In silico characterisation of the four canonical plasmodium falciparum 70 kDa heat shock proteins
- Authors: Hatherley, Rowan
- Date: 2012
- Subjects: Heat shock proteins -- Research , Plasmodium falciparum -- Research , Plasmodium -- Research , Endoplasmic reticulum , Cytosol , Mitochondria -- Formation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4026 , http://hdl.handle.net/10962/d1004086 , Heat shock proteins -- Research , Plasmodium falciparum -- Research , Plasmodium -- Research , Endoplasmic reticulum , Cytosol , Mitochondria -- Formation
- Description: The 70 kDa heat shock proteins expressed by Plasmodium falciparum (PfHsp70s) are believed to be essential to both the survival and virulence of the malaria parasite. A total of six Hsp70 genes have been identified in the genome of P. falciparum. However, only four of these encode canonical Hsp70s, which are believed to localise predominantly in the cytosol (PfHsp70-1 and PfHsp70-x), the endoplasmic reticulum (PfHsp70-2) and mitochondria (PfHsp70-3) of the parasite. These proteins bind and release peptide substrates in an ATP-dependent manner, with the aid of a J-domain protein cochaperone and a nucleotide exchange factor (NEF). The aim of this study was to identify the residues involved in the interaction of these PfHsp70s with their peptide substrates, their J-domain cochaperones and potential NEFs. These residues were then mapped to three-dimensional (3D) structures of the proteins, modelled in three different conformations; each representing a different stage in the ATPase cycle. Additionally, these proteins were compared to different types of Hsp70s from a variety of different organisms and sequence features found to be specific to each PfHsp70 were mapped to their 3D structures. Finally, a novel modelling method was suggested, in which the structures of templates were remodelled to improve their quality before they were used in the homology modelling process. Based on the analysis of residues involved in interactions with other proteins, it was revealed that each PfHsp70 displayed features that were specific to its cellular localisation and each type of Hsp70 was predicted to interact with a different set of NEFs. The study of conserved features in each PfHsp70 revealed that PfHsp70-x displayed various sequence features atypical of both Plasmodium cytosolic Hsp70s and cytosolic Hsp70s in general. Additionally, residues conserved specifically in Hsp70s of Apicomplexa, Plasmodium and P. falciparum were identified and mapped to the each PfHsp70 model. Although these residues were too numerous to reveal any information of specific value, these models may be useful for the purposes of aiding the design of drug compounds against each PfHsp70. Finally, the novel modelling approach did show some promise. Half of the models produced using the modified templates were of a higher quality than their counterparts modelled using the original templates. This approach does still require a lot of validation work and statistical evaluation. It is hoped that it could prove to be a useful approach to homology modelling when the only templates available are poor quality structures.
- Full Text:
- Date Issued: 2012
- Authors: Hatherley, Rowan
- Date: 2012
- Subjects: Heat shock proteins -- Research , Plasmodium falciparum -- Research , Plasmodium -- Research , Endoplasmic reticulum , Cytosol , Mitochondria -- Formation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4026 , http://hdl.handle.net/10962/d1004086 , Heat shock proteins -- Research , Plasmodium falciparum -- Research , Plasmodium -- Research , Endoplasmic reticulum , Cytosol , Mitochondria -- Formation
- Description: The 70 kDa heat shock proteins expressed by Plasmodium falciparum (PfHsp70s) are believed to be essential to both the survival and virulence of the malaria parasite. A total of six Hsp70 genes have been identified in the genome of P. falciparum. However, only four of these encode canonical Hsp70s, which are believed to localise predominantly in the cytosol (PfHsp70-1 and PfHsp70-x), the endoplasmic reticulum (PfHsp70-2) and mitochondria (PfHsp70-3) of the parasite. These proteins bind and release peptide substrates in an ATP-dependent manner, with the aid of a J-domain protein cochaperone and a nucleotide exchange factor (NEF). The aim of this study was to identify the residues involved in the interaction of these PfHsp70s with their peptide substrates, their J-domain cochaperones and potential NEFs. These residues were then mapped to three-dimensional (3D) structures of the proteins, modelled in three different conformations; each representing a different stage in the ATPase cycle. Additionally, these proteins were compared to different types of Hsp70s from a variety of different organisms and sequence features found to be specific to each PfHsp70 were mapped to their 3D structures. Finally, a novel modelling method was suggested, in which the structures of templates were remodelled to improve their quality before they were used in the homology modelling process. Based on the analysis of residues involved in interactions with other proteins, it was revealed that each PfHsp70 displayed features that were specific to its cellular localisation and each type of Hsp70 was predicted to interact with a different set of NEFs. The study of conserved features in each PfHsp70 revealed that PfHsp70-x displayed various sequence features atypical of both Plasmodium cytosolic Hsp70s and cytosolic Hsp70s in general. Additionally, residues conserved specifically in Hsp70s of Apicomplexa, Plasmodium and P. falciparum were identified and mapped to the each PfHsp70 model. Although these residues were too numerous to reveal any information of specific value, these models may be useful for the purposes of aiding the design of drug compounds against each PfHsp70. Finally, the novel modelling approach did show some promise. Half of the models produced using the modified templates were of a higher quality than their counterparts modelled using the original templates. This approach does still require a lot of validation work and statistical evaluation. It is hoped that it could prove to be a useful approach to homology modelling when the only templates available are poor quality structures.
