Phosphodiesterases are a superfamily of cyclic nucleotides hydrolyzing enzymes involved in transduction pathways. PDE5, a cGMP-specific family, is the targets of several drugs used to treat erectile dysfunction and pulmonary hypertension. Recently, the full-length of murine PDE5 isoforms were produced and purified from Kluyveromyces lactis. The purified activities were characterized by biochemical and structural analysis allowing the quaternary assembly of MmPDE5A1 as dimers and tetramers. This oligomeric structure was also present in physiological conditions in rat platelets. The heterologous expression of the PDE5A1 in S. cerevisiae induces an exponential growth delay indicating a precise cGMP-dependent role of A1 on the endogenous cAMP/PKA TORC1 signalling pathways. These experiments revealed thath in yeast cGMP is required in the early phases of growth to maintain the cAMP/cGMP ratio inside well-defined ranges for the metabolic adaptation of cells during proliferation. In this project, we will investigate the structure of MmPDE5A2 and MmPDE5A3 isoforms and the role of the different N-terminus in their quaternary assembly by small-angle synchrotrone X-ray scattering (SAXS) measurements. The purified proteins will be also analysed by transmission electron microscopy (TEM). In addition, the three isoforms will be expressed in the K. lactis petite-negative model organism (CBS2359 strain). This yeast is particular useful because could allow the investigation of the over-expression of the murine PDE5 isoforms on the respiratory and fermentative metabolism. Indeed, the expression of mammalian protein involved in fundamental cellular functions in simple eukaryotic model has often proved useful to understand their role. The putative knowledge acquired by this model system might be easily transferred to higher mammalian cells where could be at the basis for the control of the metabolic syndrome or other severe pathologies determined by PDE5 isoforms.
PDE5 are cGMP hydrolyzing enzymes, crucial components of transduction pathways regulating many aspects of cell life being the targets of several drugs used to treat erectile dysfunction and pulmonary hypertension. However, although very abundant and regulated in specific subset of cells of organ and tissues, the specific role of each of the three isoforms has never been determined. Since the three spliced variants are highly identical and specific antibodies against each isoform are not available, the functional role of the three proteins has never been determined. To overcome this problem, we decided to study the structural differences and the metabolic role of the three isoforms in the K. lactis lower eukaryotic model. In fact, the expression of mammalian protein involved in fundamental cellular functions in simple eukaryotic model has often proved useful to understand their role. Indeed, the expression of the PDE5A1 isoform in S. cerevisiae allowed to determine, for the first time, the role of cGMP in this yeast and the metabolic alteration produced by this protein in the host cell. The putative knowledge acquired by this model system might be transferred to higher mammalian cells where could be the basis for the control of the metabolic syndrome or other diseases.