Biomedical subjects

Robin A J Smith

Publications and source records attributed to Robin A J Smith.

7 recordsLinked to original sources

PubMed2003

Superoxide activates uncoupling proteins by generating carbon-centered radicals and initiating lipid peroxidation: studies using a mitochondria-targeted spin trap derived from alpha-phenyl-N-tert-butylnitrone.

Although the physiological role of uncoupling proteins (UCPs) 2 and 3 is uncertain, their activation by superoxide and by lipid peroxidation products suggest that UCPs are central to the mitochondrial response to reactive oxygen species. We examined whether superoxide and lipid peroxidation products such as 4-hydroxy-2-trans-nonenal act independently to activate UCPs, or if they share a common pathway, perhaps by superoxide exposure leading to the formation of lipid peroxidation products. This possibility can be tested by blocking the putative reactive oxygen species cascade with selective antioxidants and then reactivating UCPs with distal cascade components. We synthesized a mitochondria-targeted derivative of the spin trap alpha-phenyl-N-tert-butylnitrone, which reacts rapidly with carbon-centered radicals but is unreactive with superoxide and lipid peroxidation products. [4-[4-[[(1,1-Dimethylethyl)-oxidoimino]methyl]phenoxy]butyl]triphenylphosphonium bromide (MitoPBN) prevented the activation of UCPs by superoxide but did not block activation by hydroxynonenal. This was not due to MitoPBN reacting with superoxide or the hydroxyl radical or by acting as a chain-breaking antioxidant. MitoPBN did react with carbon-centered radicals and also prevented lipid peroxidation by the carbon-centered radical generator 2,2'-azobis(2-methyl propionamidine) dihydrochloride (AAPH). Furthermore, AAPH activated UCPs, and this was blocked by MitoPBN. These data suggest that superoxide and lipid peroxidation products share a common pathway for the activation of UCPs. Superoxide releases iron from iron-sulfur center proteins, which then generates carbon-centered radicals that initiate lipid peroxidation, yielding breakdown products that activate UCPs.

Animals↗

PubMed2003

Mitochondria-targeted antioxidants protect Friedreich Ataxia fibroblasts from endogenous oxidative stress more effectively than untargeted antioxidants.

Friedreich Ataxia (FRDA), the most common inherited ataxia, arises from defective expression of the mitochondrial protein frataxin, which leads to increased mitochondrial oxidative damage. Therefore, antioxidants targeted to mitochondria should be particularly effective at slowing disease progression. To test this hypothesis, we compared the efficacy of mitochondria-targeted and untargeted antioxidants derived from coenzyme Q10 and from vitamin E at preventing cell death due to endogenous oxidative stress in cultured fibroblasts from FRDA patients in which glutathione synthesis was blocked. The mitochondria-targeted antioxidant MitoQ was several hundredfold more potent than the untargeted analog idebenone. The mitochondria-targeted antioxidant MitoVit E was 350-fold more potent than the water soluble analog Trolox. This is the first demonstration that mitochondria-targeted antioxidants prevent cell death that arises in response to endogenous oxidative damage. Targeted antioxidants may have therapeutic potential in FRDA and in other disorders involving mitochondrial oxidative damage.

Antioxidants↗

PubMed2003

Delivery of bioactive molecules to mitochondria in vivo.

Mitochondrial dysfunction contributes to many human degenerative diseases but specific treatments are hampered by the difficulty of delivering bioactive molecules to mitochondria in vivo. To overcome this problem we developed a strategy to target bioactive molecules to mitochondria by attachment to the lipophilic triphenylphosphonium cation through an alkyl linker. These molecules rapidly permeate lipid bilayers and, because of the large mitochondrial membrane potential (negative inside), accumulate several hundredfold inside isolated mitochondria and within mitochondria in cultured cells. To determine whether this strategy could lead to the development of mitochondria-specific therapies, we investigated the administration and tissue distribution in mice of simple alkyltriphenylphosphonium cations and of mitochondria-targeted antioxidants comprising a triphenylphosphonium cation coupled to a coenzyme Q or vitamin E derivative. Significant doses of these compounds could be fed safely to mice over long periods, coming to steady-state distributions within the heart, brain, liver, and muscle. Therefore, mitochondria-targeted bioactive molecules can be administered orally, leading to their accumulation at potentially therapeutic concentrations in those tissues most affected by mitochondrial dysfunction. This finding opens the way to the testing of mitochondria-specific therapies in mouse models of human degenerative diseases.

Animals↗

PubMed2003

Specific targeting of a DNA-alkylating reagent to mitochondria. Synthesis and characterization of [4-((11aS)-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-on-8-oxy)butyl]-triphenylphosphonium iodide.

The selective manipulation of the expression and replication of mitochondrial DNA (mtDNA) within mammalian cells has proven difficult. In progressing towards this goal we synthesized a novel mitochondria-targeted DNA-alkylating reagent. The active alkylating moiety [(11aS)-8-hydroxy-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (DC-81)], irreversibly alkylates guanine bases in DNA (with a preference for AGA triplets), preventing its expression and replication. To target this compound to mitochondria it was covalently coupled to the lipophilic triphenylphosphonium (TPP) cation to form a derivative referred to as mitoDC-81. Incorporation of this lipophilic cation led to the rapid uptake of mitoDC-81 by mitochondria, driven by the large membrane potential across the inner membrane. This compound efficiently alkylated isolated supercoiled, relaxed-circular or linear plasmid DNA and isolated mtDNA. However mitoDC-81 did not alkylate mtDNA within isolated mitochondria or cells, even though it accessed the mitochondrial matrix at concentrations up to 100-fold higher than those required to alkylate isolated DNA. This surprising finding suggests that mtDNA within intact mitochondria may not be accessible to this class of alkylating reagent. This inability to alkylate mtDNA in situ has significant implications for the design of therapies for mtDNA diseases and for studies on the packaging, expression and turnover of mtDNA in general.

