Phosphodiesterases (PDEs) are a superfamily of evolutionary conserved cyclic nucleotides (cAMP/cGMP) hydrolysing enzymes, components of transduction pathways regulating crucial aspects of cell life. PDE5, one of these families, is the molecular target of several drugs used to treat erectile dysfunction and pulmonary hypertension. Despite its medical relevance, PDE5 macromolecular structure has only been solved for the isolated regulatory and catalytic domains. The definition of the quaternary structure of the full length PDE5 (MmPDE5A1), produced in large amounts in the yeast Kluyveromyces lactis, could greatly enhance the knowledge on its assembly/allosteric regulation and the development of new inhibitors for clinical-therapeutic applications.
Small-angle X-ray scattering (SAXS), analytical ultracentrifugation (AUC), size exclusion chromatography (SEC), native polyacrylamide gel electrophoresis (PAGE) and western blot (WB) were used to assess the assembly of PDE5A1.
The full length MmPDE5A1 isoform is a mixture of dimers and tetramers in solution. We also report data showing that dimers and tetramers also coexist in vivo in platelets, blood components naturally containing high levels of PDE5.
This is the first time that structural studies on the full length protein evidenced the assembly of PDE5 in tetramers in addition to the expected dimers.
The assembly of PDE5 in tetramers in platelets, beside the dimers, opens the possibility to alternative assembly/allosteric regulation of this enzyme, as component of large signaling complexes, in all cellular districts in which PDE5 is present.
Biochimica et biophysica acta. 2018 Jul 16 [Epub ahead of print]
Silvia Cardarelli, Adriana Erica Miele, Carlotta Zamparelli, Stefano Biagioni, Fabio Naro, Francesco Malatesta, Mauro Giorgi, Michele Saliola
Department of Biology and Biotechnology "C. Darwin", Sapienza University of Rome, Piazzale A. Moro 5, 00185 Rome, Italy. Electronic address: ., Department of Biochemical Sciences 'Rossi Fanelli', Sapienza University of Rome, Piazzale A. Moro 5, 00185 Rome, Italy; UMR 5246 ICBMS - CNRS - UCBL, Université de Lyon, Campus La Doua, 43 bd 11 Novembre 1918, 69622 Villeurbanne, Cedex, France. Electronic address: ., Department of Biochemical Sciences 'Rossi Fanelli', Sapienza University of Rome, Piazzale A. Moro 5, 00185 Rome, Italy. Electronic address: ., Department of Biology and Biotechnology "C. Darwin", Sapienza University of Rome, Piazzale A. Moro 5, 00185 Rome, Italy. Electronic address: ., Department of Anatomical, Histological, Forensic, and Orthopaedic Sciences, Sapienza University of Rome, Piazzale A. Moro 5, 00185 Rome, Italy. Electronic address: ., Department of Biochemical Sciences 'Rossi Fanelli', Sapienza University of Rome, Piazzale A. Moro 5, 00185 Rome, Italy. Electronic address: ., Department of Biology and Biotechnology "C. Darwin", Sapienza University of Rome, Piazzale A. Moro 5, 00185 Rome, Italy. Electronic address: ., Department of Biology and Biotechnology "C. Darwin", Sapienza University of Rome, Piazzale A. Moro 5, 00185 Rome, Italy. Electronic address: .