Methods of Detection of Vascular Reactive Species

Tarpey, Margaret M.; Fridovich, Irwin

doi:10.1161/hh1501.094365

Cited by 490 publications

(115 citation statements)

References 168 publications

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“…However, one has to be aware of caveats of this approach. DHE is a non-fluorescent cell permeant that undergoes oxidation to a fluorescent product 2-hydroxyethidium, that intercalates into (39), it can also oxidize DHE (20,24). Although we think the O 2 .…”

Section: Discussionmentioning

confidence: 97%

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An Unexpected Functional Link between Lysosomal Thiol Reductase and Mitochondrial Manganese Superoxide Dismutase

Bogunovic

Stojakovic

Chen

et al. 2008

Journal of Biological Chemistry

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Gamma interferon-inducible thiol reductase (GILT) is an enzyme involved in the initial steps of antigen processing and presentation. Recently we have shown that GILT is also expressed in mouse T cells, where it exerts an inhibitory role on T cell activation. In this study, we identified mitochondrial manganese superoxide dismutase (SOD2) as one of the key intermediaries affected by GILT expression in fibroblasts. Expression and activity of SOD2 is reduced in the absence of GILT because of reduced SOD2 protein stability. The forced increase in SOD2 expression in the absence of GILT restores fibroblast proliferation to wild-type levels. Thus, GILT appears to have a fundamental role in cellular proliferation mediated through its influence on SOD2 protein activity and expression.Enzymes of the thiol reductase family carry out reduction, oxidation, and isomerization of protein disulfide bonds in cytosol (for example, thioredoxin) (1, 2), mitochondria (3), endoplasmic reticulum (protein-disulfide isomerase) (2), and lysosomes (gamma interferon-inducible thiol reductase, GILT).2 The majority of these enzymes are functional at neutral or slightly alkaline conditions (4), they have similar three-dimensional structures, and all feature a conservative active site loop containing two cysteines in the sequence -CGPC-(5). GILT is a unique and unusual member of the thiol reductase family because its optimal enzymatic activity is at a low pH (4.5-5.5) (6 -8) and has an atypical active site (-CGAC-).GILT is synthesized as a 35-kDa soluble glycoprotein precursor and is transported to the endosomal compartment via the mannose-6-P receptor pathway (9). It is processed into the mature form (30 kDa) by proteolytic removal of N-and C-terminal peptides. The protein has an approximate molecular mass of 30 kDa and was therefore initially named IP-30 (6). In addition to endosomal/lysosomal localization, GILT is secreted in the tissue culture medium of the GILT-expressing cell lines (7,10), and is present in mouse sera.3 GILT is constitutively expressed in professional antigen-presenting cells (APCs), but it is also inducible by pro-inflammatory cytokines such as interferon ␥, tumor necrosis factor ␣, and interleukin 1␤ (9).Using GILT Ϫ/Ϫ mice as a model, we have shown that GILT catalyzes initial unfolding of antigenic protein (protein becomes more accessible for further processing by cathepsins) and therefore facilitates protein/peptide binding to MHC class II molecules (10). By changing the redox state of exogenous antigenic proteins with disulfide bonds, GILT initiates the adaptive immune response. However, we have shown that GILT is constitutively expressed in T cells and has a role in the regulation of T cell activation. This is so far the only known GILT function not related to MHC class II processing (11). GILT Ϫ/Ϫ T cells show increased proliferation and cytotoxic T cell activity in response to anti-CD3 stimulation. This observation suggests that GILT has a more fundamental role in cellular processes than just reduction of antigens...

