About the Project
The vast majority of eukaryotic organisms undergo senescence, from yeast to man, but bacteria do not. Many theories have been proposed to explain ageing as a universal process in animals, but few conserved genetic pathways have been shown to modulate ageing across all species. Importantly, the evolutionary origin of ageing remains a mystery.
Although archaea are prokaryotes, phylogenetic studies have demonstrated that the archaeal and eukaryotic lineages share a common ancestry. Furthermore, the archaeal protein complexes involved in DNA metabolism and protein translation and degradation are homologous to eukaryotic counterparts. In addition, recent meta-genomic sequencing has identified the Asgard archaeal species, which appear to express membrane-associated complexes indicative of eukaryotic-like endomembrane structures and vesicular trafficking systems.
The loss of protein homeostasis is a key feature of organismal ageing, from yeast to man. The ubiquitin-proteasome (UPS) and autophagy/lysosome-mediated protein degradation are two essential intracellular systems that regulate protein catabolism in living cells. Both are modulated by the Cdc48/p97/VCP AAA-ATPase, an abundant homo-oligomeric toroidal assembly that is conserved from archaea to vertebrates, but not commonly in bacteria. Cdc48/p97/VCP homologues have been found implicated in animal ageing, notably in the roundworm Caenorhabditis elegans. The recently discovered Asgard archaea may also perform autophagy/lysosomal-mediated protein degradation and could provide valuable insight into the evolutionary role of Cdc48 p97/VCP in protein homeostasis, autophagy and ageing, from prokaryotes to multicellular organisms.
This project will combine biochemical, biophysical and structural approaches in archaea and yeast (Robinson laboratory) with cell biology and physiological approaches in worms (Benedetto laboratory) to identify Cdc48 interacting partners during ageing, with a particular focus on the link between Cdc48 and autophagy in cellular ageing and neurodegeneration.
The project will start with bioinformatics analyses to identify conserved Cdc48 residues across archaeal and eukaryotic phyla. In vitro structural biochemical and biophysical approaches will then be applied to archaeal Cdc48, while the effect on protein homeostasis of mutating specific Cdc48 residues will be studied in the well-established model archaeon Sulfolobus solfataricus, to identify key residues potentially required for protein-protein interactions between Cdc48 and UPS or autophagy proteins.
Mining published genetic screens and genomics data will reveal likely binding partners for Cdc48 relating to autophagy and UPS regulation. Comparative RNAseq analyses between ageing Cdc48 mutant and control worms will identify genes whose expression is linked to Cdc48, permitting a candidate gene list to be drawn. To reveal genetic/functional links between Cdc48 and autophagy, heat-shock and starvation resistance, proteasome and autophagic activities will be measured in Cdc48 mutant and control worms exposed to RNAi targeted against those candidate genes.
Promising hits from these screens will be studied physiologically in worms (longevity, ageing pathology analyses using stress assays, microscopy, and behavioral analyses) and biochemically for yeast, Crenarchaeal and Asgard archaeal homologues, by reconstituting the relevant proteins in vitro and testing their ability to interact. Yeast, archaeal and C. elegans homologues will also be tested for their ability to genetically complement each other in transgenic yeast and worms.
This work will provide a unique window into the evolution of Cdc48 regulation and its link with ageing, while furthering our understanding of these mechanisms in both archaeal and eukaryotic species.
Applications are made by complete an application for PhD Biomedical and Life Sciences October 2017 through our online application system. Closing date, midnight 3rd April 2017.
Informal enquires about the project should be made directly to Dr Nick Robinson [Email Address Removed] or Dr Alex Benedetto [Email Address Removed] – please include a copy of your CV.
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