Primary Supervisor: Professor Flaviano Giorgini, Department of Genetics and Genome Biology
Secondary supervisor: Mariaelena Repici (Aston University)
PhD project title: Exploring the functional and pathological relevance of DJ-1 phosphorylation
University of Registration: University of Leicester
DJ-1 is a small conserved protein of 189 residues which is ubiquitously expressed and primarily localized to the cytoplasm, but also found in the nucleus and associated with mitochondria. A diverse set of potential functions have been suggested for DJ-1, including roles as an oxidative stress sensor, a protein chaperone, a protease, a RNA binding protein, a transcription regulator, a regulator of mitochondrial function, a deglycase and a regulator of autophagy. Notably, mutations in the gene encoding DJ-1 account for ~1-2% of the sporadic cases of early onset recessive Parkinson’s disease (PD), the second most common progressive neurodegenerative disorder. PD is a progressive disorder whose clinical symptoms include muscle rigidity, resting tremor, bradykinesia and postural instability due to loss of dopaminergic neurons in the substantia nigra pars compacta. Surviving neurons often show protein inclusions, known as Lewy bodies and Lewy neurites, which are key pathological hallmarks of PD. Despite the many cellular functions attributed to DJ-1, its role in PD pathogenesis is unclear.
Several post-translational modifications of DJ-1 have been described, including sumoylation, S-nitrosylation and phosphorylation. Indeed, mass spectrometry studies have identified phosphorylated threonine and serine residues of DJ-1, which have raised the possibility that these modifications could be useful as PD biomarkers. Notably, we have generated preliminary data suggesting that mutations which mimic phosphorylation at several of these residues promote protein instability, while phosphoblocking mutations have no effect. Conversely, a recent study by Ko et al. (see below) found that protein kinase A directly phosphorylates DJ-1 at the threonine 154 residue, and that a phosphoblocking mutation of this site leads to enhanced degradation of the protein via the ubiquitin proteasome system. These results suggest that DJ-1 phosphostatus is key to functionality of this protein, and that its phosphorylation at different residues can have divergent consequences. The aim of the proposed work is to understand the dynamics of DJ-1 phosphorylation in physiological and stress conditions, and to clarify the functional consequences of these post-translational modifications. This work will extend our understanding of DJ-1 biology, and may ultimately inform its role in PD pathogenesis and its potential as a biomarker for this disorder.
- Assess DJ-1 phosphostatus in physiological and stress conditions via mass spectrometry in SH-SY5Y neuroblastoma cells. Stress conditions will include oxidant treatments (H202, sodium arsenite), as well as toxins (rotenone, paraquat) and expression of transgenes (e.g. alpha-synuclein, LRRK2) used to mimic aspects of PD.
- Key residues identified above will be altered by site-directed mutagenesis to generate phosphomimetic and phosphoblocking mutants. Functional analyses will be performed to understand the biological consequences of these alterations (protein stability, DJ-1 dimerisation, cellular localization and ability to protect from oxidative stress). We will also determine the effect of single phosphoblocking/phosphomimetic mutants on the overall phosphostatus of DJ-1 in order to clarify if the dynamics of overall phosphorylation of DJ-1 are affected by changes at individual residues. Alterations in protein interaction partners will be assessed using coimmunoprecipitation/pull-downs with wild-type DJ-1 and key DJ-1 mutants identified from this work.
- Key mutations identified from these transfection studies will be replicated with the endogenous copies of the DJ-1 gene using CRISPR/Cas9 technology, which will permit more detailed analyses of the biological consequences of these manipulations.
- As a step towards translation studies, key findings from the SH-SY5Y cells above will be validated and characterised in primary rodent central nervous system cells (e.g. neurons, astrocytes) where DJ-1 expression has been previously described.
- Ko YU, Kim SJ, Lee J, Song MY, Park KS, Park JB, Cho HS, Oh YJ. Protein kinase A-induced phosphorylation at the Thr154 affects stability of DJ-1. Parkinsonism Relat Disord. 2019 Jul 23. pii: S1353-8020(19)30319-0. doi: 10.1016/j.parkreldis.2019.07.029.
- Repici M, Giorgini F. DJ-1 in Parkinson's Disease: Clinical Insights and Therapeutic Perspectives. J Clin Med. 2019 Sep 3;8(9). pii: E1377. doi: 10.3390/jcm8091377. Review.
- Repici M, Hassanjani M, Maddison DC, Garção P, Cimini S, Patel B, Szegö ÉM, Straatman KR, Lilley KS, Borsello T, Outeiro TF, Panman L, Giorgini F. The Parkinson's Disease-Linked Protein DJ-1 Associates with Cytoplasmic mRNP Granules During Stress and Neurodegeneration. Mol Neurobiol. 2019 Jan;56(1):61-77. doi: 10.1007/s12035-018-1084-y.
BBSRC Strategic Research Priority: Understanding the Rules of Life: Neuroscience and behaviour
Techniques that will be undertaken during the project:
- Microscopy techniques (confocal; electron)
- Mammalian cell culture; primary neuronal/astrocytes culturing
- Generation of site-directed mutants; mammalian cell transfections; CRISPR-Cas9
- Proteomics work (protein isolation, mass spectrometry)
- Relevant cell-based biochemical/molecular assays (viability/cell death assays, immunoblotting)
Contact: Professor Flaviano Giorgini, University of Leicester