Dataset Information

Effects of hypoxia-reoxygenation stress on mitochondrial proteome and bioenergetics of the hypoxia-tolerant marine bivalve Crassostrea gigas

ABSTRACT: Intermittent hypoxia is a common stressor in estuarine and coastal habitats due to the diurnal and tidal cycles of oxygen availability. Oxygen deficiency results in energy stress by limiting aerobic ATP production, while reoxygenation may lead to oxidative injury due to the excessive production of reactive oxygen species (ROS) in mitochondria. Mitochondria are a key intracellular target of hypoxia-reoxygenation (H/R) stress due to their central role in ATP production, ROS generation and stress signaling. Marine intertidal bivalves such as the Pacific oyster Crassostrea gigas are adapted to frequent H/R cycles in the intertidal zone and maintain mitochondrial integrity and aerobic function despite frequent oxygen fluctuations. To gain insight into the molecular responses of mitochondria of the hypoxia-tolerant Pacific oyster to H/R stress, we studied changes in oxidative phosphorylation (OXPHOS) capacity, proton leak and activity of the electron transport system enzymes (ETS) in gill mitochondria of C. gigas. We furthermore investigated shifts in mitochondrial proteome and phosphoproteome after 24 h of hypoxia and 1 h of post-hypoxic recovery. Oyster mitochondria maintained normal ETS and OXPHOS capacity during H/R stress, despite a slight but significant decline in cytochrome c oxidase (Complex IV) activity after reoxygenation. Fifty total proteins and 36 phosphoproteins were differentially abundant in mitochondria of H/R exposed oysters compared with the controls. Rearrangements of the mitochondrial (phospho)proteome during H/R stress involved upregulation of mitochondrial ETS (most notably Complexes I and IV) and iron-binding proteins (frataxin and ferrochelatase) as well as suppression of the metabolic pathways that channel electrons to ubiquinone, possibly as a mechanism to limit ROS production due to the iron overload and reverse flow of electrons through ETS. H/R stress also led to upregulation of a mitophagic activator serine/threonine protein phosphatase PGAM5 and dephosphorylation of metalloendopeptidase OMA1 indicating stimulation of mitochondrial quality control mechanisms during reoxygenation. Changes in the overall abundance and phosphorylation levels of several key proteins involved in the mitochondrial protein homeostasis (including subunits of the mitochondrial ribosome, mitochondrial inner membrane translocase and elongation factor G) are consistent with the suppression of the protein synthesis during hypoxia, likely as an energy-saving mechanism. Overall, our study indicates that shifts in the mitochondrial (phospho-)proteome play an important role in responses of oysters to H/R stress and might complement adaptations of anaerobic metabolism and metabolic rate depression in ensuring hypoxic survival and rapid recovery in this hypoxia-tolerant intertidal species.

INSTRUMENT(S): LTQ Orbitrap Classic, LTQ Orbitrap Velos

ORGANISM(S): Crassostrea Gigas (pacific Oyster) (crassostrea Angulata)

SUBMITTER: Stephanie Markert

LAB HEAD: Stephanie Markert

PROVIDER: PXD011365 | Pride | 2021-11-04

REPOSITORIES: Pride

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Publications

Effects of hypoxia-reoxygenation stress on mitochondrial proteome and bioenergetics of the hypoxia-tolerant marine bivalve Crassostrea gigas.

Sokolov Eugene P EP Markert Stephanie S Hinzke Tjorven T Hirschfeld Claudia C Becher Dörte D Ponsuksili Siriluck S Sokolova Inna M IM

Journal of proteomics 20181212

Mitochondria are key intracellular targets of hypoxia-reoxygenation (H/R) stress due to their central role in generation of ATP and reactive oxygen species (ROS). Intertidal oysters Crassostrea gigas are adapted to frequent H/R cycles and maintain aerobic function despite frequent oxygen fluctuations. To gain insight into the molecular mechanisms of H/R tolerance, we assessed changes in mitochondrial respiration and (phospho)proteome of C. gigas during hypoxia and recovery. Oyster mitochondria m ...[more]

PMID: 30550986

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Project description:MicroRNAs (miRNAs) are small non-coding RNAs that associate with Argonaute 2 protein to regulate gene expression at the post-transcriptional level in the cytoplasm. However, recent studies have reported that some miRNAs localize to and function in other cellular compartments. Mitochondria harbour their own genetic system that may be a potential site for miRNA-mediated post-transcriptional regulation. We aimed at investigating whether nuclear-encoded miRNAs can localize to and function in human mitochondria. To enable identification of mitochondrial-enriched miRNAs, we profiled the mitochondrial and cytosolic RNA fractions from the same HeLa cells by miRNA microarray analysis. Mitochondria were purified using a combination of cell fractionation and immunoisolation, and assessed for the lack of protein and RNA contaminants. We found 57 miRNAs differentially expressed in HeLa mitochondria and cytosol. Of these 57, a signature of 13 nuclear-encoded miRNAs was reproducibly enriched in mitochondrial RNA and validated by RT-PCR for hsa-miR-494, hsa-miR-1275 and hsa-miR-1974. This study provides the first comprehensive view of the localization of RNA interference components to the mitochondria. Our data outline the molecular bases for a novel layer of crosstalk between nucleus and mitochondria through a specific subset of human miRNAs that we termed ‘mitomiRs’. To assess whether nuclear-encoded miRNA are detectable in human mitochondria, we performed the following four steps approach. First, cultured HeLa cells were allowed to reach 80-100% confluence and subjected to fractionation in order to isolate the cytosolic fraction. From the same HeLa cells, mitochondria were isolated by immunomagnetic Anti-TOM22 MicroBeads from the Mitochondria Isolation Kit (Miltenyi Biotec). In total, six mitochondria preparations were perfromed, three of these were additionally treated with RNase A. Second, total RNA was extracted from the mitochondrial and cytosolic fractions. Third, mitochondrial and cytosolic RNA were respectively profiled by microRNA microarray analysis. Last, data were analyzed and normalized.Three independent assays were performed.

Dataset Information

Effects of hypoxia-reoxygenation stress on mitochondrial proteome and bioenergetics of the hypoxia-tolerant marine bivalve Crassostrea gigas

Publications

Effects of hypoxia-reoxygenation stress on mitochondrial proteome and bioenergetics of the hypoxia-tolerant marine bivalve Crassostrea gigas.

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