Dynamic protein filaments in eukaryotic cells make up cytoskeletal arrays that perform essential functions. Microtubules form part of the cytoskeleton, and impart structure, integrity and organisation to cells. End binding (EB) proteins are an evolutionarily conserved family which associate with the dynamic ends of microtubules, where they act to control microtubule growth and recruit other proteins that localise to this site (+ TIPs). EB2 is one of three vertebrate EB proteins but its role is unclear because it exhibits weak EB protein activity. This thesis describes molecular and cell . biological experiments designed to functionally characterise human EB2. Fourteen human cell lines were all shown to express EB2 at lower levels than EB 1, while isolation of EB2 in HeLa cells by siRNA-mediated knock down of EB 1 and EB3 confirmed reports that EB2 is outcompeted at microtubule ends by EB 1 and EB3. Analysis of individual microtubules corroborated proposals that EB2 has lower affinity for microtubule tips than EB 1, and also suggested that EB proteins interact more strongly with the proximal region of microtubule tips than the distal region. Furthermore, the + TIP CLIP-170 localised to this distal region independently of EB proteins, implicating an activatory rather than direct-coupling role for EB proteins in the recruitment of CLIP-170. Additionally, the functional effects of structural divergences in EB2 were examined by mutation. An N-terminal extension, EB2 Nose, was shown to attenuate + TIP binding through a functional interaction with 322pQ323 in the C- terminal tail of EB2. A putative SxIP motif, 25TIIp28, was identified within EB2 Nose which contributed to this attenuation. It is proposed from these findings that EB2 is autoinhibited for + TIP binding by intramolecular interactions, between EB2 Nose and 322pQ323, and between 25Tllp28 and the C-terminal EB domain. These data portray EB2 as an unproductive EB protein, and a '+ TIP spacer' function is postulated.

No description available.

No description available.

PTEN is a tumour suppressor protein named after its homology with phosphatase and tensin and its frequent deletion on chromosome 10. PTEN was discovered to be mutated in several solid tumours such as breast brain and ovarian cancers. This study sought to establish the role of PTEN mutation in haematological malignancies. Methods were developed to extract DNA from archival material consisting of formalin-fixed, paraffin-embedded tissue sections and methanol-fixed, May-Grunwald and Giemsa-stained bone marrow smears. To optimise amplification of extracted DNA, PCR primers were developed to produce a range of product sizes from human p-globin, prothrombin and PTEN genes. Techniques were developed for RTPCR amplification of cDNA from cultured haematological cell lines. Electrophoresis methods were developed to demonstrate heteroduplex formation using non-denaturing concentrations of denaturing agents and different gel media and running temperatures. Primers were designed to flank exon sequences and a representative group of myeloid and lymphoid malignancies were screened for PTEN mutations. Exons 5, 7 and 8 were initially amplified for heteroduplex analysis following reports of frequent mutation. No heteroduplex bands were observed in samples assayed. The possible insensitivity of heteroduplex analysis to mutations in sub populations of mutant tumour cells led to the development of SSCP techniques. These techniques were optimised for analysis of DNA that was extracted from frozen archived bone marrow. PCR reactions were developed to amplify each PTEN exon. No suitable primers were found for exon 2 analysis due to extensive non-specific amplification. A total of 912 SSCP assays were performed, screening a range of lymphoid and myeloid malignancies for mutations in all PTEN exons except exon two. Only two possible aberrant conformations were indicated, both in exon 5. The exon 5 PCR product from both positive samples were DNA sequenced using forward and reverse primers. Exon 5 of seven other leukaemia samples were sequenced as controls to detect possible lack of sensitivity of SSCP analysis to PTEN mutations. All samples showed exon 5 DNA sequences in agreement with published sequences. These results suggest that small deletions and point mutations in the PTEN gene are very rare in haematological malignancy. The expression of PTEN mRNA in cultured haematological malignancy cells was established by the earlier RTPCR amplification using cultured cells. Western blotting was performed to establish PTEN protein expression in primary malignancies. All samples assayed showed protein expression. Concerns regarding the contribution from residual normal cells to the PTEN expression picture in tumour cell samples led to the assay of protein by immunofluorescence. PTEN protein was demonstrated in all successful reactions with one CGL and one AML M3 showing higher levels of expression that control cells. These results suggest that in haematological malignancy, the PTEN gene is not mutated and PTEN protein is expressed in leukaemic cells. Research by other workers has demonstrated the importance of PTEN in normal haemopoesis. In mouse models, normal PTEN expression is essential for normal compartmentalisation and to prevent the acquisition of a leukaemic picture. This study suggests that if PTEN dysfunction is involved in the development of haematological malignancy, then it is by a subtle mechanism such as a change in expressed protein levels. It may be that, with the small number of oncogenic mutations necessary to promote a leukaemic phenotype, mutated PTEN does not provide a selective advantage to promote further the survival of the malignant cell population.

