Cutaneous leishmaniasis affects around 1 million people worldwide, where Leishmania mexicana is one of several Leishmania spp., which cause this form of the disease. At present, there are several treatments for leishmaniasis, however, these are far from ideal due to high cost, toxicity, severe side effects and coherent parasite resistance.
Protein kinases are potential drug targets for many diseases as they are central players in the homeostasis of all cells. In Leishmania the mitogen-activated protein kinase LmxMPK2 is of particular significance as double allele null mutants highlighted this kinase as crucial for parasite survival in mammalian hosts. In promastigotes, LmxMPK2 localisation has been pinpointed to both the posterior and anterior cell tip using GFP-tagged LmxMPK2. Moreover, this kinase might be involved in the cell cycle and/or cytokinesis affecting the cytoskeletal network. However, the LmxMPK2 signal transduction pathway has not yet been uncovered as kinase-substrate interactions are transient and difficult to prove in vivo.
This project focused on detecting interaction partners of LmxMPK2 by use of proximity-dependent biotin identification (BioID). BioID uses a mutant biotin ligase from E. coli known as BirA*, which catalyses the attachment of biotin to lysine amide groups of proteins in the vicinity (~10 nm radius). Analysis consisted of purifying proteins using streptavidin, immunoblotting, and mass spectrometry for identification of biotinylated proteins.
To verify BirA* activity an in vitro BioID assay using L. mexicana wild type cell lysates and recombinantly expressed BirA*-fusion peptides containing a phosphorylation site from the glucose transporter LmxGT2, and two kinesins LmxKin19 and LmxKin29 was used to identify the corresponding protein kinases. The BirA*HALmxKin29p fusion peptide showed biotinylation in the cell lysate establishing BirA* activity. The same BirA*-fusions were also expressed in L. mexicana promastigotes. Cell lines expressing BirA*HALmxKin19ps and BirA*HALmxKin29p exhibited biotinylation not observed in the wild type control samples following in vivo BioID and subsequent immunoblotting with anti-biotin antiserum. Further analysis revealed BirA*HALmxKin19ps biotinylated an unidentified protein consistently detected when blotting with anti-LmxMPK3 antiserum. This result suggests interaction between the protein and the LmxKin19ps region of the fusion peptide. BirA* was fused to the N-terminus of LmxMPK2 and fusion protein expression in L. mexicana promastigotes was confirmed by immunoblotting using antiserum specific for MPK2. Immunoblotting using anti-biotin antiserum of the detergent-soluble protein fraction following incubation with streptavidin displayed bands not present in the wild type control samples. To confirm that the addition of BirA* to LmxMPK2 had no effect on the localisation of the protein GFP was fused to the N-terminus of BirA*-tagged LmxMPK2 and shown in fluorescence microscopy to reside in the exact localisation of LmxMPK2-GFP. Seven candidate LmxMPK2 interaction partners were identified by mass spectrometry of the enriched biotinylated proteins using additional information about their localisation in T. brucei.
So far BirA has only been expressed in vivo in Leishmania tarentolae, however, biotin identification experiments have not been performed. Therefore, to my knowledge, this is the first successful BioID study in Leishmania, however, further study is required before any candidate(s) can be finally confirmed as interaction partner(s) and/or substrate(s) of LmxMPK2.
|Date of Award||25 Jun 2020|
- University Of Strathclyde
|Supervisor||Martin Wiese (Supervisor)|