerate TNF-induced apoptosis including mitochondrial integrity (ATP/ADP levels and permeability transition), alterations in calcium homoeostasis and transcription factor activation. Of these, only the transcription factor NF-kappaB (nuclear factor kappaB) was implicated. TNF caused maximal nuclear translocation of NF-kappaB within 15 min, compared with 1 h in cells pretreated with MitoVit E. Thus the accumulation of an antioxidant within the mitochondrial matrix enhances TNF-induced apoptosis by decreasing or delaying the expression of the protective antiapoptotic proteins. These results demonstrate that mitochondrial ROS production is a physiologically relevant component of the TNF signal-transduction pathway during apoptosis, and reveal a novel functional role for mitochondrial ROS as a temporal regulator of NF-kappaB activation and NF-kappaB-dependent antiapoptotic signalling.

Antioxidants↗
PubMed2005

A targeted antioxidant reveals the importance of mitochondrial reactive oxygen species in the hypoxic signaling of HIF-1alpha.

Exposure to limiting oxygen in cells and tissues induce the stabilization and transcriptional activation of the hypoxia-inducible factor 1 alpha (HIF-1alpha) protein, a key regulator of the hypoxic response. Reactive oxygen species (ROS) generation has been implicated in the stabilization of HIF-1alpha during this response, but this is still a matter of some debate. In this study we utilize a mitochondria-targeted antioxidant, mitoubiquinone (MitoQ), and examine its effects on the hypoxic stabilization of HIF-1alpha. Our results show that under conditions of reduced oxygen (3% O(2)), MitoQ ablated the hypoxic induction of ROS generation and destabilized HIF-1alpha protein. This in turn led to an abrogation of HIF-1 transcriptional activity. Normoxic stabilization of HIF-1alpha, on the other hand, was unchanged in the presence of MitoQ suggesting that ROS were not involved. This study strongly suggests that mitochondrial ROS contribute to the hypoxic stabilization of HIF-1alpha.

Antioxidants↗