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No description available.

No description available.

The aim of this project was to investigate into the effect of the Loa mutation in the function of the cytoplasmic dynein complex. Protein-protein interaction assays were performed on NIH3T3, NSC34 and HEK293T cells transfected with a 2 kb fragment of the wild type or mutant dynein heavy chain gene 1 bearing the Loa mutation and seven different intermediate chain isoforms. The results indicated a 2.5 fold increase in the binding affinity of the mutant dynein heavy chain 1 727 amino acid fragment for the dynein intermediate chain gene 2 isoform A, compared to the wild type.

Secreted phosphoprotein 24 (spp24) is a member of the cystatin superfamily.;This study identifies a rare single-amino acid polymorphism (p.S38F) of human spp24 and its importance has been assessed by comparing the sequence of human spp24 with that of eight other species. The gene encoding spp24 in mouse (Spp2), like its human ortholog, comprises eight exons with an apparently TATA-les promoter. The exon-intron structure is identical in mouse and human and the size and location of intron 1 is conserved between many species. Using several strategies, the gene encoding spp24 in mouse has been mapped to 88832387-88853226 bp of the mouse chromosome 1. An extensive expression study was carried out on the mouse and human genes encoding spp24. These studies indicated that the gene has an expression pattern of a tissue-specific and cell-specific nature, being expressed predominantly in liver and its expression is down-regulated by lipopolysaccharide (LPS) and tumour necrosis factor alpha (TNFalpha). In an attempt to elucidate the function of the spp24 protein in mouse, a pooled-tissues cDNA library was constructed in a yeast two-hybrid vector. Two different constructs comprising the entire spp24 protein and the C-terminal non-cystatin like domain of the protein were used individually as baits in the yeast two-hybrid system to screen the constructed library. Seven potential interacting proteins were identified including granulin precursor also known as acrogranulin/epithelin (Grn), tissue specific transplantation antigen P35B (Tsta3), keratin complex 1, acidic, gene 18 (Krt1-18), keratin complex 1, acidic, gene 13 (Krtl-13 ), vimentin (Vim), similar to protein phosphatase 1, regulatory (inhibitor) subunit 12C (no gene symbol yet assigned) or myosin binding subunit 85 and alpha-actinin-4 (Actn4).

The role of B-Raf in cell growth and survival was investigated using primary MEFs lacking B-Raf. Cell growth and proliferation were found to be reduced and this coincided with alterations in the expression levels of various proteins involved in the G1 to S phase of the cell cycle. Analysis of B-raf cells upon serum withdrawal-induced apoptosis showed this was associated with decreased phosphorylation levels of phospho-T125-caspase 9. A B-raf allele containing a conditional kinase inactivating D594A mutation was generated. This was produced by designing a vector containing loxP sites flanking exons 15-18 of B-raf upstream of a second B-raf exon 15 containing the mutation. Upon gene targeting homologous recombination was successfully achieved for three ES clones and these were used to generate chimeric mice. Two of these targeted ES clones led to successful germline transmission. Mice carrying the mutation were mated with Cre-expressing mice, allowing the generation of mice heterozygous for the D594A mutation, B-raf+Lox-D594A. These mice were shown to have neoplasia due to splenomegaly and this was attributed to the elevated phospho-ERK levels observed. Intercrossing of B-raf+/Lox-D594A mice did not generate any viable B-rafLox-D594A/Lox-D594A mice, suggesting the D594A mutation was embryonic lethal. Analysis of B-rafLox-D594A/Lox-D594A MEFs, phospho-MEK and phospho-ERK levels were significantly elevated. This was attributed to increased C-Raf kinase activity observed within these cells. Further analysis indicated increased survival and increased cell growth and proliferation in these cells.

