African trypanosomiasis is caused by a unicellular eukaryote that parasitizes multicellular organisms and causes medically and economically important diseases in humans (Human African Trypanosomiasis) and their domestic animals (African Animal Trypanosomiasis). Incidence is currently declining due to the application of present chemotherapy, although the drugs are old, toxic, difficult to administer and in some cases expensive, and of diminishing efficacy due to resistance. However, this trend needs to be sustained with the discovery of new compounds active against the resistant strains. These new treatment options must meet the current pharmacological requirements, must be parasite specific and must be relatively cheap to produce. Pharmacological manipulation of phosphodiesterases (PDEs), which hydrolyse cyclic Adenosine Monophosphate (cAMP), have been extensively studied in humans and found to have great therapeutic effect. Kinetoplastid genomes code for the same set of cyclic nucleotide-specific class 1-type phosphodiesterases, with catalytic domains similar to those of human PDEs. The locus of Trypanosoma brucei PDEB1/2 was found to be essential, by either genetic manipulation or the use of the pharmacological inhibitor CpdA, but therapeutic exploitation of TbPDEB1 has so far been hampered by its catalytic domain similarity to human PDEs. However, investigating the unique downstream cAMP signalling cascade, which includes the recently identified cAMP Response Proteins (CARPs), could reveal potentially new trypanosome-specific therapeutic targets. In this study we show that single knockout (sKO) of the CARP genes causes a decreased susceptibility to CpdA, and that null mutants of CARP2-4 display significantly increased intracellular and extracellular cAMP levels. A double knockout (dKO) of CARP2 also shows a significant growth defect. Conversely, overexpression of CARP3 causes a growth delay in both WT s427 and CpdA resistant (R0.8) cells, when exposed to CpdA, and were more sensitive to CpdA compared to other CARP overexpressing cells as well as the WT s427 and R0.8 controls. These cells also have significantly higher intracellular and extracellular cAMP levels relative to the control lines and the other CARP overexpressors. In the cells overexpressing CARP3, whether WT s427 or R0.8, the cellular content of both CARP3 messenger Ribonucleic Acid (mRNA) and protein decreases extensively within 6 h of CpdA exposure. The CARP3 protein has domains that are indicative of a role in protein-protein interaction, signalling, regulation and degradation and probably undergo acylation. Some experimental confirmation of these traits was obtained, using Co-immunoprecipitation (Co-IP) and Mass Spectrometry (MS), with the identification of Adenylyl cyclase (AC) GRESAG4s (also found through RNAinterference Target Sequencing (RITseq) and confirmed by quantitative Reverse Transcription PCR), and proteasome regulatory proteins (PRNs) in addition to membrane and flagellar binding proteins, as potential interactors. Preliminary Immunofluorescence (IF) microscopy showed that CARP3 localizes to plasma membrane ad the flagellum, CARP2 to specific bodies/organelles in the cytosol and CARP1 in the cytosol. RNA sequencing of overexpressing CARP3 reveals differentially expressed proteins involved in cell cycle and cytokinesis as well as transport proteins with several transmembrane domains, consistent with the proposed acylated membrane localisation, and with interaction of CARP3 with membrane proteins and ACs (GRESAG4 isoforms).Thus CARP3 has the domains, interactions and localization consistent with a regulatory role in cAMP metabolism. Thus the CARPs and especially CARP3 are interesting biological molecules providing key new insights into signalling and the cell biology of the trypanosome.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:681838 |
| Date | January 2015 |
| Creators | Tagoe, Daniel Nii Aryee |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/6819/ |
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