- Full Text:
- Date Issued: 2012
Structural analysis of effects of mutations on HIV-1 subtype C protease active site
- Mathu, Alexander Muchugia Nganga
- Authors: Mathu, Alexander Muchugia Nganga
- Date: 2012
- Subjects: HIV (Viruses) -- Research , HIV infections -- Treatment -- Research , Protease inhibitors -- Research , Viruses -- Effect of drugs on -- Research
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4013 , http://hdl.handle.net/10962/d1004073 , HIV (Viruses) -- Research , HIV infections -- Treatment -- Research , Protease inhibitors -- Research , Viruses -- Effect of drugs on -- Research
- Description: HIV/AIDS is a global pandemic that poses a great threat especially in Sub-Saharan Africa where the highest population of those infected with the virus is found. It has far reaching medical, socio-economic and scientific implications. The HIV-1 protease enzyme is a prime therapeutic target that has been exploited in an effort to reduce morbidity and mortality. However problems arise from drug toxicity and drug-resistant mutations of the protease which is a motivation for research for new, safer and effective therapies. Evidence exists to show that there are significant genomic differences in Subtype B and C that have a negative effect on the intrinsic binding of inhibitors. It is imperative to look at all perspectives from epidemiological, molecular to the pharmacological ones so as to achieve rational design of therapeutic agents. This study involved the use of in silico structural analysis of the effects of mutations in the active site. The data was provided by the National Institute of Communicable Diseases consisting of HIV-1 Subtype C protease sequences of 29 infants exhibiting drug-resistance to ritonavir and lopinavir. The major active site mutations causing drug resistance identified in this study were M46I, I54V and V82A using the Stanford HIV database tool. Homology modeling without extra restraints produced models with improved quality in comparison to those with restraints. MetaMQAPII results differed when models were visualized as dimers giving erroneous modeled regions in comparison to monomers. A broader study with a larger dataset of HIV-1 subtype C protease sequences is required to increase statistical confidence and in order to identify the pattern of drug resistant mutations. Homology modeling without extra restraints is preferred for calculating homology models for the HIV-1 subtype C. Further investigations needs to be done to ascertain the accuracy of validation results for dimers from MetaMQAPII as it is designed for evaluation of monomers.
- Full Text:
- Date Issued: 2012
- Authors: Mathu, Alexander Muchugia Nganga
- Date: 2012
- Subjects: HIV (Viruses) -- Research , HIV infections -- Treatment -- Research , Protease inhibitors -- Research , Viruses -- Effect of drugs on -- Research
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4013 , http://hdl.handle.net/10962/d1004073 , HIV (Viruses) -- Research , HIV infections -- Treatment -- Research , Protease inhibitors -- Research , Viruses -- Effect of drugs on -- Research
- Description: HIV/AIDS is a global pandemic that poses a great threat especially in Sub-Saharan Africa where the highest population of those infected with the virus is found. It has far reaching medical, socio-economic and scientific implications. The HIV-1 protease enzyme is a prime therapeutic target that has been exploited in an effort to reduce morbidity and mortality. However problems arise from drug toxicity and drug-resistant mutations of the protease which is a motivation for research for new, safer and effective therapies. Evidence exists to show that there are significant genomic differences in Subtype B and C that have a negative effect on the intrinsic binding of inhibitors. It is imperative to look at all perspectives from epidemiological, molecular to the pharmacological ones so as to achieve rational design of therapeutic agents. This study involved the use of in silico structural analysis of the effects of mutations in the active site. The data was provided by the National Institute of Communicable Diseases consisting of HIV-1 Subtype C protease sequences of 29 infants exhibiting drug-resistance to ritonavir and lopinavir. The major active site mutations causing drug resistance identified in this study were M46I, I54V and V82A using the Stanford HIV database tool. Homology modeling without extra restraints produced models with improved quality in comparison to those with restraints. MetaMQAPII results differed when models were visualized as dimers giving erroneous modeled regions in comparison to monomers. A broader study with a larger dataset of HIV-1 subtype C protease sequences is required to increase statistical confidence and in order to identify the pattern of drug resistant mutations. Homology modeling without extra restraints is preferred for calculating homology models for the HIV-1 subtype C. Further investigations needs to be done to ascertain the accuracy of validation results for dimers from MetaMQAPII as it is designed for evaluation of monomers.
- Full Text:
- Date Issued: 2012
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