M.Sc. / Statistics provided by the World Health Organization state that cancer accounted for 7.9 million deaths worldwide in 2007, with numbers expected to increase to over 12 million by the year 2030. The transformation of a normal cell to a malignant tumour is known to be the result of a set of several key mutations in the genome of a normal cell, resulting in several unique properties including the evasion of programmed cell death, or apoptosis. Exacerbation of this cell death evasion can occur by overexpression of cell survival effectors such as heat shock proteins (Hsps), which are a family of highly conserved proteins that are rapidly induced in response to a variety of stresses in order to protect the cell from death. These proteins perform this function both by assisting in protein folding and therefore acting as molecular chaperones and also by directly interacting with the apoptotic machinery to prevent the initiation of cellular death. Various Hsps interfere at a range of sites in the intrinsic apoptotic pathway, both upstream of the mitochondria, and downstream at the sites of caspase activation. Similarly, Hsps also interfere at various sites in the extrinsic pathway, the caspase-independent pathways, and also function to promote the activity of survival pathways. Nonsteroidal anti-inflammatory drugs (NSAIDs) are well known for their anti-inflammatory properties via inhibition of cyclooxygenase (COX) enzymes. These drugs have also been shown to induce apoptosis in a variety of cancer cell lines as well as decrease the risk of the development of various cancers. Interestingly, NSAIDs have additionally been shown to have the curious property of activating the heat shock transcription factor (HSF1) at concentrations much higher than that required for inhibition of COX activity. The combination of NSAIDs and hyperthermia has resulted in seemingly contradictory evidence, where some studies show that this combination leads to thermotolerance and resistance to further treatments, whilst other studies have shown that this combination directly leads to cell death or indirectly sensitizes cells to subsequent stress.

Programmed cell death (PCD) is a universal event experienced by all eukaryotes and is an essential process for the maintenance of regular homeostasis. The careful regulation of PCD pathways is beneficial to all organisms and a better understanding of various PCD components and their interactions may enhance overall quality of life and increase longevity of defective organisms. Since a boom of interest was shown towards the study of PCD approximately thirty years prior, a comprehensive understanding of the underlying mechanisms of apoptosis currently exists. Many of the key features involved in PCD have been shown to be conserved across the animal and plant kingdoms and although the overall process of cell death appears to occur in a similar manner in both plants and animals, subtle variations have been identified between these two kingdoms regarding various mechanisms of apoptosis. The major component of cell death in plants and animals appears to be the mitochondria where most of the PCD points of control converge. Heat shock proteins (Hsps) play a vital role in the regulation of PCD and act at an array of points along the PCD pathway. One of the crucial events of PCD is the release of cytochrome c from the mitochondria which proceeds to activate the protease cascade. Hsp70 has been shown to attenuate apoptosis by specifically preventing cytochrome c release into the cytosol of animal cells. A similar correlation between increases of Hsp70 and decreased PCD has been identified in plants, the mechanism of which remains unclear. The objective of this study was to investigate the interaction between increased Hsp70 accumulation and decreased PCD, as indicated by reduced cytochrome c translocation and DNA laddering. The major observations of this study proceeded to show that an increased expression of Hsp70, induced by a mild heat shock, was able to protect tobacco cells against H2O2-mediated cell death. This protection appeared to commence at a mitochondrial level as cytochrome c translocation was clearly inhibited and was confirmed by the absence of downstream DNA laddering. The major findings obtained from this study provided a clearer picture of the mechanisms surrounding the cytoprotective properties of thermotolerant cells, the implications of which are beneficial to post harvest industries, as the ability to postpone PCD provides an advantage enables prolonged shelf lives of various crops. / Dr. Marianne J. Cronjé

M.Sc. / The induction of heat shock proteins (HSPs) in human monocytes during a pathogen challenge is a sophisticated selective response and plays an important role in cytoprotection from inflammation-related stress, including oxidative injury. We investigated the accumulation of the inducible isoform of the 70 kDa HSP, Hsp70, in peripheral blood monocytes from 12 healthy donors in response to Mycobacterium tuberculosis (M.tb) using flow cytometry, biometabolic labeling or Western blot analysis. Cells from each donor, prepared on two different occasions, were exposed to virulent (H37Rv) and attenuated (H37Ra) strains of M.tb at two bacterium : monocyte ratios (1:1 and 10:1) for 3 h and allowed to recover for an additional 2 h or 24 h. In spite of a prominent inter-individual variation, H37Ra (1:1, 2 h) significantly induced the mean Hsp70 accumulation (p<0.05) compared to normal cells, while H37Rv (10:1, 24 h) significantly suppressed the mean Hsp70 levels (p<0.001) in monocyte compared to normal monocytes or monocytes exposed to H37Ra. Survival of H37Rv-infected monocytes showed a significant correlation with Hsp70 levels. These results suggest a protective role of Hsp70 in host defense against mycobacterial infection. Cell death due to insufficient endogenous levels of Hsp70 implies a novel pathogenic strategy for virulence of M. tuberculosis.

