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Quantitative Proteomic Studies Of Protein Misfolding & Disease

Cryar, AJ; (2015) Quantitative Proteomic Studies Of Protein Misfolding & Disease. Doctoral thesis , UCL (University College London). Green open access

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Abstract

In the post-genomic era a major paradigm shift occurred in biological research. The ability to catalogue all known genes and corresponding nucleotide sequences and to correlate this information with disease state, cellular function and phenotype, promised to change scientific approaches. As the wealth of genomic data increased, however, it became clear that far more information, in the form of the gene products (proteins), was required to put these new data into context. To achieve this, proteomics; the systematic analysis of the protein complement to the genome, became key. The protein sequence database information derived from gene sequencing heralded mass spectrometry-based proteomics emergence as an indispensable tool in biological studies. No other analytical tool offers the ability to determine the identity, quantity, modification status and cellular stability of hundreds to thousands of proteins in relatively short time periods. Continued technological and bioinformatics developments have increased the ability of mass spectrometry to catalogue proteomes and we are now at a point where close to comprehensive coverages are possible for simple organisms. An ever increasingly aged western society has seen a steady increase in the prevalence of age-related diseases. Neurodegenerative diseases, are, like many other age-related diseases associated with protein misfolding. The cellular responses to protein misfolding and how they relate to disease clinical symptoms are not fully understood. It is, however, commonly accepted that protein misfolding is a key component in disease aetiology. Proteome alterations as a result of protein misfolding, therefore, represent an important avenue of research to better understand this debilitating category of diseases. This work describes the use of label-free quantitative proteomics to expand upon currently known cellular responses to two misfolding diseases, Alzheimer’s disease and α1-antitrypsin deficiency. Initially a GeLC-MSE workflow was developed and successfully utilised to identify proteins that were potentially implicated in Aβ42 toxicity and age-related vulnerability to Aβ42 in D. melanogaster. It is hoped that follow-up studies will provide more information on how these proteins are functionally implicated in the link between ageing and Alzheimer’s disease. [xvi] Following this, a recently developed proteomic technology, that uses ion mobility to increase system peak capacity on the fly, HDMSE, was employed to conduct time-course analysis of cellular responses to wild-type ageing and chronic Aβ toxicity in D. melanogaster. Using this more comprehensive approach, quantitative protein profiles across two lifespans were recorded. The data successfully identified large number of proteins that were significantly altered in abundance in relation to age and Aβ42 misfolding. The data represent a unique resource for follow-up reductionist and functional studies on the link between ageing and Alzheimer’s disease. Finally, a subcellular GeLCMSE approach was successful at characterising ER enriched stress responses to the misfolding of two α1-antitrypsin variants in relation to the wild-type, well folded and fully secreted variant. The present study expands on current pathways implicated in ER stress which have been largely characterised by reductionist studies. Interestingly co-fractionating bioenergetic components were quantified with large fold changes measured between variants. Follow-up live cell confocal imaging correlated these changes with mitochondrial elongation as a result of protein misfolding. This represents a novel finding not previously documented for α1-antitrypsin deficiency.

Type: Thesis (Doctoral)
Title: Quantitative Proteomic Studies Of Protein Misfolding & Disease
Event: University College London
Open access status: An open access version is available from UCL Discovery
Language: English
Keywords: Proteomics, Ageing, Alpha one antitrypsin deficiency, Alzheimer's Disease, HDMSE
UCL classification: UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Genetics, Evolution and Environment
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Structural and Molecular Biology
URI: https://discovery.ucl.ac.uk/id/eprint/1473110
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