The Role of Dihydroorotate Dehydrogenase in Apoptosis Induction in Response to Inhibition of the Mitochondrial Respiratory Chain Complex III

Abstract

A mechanism for the induction of programmed cell death (apoptosis) upon dysfunction of the mitochondrial respiratory chain has been studied. Previously, we had found that inhibition of mitochondrial cytochrome bc1, a component of the electron transport chain complex III, leads to activation of tumor suppressor p53, followed by apoptosis induction. The mitochondrial respiratory chain is coupled to the de novo pyrimidine biosynthesis pathway via the mitochondrial enzyme dihydroorotate dehydrogenase (DHODH). The p53 activation induced in response to the inhibition of the electron transport chain complex III has been shown to be triggered by the impairment of the de novo pyrimidine biosynthesis due to the suppression of DHODH. However, it remained unclear whether the suppression of the DHODH function is the main cause of the observed apoptotic cell death. Here, we show that apoptosis in human colon carcinoma cells induced by the mitochondrial respiratory chain complex III inhibition can be prevented by supplementation with uridine or orotate (products of the reaction catalyzed by DHODH) rather than with dihydroorotate (a DHODH substrate). We conclude that apoptosis is induced in response to the impairment of the de novo pyrimidine biosynthesis caused by the inhibition of DHODH. The conclusion is supported by the experiment showing that downregulation of DHODH by RNA interference leads to accumulation of the p53 tumor suppressor and to apoptotic cell death.

A sustained deficiency of mitochondrial respiratory complex III induces an apoptotic cell death through the p53-mediated inhibition of pro-survival activities of the activating transcription factor 4

“…Treatment with myxothiazol did not induce ATF4 transcripts in these cells ( Figure 6a ). In agreement with our previous results, 8 , 12 the treatment induced apoptosis and led to approximately threefold increase in caspase-3/7 activity ( Figure 6b ). We used this value as a positive control.…”

“…We used this value as a positive control. As expected, 12 uridine completely prevented the activation of caspases-3/7 in the cells with normal ATF4 levels, but a substantial activity of caspase-3/7 (73–76% of the positive control) was observed in the cells with ATF4 knockdown ( Figure 6b ). We conclude that if p53 activation is prevented by uridine, ATF4 plays a pro-survival role and protects the cells from apoptosis induced by complex III inhibition.…”

“…Blocking the p53 activation by uridine dramatically reduced the expression of pro-apoptotic genes including AIFM3, BBC3 (PUMA), BCL2L11, Casp3, Casp7, FAS, Lrdd (PIDD), PMAIP1 (NOXA) and TNFRSF10B (KILLER/DR5), which is consistent with the prevention of myxothiazol-induced apoptosis upon uridine supplementation. 12 …”

Mild phenotypes and proper supercomplex assembly in human cells carrying the homoplasmic m.15557G > A mutation in cytochromebgene

“…These data further support the idea that in human cells the p.Glu271Lys change has only a mild impact on CIII function. In fact, mitochondrial defective cells and in particular those carrying CIII defects rely on uridine supplementation for pyrimidine biosynthesis (Grégoire, Morais, Quilliam, & Gravel, 1984;Khutornenko, Dalina, Chernyak, Chumakov, & Evstafieva, 2014;King & Attardi, 1996). Besides its direct role in Q o site catalytic mechanism, Glu271 has been suggested to be important for the prevention of electron short-circuit reactions, which would limit the efficiency of the enzyme and would produce deleterious ROS.…”

Human neural progenitor cells decrease photoreceptor degeneration, normalize opsin distribution and support synapse structure in cultured porcine retina

“…In mammals, it is located in the inner mitochondrial membrane where it is known to catalyze the oxidation of dihydroorotate to orotate in the presence of the cofactor flavin mononucleotide (FMN) by the transfer of electrons to ubiquinone (CoQ) to form ubiquinol (CoQH2). Ubiquinol is used by complex III of the electron transport chain (117,119). DHODH has two domains; a large catalytic domain (CAT) which contains the active site, and a small domain referred to as the quinone tunnel (QT) where ubiquinone binds (117).…”