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Biallelic mutations in CAD, impair de novo pyrimidine biosynthesis and decrease glycosylation precursors.

ABSTRACT: In mitochondria, carbamoyl-phosphate synthetase 1 activity produces carbamoyl phosphate for urea synthesis, and deficiency results in hyperammonemia. Cytoplasmic carbamoyl-phosphate synthetase 2, however, is part of a tri-functional enzyme encoded by CAD; no human disease has been attributed to this gene. The tri-functional enzyme contains carbamoyl-phosphate synthetase 2 (CPS2), aspartate transcarbamylase (ATCase) and dihydroorotase (DHOase) activities, which comprise the first three of six reactions required for de novo pyrimidine biosynthesis. Here we characterize an individual who is compound heterozygous for mutations in different domains of CAD. One mutation, c.1843-1G>A, results in an in-frame deletion of exon 13. The other, c.6071G>A, causes a missense mutation (p.Arg2024Gln) in a highly conserved residue that is essential for carbamoyl-phosphate binding. Metabolic flux studies showed impaired aspartate incorporation into RNA and DNA through the de novo synthesis pathway. In addition, CTP, UTP and nearly all UDP-activated sugars that serve as donors for glycosylation were decreased. Uridine supplementation rescued these abnormalities, suggesting a potential therapy for this new glycosylation disorder.

SUBMITTER: Ng BG

PROVIDER: S-EPMC4424951 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Biallelic mutations in CAD, impair de novo pyrimidine biosynthesis and decrease glycosylation precursors.

Ng Bobby G BG Wolfe Lynne A LA Ichikawa Mie M Markello Thomas T He Miao M Tifft Cynthia J CJ Gahl William A WA Freeze Hudson H HH

Human molecular genetics 20150212 11

In mitochondria, carbamoyl-phosphate synthetase 1 activity produces carbamoyl phosphate for urea synthesis, and deficiency results in hyperammonemia. Cytoplasmic carbamoyl-phosphate synthetase 2, however, is part of a tri-functional enzyme encoded by CAD; no human disease has been attributed to this gene. The tri-functional enzyme contains carbamoyl-phosphate synthetase 2 (CPS2), aspartate transcarbamylase (ATCase) and dihydroorotase (DHOase) activities, which comprise the first three of six rea ...[more]

PMID: 25678555

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Project description:The use of amphotericin B (AmB) in conjunction with 5-fluorocytosine (5-FC) is known to be the optimal therapy for treating cryptococcosis, but the mechanism by which 5-FC synergizes with AmB is unknown. In this study, we generated a Cryptococcus neoformans ura1? mutant lacking dihydroorotate dehydrogenase (DHODH), which demonstrated temperature-sensitive growth due to a defect in cell integrity and sensitivity to cell wall-damaging agents. In addition, sensitivity to AmB was greatly increased. Inclusion of uracil or uridine in the medium did not suppress the cell wall or AmB phenotype, whereas complementation with the wild-type URA1 gene complemented the mutant phenotype. As a measure of membrane accessibility, we assayed the rate of association of the lipid-binding dye 3,3'-dihexyloxacarbocyanine iodide (DiOC6) and saw more rapid association in the ura1? mutant. We likewise saw an increased rate of DiOC6 association in other AmB-sensitive mutants, including a ura- spontaneous URA5 mutant made by 5-fluoroorotic acid (5-FOA) selection and a bck1? mutant defective in cell integrity signaling. Similar results were also obtained by using a specific plasma membrane-binding CellMask live stain, with cell integrity mutants that exhibited increased and faster association of the dye with the membrane. Chitin synthase mutants (chs5? and chs6?) that lack any reported cell wall defects, in turn, demonstrate neither any increased susceptibility to AmB nor a greater accessibility to either of the dyes. Finally, perturbation of the cell wall of the wild type by treatment with the ?-1,6-glucan synthase inhibitor caspofungin was synergistic with AmB in vitro. IMPORTANCE Synergy between AmB and nucleotide biosynthetic pathways has been documented, but the mechanism of this interaction has not been delineated. Results from this study suggest a correlation between uridine nucleotide biosynthesis and cell integrity likely mediated through the pool of nucleotide-sugar conjugates, which are precursor molecules for both capsule and cell wall of C. neoformans. Thus, we propose a mechanism by which structural defects in the cell wall resulting from perturbation of pyrimidine biosynthesis allow faster and increased penetration of AmB molecules into the cell membrane. Overall, our work demonstrates that impairment of pyrimidine biosynthesis in C. neoformans could be a potential target for antifungal therapy, either alone or in combination with AmB.

Dataset Information

Biallelic mutations in CAD, impair de novo pyrimidine biosynthesis and decrease glycosylation precursors.

Publications

Biallelic mutations in CAD, impair de novo pyrimidine biosynthesis and decrease glycosylation precursors.

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