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Section: Discussionmentioning

confidence: 97%

“…DCF-DA reacts even faster with various cellular radicals (RO 2 . , RO ⅐ , NO 2 ⅐ , CO 3 ⅐ , OH ⅐ , and ONOO Ϫ ); thus, it is not a very specific probe and easily oxidizes (20,24). Fibroblasts were incubated with DHE in RPMI for 5 min and immediately analyzed by flow cytometry.…”

Section: Gilt-deficient Mouse Fibroblast Cell Line Has Increased Supementioning

confidence: 99%

An Unexpected Functional Link between Lysosomal Thiol Reductase and Mitochondrial Manganese Superoxide Dismutase

Bogunovic

Stojakovic

Chen

et al. 2008

Journal of Biological Chemistry

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“…When required, superoxide dismutase (SOD) (Cu-Zn-SOD from bovine erythrocytes; Sigma) was added to 10 units ml Ϫ1 . H 2 O 2 was quantified by using the horseradish peroxidase (2 units ml Ϫ1 ; MP Biomedicals, Aurora, OH) dependent oxidation of Amplex Red (10 M; Invitrogen) to resorufin ( 557-620 ϭ 51.6 Ϯ 2.5 mM Ϫ1 cm Ϫ1 at pH 7.5; Sigma) (16,48). Note that [NADH] Ͼ30 M interferes with the Amplex Red assay.…”

Section: Methodsmentioning

confidence: 99%

The mechanism of superoxide production by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria

Kußmaul

Hirst

2006

Proc. Natl. Acad. Sci. U.S.A.

644

561

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NADH:ubiquinone oxidoreductase (complex I) is a major source of reactive oxygen species in mitochondria and a significant contributor to cellular oxidative stress. Here, we describe the kinetic and molecular mechanism of superoxide production by complex I isolated from bovine heart mitochondria and confirm that it produces predominantly superoxide, not hydrogen peroxide. Redox titrations and electron paramagnetic resonance spectroscopy exclude the iron-sulfur clusters and flavin radical as the source of superoxide, and, in the absence of a proton motive force, superoxide formation is not enhanced during turnover. Therefore, superoxide is formed by the transfer of one electron from fully reduced flavin to O2. The resulting flavin radical is unstable, so the remaining electron is probably redistributed to the iron-sulfur centers. The rate of superoxide production is determined by a bimolecular reaction between O2 and reduced flavin in an empty active site. The proportion of the flavin that is thus competent for reaction is set by a preequilibrium, determined by the dissociation constants of NADH and NAD ؉ , and the reduction potentials of the flavin and NAD ؉ . Consequently, the ratio and concentrations of NADH and NAD ؉ determine the rate of superoxide formation. This result clearly links our mechanism for the isolated enzyme to studies on intact mitochondria, in which superoxide production is enhanced when the NAD ؉ pool is reduced. Therefore, our mechanism forms a foundation for formulating causative connections between complex I defects and pathological effects.flavin ͉ iron-sulfur cluster ͉ semiquinone ͉ oxidative stress T he production of reactive oxygen species, such as superoxide, by mitochondria is a major cause of cellular oxidative stress. It contributes to many pathological conditions such as Parkinson's and other neurodegenerative diseases, ischemia reperfusion injury, atherosclerosis, and aging (1-3). In mammalian mitochondria, most of the superoxide originates from NADH:ubiquinone oxidoreductase (complex I) and ubiquinol: cytochrome c oxidoreductase (complex III) of the electron transport chain, but there is increasing evidence that superoxide production by complex I, into the mitochondrial matrix, is predominant (4, 5). Indeed, complex I deficiencies have been identified across a wide spectrum of pathologies and linked to enhanced superoxide production as well as to deficiencies in energy production (6-10). Therefore, it is imperative to define how, why, and when superoxide is produced by complex I to formulate causative connections with pathological effects and rational proposals for how defects may be addressed.Many previous studies have addressed the question of how superoxide is produced by complex I (11-18). Most of these studies examined intact mitochondria or submitochondrial particles, in which it is difficult to correlate observations directly to complex I, or to define and control the conditions precisely (NADH, NAD ϩ , ubiquinone, and ubiquinol concentrations, redox status, proton motive...

