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.

Mitochondrial protein biogenesis depends on the import of nucleus-encoded precursors from the cytosol. Import is highly regulated and specific for different subcompartments, with intermembrane space (IMS) import driven by an oxidative mechanism on conserved cysteine residues. Oxidative folding in the IMS is facilitated by the mitochondria import and assembly (MIA) pathway. Proteins can only be imported into the IMS in Cys-reduced unfolded forms, as oxidation prevents translocation into the IMS. How the import-competent forms are maintained in the cytoplasm is lesser characterised compared to the MIA pathway. Two recent studies suggest that the cytosolic Thioredoxin (Trx) and Glutaredoxin (Grx) reductase systems play a role in facilitating IMS protein import, with previous evidence identifying a role for yeast Trxs in small Tim protein biogenesis. In this study, the redox properties of the yeast Trx and Grx systems were investigated, as well as whether the yeast Grx system play a role in the biogenesis of two typical types of IMS precursor proteins. First, in vitro studies were carried out to determine the standard redox potentials (E°’) of the Trx and Grx enzymes. This was a quantifiable parameter of reducing activity and the results were described in Chapter 3. This study determined the E°’Trx1 value, which was shown to be a more effective reductant compared to other orthologs. Experimental limitations prevented the Grx system E°’ values being determined. Next, whether the Grx plays a role in the biogenesis of the CX3C motif-containing small Tim proteins were investigated using yeast genetic in vivo and biochemical analysis methods. The results were described in Chapter 4. There, Grxs were observed to not affect cell growth, but in using overexpressed Tim9 as an import model, significant differences were observed for the Grx system as GRX deletion significantly decreased overexpressed Tim9 levels. Study on the isolated mitochondria and cytosol with overexpressed Tim9 was unclear however. This study also observed a genetic interaction between GRX andYME1 that recovered cell functioning under respiratory conditions. Finally, whether the Grx system plays a role in the biogenesis of CX9C motif-containing proteins (Mia40, Mia40C and Cox17) was studied in Chapter 5. Whilst Mia40C (C-domain of Mia40) and Cox17 are imported into mitochondria via the MIA pathway, the full-length Mia40 is a substrate of the presequence-targeted TIM23 pathway. The results indicated that import of the full-length Mia40 was unaffected by GRX deletion. However, studies of an overexpressed Mia40C as a substrate of the MIA pathway, showed strong mitochondrial protein level decreases caused by deletion of the Grx proteins. This decrease was also accompanied by an accumulation of unimported Mia40C in the cytosol. Cox17 as an alternative MIA pathway substrate also showed decreased mitochondrial levels in the GRX deletion mutants. These results altogether suggest that the cytosolic Grx system can function in the biogenesis of CX9C motif-containing IMS proteins imported through the MIA pathway, as well as the CX3C small Tim proteins. The topic of how IMS proteins are degraded in the cell was also raised by the study of Yme1.

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.

Plamodium falciparum heat shock protein (PfHsp70) is believed to be involved in the cytoprotection of the malaria parasite through its action as a molecular chaperone. Bioinformatic analysis reveal that PfHsp70 consists of the three canonical Hsp70 domains; an ATPase domain of 45 kDa, Substrate binding domain of 15 kDa and a C-terminal domain of 10 kDa. At the C-terminus there is a GGMP repeat motif that is commonly found in Hsp70s of parasitic origins. Plasmodium falciparum genome is 80% A-T rich, making it difficult to recombinantly express its proteins in Escherhia coli (E. coli) as a result of rare codon usage. In this study we carried out experiments to improve expression in E. coli by inserting the PfHsp70 coding region into the pQE30 expression vector. However multiple bands were detected by Western analysis, probably due to the presence of rare codons. The RIG plasmid, which encodes tRNAs for rare codons in particular Arg (AGA/AGG), Ile (AUA) and Gly (GGA) was engineered into the E. coli strain resulting in production of full length PfHsp70. Purification was achieved through Ni²⁺ Chelating sepharose under denaturing conditions. PfHsp70 was found to have a very low basal ATPase activity of 0.262 ± 0.05 nmoles/min/mg of protein. In the presence of reduced and carboxymethylated lactalbumin (RCMLA) a 11-fold increase in ATPase activity was noted whereas in the presence of both RCMLA and Trypanosoma cruzi DnaJ (Tcj2) a 16-fold was achieved. For ATP hydrolysis kcat value of 0.003 min⁻¹ was obtained whereas for ADP release a greater kcat value of 0.8 min⁻¹ was obtained. These results indicated that rate of ATP hydrolysis maybe the rate-determining step in the ATPase cycle of PfHsp70.

