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Endocrine Abstracts (2015) 37 GP05.02 | DOI: 10.1530/endoabs.37.GP.05.02

ECE2015 Guided Posters Developmental and paediatric endocrinology (10 abstracts)

The differential impact of PAPS synthase isoforms on DHEAS may be explained by an isoform-specific interaction of SULT2A1 with PAPSS2, but not PAPSS1

Jon W Mueller 1 , Jan Idkowiak 1 , Rebecca E Hardman 1 , Philip J House 1 , Joanne C McNelis 1 , Ian T Rose 1 , Vivek Dhir 1 , Edina Rosta 2 & Wiebke Arlt 1

1CEDAM, University of Birmingham, Birmingham, UK; 2Department of Chemistry, King’s College London, London, UK.

Human sulfation depends on provision of the universal sulfate donor PAPS by the two PAPS synthase isoforms PAPSS1 and PAPSS2. Mutations in PAPSS2 have been identified as a monogenic cause of androgen excess presenting with premature adrenarche and polycystic ovary syndrome, due to decreased sulfation of the androgen precursor DHEA by DHEA sulfotransferase (SULT2A1) and hence increased conversion of DHEA to active androgens (New Eng J Med 2009 360 (22) 2310–2318 and J Clin Endocrinol Metab 2015 jc20143556). Here, we examined why ubiquitously expressed PAPSS1 cannot compensate for the impact of PAPSS2 deficiency on DHEAS. First, we carried out siRNA-mediated knockdown of PAPSS1/2 in the adrenal cell line NCI-H295R1. Real-time PCR confirmed >90% knockdown and the impact at protein level was assessed by western blot and SULT2A1 activity assays. Efficient knockdown of PAPSS2 reduced DHEAS to 30±5%. Strikingly, PAPSS1 knockdown did not impact on DHEAS, providing in vitro evidence for non-overlapping functionality of the two PAPSS isoforms. To test whether subcellular localisation impacts on isoform-specific capacity to support DHEAS, we used HEK293 cells to co-express SULT2A1 with either WT PAPSS1/2 or exclusively nuclear or cytoplasmic PAPSS1/2 variants. WT and nuclear PAPSS1/S2 equally supported DHEAS; however, SULT2A1 activity with exclusively cytoplasmic expression of PAPSS2 was 1.6-fold higher than with cytoplasmic PAPSS1; suggesting an isoform-specific protein-protein interaction of SULT2A1 with PAPSS2, but not PAPSS1. To examine this further, we carried out docking studies using ClusPro, Z-dock and gramm-X servers employing the APS kinase protein structures 2ofx and 2ax4 for PAPSS1/2 and three different structures for human SULT2A1 (1efh, 3f3y, and 4ifb). Docking of SULT2A1 to PAPSS2 unanimously resulted in more homogeneous complexes than for SULT2A1–PAPSS1, yielding significantly higher scores by the associated docking algorithms. Such an isoform-specific interaction with SULT2A1 may be the underlying mechanism explaining why PAPSS2 deficiency results in disruption of DHEAS despite ubiquitous expression of PAPSS1. Follow-up studies to prove direct interaction of PAPSS2 and SULT2A1, employing pulldown assays and FRET analyses, are currently underway.

Disclosure: Marie Curie IEF award 625451 SUPA-HD (to J W Mueller) and Wellcome Trust project grant 092283 (to W Arlt).

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