TcPDE4, a novel membrane-associated cAMP-specific phosphodiesterase from Trypanosoma cruzi.

A. SCHOIJET; H. TORRES; M. FLAWIÁ; G. ALONSO

Glasgow, UK.

Congreso; XI International Congress of Parasitology (ICOPA XI).; 2006

Cyclic nucleotide phosphodiesterases (PDEs) catalyze the degradation of the second messengers cAMP and cGMP and constitute the only known mechanism for the rapid down regulation of cyclic nucleotide signals. PDEs are members of a superfamily comprising 11 different families, each composed of several genes and many isoforms generated by alternative splicing, indicating the high complexity of this superfamily. In Trypanosoma cruzi, cAMP has been shown to downregulate cell proliferation. In our laboratory a cAMP-specific phosphodiesterase associated to the flagellar apparatus, named TcPDE1, has been identified in T. cruzi. By using the catalytic domain sequence of TcPDE1 to screen a T. cruzi genomic database, a novel phosphodiesterase was found. TcPDE4 encodes a 925-amino acid protein and presents three conserved domains, FYVE, phosphohydrolase and PDEaseI. Sequence analysis shows all characteristic domains present at the type-4 phosphodiesterases specific for cAMP. Moreover, TcPDE4 shows the inhibition profile characteristic for PDE4 subfamily, with an IC50 of 10.46 mM for rolipram and 1.3 mM for etazolate. TcPDE4 is able to complement a heat-shock-sensitive yeast mutant deficient in phosphodiesterase genes. The enzyme is specific for cAMP (Km value of about 20mM), Mg2+-dependent and cGMP or Ca2+ does not affect its activity. The association of TcPDE4 with membranes was studied by subcellular fractionation of recombinant yeast and extraction under several conditions. Most of the enzyme remained associated to the membrane fraction after treatment with high salt concentration, detergent, or chaotropic agents. Finally, confocal laser scanning microscopy indicates that TcPDE4 seems to be located in endosome-like vesicles.