Heat shock proteins (HSP), although not considered classical defence proteins, have general cytoprotective properties, which promote survival of cells and organisms. Hsp70, in particular, provides resistance to the harmful consequences of various forms of otherwise damaging or even lethal stress including pathogen infection. Increased levels of Hsp70, due to stable transfection of cells with hsp70 genes, or elevated expression in response to stress, generally correlate with the hindrance of cell death processes triggered by a variety of noxious stimuli or toxic agents. The effect of lipopolysaccharides (LPS), the major constituent of the outer membrane of the cell wall (envelope) of almost all Gram-negative bacteria, on Hsp70/Hsc70 expression in plants is unknown. In various mammalian systems, LPS has been shown to induce Hsp70 accumulation, along with programmed (apoptotic) cell death. Contrary to the effects of LPS on animal hosts however, LPS does not elicit cell death in plants, but rather pre-treatment with LPS fraction can prevent or delay the so-called hypersensitive response (HR), thus sensitizing plant tissue to respond more rapidly, or to a greater extent, to subsequently inoculated phytopathogenic bacteria. Elevated levels of reactive oxygen species (ROS) reportedly contribute to stress sensing and hsp gene activation, and subsequent Hsp70 induction, during the stress response. Increased ROS production can also trigger cell death via either programmed cell death (PCD), an active (i.e., energy-dependent) physiological suicide mechanism that is genetically regulated, or uncontrolled necrosis, an accidental, lytic form of cell destruction passively triggered by severe trauma or injury. In plants specifically, ROS may be involved in PCD activation during the HR. As a pathogen-associated molecular pattern (PAMP) or general elicitor of defence or resistance-related responses, LPS may trigger some defence-related responses, including an oxidative burst (manifest as increased levels of reactive oxygen species or ROS) in certain plant cells as it does in animal systems. However, there is conflicting evidence that shows that LPS treatment does not elicit an oxidative burst in plants. The aim of this study was to determine the effect of LPS isolated from Ralstonia solanacearum, an extremely harmful soil-borne bacterium that causes Southern wilt in over 200 plant species by infecting the host’s roots and invading the xylem vessels, on Hsp70/Hsc70 accumulation, cell death and ROS production in suspension-cultured tobacco (Nicotiana tabacum) cells, in order to gain a better understanding of the inter-relationship between these three phenomena in plant cells responding to LPS(Ralstonia). Western (immuno)blot analysis was used to study the unknown effect of LPS(Ralstonia) on Hsp70/Hsc70 accumulation in tobacco cell suspensions. LPS(Ralstonia) (all concentrations and time periods studied) generally suppressed Hsp70/Hsc70 accumulation. However, only exposure to 100 μg LPS/ml for 3 h caused a significant reduction (P < 0.05). Therefore, there was an early suppression of Hsp70/Hsc70 accumulation in response to 100 μg LPS(Ralstonia)/ml. To determine whether the observed LPS-mediated attenuation in Hsp70/Hsc70 accumulation was due to an increase in cell death in these cells, we investigated the effect of LPS(Ralstonia) on i) the general viability of the cells, and ii) the integrity of nuclear or genomic DNA extracted from LPS-treated suspension-cultured tobacco cells. The AlamarBlue™ (AB) assay was used to investigate the general cell viability in response to LPS(Ralstonia) treatment. LPS(Ralstonia) (all concentrations and time intervals studied) did not significantly affect the overall viability of the cells. Because treatment of tobacco cell suspensions with LPS(Ralstonia) did not result in a significant decrease (P < 0.05) in AB reduction, it was presumed that LPS(Ralstonia) did not appreciably compromise metabolic activity and was therefore not particularly toxic to these cells. Genomic DNA from cells undergoing PCD-associated internucleosomal DNA fragmentation (IDF) typically runs as a ladder of internucleosomal-sized DNA fragments corresponding to multimers of ca. 180 bp in agarose gels. In contrast, random DNA cleavage, usually manifest as smearing of nuclear DNA