Principal Supervisor: Professor Bibek Gooptu, Department of Molecular and Cell Biology
Co-supervisor: Dr Salvador Macip
PhD project title: Proteostatic challenge, senescence and metabolic reprogramming
University of Registration: University of Leicester
This project aims to explore the links between three major cellular behaviours under stress. Proteostasis refers to the suite of dynamic cellular processes required to maintain protein synthesis, to chaperone correct protein folding, and to detect and respond appropriately to protein misfolding. Impaired proteostasis is associated with accelerated ageing, resulting in the accumulation of misfolded proteins in tissue. This is well-recognised in one group of ageing-related diseases – dementias such as Alzheimer’s and Lewy Body dementias where protein misfolding is associated with neurodegenerative responses in the brain. However various emerging data indicate it may also play a role in another set of conditions that reflect a dysfunctional age-related response – scarring (fibrosing) disorders e.g. of liver and lung.
The proteostatic challenge posed by misfolding of the major secreted antiprotease a1-antitrypsin in the endoplasmic reticulum (ER) of hepatocytes causes liver fibrosis. Remarkably recent data within the Gooptu group indicate that this ER event is closely coupled with alterations in mitochondrial morphologic behaviour and bioenergetic pathways within the cell. However such changes are consistent with other groups’ work in the last few years demonstrating how lung fibrosis is associated with dramatic ‘metabolic reprogramming’ similar to that seen in cancer, for which fibrosis can be a precursor event. The molecular mechanisms of cross-talk between proteostatic challenge, metabolic reprogramming and fibrosis remain to be clarified. Moreover fibrosis is associated with a senescent cell phenotype, and the Macip group are at the forefront of studies defining this.
This project will study cell models of liver (a1-antitrypsin) and lung (surfactant protein (SP-)C, mucin5B) fibrosis across various levels of proteostatic challenge (varying expression levels, varying severity of mutation, -/+ ‘second hit’ challenges). It will assess the consequences of these challenges in terms of bioenergetics pathways, mitochondrial phenotype and function, and senescence and other phenotypic markers of pro-fibrotic behaviour (e.g. galectin-3 expression, EMT). The relationship between the different phenotypic responses will also be explored. These systems will then be probed with treatments that can directly modify mitochondrial dynamics (e.g. siRNA-mediated knockdown of mitofusin), senescence (senolytic agents), galectin-3-mediated fibrosis (galectin-3 inhibitors). Such approaches will differentiate upstream from downstream effects, identify feedback loops and define critical interface interactions between proteostasis, metabolic flux, cellular senescence, and pro-fibrotic responses.
BBSRC Strategic Research Priority: Bioenergy and Industrial Biotechnology
Techniques that will be undertaken during the project:
- Cell culture
- Transfections (most required lines already exist)
- siRNA-mediated knockdown
- Fluorescence microscopy and quantitative image analysis (ImageJ, Volocity) - immunofluorescence and MitoTracker dyes: blinded, automated quantification of intensity, measurement of extended v punctate mitochondrial morphology by automated ‘sphericity’ assessment
- ELISA (quantitative analyses) for proteins involved in glycolysis, oxidative phosphorylation, TCA cycle, redox, unfolded protein response, senescence
- Epithelial-mesenchymal transition characterisation (blinded quantitative analyses)
- ROS assays (blinded, quantitative)
- Analyses of dose:response relationships of different treatments v readouts
- Seahorse and/or Ourobouros respirometer quantitative analysis of mitochondrial function
- Statistical analysis
- MS focused proteomic analyses of secreted senescence markers (normalisation, ANOVA and t-test with corrections for multiple comparisons)