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“…Production of superoxide by TtPRODH was studied by measuring reduction of cytochrome c as described previously (36). Reduction of cytochrome c was indicated by an increase in absorbance at ϭ 550 nm.…”

Section: Methodsmentioning

confidence: 99%

Structure and Kinetics of Monofunctional Proline Dehydrogenase from Thermus thermophilus

White¹,

Krishnan

Becker

et al. 2007

Journal of Biological Chemistry

145

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Proline dehydrogenase (PRODH) and ⌬ 1 -pyrroline-5-carboxylate dehydrogenase (P5CDH) catalyze the two-step oxidation of proline to glutamate. They are distinct monofunctional enzymes in all eukaryotes and some bacteria but are fused into bifunctional enzymes known as proline utilization A (PutA) in other bacteria. Here we report the first structure and biochemical data for a monofunctional PRODH. The 2.0-Å resolution structure of Thermus thermophilus PRODH reveals a distorted (␤␣) 8 barrel catalytic core domain and a hydrophobic ␣-helical domain located above the carboxylterminal ends of the strands of the barrel. Although the catalytic core is similar to that of the PutA PRODH domain, the FAD conformation of T. thermophilus PRODH is remarkably different and likely reflects unique requirements for membrane association and communication with P5CDH. Also, the FAD of T. thermophilus PRODH is highly solvent-exposed compared with PutA due to a 4-Å shift of helix 8. Structurebased sequence analysis of the PutA/PRODH family led us to identify nine conserved motifs involved in cofactor and substrate recognition. Biochemical studies show that the midpoint potential of the FAD is ؊75 mV and the kinetic parameters for proline are K m ‫؍‬ 27 mM and k cat ‫؍‬ 13 s ؊1 .3,4-Dehydro-L-proline was found to be an efficient substrate, and L-tetrahydro-2-furoic acid is a competitive inhibitor (K I ‫؍‬ 1.0 mM). Finally, we demonstrate that T. thermophilus PRODH reacts with O 2 producing superoxide. This is significant because superoxide production underlies the role of human PRODH in p53-mediated apoptosis, implying commonalities between eukaryotic and bacterial monofunctional PRODHs.Oxidation of amino acids is a central part of energy metabolism. The oxidative pathway for proline consists of two enzymatic steps and an intervening nonenzymatic equilibrium (Scheme 1) (1, 2). The first enzymatic step transforms proline to ⌬ 1 -pyrroline-5-carboxylate (P5C), 2 which is non-enzymatically hydrolyzed to glutamic semialdehyde. The semialdehyde is oxidized in the second enzymatic step to glutamate.This 4-electron transformation of proline is common to all organisms, but the enzymes of proline catabolism differ widely among the three kingdoms of life. Amino acid sequence analysis shows that bacteria and eukaryotes share a common set of proline catabolic enzymes called proline dehydrogenase (PRODH) and P5C dehydrogenase (P5CDH). Studies of the bacterial enzymes have shown that PRODH is an FAD-dependent enzyme with a (␤␣) 8 barrel catalytic core (3, 4), and P5CDH is an NAD ϩ -dependent Rossmann fold enzyme featuring a nucleophilic Cys (5). These enzymes are unrelated in sequence and structure to hyperthermophilic archaeal proline catabolic enzymes, which appear in unique hetero-tetrameric and hetero-octameric complexes (6).An intriguing aspect of proline catabolism in eukaryotes and bacteria is that PRODH and P5CDH are separate enzymes in some organisms, whereas the two enzymes are fused in other organisms. The traditional view has been that P...

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Methods of Detection of Vascular Reactive Species

Cited by 490 publications

References 168 publications

An Unexpected Functional Link between Lysosomal Thiol Reductase and Mitochondrial Manganese Superoxide Dismutase

An Unexpected Functional Link between Lysosomal Thiol Reductase and Mitochondrial Manganese Superoxide Dismutase

The mechanism of superoxide production by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria

Structure and Kinetics of Monofunctional Proline Dehydrogenase from Thermus thermophilus

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