cUMP hydrolysis by PDE3B

Abstract

Previous results indicate that the phosphodiesterase PDE3B hydrolyzes cUMP. Also, almost 50 years ago, cUMP-hydrolytic activity was observed in rat adipose tissue. We intended to characterize the enzyme kinetics of PDE3B-mediated cUMP hydrolysis, to determine the PDE3B binding mode of cUMP, and to analyze cUMP hydrolysis in adipocyte preparations. Educts (cNMPs) and products (NMPs) of the PDE reactions as well as intracellular cNMPs were quantitated by HPLC-coupled tandem mass spectrometry. PDE3B expression was determined by qPCR and Western blot. Docking studies were performed with the PDE3B crystal structure PDB ID 1SO2 (complex with a dihydropyridazine inhibitor). PDE3B hydrolyzed cUMP (K ~ 550 μM, V ~ 76 μmol/min/mg) and cAMP (K ~ 0.7 μM, V ~ 4.3 μmol/min/mg) in a milrinone (PDE3-selective inhibitor)-sensitive manner (K for inhibition of cUMP hydrolysis: 205 nM). cUMP forms one hydrogen bond with PDE3B (uracil 3-NH with side chain oxygen of Q988). Two hydrogen bonds stabilize cAMP binding. cCMP does not interact with PDE3B. Possibly, the cytosine base cannot form hydrogen bonds with PDE3B, and the 4-NH group clashes with L987 of the enzyme. Adipocyte differentiation of 3T3-L1 MBX cells increased mRNA of PDE3B, but not of PDE3A. Significant amounts of cUMP were detected in differentiated and undifferentiated 3T3-L1 MBX cells. 3T3-L1 MBX adipocyte lysates and rat epididymal adipose tissue membranes contained milrinone-sensitive cUMP-hydrolytic activity. PDE3B is a low-affinity and high-velocity phosphodiesterase for cUMP. The cUMP-hydrolyzing activity described almost 50 years ago for rat adipose tissue is caused by PDE3, probably by the isoform PDE3B.

Apoptotic and anti-proliferative effect of guanosine and guanosine derivatives in HuT-78 T lymphoma cells

“…Moreover, the pyrimidine nucleoside uridine activates adenosine receptors (Yilmaz et al 2008). Thus, the non-canonical cyclic nucleotide cUMP, which is currently discussed as a potential second messenger (Seifert et al 2015;Berrisch et al 2017;Ostermeyer et al 2018;Scharrenbroich et al 2019), may as well be exported and degraded to biologically active products. Preliminary results suggest that first-and second messenger effects of cyclic nucleotides are highly dependent on the investigated cell type (Schneider et al 2015).…”