Ph.D. / In plants, salicylic acid (SA) is a signaling molecule that regulates disease resistance responses such as systemic acquired resistance (SAR) and the hypersensitive response (HR), and has been implicated in both basal and acquired thermotolerance. It has also been shown that SA enhances heat-induced Hsp/Hsc70 accumulation in plants. In this study, temperature studies revealed that heat shock (HS) at 40 °C for 30 min significantly induced Hsp/Hsc70 accumulation in 3-week old tomato (UC82B) seedlings. Time- and dose-responsive studies showed that 0.1 mM SA for 17 hrs was unable to induce Hsp/Hsc70 but in combination with HS significantly (P > 0.001) potentiated this response. To investigate the mechanism of SA-mediated, heat-induced Hsp/Hsc70 potentiation, tomato seedlings were treated with either SA alone, HS or both, before analyses of hsp70 mRNA, Hsf DNA-binding and gene expression of hsp70, hsfAl, hsfA2 and hsfEll. SA alone established Hsf DNA-binding, but was not accompanied by increased Hsp70 accumulation or expression of hsp70 mRNA. SA had no significant effect on hsfA2 and hsf81 gene expression, but increased the basal levels of hsfAl. In heat-shocked plants, Hsf DNA-binding was enhanced, and increased hsfAl, hsfA2 and hsfB1 expression preceded accumulation of Hsp70. The combined treatment of SA and HS resulted in potentiated Hsf DNA-binding, enhanced expression of hsp70, hsfAl, hsfA2 and hsfB1, leading to potentiated levels of Hsp/Hsc70. Since increased hsp70 and hsf gene expression coincided with increased levels of Hsp70 accumulation, it is likely that the SA-mediated potentiation of Hsp70 is due to the ability of SA to regulate Hsfs during HS. This study therefore proposes a mechanism for the potentiation of Hsp70 by SA in the presence of heat, which might contribute to our understanding of the role SA plays in the heat shock response and thermotolerance.

Using iron super-paramagnetic and ferromagnetic nanoparticles composed of Fe3O4 molecules, scientists analyze the effectiveness and practicality of this new treatment theory, hyperthermia. The problems of magnetic particle density, isothermal barriers/cellular cooling thresholds, and nanoparticle specific targeting are addressed in this review. Iron magnetic nanoparticles were chosen due to their relatively low biological reactivates and lack of subsequent cellular toxicity. However, there are significant heating problems associated with these magnetic nanoparticles due to their relative size and short thermal time constants or thermal half-lives. Effectively, these aforementioned issues create a phenomenon where cancerous cells, surrounded by unheated healthy tissue, exhibit properties similar to those of an isothermal barrier. As a result, target cells experience limited gross heating, which is localized to the area directly surrounding the active magnetic nanoparticle within the cytoplasm. The effects of isothermal barriers and HSP up regulation on particle-based hypothermia are profound and prevent therapeutic temperatures from being achieved in single cell heating limiting the applications for Fe3O4 magnetic nanoparticle hyperthermia applications. It has been shown that reaching a certain magnetic nanoparticle density within the cell can result in a larger heating capacity, though this effect is also dependent on the particle dispersion pattern within cytoplasm. It has yet to be concluded whether ferromagnetic particles or super-paramagnetic particles are superior or more practical for hyperthermic treatments as they each have distinct benefits, and further study is needed. Finally, the popular targeting mechanism associated with magnetic nanoparticle research, monoclonal antibodies, require that they have an organic coating (such as starch) as a means of both providing an organic binding point and as camouflage for avoiding host filtration pathways. Forgoing this organic coating could lead to increased particle density within the cell and the adoption of a more specific targeting mechanism such as virus like particles (VLPs) altered to target HSP’s could lead to an increase in yield. Furthermore the up regulation of HSPs in response to therapeutic temperature is problematic for the therapies practically.

