No description available.

No description available.

No description available.

The transforming growth factor-β (TGF β) superfamily of ligands controls a wide range of biological processes, including cellular differentiation, proliferation, motility, adhesion and apoptosis. It exerts its biological processes mainly through the TGF β receptor and downstream Smad proteins. Deregulation of TGF β signalling is associated with a variety of human diseases, including cancer. Ubiquitination is a reversible post-translational modification implicated In a variety of cellular processes, including proteasome-mediated protein degradation, DNA repair, regulation of transcription, endocytosis and receptor trafficking. Reversible ubiquitination tightly regulates the TGF β signaling pathway by controlling the basal expression level of TGF β pathway components, terminating signals after prolonged activation of signalling cascade or regulating activity of transcription factors involved in regulation of TGF β target gene expression. In this study, I assessed the involvement of two deubiquitinating enzymes (DUBs) AMSH (~ssociated molecule with the SH3 domain of STAM) and AMSH-LP (AMSH like protein) in the signalling of the TGF~ superfamily of ligands. I compared cellular localisation of both DUBs in HeLa cells as well as their relative protein levels in HEK293T cells. I performed a comparative analysis of the interactions of both AMSH and AMSH-LP with the components of the Smad family of signal transducers using a yeast two- hybrid approach. I also demonstrated evidence for the role of AMSH and AMSH-LP in the regulation of signalling by the TGF~ superfamily ligands, using a luciferase reporter driven by the TGF β or BMP-responsive promoters. Importantly, I showed differential dependence of AMSH and AMSH-LP effect on TGF~ signalling on their DUB activity. I then tested the importance of endosomal localisation for the role of AMSH and AMSH-LP in TGF β signalling and presented evidence for AMSH regulation of TGF β type I receptor levels. Finally, I report on a comprehensive siRNA screen testing the involvement of other DUBs in these signalling pathways.

This thesis is focused on elucidating the structure/function relationships of two copper proteins, nitrite reductase and rusticyanin using a combined approach of site-directed mutagenesis and X-ray crystallography. Dissimilatory nitrite reductase (NiR) catalyses the reduction of nitrite to nitric oxide (NO) as part of the key biological process of denitrification. A 'hydrophobic pocket' on the protein surface has been identified as the channel through which the substrate, nitrite, may be guided to the catalytic type 2 Cu site. The residues Glu133 and His313 are at the opening of this pocket and the latter was mutated to a Gin in Alcaligenes xylosoxidans NiR to investigate the role of this residue in substrate guidance. The structures of His313Gln and substrate-bound His313Gln have been determined to 1.65 A and 1.72 A, respectively. These structures confirm that His313 is the port of entry for the substrate and reveal an asymmetric bidentate oxy-co-ordinate binding of nitrite. Residue Trp138, adjacent to the type 1 Cu ligand His139 is one of the residues surrounding the small depression speculated to be important in the complex formation with the physiological redox partners, azurin I and azurin II. This Trp residue was mutated to a His and its structure determined to 1.60 A. The ability of the Trp138His mutant to reduce nitrite was investigated using both an artificial donor and a physiological partner, azurin I. These enzymatic activity experiments revealed that whilst the protein is essentially fully active using methyl viologen as the electron donor, there is a significant decrease in the activity using azurin I when compared to the native protein. These observations suggest that Trp138 is an important residue in complex formation and this with residues in the vicinity represent a likely docking surface for azurin. A simultaneous Asp92His/Met144Leu mutation in NiR was undertaken to investigate the role of redox coupling between the two copper centres and proton ii extraction. The residue Asp92 has been demonstrated to be an important residue in a water-mediated pathway for delivering protons to the T2Cu site for the reduction of nitrite. The mutation of Asp92 has a significant effect on the protein, reducing the activity to -10%. The structure determination of Asp92His/Met144Leu NiR to 1 .65 A revealed that despite the perturbation of Asp92 a route exists from the surface of the protein to deliver protons to the T2Cu centre. The blue-copper protein rusticyanin has attracted considerable interest due to its unique properties of high redox potential and stability to extremely high pH. The copper site of rusticyanin is similar to other blue-copper proteins with three strong ligands His85, Cys138, His143 and a relatively weaker Met148 ligand in an axial position. A mutation of His143 to a Met yielded a structure determination to 1.1 A from which refinement using anisotropiC displacement parameters provides an exceptional basis to examine the protein in detail. Furthermore, the metrical accuracy obtained from such refinement coupled with EXAFS analysis suggests that the Cu site alone is not wholly responsible for the properties of rusticyanin. A 2.3 A structure of His143Met demonstrates that the molecules pack in a head to head fashion thus providing further structural evidence to the hypothesis that this interaction is important for electron transfer from a partner protein. The interaction is similar to those present in Met148Leu and Met148Gln rusticyanin crystal structures although the replacement of His with Met disrupts the solvent mediated hydrogen bonding observed in those structures. In His143Met the two molecules are now associated via non-bonded interactions. In conclusion, the crystal structures of the NiR mutants presented in this thesis provide direct evidence for both the point of interaction with its proposed physiological redox partner and the probable mode of nitrite binding in blue CuNiR's as well as confirming the role of Asp92 in proton abstraction. The two crystal structures of His143Met rusticyanin have shed light on particular structural iii features that may account for the proteins unique properties which can now be examined with a future site-directed mutagenesis programme.

No description available.

