Analysis of Hsp90 in nematodes and its role in drug resistance

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

Long-chain polyunsaturated fatty acid biosynthesis in a land-crab with advanced terrestrial adaptations: Molecular cloning and functional characterization of two fatty acyl elongases

Depending on the presence and activities of the front-end fatty acyl desaturases and elongation of very long-chain fatty acid (Elovl) enzymes, animals have different capacities for long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA) biosynthesis. Successful land colonisation in brachyuran crabs requires a shift towards terrestrial food chain with limited LC-PUFA availability. We cloned and functionally characterised two elovl genes from the purple land crab Gecarcoidea lalandii. The two Elovl contained all the necessary motifs of a typical polyunsaturated fatty acids (PUFA) Elovl and phylogenetically clustered in the Elovl1 and Elovl6 clades, respectively. The G. lalandii Elovl1 elongated saturated fatty acids, with low activities towards C20 and C22 PUFA substrates. Moreover, the G. lalandii Elovl6 was particularly active in the elongation of C18 PUFA, although it also recognised monounsaturated fatty acids as substrates for elongation. Collectively, the herein characterised G. lalandii elovl paralogues fulfil all the elongation steps involved in the LC-PUFA biosynthetic pathways. Tissue distribution of the G. lalandii elovl genes, along with the FA composition analyses, suggest the hepatopancreas and gill as key metabolic sites for fatty acid elongation. However, current data suggest that G. lalandii is unable to rely solely on biosynthesis to fulfil LC-PUFA requirements, since front-end desaturase appears to be absent in this species and other decapods.We are grateful to the Ministry of Higher Education Malaysia for Fundamental Research Grant Scheme with Project Code: FRGS/1/2018/STG05/USM/01/4. Moreover, this study was partly funded through the project IMPROMEGA of the Ministry of Science, Innovation and Universities, Spanish Government (RTI2018-095119-B-100, MCIU/AEI/FEDER/UE). We also thank Universiti Sains Malaysia for the appointment of Óscar Monroig as Adjunct Academic Fellow USM. 9/1/v Jld. XIII)

The Effect of Macrocyclic Lactones-Ivermectin Exposure on Egg Hatching and Larval Development of Caenorhabditis elegans

Abstract: To date, the ivermectin resistance in nematode parasites has been reported and many studies are carried out to determine the causes of this problem. A free-living Caenorhabditis elegans is used as a model system for this study to investigate the response of C. elegans to ivermectin exposure by using larval development assay. Worms were exposed to ivermectin at concentration from 1 ng/mL to 10 ng/mL and dimethyl sulphoxide (DMSO) as a control. The developments of the worms were monitored for 24, 48, 72, and 96 hours until the worms become adults. Results indicated that worms&apos; growth began to be affected by ivermectin at a concentration of 5 ng/mL, while at the concentration of 6, 7, 8, 9, and 10 ng/mL, the growth of worms were inhibited compared to control worms. Further study of the protein expression in C. elegans should be done to investigate the up-regulated and down-regulated proteins involve in ivermectin resistance