Dihydro-orotate dehydrogenase is physically associated with the respiratory complex and its loss leads to mitochondrial dysfunction

Abstract

Some mutations of the DHODH (dihydro-orotate dehydrogenase) gene lead to postaxial acrofacial dysostosis or Miller syndrome. Only DHODH is localized at mitochondria among enzymes of the de novo pyrimidine biosynthesis pathway. Since the pyrimidine biosynthesis pathway is coupled to the mitochondrial RC (respiratory chain) via DHODH, impairment of DHODH should affect the RC function. To investigate this, we used siRNA (small interfering RNA)-mediated knockdown and observed that DHODH knockdown induced cell growth retardation because of G2/M cell-cycle arrest, whereas pyrimidine deficiency usually causes G1/S arrest. Inconsistent with this, the cell retardation was not rescued by exogenous uridine, which should bypass the DHODH reaction for pyrimidine synthesis. DHODH depletion partially inhibited the RC complex III, decreased the mitochondrial membrane potential, and increased the generation of ROS (reactive oxygen species). We observed that DHODH physically interacts with respiratory complexes II and III by IP (immunoprecipitation) and BN (blue native)/SDS/PAGE analysis. Considering that pyrimidine deficiency alone does not induce craniofacial dysmorphism, the DHODH mutations may contribute to the Miller syndrome in part through somehow altered mitochondrial function.

De NovoPyrimidine Biosynthesis Connects Cell Integrity to Amphotericin B Susceptibility in Cryptococcus neoformans

“…DHODH is a mitochondrial enzyme, and depletion of DHODH in mammalian cells induces mitochondrial dysfunction (30, 31). To determine if the ura1 Δ mutant exhibited mitochondrial defects, we assayed sensitivity to compounds that perturb mitochondrial function, including the respiratory chain inhibitor antimycin A and oxidative stress inducer H 2 O 2 .…”

Functional and regulatory profiling of energy metabolism in fission yeast

“…For example, the mutants only identified in the auxotroph background require the presence of auxotroph mutants for the respiratory phenotype to manifest, pointing to negative genetic interactions with the auxotroph markers. The ura4 deletion mutant, defective in uracil synthesis, is likely the main cause of this effect for the following reasons: 1) this deletion results in decreased growth on glycerol [24]; 2) the pyrimidine synthesis pathway is linked to reduction of coenzyme Q which may directly impact the ETC and antioxidant defence [49, 60]; and 3) this deletion affects cell wall integrity [61], which could indirectly compromise respiratory metabolism. Such genetic interactions may complicate functional analyses of the corresponding respiratory genes.…”

Pyrimidine Pathway-Dependent and -Independent Functions of the Toxoplasma gondii Mitochondrial Dihydroorotate Dehydrogenase

“…Recent studies on mammalian DHODH have shown that DHODH is physically associated with the respiratory chain complexes III and II (42). Moreover, depletion of DHODH induced cell cycle arrest and mitochondrial dysfunction (42)(43)(44). Here, we show that wild-type and mutant TgDHODH enzymes localized to the parasite mitochondria and that T. gondii expressing catalytically deficient DHODH retained an intact mitochondrial membrane potential even with reduced expression levels of catalytically deficient TgDHODH enzymes.…”

“…T. gondii and blood stages of malaria parasites both require mitochondrial metabolism, because these parasites are highly susceptible to inhibitors of cytochrome bc 1 (complex III), such as atovaquone (36)(37)(38), that inhibit electron transport through the essential respiratory chain (39)(40)(41). Recent studies on mammalian DHODH have shown that DHODH is physically associated with the respiratory chain complexes III and II (42). Moreover, depletion of DHODH induced cell cycle arrest and mitochondrial dysfunction (42)(43)(44).…”