Human glyoxylate reductase/hydroxypyruvate reductase (GRHPR) is a D-2-hydroxyacid dehydrogenase found predominantly within the liver, and is essential for the removal of the reactive metabolic product glyoxylate. Primary hyperoxaluria type 2 (PH2) disease, which is characterised by increased urinary oxalate and L-glycerate levels leading to calcium oxalate deposition and decreased renal function, is caused by mutations in the gene for GRHPR. In this study the first crystal structures of the human GRHPR enzyme are reported. Structures of apo, binary and ternary forms of GRHPR are reported, the latter being the first structure of a true ternary complex of an enzyme from the D-2-hydroxyacid dehydrogenase family.

No description available.

Proteinase-activated receptors (PARs) are a novel family of G-protein coupled receptors (GPCRs) that mediate many of the diverse biological effects of proteinases on target cells. N-linked glycosylation is a common post-translational modification in the GPCR superfamily that enables the cell to regulate protein function without recourse to the genome. Previous pharmacological studies have found that activation of hPAR-2 by mast cell tryptase can be regulated by receptor N-terminal glycosylation. However, the role of N-linked glycosylation in regulating the expression and function of other PARs is not known.

Muscle atrophy is a feature of many chronic diseases and contributes to both morbidity and mortality, emphasising the importance of understanding the molecular pathways involved. Zinc finger protein 216 (ZNF216) is an atrogene, a gene which is up-regulated during and directly mediates skeletal muscle atrophy, and encodes the ubiquitin (Ub) receptor protein ZNF216. Herein it is demonstrated that ZNF216 mRNA levels increase in the extensor digitorum longus (EDL) in a lipopolysaccharide (LPS)-infusion rat model of muscle atrophy, relative to saline control. However, combined administration of low level dexamethasone (Dex) with LPS, although sparing muscles from atrophy, did not blunt ZNF216 expression which parallels previous observations for the atrogenes muscle atrophy F-box protein (MAFbx) and muscle RING-finger 1 (MuRF1). ZNF216 expression levels were further elevated in biceps femoris muscle in rats dosed with the statin drug simvastatin (in which severe muscle damage and atrophy occurs), relative to control rats. The ZNF216 protein’s Ub-binding ability and its reported association with the 26S proteasome indicates it may shuttle proteins targeted for degradation to the proteasome as part of the atrophy programme. We utilised immobilised recombinant ZNF216 protein and its Ub-binding Znf_A20 domain alone to capture Ub-modified proteins from rat skeletal muscle that may represent ZNF216’s substrates. Bound proteins specifically eluted by deubiquitination were identified via liquid chromatography tandem mass spectrometry (LC-MS/MS) and included adenylate kinase 1 (AK1) and actin, both previously proposed as substrates of MuRF1. However, ion scores for all candidates were below the accepted threshold of significance and immunoblotting failed to validate LC-MS/MS data. This approach also revealed an increase in a low molecular weight Ub-positive protein from EDL muscle after 24hrs of LPS infusion. Retrospective analysis revealed this Ub-positive protein was consistently captured in other experiments and confirmed by protein MS and immunoblotting to represent an unmodified and unanchored (i.e. not attached to a substrate) K48-linked Ub dimer. Subsequent capture of the Ub dimer using the Znf_UBP domain of isopeptidase T (isoT), a Ub-binding domain selective for the free C-terminus of Ub, confirmed the dimer was unanchored and also revealed a ladder of longer endogenous unanchored poly-Ub chains. Optimised affinity capture conditions has afforded the first opportunity to purify longer free poly-Ub chains and perform the initial molecular analyses of endogenous unanchored poly-Ub purified from in vivo sources.

