The cellular levels of a number of proteins have been found to be regulated by the ubiquitin-proteasome pathway. In this pathway, proteins are covalently tagged (&ldquo / ubiquitinated&rdquo / ) by ubiquitin, which acts as a signal for degradation by the proteasome. A number of key cellular processes, including cell-cycle progression, transcription and DNA repair, are regulated in this way. In recent years a number of cellular proteins resembling ubiquitin in structure or function, the so-called ubiquitin-like proteins, have been identified. Ubiquitin-like proteins can be divided into two classes-the so-called &ldquo / ubiquitin-like modifiers&rdquo / , which consist of a single domain that structurally resembles ubiquitin, and &ldquo / ubiquitin-domain&rdquo / proteins, which are multi-domain proteins, which include domains that resemble ubiquitin.<br /> <br /> This thesis describes the recombinant expression, purification and full backbone assignment of the human DWNN domain, a novel ubiquitin-like domain. The DWNN domain occurs at the N-terminus of RBBP6, a protein that has been shown to interact with p53 and Rb as well as to be involved in mRNA processing and apoptosis. A bacterial expression system was used to overexpress the DWNN domain as a GST fusion protein. The domain was labelled with 15N and 13C to perform triple-resonance heteronuclear NMR experiments, from which full backbone assignments were obtained.<br /> <br /> Although full structure determination of the DWNN domain falls outside the scope of this thesis, the backbone assignments formed the basis for the subsequent structure determination, which confirmed that the DWNN domain is indeed a novel ubiquitin-like domain. The RBBP6 protein may therefore represent a novel E3 ubiquitin ligase that plays a role in regulating the cellular levels of p53 and Rb.

The HIV/AIDS still remains a global health challenge with 42 million people infected with the virus. An alarming 70% of these people reside in sub-Saharan Africa with HIV-1 subtype C being the most prevalent subtype in this region and globally. HIV-1 protease (PR) is an obligate homodimer which plays a pivotal role in the maturation and hence propagation of the HI virus. Although successful developments on PR active site inhibitors have been achieved, the major limiting factor has been the emergence of HIV drug resistant strains. It has been postulated that disruption/dissociation of the dimer interface may lead to an inactive enzyme. The development of small molecules and peptides has been a major research area with the key target being the N- and C-termini antiparallel β-sheet. This is due to its highly conserved nature and because it consists of a cluster of amino acids that contribute most of the binding energy and stability of the dimer interface. Hence it is referred to as a ‘hot-spot’. Therefore, binding of protease inhibitors at this site could cause destabilisation and/or dissociation of the enzyme. The terminal residue, F99, was mutated to an alanine disrupting the presumed lock-and-key motif it forms and in turn creating a cavity at the N- and C-termini antiparallel β-sheet. A second mutant, W42F/F99A, was created for monitoring tertiary structural changes exclusively at the N- and C-termini antiparallel β-sheet. The F99A and W42F/F99A, compared to the wild-type, showed a higher expression yield and also migrated further when separated using tricine SDS-PAGE. Wild-type protease CD spectra showed a minimum at 214 nm and a local maximum at 230 nm, while the mutants exhibited minima at 203 nm and absence of the local maxima. A 50% higher fluorescence intensity and a 2 nm red-shift for the mutants versus the wild-type was observed. According to SE-HPLC data the relative molecular weight of the wild-type, F99A and W42F/F99A are 16.4 kDa, 20.7 kDa and 18.1 kDa, respectively. Although the thermal unfolding of all three proteases was irreversible, the unfolding transition of the wild-type was clearly defined between 55 °C and 63 °C. The F99A and W42F/F99A unfolding curves were linear without clearly defined transition states. The specific activity of the F99A (0.13 μmol/min/mg) amounted to a ten-fold reduction compared to the wild-type (1.5 μmol/min/mg). The substrate binding affinity (KM) for the F99A was 41% lower than the wild-type when 2 μM of protein was used. The Vmax and kcat values were about 30-fold and two-fold, respectively, higher for the wild-type when compared to the F99A. Therefore, the tricine SDS-PAGE analysis, secondary and tertiary structural characterisation and thermal denaturation curve showed that the F99A mutation has altered the structure causing ‘partial’ unfolding of the protein. But, the protein still maintained minute activity. The overlap between the ANS binding spectra of the wild-type and variants suggests that the dimeric form still exists.