Currently the mammalian endoplasmic reticulum (ER) is known to contain at least 20 different protein disulphide isomerase (PDI) family members. The oxidoreductases in the PDI family are thought to catalyse the formation and rearrangement of disulphide bonds in newly synthesised proteins. The focus of this work was to characterise two of the PDI family members: PDI and ERp46. In vitro translation reactions of major histocompatibility complex (MHC), β1-integrin (β1-I), haemagglutinin (HA), procollagen α1(III) and preprolaction (pPL) were carried out in untreated or PDI-depleted cells. The depletion of PDI decreased the rate of folding of MHC and β1-I and also prevented the oligomerisation of HA, suggesting a role for PDI in folding these putative substrates. However, when PDI was depleted neither the folding of pPL or HA was affected, implying that they may not be substrates for PDI. To determine the role of ERp46 in the cell, a substrate-trapping approach was used. Here substrates interacting with ERp46 were trapped as mixed disulphides isolated by immunoprecipitation, separated by 2D SDS-PAGE and identified by mass spectrometry. It was demonstrated that ERp46 forms mixed disulphides with at least 23 proteins, including heavily secreted proteins such as laminins, integrins and collagens. In particular, interactions with Ero1, Prx IV, EDEM3 and ERAP2 were found and confirmed by immunoprecipitation of radiolabelled in vitro translated protein. Notably nine of these clients of ERp46 have previously been identified as substrates of ERp57 (Jessop, Watkins et al. 2009). This would support the hypothesis that several different oxidoreductases, working in concert, are required to fold certain substrate proteins. Also, it was confirmed that Prx IV and Ero1 each form a mixed disulphide with PDI. These results highlight the importance of PDI family members in recruiting co-factors to substrates. Additionally, the over-expression of ERp46 led to increased cell survival following DTT treatment, yet after depletion of ERp46, cells were less able to grow, perhaps suggesting a role for ERp46 in maintaining ER redox homeostasis and cell survival. This suggestion was supported by the finding that ERp46 is able to catalyse the reduction of Prx IV in the presence of glutathione. These results suggest that Prx IV provides a novel mechanism for the transfer of disulphide bonds to nascent proteins in the ER via PDI family members such as ERp46 and PDI.

A study of local context has revealed that universal relief strategies are failing to meet the needs of those who have just lived through the traumatic experience of losing their primary dwelling. Where as conventional ‘donor’ structures may economically shelter the body, they neglect to address issues of home and belonging. The hypothesis argues that shelter after disaster is not just a temporary solution but rather a ‘starter kit’ with the potential of becoming a home. Hence shelter is the beginning of a process, that involves first a sign of the event of dwelling before it can host a more complex scope of concerns. While acknowledging that the design cannot be site specific, the proposal responds to regional disasters within greater Tshwane region, through a comprehensive investigation of context, climate and selected case studies. Set within the reality of monotonous modular design the project seeks to provide a flexible and innovative shelter typology that can remain on site, providing a period of grace. Thus enabling the displaced to focus on rebuilding their homes without living with the fear of their tent being reclaimed. The project conducts a critical investigation into rapidly deployable structure. The object of the study is to highlight the potential of cardboard as an alternative building material. Copyright / Dissertation (MInt(Prof))--University of Pretoria, 2010. / Architecture / unrestricted

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