Endocrine Abstracts

Mutations in the gene for 3′-phospho-adenosine-5′-phosphosulfate synthase 2 (PAPSS2) are linked to bone and cartilage mal-formation. More recently, we could identify PAPSS2-mutations as mono-genetic cause for androgen excess in women due to apparent SULT2A1 deficiency, the enzyme responsible for sulfation of the testosterone precursor DHEA that relies on PAPS provision by PAPS synthases. The only human orthologue, PAPSS1, is expressed in the affected tissues at comparable levels to PAPSS2, suggesting non-overlapping functionality of the two proteins. Here, we characterised specific activity of the sulfurylase and APS-kinase activities by coupled enzyme assays as well as ligand-binding affinity by fluorescence titrations using fluorescently-labelled APS for both PAPS synthases and found remarkable similarity. We then characterised expression levels of PAPS synthases by RT-PCR in hepatic and adrenal cell lines. From this analysis, we chose the adrenal cell line NCI-H295R1 for siRNA-mediated knockdown of PAPSS1/S2 as these two genes were expressed at similar levels. siRNA-knockdown was confirmed by real-time PCR and western blot for PAPSS1, PAPSS2 and SULT2A1. Functionally, SULT2A1 knockdown reduced DHEA-sulfation to 19±6% of the control reaction and a knockdown of PAPSS2 resulted in 30±5% conversion, while a PAPSS1-knockdown did not reduce DHEA-sulfation. Next, we analysed the influence of subcellular localisation of PAPS synthases on their ability to support DHEA-sulfation by co-expressing nuclear and cytoplasmic variants of PAPSS1/S2 in HEK293 cells with SULT2A1. While WT and nuclear PAPSS1/S2 proteins supported DHEA-sulfation to a similar extent, cytoplasmic PAPSS2 caused significantly higher SULT2A activity (160%) than cytoplasmic PAPSS1. Finally, we conducted a proteomics screen for PAPSS2-interaction partners in HEK293-FlpIn cells expressing PAPSS2-Strep-HA followed by pull-down and LC-MS/MS analysis. Proteasomal subunits are significantly enriched in our dataset, suggesting active protein turn-over of PAPSS2 involving the proteasomal machinery. Whether this is responsible for the functional differences between PAPS synthases remains to be elucidated in the future.