“Identification of Parvulin type Peptidyl-prolyl isomerases in Trypanosoma cruzi”

ERBEN ESTEBAN D., VALGUARNERA EZEQUIEL P.,TÉLLEZ-IÑÓN MARÍA T.

Rosario, Argentina

Congreso; VIII Congreso Argentino de Protozoología y Enfermedades Parasitarias,; 2008

Sociedad Argentina de Protozoologia

In Trypanosoma cruzi two Cyclin-Related Kinases genes, TcCRK1 and TcCRK3 have been cloned. TcCRK1 levels and localization do not vary during the cell cycle, being highly concentrated in the kinetoplast, suggesting a putative function in this organelle. TzCRK3 expression and activity was present throughout three life cycle stages of the parasite. In synchronized epimastigotes, TzCRK3 activity expression was constant during the cell cycle and PK activity increases at G2/M, showing the common pattern of CDK regulation. The results allow us to postulate that CRK3 shares functional homology with CDK1, and that it has a role controlling the G2/M transition in T. cruzi . The activity of CDKs is regulated by multiple regulatory mechanisms. The peptidyl- prolyl cis/trans isomerases (PPIases) are a conserved family that catalyses the cis/trans isomerization of the peptide bonds preceding Proline residues. Prolyl isomerases include three major subfamilies: the cyclophilins, FK506 binding proteins and the parvulins. Studies in multiple systems have suggested that a parvulin, PIN1 PPIase plays a critical role for mitosis progression., participate in the phosphorylation-dependent prolyl isomerase that changes the conformation of CDKs substrates controlling cell-cycle progression. In T.cruzi we identified three PPIases. The first described was TcPIN1, an as a homologue of the essential hPin1 parvulin type. Based on functional assays, subcellular localization and enzymatic PPIase activity, we showed that TcPIN1 is a member of the Pin1-type PPIases, As all plants homologues identified, TcPin1 have not WW domain at the N-terminus or analogous module. Nevertheless, TcPin1 is able to rescue the temperature sensitive phenotype of a mutation in the hPin1 homologue ESS1/PTF1 in S. cerevisiae. However, the overexpression of PIN1 in T. cruzi or the down regulation by RNAi in procyclics forms of T. brucei gene did not modified the growth of the parasites. Searching in the GeneDB trypanosomatid database, a second member of the parvulin family of PPIase, TcPar14, was identified. The encoding region of the gene 375 bp, codified a protein of 124 amino acids. The analysis of the sequence of TcPar14 revealed the presence of motifs typical of the parvulin catalytic domain. Like most hPar14 homologue parvulins, TcPar14 has an N-terminal extension, but in contrast to hPar14, a WW domain is absent in this parvulin. The TcPar14 PPIase-activity was investigated using the protease-free method. A comparison of the relative specificity constants for various substrates shows a strong preference for a substrate with the basic Arginine residue preceding proline. A new member of the parvulin family of PPIases was identified as a 46 kDa protein named TcFHAPIN. The protein contains an N-terminal forkhead-associated motif (FHA) and a C-terminal parvulin-like catalytic domain. The FHA domain is a phosphopeptide recognition domain found in many regulatory proteins with a striking specificity for phospho- threonine-containing epitopes. The domain is present in a wide range of proteins, such as kinases, phosphatases, kinesins, transcription factors, RNA-binding proteins and metabolic enzymes, which participate in many different cellular processes. To our knowledge, TcFHAPIN is the first example of a FHA domain present in the same polypeptide chain with a PPIase catalytic domain. Searching in the genome databases reveal that putative homologues are present in all trypanosomatids but not in other organisms. The presence of an FHA domain strongly indicates that the FHA-containing protein will interact with a protein partner in a process regulated by reversible protein phosphorylation. The results obtained with these PPIases, suggest the existence of an additional conserved level of post-translational control in trypanosomatids. Supported by FONCYT, CONICET, UBA.