following agarose gel electrophoresis, is a token of uncontrolled necrosis. Therefore, if so-called “DNA laddering” is observed following agarose gel electrophoresis of genomic DNA extracted from suspension-cultured tobacco cells exposed to LPS(Ralstonia) then it can be assumed that LPS(Ralstonia) induced PCD. Alternatively, if a long, continuous “necrotic smear” is evident after electrophoretic separation of nuclear DNA from LPS-treated cells then LPS(Ralstonia) clearly induced uncontrolled necrosis. Whether or not LPS(Ralstonia) induced PCD-associated IDF or necrotic smearing was determined by investigating genomic DNA fragmentation (or DNA integrity) in response to LPS(Ralstonia) iii treatment. Although no typical DNA ladders were detected following electrophoresis of DNA isolated from LPS-treated cells, PCD may still have transpired. However, this is highly unlikely. No necrotic smearing was evident in LPS-treated samples either, which verifies the hypothesis that LPS(Ralstonia) (25–100 μg/ml) did not induce uncontrolled necrosis in suspension-cultured tobacco cells. In fact, these concentrations of LPS(Ralstonia) did not seem to significantly compromise DNA integrity given that LPS(Ralstonia) (25–100 μg/ml) generally had no appreciable effect on genomic DNA fragmentation (compared to untreated control samples). Incidentally, 24-h exposure of tobacco cell suspensions to higher concentrations of LPS(Ralstonia) (500 and 1000 μg/ml) may have resulted in partial DNA cleavage and/or degradation. Exposure of tobacco cell suspensions to 400 μg LPS(Burkholderia)/ml for 7 days may also have evoked partial DNA cleavage and/or degradation. Whether this cleavage and/or degradation occurred deliberately by means of a fixed or predetermined mechanism or randomly by an uncontrolled mechanism remains uncertain. Finally, the H2DCF-DA (2′, 7′-dihydrodichlorofluorescein-diacetate) fluorescence assay was used to investigate the effect of LPS(Ralstonia) on ROS production, a common factor in the regulation of HSP expression and cell death activation. LPS(Ralstonia) treatment (25–100 μg/ml) generally increased ROS levels in suspension-cultured tobacco cells (compared to untreated control cells). Exposure to 75 μg LPS(Ralstonia)/ml resulted in a particularly prominent elevation in ROS levels almost instantaneously. Incidentally, higher concentrations of LPS(Ralstonia) (500 and 1000 μg/ml) resulted in decreased ROS levels at some point during the assay. Although LPS(Ralstonia) (100 μg/ml for 3 h) significantly decreased Hsp70/Hsc70 accumulation in tobacco cell suspensions, cell death did not appear to be induced. In fact, LPS(Ralstonia) had no effect on general cell viability and appeared to be ineffective at causing PCD-associated IDF (DNA laddering) or necrotic smearing regardless of concentration or time of exposure. Despite these findings, treatment of suspension-cultured tobacco cells with LPS(Ralstonia) (≤ 100 μg/ml) resulted in a mild increase in ROS production. Although the exact mechanism(s) by which LPS(Ralstonia) suppressed Hsp70/Hsc70 accumulation is elusive, our results suggest that the suppression is not related to excessive LPS-mediated injury caused by excessively high ROS levels or increased cell death. We speculate that the prevention of HR-related PCD often observed in plants that are pre-treated with LPS and subsequently inoculated with phytopathogenic bacteria may be dependent on the LPS-mediated suppression of cytosolic Hsp70 expression. / Dr. M.J. Cronje

Background: Heat-shock (HS) proteins (HSP) are induced or increasingly expressed to protect against lethal environmental stresses. Hsp70 in particular, provides protection against various stresses including oxidative stress, is implicated in thermotolerance and appears to have an anti-apoptotic function. Anti-inflammatory salicylates potentiate the induction of the 70 kDa HSP (Hsp70) in mammals in response to HS, enhance thermotolerance and induce apoptosis. In plants, salicylic acid (SA) is a natural signalling molecule, mediating resistance in response to avirulent pathogens. The effects of salicylic acid-mediated increases in Hsp70/Hsc70 expression and its relation to events associated with PCD/ apoptosis in plants are unknown. Hypothesis and Objectives: The hypothesis studied in this investigation was that SA