Understanding the role of post-translational modification in age-related protein aggregation

Supervisors: David Clarke, Tilo Kunath

Project description:

This project will apply state-of-the-art-techniques in protein mass spectrometry to study the structure and function of the amyloid-forming protein alpha-synuclein. The overall aim is to understand the molecular mechanisms which lead to misfolding and aggregation in amyloid forming proteins.

Amyloids are aggregates of proteins that, under the correct physiological conditions, fold into a conformations that allows many copies of that protein to aggregate forming fibrils. Amyloid formation is implicated in various age-related diseases, as previously healthy proteins lose their normal physiological functions and form fibrous deposits in plaques around cells that disrupt the healthy function of tissues and organs. The molecular determinates which lead to this transition of a healthy protein to an amyloid-forming conformation are not fully understood. In some cases specific polymorphisms have been implicated in increasing susceptibility to amyloid formation. More recently, evidence has suggested that post-translational modification (PTM) may be an important trigger for amyloid formation. The protein alpha-synuclein has been shown to form aggregates, known as Lewy bodies, in neurons and is implicated in Parkinson’s disease. Several PTMs are thought to be enriched within Lewy bodies and exist prior to formation of these structures as early drivers of synucleinopathy (1). This observation suggests that certain proteoforms (a specific modified forms of the protein) might be more relevant biomarkers than the total alpha synuclein levels.

Over the last decade, top-down mass spectrometry has emerged as a powerful technique for the footprinting of protein PTMs and the detailed characterisation of individual proteoforms (2). In this project, this top-down technique will be used to characterise the PTM-patterns which exist in proteoforms of alpha-synuclein derived from neurons in the early stages of pathology and Lewy bodies. Once proteoforms have been characterised, the structural consequences of specific PTMs will be analysed by native mass spectrometry to understand the structural effects of specific protein PTMs. This study will allow us to assemble a picture of the protein’s structural changes which occur in the early stages of alpha-synuclein fibril formation. 

The project will be based in both the School of Chemistry and Centre for Regenerative Medicine at the University of Edinburgh. The School of Chemistry houses state-of-the-art mass spectrometry instrumentation including a 12 Telsa SolariX FT-ICR MS (Bruker), an UltrafleXtreme MALDI ToF/ToF MS (Bruker), and an ion mobility equipped Synapt-G2 (Waters). Training on these instruments will be provided and these facilities will be made available throughout the project. The student will learn skills in protein mass spectrometry, recombinant protein production, cell biology, and biochemistry. The ideal candidate will have a strong interest in mass spectrometry, protein chemistry and structural biology and an enthusiasm for multidisciplinary research.

References:

(1) Schmid et al. Mol Cell Proteomics. 2013, 12, 3543-58.

(2) Lanucara et al. Mass Spec Reviews. 2013, 32, 27-42.

Other: