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Unraveling the cellular responses to aberrantly-folded and aggregated proteins

Final Report Summary - PROTEOTOXNET (Unraveling the cellular responses to aberrantly-folded and aggregated proteins)

Protein aggregation diseases such as Parkinson’s disease (PD) are associated with the intracellular
accumulation of aggregates of specific aggregation-prone proteins (APPs). These structures have
been associated to cellular toxicity and cell degeneration. Recent genetic screens and genome-wide
association studies uncovered sets of genes that significantly reduce APP toxicity. These modulators
are very promising for therapeutic purposes, but their mechanisms of action are currently unknown.

To analyse the intracellular consequences of protein aggregation, we applied proteomics screens
coupled with molecular biology follow-ups to a set of yeast and neuronal models of proteotoxicity,
Our data indicated that intracellular responses depend on the sequence of the protein forming the
aggregates and that APPs activate compartment-specific responses aimed at counteracting the damage.

We also identified a novel cellular mechanism of clearance of pathological aggregates of alpha-synuclein (aSyn), a protein involved in PD, which counteracts its toxicity and pathological transcellular spread in vitro and in vivo. This machinery, from the family of cullin-RING ubiquitin ligases, could constitute a promising new therapeutic target for PD.

To evaluate the mechanism of action of genetic modifiers and risk factors of PD, we developed a novel mass spectrometric method, the sentinel assay, which relies on a set of marker proteins that cumulatively report on the activation state of ~200 biological processes. Using this approach we characterized the mechanisms of action of 33 known genetic modulators of aSyn toxicity and identified a lipid metabolic enzyme as a common downstream effector of rescue from toxicity. Follow-up studies demonstrated that inhibition of the diacylglycerol branch of lipid metabolism ameliorates the toxic effects of a-Syn.

Last, we developed a novel structural proteomics approach (which we termed LiP-MS) to characterize the structural conversions of APPs directly in their cellular or tissue matrix. We applied LiP-MS to characterize the structural features of aSyn in cell models of PD pathogenesis. This structural proteomics approach finds also application in the fields of drug target deconvolution and for the identification of novel allosteric interactions.