Characterisation of a plasmodium falciparum type II Hsp40 chaperone exported to the cytosol of infected erythrocytes
- Authors: Maphumulo, Philile Nompumelelo
- Date: 2013
- Subjects: Erythrocytes , Heat shock proteins , Plasmodium falciparum , Molecular chaperones , Malaria -- Prevention -- Research , Protein folding , Proteins -- Analysis , Malaria -- Immunological aspects
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4128 , http://hdl.handle.net/10962/d1015681
- Description: Heat Shock 40 kDa proteins (Hsp40s) partner with heat shock 70 kDa proteins (Hsp70s) in facilitating, among other chaperone activities; correct protein transport, productive protein folding and assembly within the cells; under both normal and stressful conditions. Hsp40 proteins regulate the ATPase activity of Hsp70 through interaction with the J-domain. Plasmodium falciparum Hsp70s (PfHsp70s) do not contain a Plasmodium export element (PEXEL) sequence although PfHsp70-1 and PfHsp70-3 have been located outside of the parasitophorous vacuole. Studies reveal that a type I P. falciparum (PfHsp40) chaperone (PF14_0359) stimulates the rate of ATP hydrolysis of the cytosolic PfHsp70 (PfHsp70-1) and that of human Hsp70A1A. PFE0055c is a PEXEL-bearing type II Hsp40 that is exported into the cytosol of P. falciparum-infected erythrocytes; where it potentially interacts with human Hsp70. Studies reveal that PFE0055c associates with structures found in the erythrocyte cytosol termed “J-dots” which are believed to be involved in trafficking parasite-encoded proteins through the erythrocyte cytosol. If P. falciparum exports PFE0055c into the host cytosol, it may be proposed that it interacts with human Hsp70, making it a possible drug target. The effect of PFE0055c on the ATPase activity of human Hsp70A1A has not been previously characterised. Central to this study was bioinformatic analysis and biochemical characterisation PFE0055c using an in vitro (ATPase assay) approach. Structural domains that classify PFE0055c as a type II Hsp40 were identified with similarity to two other exported type II PfHsp40s. Plasmids encoding the hexahistidine-tagged versions of PFE0055c and human Hsp70A1A were used for the expression and purification of these proteins from Escherichia coli. Purification was achieved using nickel affinity chromatography. The urea-denaturing method was used to obtain the purified PFE0055c whilst human Hsp70A1A was purified using the native method. PFE0055c could stimulate the ATPase activity of alfalfa Hsp70, although such was not the case for human Hsp70A1A in vitro.
- Full Text:
- Date Issued: 2013
Characterisation of Human Hsj1a : an HSP40 molecular chaperone similar to Malarial Pfj4
- Authors: McNamara, Caryn
- Date: 2007
- Subjects: Heat shock proteins , Protein folding , Proteins -- Analysis , Proteins -- Structure , Plasmodium , Malaria , Molecular chaperones
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4083 , http://hdl.handle.net/10962/d1007603
- Description: Protein folding, translocation, oligomeric rearrangement and degradation are vital functions to obtain correctly folded proteins in any cell. The constitutive or stress-induced members of each of the heat shock protein (Hsp) families, namely Hsp70 and Hsp40, make up the Hsp70/Hsp40 chaperone system. The Hsp40 J-domain is important for the Hsp70-Hsp40 interaction and hence function. The type-II Hsp40 proteins, Homo sapiens DnaJ 1a (Hsj1a) and Plasmodium falciparum DnaJ 4 (Pfj4), are structurally similar suggesting possible similar roles during malarial infection. This thesis has focussed on identifying whether Hsj1a and Pfj4 are functionally similar in their interaction with potential partner Hsp70 chaperones. Analysis in silico also showed Pfj4 to have a potential chaperone domain, a region resembling a ubiquitin-interacting motif (UIM) corresponding to UIM1 of HsjIa, and another highly conserved region was noted between residues 232-241. The highly conserved regions within the Hsp40 J-domains, and those amino acids therein, are suggested to be responsible for mediating this Hsp70-Hsp40 partner interaction. The thermosensitive dnaJ cbpA Escherichia coli OD259 mutant strain producing type-I Agrobacterium tumefaciens DnaJ (AgtDnaJ) was used as a model heterologous expression system in this study. AgtDnaJ was able to replace the lack of two E coli Hsp40s in vivo, DnaJ and CbpA, whereas AgtDnaJ(H33Q) was unable to. AgtDnaJ-based chimeras containing the swapped J-domains of similar type-II Hsp40 proteins, namely Hsj1Agt and Pfj4Agt, were also able to replace these in E. coli OD259. Conserved J-domain amino acids were identified and were substituted in these chimeras. Of these mutant proteins, Hsj IAgt(L8A), Hsj1Agt(R24A), Hsj1Agt(H31Q), Pfj4Agt(L 11A) and Pfj4Agt(H34Q) were not able to replace the E. coli Hsp40s, whilst Pfj4Agt(Y8A) and Pfj4Agt(R27A) were only able to partially replace them. This shows the leucine of helix I and the histidine of the loop region are key in the in vivo function of both proteins and that the arginine of helix II is key for Hsj1a. The histidine-tagged Hsj1a protein was also successfully purified from the heterologous system. The in vitro stimulated ATPase activity of human Hsp70 by Hsj1a was found to be approximately 14 nmol Pí[subscript]/min/mg, and yet not stimulated by Pfj4, suggesting a possible species-specific interaction is occurring.
