No description available.

No description available.

No description available.

No description available.

Fused-in-sarcoma (FUS) is an RNA binding protein, thought to be involved in a wide variety of cellular processes, and mutations in FUS are known to be causative for amyotrophic lateral sclerosis (ALS). The mechanism of pathogenesis for ALS has not been established but it has been proposed that dysfunction in cellular functions involving RNA could be responsible. Investigations into a FUS-ALS patient cell line showed sensitivity to the transcriptional inhibitor camptothecin (CPT) and demonstrated constitutively fragmented nucleoli, a phenotype that has been associated with rRNA dysfunction, as well as a possible defect in ribosomal RNA (rRNA) maturation. In addition a reversible relocalisation of FUS to the nucleolus in response to inhibition of RNA polymerase II was observed in all cell lines examined. This relocalisation appeared to be dependent on the activity of phosphodiesterase 8 (PDE8) and on the presence of rRNA, as pre-inhibition of RNAP I (which produces rRNA) prevented relocalisation of FUS. However treatment of both RNAP I and RNAP II at the same time resulted in FUS relocalisation and the protein remaining in the nucleolus for hours if inhibition was maintained - long after RNA would be depleted at the site were RNAP I inhibited in isolation. These findings suggest that FUS may have a role in protecting pre-rRNA transcripts from degradation during transcriptional stress.

No description available.

Linear DNA is packaged into higher-order structures termed chromatin, in which the majority of DNA sequences are structurally inaccessible and functionally inactive. Hence, chromatin must exist in a dynamic state to govern the accessibility of the DNA to various regulatory factors that control nuclear processes, such as transcription, DNA replication, and DNA repair. Accessibility in turn is mediated by post-translational modifications, histone variants, histone chaperones, and ATP-dependent chromatin remodelling complexes. In particular, these complexes, such as the multi-subunit INO80 complex, are characterized by their ability to utilise ATP hydrolysis to alter histone-DNA contact by the sliding, eviction, or exchange of histones or nucleosomes. Since its identification in 1999, many INO80 subunits have been purified and structurally characterized. Here, we structurally and biochemistry characterized the recently identified human INO80 YY1 subunit and demonstrated a role for YY1 together with RUVBL2, another INO80 subunit, in promoting DNA repair by homologous recombination (HR). Consequential to its chromatin-remodelling activity, INO80 plays multiple roles in cellular metabolism including DNA repair and chromosomal stability. Although evidence from yeast and mammals has indicated the involvement of INO80 in HR repair, the exact mechanism affected by INO80 therein remained unclear. Through a combination of live cell imaging and in vivo techniques, we revealed that in human cells the histone variant H2AZ is rapidly removed from damaged chromatin by INO80. Furthermore, we found that INO80 together with the histone chaperone, ANP32E, promotes HR by removing H2AZ from damaged chromatin. Finally, we verified that INO80 is required for the maintenance of chromosomal stability and that loss of INO80 in CIN+ tumour cells induced cell death. Therefore, INO80 may serve as a therapeutic target for the selective elimination of CIN+ tumour cells.

Small ubiquitin like modifier (SUMO) is post-translationally attached to target proteins, forming a covalent bond between its C-terminal glycine and one or more lysine residues on the target protein. SUMO modification of target proteins can affect protein-protein interactions, protein activity, localistation and stability. This study set out to develop an efficient in vitro SUMOylation system to enable the identification of target lysine residues in S. pombe proteins by mass spectrometry. This involved incorporating a trypsin cleavage site adjacent to the SUMO di-glycine motif to improve peptide coverage during mass spectrometry. Several SUMOylated target proteins were identified here, including the E2 SUMO conjugating enzyme Hus5, the E3 SUMO ligase Nse2 and PCNA. The second part of this study focused on the characterisation of unSUMOylatable E3 SUMO ligase nse2 mutants. Integration of lysine to arginine mutations into the genome did not result in any mutant phenotypes and a function for auto-SUMOylation of Nse2 was not identified. During this study, human patients with mutations in the nse2 gene were reported and the equivalent mutations were integrated into the S. pombe nse2 gene to investigate the effect of the mutations. The final part of this work involved the analysis of the SUMOylation of S. pombe PCNA. Using the in vitro system, four target lysine residues for SUMO were identified. SUMOylation of PCNA was also observed in vivo following pull-down studies and 2D gel analysis of wild type and unSUMOylatable mutants. Extensive epistasis analysis was undertaken using these mutants to investigate the role of SUMOylation of S. pombe PCNA.

