STRUCTURE AND FUNCTION OF PROTEINS IMPLICATED IN NEURODEGENERATION
Our goal is to understand how proteins implicated in recessive forms of Parkinson's disease (PD) normally protect neurons, and how they are inactivated in PD. Our approach consists of elucidating their structures using a wide-range of techniques such as X-ray crystallography, nuclear magnetic resonance and mass spectrometry. The 3D structures obtained will be used to understand the mode of action of these proteins. Moreover, these structures can be used as scaffolds for designing new drugs that will enhance their activity and therefore could help slow down or even stop the degeneration of neurons causing PD.
PROTEOMICS
We use mass spectrometry to determine how proteins are modified, either by phosphorylation, ubiquitination, or proteolysis. As the co-director of the Pharmacology SPR-MS unit (https://www.mcgill.ca/pharma/core-facilities), Dr Trempe supervises the operation of a Bruker Impact II Q-TOF, coupled to a Dionex 3000 HPLC (CFI 2014). We routinely analyze intact proteins, resolved on a C4 reverse-phase column, as well as tryptic digests on a C18 nano-column, which we use to characterize protein phosphorylation. Since Feb 2020, Dr Trempe is the Scientific Director of the Proteomics platform at the Research Institute of the McGill University Health Centre (https://rimuhc.ca/clinical-proteomics).
STRUCTURAL BIOLOGY
To understand how proteins function, we use biophysical methods such as NMR spectroscopy X-ray crystallography to solve the 3D structure of proteins, which reveal how they catalyze reactions or interact with each other.
STRUCTURE AND FUNCTION OF PINK1
Mutations in PINK1 causes autosomal recessive Parkinson's disease. The protein is a mitochondrial kinase that phosphorylates ubiquitin. We published a biophysical analysis of PINK1, which revealed how the protein binds ubiquitin, and how it must auto-phosphorylate in trans in order to recognize ubiquitin (Rasool et al, EMBO Rep, 2018).
More recently, we have solved the structure of PINK1 in its dimeric form (movie on the left, PDB 7MP9). The structure reveals the mechanism of PINK1 activation via autophosphorylation, and provide insights into binding to the TOM complex on mitochondria (Rasool et al, Mol Cell, 2022).
FINDING NEW PARKIN ACTIVATORS
Loss of Parkin activity cause early-onset familial PD through inherited mutations. Numerous studies have shown that Parkin activity is neuroprotective in cell and animal models of PD. Based on structural studies, we have demonstrated that mutation of W403 increase Parkin’s activity in cells (Trempe et al. 2013), and can rescue inactive and PD mutations (Tang, Vranas et al. 2017; Yi et al. 2019). More recently, we characterized novel activating mutations in Parkin (Stevens, Croteau, Eldeeb et al. 2023). In collaboration with Biogen and the lab of Kalle Gehring, we determined the mechanism of action of molecular glue that activates Parkin and rescue pathogenic mutations in the Ubiquitin-Like (UBL) domain (Sauve et al., 2024).
PINK1 INHIBITORS
Loss-of-function mutations in the PINK1 kinase cause early onset Parkinson's Disease. There is currently a need for tool compounds for PINK1 to better understand its function in mammalian cells and in vivo. We identified and characterized the small-molecule inhibitor PRT062607 as a PINK1 inhibitor, with an IC50 around 1 uM (Rasool, Shomali et al 2024). This molecule can be used as a tool compound to investigate the effect of acute PINK1 inhibition in cells. Since PRT062607 is also a potent SYK inhibitor (IC50 = 1 nM), we are currently developing more potent and selective derivatives of PRT062607.
MTSviewer
The localization and import of mitochondrial proteins are driven by N-terminal mitochondrial targeting sequences (MTS's). The recent discovery of internal MTS's has expanded their role beyond conventional N-terminal regulatory pathways. Still, the global mutational landscape of MTS's remains poorly characterized, both from genetic and structural perspectives. To this end, we have integrated a variety of tools into one harmonized database called MTSviewer (https://mtsviewer.neurohub.ca/), which combines MTS predictions, cleavage sites, genetic variants, pathogenicity predictions, and N-terminomics data with structural visualization using AlphaFold models of human and yeast mitochondrial proteomes. The details are reported in a recent publication (Bayne et al, 2023)
