title: Protein instability and functional defects caused by mutations of dihydro-orotate dehydrogenase in Miller syndrome patients.
abstract: Miller syndrome is a recessive inherited disorder characterized by postaxial acrofacial dysostosis. It is caused by dysfunction of the DHODH (dihydroorotate dehydrogenase) gene, which encodes a key enzyme in the pyrimidine de novo biosynthesis pathway and is localized at mitochondria intermembrane space. We investigated the consequence of three missense mutations, G202A, R346W and R135C of DHODH, which were previously identified in patients with Miller syndrome. First, we established HeLa cell lines stably expressing DHODH with Miller syndrome-causative mutations: G202A, R346W and R135C. These three mutant proteins retained the proper mitochondrial localization based on immunohistochemistry and mitochondrial subfractionation studies. The G202A, R346W DHODH proteins showed reduced protein stability. On the other hand, the third one R135C, in which the mutation lies at the ubiquinone-binding site, was stable but possessed no enzymatic activity. In conclusion, the G202A and R346W mutation causes deficient protein stability, and the R135C mutation does not affect stability but impairs the substrate-induced enzymatic activity, suggesting that impairment of DHODH activity is linked to the Miller syndrome phenotype.
authors.affiliation: Department of Clinical Chemistry and Laboratory Medicine, Graduate School of Medical Sciences, Kyushu University, Maidashi, Higashi-ku, Fukuoka, Japan.
authors.forename: JingXian
authors.initials: J
authors.lastname: Fang
authors.forename: Takeshi
authors.initials: T
authors.lastname: Uchiumi
authors.forename: Mikako
authors.initials: M
authors.lastname: Yagi
authors.forename: Shinya
authors.initials: S
authors.lastname: Matsumoto
authors.forename: Rie
authors.initials: R
authors.lastname: Amamoto
authors.forename: Toshiro
authors.initials: T
authors.lastname: Saito
authors.forename: Shinya
authors.initials: S
authors.lastname: Takazaki
authors.forename: Tomotake
authors.initials: T
authors.lastname: Kanki
authors.forename: Haruyoshi
authors.initials: H
authors.lastname: Yamaza
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Nonaka
authors.forename: Dongchon
authors.initials: D
authors.lastname: Kang
mesh_terms: D000015:Abnormalities, Multiple / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D000090722:Dihydroorotate Dehydrogenase; D014450:Electron Transport Complex III / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D017880:Limb Deformities, Congenital / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D008342:Mandibulofacial Dysostosis / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D008844:Micrognathism / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism; D020125:Mutation, Missense; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D055550:Protein Stability; D013385:Succinate Dehydrogenase / Q000378:metabolism; D014451:Ubiquinone / Q000378:metabolism
chemical_list: D000090722:Dihydroorotate Dehydrogenase; D014451:Ubiquinone; D044925:Oxidoreductases Acting on CH-CH Group Donors; D013385:Succinate Dehydrogenase; D014450:Electron Transport Complex III