- Full Text:
- Date Issued: 2007
Identification of cis-elements and transacting factors involved in the abiotic stress responses of plants
- Authors: Maclear, Athlee
- Date: 2005 , 2013-06-10
- Subjects: Plants -- Effect of stress on , Proteins -- Analysis , Bioinformatics , DNA , Plant genetics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4074 , http://hdl.handle.net/10962/d1007236 , Plants -- Effect of stress on , Proteins -- Analysis , Bioinformatics , DNA , Plant genetics
- Description: Many stress situations limit plant growth, resulting in crop production difficulties. Population growth, limited availability and over-utilization of arable land, and intolerant crop species have resulted in tremendous strain being placed on agriculturalists to produce enough to sustain the world's population. An understanding of the principles involved in plant resistance to environmental stress will enable scientists to harness these mechanisms to create stress-tolerant crop species, thus increasing crop production, and enabling the farming of previously unproductive land. This research project uses computational and bioinformatics techniques to explore the promoter regions of genes, encoding proteins that are up- or down-regulated in response to specific abiotic stresses, with the aim of identifying common patterns in the cis-elements governing the regulation of these abiotic stress responsive genes. An initial dataset of fifty known genes encoding for proteins reported to be up- or down-regulated in response to plant stresses that result in water-deficit at the cellular level viz. drought, low temperature, and salinity, were identified, and a postgreSQL database created to store relevant information pertaining to these genes and the proteins encoded by them. The genomic DNA was obtained where possible, and the promoter and intron regions identified. The Neural Network Promoter Prediction (NNPP) software package was used to predict the transcription start signal (TSS) and the promoter searching software tool, TESS (Transcription Element Search Software) used to identify known and user-defined cis-elements within the promoter regions of these genes. Currently available promoter prediction software analysis tools are reported to predict one promoter per kilobase of DNA, whilst functional promoters are thought to only occur one in 30-40 kilobases, which indicates that a large perccntage of predictions are likely to be false positives (pedersen et. al., 1999). NNPP was chosen as it was rated as the highest performing promoter prediction software tool by Fickett and Hatzigeorgiou (1997) in a thorough review of eukaryotic promoter prediction algorithms, however results were less than promising as very few predicted TSS were identified in the area 50 bps up- and downstream of the gene start site, where biologically functional TSSs are known to occur (Reese, 2000; Fickett and Hatzigeorgiou, 1997). TESS results seemed to support the hypothesis that drought, low-temperature and high salinity plant stress response proteins have similar as-elements in their promoter regions, and suggested links to various other gene regulation mechanisms viz. gibberellin-, light-, auxin- and development-regulated gene expression, highlighting the vast complexity of plant stress response processes. Although far from conclusive, results provide a valuable basis for future comparative promoter studies that will attempt to deduce possible common transcriptional initiation of abiotic stress response genes. , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Date Issued: 2005
Stress-inducible protein 1: a bioinformatic analysis of the human, mouse and yeast STI1 gene structure
- Authors: Aken, Bronwen Louise
- Date: 2005
- Subjects: Molecular chaperones , Proteins -- Analysis , Heat shock proteins , Bioinformatics , Genetics -- Data processing
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3990 , http://hdl.handle.net/10962/d1004049 , Molecular chaperones , Proteins -- Analysis , Heat shock proteins , Bioinformatics , Genetics -- Data processing
- Description: Stress-inducible protein 1 (Sti1) is a 60 kDa eukaryotic protein that is important under stress and non-stress conditions. Human Sti1 is also known as the Hsp70/Hsp90 organising protein (Hop) that coordinates the functional cooperation of heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) during the folding of various transcription factors and kinases, including certain oncogenic proteins and prion proteins. Limited studies have been conducted on the STI1 gene structure. Thus, the aim of this study was to develop a comprehensive description of human STI1 (hSTI1), mouse STI1 (mSTI1), and yeast STI1 (ySTI1) genes, using a bioinformatic approach. Genes encoded near the STI1 loci were identified for the three organisms using National Centre for Biotechnology Information (NCBI) MapViewer and the Saccharomyces Genome Database. Exon/intron boundaries were predicted using Hidden Markov model gene prediction software (HMMGene) and Genscan, and by alignment of the mRNA sequence with the genomic DNA sequence. Transcription factor binding sites (TFBS) were predicted by scanning the region 1000 base pairs (bp) upstream of the STI1 orthologues’ transcription start site (TSS) with Alibaba, Transcription element search software (TESS) and Transcription factor search (TFSearch). The promoter region was defined by comparing the number, type and position of TFBS across the orthologous STI1 genes. Additional putative TFBS were identified for ySTI1 by searching with software that aligns nucleic acid conserved elements (AlignACE) for over-represented motifs in the region upstream of the TSS of genes thought to be co-regulated with ySTI1. This study showed that hSTI1 and mSTI1 occur in a region of synteny with a number of genes of related function. Both hSTI1 and mSTI1 comprised 14 putative exons, while ySTI1 was encoded on a single exon. Human and mouse STI1 shared a perfectly conserved 55 bp region spanning their predicted TSS, although their TATA boxes were not conserved. A putative CpG island was identified in the region from -500 to +100 bp relative to the hSTI1 and mSTI1 TSS. This region overlapped with a region of high TFBS density, suggesting that the core promoter region was located in the region approximately 100 to 200 bp upstream of the TSS. Several conserved clusters of TFBS were also identified upstream of this promoter region, including binding sites for stimulatory protein 1 (Sp1), heat shock factor (HSF), nuclear factor kappa B (NF-kappaB), and the cAMP/enhancer binding protein (C/EBP). Microarray data suggested that ySTI1 was co-regulated with several heat shock proteins and substrates of the Hsp70/Hsp90 heterocomplex, and several putative regulatory elements were identified in the upstream region of these co-regulated genes, including a motif for HSF binding. The results of this research suggest several avenues of future experimental work, including the confirmation of the proposed core promoter, upstream regulatory elements, and CpG island, and the investigation into the co-regulation of mammalian STI1 with its surrounding genes. These results could also be used to inform STI1 gene knockout experiments in mice, to assess the biological importance of mammalian STI1.
