Nik Him, Nik Ahmad Irwan Izzauddin
Analysis of Hsp90 in nematodes and its role in drug resistance.
PhD thesis, University of Glasgow.
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The exposure of cells to environmental stresses, including heat shock, heavy metals, alcohols or oxidative stress results in the cellular accumulation of a number of chaperones, commonly known as heat shock proteins (Hsps). Hsps maintains the correct folding and conformation of proteins and are vital in regulating the stability between protein synthesis and degradation. Hsps are not only fundamental in the cellular stress response but are also important in regulating numerous important normal functions, such as cell proliferation and apoptosis. The results presented in the thesis focus on Hsp90 in nematodes, its possible roles in acquired drug resistance and further characterisation of Brugia pahangi Hsp90.
Hsp90 plays a key role in the cellular stress response by interacting with proteins after their native conformation has been altered by stress. Hsp90 is essential in all eukaryotes and plays a central role in multiple cellular processes. Since knock-out of hsp90 is lethal to most eukaryotes, inhibitors of Hsp90 have been widely used to study its function. The most widely used inhibitor is geldanamycin (GA). GA binds to the N-terminal/ATP binding site of Hsp90 which results in conformational changes and the degradation of client proteins. Although the gene encoding hsp90 has been characterized, little is known regarding its function in parasitic nematodes. Previous studies have shown that Caenorhabditis elegans Hsp90 is unique amongst eukaryotes because it fails to binds GA. This was suggested as an example of ‘adaptive evolution’ because C. elegans inhabits the same ecological niche of the soil as the Streptomyces species which produces GA. In contrast B. pahangi Hsp90 does bind to GA. The first objective of this project was to determine whether the resistance of C. elegans Hsp90 to GA is the norm or the exception amongst nematodes and to determine whether GA binding is associated with particular clades of nematodes. The results showed that the ability of Hsp90 to bind GA is associated with the life-cycle of the nematode. Free-living species or those that have a free-living larval stage in the soil do not bind GA, while those species which are obligate parasites (Trichinella and the filarial worms), or which are enclosed within a protective egg shell while in the environment (Ascarids), possess an Hsp90 that binds GA. These data support the adaptive evolution hypothesis.
Further characterisation of B. pahangi Hsp90 showed that it was expressed in all life-cycle stages but in the pull-down assay, only adult B. pahangi Hsp90 bound to GA, not Hsp90 from L3 or Mf. The inability to detect L3 or Mf Hsp90 binding to GA beads could perhaps reflect a lack of sensitivity of the assay, or alternatively may suggest that Hsp90 from these stages is processed in such way that it fails to bind to GA. In addition, only a small amount (6.3%) of B. pahangi Hsp90 bound to GA after a series of pull-down assays. Whether these data represents an accurate reflection of the amount of Hsp90 that can bind to GA is not known, but if correct, the results suggest that it might be necessary to inhibit only a small percentage of total Hsp90 to kill the worm.
Attempts were make to identify proteins that associate with Hsp90 by comparing the proteome of GA-treated adult B. pahangi with control worms. In this analysis, several proteins with structural functions were identified. These proteins were down-regulated after exposure to GA at 1.0 µM. Comparison of Hsp90 levels in B. pahangi and C. elegans showed that Hsp90 was expressed at relatively higher levels in B. pahangi (p<0.05) but there was no significant difference in the affinity of B. pahangi or C. elegans for Hsp90 binding to ATP beads. Results in Chapter 4 showed that the binding of Hsp90 to GA beads was competed by free GA or ATP. In addition, novobiocin, another Hsp90 inhibitor which binds at C-terminal domain of Hsp90 also inhibited binding of B. pahangi Hsp90 to GA beads, suggesting an interaction between the N-terminal and C-terminal of Hsp90.
Hsp90 has also been shown to be involved in the acquisition of drug resistance in fungi. A previous study on fungi showed that the emergence of fluconazole resistance depended on high levels of Hsp90 and was abolished when Hsp90 expression was reduced. So, given the conservation of Hsp90 it is possible that Hsp90 may be involved in the acquired resistance of nematodes to anthelmintic. The analysis carried out did not produce any correlation between expression levels of Hsp90 and drug resistance in nematodes. RNAi experiments were carried out to reduce Hsp90 levels in C. elegans and to investigate whether Hsp90 may play a role in the tolerance of ivermectin-resistant C. elegans to drug. Results showed that reducing Hps90 levels altered the sensitivity of ivermectin-resistant C. elegans to drug.
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