|M.Sc Thesis||Department of Biology|
|Supervisor:||Prof. Zilberstein Dan|
|Full Thesis text - in Hebrew|
Protozoan parasites of the genus Leishmania are the causative agents of a wide range of visceral and cutaneous diseases in humans. These organisms cycle between extracellular flagellated promastigotes in sand flies, the vectors that transmit the parasites, and the intracellular non flagellated amastigotes in phagolysosomes of mammalian macrophages, to whom they are pathogenic. Hence, similar to other parasites, Lieshmania undergo a developmental program, which results from changes they encounter in the environments of their vectors to hosts. An experimental system has been developed in our laboratory which creates an intralysosmal-like environment (e.g. 370c and pH 5.5 in a 5-7% CO2) and thereby induces promastigotes to undergo differentiation into amastigotes in a host free culture. Using this system, our group found that ethanol and azetidine-2-carboxylic acid (AZC), at sub lethal concentrations, induce promastigote to amastigote differentiation at acidic pH without raising the medium temperature. AZC and ethanol are well-established modulators of heat shock response by increasing cellular protein misfolding. We hypothesized that protein misfolding play a role in mediating the L. donovani differentiation signal. Protein misfolding in the endoplasmic reticulum of eukaryotes activates the unfolded protein response (UPR), a pathway that attenuates protein synthesis by phosphorylating the alfa subunit of the translation initiation factor eIF2. In the present study we explored eIF2's activity and protein synthesis during L. donovani differentiation.
L. donovani`s eIF2a was cloned and sequenced. Analysis of the predicted protein , revealed a unique, Trypanosomatidae-specific, phosphorylaton site in a highly conserved phosphorylation residue. The most striking difference was that the conserved serine 51, whose phosphorylation inhibits translation, was replaced by threonine. Additional changes in amino acids in the conserved site have been identified, which made the L. donovani phosphorylation box unique.
Assessment of protein synthesis during differentiation, using 35S-methionine incorporation, indicated that during the first three to five hours of differentiation protein synthesis underwent a 3-fold increase. The rate of protein synthesis gradually returned to that measured in promastigotes at approximately 24 hours after differentiation itnitiated. To confirm these results we isolated polysomes at various differentiation time points. Surprisingly, in the initial steps of differentiation, we detected a gradual decrease in polysomal quantity, an observation which conflicted the transient increase in protein synthesis. Furthermore, previous studies indicated that at heat shock promastigotes undergo cessation in protein synthesis. , We conclude that if protein misfolding mediates the differentiation via UPR, its mechanism is different from the classical heat shock response. Further studies are required to reveal this pathway.