Talin is a conserved cytoskeletal protein consisting of two subdomains: the talin head and the talin rod. Talin physically connects integrins to the actin cytoskeleton and regulates the inside-out activation of integrins. Binding partners of the talin head include integrins, focal adhesion kinase (FAK) and phosphatidylinositol-4-phosphate 5 kinases type 1 gamma isoform (PIPK1gamma). Along the talin rod, two actin binding sites, one integrin binding site and a minimum of three vinculin-binding sites have so far been mapped.;Vinculin is a cytoskeletal protein, which localises to focal adhesions. It has a molecular weight of 120kDa and consists of two parts: the head and a C-terminal tail. The talin binding site is located in the vinculin head between residues 1-258 (Vh').;In this thesis, the crystal structures of talin rod fragments 482-655 and 482-789 are presented. The structures are classified and compared with structural homologues, demonstrating that the talin rod structural organisation resembles the vinculin tail, alpha-catenin and apolipoproteins A and E. Talin 482-655 is a five helix bundle and talin 656-789 a four helix bundle. All helices are amphipathic and the predicted vinculin-binding site 1 (VBS1) is found buried in the hydrophobic core of the 5-helix bundle of talin 482-655. With supporting evidence from the two structures, mutant binding studies, alanine substitution experiments, NMR spectroscopy, limited proteolysis and the crystal structure of Vh' in complex with talin 605-628, a mechanism involving extensive conformational changes for both talin and vinculin is proposed to be required, in order to achieve vinculin/talin interaction.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most recently characterized Ca2+ mobilizing second messenger, joining inositol 1,4,5-trisphosphate (IP3) and cyclic ADP-ribose (cAD PR). Previous pharmacological and biochemical studies have suggested that NAADP targets acidic Ca2+ stores rather than the ER, which releases Ca2+ in response to IP3 or cAD PR. Although NAADP receptors are proposed to be distinct from those for IP3 and cADPR, the molecular identity of NAADP receptors has remained elusive. This study focuses on establishing whether two-pore channels (TPCs), a family of intracellular Ca2+-release channels, function as NAADP receptors. In this thesis, TPC proteins have been investigated as potential NAADP receptors in the sea urchin, Strongylocentrotus purpuratus, whose genome encodes three potential TPC proteins, namely SpTPCl, SpTPC2 and SpTPC3, and in the social amoeba, Dictyostelium discoideum, which encodes only one TPC2 (DdTPC2). These channels were analyzed in multiple model systems namely the sea urchin egg, human cell lines (HEK cells and HeLa cells), an insect cell line (sf9) and Dictyostelium. Analytical approaches including the generation of anti- TPC antibodies, localization studies, ligand binding assays and Ca2+ release studies were employed. SpTPCl and SpTPC2 were predominantly localized to the ER and the lysosome, respectively. Endogenous sea urchin protein immunoprecipitated with anti-SpTPC3 antibody showed enhanced NAADP binding. However, no correlation between NAADP binding and expression levels of TPCs in HEK cells or Sf9 cells was detectable. In Dictyostelium, TPC2 was localized to the contractile vacuole and the ER, whereas DdTPC2 localized to the lysosome and Golgi apparatus when expressed in HeLa cells. Protein expression of DdTPC2 is regulated developmentally, reaching a peak in the late stages of development. A tpc2 null strain exhibited abnormal post- aggregative development, consistent with a role for DdTPC2 at later stages of development. No high affmity NAADP binding sites or NAADP-induced Ca2+ release were detected in Dictyostelium membranes isolated either at the early or late development stages in these studies. However, NAADP-induced Ca2+ release from vesicle fractions prepared from vegetatively growing cells was observed. On the other hand, arachidonic acid (AA) induced Ca2+ release from vesicles prepared from growing cells and from cells developed for 4 hours or 20 hours. AA-induced Ca2+ release was altered in tpc2 null cells. In sea urchin egg homogenate, AA also elicited Ca2+ release with an ECso value of approximately 8 f.!M. Low concentrations of AA effectively inhibited NAADP-induced Ca2+ release. It was not possible to produce evidence of TPCs being NAADP-sensitive Ca2+ channels in the systems investigated. In Dictyostelium, TPC2 does not appear to be responsible for NAADP-induce Ca2+ release. However, TPC2 may play a role in AA- induced ci+ release.

The SALMFamides SI (GFNSALMFamide) and S2 (SGPYSFNSGLTFamide) are neuropeptides that act as muscle relaxants in the starfish, with S2 ten times more potent than S 1. The aim of this study was to investigate the structural basis for this difference in potency. Synthetic analogues of SI and S2 were synthesized with the N-terminal SGPY sequence of S2 removed from S2 (SS2) and added to SI (LS 1) or with the penultimate amino-acid residues exchanged between SI and S2, SI (T) and S2(M). Analysis of the effects of SI, S2, LS 1, SS2, SI (T) and S2(M) on starfish cardiac stomach and tube preparations indicated that sequence differences in the C-terminal region of S 1 and S2 are the primary determinants ofS2's increased potency compared to SI and not the presence and absence of the N-terminal SGPY sequence in S2 and SI, respectively. To further investigate the structural basis for the differing potencies of SI and S2, the solution conformations of SI, S2, LS 1, SS2, SI (T) and S2(M) were investigated using CD and IH NMR spectroscopy. All the peptides except S2 and LSl have a random coil conformation in water. Interestingly, however, S2 has a single well-defmed conformation with a main-chain RMSD of 0.s03A for residues 2-11, comprising three loops in which aromatic side-chains form hydrophobic clusters with side-chains of other non-polar amino acids. In contrast, LS 1 has a single loop in its C-terminal region with an RMSD of 0.909 A for residues 6-12, which results from clustering of the hydrophobic side-chains ofPhe12, Met 11 , LeulO and Phe6• The remarkably well-defmed conformation of S2 is unusual amongst neuropeptides of comparable size without disulphide bridges. The N-terminal region of S2 (SGPy) may be important for induction and stabilisation of the conformation adopted by S2 in water.

Colicins are a fascinating group of proteins that bind to their cognate immunity proteins with femtomolar affinity, but are also highly cross-reactive binding to non-cognate immunity proteins over a wide range of affinities.