Peroxisomes are ubiquitous and are considered to be vital organelles in eukaryotic cells; however, unlike mitochondria and chloroplast, they lack DNA and a protein secretory apparatus. Therefore, peroxisome biogenesis requires a group of proteins called peroxins encoded by the pex genes. Out of the thirty two known peroxins discovered so far, a subset of peroxins including enzyme IDP3 and proteins namely, PEX18, PEX21 and PEX6 were chosen for this research. IDP3 plays a vital role in peroxisomal metabolism where it generates NADPH which in turn is needed by the peroxisomal enzymes to degrade unsaturated fatty acids. PEX18 and PEX21 are mutually redundant but essential for the transport of PTS2 targeted proteins into the peroxisome. PEX6 is involved in the ATP-dependent recycling of the protein receptor from the peroxisomal membrane to the cytosol. Expression plasmids were constructed that encoded each of these proteins in tandem with a histidine tag at either or both the amino and carboxy terminals of the protein. The purification of IDP3 was achieved using affinity chromatography on a nickel resin. After several unsuccessful attempts using ion exchange and size exclusion chromatography, PEX18 and PEX21 were purified by nickel affinity chromatography after denaturation to expose their His tags. The expression of PEX6 was poor by comparison with the other proteins and the low amount of protein precluded a complete purification. Future work will involve crystal screen trials, X-ray diffraction and structure refinement.

Peroxisomes are virtually found in all eukaryotic cells, but unlike mitochondria and chloroplasts, they do not contain DNA or a protein secretory apparatus. Therefore, all of their proteins must be imported by a process called peroxisomal biogenesis. This requires a group of protein factors referred to as peroxins which are encoded by the pex genes. Currently, there are approximately thirty-two known peroxisomal proteins. Among all the peroxisomal proteins, two enzymes namely GPD1, LYS1 and a peroxin, PEX7 were selected for the research. GPD1 is a NAD+ -dependent glycerol 3-phosphate dehydrogenase1 that catalyzes the conversion of dihydroxyacetone phosphate (DHAP) to glycerol 3-phosphate which is crucial for growth under osmotic stress. Its purification was achieved using ion exchange chromatography and the pure protein was crystallized for structure determination. Diffraction data sets were obtained to a resolution of 2.2 Å which were used to solve the C-terminal portion of the structure. Unfortunately, the N-terminal portion remained disordered. LYS1 is the terminal enzyme of α-aminoadipate pathway and catalyzes the reversible NAD-dependent oxidative cleavage of saccharopine to yield L-lysine and α-ketoglutarate. The purification of LYS1 was carried out using affinity chromatography. Another protein, PEX7 is responsible for peroxisome biogenesis by importing newly synthesized proteins bearing PTS2 (peroxisome targeting signal sequence2) into peroxisomes. PEX7 presented the greatest challenge among the three proteins at both the expression stage and the purification stage. Its soluble fraction was purified using ion exchange and affinity chromatographies, although the final yield was too low for crystallization trials. A much large proportion of the protein was found in the insoluble cell debris and attempts were made to purify this fraction after denaturation. An alternative, protocol involving the formation of a GPD1-PEX7 complex proved to be effective route to co-purification of the two proteins and crystallization trials are proceeding. Having known the structures of peroxisomal proteins, it would be helpful for studying the development and maintenance of the organelle related to its metabolic diseases in the eukaryotic cells.

