Structural Characterization of Murine Phosphodiesterase 5 Isoforms and Involvement of Cysteine Residues in Supramolecular Assembly

Mauro Giorgi; Adriana Erica Miele; Silvia Cardarelli; Alessandra Giorgi; Mara Massimi; Stefano Biagioni; Michele Saliola

doi:10.3390/ijms24021108

Structural Characterization of Murine Phosphodiesterase 5 Isoforms and Involvement of Cysteine Residues in Supramolecular Assembly

Int J Mol Sci. 2023 Jan 6;24(2):1108. doi: 10.3390/ijms24021108.

Authors

Mauro Giorgi¹, Adriana Erica Miele^{2

3}, Silvia Cardarelli¹, Alessandra Giorgi², Mara Massimi⁴, Stefano Biagioni¹, Michele Saliola¹

Affiliations

¹ Department of Biology and Biotechnology "C. Darwin", Sapienza University of Rome, Piazzale A. Moro 5, 00185 Rome, Italy.
² Department of Biochemical Sciences, Sapienza University of Rome, Piazzale A. Moro 5, 00185 Rome, Italy.
³ UMR 5280 ISA-CNRS-UCBL, Université de Lyon, 5 Rue de La Doua, 69100 Villeurbanne, France.
⁴ Department of Life, Health and Environmental Sciences, University of L'Aquila, Via Vetoio, 67100 L'Aquila, Italy.

Abstract

Phosphodiesterases (PDEs) are a superfamily of evolutionarily conserved cyclic nucleotide (cAMP/cGMP)-hydrolyzing enzymes, components of transduction pathways regulating crucial aspects of cell life. Within this family, the cGMP-dependent PDE5 is the major hydrolyzing enzyme in many mammalian tissues, where it regulates a number of cellular and tissular processes. Using Kluyveromyces lactis as a model organism, the murine PDE5A1, A2 and A3 isoforms were successfully expressed and studied, evidencing, for the first time, a distinct role of each isoform in the control, modulation and maintenance of the cellular redox metabolism. Moreover, we demonstrated that the short N-terminal peptide is responsible for the tetrameric assembly of MmPDE5A1 and for the mitochondrial localization of MmPDE5A2. We also analyzed MmPDE5A1, A2 and A3 using small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM), structural mass spectrometry (MS) and polyacrylamide gel electrophoresis in their native conditions (native-PAGE) and in the presence of redox agents. These analyses pointed towards the role of a few specific cysteines in the isoforms' oligomeric assembly and the loss of enzymatic activity when modified.

Keywords: MS; PDE5; SAXS; TEM; native-PAGE; quaternary structure; reactive Cys.

MeSH terms

Animals
Cyclic GMP* / metabolism
Cyclic Nucleotide Phosphodiesterases, Type 5 / metabolism
Cysteine*
Mammals / metabolism
Mice
Protein Isoforms
Scattering, Small Angle
X-Ray Diffraction

Substances

Cyclic Nucleotide Phosphodiesterases, Type 5
Cysteine
Protein Isoforms
Cyclic GMP

Grants and funding

This work used the EM facilities at the Grenoble Instruct-ERIC Center (ISBG; UMS 3518 CNRS CEA-UGA-EMBL) with support from the French Infrastructure for Integrated Structural Biology (FRISBI; ANR-10-INSB-05-02) and GRAL, a project of the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EURGS (ANR-17-EURE-0003) within the Grenoble Partnership for Structural Biology. The IBS electron microscope facility is supported by the Auvergne–Rhône–Alpes Region, the Fonds Feder, the Fondation pour la Recherche Médicale and GIS-IBiSA. This research was funded by Sapienza University of Rome Progetti Ateneo 2018–2019 to S.B.; by University of L’Aquila 06FFO_2021 to M.M.; by Auvergne-Rhone Alpes SCUSI2018 (P010O003) to A.E.M.