Analysis of 4E-BP effectors with cellular protective potential in vitro and in vivo by quantitative mass spectrometry

Translation initiation factor 4E binding proteins (4E-BP) are crucial for stress resistance and the survival of cells upon encountering different stressors. In particular, they are cell protective and able to rescue the Parkinson’s
disease phenotype of patient cells and in Drosophila models. 4E-BPs are inhibitors of cap-dependent translation, the predominant form of translation in eukaryotes. However, an estimated 10 - 15 % of mRNAs can initiate translation
cap-independently via internal ribosome entry sequences (IRES), which become predominant when 4E-BPs block cap-dependent translation. Although a lot is already known about the upstream regulation of 4E-BP activity, much less is known about its downstream effectors, and the mechanism
by which they are responsible for the protective effect. Here, I developed two inducible stable cell lines overexpressing wildtype 4E-BP1 (4E-BP1[WT]),the most abundant isoform in humans or a constitutively active form carrying two point mutations (4E-BP1[TA]). Likewise, two Drosophila lines were generated overexpressing the wildtype orthologue of 4E-BP1 (d4E-BP[WT]) or its constitutively active form (d4E-BP[TA]). Stable isotope labelling of
these cells and flies allowed the quantification of individual protein abundances after 4E-BP overexpression and the identification of upregulated proteins by mass spectrometry. Bioinformatic analyses of the mass spectrometry data revealed that many antioxidant, mitochondrial and lipid metabolic proteins were enriched among the proteins upregulated upon 4E-BP overexpression.
Cell viability assays confirmed that 4E-BP1 overexpression rescues cells from the toxic effects of the mitochondrial complex I inhibitor rotenone. The effect was reduced after knockdown of different 4E-BP1 effectors identified
by prior quantitative proteomics.