Edmonds, Matthew
A systematic study of palmitoylation using the model organism Caenorhabditis elegans.
PhD thesis, University of Liverpool.
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Abstract
Palmitoylation is a reversible post-translational modification of proteins which involves the addition of the C16 saturated fatty acid palmitic acid to sulfhydryl groups on cysteine residues, forming a thioester linkage. The addition of palmitate allows proteins to associate with different cellular membranes and membrane subdomains. Palmitoylation is catalysed by the DHHC family of palmitoyl-acyl transferases (PATs), named for their characteristic DHHC motif in a cysteine-rich domain (CRD). Reversibility is conferred by palmitoyl-protein thioesterases (PPTs), which cleave the thioester linkage. The study of palmitoylation has recently gathered pace with the development of methods which allow proteome-scale identification of candidate palmitoyl-proteins. Despite the importance of model organisms in several key studies in the field, palmitoylation has barely been studied in the simple eukaryote Caenorhabditis elegans, the nematode worm. This study commenced with the use of the C. elegans genome data to identify its PATs and PPTs, using the DHHC-CRD and homology respectively. The 15 DHHC PATs were officially named using a dhhc-x system and the two previously known PPTs were confirmed as the only ones with homology to other known PPTs. The current knowledge on palmitoylation enzymes and substrates was collated and analysed to predict possible phenotypes resulting from mutation or knockdown of the enzymes and potential substrates. All available C. elegans strains containing a mutation in an individual PAT or PPT were obtained, covering about half of the PATs and both PPTs, and assayed for various gross phenotypes. In addition a complete library of bacteria able to express double-stranded RNA against PAT or PPT genes was sourced and used to perform similar assays using feeding RNA interference (RNAi). A number of small but significant differences were seen both with mutant and RNAi-treated strains, especially in lifespan assays. To test for possible redundancy and compensation amongst the enzymes, double RNAi was performed against selected closely related PATs and both PPTs. This resulted in the largest phenotype seen: a reduction in lifespan after simultaneous knockdown of both ppt-1 and ath-1. As there are no known palmitoyl-proteins in C. elegans, the proteomic approaches acyl-biotin exchange (ABE) and acyl-resin-assisted capture (acyl-RAC) were employed to provide a list of candidates. These were first optimised using rat brain material and the results compared with previous proteomic studies to find that two-thirds of the hits had been previously found. With this validation, both methods were applied to wild-type C. elegans lysates to give 91 hits as putative palmitoyl-proteins. Mutants for the PPT ath-1 were also profiled by ABE, showing 33 hits which were not present in the wild-type profile. These are potential ATH-1 substrates whose lack of depalmitoylation in the mutant leads to their enrichment relative to wild-type. However, further repeats of these analyses are required for rigorous statistical testing. Taken together, this study shows the first characterisation of palmitoylation in C. elegans, encompassing all of the DHHC PAT and PPT enzymes, putative palmitoyl-proteins and potential substrates of the PPT enzyme ATH-1.
Item Type: | Thesis (PhD) |
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Additional Information: | Date: 2013-04 (completed) |
Subjects: | ?? QH301 ?? |
Divisions: | Faculty of Health and Life Sciences > Institute of Systems, Molecular and Integrative Biology |
Depositing User: | Symplectic Admin |
Date Deposited: | 13 Feb 2014 11:18 |
Last Modified: | 16 Dec 2022 04:39 |
DOI: | 10.17638/00012253 |
URI: | https://livrepository.liverpool.ac.uk/id/eprint/12253 |