Thesis

Analysis of LmxMPK2 and LmxGSK3-β, two protein kinases important for the survival of Leishmania mexicana

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Awarding institution
  • University of Strathclyde
Date of award
  • 2013
Thesis identifier
  • T13786
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Department, School or Faculty
Abstract
  • Leishmaniasis is a tropical disease affecting around 12 million people worldwide with a further 350 million people at risk. Two million new cases are identified every year with an estimated 60,000 fatalities. Treatments have changed little in the past century and often patients are treated with increasingly ineffective and highly toxic drugs, thus highlighting the requirement for safer and more effective medication. Protein kinases have attracted a lot of attention as potential targets for treating a variety of human diseases, including leishmaniasis. LmxMPK2 is a mitogen-activated protein (MAP) kinase homologue in Leishmania mexicana that is expressed in both the amastigote and promastigote life stages. Generation of homozygous gene knock out mutants revealed a reduction in cell proliferation and a range of morphological alterations with cells showing multiple flagella, kinetoplasts and nuclei, lobed cell bodies, spiked posterior ends and division furrow ingression from the posterior end. Localisation studies using GFP-tagged LmxMPK2 revealed localisation at both poles of the cell. LmxMPK2 might play a role in the organisation of microtubules influencing cell shape and cytokinesis. Recombinant expression of LmxMPK2 resulted in an active enzyme already phosphorylated on tyrosine and threonine which is able to phosphorylate myelin basic protein (MBP) despite the absence of activation by a MAP kinase kinase. Co-expression with different phosphatases led to LmxMPK2 being dephosphorylated on tyrosine but not threonine residues, yet retaining the ability of tyrosine autophosphorylation maintaining equal levels of MBP phosphorylation. This suggests that LmxMPK2 is an unusual MAP kinase which is able to autophosphorylate on threonine and tyrosine residues of unknown localisation without affecting the activity of the enzyme. Investigations to determine whether a relationship existed between LmxMPK2 and LmxDIP13 (Deflagellation Inducible Protein), a protein shown to associate with microtubules and likely to be involved in cell division, were undertaken. N-terminally GFP-tagged LmxDIP13 was expressed in promastigotes of wild type and LmxMPK2 null mutant Leishmania. Localisation was punctate and found in singular or multiple spots following a discrete line throughout the cell. An increased percentage of null mutant promastigotes with an anterior localisation of GFPLmxDIP13 suggested a functional link between LmxDIP13 and LmxMPK2. Glycogen Synthase Kinase 3 (LmxGSK3-β) was investigated as a potential drug target. The phosphatase co-expression system was applied to recombinant LmxGSK3-β. However, it was not possible to fully dephosphorylate LmxGSK3-β on either threonine or tyrosine. Activity of LmxGSK3-β was discovered to be similar to that of human GSK3-β. Natural compounds Malabaricone B and C were isolated from Myristica plants and used for screening against LmxGSK3-β. Both compounds were identified as inhibitors of LmxGSK3-β and possess potent anti-leishmanial activity.
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DOI
Date Created
  • 2013
Former identifier
  • 1036468

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