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Elucidating the mechanism of ferrocytochrome c heme disruption by peroxidized cardiolipin.

ABSTRACT: The interaction of peroxidized cardiolipin with ferrocytochrome c induces two kinetically and chemically distinct processes. The first is a rapid oxidation of ferrocytochrome c, followed by a slower, irreversible disruption of heme c. The oxidation of ferrocytochrome c by peroxidized cardiolipin is explained by a Fenton-type reaction. Heme scission is a consequence of the radical-mediated reactions initiated by the interaction of ferric heme iron with peroxidized cardiolipin. Simultaneously with the heme c disruption, generation of hydroxyl radical is detected by EPR spectroscopy using the spin trapping technique. The resulting apocytochrome c sediments as a heterogeneous mixture of high aggregates, as judged by sedimentation analysis. Both the oxidative process and the destructive process were suppressed by nonionic detergents and/or high ionic strength. The mechanism for generating radicals and heme rupture is presented.

SUBMITTER: Musatov A

PROVIDER: S-EPMC3536883 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Elucidating the mechanism of ferrocytochrome c heme disruption by peroxidized cardiolipin.

Musatov Andrej A Fabian Marian M Varhač Rastislav R

Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry 20121118 1

The interaction of peroxidized cardiolipin with ferrocytochrome c induces two kinetically and chemically distinct processes. The first is a rapid oxidation of ferrocytochrome c, followed by a slower, irreversible disruption of heme c. The oxidation of ferrocytochrome c by peroxidized cardiolipin is explained by a Fenton-type reaction. Heme scission is a consequence of the radical-mediated reactions initiated by the interaction of ferric heme iron with peroxidized cardiolipin. Simultaneously with ...[more]

PMID: 23160757

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Project description:BackgroundBoth TGFβ and estradiol (E2), a form of estrogen, are pro-fibrotic in the skin. In the connective tissue disease, systemic sclerosis (SSc), both TGFβ and E2 are likely pathogenic. Yet the regulation of TGFβ in E2-induced dermal fibrosis remains ill-defined. Elucidating those regulatory mechanisms will improve the understanding of fibrotic disease pathogenesis and set the stage for developing potential therapeutics. Using E2-stimulated primary human dermal fibroblasts in vitro and human skin tissue ex vivo, we identified the important regulatory proteins for TGFβ and investigated the extracellular matrix (ECM) components that are directly stimulated by E2-induced TGFβ signaling.MethodsWe used primary human dermal fibroblasts in vitro and human skin tissue ex vivo stimulated with E2 or vehicle (ethanol) to measure TGFβ1 and TGFβ2 levels using quantitative PCR (qPCR). To identify the necessary cell signaling proteins in E2-induced TGFβ1 and TGFβ2 transcription, human dermal fibroblasts were pre-treated with an inhibitor of the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway, U0126. Finally, human skin tissue ex vivo was pre-treated with SB-431542, a TGFβ receptor inhibitor, and ICI 182,780, an estrogen receptor α (ERα) inhibitor, to establish the effects of TGFβ and ERα signaling on E2-induced collagen 22A1 (Col22A1) transcription.ResultsWe found that expression of TGFβ1, TGFβ2, and Col22A1, a TGFβ-responsive gene, is induced in response to E2 stimulation. Mechanistically, Col22A1 induction was blocked by SB-431542 and ICI 182,780 despite E2 stimulation. Additionally, inhibiting E2-induced ERK/MAPK activation and early growth response 1 (EGR1) transcription prevents the E2-induced increase in TGFβ1 and TGFβ2 transcription and translation.ConclusionsWe conclude that E2-induced dermal fibrosis occurs in part through induction of TGFβ1, 2, and Col22A1, which is regulated through EGR1 and the MAPK pathway. Thus, blocking estrogen signaling and/or production may be a novel therapeutic option in pro-fibrotic diseases.

Project description:BackgroundWhile transmission ratio distortion, TRD, (a deviation from Mendelian ratio) is extensive in humans and well-documented in mice, the underlying mechanisms are unknown. Our earlier studies on carriers of spontaneous mutations of mouse Sperm Adhesion Molecule 1 (Spam1) suggested that TRD results from biochemically different sperm, due to a lack of transcript sharing through the intercellular cytoplasmic bridges of spermatids. These bridges usually allow transcript sharing among genetically different spermatids which develop into biochemically and functionally equivalent sperm.ObjectivesThe goals of the study were to provide support for the lack of sharing (LOS) hypothesis, using transgene and null carriers of Spam1, and to determine the mechanism of Spam1-associated TRD.MethodsCarriers of Spam1-Hyal5 BAC transgenes were mated with wild-type female mice and the progeny analyzed for TRD by PCR genotyping. Sperm from transgene and Spam1 null carriers were analyzed using flow cytometry and immunocytochemistry to detect quantities of Spam1 and/or Hyal5. Transgene-bearing sperm with Spam1 overexpression were detected by fluorescence in situ hybridization. In wild-type animals, EM studies of in situ transcript hybridization of testis sections and Northern analysis of biochemically fractionated testicular RNA were performed to localize Spam1 transcript. Finally, AU-rich motifs identified in the 3' UTR of Spam1 RNA were assayed by UV cross-linking to determine their ability to interact with testicular RNA binding proteins.ResultsThe Tg8 line of transgene carriers had a significant (P < 0.001) TRD, due to reduced fertilizing ability of transgene-bearing sperm. These sperm retained large cytoplasmic droplets engorged with overexpressed Spam1 or Hyal5 protein. Caudal sperm from transgene carriers and caput sperm of null carriers showed a bimodal distribution of Spam1, indicating that the sperm in a male were biochemically different with respect to Spam1 quantities. Spam1 RNA was absent from the bridges, associated exclusively with the ER, and was shown to be anchored to the cytoskeleton. This compartmentalization of the transcript, mediated by cytoskeletal binding, occurs via protein interactions with 3' UTR AU-rich sequences that are likely involved in its stabilization.ConclusionWe provide strong support for the LOS hypothesis, and have elucidated the mechanism of Spam1-associated TRD.

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Elucidating the mechanism of ferrocytochrome c heme disruption by peroxidized cardiolipin.

Publications

Elucidating the mechanism of ferrocytochrome c heme disruption by peroxidized cardiolipin.

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