Background to the project
Initially identified in human diseases such as Alzheimer’s, Parkinson’s, type II diabetes and dialysis related amyloidosis, amyloid fibrils also perform functional roles in mammalian cells and are being developed as nanomaterials. Understanding how cells respond to amyloid fibrils therefore represents a fundamental biological question. We have shown that amyloid fibrils exhibit length-dependent toxicity, with nanoscale amyloid fibrils being endocytosed into lysosomes, whereupon they inhibit the function of this organelle and disrupt trafficking in the endolysosomal pathway. We have also found that HSP70 can protect against amyloid fibril toxicity. These observations highlight that not only is access to intracellular compartments is important in amyloid toxicity, but also that cellular proteins may protect against the deleterious effects of amyloid.
This project will identify cellular proteins in the endolysosomal pathway that bind to amyloid fibrils and determine the role of these interactions in amyloid toxicity. Objective 1 will use proteomics to identify cellular proteins that bind to amyloid fibrils. Cells will be incubated with nanaoscale fibrils produced from the model amyloid protein beta-2-microglobulin and other disease relevant amyloid fibrils; cellular proteins that bind to amyloid fibrils will be isolated in pull-downs and identified by mass spectrometry. Objective 2 will analyse the role of fibril-cellular protein interactions, by testing whether binding to fibrils impairs the function of proteins in the endolysosomal pathway and/or if protein binding reduces fibril toxicity. The project will use a wide range of experimental techniques, including protein expression, protein purification, confocal microscopy, flow cytometry, subcellular fractionation and proteomics.
Relationship to other cutting edge studies
This project aligns with recent genetic studies that have implicated the disruption of lysosome function and trafficking in the endolysosomal pathway as factors in amyloid disease. Moreover the identification of functional amyloids suggests that cells can also protect themselves against the deleterious effects of amyloid fibrils.
Overview of the interdisciplinary approach and the supervisors
An interdisciplinary approach is required to understand cellular responses amyloid. This project will therefore use a combination of protein chemistry, structural biology, proteomics and cell biology to determine how dissect cellular interactions with amyoid. Thus this project will embrace new ways of working, by providing multidisciplinary training that spans these key disciplines to enable the successful completion of the experimental objectives. Central to the success of the project is the collaboration between Hewitt, an expert in cell biology, and Radford, a world leader in the biophysical characterisation of protein folding and misfolding. This collaboration has to date has produced 10 research articles that are co-authored by PhD students jointly supervised by Hewitt and Radford. The PhD student will work in the research labs of both supervisors where he/she will receive training in an array of specialist techniques: protein expression and purification, the analysis of amyloid structure, cell culture, imaging of amyloid interactions with cells and proteomics.
Self-funded students: International or domestic self-funded or scholarship/fellowship PhD students are always welcome to apply. International students must have a good command of both written and spoken English. In addition to University fees, laboratory fees will be required if you are self-funded. Applications can be made throughout the year.
The project builds upon a successful collaboration between Dr Eric Hewitt and Professor Sheena Radford. Below are some of their recent publications that are relevant to this project.
Xue, WF, Hellewell, AL, Gosal, WS, Homans, SW, Hewitt, EW, Radford, SE (2009) Fibril fragmentation enhances amyloid toxicity. J. Biol Chem. 284, 34272-82
Porter MY, Routledge KE, Radford SE, Hewitt EW. (2011). Characterization of the response of primary cells relevant to dialysis-related amyloidosis to β2-microglobulin monomer and fibrils.PLoS One. 6:e27353.
Goodchild SC, Sheynis T, Thompson R, Tipping KW, Xue WF, Ranson NA, Beales PA, Hewitt EW, Radford SE. (2014) β2-Microglobulin amyloid fibril-induced membrane disruption is enhanced by endosomal lipids and acidic pH. PLoS One. 9, e104492.
Jakhria, T, Hellewell, AL, Porter, MY, Jackson, M, Tipping, KW, Xue, WF Radford, SE, Hewitt, EW (2014). β2-microglobulin amyloid fibrils are nanoparticles that disrupt
lysosomal membrane protein trafficking and inhibit protein degradation by lysosomes. J. Biol. Chem. 289, 35781-94
Tipping KW, Karamanos TK, Jakhria T, Iadanza MG, Goodchild SC, Tuma R, Ranson NA, Hewitt EW, Radford SE. (2015). pH-induced molecular shedding drives the formation of amyloid fibril-derived oligomers. Proc Natl Acad Sci U S A. 112:5691-6.
Tipping KW, van Oosten-Hawle P, Hewitt EW and Radford, SE. (2015) Amyloid Fibres: Inert End-Stage Aggregates or Key Players in Disease? Trends in Biochemical Sciences 40, 719-727
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FTE Category A staff submitted: 60.90
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