Ubiquitination and phosphorylation are among the most widespread posttranslational protein modifications which control most cellular processes. Recent research indicates that there is an important cross-talk between ubiquitination and phosphorylation which contributes to dynamic regulation of signalling pathways. In this project we focus on gaining detailed understanding of the structural dynamics of interconnected regulation of signalling controlled by ubiquitination and phosphorylation. As example we will study two enzymes at the crossroads of ubiquitination and phosphorylation - Parkin and PINK1, which are involved in cellular mitochondrial autophagy.
The E3 ubiquitin ligase Parkin and the protein kinase PINK1 are involved in mitochondrial quality control by inducing removal of damaged mitochondria by autophagy - a process termed mitophagy. In response to depolarised mitochondria, PINK1 phosphorylates both Parkin and ubiquitin, which leads to Parkin activation and subsequent polyubiquitination of substrates on the mitochondrial membrane (Wauer et al, 2015) and eventually to mitophagy. Understanding of molecular mechanisms of Parkin activity and identification of its modulators is of critical importance for normal cell function and in neurodegeneration (LS Chin and L Li, 2016). Recent reports shed light on certain aspects of this process, however we are only beginning to understand the underlying mechanisms.
This project will start with development of a ubiquitination/phosphorylation assay for Parkin activity using our innovative on-bead confocal imaging technology (Auer, Koszela and Tyers, 2014). This new assay will be used to quantitatively reveal the kinetic and thermodynamic aspects of how phosphorylation of Parkin affects its autoubiquitination and as a consequence, its activity as an E3 ligase. The hypotheses generated during the in vitro studies will be followed up in cellular assays using neuronal cells. Once established and optimised for high-throughput screening, the in vitro assay will be used to search for peptidic and biosimilar modulators of Parkin enzymatic activity.
While not a drug discovery project, this basic science chemical biology effort will lead to the identification of chemical and biological modulators, which will then be validated in neurons. The combined technology development and chemical biology project will lead to probes of Parkin function, which will be of high value for the research community. Within a highly challenging field of timely connected multi parameter signal transduction, the project aims to exemplify the mechanism of collaborative phosphorylation and ubiquitination reactions on one high value system and thereby pave the route to follow up studies in other areas.
This cross-disciplinary project, encompassing areas of molecular and cellular biology, biochemistry, biophysics, fluorescence spectroscopy, imaging and assay technology development, will allow the PhD candidate to acquire skills in a vast repertoire of techniques. Besides classical cell and molecular techniques, including mammalian and bacterial cell culture and manipulation, protein expression and purification, enzymatic assays, a range of cutting-edge technologies and advanced equipment will be used, such as the high content - high speed confocal imaging on the Opera system (PerkinElmer) and the laser-enabled analysis and processing (LEAP) of live cells in situ.
Wauer T, Simicek M, Schubert A, Komander D (2015). Mechanism of phospho-ubiquitin-induced PARKIN activation. Nature. 2015 Aug 20;524(7565):370-4.
LS Chin and L Li (2016). Ubiquitin phosphorylation in Parkinson’s disease: Implications for pathogenesis and treatment. Translational Neurodegeneration 2016 Jan 6; 5:1.
Auer M, Koszela J, Tyers M (2014). UPS-CONA On-bead ubiquitination assay: A high-throughput enzymatic on-bead assay for interrogation of the ubiquitin-proteasome system in vitro by confocal nanoscanning. European Patent Application, WO 2016/034895 A1.