In this thesis molecular dynamics simulations, in conjunction with the complementary methods of docking and QM-MM, are used, and further developed, to study two unusual polypeptide systems: the conformational preferences of isomers of an antibiotic peptide and the binding behaviour of a human transporter protein. The antibiotic peptides are analogues of a naturally occurring antibacterial called nisin which has a biological function dependent on the formation of five macrocyclic rings closed by a thioether bond between modified L-amino and D-amino residues. We propose analogues where the thioether bond is replaced by a disulfide bond between cysteine residues and the chirality of the cysteines is altered. The conformational preferences of the nisin analogues, and the dependence of ring formation on cysteine chirality, are characterised using molecular dynamics. An analogue (D-Cys3-D-Cys7-L-Cys8-L-Cys11) is identified that favours the simultaneous formation of the S3-S7 and S8-S11 disulfide bonds and has an RMSD of 0.6 Å to 1.7 Å between the centroids from clustering the MD trajectories and an NMR structure of wt-nisin. The nisin analogues contain unusual D-amino residues and using explicit solvent MD simulations of four polypeptides, it is shown that the (φ, ψ) → (-φ, -ψ) transformation of the CMAP term in the CHARMM potential energy function leads to sampling of conformations which are closest to X-ray crystallographic structures for D-amino residues and that the standard CMAP correction destabilises D-amino β-sheets and β-turns. The ileal lipid binding protein (ILBP) shows cooperative binding comparable to haemoglobin and unusual site selectivity where one ligand will completely displace another from a binding site, despite both sites having an affinity for each ligand type and the ligands only differing by a single hydroxyl group. A probable location of the third binding site of ILBP is identified which has a role in the allosteric binding mechanism. MD simulations indicate that binding to this exterior site induces changes in the orientation of the α-helices with respect to the β-barrel by ~10°. An energetic mechanism of site selectivity for ILBP is proposed using evidence from MD simulations. The higher hydrophobicity of chenodeoxycholic acid leads it to sit deeper in the binding cavity and interact with Gln-51. This causes the cholic acid ligand to be deeper and induces the helices to move closer to the β-barrel, preventing further ligand exchange.

The availability of large scale protein interaction networks for several species has motivated many comparative studies in recent years. These studies typically employ network alignment algorithms for the task and use the sequence similarity of proteins to aid the alignment process. In this thesis I use a quantitative measure of protein functional similarity and show that the results are superior to sequence based network alignment. I present a method for module detection that combines results from network alignments with clustering measures to achieve superior results over several existing methods. Next, I address the issue of generally low conservation detected by alignments of interaction networks from model organisms. By explicitly modelling evolutionary mechanisms on pairs of networks I test the hypothesis that divergent evolution alone may be the cause. I use a distance metric based on graph summary statistics to assess the fit between experimental and simulated network alignments. Our results indicate that network evolution alone is unlikely to account for the poor quality alignments given by real data. We also find that false positives appear to affect network alignments little compared to false negatives indicating that incompleteness, not spurious links, is the major challenge for interactome-level comparisons. Finally, I focus on the comparative analysis of a subset of the interaction network related to mitosis in Yeast, Human and Fly. Manual ordering of mitosis-related functional annotations allows the study of temporal aspects of the network. I also use a Markov random field approach to infer temporal labels for unlabelled proteins. Sequence based network alignment of the mitotic networks in the three species finds little conservation despite the proteins being functionally very similar. Further investigation suggests a fuzzy relationship between protein sequence and function that may have implications for future network alignment studies.