Background
Autophagy is the major intracellular degradation and recycling route in mammalian cells. The key player is a unique organelle, the autophagosome, which engulfs and delivers cytoplasmic materials for clearance in lysosomes. Autophagosome formation requires the concerted interplay of autophagy-related (ATG) proteins that operate ubiquitin-like conjugation cascades to critically regulate cell homeostasis.
Congenital defects of autophagy are extremely rare, although we recently evidenced the first pathogenic variants in ATG7, the central regulator of the ubiquitin-like cascades, in adults with a rare neurodevelopment disorder. The conundrum that deficiency of an essential autophagy protein is compatible with human life, forms the basis of this multi-disciplinary study, to yield novel molecular insights into autophagy and guide the development of treatment approaches.
Objectives
Combining established biochemical, structural, cell-based and microscopy techniques from two world-leading laboratories, this project (i) investigates effects of newly-discovered ATG7 variants on protein folding, stability, and enzymatic reactions; (ii) uncovers novel protein-protein interactions involving ATG7 in the wild-type as well as autophagy-defective disease states. The overall objective is towards the molecular basis that drives congenital autophagy defects from recessive and de novo ATG7 variants. As such the project will also answer fundamental questions on the role of ATG7 and its associated protein complexes in autophagy.
Novelty
To date, any molecular and functional understanding of human ATG7 in the early autophagy process is extrapolated from characterisation of the yeast orthologue, sharing only ~38% sequence identity. Therefore, obtaining structural information of human ATG7 coupled with detailed biochemical analysis of variant proteins, alone and in complex with binding partners, are key to a deeper understanding of ATG7 defects. This forms the much-needed starting point for deciphering the role of ATG7 in autophagy, how its dysfunction is involved in monogenic and complex diseases, and therapeutic development of autophagy modulation.
Experimental Approach and Timeline
The student will carry out five key steps:
· Production of recombinant ATG7 wild-type and disease variants, and binding partners using various expression hosts (year 1)
· Isolation of endogenous ATG7-associated complexes through CRISPR knock-in of fusion tags and disease-causing variants in relevant cell lines (years 1-2)
· Characterisation of ATG7 variants in vitro - enzymatic activity, protein stability, protein-protein interaction (years 1-2)
· Structural determination of human ATG7/complexes using x-ray crystallography and cryo-electron microscopy (years 2-3)
· Characterisation of ATG7 variants using cell-based assays, mass spec-proteomics, and single-molecule microscopy (year 3).
This project presents a rare opportunity to work at the interface of clinicopathological studies led within the Wellcome Centre for Mitochondrial Research (Taylor and Oláhová) and fundamental structural biology research in the Yue lab at the Biosciences Institute (Yue and McCorvie). The student will be trained in cloning and CRISPR editing, protein expression systems (bacterial, insect, and mammalian), biophysical activity and binding assays (ITC, SPR, DSF, fluorescence), cell-based interaction assays, x-ray crystallography and cryo-electron microscopy. The student will be supported in writing scientific manuscripts and presenting research findings at national and international conferences.
Weblinks:
https://newcastle-mitochondria.com
https://www.staff.ncl.ac.uk/yuelab/
Twitter:
@TheYueLab
@mitoresearch
Benefits of being in the DiMeN DTP:
This project is part of the Discovery Medicine North Doctoral Training Partnership (DiMeN DTP), a diverse community of PhD students across the North of England researching the major health problems facing the world today. Our partner institutions (Universities of Leeds, Liverpool, Newcastle, York and Sheffield) are internationally recognised as centres of research excellence and can offer you access to state-of the-art facilities to deliver high impact research.
We are very proud of our student-centred ethos and committed to supporting you throughout your PhD. As part of the DTP, we offer bespoke training in key skills sought after in early career researchers, as well as opportunities to broaden your career horizons in a range of non-academic sectors.
Being funded by the MRC means you can access additional funding for research placements, international training opportunities or internships in science policy, science communication and beyond. See how our current DiMeN students have benefited from this funding here: http://www.dimen.org.uk/overview/student-profiles/flexible-supplement-awards
Further information on the programme and how to apply can be found on our website:
http://www.dimen.org.uk/how-to-apply/application-overview
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