- Full Text:
- Date Issued: 2005
Electrochemical studies of metal-ligand interactions and of metal binding proteins
- Authors: Limson, Janice Leigh
- Date: 1999
- Subjects: Electrochemical analysis , Metals -- Analysis , Proteins -- Analysis , Electrochemistry -- Technique
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4551 , http://hdl.handle.net/10962/d1018239
- Description: Electrochemical methods were researched for the analysis of metals, proteins and the identification of metal binding proteins. Adsorptive cathodic stripping voltamrnetry for metal analysis combines the inherent sensitivity of electrochemical techniques with the specificity of ligands for the nonfaradaic preconcentration of analytes at the electrode. The utility of catechol, resorcinol, 4-methylcatechol and 4-t-butylcatechol as ligands was explored for the sensitive analysis of copper, bismuth, cadmium and lead on a mercury film glassy carbon electrode. Metal complexes of lead, copper and bismuth with resorcinol showed the largest increase in current with increase in metal concentration, whereas complexes of these metals with 4-t-butylcatechol showed the lowest current response. Cadmium showed the highest current responses with 4-methylcatechol. The four metals could be determined simultaneously in the presence of resorcinol, although considerable interference was observed between bismuth and copper. The electroanalysis of cysteine and cysteine containing proteins at carbon electrodes are impaired by slow electron transfer rates at carbon electrodes, exhibiting high overpotentials, greater than 1 V vs Ag! Agel. Metallophthalocyanines have been shown to promote the electrocatalysis of cysteine at lowered potentials. Chemical modification of electrodes with appropriate modifiers is a means of incorporating specificity into electroanalysis, with applications in electrocatalysis. A glassy carbon electrode was modified by electrodeposition of cobalt (II) tetrasulphophthalocyanine [Co(II)TSPct to produce a chemically modified glassy carbon electrode (CMGCE). The CoTSPc-CMGCE catalysed the oxidation of cysteine in the pH range 1 to 10. The significance of this electrode is an application for analysis of proteins at biological pH's. A biscyanoruthenium(II) phthalocyanine CMGCE catalysed the oxidation of cysteine at 0.43 V vs Ag/AgCl a significant lowering in the overpotential for the oxidation of cysteine. Metallothionein, a metal binding protein, is believed to be involved in metal homeostasis and detoxification in the peripheral organs of living systems. A method for the quantitative determination of this protein utilising its high cysteine content was presented. At pH 8.4 Tris-HCl buffer, and using a CoTSPc-CMGCE modified by electrodeposition of the modifier, the anodic peaks for the oxidation of metallothionein was observed at 0. 90 V vs Ag/ AgCI. Ferredoxin is a simple iron-sulphur protein. One tenth of its residues are cysteine. Ferredoxin is involved in simple electron transfer processes during photosynthesis and respiration. Electrochemical studies of spinach ferredoxin were conducted at a CoTSPc-CMGCE. Anodic currents for the oxidation of the cysteine fragment of ferredoxin was observed at 0.85 V vs Ag/AgCl in HEPES buffer at pH 7.4, representing a new method for analysis of this protein. Voltammetric studies of its ferric/ferrous transition have shown quasi-reversible waves atE~ -0.62 V vs Ag/AgCl only in the presence of promoters. At a CoTSPc-CMGCE, a cathodic wave attributed to the reduction of Fe(III)/Fe(II) was observed at Epc -0.34 V vs Ag/AgCl. This represents an alternative method for voltammetric studies of the ferric/ferrous transition at significantly lowered potentials. Melatonin, a pineal gland hormone functions m setting and entraining circadian rhythms and in neuroprotection as a free radical scavenger and general antioxidant. Using adsorptive cathodic stripping voltammetry, the binding affinities of melatonin, serotonin and tryptophan for metals, were measured. The results showed that the following metal complexes were formed: aluminium with melatonin, serotonin and tryptophan; cadmium with melatonin and tryptophan; copper with melatonin and serotonin; iron (III) with melatonin and serotonin; lead with melatonin, tryptophan and serotonin, zinc with melatonin and tryptophan and iron (II) with tryptophan. The studies suggest a further role for melatonin in the reduction of free radical generation and in metal detoxification and may explain the accumulation of aluminium in Alzheimer's disease.
- Full Text:
- Date Issued: 1999
A study of the molecular variation between orbivirus proteins
- Authors: Whistler, Toni
- Date: 1985 , 2013-03-13
- Subjects: Proteins -- Analysis , Polypeptides , Bluetongue virus , Orbivirus infections
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3892 , http://hdl.handle.net/10962/d1003290 , Proteins -- Analysis , Polypeptides , Bluetongue virus , Orbivirus infections
- Description: The aim of this study was to initiate a structural analysis of the capsid polypeptides from several serotypes of bluetongue virus in order to provide insight into the relatedness and possible origins of the different serotypes. Tryptic peptide mapping of ¹²⁵I-labelled group antigen by ion exchange chromatography was used to assess the structural relatedness of seven BTV serotypes from Southern Africa, North America and Australia. Each serotype had several tyrosine containing tryptic peptides which were unique, but approximately 35% of the peptides analyzed were found to be highly conserved between all 7 serotypes. BTV-20 appeared to be closely related to BTV-B and these two serotypes with BTV-4 and BTV-17 appeared to form a closely knit central cluster. , KMBT_363 , Adobe Acrobat 9.53 Paper Capture Plug-in
- Full Text:
- Date Issued: 1985