Suppression of reactive oxygen species generation in heart mitochondria from anoxic turtles: the role of complex I S-nitrosation.

Freshwater turtles (Trachemys scripta) are among the very few vertebrates capable of tolerating severe hypoxia and re-oxygenation without suffering from damage to the heart. As myocardial ischemia and reperfusion causes a burst of mitochondrial reactive oxygen species (ROS) in mammals, the question arises as to whether, and if so how, this ROS burst is prevented in the turtle heart. We find that heart mitochondria isolated from turtles acclimated to anoxia produce less ROS than mitochondria from normoxic turtles when consuming succinate. As succinate accumulates in the hypoxic heart and is oxidized when oxygen returns, this suggests an adaptation to lessen ROS production. Specific S-nitrosation of complex I can lower ROS in mammals and here we show that turtle complex I activity and ROS production can also be strongly depressed in vitro by S-nitrosation. We detect in vivo endogenous S-nitrosated complex I in turtle heart mitochondria, but these levels are unaffected upon anoxia acclimation. Thus, while heart mitochondria from anoxia-acclimated turtles generate less ROS and have a lower aerobic capacity than those from normoxic turtles, this is not due to decreases in complex I activity or expression levels. Interestingly, in-gel activity staining reveals that most complex I of heart mitochondria from normoxic and anoxic turtles forms stable super-complexes with other respiratory enzymes and, in contrast to mammals, these are not disrupted by dodecyl maltoside. Taken together, these results show that although S-nitrosation of complex I is a potent mechanism to prevent ROS formation upon re-oxygenation after anoxia in vitro, this is not a major cause of the suppression of ROS production by anoxic turtle heart mitochondria

Metabolic adaptations during extreme anoxia in the turtle heart and their implications for ischemia-reperfusion injury.

ATP depletion and succinate accumulation during ischemia lead to oxidative damage to mammalian organs upon reperfusion. In contrast, freshwater turtles survive weeks of anoxia at low temperatures without suffering from oxidative damage upon reoxygenation, but the mechanisms are unclear. To determine how turtles survive prolonged anoxia, we measured ~80 metabolites in hearts from cold-acclimated (5 °C) turtles exposed to 9 days anoxia and compared the results with those for normoxic turtles (25 °C) and mouse hearts exposed to 30 min of ischemia. In turtles, ATP and ADP decreased to new steady-state levels during fasting and cold-acclimation and further with anoxia, but disappeared within 30 min of ischemia in mouse hearts. High NADH/NAD+ ratios were associated with succinate accumulation in both anoxic turtles and ischemic mouse hearts. However, succinate concentrations and succinate/fumarate ratios were lower in turtle than in mouse heart, limiting the driving force for production of reactive oxygen species (ROS) upon reoxygenation in turtles. Furthermore, we show production of ROS from succinate is prevented by re-synthesis of ATP from ADP. Thus, maintenance of an ATP/ADP pool and low succinate accumulation likely protects turtle hearts from anoxia/reoxygenation injury and suggests metabolic interventions as a therapeutic approach to limit ischemia/reperfusion injury in mammals

Let-7 microRNA controls invasion-promoting lysosomal changes via the oncogenic transcription factor myeloid zinc finger-1

Cancer cells utilize lysosomes for invasion and metastasis. Myeloid Zinc Finger1 (MZF1) is an ErbB2-responsive transcription factor that promotes invasion of breast cancer cells via upregulation of lysosomal cathepsins B and L. Here we identify let-7 microRNA, a well-known tumor suppressor in breast cancer, as a direct negative regulator of MZF1. Analysis of primary breast cancer tissues reveals a gradual upregulation of MZF1 from normal breast epithelium to invasive ductal carcinoma and a negative correlation between several let-7 family members and MZF1 mRNA, suggesting that the inverse regulatory relationship between let-7 and MZF1 may play a role in the development of invasive breast cancer. Furthermore, we show that MZF1 regulates lysosome trafficking in ErbB2-positive breast cancer cells. In line with this, MZF1 depletion or let-7 expression inhibits invasion-promoting anterograde trafficking of lysosomes and invasion of ErbB2-expressing MCF7 spheres. The results presented here link MZF1 and let-7 to lysosomal processes in ErbB2-positive breast cancer cells that in non-cancerous cells have primarily been connected to the transcription factor EB. Identifying MZF1 and let-7 as regulators of lysosome distribution in invasive breast cancer cells, uncouples cancer-associated, invasion-promoting lysosomal alterations from normal lysosomal functions and thus opens up new possibilities for the therapeutic targeting of cancer lysosomes.Peer reviewe

Stable mitochondrial CICIII2 supercomplex interactions in reptiles versus homeothermic vertebrates.

