RESUMO
DJ-1, a causative gene for hereditary recessive Parkinsonism, is evolutionarily conserved across eukaryotes and prokaryotes. Structural analyses of DJ-1 and its homologs suggested the 106th Cys is a nucleophilic cysteine functioning as the catalytic center of hydratase or hydrolase activity. Indeed, DJ-1 and its homologs can convert highly electrophilic α-oxoaldehydes such as methylglyoxal into α-hydroxy acids as hydratase in vitro, and oxidation-dependent ester hydrolase (esterase) activity has also been reported for DJ-1. The mechanism underlying such plural activities, however, has not been fully characterized. To address this knowledge gap, we conducted a series of biochemical assays assessing the enzymatic activity of DJ-1 and its homologs. We found no evidence for esterase activity in any of the Escherichia coli DJ-1 homologs. Furthermore, contrary to previous reports, we found that oxidation inactivated rather than facilitated DJ-1 esterase activity. The E. coli DJ-1 homolog HchA possesses phenylglyoxalase and methylglyoxalase activities but lacks esterase activity. Since evolutionary trace analysis identified the 186th H as a candidate residue involved in functional differentiation between HchA and DJ-1, we focused on H186 of HchA and found that an esterase activity was acquired by H186A mutation. Introduction of reverse mutations into the equivalent position in DJ-1 (A107H) selectively eliminated its esterase activity without compromising α-oxoaldehyde hydratase activity. The obtained results suggest that differences in the amino acid sequences near the active site contributed to acquisition of esterase activity in vitro and provide an important clue to the origin and significance of DJ-1 esterase activity.
Assuntos
Escherichia coli , Doença de Parkinson , Proteína Desglicase DJ-1 , Proteína Desglicase DJ-1/metabolismo , Proteína Desglicase DJ-1/genética , Proteína Desglicase DJ-1/química , Humanos , Escherichia coli/genética , Escherichia coli/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Esterases/metabolismo , Esterases/genética , Esterases/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Evolução Molecular , OxirreduçãoRESUMO
OBJECTIVE: To establish a threshold value of bovine leukemia virus (BLV) proviral load (PVL) to identify increased risk of severe clinical mastitis, and to examine the prognosis and economic loss of clinical mastitis based on the newly established PVL cut-off value. ANIMALS: 97 lactating Holstein cows with clinical mastitis. METHODS: Blood and milk samples were collected aseptically from each cow. Youden index was used for receiver-operating characteristic curve analysis with the severity rate of clinical mastitis as the dependent variable and PVL as an independent variable. PVL cut-off value was used as a criterion to compare the severity rate of clinical mastitis, percentage of cows with and without systemic treatments, number of treatments, cost of treatment, and prognosis. RESULTS: PVL cut-off value was 17.8 copies/10 ng DNA for the dependent variable MILD vs SEVERE. The severity rate of clinical mastitis, percentage of cows given systemic treatments, and technical fees for medical treatment were significantly higher in the group above the PVL cut-off value than in the group below the PVL cut-off value and the negative group. Number of treatments was significantly higher in the group above the cut-off value than in the group below the cut-off value. There was no significant difference in prognosis after mastitis among the 3 groups. CLINICAL RELEVANCE: These results suggested that PVL cut-off value of 17.8 copies/10 ng DNA was a useful threshold for increased economic losses in BLV-infected cows; it may also serve as a new standard value for the detection and culling of BLV-infected cows in Japan.