Characterisation of metacaspases of Trypanosoma brucei

Helms, Matthew John (2004) Characterisation of metacaspases of Trypanosoma brucei. PhD thesis, University of Glasgow.

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Metacaspases are proteins, with some sequence similarity to caspases, that have now been identified in plants, fungi and parasitic protozoa, but not mammals. Caspases are a conserved family of central effector cysteine peptidases involved in inflammatory disease, neurodegenerative disorders and apoptosis. Little was known about metacaspase function in any organism, although several studies have linked the metacaspases to apoptotic-like phenomena in both yeast and plants. Five metacaspase genes were identified in Trypanosoma brucei (MCA1-MCA5) whereas only one, syntenic with MCA5, has been identified in Leishmania major. MCA2, MCA3 and MCA5 encode for predicted cysteine peptidases, while MCA1 and MCA4 have substitutions at the putative active site residues which suggest that they may have a peptidase-independent function. MCA2, MCA3 and MCA5 also contain predicted WW binding domain motifs, suggesting the metacaspases may interact with other proteins. Antibodies against MCA2, MCA3 and MCA5 were raised, and Western blotting revealed that MCA5 is expressed in both the bloodstream form (BSF) and procyclic form (PCF) while MCA2 and MCA3 expression is BSF specific in vitro. None of the proteins showed any large scale processing events, all being detected at around their predicted full-length masses. MCA2, MCA3 and MCA5 localised to the same compartment - vesicles located primarily between the nucleus and kinetoplast. Analysis revealed that the metacaspases co-localise with Rab11 in both PCF and BSF parasites. In BSF T. brucei, Rab11 positive recycling endosomes are involved in the recycling of both variable surface glycoprotein (VSG) and the transferrin receptor. Metacaspase function was investigated by RNAi and subsequently gene knockouts. While the RNAi results were inconclusive, the gene knockouts revealed that MCA2, MCA3 and MCA5 are non-essential genes, both in vitro and in vivo. Upon generation of BSF Deltamca2/Δmca5 parasites, an initial reduced growth rate was observed, although after successive passages growth rate returned to wild type levels. A comparative analysis of both VSG and transferrin receptor recycling processes revealed that these were the same in wild type and Δmca2/Δmca5 BSF parasites. The possibility of metacaspase involvement in cell death processes was also examined. Parasites were exposed to heat shock and salt stress, but no differences between wild type and Δmca2/3Δmca5 parasites were detected. These data suggest that the metacaspases are associated with Rab11 positive recycling endosomes, but are not essential for either VSG or transferrin receptor recycling, nor anti-VSG antibody or transferrin degradation.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Molecular biology, parasitology.
Colleges/Schools: College of Medical Veterinary and Life Sciences
Supervisor's Name: Mottram, Prof. Jeremy and Coombs, Prof. Graham
Date of Award: 2004
Depositing User: Enlighten Team
Unique ID: glathesis:2004-71262
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 10 May 2019 10:49
Last Modified: 20 Jul 2021 13:35

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