Trypanosomiases and leishmaniases are amongst the world’s most neglected infectious diseases. Trypanosoma brucei, Trypanosoma cruzi and Leishmania major are the primary human pathogens of the trypanosomatidae family and the causative agents of African sleeping sickness, Chagas’ disease and cutaneous leishmaniasis, respectively. The molecular mechanism controlling each parasite’s life cycle and virulence is poorly understood however protein phosphatases are expected to play a critical role. This study presents the biochemical characterisation data of two groups of phosphatases from the trypanosomatids: the LRR-DSP and LM phosphatases. The LRR-DSPs are a group of twelve proteins, characterised by a unique domain architecture: a leucine-rich repeat (LRR) domain together with a dual- specificity phosphatase (DSP) catalytic domain. In this study, the recombinant expression of a representative LRR-DSP orthologue from T. brucei (TbLRR-DSP), proved to be highly problematic in E. coli. Most full-length and catalytic domain constructs were expressed at very low levels or as insoluble proteins. Soluble protein was obtained by denaturation, treatment with detergents, non-denaturing extraction from inclusion bodies and fusion to solubility-enhancing proteins. However, no method yielded protein with catalytic activity or detectable secondary structure. Soluble expression of TbLRR-DSP was achieved using baculovirus-infected insect cells, but the protein co-purified with endogenous chaperones and exhibited no catalytic activity thus implying a lack of correct folding. In the second part of this study, two phosphatases specific to Leishmania major, LM1 and LM2, were characterised and structural studies were initiated. LM2 was shown to readily hydrolyse phospho-tyrosine substrates in vitro, but not phosphoinositides like its homologue, LM1. Both proteins therefore have a differentiated catalytic profile and are likely to have different functions in vivo. Purification protocols for both proteins were established and crystallisation screenings set up. Preliminary hits were obtained for LM2 and a mutagenesis strategy was developed to improve chances of obtaining diffraction quality crystals. Recombinant LM1 samples exhibited heterogeneity and therefore will require additional engineering to improve chances of crystallization. Promising pilot NMR data was also obtained for both phosphatases. In conclusion, this study demonstrates that the recombinant expression of multi- domain trypanosomatid proteins (like the LRR-DSPs) can be highly problematic and may pose a challenge for their biochemical characterisation and functional elucidation. Future work into trypanosomatid phosphatases, however challenging, will improve our understanding of their cell biology and potentially identify therapeutic targets.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:617952 |
| Date | January 2013 |
| Creators | Currin, Andrew |
| Contributors | Tabernero, Lydia; Bella, Jordi |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/a-study-of-protein-phosphatases-from-the-genomes-of-trypanosomatid-parasites(6afd7092-b7e6-4816-903b-647d77942e95).html |
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