Disease-Related Protein Variants of the Highly Conserved Enzyme PAPSS2 Show Marginal Stability and Aggregation in Cells

Abstract

Cellular sulfation pathways rely on the activated sulfate 3'-phosphoadenosine-5'-phosphosulfate (PAPS). In humans, PAPS is exclusively provided by the two PAPS synthases PAPSS1 and PAPSS2. Mutations found in the PAPSS2 gene result in severe disease states such as bone dysplasia, androgen excess and polycystic ovary syndrome. The APS kinase domain of PAPSS2 catalyzes the rate-limiting step in PAPS biosynthesis. In this study, we show that clinically described disease mutations located in the naturally fragile APS kinase domain are associated either with its destabilization and aggregation or its deactivation. Our findings provide novel insights into possible molecular mechanisms that could give rise to disease phenotypes associated with sulfation pathway genes.

Keywords: PAPS synthase; in-cell spectroscopy; protein folding; stability and aggregation; sulfation pathways.

FIGURE 1

In-cell thermal unfolding of APSK37 wt using Fast Relaxation Imaging. (A) Schematic representation of the Fast Relaxation Imaging setup. (B) Induced temperature profile calculated by the calibration procedure published in Büning et al., 2017. (C) Change in fluorescence according to the temperature profile in B of the APSK37 AcGFP1 FRET donor (D) and the mCherry FRET acceptor (A). (D) D/A ratio calculated from the intensity data in (C). The thermal unfolding region is shaded in blue and the calculated T_M is displayed by a dash blue line. (E) Crystal structure of the APSK domain of PAPSS2 (PDB:2AX4). Studied mutations are shown as red spheres. ADP/ATP (green) and APS/PAPS (yellow) binding sites are indicated by the substrates surface representation.

FIGURE 2

Thermal unfolding curve APSK37 wt and mutants (G78R). (A) Exemplary temperature induced thermal unfolding curves of wt (data shown from (Brylski et al., 2021)). (B) Exemplary temperature induced thermal unfolding curves of G78R. (C) Exponential unfolding curves of single temperature jumps from panel (B) showing the relaxations kinetics at the respective temperatures. (D) Kinetic amplitudes as a function of temperature to determine the T_m (dashed line). (E) Thermal stability comparison of the mutant G78R with APSK37 wt: Melting points of APSK37 (green) and melting point of G78R (blue) derived from FReI measurrement showing average ± s.d. (F) Folding free energy ∆G_f ^0’ for both APSK37 wt and G78R mutant with mean ± s.d. There are no statistically significant differences between wt and G78R. Significance were tested via one-way ANOVA with a post-hoc Holm-Sidak test correcting for multiple comparisons (no significant changes observed).

FIGURE 3

Exemplary temperature-induced thermal unfolding curves of the L76Q, T48RM, and C43Y mutants are visible in Figures 3A–C, respectively.

FIGURE 4

Expression and distribution of recombinant human PAPSS in HEK293 cells. (A) Exemplary fluorescence image of HEK293 cells showing fluorescence spots classified according to three categories. (B) Even distribution; no speckles: grade 1, 1–3 speckles: grade 2, 4–10 speckles: grade 3 and more than 10 speckles or large clumps: grade 4. Data is presented as average ± s.e.m. Cells were counted from four different slides (N = 4) with n > 200 cells in total for each protein variant. Asterisks indicate significant differences of the fraction of cells showing no speckles compared to PAPSS2 wt (*p < 0.05, ***p < 0.001). Additional statistical significance within grade 1 was found between G78R and T48R(***) or G78R and L76Q(*).