The mesenchyme homeobox genes, MEOX1 and MEOX2, encode homeodomain transcription factors. Studies of Meox1/Meox2 knockout mice established that these proteins are partially redundant during development, suggesting that they may regulate common target genes. In the adult vasculature, MEOX2 is expressed in vascular smooth muscle and endothelial cells. MEOX2 has been demonstrated to: i) inhibit proliferation, ii) activate apoptosis and iii) induce senescence. In contrast, the role of MEOX1 has not been studied in the vasculature. Currently, there are two known target genes of MEOX2: cyclin-dependent kinase inhibitor 1A (CDKN1A/p21CIP1/WAF1) and cyclin-dependent kinase inhibitor 2A (CDKN2A/p16INK4a), which regulate transient (quiescent) and permanent (senescent) cell cycle arrest. Senescence is postulated to contribute to the development of atherosclerotic vascular disease by promoting endothelial dysfunction. We hypothesized that MEOX1 and MEOX2 would activate both p21CIP1/WAF1 and p16INK4a expression, as well as induce apoptosis, cell cycle arrest and senescence in endothelial cells. Furthermore, we postulated that the majority of newly identified MEOX target genes in endothelial cells would be regulated by both MEOX1 and MEOX2. MEOX proteins were expressed in human endothelial cells via adenoviral transduction. Levels of target gene expression were measured by luciferase reporter gene assays, western blot and quantitative real-time PCR. Electrophoretic mobility shift assays were used to demonstrate MEOX binding to DNA. Cellular proliferation, senescence, and apoptosis were evaluated. For the identification of novel target genes, microarrays were used to compare levels of gene expression in endothelial cells transduced with MEOX constructs or control virus. Both MEOX1 and MEOX2 activated p21CIP1/WAF1 and p16INK4a gene transcription, inhibited proliferation and induced apoptosis and senescence in endothelial cells. MEOX activation of p21CIP1/WAF1 transcription occurs via a DNA-binding independent mechanism that requires the SP1 transcription factor. In contrast, MEOX activation of p16INK4a transcription is dependent upon DNA-binding. Microarray analysis revealed that both MEOX1 and MEOX2 increased the expression of intercellular adhesion molecule 1 (ICAM-1) and decreased the expression of nitric oxide synthase 3 (NOS3/eNOS). Taken together, we conclude that MEOX1 and MEOX2 have similar target genes in endothelial cells including p21CIP1/WAF1, p16INK4a and eNOS. As increased endothelial senescence and decreased nitric oxide production are hallmarks of endothelial dysfunction, this study proposes a role for the MEOX proteins in the progression of atherosclerotic vascular disease.

Previous experimentation using differential immunological screening identified Tp0326, a protein predicted to be located in the outer membrane (OM) of the bacterium Treponema pallidum. This protein is homologous to BamA members of the β-barrel assembly machinery (BAM) family of proteins, which are conserved throughout pathogenic Gram-negative bacteria. In Escherichia coli the BAM proteins are found as a complex composed of five proteins: BamA, which is an integral membrane protein, and four accessory lipoproteins, BamB - BamE, which localize to the inner leaflet of the outer membrane. In E. coli BamA has been shown to mediate the insertion and assembly of proteins in the OM via interaction with the BAM complex and periplasmic chaperones (SurA, Skp, and DegP). We hypothesize that a similar OMP translocation complex exists within T. pallidum and that this complex is responsible for ushering T. pallidum OMPs to the bacterial surface. Characterization of the putative T. pallidum OMP transport machinery was performed by bioinformatic analyses and protein-protein interaction studies. Protein-protein interaction studies included screening a T. pallidum Lambda genomic expression library with recombinant T. pallidum protein Tp0326 and Far-Western blotting techniques. Using bioinformatic analyses we have identified putative T. pallidum homologues of the E. coli lipoproteins BamD (Tp0622) and BamB (Tp0133) as well as putative homologues of the E. coli chaperone proteins Skp (Tp0327) and DegP (Tp0773). The T. pallidum Lambda genomic expression library screen identified the putative E. coli BamD homologue (Tp0622), which was originally discovered through bioinformatic analyses. The expression library screen also identified two putative T. pallidum OMPs (Tp0750 and Tp0751) as potential interaction partners of Tp0326. Combined bioinformatic analyses and protein-protein interaction studies provide evidence a BAM complex may exist within T. pallidum, and similar to E. coli, this complex may be involved in ushering T. pallidum OMPs to the bacterial surface. / Graduate