HSP70 is one of the most important families of molecular chaperone that regulate the folding and transport of client proteins in an ATP dependent manner. The ATPase activity of HSP70 is stimulated through an interaction with its family of HSP40 co-chaperones. There is evidence to suggest that specific partnerships occur between the different HSP40 and HSP70 isoforms. While some of the residues involved in the interaction are known, many of the residues governing the specificity of HSP40-HSP70 partnerships are not precisely defined. It is not currently possible to predict which HSP40 and HSP70 isoforms will interact. We attempted to use bioinformatics to identify residues involved in the specificity of the interaction between the J domain from HSP40 and the ATPase domain from the HSP70 isoforms from humans. A total of 49 HSP40 and 13 HSP70 sequences from humans were retrieved and used for subsequent analyses. The HSP40 J domains and HSP70 ATPase domains were extracted using python scripts and classified according to the subcellular localization of the proteins using localization prediction programs. Motif analysis was carried out using the full length HSP40 proteins and Multiple Sequence Alignment (MSA) was performed to identify conserved residues that may contribute to the J domain – ATPase domain interactions. Phylogenetic inference of the proteins was also performed in order to study their evolutionary relationship. Homology models of the J domains and ATPase domains were generated. The corresponding models were docked using HADDOCK server in order to analyze possible putative interactions between the partner proteins using the Protein Interactions Calculator (PIC). The level of residue conservation was found to be higher in Type I and II HSP40 than in Type III J proteins. While highly conserved residues on helixes II and III could play critical roles in J domain interactions with corresponding HSP70s, conserved residues on helixes I and IV seemed to be significant in keeping the J domain in its right orientation for functional interactions with HSP70s. Our results also showed that helixes II and III formed the interaction interface for binding to HSP70 ATPase domain as well as the linker residues. Finally, data based docking procedures, such as applied in this study, could be an effective method to investigate protein-protein interactions complex of biomolecules.

Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

>Magister Scientiae - MSc / Salvia hispanica L. is an annual pseudocereal food crop, locally known as chia that has the ability to grow in water stress environments. The importance of chia dates back to the pre-columbian era where it was consumed as staple food by the indigenous South Americans due to its high nutritional and medicinal benefits. A single chia plant produces two seed variants: white seed genotype (denoted as WSG) and black seed genotype (denoted as BSG). Chia seeds have been proven to have a huge potential as a healthy food source and contained various medicinal properties. However, these plants are still prone to environmental stress conditions such as salinity that is one of the major abiotic stresses that influence crop production and yield worldwide. Despite the nutritional impact of the chia seeds, limited information regarding their molecular responses to abiotic stress conditions are known. This study was divided into two distinct parts. Firstly, the study comparatively analysed the leaf proteomes of two chia genotypes using gelbased proteomic analysis coupled with mass spectrometry. Total soluble proteins were extracted from chia leaves and subjected to 2-D PAGE analysis. Proteins were visualized by CBB and identified by MALDI-TOF MS/MS. A total of 284 and 209 spots were detected in WSG and BSG, respectively. Using mass spectrometry, 36 differentially expressed protein spots were successfully identified based on their protein abundance using homology database searches. Interestingly, two defensive-related proteins (osmotin-like protein and the chalcone isomerase) were only present in WSG and absent in BSG. In light of previous information regarding the nutritional profiles (no significant difference) of these two genotypes, this study has shown that there are distinct molecular differences between these genotypes. Therefore, WSG will be used in further downstream analysis. The second part of this study focused on the influence of salt stress (imposed by 100 mM NaCl) on the leaf proteome of WSG. Using gel-based proteomic analysis, 61 differentially expressed proteins were identified and classified into nine functional categories. Most of the proteins identified in this study were upregulated by salt stress. Interesting to note, 12 proteins identified in this study were only present in response to salt stress but were absent in the control. These proteins include ATP-dependent zinc metalloprotease FTSH 2 (spot 48), HSP70 proteins (spots 46 and 47), superoxide dismutases (spots 10, 41 and 42) and an ascorbate peroxidase (spot 56). All these proteins are important antioxidants that play a significant role in scavenging reactive oxygen species (ROS). Previous studies have shown that these antioxidants play vital roles in stress tolerance. These proteins could serve as potential biomarkers that could be used to enhance salt stress tolerance in pseudocereals and cereal food crops. / National Research Foundation (NRF) and Agricultural Research Council