Alkylating Agents↗

PubMed2002

Superoxide activates mitochondrial uncoupling protein 2 from the matrix side. Studies using targeted antioxidants.

Superoxide activates nucleotide-sensitive mitochondrial proton transport through the uncoupling proteins UCP1, UCP2, and UCP3 (Echtay, K. S., et al. (2002) Nature 415, 1482-1486). Two possible mechanisms were proposed: direct activation of the UCP proton transport mechanism by superoxide or its products and a cycle of hydroperoxyl radical entry coupled to UCP-catalyzed superoxide anion export. Here we provide evidence for the first mechanism and show that superoxide activates UCP2 in rat kidney mitochondria from the matrix side of the mitochondrial inner membrane: (i) Exogenous superoxide inhibited matrix aconitase, showing that external superoxide entered the matrix. (ii) Superoxide-induced uncoupling was abolished by low concentrations of the mitochondrially targeted antioxidants 10-(6'-ubiquinonyl)decyltriphenylphosphonium (mitoQ) or 2-[2-(triphenylphosphonio)ethyl]-3,4-dihydro-2,5,7,8-tetramethyl-2H-1-benzopyran-6-ol bromide (mitoVit E), which are ubiquinone (Q) or tocopherol derivatives targeted to the matrix by covalent attachment to triphenylphosphonium cation. However, superoxide-induced uncoupling was not affected by similar concentrations of the nontargeted antioxidants Q(o), Q(1), decylubiquinone, vitamin E, or 6-hydroxy-2,5,7,8-tetramethylchroman 2-carboxylic acid (TROLOX) or of the mitochondrially targeted but redox-inactive analogs decyltriphenylphosphonium or 4-chlorobutyltriphenylphosphonium. Thus matrix superoxide appears to be necessary for activation of UCP2 by exogenous superoxide. (iii) When the reduced to oxidized ratio of mitoQ accumulated by mitochondria was increased by inhibiting cytochrome oxidase, it induced nucleotide-sensitive uncoupling that was not inhibited by external superoxide dismutase. Under these conditions quinols are known to produce superoxide, and because mitoQ is localized within the mitochondrial matrix this suggests that production of superoxide in the matrix was sufficient to activate UCP2. Furthermore, the superoxide did not need to be exported or to cycle across the inner membrane to cause uncoupling. We conclude that superoxide (or its products) exerts its uncoupling effect by activating the proton transport mechanism of uncoupling proteins at the matrix side of the mitochondrial inner membrane.

Aconitate Hydratase↗

PubMed2002

Specific modification of mitochondrial protein thiols in response to oxidative stress: a proteomics approach.

Mitochondria play a central role in redox-linked processes in the cell through mechanisms that are thought to involve modification of specific protein thiols, but this has proved difficult to assess. In particular, specific labeling and quantitation of mitochondrial protein cysteine residues have not been achieved due to the lack of reagents available that can be applied to the intact organelle or cell. To overcome these problems we have used a combination of mitochondrial proteomics and targeted labeling of mitochondrial thiols using a novel compound, (4-iodobutyl)triphenylphosphonium (IBTP). This lipophilic cation is accumulated by mitochondria and yields stable thioether adducts in a thiol-specific reaction. The selective uptake into mitochondria, due to the large membrane potential across the inner membrane, and the high pH of the matrix results in specific labeling of mitochondrial protein thiols by IBTP. Individual mitochondrial proteins that changed thiol redox state following oxidative stress could then be identified by their decreased reaction with IBTP and isolated by two-dimensional electrophoresis. We demonstrate the selectivity of IBTP labeling and use it to show that glutathione oxidation and exposure to an S-nitrosothiol or to peroxynitrite cause extensive redox changes to mitochondrial thiol proteins. In conjunction with blue native gel electrophoresis, we used IBTP labeling to demonstrate that thiols are exposed on the matrix faces of respiratory Complexes I, II, and IV. This novel approach enables measurement of the thiol redox state of individual mitochondrial proteins during oxidative stress and cell death. In addition the methodology has the potential to identify novel redox-dependent modulation of mitochondrial proteins.

Animals↗

PubMed2002

Prevention of mitochondrial oxidative damage using targeted antioxidants.

Mitochondrial-targeted antioxidants that selectively block mitochondrial oxidative damage and prevent some types of cell death have been developed. These antioxidants are ubiquinone and tocopherol derivatives and are targeted to mitochondria by covalent attachment to a lipophilic triphenylphosphonium cation. Because of the large mitochondrial membrane potential, these cations accumulated within mitochondria inside cells, where the antioxidant moiety prevents lipid peroxidation and protects mitochondria from oxidative damage. The mitochondrially localized ubiquinone also protected mammalian cells from hydrogen peroxide-induced apoptosis while an untargeted ubiquinone analogue was ineffective against apoptosis. When fed to mice these compounds accumulated within the brain, heart, and liver; therefore, using these mitochondrial-targeted antioxidants may help investigations of the role of mitochondrial oxidative damage in animal models of aging.

Animals↗