Fig 2: Pyrimidine biosynthesis is suppressed in the presence of CIIlow. a Label-free quantitative proteomic analysis of downregulated proteins in SDHBKO MDAMB231 cells and the percentage of proteins identified per pathway relative to genes with significantly altered expression in SDHBKO cells. b Scheme of the de novo pyrimidine nucleotide synthesis pathway. c SDS-PAGE following WB analysis of MDA231 sublines for proteins of the de novo pyrimidine pathway. d Densitometric evaluation of the level of the CAD and DHODH proteins by WB. Relative isotopomer amounts (M+1) of CTP, CDP, UTP, and UDP were assessed by LC-MS/MS using [U-13C] glucose (e) and [U-13C] glutamine (f) as tracers. Data shown are mean values ± SD (n = 3); images are representative of at least three independent experiments. The symbol ** indicates differences with p < 0.05 and ‘ns’ indicates non-significant differences, as assessed by the two-tailed unpaired Student’s t-test

Fig 3: Clinical summary for ten unreported CAD-deficient individuals.Clinical information for ten of the available subjects was collected and summarized as % affected.

Fig 4: Schematic of the pathway for de novo biosynthesis of the pyrimidine nucleotide uridine 5-monophosphate (UMP).The initial enzymatic activities, glutaminase (GLN), carbamoyl phosphate synthetase (SYN), aspartate transcarbamoylase (ATC), and dihydroorotase (DHO) are fused into the multifunctional protein CAD. The next reaction after CAD is catalyzed by dihydroorotate dehydrogenase (DHODH), an enzyme anchored to the inner mitochondrial membrane. The last two steps are catalyzed by UMP synthase (UMPS), a bifunctional enzyme with orotate phosphoribosyl transferase (OPRT) and orotidine decarboxylase (ODC) activities. Alternatively, UMP can be obtained from uridine through salvage pathways (depicted in cyan).

Fig 5: Assessing the pathogenicity of CAD variants.(a–d) Growth complementation assay of CAD-knockout (KO) cells grown in absence of uridine and transfected with GFP-CAD bearing point variants in the GLN (a), SYN (b), DHO (c), or ATC (d) domains. Variants in the loop connecting the DHO and ATC domains are included in (d). Cell proliferation is represented as % confluence with respect to cells transfected with GFP-CAD wild type (WT). Each point represents the mean and standard deviation of three measurements, and all mutants were tested in at least two independent experiments. Variants compromising CAD activity are colored in red. (e) Linear representation of CAD, mapping the inactivating (in red) and benign (in black) variants.