NEW INSIGHTS INTO THE STRUCTURAL AND BIOCHEMICAL PROPERTIES OF HUMAN RENALASE: A NOVEL FLAVOENZYME INVOLVED IN BLOOD PRESSURE REGULATION

Abstract Renalase is a flavoprotein recently discovered in humans, which is ubiquitous in vertebrates and conserved in some other phyla. In 2005, it was identified within a project aimed to determine novel proteins secreted by the kidney, whose defect could explain the high incidence of cardiovascular complications in patients with chronic kidney disease (Xu et al., 2005). The protein is preferentially expressed in the renal proximal tubules and heart, and it’s secreted in blood and urine. Genetic, epidemiological, clinical studies and animal experimental models have constantly accumulated evidence of the important role played by renalase in lowering blood pressure, decreasing the catecholaminergic tone and control heart function. A renalase knockout mouse model resulted in increased levels of catecholamines in plasma and heart, cardiac ischemia and myocardial necrosis more severe than WT littermates (Wu et al., 2011). However, the possible molecular mechanism, the nature of the in vivo catalyzed reaction and the identity of renalase substrate(s) are still unclear. Based on these premises, the main aim of the project was to provide a detailed biochemical and structural characterization of renalase in order to better elucidate its physiological function. We solved the crystallographic structure of recombinant human renalase at 2.5 Å resolution. The general fold classified it as a member of the p-hydroxybenzoate hydroxylase family. Renalase contains non-covalently bound FAD with redox features suggestive of a oxidase or NAD(P)H-dependent monooxygenase activity (Milani et al., 2011), in contrast with the proposed activity of catecholamine degradation via a superoxide (O2-)-dependent mechanism (Farzaneh-Far et al., 2010). Furthermore, structural evidence indicates that the proposed secretion signal of renalase could not be cleaved without disrupting the protein native conformation, suggesting that renalase trafficking occurs through an atypical secretory pathway. The resolution of renalase crystallographic structure and the biochemical data available will hopefully provide the basis towards the understanding of the molecular mechanism of renalase physiological action, which is expected to favor the development of novel therapeutic tools for the treatment of kidney and cardiovascular diseases. During the PhD program, I was also involved in a side project focused on the elucidation of the role of Y258 residue of P. falciparum Ferredoxin-NADP+ reductase (PfFNR) in the control of NADPH specificity. PfFNR is a FAD-containing enzyme able to promote the transfer of two electrons from NADPH to ferredoxin and represents a promising target of novel antimalarial drugs. Rapid reactions kinetics, active site titrations with NADP+ and anaerobic photoreduction experiments allowed us to conclude that the Y258 side chain favors the stabilization of the catalytically competent conformation of the MNM moiety of NADPH, enhancing the hydride transfer between the nicotinamide nucleotide and the FAD prosthetic group. The almost complete abolishment of NADPH selectivity has never been accomplished before through a single mutation.