Outline
Maintenance of functional mitochondria is essential for life and an important therapeutic target in several diseases, including age-related neurodegenerative diseases. Mitochondrial dysfunction is linked to redox damage and dysregulation of mitochondrial protein homeostasis mechanisms. The burden of these to mitochondria can often lead to cell death. Mechanisms that control import of proteins and elimination of damaged proteins, redox regulation and protein homeostasis are therefore critical for safeguarding mitochondria under stress conditions and key to cell physiology. Despite their importance, these mechanisms remain elusive.
Our recent work has revealed that under stress conditions a number of proteins, many of them with critical antioxidant and chaperone functions, are targeted to mitochondria (particularly in the intermembrane space) via unconventional pathways. Once inside the mitochondria, these proteins protect the organelles from oxidative damage and protein aggregation, which pose a dual threat to cell homeostasis. It is critical to understand the key players and how they are imported to mitochondria to exert a pro-survival function, how they affect the function of critical proteins like the inner membrane metabolite transporters that are critical for metabolism and how they inhibit the onset of diseases like neurodegeneration.
Recent evidence from our lab has identified several proteins that undergo stress-dependent targeting to mitochondria where they either assist the physiological mitochondria biogenesis process or facilitate the elimination of damaged proteins by proteolytic systems like the proteasome. The project will therefore address how stress-responsive alterations of mitochondria biogenesis ensure mitochondria fitness and metabolic rewiring as a protection against irreversible damage.
Aims
Our overarching goal is to determine a previously hidden aspect of cells undergoing oxidative stress whereby their capacity to target proteins to mitochondria is substantially altered to benefit the cell (for example through control of mitochondrial Reactive Oxygen Species). The broader effects on function of resident mitochondrial transporters is also key to understand as a mechanism of stress-induced metabolic rewiring.
This will be achieved by three specific aims to be addressed in this project:
- To characterize new mitochondrial targeting mechanisms of proteins that become alternatively translated under oxidative stress
- To elucidate specific stress-induced modifications on the mitochondrial protein import components and inner membrane metabolite transporters that underpin their expanded transport capacity under stress
- To explore how misfolded and aggregated mitochondrial proteins are exported for clearance by the proteasome as a means to maintain a healthy protein balance within mitochondria and influence cell death pathways.
Training outcomes:
The student will be trained in several complementary biological disciplines in the laboratories of the supervisory team:
- Molecular biology and genetics approaches will be used to generate several yeast (S. cerevisiae) models for the project. Further, shRNA and CRISPR/Cas-9 approaches will be used for gene manipulations in mammalian cells (fibroblasts and cancer cell lines).
- Biochemistry and Cell Biology techniques will be employed to assess protein import, protein-protein interactions, intramitochondrial localization using FRET, split GFP and super-resolution microscopy and mitochondrial function using high-resolution respirometry
- Biophysical and structural biology assays will be applied for purified proteins in a reconstituted system using microscale thermophoresis and isothermal titration calorimetry for measuring binding affinities between proteins and NMR for structural analysis
- Bioinformatic and proteomics approaches will assist the student to elucidate unknown mitochondrial targeting motifs and identify redox-related PTMs on protein translocon components. Omics approaches will be performed in collaboration with experts (Redox modifications group at the National Proteome Facility, Beijing, China) and professional services. The student will learn to prepare the samples (including experimental design) for analysis as well as to analyse and interpret the results.
- Transferable skills (eg. communication, networking, time management) will be provided by the MVLS Researcher Development Plan and by tailor-made activities within the participating labs which are very active in KE activities (public engagement, Industry liaison events) and extended interactions with the EMBO, FEBS and international mitochondria research community.
References
- Kritsiligkou P et al (2017) Unconventional targeting of a thiol peroxidase to mitochondrial intermembrane space facilitates oxidative protein folding Cell Reports 18(11):2729-2741
- Banci L et al (2009) MIA40 is an oxidoreductase catalyzing oxidative protein folding in mitochondria, Nature Structural and Molecular Biology 16(2), 198-206
- Scialo, F. et al. (2016) Mitochondrial ROS Produced via Reverse Electron Transport Extend Animal Lifespan Cell Metab 23, 725-734
- Giampazolias et al (2017) Mitochondrial permeabilization engages NF-κB-dependent anti-tumour activity under caspase deficiency Nat Cell Biol. 2017 Sep;19:1116-1129
