Genomic and experimental evidence for multiple metabolic functions in the RidA/YjgF/YER057c/UK114 (Rid) protein family

Background

It is now recognized that enzymatic or chemical side-reactions can convert normal metabolites to useless or toxic ones and that a suite of enzymes exists to mitigate such metabolite damage. Examples are the reactive imine/enamine intermediates produced by threonine dehydratase, which damage the pyridoxal 5''-phosphate cofactor of various enzymes causing inactivation. This damage is pre-empted by RidA proteins, which hydrolyze the imines before they do harm. RidA proteins belong to the YjgF/YER057c/UK114 family (here renamed the Rid family). Most other members of this diverse and ubiquitous family lack defined functions.

Results

Phylogenetic analysis divided the Rid family into a widely distributed, apparently archetypal RidA subfamily and seven other subfamilies (Rid1 to Rid7) that are largely confined to bacteria and often co-occur in the same organism with RidA and each other. The Rid1 to Rid3 subfamilies, but not the Rid4 to Rid7 subfamilies, have a conserved arginine residue that, in RidA proteins, is essential for imine-hydrolyzing activity. Analysis of the chromosomal context of bacterial RidA genes revealed clustering with genes for threonine dehydratase and other pyridoxal 5''-phosphate-dependent enzymes, which fits with the known RidA imine hydrolase activity. Clustering was also evident between Rid family genes and genes specifying FAD-dependent amine oxidases or enzymes of carbamoyl phosphate metabolism. Biochemical assays showed that Salmonella enterica RidA and Rid2, but not Rid7, can hydrolyze imines generated by amino acid oxidase. Genetic tests indicated that carbamoyl phosphate overproduction is toxic to S. enterica cells lacking RidA, and metabolomic profiling of Rid knockout strains showed ten-fold accumulation of the carbamoyl phosphate-related metabolite dihydroorotate.

Conclusions

Like the archetypal RidA subfamily, the Rid2, and probably the Rid1 and Rid3 subfamilies, have imine-hydrolyzing activity and can pre-empt damage from imines formed by amine oxidases as well as by pyridoxal 5''-phosphate enzymes. The RidA subfamily has an additional damage pre-emption role in carbamoyl phosphate metabolism that has yet to be biochemically defined. Finally, the Rid4 to Rid7 subfamilies appear not to hydrolyze imines and thus remain mysterious.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1584-3) contains supplementary material, which is available to authorized users.     

酵母双杂交系统筛选与RapGAP相互作用的蛋白质

目的筛选与Rap GAP相互作用的蛋白质,为进一步研究人源Rap1GAP介导的信号转导通路、揭示其与肿瘤的关系提供实验依据。方法选用与Rap1GAP同源的来自美丽线虫的Rap GAP作为饵蛋白,以来源于美丽线虫的c DNA文库作为靶蛋白,应用p PC97、p PC86组成的酵母双杂交系统筛选c DNA文库中与Rap GAP相互作用的蛋白质。结果通过营养缺陷平板(-LTH)筛选出63个拟似阳性菌落。经过Lac Z鉴定,19个菌落为阳性,其中7个为强阳性。提取来自19个酵母菌落中的重组DNA,经PCR扩增,12个菌落出现阳性结果。将该19个重组DNA分别电转化入DH5α细菌,涂板培养后,每板挑取4~10个克隆,通过Sal I和Not I双酶切鉴定进行阳性克隆筛选。将阳性克隆的重组DNA进行序列测定。测序结果与Gen Bank比较,其中4个克隆的DNA片段为Y39b6a基因片段、2个为Rap GAP、1个为苯丙氨酸-4-羟化酶、1个为细胞色素C氧化酶,还有1个DNA片段编码美丽线虫特有的小分子蛋白的基因片段,其余11个DNA片段不编码已知蛋白质。结论初步筛选出与Rap GAP相互作用的蛋白质,特别是其中有2个克隆为Rap GAP,提示Rap GAP可能以二聚体的方式存在。     

Delayed blockade of the kinin B1 receptor reduces renal inflammation and fibrosis in obstructive nephropathy

Renal fibrosis is the common histological feature of advanced glomerular and tubulointerstitial disease leading to end-stage renal disease (ESRD). However, specific antifibrotic therapies to slow down the evolution to ESRD are still absent. Because persistent inflammation is a key event in the development of fibrosis, we hypothesized that the proinflammatory kinin B1 receptor (B1R) could be such a new target. Here we show that, in the unilateral ureteral obstruction model of renal fibrosis, the B1R is overexpressed and that delayed treatment with an orally active nonpeptide B1R antagonist blocks macrophage infiltration, leading to a reversal of the level of renal fibrosis. In vivo bone marrow transplantation studies as well as in vitro studies on renal cells show that part of this antifibrotic mechanism of B1R blockade involves a direct effect on resident renal cells by inhibiting chemokine CCL2 and CCL7 expression. These findings suggest that blocking the B1R is a promising antifibrotic therapy.     

Positive selection of novel peroxisome biogenesis-defective mutants of the yeast Pichia pastoris

We have developed two novel schemes for the direct selection of peroxisome-biogenesis-defective (pex) mutants of the methylotrophic yeast Pichia pastoris. Both schemes take advantage of our observation that methanol-induced pex mutants contain little or no alcohol oxidase (AOX) activity. AOX is a peroxisomal matrix enzyme that catalyzes the first step in the methanol-utilization pathway. One scheme utilizes allyl alcohol, a compound that is not toxic to cells but is oxidized by AOX to acrolein, a compound that is toxic. Exposure of mutagenized populations of AOX-induced cells to allyl alcohol selectively kills AOX-containing cells. However, pex mutants without AOX are able to grow. The second scheme utilizes a P. pastoris strain that is defective in formaldehyde dehydrogenase (FLD), a methanol pathway enzyme required to metabolize formaldehyde, the product of AOX. AOX-induced cells of fld1 strains are sensitive to methanol because of the accumulation of formaldehyde. However, fld1 pex mutants, with little active AOX, do not efficiently oxidize methanol to formaldehyde and therefore are not sensitive to methanol. Using these selections, new pex mutant alleles in previously identified PEX genes have been isolated along with mutants in three previously unidentified PEX groups.     

The dermatoglyphics of American Negroes

Digital and palmar dermatoglyphics of 184 male and 224 female normal American Negroes were evaluated for digital patterns, digital ridge counts, palmar patterns, palmar main line terminations, accessory triradii and palmar creases. All subjects were seven year olds examined and found free of chronic or other genetic diseases. The results were presented for the left and right hand for the most part comparable to those of the African and other American Negro groups reported earlier. The distributions of the various dermatoglyphic features among the Negroes taken as a group were compared to those of the other racial groups and their differences were discussed.