influences Hsp70 expression similar to that found in mammalian cells and may influence the choice between survival or death, whether apoptosis or necrosis. In order to verify this hypothesis the effect of SA alone or in combination with HS on Hsp70/Hsc70 accumulation and events associated with apoptosis were investigated through three main objectives: 1) Determine whether SA in plants, as in mammalian cells, can potentiate heat-induced Hsp70/Hsc70 accumulation or induce Hsp70/Hsc70 by itself at elevated levels. This was done by investigating the effect of SA at various concentrations on Hsp70/Hsc70 expression at normal temperatures or following heat. 2) Establish flow cytometry as a rapid and quantitative alternative for the evaluation of Hsp70 accumulation in plant protoplasts to be evaluated in concert with various parameters indicative of cellular integrity. 3) Investigate whether Hsp70/Hsc70 expression modulated by SA influences cell death (apoptosis/necrosis) or associated events such as mitochondrial membrane permeability (MMP) or reactive oxygen species (ROS) in plant protoplasts using flow cytometry. Materials and Methods: The effect of SA alone or in combination with HS on Hsp70/Hsc70 levels in tomato cells was investigated using biometabolic labelling and Western blotting. A flow cytometric assay was developed to determine Hsp70/Hsc70 levels in tobacco protoplasts. MMP and ROS were monitored by the fluorescent probes DiIC1(5) and H2DCFDA respectively, phosphatidylserine externalisation by annexin V binding and DNA fragmentation by the TUNEL assay in protoplasts treated with SA and/or HS. Results: Results obtained in the attainment of the three main objectives were: 1) In plants, as in mammals, low concentrations of SA do not induce Hsp70/Hsc70 but significantly potentiate heat-induced Hsp70/Hsc70 levels while cytotoxic levels significantly induce Hsp70/Hsc70. In cell suspension cultures, this induction was preceded by increased membrane permeability. 2) Flow cytometry can be implemented as a rapid, quantitative alternative to detect intracellular Hsp70/Hsc70 accumulation in protoplasts. 3) In protoplasts exposed to low doses of SA at normal temperatures, PCD/apoptosis is increased as reflected by increased DNA fragmentation and phosphatidylserine externalisation, but not by increased MMP or ROS. High doses of SA were associated with increased levels of necrosis. Exposure of protoplasts to low doses of SA in combination with HS showed suppression of PCD/apoptosis (reflected by decreased DNA fragmentation and phosphatidylserine externalisation), accompanied by decreased levels of ROS and increased MMP. Discussion: These results suggest that SA-mediated increases in Hsp70/Hsc70 accumulation at normal temperatures are associated with cellular damage and protect cells against necrosis. On the other hand, low doses of SA that potentiate heat-induced Hsp70/Hsc70 accumulation abrogated the induction of apoptosis that was induced by low doses of SA at normal temperatures. The anti-apoptotic effects of Hsp70 could therefore influence plant resistance by interfering with the execution of PCD. These results could contribute to our understanding of heat-induced disease susceptibility, and the manipulation of SA-modulated Hsp70/Hsc70 should be carefully considered in the light of its ability to affect cell death, which may be advantageous or deleterious to the plant cell. / Prof. L. Bornman

M.Sc. / Breast cancer is the most commonly diagnosed cancer and cause of death in women world wide as well as in South Africa. Cancer is characterized by over-proliferation of cells or the inhibition of programmed cell death known as apoptosis, a well coordinated process that results in the activation of several proteases and other hydrolytic enzymes. Apoptosis is regulated by enhancers and inhibitors, such as heat shock proteins (Hsps) that modulate the apoptotic process according to the demands of specific cells. Hsps can regulate the release of pro-apoptotic factors from the mitochondria as well as inhibit key steps in the apoptotic cascade such as activation of caspases. The Hsp70 family constitutes the most conserved and best studied class of Hsps and the stress-induced Hsp70 also blocks the apoptotic pathway at different levels. Hsp70 is furthermore overexpressed in several tumor cells and can effectively inhibit cell death induced by a wide range of stimuli including several cancer related stresses such as hyperthermia, chemotherapeutic agents and nonsteroidal anti-inflammatory drugs (NSAIDs) i.a. aspirin (acetylated salicylic acid) In addition to their potent analgesic, antipyretic and anti-inflammatory activity, NSAIDs can inhibit cell proliferation and induce apoptosis in many cancer cell lines. However, NSAIDs can also lower the temperature threshold for Hsp70 induction and induce a transcriptionally inert intermediate of Hsp70 that can be converted to a transcriptionally active state by a subsequent exposure to heat shock. This suggests that NSAIDs act differently under heat stress, possibly increasing cellular protection through the heat shock response in cancer cells with already elevated levels of Hsps. It is therefore hypothesized that the synergistic use of heat shock with salicylic acid (SA) treatment will increase Hsp70 expression and protein accumulation and further enhance the resistance of breast cancer cells to apoptosis. The effects of SA on its own or in combination with HS on the viability of MCF-7 breast cancer cells as well as Hsp70 protein levels and gene expression were therefore investigated. SA treatments were found to induce cell death in a dose-dependent manner with the most significant decrease in viability observed after treatment with 20 mM SA.

Acclimatization is a process which occurs as a result of repeated mild increases in core temperature, which allows an organism to carry out increased work in the heat due to better heat dissipation (Moseley, 1997). In order to prevent the occurrence of heat illness, it is necessary for individuals who perform work in hot, humid environments to undergo acclimatization. Exposure to different types of stimuli, such as heat exercise and oxidative stress, results in the formation of proteins in the cells which are known as heat shock protein (HSP) (Powers et al, 2001). The main function of HSP is to act as molecular chaperones. Specifically, the 70 kDa HSP, known as Hsp70, play an important part in cellular protection and adaptation during and following exposure to stressful events. Two prominent members of the Hsp70 family are Hsp72, which is the inducible form of Hsp70, and Hsp73, which is the constitutively synthesized form of the protein. Acquisition of thermotolerance in vitro in Chinese hamster fibroblast cells is accompanied by increased Hsp72 levels during a heating episode, indicating that Hsp72 tends to target proteins which have been damaged by a stress situation more than Hsp73 (Li and Werb, 1982). A number of different hsp70 genes are found, which include hsp70-1, hsp70-2 (3¢utr and pst I) and hsp70-hom (Milner and Campbell, 1990). Both the hsp70-1 and hsp70-2 genes encode the primary heat-induced Hsp70 protein as an identical protein product (Hunt and Morimoto, 1985). Following heat shock, no increase in the hsp70-hom mRNA levels is observed (Milner and Campbell, 1990). The use of the Hsp70 protein and hsp70 gene polymorphisms as markers of acclimatization were investigated by subjecting twenty-two individuals to exercise in a hot, humid environment. These individuals were exposed to an initial heat stress, where they participated in a step test at an external work rate of 70 W, followed by participation in an acclimatization program which involved exercising at various combinations of 35 W and 70 W of the external work rate. After acclimatization, the individuals were exposed to a second heat stress, identical to the initial heat stress. The Hsp70 levels both before and after acclimatization were determined in response to heat stress in blood serum by means of the StressXpress ELISA Kit (Stressgen Biotechnologies) and in human monocytes by means of Western blots, using a mouse antiñHsp70 monoclonal primary antibody (Stressgen Biotechnologies) and a goat antiñmouse IgG (H+L) peroxidase conjugated secondary antibody. The hsp70 gene polymorphisms were determined by means of polymerase chain reaction (PCR), using primers specific to the hsp70-1, hsp70-2 (3¢utr), pst I and hsp70-hom genes, so that the genotype combinations for each individual were determined. Blood type was also assessed. It was found that the basal serum Hsp70 levels in individuals who exhibited