Plasmodium falciparum is a protozoan parasite that causes a severe form of malaria, a mosquito-borne infectious disease in humans. P. falciparum encodes a number of proteins to facilitate its life-cycle, including a type II heat shock protein 40 (Hsp40), Pfj2. Pfj2 shows a degree of homology to human ERdj5, a resident protein of the endoplasmic reticulum (ER) that promotes protein quality control by facilitating the degradation of misfolded proteins. The overall aim of this study was to further understand the function of Pfj2 in the P. falciparum cell by characterising it biochemically. A bioinformatic analysis of Pfj2 was carried out to enable the identification of a potential ER signal sequence and cleavage site. Furthermore, an analysis of Pfj2 protein sequence was performed to compare domain similarities and identities with typical type II Hsp40s namely, human ERdj5, S. cerevisiae Sis1, human Hsj1a and human DnaJB4. The method used included the insertion of the codon-optimised coding sequence for the processed ER form of Pfj2 into the prokaryotic expression vector, pQE30, to enable overproduction of a histidine-tagged protein. A 62 kDa His₆-Pfj2 was successfully expressed in Escherichia coli and purified using denaturing nickel affinity chromatography. ATPase assays were performed to determine the ability of His₆- Pfj2 to stimulate the chaperone activity of the ER Hsp70, also called immunoglobulin binding protein (BiP). Initial studies were conducted on readily available mammalian His₆-BiP as a control, which was shown to have an intrinsic activity of 12.07±3.92 nmolPi/min/mg. His₆- Pfj2 did not stimulate the ATPase activity of mammalian His₆-BiP, suggesting that it either could not act as a co-chaperone of mammalian His₆-BiP (specificity), or it required a misfolded substrate in the system. Therefore, ongoing studies are addressing the interaction of Pfj2 and misfolded substrates with P. falciparum BiP. The results of these studies will further our understanding of a poorly-studied parasite chaperone that represents a potential drug target for development of novel strategies for the control of a serious human disease

The heat shock proteins of ~ 70 kDa (Hsp70s) are a conserved group of molecular chaperones important in maintaining the protein homeostasis in cells, carrying out functions including refolding of misfolded or unfolded proteins. Hsp70s function in conjunction with a number of other proteins including Hsp40 cochaperones. Central to the regulation Hsp70 activity is the Hsp70 ATPase cycle, involving ATP hydrolysis by Hsp70, and stimulation of this ATP hydrolysis by Hsp40. PfHsp70-1, the major cytosolic Hsp70 in the malaria parasite, Plasmodium falciparum, and PfHsp70-x, a novel malarial Hsp70 recently found to be exported to the host cell cytosol during the erythrocytic stages of the P. falciparum lifecycle, are both thought to play important roles in the malaria parasite’s survival and virulence, and thus represent novel antimalarial targets. Modulation of the function of these proteins by small molecules could thus lead to the development of antimalarials with novel targets and mechanisms. In the present study, malarial Hsp70s (PfHsp70-1 and PfHsp70-x), human Hsp70 (HSPA1A), malarial Hsp40 (PfHsp40) and human Hsp40 (Hsj1a) were recombinantly produced in Escherichia coli. In a characterisation of the chaperone activity of recombinant PfHsp70-x, the protein was found to have a basal ATPase activity (15.7 nmol ATP/min/mg protein) comparable to that previously described for PfHsp70-1, and an aggregation suppression activity significantly higher than that of PfHsp70-1. In vitro assays were used to screen five compounds of interest (lapachol, bromo-β-lapachona and malonganenones A, B and C) belonging to two compound classes (1,4 naphthoquinones and prenylated alkaloids) for modulatory effects on PfHsp70-1, PfHsp70-x and HsHsp70. A wide range of effects by compounds on the chaperone activities of Hsp70s was observed, including differential effects by compounds on different Hsp70s despite high conservation (≥ 70 % sequence identity) between the Hsp70s. The five compounds were shown to interact with all three Hsp70s in in vitro binding studies. Differential modulation by compounds was observed between the Hsj1a-stimulated ATPase activities of different Hsp70s, suggestive of not only a high degree of specificity of compounds to chaperone systems, but also distinct interactions between different Hsp70s and Hjs1a. The effects of compounds on the survival of P. falciparum parasites as well as mammalian cells was assessed. Bromo-β-lapachona was found to have broad effects across all systems, modulating the chaperone activities of all three Hsp70s, and showing significant toxicity toward both P. falciparum parasites and mammalian cells in culture. Malonganenone A was found to modulate only the malarial Hsp70s, not human Hsp70, showing significant toxicity toward malarial parasites (IC₅₀ ~ 0.8 μM), and comparatively low toxicity toward mammalian cells, representing therefore a novel starting point for a new class of antimalarials potentially targeting a new antimalarial drug target, Hsp70.

