Time‐Resolved NMR Analysis of Proteolytic α‐Synuclein Processing in vitro and in cellulo

Abstract

Targeted proteolysis of the disordered Parkinson's disease protein alpha-synuclein (αSyn) constitutes an important event under physiological and pathological cell conditions. In this work, site-specific αSyn cleavage by different endopeptidases in vitro and by endogenous proteases in extracts of challenged and unchallenged cells was studied by time-resolved NMR spectroscopy. Specifically, proteolytic processing was monitored under neutral and low pH conditions and in response to Rotenone-induced oxidative stress. Further, time-dependent degradation of electroporation-delivered αSyn in intact SH-SY5Y and A2780 cells was analyzed. Results presented here delineate a general framework for NMR-based proteolysis studies in vitro and in cellulo, and confirm earlier reports pertaining to the exceptional proteolytic stability of αSyn under physiological cell conditions. However, experimental findings also reveal altered protease susceptibilities in selected mammalian cell lines and upon induced cell stress.

In-Cell NMR Study of Tau and MARK2 Phosphorylated Tau

“…However, we did not observe the additional resonances for Tau40 in HEK-293T cells which was previously identified as a possible phosphorylation resonance of Tau40 modified in Xenopus laevis oocytes. A recent in-cell NMR study revealed that cell type specifically contributes to the biological and pathological behaviors of α-syn in different intracellular environments [6,50]. Therefore, different cell types may feature distinct intracellular environment as well as distinct post-translational modification systems.…”

Real-time nuclear magnetic resonance spectroscopy in the study of biomolecular kinetics and dynamics

“…The investigation of protein modification and in particular phosphorylation was put forward by Selenko in 2010 and has been used since then in a couple of applications (Kosten et al, 2014, p. 129;Landrieu et al, 2006;Liokatis et al, 2010;Mylona et al, 2016). Such studies are even possible in cellular environment by time-resolved in-cell NMR (Theillet et al, 2013).…”

Real-time NMR spectroscopy in the study of biomolecular kinetics and dynamics

“…Such studies are even possible in cellular environment by time-resolved in-cell NMR (Theillet et al, 2013). Other applications of time-resolved in-cell NMR are the proteolytic alpha-synuclein processing (Limatola et al, 2018), the methylation of lysines in cell, the investigation of ligand binding in cellular environment (Luchinat et al, 2020a(Luchinat et al, , 2020b) and the modulation of bound GTP levels of RAS (Zhao et al, 2020). Time-resolved NMR experiments are now also pursued to characterize metabolic flux in patient-derived primary cells (Alshamleh et al, 2020;Reed et al, 2019).…”