Self-assembling molecules are central to a plethora of processes found in nature, biotechnology and even disease. The importance of the non-covalent interaction of monomers to the formation of fibrillar assemblies is evident in the repeated use of this mechanism throughout nature, from essential cellular processes such as the formation of the cytoskeleton to the production of silk. Further, it has been recognised in the last two decades that a self-assembly mechanism, that is the formation of amyloid, underpins the pathology of protein misfolding diseases; it is therefore essential to dissect these mechanisms. Despite recent technological and model system developments, self-assembling molecules remain challenging to investigate. Using combined structural and biophysical characterisations of penta- and hexa-peptide self-assembling model systems these investigations shed further light on the structure of amyloid-like fibrils. The elucidation of the structures of these fibrillar systems not only has implications for disease but also makes them well placed for consideration for biotechnological applications. In reflecting upon how cross-ß structural architectures can be organised in the fibrillar state, a molecular and supramolecular model of fibrils formed by a fragment of !-synuclein is reported. The fibrils are found to consist of a novel and elaborate cross-ß architecture that leads to a helical supramolecular assembly spanning length scales previously unobserved for such a system. Where self-assembly is a useful route to supramolecular structure formation, the use of low molecular weight gelator (LMWG) peptides to create fibrillar structures with defined material properties is also explored. The complex link between molecular structure, self-assembled architecture, fibril formation, fibril interaction and ultimately bulk material properties is described. It is found that the determinants of self-assembly are distinct from the determinants of gelation and so future LMWG design will have to consider both individually. This work presents methodological advances in the characterisation of self-assembled structures. The investigations presented here have relevance for disease related processes but also to the technological use of these systems as materials. Finally, this work emphasises the beauty of the extravagant, yet elegant connection between molecular interaction and supramolecular selfassembly.

Amyloid fibrils are associated with a large number of diseases in which proteins and peptides abnormally assemble to form insoluble amyloid that deposit in the tissues. However, oxidative stress has been implicated in the pathogenesis of a number of neurodegenerative diseases and is believed to play an important role in the amyloid deposition through protein cross-linkings. Under oxidative stress conditions, tyrosyl radicals can be formed and coupled to form dityrosine cross-linkage. The formation of dityrosine cross-linked oligomers is one of the oxidative modifications that may mediate the toxicity of amyloid β (Aβ) and α-synuclein (α-syn) in Alzheimer's disease (AD) and Parkinson's disease (PD) respectively. In this thesis, I explored the oxidative modification of two short peptides, HYFNIF and VIYKI, using a Cu2+/H2O2 oxidation system, and studied the morphological and conformational changes of these amyloid fibrils during the oxidation process. These peptides were selected as simple amyloid model systems that have been previously structurally characterised, to better understand the dityrosine formation at a structural level and to optimise the oxidation conditions. Oxidative stress has been implicated in AD. Here, I have explored the formation of dityrosine cross-linked Aβ42 in vitro. We have shown that dityrosine is generated in internalised Aβ in cell cultures. Results also revealed the prevalence of dityrosine crosslinks in amyloid plaques in brain tissue and cerebrospinal fluid from AD patients, indicating that dityrosine could be used as a biomarker of oxidative stress in AD. The ability of the Cu2+ ion to promote the formation of in vitro dityrosine cross-linked α-syn was also explored and the effect on α-syn fibrillogenesis and conformation induced by Cu2+ was investigated. The results revealed the possibility of involvement the dityrosine cross-linked α-syn dimer as a nucleus to initiate the polymerisation process of α-syn to form amyloid fibrils. Dityrosine cross-linkages can be generated in vitro using oxidation system of Cu2+/H2O2, and might play an important role in the solubility and assembly of amyloidogenic peptides and proteins that are associated in the pathogenesis of many neurodegenerative disease including AD and PD. Dityrosine cross-linkages can lend a further stability to the already stable amyloid fibrils, and this may explain their protease resistance. Dityrosine cross-links formation represents one of the possible pathways by which oligomers can be formed. Dityrosine cross-linked oligomers represent a good bio-index of oxidatively coupled tyrosine-contained proteins due to their high stability.