The Human Immunodeficiency Virus (HIV) subtype C accounts for the majority of infections in Southern Africa. The HIV protease is one of the targets in HIV treatment due to its pivotal role in HIV maturation in the host cell. However, because of polymorphisms in the HIV genome, drug resistance becomes a major problem in HIV treatment. Polymorphisms in the HIV protease gene result in altered substrate cavities, and /or flap hinge modifications leading to unfavourable drug interaction with the enzyme. The most common form of drug resistant mutations is single amino acid substitutions. Although, amino acid insertions have been reported, this form of mutation in the HIV protease is rare. L38↑N↑L insertion is a unique form of HIV protease polymorphism that was isolated from a patient failing drug therapy in South Africa. The objective of this research was to assess the impact of the L38↑N↑L insertions, with accompanying background mutations, on the structure and function of this form of polymorphism in HIV-1 South African subtype C protease. The far-UV circular dichroism (CD) spectra of L38↑N↑L protease shows a trough at 203 nm, suggesting alterations in the secondary structure content of this mutant. Whereas the wild type (WTCSA-HIVPR) displays a trough at 215 nm. However, tertiary structure characterisation using fluorescence spectroscopy did not detect changes within the local tryptophan environment of L38↑N↑L protease in comparison with the wild type due to no significant shift in emission wavelength. The specific activity of L38↑N↑L protease and wild type was 28.0±1.3 μmol.min-1.mg-1 and 123.45±6.4 μmol.min-1.mg-1 respectively. The turn-over number for L38↑N↑L protease and wild type was 1.0 × 10-3 ± 6.0 × 10-5 and 7.7 × 10-3 ± 5.6 × 10-4 respectively. As much as the presence of known drug resistance mutations in L38↑N↑L can be attributed to drug resistance, it should also be noted that the insertions may have also caused local structural alterations that may have enhance drug resistance of L38↑N↑L. These changes could have lead to the decreased catalytic activity of the L38↑N↑L protease. Homology modelling studies show that the insertions in L38↑N↑L protease may have resulted in a fold similar to 2HS1 (PDB code), which has a modification on the flap hinge. In addition, the homology modelling studies suggest that L38↑N↑L protease may have a second inhibitor binding site next to one of the flap hinge regions as seen in the 2HS1 model. In conclusion, the L38↑N↑L insertions and accompanying background mutations may have contributed to the local structural modifications that lead to drug resistance in L38↑N↑L protease.

Pollen grains are ubiquitous triggers of allergic asthma and seasonal rhinitis. Proteolytic enzymes in pollen as well as other sources are capable of disrupting airway epithelial integrity in vivo and in vitro. This provides a plausible mechanism for the initiation of sensitisation of the respiratory immune system to inhaled pollen allergens, comparable to that suggested for Group 1 allergens from house dust mites and cat dander, which are known to possess intrinsic proteolytic activity. This thesis explores the relationship between pollen allergens and proteolytic enzymes. It describes the different strategies used for the characterisation, purification and identification of immunogenic and proteolytic proteins in the complex mixtures of pollen diffusates. The peptidases in the diffusates of Kentucky blue grass, ryegrass and Bermuda grass pollens were characterised by a sensitive fluorescence assay and gelatin zymography. Among these, Bermuda grass pollen demonstrated the presence of a serine peptidase at Mr ~30,000 Da, which corresponded to an intense band by Western blotting using a monoclonal antibody to the timothy grass (Phleum pratense) group 1 allergen, Phl p 1. Purification of this enzyme from Bermuda grass was complicated by the low levels of the enzyme present in the diffusate, as well as by its autohydrolysis. Partial purification of the serine peptidase activity by affinity chromatography using Concanavalin A Sepharose demonstrated that the diffusate contained a trypsin-like peptidase, detected by the fluorescent assay, in addition to the ~30,000 Da serine endopeptidase, detected on gelatin zymography. Proteomic analysis of the ~30,000 Da protein using one- and two-dimensional electrophoresis and mass spectrometry identified it as the major pollen allergen of Bermuda grass, Cyn d 1. The studies reported here provide, for the first time, evidence that a pollen allergen may possess intrinsic proteolytic activity. This activity may play a role in the initiation of airway inflammation and allergic sensitisation.

The mechanisms of substrate recognition by regulatory proteases are not well understood. Presently, two opposing models have arisen to describe E. coil Lon's ability to discriminate between substrates: one suggests the N-terminus involvement while the second suggests the C-terminus involvement. In this project, the role of the C-terminal domain as it relates to the recognition of Lon's normal physiological substrates RcsA, an activator of colanic acid capsular polysaccharide, and SulA, an inhibitor of cell division, was addressed. Using site-directed mutagenesis, five mutations in Lon (R537G, E538A, GS40W, R542G, R542P) were isolated. Their phenotypic impact was either similar in character to wildtype Lon (R537G, E538A) or ��lon cells (G540W, R542G, R542P). The stabilization of both RcsA and SulA based on phenotypic assays and immunological detection of lon* strains (G540W, R542G, R542P) suggests the C-terminal domain may be involved in substrate degradation as opposed to discriminator activity. / Graduation date: 2002

Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.

Thesis (Ph. D.)--University of Chicago, Department of Molecular Genetics and Cell Biology, June 2001. / Includes bibliographical references. Also available on the Internet.

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