Date of Award
5-2012
Document Type
Thesis
Degree Name
Master of Science (MS)
College/School
College of Science and Mathematics
Department/Program
Chemistry and Biochemistry
Thesis Sponsor/Dissertation Chair/Project Chair
John J. Siekeirka
Committee Member
Nina M. Goodey
Committee Member
Elena Petroff
Abstract
The disease, Leishmaniasis, is caused by the protozoal parasite Leishmania, which is transmitted by the bite of an infected Phlebotomine female sandfly. It is a significant health problem in tropical regions of the world and new therapeutic approaches for treating this disease are urgently needed.
Protozoal MAPKs (Mitogen Activated Protein Kinases) play important roles in parasite viability and infectivity and as such, represent viable drug targets. It has been demonstrated that LmxMPKl is an essential MAPK required for the parasite to establish infection and for proliferation of the amastigote stage (the mammalian stage of the parasite) of the parasite. In our study we focused on LmxMPKl (Leishmania mexicana mitogen-activated protein kinase 1), which shares sequence homology to human ERK8 and p38 MAPK.
In our study we have successfully expressed LmxMPKl enzyme in HEK293T cells and BL21-AI cells. Mammalian expressed LmxMPKl was largely inactive but demonstrated low level basal activity as assessed by incorporation of the [y p] labeled ATP into MBP. In this assay activity was inhibited by BIRB796. On the other hand, bacterial expressed LmxMPKl, when assayed using an assay measuring consumption of ATP, was active but did not phosphorylate MBP which suggests we are measuring autophosphorylation of the enzyme itself. Interestingly, BIRB796 did not exhibit inhibitory activity in this assay.
In collaboration with Dr. Goodey, the direct binding of BIRB796 to active and inactive (dephosphorylated) LmxMPKl was assessed using tryptophan fluorescence quenching. Surprisingly, BIRB796 exhibited high affinity binding only to active LmxMPKl.
My results suggest that BIRB796 binds to active LmxMPKl with high affinity, but does not result in inhibition of the enzyme. This unusual result may indicate varying conformational or phosphorylation states of the enzyme depending upon the source (i.e. mammalian or bacterial) of recombinant enzyme.
File Format
Recommended Citation
Sayakci, Aysenur, "A Leishmania Mitogen Activated Protein Kinase as a Potential Anti-Parasitic Drug Target : Purification, Characterization and Inhibitor Interactions" (2012). Theses, Dissertations and Culminating Projects. 973.
https://digitalcommons.montclair.edu/etd/973