Dihydro-orotate dehydrogenase is physically associated with the respiratory complex and its loss leads to mitochondrial dysfunction
| Autor: | Jing Xian Fang, Haruyoshi Yamaza, Kazuaki Nonaka, Rie Amamoto, Takeshi Uchiumi, Mikako Yagi, Shinya Matsumoto, Dongchon Kang, Shinya Takazaki |
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| Rok vydání: | 2013 |
| Předmět: |
CCCP
carbonyl cyanide m-chlorophenylhydrazone Small interfering RNA Ubiquinone OXPHOS oxidative phosphorylation lcsh:Life lcsh:QR1-502 Dihydroorotate Dehydrogenase Respiratory chain DCPIP 2 6-dichlorophenol-indophenol Mitochondrion UMP uridine monophosphate Biochemistry lcsh:Microbiology Oxidative Phosphorylation DMEM Dulbecco’s modified Eagle’s medium chemistry.chemical_compound 2D two-dimensional DHODH dihydro-orotate dehydrogenase pyrimidine pathway RNA Small Interfering Miller syndrome Membrane Potential Mitochondrial HA haemagglutinin Gene knockdown Electron Transport Complex II TFAM mitochondrial transcription factor A IP immunoprecipitation Mitochondria SDHA succinate dehydrogenase complex subunit A COX cytochrome c oxidase UMPS UMP synthase Pyrimidine metabolism Oxidoreductases Acting on CH-CH Group Donors DCF 2′7′-dichlorofluorescein Micrognathism DOX doxycycline Limb Deformities Congenital IgG immunoglobulin G Biophysics Oxidative phosphorylation Biology S4 ROS reactive oxygen species FBS fetal bovine serum LFN leflunomide mitochondrial dysfunction medicine Humans Abnormalities Multiple Molecular Biology Original Paper RC respiratory chain dihydro-orotate dehydrogenase (DHODH) DCFH-DA 2′7′-dichlorodihydrofluorescein diacetate Cell Biology medicine.disease Molecular biology Uridine mtDNA mitochondrial DNA lcsh:QH501-531 Pyrimidines BN Blue native chemistry siRNA small interfering RNA Mutation Reactive Oxygen Species DHO dihydro-orotate Mandibulofacial Dysostosis HeLa Cells |
| Zdroj: | Bioscience Reports Bioscience Reports, Vol 33, Iss 2, p e00021 (2013) |
| ISSN: | 1573-4935 0144-8463 |
| DOI: | 10.1042/bsr20120097 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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