the ability to acclimatize decreased in response to the acclimatization program, which allowed more Hsp70 to be induced in response to the second heat stress compared to the initial heat stress. The individuals who were unable to acclimatize showed increased basal serum Hsp70 levels in response to acclimatization, which prevented these individuals from inducing high Hsp70 levels in response to the second heat stress. The Hsp70 induced in the monocytes during this program followed the same pattern in both the individuals able to acclimatize and those who were unable to acclimatize, and can therefore not be used as a marker of acclimatization. For the female participants, the current menstrual phase of each woman had to be taken into account, as this had an affect on the core temperature and therefore influenced the division of the female participants into their respective groups. These were the group of individuals who demonstrated the ability to acclimatize or the group of individuals who were unable to acclimatize. The use of oral contraceptives also had to be taken into account, as this too had an influence on the core temperature and therefore also affected the division of the individuals into the group who demonstrated the ability to acclimatize or those who were unable to acclimatize. Cyclic changes during the menstrual cycle may have also changed the Hsp profile. Regarding the hsp70 gene polymorphisms, the A/A, P2 P2 and A1 A1 genotype combination was not present in any of the individuals who were unable to acclimatize, however six of the individuals who showed the ability to acclimatize possessed this genotype combination. The level of induced Hsp70 levels present in the serum of individuals able to acclimatize and the presence or absence of the A/A, P2 P2 and A1 A1 genotype combination therefore have the potential to be used as markers of acclimatization. / Dr. M. Cronje

The 90-KDa heat shock protein (Hsp90) is part of the molecular chaperone family, and as such it is involved in the regulation of protein homeostasis within cells. Specifically, Hsp90 aids in the folding of nascent proteins and re-folding of denatured proteins. It also plays an important role in the prevention of protein aggregation. Hsp90’s functionality is attributed to its several staged, multi-conformational ATPase cycle, in which associated client proteins are bound and released. Hsp90 is known to be associated with a wide array of client proteins, some of which are thought to be involved in multiple oncogenic processes. Indeed Hsp90 is known to be directly involved in perpetuating the stability and function of multiple mutated, chimeric and over-expressed signalling proteins that are known to promote the growth and survival of cancer cells. Hsp90 inhibitors are thus thought to be promising therapeutic agents for cancer treatment. A lack of a 3D structure of human Hsp90 however has restricted Hsp90 inhibitor development in large to in vivo investigations. This study, aims to investigate and calculate hypothetical homology models of the full human Hsp90 protein, and to probe these structural models for novel drug target sites using several in silico techniques. A multi-template homology modelling methodology was developed and in conjunction with protein-protein docking techniques, two functionally important human Hsp90 structural models were calculated; the nucleotide free “v-like” open and nucleotide bound closed conformations. Based on the conservation of ligand binding, virtual screening experiments conducted on both models using 316 natural compounds indigenous to South Africa, revealed three novel putative target sites. Two binding pockets in close association with important Hsp90-Hop interaction residues and a single binding pocket on the dimerization interface in the C-terminal domain. Targeted molecular docking experiments at these sites revealed two compounds (721395-11-5 and 264624-39-7) as putative inhibitors, both showing strong binding affinities for at least one of the three investigated target sites. Furthermore both compounds were found to only violate one Lipinski’s rules, suggesting their potential as candidates for further drug development. The combined work described here provides a putative platform for the development of next generation inhibitors of human Hsp90.

