TcRho1, a Farnesylated Rho Family Homologue fromTrypanosoma cruzi

Abstract

Rho GTPases are members of the Ras superfamily and are involved in signal transduction pathways, including maintenance of cell morphology and motility, cell cycle progression, and transcription activation. We report the molecular identification in trypanosomatids (Trypanosoma cruzi) of the first member of the Rho family. The cloned Rho protein, TcRho1, shares ϳ40% homology with other members of the Rho family. Southern blot analysis revealed that TcRHO1 is a single copy gene per haploid genome, and Northern blot assays showed a transcript of 1200 nucleotides in length. Mapping the 5-untranslated region of TcRHO1 transcripts revealed at least five different transcripts derived from differential trans-splicing. Three of the five transcripts contain the trans-splicing site within the coding region of the TcRHO1 gene. TcRho1 also contains the C-terminal sequence CQLF (CAAX motif), which is predicted to direct post-translation prenylation of the cysteine residue. A synthetic peptide containing this C-terminal motif, when tested against Q-Sepharose chromatography fractions from T. cruzi cytosol, was shown to be efficiently farnesylated, but not geranylgeranylated, despite the fact that the CAAX motif with X ‫؍‬ Phe specifies geranylgeranylation by mammalian protein geranylgeranyltransferase I. Furthermore, immunoblot analyses of epimastigote protein with anti-S-farnesylcysteine methyl ester and anti-TcRho1 antisera strongly suggested that TcRho1 is farnesylated in vivo. The farnesylation of proteins such as Rho GTPases could be the basis for the selective cytotoxic action of protein farnesyltransferase inhibitors on trypanosomatids versus mammalian cells.

Protein geranylgeranyltransferase-I of Trypanosoma cruzi

“…As shown in Table 2, the search revealed at least 5 small GTPase-like proteins that have C-terminal CaaX tetrapeptide sequences, which include previously reported TcRho1 GTPase with C-terminal CQLF [26]. By sequence alignment of these putative T. cruzi GTPase-like proteins with human GTPases (not shown), XP_818107 was found to be highly similar to human H-, K-, N-Ras and Rap proteins (42-47% sequence identity).…”

“…The significance of the first short ORF is unknown. A very low level of PGGT-I activity was detectable in the cytosol fraction of T. cruzi epimastigotes [26]. It is possible that this gene architecture may allow for low expression levels of the PGGT-I β since the ribosome could preferentially begin translation at the first start codon.…”

“…Among five GTPase-like proteins found to have the CaaX motif, two of those contain the C-terminal Leu ( Table 2) that are predicted to be T. cruzi PGGT-I substrates. One putative Rab GTPase with CWLM [37] may be a PGGT-II substrate, and other two GTPase-like proteins (TcRho1-CQLF [26] and one with CHFM) seem to serve as selective T. cruzi PFT substrates (Table 3). Four of the T. cruzi PRL protein tyrosine phosphatase homologs and five of the DNAJ homologs were found to have the CaaX ending with Met and Gln (Table 2), respectively, and thus these are predicted to be farnesylated (Table 3).…”

“…Two proteins in T. cruzi, TcRho1 GTPase [26] and TcPRL-1 protein tyrosine phosphatase [27] have so far been shown to be farnesylated. Here we describe the cloning of a putative PGGT-I β subunit and co-expression of this gene in combination with the T. cruzi PFT α gene to yield a functional PGGT-I.…”

Trypanosomatid EST: a neglected information resource regarding flagellated protozoa?

“…The immature polycistronic transcript is processed via a trans-splicing mechanism, which adds a small, spliced, 39 nucleotide RNA leader to the 5' end of the newly generated monocistronic messengers (Campbell et al 2003). Variant mRNAs can be found in trypanosomes due to different transcription initiation sites that generate distinct 5' UTRs lengths (alternative transsplicing) (Nepomuceno-Silva et al 2001). Conversely, alternative polyadenylation is also a mechanism for the generation of transcriptional diversity (different 3' UTR lengths) from the same gene (Kubo et al 2006).…”