In procyclic trypanosomes, it is homogeneously distributed throughout the entire cytoplasm, with no evidence for specific co-localization with the acidocalcisomes. This is similar to the subcellular localization observed with its homologue in L. major [14]. In Sotrastaurin price the bloodstream form, TbrPPX1 is localized

in more granular structures throughout the cytoplasm, suggesting that its subcellular organization might be lifecycle stage dependent. Nevertheless, these granules exhibit no specific co-localization with the acidocalcisomes. In both stages, TbrPPX1 is excluded from the flagellum. Upon cell fractionation of either procyclic or bloodstream cells with the non-ionic detergent Triton X-100, TbrPPX1 partitions quantitatively into the soluble phase, demonstrating that it is not firmly associated to cytoskeletal structures in either life cycle stage. This is in agreement with the observation that TbrPPX1, similar to LmPPX

[14], lacks an N-terminal signal sequence, suggesting that it does not enter the endoplasmic reticulum-mediated secretory pathway, but is synthesized on free polysomes and then kept in the cytosol. TbrPPX1 is an active exopolyphosphatase that accepts inorganic pentasodium triphosphate as a substrate, but selleck screening library neither nucleoside triphosphates nor inorganic pyrophosphate. The marked inhibition of TbrPPX1 by Zn2+ ions even in the presence of a large excess of Mg2+ is reminiscent to what was reported for its L. major [14] and T. cruzi [15] homologues. Several experimental approaches learn more have demonstrated that TbrPPX1 definitely does not contain an endogenous cAMP-phosphodiesterase activity. This is in agreement with recent similar findings with human prune [9] for which such an activity

had initially been postulated [17]. Also, the exopolyphosphatase activity of TbrPPX1 is not inhibited by several inhibitors with specificities against different human cyclic nucleotide-specific phosphodiesterases. These findings support the central paradigm of cAMP signaling in eukaryotes which posits that PLEK2 the cyclic nucleotide-specific phosphodiesterases represent the only mechanism for a rapid disposal of cAMP. TbrPPX1 is not essential in T. brucei, neither in the procyclic nor in the bloodstream form. Gene ablation by genetic knock-out or knock-down by RNAi only slightly prolonged the generation time. Furthermore, in-vivo RNAi in a mouse model did not abolish the virulence of two independent RNAi clones. The absence of a dramatic phenotype is in agreement with the observation that the overall polyphosphate content of wild type versus TbrPPX1-knockout cells was not changed, suggesting that TbrPPX1 is not involved in the quantitative management of polyphosphate stores. The overall polyphosphate content measured for T. brucei in this study is in good agreement with earlier findings with T. cruzi [11].