Properdin is a serum glycoprotein that upregulates the alternative pathway of complement by stabilizing the C3bBb complex. It also binds sulphated glycoconjugates, such as sulphatide, in vitro. Properdin is composed of cyclic dimers, trimers and tetramers of a 53 kDa monomeric subunit. The monomer contains an N-terminal region of no known homology and six thrombospondin type 1 repeats (TSRs) of approximately sixty amino acids. The sixth TSR of properdin contains an insertion of approximately 30 amino acids which corresponds to the position of an intron in the human properdin gene. In order to identify the regions of properdin important for function, human properdin, and mutant forms each lacking a single TSR, were expressed in Chinese Hamster Ovary cells. In addition, limited tryptic digestion yielded "nicked" properdin by the cleavage of one peptide bond in TSR5. The structural and functional properties of the normal and altered forms of properdin were investigated. Wild type recombinant properdin is similar to properdin purified from plasma in size, immunoreactivity, N-terminal sequence, possession of N-linked sugar, oligomerization (as determined by electron microscopy and gel exclusion chromatography), and functional activity in an alternative pathway haemolytic assay, and in C3b and sulphatide binding assays. Properdin "nicked" in TSR5 is unable to bind C3b, while retaining its overall structure and its ability to bind sulphatide. The removal of TSRS prevents C3b and sulphatide binding. Properdin lacking TSR4 is unable to stabilize the C3bBb complex, but is able to bind C3b and sulphatide, and shows the presence of monomers and dimers in the electron microscope. Properdin without TSR3 is able to stabilize the C3bBb complex, to bind CSb and sulphatide, and forms dimers, trimers and tetramers. Properdin lacking TSR6 is unable to form oligomers. The N-linked carbohydrate of properdin is not required for oligomerization or stabilization of the C3bBb complex. Monoclonal antibodies which bind to the N-terminal region, TSR1, or TSR2 are able to inhibit properdin binding to CSb. A monoclonal antibody which binds TSR4 is able to inhibit properdin binding to sulphatide, but not to CSb. The results confirm that TSRs are folded as independent units. The N-terminal end and TSR5 of properdin are implicated in CSb binding. The vertices of properdin oligomers may be important for interaction with CSb. TSR4 may also be involved in stabilization of the C3bBb complex. The sulphatide binding site is distinct from the CSb binding site, but TSR5, which contains many basic residues, may be important for both activities.

No description available.

The IL-6 family of cytokines includes IL-6, ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), oncostatin M (OSM), cardiotrophin-1, and IL-11. Functioning in a pleiotropic and redundant manner, these cytokines play an important role in the regulation of complex cellular processes such as gene activation, proliferation and differentiation, by signalling through homo- or heterodimers of gp130. This thesis describes the characterization of the interactions between the cytokine oncostatin M (OSM) and the cytokine-binding homology region (CHR) of its receptor gp130. Three forms of OSM were expressed, the native form and two truncated forms. Both mutations were obtained by C-terminal truncation. The first, OSM185, has an 11 amino residue deletion and the second, OSM187, has a 9-residue deletion. A variety of biophysical techniques were applied to investigate the complex. Analytical ultra-centrifugation (AUC), surface Plasma Resonance (SPR) and isothermal titration calorimetry (ITC) studies indicated that the purified proteins were stable in monomeric form and can form a 1:1 complex with affinity in the 0.1 μM range. One of the C-terminal truncated forms, the 187 residues version, showed higher stability than the native OSM (196 residues), but still demonstrated similar binding properties to the gp130-CHR. A ¹⁵N and ¹³C double-labelled OSM187 sample was produced for NMR studies. Due to the size of these two proteins, OSM187 (21.5 kDa) and gp130-CHR (25.2 kDa), the NMR studies of the complex are challenging. Applying the TROSY technique, data were obtained from the labelled OSM187 when it is in complex with gp130-CHR. The data could be compared with the free form OSM187 and several shifted peaks were detected. The binding site mapping work has just begun. The characterized binding properties and methods established for sample preparation provide a solid starting point for later studies. The thesis also contains an exploratory study of interactions between interleukin-2 (IL-2) and the IL-2 receptor β chain.

No description available.

No description available.

No description available.