Abstract
MUL1 is a multifunctional E3 ubiquitin ligase anchored in the outer mitochondrial membrane with its RING finger domain facing the cytoplasm. MUL1 participates in various biological pathways involved in apoptosis, mitochondrial dynamics, and innate immune response. The unique topology of MUL1 enables it to “sense” mitochondrial stress in the intermembrane mitochondrial space and convey these signals through the ubiquitination of specific cytoplasmic substrates. We have identified UBXN7, the cofactor protein of the CRL2VHL ligase complex, as a specific substrate of MUL1 ligase. CRL2VHL ligase complex regulates HIF-1α protein levels under aerobic (normoxia) or anaerobic (hypoxia) conditions. Inactivation of MUL1 ligase leads to accumulation of UBXN7, with concomitant increase in HIF-1α protein levels, reduction in oxidative phosphorylation, and increased glycolysis. We describe a novel pathway that originates in the mitochondria and operates upstream of the CRL2VHL ligase complex. Furthermore, we delineate the mechanism by which the mitochondria, through MUL1 ligase, can inhibit the CRL2VHL complex leading to high HIF-1α protein levels and a metabolic shift to glycolysis under normoxic conditions.
Highlights
Eukaryotic cells are dependent on oxygen levels as well as the presence of functional mitochondria in order to efficiently generate ATP through oxidative phosphorylation (OXPHOS)
We investigated if K48-linked polyubiquitination of UBXN7 is involved, which would target the protein for proteasomal degradation, we transfected HEK293 cells with a vector that expresses the full length MUL1 or two different mutants of MUL1 where an amino acid substitution abolishes its ligase activity[19]
In HEK293 MUL1(+/+) cells the UBXN7 and Hypoxia-inducible factor 1α (HIF-1α) levels increase with hypoxia as previously described, and there seems to be a biphasic regulation of HIF-1α after 9 or 24 hours of incubation (Fig. 5C), which is not seen with UBXN7 (Fig. 5B). These results suggest that the HEK293 MUL1(−/−) cells have lost the regulation of HIF-1α during hypoxia, and they express the highest possible level of HIF-1α protein both during normoxia and hypoxia
Summary
Eukaryotic cells are dependent on oxygen levels as well as the presence of functional mitochondria in order to efficiently generate ATP through oxidative phosphorylation (OXPHOS). Accumulation of HIF-1α protein and its translocation to the cell nucleus leads to the transcriptional activation of several hundred genes that carry a hypoxia response element (HRE) in their promoters[5,6] This leads to HIF-1α-dependent reprogramming of cellular metabolism that shifts the ATP production from oxidative phosphorylation, that is limited under low oxygen levels, to glycolysis[7,8]. We have uncovered a new pathway that regulates HIF-1α protein levels and involves the mitochondrial MUL1 E3 ubiquitin ligase. The accumulation of HIF-1α is functional and is accompanied by activation of GLUT1, a known target of HIF-1α38 This deregulation of HIF-1α affects the metabolic state of cells with glycolysis becoming the predominant energy production pathway even during aerobic conditions. We describe a new mitochondrial-initiated pathway that interferes with the process of HIF-1α regulation and reprograms cellular metabolism to induce aerobic glycolysis
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