Search for: All records

Xanthine dehydrogenase (XDH) is a molybdenum cofactor (Moco) requiring enzyme that catabolizes hypoxanthine into xanthine and xanthine into uric acid, the final steps in purine catabolism. Human patients with mutations in XDH develop xanthinuria which can lead to xanthine stones in the kidney, recurrent urinary tract infections, and renal failure. Currently, there are no therapies for treating human XDH deficiency. Thus, understanding mechanisms that maintain purine homeostasis is an important goal of human health. Here, we used the nematodeCaenorhabditis elegansto model human XDH deficiency using two clinically relevant paradigms: Moco deficiency or loss-of-function mutations inxdh-1,theC. elegansortholog of XDH.Both Moco deficiency andxdh-1loss of function caused the formation of autofluorescent xanthine stones inC. elegans. Surprisingly, only 2% ofxdh-1null mutantC. elegansdeveloped a xanthine stone, suggesting additional pathways may regulate this process. To uncover such pathways, we performed a forward genetic screen for mutations that enhance the penetrance of xanthine stone formation inxdh-1null mutantC. elegans. We isolated multiple loss-of-function mutations in the genesulp-4which encodes a sulfate permease homologous to human SLC26 anion exchange proteins. We demonstrated that SULP-4 acts cell-nonautonomously in the excretory cell to limit xanthine stone accumulation. Interestingly,sulp-4mutant phenotypes were suppressed by mutations in genes that encode for cystathionase (cth-2)or cysteine dioxygenase (cdo-1), members of the sulfur amino acid catabolism pathway required for production of sulfate, a substrate of SULP-4. We propose that sulfate accumulation caused bysulp-4loss of function promotes xanthine stone accumulation. We speculate that sulfate accumulation causes osmotic imbalance, creating conditions in the intestinal lumen that favor xanthine stone accumulation. Supporting this model, a mutation inosm-8that constitutively activates the osmotic stress response also promoted xanthine stone accumulation in anxdh-1mutant background. Thus, our work establishes aC. elegansmodel for human XDH deficiency and identifies the sulfate permeasesulp-4as a critical player controlling xanthine stone accumulation. 

Abstract Engineering plant vegetative tissue to accumulate triacylglycerols (TAG, e.g. oil) can increase the amount of oil harvested per acre to levels that exceed current oilseed crops. Engineered tobacco (Nicotiana tabacum) lines that accumulate 15% to 30% oil of leaf dry weight resulted in starkly different metabolic phenotypes. In-depth analysis of the leaf lipid accumulation and 14CO2 tracking describe metabolic adaptations to the leaf oil engineering. An oil-for-membrane lipid tradeoff in the 15% oil line (referred to as HO) was surprisingly not further exacerbated when lipid production was enhanced to 30% (LEAFY COTYLEDON 2 (LEC2) line). The HO line exhibited a futile cycle that limited TAG yield through exchange with starch, altered carbon flux into various metabolite pools and end products, and suggested interference of the glyoxylate cycle with photorespiration that limited CO2 assimilation by 50%. In contrast, inclusion of the LEC2 transcription factor in tobacco improved TAG stability, alleviated the TAG-to-starch futile cycle, and recovered CO2 assimilation and plant growth comparable to wild type but with much higher lipid levels in leaves. Thus, the unstable production of storage reserves and futile cycling limit vegetative oil engineering approaches. The capacity to overcome futile cycles and maintain enhanced stable TAG levels in LEC2 demonstrated the importance of considering unanticipated metabolic adaptations while engineering vegetative oil crops.