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Published on: July 22, 2014
Novel kinetoplastid-specific cAMP binding proteins identified by RNAi screening for cAMP resistance in Trypanosoma
Sabine Bachmaier1, Matthew K Gould1, Eleni Polatoglou1
1Faculty of Biology, Genetics, Ludwig-Maximillians University Munich (LMU), Martinsried, Germany.
Abstract:
Cyclic AMP signalling in trypanosomes differs from most eukaryotes due to absence of known cAMP effectors and cAMP independence of PKA. We have previously identified four genes from a genome-wide RNAi screen for resistance to the cAMP phosphodiesterase (PDE) inhibitor NPD-001. The genes were named cAMP Response Protein (CARP) 1 through 4. Here, we report an additional six CARP candidate genes from the original sample, after deep sequencing of the RNA interference target pool retrieved after NPD-001 selection (RIT-seq). The resistance phenotypes were confirmed by individual RNAi knockdown. Highest level of resistance to NPD-001, approximately 17-fold, was seen for knockdown of CARP7 (Tb927.7.4510). CARP1 and CARP11 contain predicted cyclic AMP binding domains and bind cAMP as evidenced by capture and competition on immobilised cAMP. CARP orthologues are strongly enriched in kinetoplastid species, and CARP3 and CARP11 are unique to Trypanosoma. Localization data and/or domain architecture of all CARPs predict association with the T. brucei flagellum. This suggests a crucial role of cAMP in flagellar function, in line with the cell division phenotype caused by high cAMP and the known role of the flagellum for cytokinesis. The CARP collection is a resource for discovery of unusual cAMP pathways and flagellar biology.
Insights
Researchers identified new cAMP-binding proteins (CARPs) in trypanosomes, revealing unusual cyclic AMP signaling pathways. These proteins are crucial for flagellar function and parasite survival.
Area of Science:
- * Parasitology
- * Molecular Biology
- * Biochemistry
Background:
- * Cyclic AMP (cAMP) signaling in trypanosomes is distinct from other eukaryotes, lacking typical effectors and PKA dependence.
- * Previous studies identified four cAMP Response Protein (CARP) genes conferring resistance to a cAMP phosphodiesterase inhibitor.
- * The unique nature of cAMP signaling in trypanosomes necessitates further investigation into its components and functions.
Purpose of the Study:
- * To identify additional genes involved in cAMP signaling in trypanosomes using deep sequencing.
- * To characterize the function and localization of newly identified CARP proteins.
- * To explore the role of cAMP in trypanosome flagellar biology and cell division.
Main Methods:
- * RNA interference (RNAi) screening and deep sequencing (RIT-seq) to identify resistance genes.
- * Individual RNAi knockdown to confirm resistance phenotypes.
- * cAMP binding assays (capture and competition) to validate protein-ligand interactions.
- * Bioinformatic analysis of CARP orthologues and domain architecture.
- * Protein localization studies.
Main Results:
- * Six additional CARP candidate genes were identified through RIT-seq.
- * RNAi knockdown of CARP7 conferred the highest resistance (approx. 17-fold) to the PDE inhibitor.
- * CARP1 and CARP11 possess cyclic AMP binding domains and directly bind cAMP.
- * CARP orthologues are prevalent in kinetoplastids; CARP3 and CARP11 are specific to *Trypanosoma*.
- * All identified CARPs are predicted to localize to the flagellum of *T. brucei*.
Conclusions:
- * The CARP protein family represents novel components of trypanosome cAMP signaling pathways.
- * cAMP signaling is intrinsically linked to flagellar function, impacting cell division.
- * The identified CARPs are essential for understanding unique cAMP pathways and flagellar biology in trypanosomes.
- * This CARP collection serves as a valuable resource for future research in these areas.