Heat shock protein 70 (called DnaK in prokaryotes) is one of the most prominent groups of chaperones whose role is to prevent and reverse protein misfolding. PfHsp70 is a heatinducible cytoplasm/nuclear localised Plasmodium falciparum Hsp70. PfHsp70 is thought to confer chaperone cytoprotection to P. falciparum during the development of malaria fever. The objective of this study was to examine the chaperone properties of PfHsp70 using a bioinformatics approach, coupled to in vivo and in vitro analysis. Structural motifs that qualify PfHsp70 as a typical Hsp70 chaperone were identified. Although PfHsp70 has a higher similarity to human Hsc70 than E. coli DnaK, in vivocomplementation assays showed that PfHsp70 was able to reverse the thermosensitivity of E. coli dnaK756 (a temperature sensitive strain whose DnaK is functionally compromised). Two residues (V401 and Q402) in the linker region of PfHsp70 that are critical for its in vivo function were identified. Constructs were generated that encoded the ATPase domain of PfHsp70 and the peptide binding domain of E. coli DnaK (to generate PfK chimera); and the ATPase domain of E. coli DnaK fused to the peptide binding domain of PfHsp70 (KPf). The two chimeras were tested for their ability to reverse the thermosensitivity of E. coli dnaK756 cells. Whilst KPf was able to reverse the thermosensitivity of the E. coli dnaK756 cells, PfK could not. Previously, PfHsp70 purification involved urea denaturation. Using a detergent, polyethylenimine (PEI), PfHsp70 was natively purified. Natively purified PfHsp70 had a basal ATPase activity approximately two times higher than the previously reported activity for the protein purified through urea denaturation. PfJ4, a type II Hsp40, could not stimulate the ATPase activity of PfHsp70 in vitro. Arch and hydrophobic pocket substitutions (A419Y, Y444A and V451F) were introduced in the PfHsp70 peptide binding domain. Similar substitutions were also introduced in the KPf chimera. PfHsp70-V451F (hydrophobic pocket mutant) had marginally compromised in vivo function. However, a similar mutation (V436F), introduced in KPf abrogated the in vivo function of this chimera. The arch and hydrophobic pocket derivatives of PfHsp70 exhibited marginally compromised in vivo function, whilst equivalent mutations in KPf did not affect its in vivo function. The ability of PfHsp70 and its arch/hydrophobic pocket mutants to suppress the heatinduced aggregation of malate dehydrogenase (MDH) in vitro was investigated. Whilst PfHsp70 arch mutants displayed marginal functional loss in vivo, data from in vitro studies revealed that their functional deficiencies were more severe. This is the first study in which an Hsp70 from a parasitic eukaryote was able to suppress the thermosensitivity of an E. coli DnaK mutant strain. Findings from the in vivo and in vitro assays conducted on PfHsp70 suggest that this protein plays a key role in the life-cycle of P. falciparum. Furthermore, this study raised insights that are pertinent to the current dogma on the Hsp70 mechanism of action.

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.

Initially, protoplasts were isolated to detect various parameters using flow cytometric analysis. The most efficient ratio of cells to enzyme solution, for digestion of cell walls, needed to be established. To detect whether the time of incubation with the enzyme solution influenced the state or viability of the protoplasts, they were observed periodically under the light microscope during digestion at different concentrations of enzyme solution. After 2 h digestion with light swirling every 20 min, the protoplasts were still intact (Figure 1), and viable as detected with Trypan blue staining (results not shown). Increasing the digestion period led to a decrease in cell membrane integrity. The size of the protoplasts varied between 60 mm and 90 mm. Figure 1 shows the difference between cells before digestion with an enzyme solution and protoplasts after digestion. Size determination of protoplasts was important since the flow tip of the flow cytometer is limited to 100 mm and if the protoplasts exceeded this size, could lead to blockages in the flow tip of the flow cytometer, with ineffective readings and a time consuming clean-up process. / Dr. Marianne J. Cronje