Molecular characterisation of the thioredoxins in the parasitic nematode haemonchus contortus

Sotirchos, Irene M

Molecular characterisation of the thioredoxins in the parasitic nematode haemonchus contortus

Sotirchos, Irene M

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Publication Type:: Thesis
Issue Date:: 2008

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Field	Value	Language
dc.contributor.author	Sotirchos, Irene M
dc.date.accessioned	2016-04-20T03:54:17Z
dc.date.available	2016-04-20T03:54:17Z
dc.date.issued	2008
dc.identifier.uri	http://hdl.handle.net/10453/43677
dc.description	University of Technology, Sydney. Faculty of Science.	en_AU
dc.description.abstract	Thioredoxins are a family of small proteins conserved through evolution, essential for cellular homeostasis. The 'classic' thioredoxin, identified in most species, is a 12 kDa protein with a Cys-Gly-Pro-Cys (CGPC) active site. The thioredoxin system, composed of thioredoxin, thioredoxin reductase and peroxiredoxin, is essential to protect cells from metabolically produced reactive oxygen. This and the diversification of this system through evolution identified it as a target for the control of many diseases, including parasitic infections. This work characterises the thioredoxins of Haemonchus contortus, a parasite with increasing economic impact on sheep and wool production in Australia. Five thioredoxin proteins were identified, expressed and characterised (HcTrx1-5). H. contortus contained the classic thioredoxin (HcTrx1), but the major thioredoxin was a 16 kDa protein (HcTrx3) with a Cys-Pro-Pro-Cys (CPPC) active site, which is related to tryparedoxin, a unique protein in Trypanosomes. Both proteins were expressed through the lifecycle and both had a similar ability to reduce the disulphide bonds of insulin compared to the classic thioredoxins in Escherichia coli and sheep. Both proteins were regenerated by thioredoxin reductase, but unlike the ovine thioredoxin, both were also able to reduce oxidised glutathione, directly reduce hydrogen peroxide and indirectly reduce hydrogen peroxide coupled with H.contortus peroxiredoxin. Two thioredoxin-like proteins were identified with homology to thioredoxins reported in human cells, a 31 kDa protein with a CGPC classic active site (HcTrx2) and a 28 kDa protein with a Cys-Pro-Ala-Cys (CPAC) active site, a transmembrane domain and an endoplasmic reticulum localisation signal (HcTFx4). These had different activities to the classic HcTrx1 in that HcTrx2 could not directly reduce insulin, but could when coupled to thioredoxin reductase. In contrast, HcTrx4 could directly reduce insulin, but could not react with thioredoxin reductase. The results suggest that HcTrx4 is not a thioredoxin, but acts as a protein-disulphide isomerase (PDI). Other characterised PDis, contain at least two active sites, in contrast to the one active site in the H.contortus protein. HcTrx5 is a 20 kDa protein with a unique active site Cys-Arg-Ser-Cys (CRSC). Although this active site has a charge change, HcTrx5 was able to reduce insulin, be regenerated by thioredoxin reductase and react with H.contortus peroxiredoxin. However, HcTrx5 was also regenerated by glutathione reductase coupled with glutathione, showing it had the activity of a glutaredoxin as well as a thioredoxin, an activity not reported for any thioredoxin. This study characterised the thioredoxins of a parasitic nematode. The differences identified may provide new drug targets for the control of many tropical diseases for which drug resistance is emerging as a major problem. Preliminary investigations showed increased H.contortus thioredoxin expression in a drug resistant strain. HcTrxl was highly increased in ivermectin resistant parasites and may provide a marker of drug resistance.	en_AU
dc.format	Thesis (PhD)
dc.language.iso	en_AU	en_AU
dc.relation	https://opus.lib.uts.edu.au/bitstream/10453/43677/7/02Whole.pdf
dc.rights	The author owns the copyright in this thesis including all reproduction and reuse rights for the work. The work may not be altered without the permission of the copyright owner. Attribution is essential when quoting or paraphrasing from this thesis.
dc.rights	au.edu.uts.lib/ppc
dc.rights	info:eu-repo/semantics/openAccess
dc.title	Molecular characterisation of the thioredoxins in the parasitic nematode haemonchus contortus	en_AU
dc.type	Thesis	en_AU
utslib.copyright.status	open_access

Abstract:

Thioredoxins are a family of small proteins conserved through evolution, essential for cellular homeostasis. The 'classic' thioredoxin, identified in most species, is a 12 kDa protein with a Cys-Gly-Pro-Cys (CGPC) active site. The thioredoxin system, composed of thioredoxin, thioredoxin reductase and peroxiredoxin, is essential to protect cells from metabolically produced reactive oxygen. This and the diversification of this system through evolution identified it as a target for the control of many diseases, including parasitic infections. This work characterises the thioredoxins of Haemonchus contortus, a parasite with increasing economic impact on sheep and wool production in Australia. Five thioredoxin proteins were identified, expressed and characterised (HcTrx1-5). H. contortus contained the classic thioredoxin (HcTrx1), but the major thioredoxin was a 16 kDa protein (HcTrx3) with a Cys-Pro-Pro-Cys (CPPC) active site, which is related to tryparedoxin, a unique protein in Trypanosomes. Both proteins were expressed through the lifecycle and both had a similar ability to reduce the disulphide bonds of insulin compared to the classic thioredoxins in Escherichia coli and sheep. Both proteins were regenerated by thioredoxin reductase, but unlike the ovine thioredoxin, both were also able to reduce oxidised glutathione, directly reduce hydrogen peroxide and indirectly reduce hydrogen peroxide coupled with H.contortus peroxiredoxin. Two thioredoxin-like proteins were identified with homology to thioredoxins reported in human cells, a 31 kDa protein with a CGPC classic active site (HcTrx2) and a 28 kDa protein with a Cys-Pro-Ala-Cys (CPAC) active site, a transmembrane domain and an endoplasmic reticulum localisation signal (HcTFx4). These had different activities to the classic HcTrx1 in that HcTrx2 could not directly reduce insulin, but could when coupled to thioredoxin reductase. In contrast, HcTrx4 could directly reduce insulin, but could not react with thioredoxin reductase. The results suggest that HcTrx4 is not a thioredoxin, but acts as a protein-disulphide isomerase (PDI). Other characterised PDis, contain at least two active sites, in contrast to the one active site in the H.contortus protein. HcTrx5 is a 20 kDa protein with a unique active site Cys-Arg-Ser-Cys (CRSC). Although this active site has a charge change, HcTrx5 was able to reduce insulin, be regenerated by thioredoxin reductase and react with H.contortus peroxiredoxin. However, HcTrx5 was also regenerated by glutathione reductase coupled with glutathione, showing it had the activity of a glutaredoxin as well as a thioredoxin, an activity not reported for any thioredoxin. This study characterised the thioredoxins of a parasitic nematode. The differences identified may provide new drug targets for the control of many tropical diseases for which drug resistance is emerging as a major problem. Preliminary investigations showed increased H.contortus thioredoxin expression in a drug resistant strain. HcTrxl was highly increased in ivermectin resistant parasites and may provide a marker of drug resistance.

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