Fig. 3: Characterization of PAICS variants: structural, biochemical, and functional analyses in fibroblasts and recombinant proteins.

A Structural positioning of variants in PAICS (PDB ID 2H31). On the left is octameric assembly of PAICS with one subunit highlighted in yellow. The positions of the identified variants are shown in red. On the right, a close-up view shows three subunits of PAICS octamer in different colors. SAICARs active site is highlighted by spheres. Positions of variants are highlighted in red color. B Western blot analysis in skin fibroblasts with antibodies against PPAT, GART, PFAS, PAICS, ADSL, ATIC, and HGPRT revealed a decrease in PAICS, ATIC and PFAS content, while the content of PPAT was increased. The content of other DNPS enzymes was similar to that in control fibroblasts. Protein amounts were normalized to GAPDH and actin. C PAICS enzyme activities in fibroblasts and recombinant proteins. PAICS enzyme activity was not detectable in skin fibroblasts compared to controls. Enzyme activity of mutated maltose binding-PAICS fusion proteins MBP-PAICS_S179P, MBP-PAICS_R403Ter and MBP-PAICS_K53R were reduced compared to the wild type to 76, 21 and 12%, respectively. C1-3 denote control skin fibroblast samples, while P1-2 represent patient samples. MBP-PAICS fusion proteins are indicated by their respective variant abbreviations. The MBP represents activity of the recombinant maltose binding protein devoid of PAICS. Standardized boxplot graphs with box from the first to third quartiles, whiskers to minimum and maximum, all displayed points, and the median represented by a line. The experiments were performed at least three times (n ≥ 3). One-way ANNOVA statistical tests were calculated by GraphPad software and p-values are shown: ** for P ≤ 0.01 and **** for P ≤ 0.0001. D Blue native electrophoresis of PAICS. Recombinant maltose binding-PAICS fusion proteins were expressed in E.coli, purified on amylose resin and cleaved with Factor Xa protease. Products of cleavage were separated on BN-PAGE, blotted and immunodetected with anti-PAICS antibodies. The wild type forms two high molecular weight structures. Both PAICS variants, Ser179Pro and Arg403Ter, individually as well as in mixture, exhibit significantly reduced octamer formation. The graph shows the relative amounts of PAICS octamer in each variant compared to the wild type (wt) (n = 3). PAICS protein variants are indicated by their respective abbreviations. The MBP represents immunodetection signal of the recombinant maltose binding protein devoid of PAICS. E Immunofluorescent detection of PPAT and GART in skin fibroblasts. Patients and control skin fibroblasts were cultured in purine-depleted medium and in both cases PPAT co-localized with GART in finely granular cytoplasmic structures that are characteristic of purinosome bodies. Colocalization values are converted to pseudo color, with the scale displayed in a corresponding lookup table (LUT) at the bottom right.