According to a paper published in Molecular Cell, researchers have developed a new technique to analyze how cells initiate the autophagy process to remove defective mitochondria with unprecedented detail. The methodology was used to study this process in human neurons derived from stem cells. Malfunctions in a cell’s ability to remove damaged mitochondria have been linked to numerous disease including cancer and neurodegenerative diseases such as Parkinson’s, and application of this technique could offer insights into these diseases and novel treatment targets.
Mitochondrial quality control involves the enzymes PINK1, which chemically modifies proteins with phosphate, and PARKIN, which marks target proteins with ubiquitin. Healthy mitochondria have little PINK1 on their outer surface, but damaged mitochondria accumulate PINK1, causing phosphate to transfer to PARKIN, thus activating it. Activated PARKIN then transfers ubiquitin to a variety of proteins, coating the mitochondrial surface leading to the activation of autophagy machinery and mitochondrial death.
In the current study, Harvard Medical School researchers developed a new method, called PARKIN target-parallel reaction monitoring (Pt-PRM), to assess the protein ubiquitylation landscape involved in mitochondrial quality control. They also created a reference peptide library to measure precise abundance of PARKIN targets and individual ubiquitylation events on damaged mitochondria.
This allowed them to use mass spectrometry to “count” ubiquitylation events in a site-specific manner, across multiple mitochondrial surface proteins simultaneously, creating a “digital snapshot” of ubiquitylation. According to the authors, this offers an improvement over previous methods for measuring protein ubiquitylation events that are nonquantitative and do not provide site-specificity.
"We can measure over three orders of magnitude to better understand kinetics, stoichiometry, spatial organization, integration with phosphorylation and many other features," said senior study author J. Wade Harper, the HMS Bert and Natalie Vallee Professor of Molecular Pathology and Chair of the Department of Cell Biology.
The technique was applied to do the first quantitative analysis of native ubiquitylation-related events in neurons from genetically modified human stem cells, including analysis of dopaminergic neurons, which are known to be lost in Parkinson’s disease. So far, most research into this area has been conducted on HeLA cells engineered to express PARKIN at unnaturally high levels.
The team was able to shed new light on questions, such as whether ubiquitin chains target mitochondrial proteins indiscriminately or in a targeted manner and examine mechanistic aspects of the pathway.
These mechanistic insights could enable discovery of new drug targets or ways to bypass disease-causing mutations. The method can be applied not only to Parkinson’s studies, but to many other diseases related to defective mitochondrial clearing pathways.