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Time‐Resolved NMR Analysis of Proteolytic α‐Synuclein Processing in vitro and in cellulo
Author(s) -
Limatola Antonio,
Eichmann Cédric,
Jacob Reeba Susan,
BenNissan Gili,
Sharon Michal,
Binolfi Andres,
Selenko Philipp
Publication year - 2018
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201800056
Subject(s) - proteolysis , in vitro , proteases , biochemistry , protease , proteolytic enzymes , cell culture , proteasome , chemistry , alpha synuclein , microbiology and biotechnology , biology , oxidative stress , proteomics , nuclear magnetic resonance spectroscopy , biophysics , enzyme , parkinson's disease , stereochemistry , genetics , medicine , pathology , disease , gene
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.