This lab has previously shown that calyculin A produces two dose-dependent phenotypes in Trypanosoma cruzi. At low doses, it abrogates cell division while allowing organellar duplication to continue. At higher doses (or at longer incubation times), it causes the loss of the characteristic elongated typanosome cell shape. Calyculin A presumably acts through inhibition of type 1 protein phosphatase activity. We have cloned two genes encoding PPl-like enzymes, TcPPl alpha and TcPPl beta. One of these, TcPPl beta, expressed as a recombinant protein, is an active phosphatase that displays a classic PP1 inhibitor profile to small molecule inhibitors, but is unique among PP1 enzymes characterized to date (in greater than 20 species widely separated in evolution) in that it expressed absolute resistance to the heat-stable protein inhibitors Protein Phosphatase Inhibitor-1 and Inhibitor-2, as well as 200-fold resistance to the polyanion heparin. Molecular modeling based on the crystal structure of mammalian PP1 suggest that this may be due to non-conservation of the regulatory peptide-PP1 interacting domains. In mammalian systems, multiple attempts to engineer or select mutant PPls that totally abrogate this interaction have not been successful. TcPP1 beta represents a naturally occurring null mutant of this interacting domain. We propose to systematically mutate divergent amino acids in TcPPl beta back to the evolutionary PP1 consensus to determine which amino acids are responsible for PPl-regulatory protein interactions. In addition, we plan to transfect trypanosomes with a variety of phosphatase constructs. As part of this effort, we plan to adapt a tetracycline-derepressible gene expression system for use in T. cruzi.
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