An understanding of the action of the helical antimicrobial peptide magainin II has significance clinically, since the peptide has broad spectrum antimicrobial, tumouricidal and antifungal activity through its ability to create pores in membranes. To better understand the mechanism of action of this peptide, a joint approach of simulations and experiment was used to determine the factors involved in pore formation by magainin II. Previous studies involving a cysteine-crosslinked mutant indicated that dimerisation of magainin II may be a key step in the insertion mechanism, and atomic detail molecular dynamics simulations have supported the theory that this is due to an inability of the membrane to accommodate the larger bulk of a parallel dimer without significant distortion.

The WASP family of proteins plays a pivotal role in the cell, integrating signals from extracellular stimuli to generate dynamic changes in the actin cytoskeleton. The WASP family of proteins consists of three subfamilies; the WASP subfamily which contains the haematopoietically expressed WASP and its ubiquitously expressed homologue N-WASP, the WAVE subfamily comprising WAVE1-3, and the recently identified WASH subfamily. All three sub-families contain a C-terminal VCA domain, through which they nucleate actin polymerisation via activation of the Arp2/3 complex. Regulation of WASP family proteins differs between sub-families, however a rapidly emerging theme in the WASP and WAVE sub-families is regulation by phosphorylation.

Anatomical evidence describes both extensive serotonergic input and local GABAergic modulation in the hippocampus and synaptic GABA release is modulated by 5-HT in vitro. The majority of extracellular GABA mediating tonic inhibition originates from synaptic spillover suggesting serotonergic modulation of GABA output will have concomitant effects upon tonic inhibition. Therefore, microdialysis coupled to electrochemical detection was utilised to measure extracellular concentrations of 5-HT, 5-HlAA and GABA to examine hippocampal regulation of tonic inhibition. The augmentation of hippocampal 5-HT transmission by the infusion of the selective serotonin reuptake inhibitor citalopram revealed a negative modulation upon ambient GABA levels. Subsequently, local infusion of selective pharmacological compounds was used to examine the receptors mediating this effect. Infusions of the 5-HT1A receptor agonist, 8-0H- DPAT and the 5-HT1A receptor antagonist, WAY 100,635, demonstrated that the citalopram- induced suppression of GABA was dependent upon 5-HT1A receptor activation and its activation evoked comparable decreases. Conversely, activation of hippocampal 5-HT 2A receptors by TCB-2 infusion caused transient increases in detected GABA. No GABA alterations were seen in response to local activation of 5-HT2C or 5-HT3 receptors and no tonic serotonergic modulation was revealed via suppressions of 5-HT in response to the 5-HT1B agonist CP 93,129. Hippocampal endocannabinoids function as inhibitory retrograde messengers suppressing synaptic GABAergic transmission via activation of CB1 receptors in response to postsynaptic triggers. However, the current examination of the influence of CB1 receptor activation upon hippocampal extracellular GABA levels revealed a complex biphasic effect. During drug infusions, both exogenous and endogenous activation of CB1 receptors induced increases in extracellular GABA concentrations which were subsequently followed by long-term suppressions. Hippocampal tonic inhibition is implicated in apt behavioural and neuronal responding, cognition and mood. Therefore, the current demonstration of novel, local modulatory roles for 5-HT and the endocannabinoids upon tonic inhibition is both physiologically important and pathologically relevant.

This study used extracellular field potential recordings (fEPSP) to investigate the role of kainate receptors (containing GLUK5) in short- and long-term potentiation (S-/L-TP) in the mossy fibre (MF) pathway. In vitro, the role of GLUK5 in synaptic plasticity is dependent on extracellular calcium concentration. So the physiological importance was investigated in vivo. In a single experiment GLUK5 selective antagonist, LY382884 (10 mg.kg-l, i.v.), attenuated STP. Notwithstanding, there is continued controversy regarding the role of GLUK5 in MF synaptic plasticity. Using GLUK5 selective antagonists, LY382884 and ACET, the method of hippocampal slice preparation is identified as deterministic. In parasagittal slices prepared in standard aCSF (PsH), STP, measured as ratio of 1st and 5th pulse (P5:P1) evoked at 25Hz, was antagonised by 10uM LY382884; NMDAR independent L TP, evoked by 100 pulses at 100Hz in 50uM AP5, was reversibly antagonised by 50nM ACET. Transverse hippocampal slices prepared in standard aCSF were not viable. In transverse slices prepared in high sucrose aCSF (TH), 10uM LY382884 had no effect on P5:P1-25Hz or P5:P1-50Hz; furthermore 100nM ACET had no effect on P5:P1-50Hz, or on the induction of NMDAR independent LTP. The depression of transmission by Group II mGluR agonists is reportedly a characteristic MF property. In PsH, DCG-IV had no effect on MF fEPSP but depressed P5:Pt-25Hz. In TH, DCG-IV and LY395756 (mGlu2 selective agonist) transiently depressed MF fEPSP. This was attenuated by a Group U competitive antagonist L Y341495. DCG-IV and LY395756 induced a concentration dependent long-term depression (LTD). While 100nM L Y341495 had no effect on OCGI-IV L TO, 300nM transiently reversed L Y395756 L TO. In conclusion, the pharmacology of synaptic plasticity in vitro is critically dependent on slice preparation; preliminary data suggest that GLUK5 receptors contribute to MF plasticity in vivo. Disparity in EC50 values for DCG-IV and L Y395756 induced depression suggests the possible involvement of mGlu3.