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

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.

Cysteine Redox Chemistry in Peptide Self-Assembly to Modulate Hydrogelation

“…There, they are exposed to harsher conditions, and disulfide bridges can exert an important stabilizing role to preserve their function [ 10 ]. Important examples include: Hormones, such as insulin, whose function is strongly dependent on correct disulfide formation [ 11 , 12 ]; Functional proteins of the immune system, such as antigen-presenting major histocompatibility complexes (MHCs) [ 13 ] and antibodies [ 14 ]; Natural antimicrobial peptides [ 15 ], such as defensins [ 16 , 17 ]; Respiratory complexes that are key for cell metabolism, such as cytochrome c [ 18 ]; Proteins of the extracellular matrix, such as collagen [ 19 ]; Focal adhesion complexes that link integrins to the cytoskeleton in key processes, such as cell adhesion and migration [ 20 , 21 ]; Several toxins and venom peptides [ 22 , 23 , 24 ]; Ubiquitin transfer between catalytic cysteines leading to protein degradation [ 25 , 26 ]; Enzymes controlling transduction pathways, such as phospodiesterases [ 27 ]. …”