The association of complex I (CI), complex III (CIII) and complex IV (CIV) of the mitochondrial electron transport chain into stable high molecular weight supercomplexes (SCs) has been observed in several prokaryotes and eukaryotes, but among vertebrates it has only been examined in mammals. The biological role of these SCs is unclear but suggestions so far include enhanced electron transfer between complexes, decreased production of the reactive oxygen species (ROS) O2- and H2O2, or enhanced structural stability. Here, we provide the first overview on the stability, composition and activity of mitochondrial SCs in representative species of several vertebrate classes to determine patterns of SC variation across endotherms and ectotherms. We found that the stability of the CICIII2 SC and the inclusion of CIV within the SC varied considerably. Specifically, when solubilized by the detergent DDM, mitochondrial CICIII2 SCs were unstable in endotherms (birds and mammals) and highly stable in reptiles. Using mass-spectrometric complexomics, we confirmed that the CICIII2 is the major SC in the turtle, and that 90% of CI is found in this highly stable SC. Interestingly, the presence of stable SCs did not prevent mitochondrial H2O2 production and was not associated with elevated respiration rates of mitochondria isolated from the examined species. Together, these data show that SC stability varies among vertebrates and is greatest in poikilothermic reptiles and weakest in endotherms. This pattern suggests an adaptive role of SCs to varying body temperature, but not necessarily a direct effect on electron transfer or in the prevention of ROS production

Stable mitochondrial CICIII2 supercomplex interactions in reptiles compared to homeothermic vertebrates.

The association of complex I (CI), complex III (CIII) and complex IV (CIV) of the mitochondrial electron transport chain into stable high-molecular weight supercomplexes (SCs) has been observed in several prokaryotes and eukaryotes, but among vertebrates it has only been examined in mammals. The biological role of these SCs is unclear but suggestions so far include enhanced electron transfer between complexes, decreased production of the reactive oxygen species (ROS) O2·- and H2O2, or enhanced structural stability. Here, we provide the first overview on the stability, composition and activity of mitochondrial SCs in representative species of several vertebrate classes to determine patterns of SC variation across endotherms and ectotherms. We found that the stability of the CICIII2 SC and the inclusion of CIV within SC varied considerably. Specifically, when solubilized by the detergent DDM, mitochondrial CICIII2 SCs were unstable in endotherms (birds and mammals) and highly stable in reptiles. Using mass-spectrometric complexomics, we confirmed that the CICIII2 is the major SC in the turtle, and that 90% of CI is found in this highly stable SC. Interestingly, the presence of stable SCs did not prevent mitochondrial H2O2 production and was not associated with elevated respiration rates of mitochondria isolated from the examined species. Together, these data show that SC stability varies among vertebrates and is greatest in poikilothermic reptiles and weakest in endotherms. This pattern suggests an adaptive role of SCs to varying body temperature, but not necessarily a direct effect on electron transfer or in the prevention of ROS production

Intrinsic Mechanisms Underlying Hypoxia-Tolerant Mitochondrial Phenotype During Hypoxia-Reoxygenation Stress in a Marine Facultative Anaerobe, the Blue Mussel Mytilus edulis

Hypoxia is common in marine environments and a major stressor for marine organisms inhabiting benthic and intertidal zones. Several studies have explored the responses of these organisms to hypoxic stress at the whole organism level with a focus on energy metabolism and mitochondrial response, but the instrinsic mitochondrial responses that support the organelle’s function under hypoxia and reoxygenation (H/R) stress are not well understood. We studied the effects of acute H/R stress (10 min anoxia followed by 15 min reoxygenation) on mitochondrial respiration, production of reactive oxygen species (ROS) and posttranslational modifications (PTM) of the proteome in a marine facultative anaerobe, the blue mussel Mytilus edulis. The mussels’ mitochondria showed increased OXPHOS respiration and suppressed proton leak resulting in a higher coupling efficiency after H/R stress. ROS production decreased in both the resting (LEAK) and phosphorylating (OXPHOS) state indicating that M. edulis was able to prevent oxidative stress and mitochondrial damage during reoxygenation. Hypoxia did not lead to rearrangement of the mitochondrial supercomplexes but impacted the mitochondrial phosphoproteome including the proteins involved in OXPHOS, amino acid- and fatty acid catabolism, and protein quality control. This study indicates that mussels’ mitochondria possess intrinsic mechanisms (including regulation via reversible protein phosphorylation) that ensure high respiratory flux and mitigate oxidative damage during H/R stress and contribute to the hypoxia-tolerant mitochondrial phenotype of this metabolically plastic species