The 70 kDa heat shock proteins (Hsp70s) are vital for normal protein folding, as they stabilise the unfolded state of nascent polypeptides, allowing these sufficient time to attain a correct tertiary structure. Hsp70s are aided by the DnaJ-like family of proteins, which interact with Hsp70s in order to enhance the chaperone activity of these proteins. DnaJ-like proteins contain a J domain, a seventy amino acid domain consisting of four α-helices, which is defined by the presence of an invariant tripeptide of histidine, proline and aspartic acid (HPD motif). This motif is key to the interaction between DnaJ-like proteins and Hsp70s. This thesis has focused on determining the presence of other conserved residues in the J domain and their role in mediating the interaction of DnaJ-like proteins with partner Hsp70s. DnaJ-like proteins from Agrobacterium tumefaciens RUOR were isolated and used as a model system. A. tumefaciens DnaJ (Agt DnaJ) was able to replace the lack of E. coli DnaJ in an E. coli null mutant strain, however, additional A. tumefaciens DnaJ-like proteins Agt DjC1/DjlA, Agt DjC2 and Agt DjC5 were unable to complement for the lack of E. coli DnaJ. Replacement of the Agt DnaJ J domain with J domains from these proteins resulted in non-functional chimeric proteins, despite some sequence conservation. The kinetics of the basal specific ATPase activity of Agt DnaK, and its ability to have this activity stimulated by Agt DnaJ and Agt DnaJ-H33Q were also investigated. Stimulation of the ATPase activity by Agt DnaJ ranged between 1.5 to 2 fold, but Agt DnaJ-H33Q was unable to stimulate the basal ATPase activity. Conserved amino acids in the J domain were identified in silico, and these residues were substituted in the J domain of Agt DnaJ. The ability of these derivative proteins to replace E. coli DnaJ was investigated. Alterations in the HPD motif gave rise to proteins unable to complement for lack of E. coli DnaJ, consistent with literature. Agt DnaJ-R26A was unable to replace E. coli DnaJ suggesting that Arg26 could be key to the interaction with partner Hsp70s. Agt DnaJ-D59A was unable to replace E. coli DnaJ; substitutions in Asp59 have not previously been shown to impact on the function of DnaJ. Substituting Arg63 in Agt DnaJ abrogated the levels of complementation. Substitution of several structural residues was also found to disrupt the in vivo function of Agt DnaJ suggesting that the maintenance of the structural integrity of the J domain was important for function. This study has identified a number of residues critical to the structure and function of the J domain of Agt DnaJ, and potentially of general importance as molecular determinants for DnaJ-Hsp70 interaction.

Hop (Hsp70-Hsp90 Organizing Protein) is a co-chaperone of two major molecular chaperones, Hsp70 and Hsp90, and acts by transferring substrates from Hsp70 to Hsp90. Although under normal conditions Hop is predominantly localized within the cytosol, Hop has been detected in the nucleus under certain conditions including cell cycle arrest. A putative nuclear localization signal (NLS) has been identified within Hop, which overlaps with the TPR2A domain (previously shown to be critical for Hop-Hsp90 interactions). Hop is phosphorylated in vitro by two cell cycle kinases, namely, casein kinase II (CKII) at S189 and cdc2-kinase at T198; both residues are found upstream of the putative NLS and TPR2A domain. Mimicking phosphorylation at either phosphorylation site appeared to affect the subcellular localization of Hop. The aim of this study was to characterize Hop with respect to its phosphorylation status in vivo, as well as its subcellular localization pattern under heat stress and determine how these properties affected its interaction with Hsp90 as a co-chaperone. Dephosphorylation of proteins under normal and heat shock conditions changed the isoform composition of Hop, providing strong evidence that Hop was phosphorylated in vivo. Surface plasmon resonance (SPR) and glutatione-S-transferase (GST) co-precipitation studies showed that a cdc2-kinase phosphorylated mimic of Hop disrupted Hop-Hsp90 binding. A full length Hop-EGFP construct, as well as substitution mutants of the predicted NLS residues within the Hop-EGFP construct, were transfected into baby hamster kidney (BHK)-21 cells in order to establish the subcellular localization of Hop under heat stress and to test whether predicted residues were critical for nuclear localization of Hop. Under normal conditions, both Hop-EGFP and the NLS mutants were predominantly cytosolic, but when the cells were subjected to heat stress, Hop and its NLS-mutants were localized to both the cytosol and the nucleus. SPR and GST co-precipitation studies showed that substitution of the residues within the major arm of the putative NLS abrogated Hop-Hsp90 interactions. The data obtained from this study, showed for the first time, that Hop was phosphorylated in vivo and suggested that phosphorylation of Hop by cdc2-kinase could inhibit Hop-Hsp90 interactions. Moreover, these results suggested that the subcellular localization of Hop was dependent on stress levels of the cell, particularly heat stress. We propose that the nuclear localization of Hop may be primarily regulated by stress and secondarily by cell cycle arrest. The major arm of the putative NLS did not affect the localization of Hop directly, but was shown to be critical for Hop-Hsp90 binding in vitro. The results of this study suggested that binding of Hop to Hsp90 sequestered Hop within the cytosol and that Hsp90 acted as a cytosolic retention factor for Hop. Both phosphorylation of Hop, and its subcellular localization, appeared to be intimately related to its interaction with Hsp90 as a co-chaperone.