Targeted proteomics addresses selectivity and complexity of protein degradation by autophagy.

ABSTRACT

Macroautophagy/autophagy is a constitutively active catabolic lysosomal degradation pathway, often found dysregulated in human diseases. It is often considered to act in a cytoprotective manner and is commonly upregulated in cells undergoing stress. Its initiation is regulated at the protein level and does not require de novo protein synthesis. Historically, autophagy has been regarded as nonselective; however, it is now clear that different stimuli can lead to the selective degradation of cellular components via selective autophagy receptors (SARs). Due to its selective nature and the existence of multiple degradation pathways potentially acting in concert, monitoring of autophagy flux, i.e. selective autophagy-dependent protein degradation, should address this complexity. Here, we introduce a targeted proteomics approach monitoring abundance changes of 37 autophagy-related proteins covering process-relevant proteins such as the initiation complex and the Atg8-family protein lipidation machinery, as well as most known SARs. We show that proteins involved in autophagosome biogenesis are upregulated and spared from degradation under autophagy-inducing conditions in contrast to SARs, in a cell-line dependent manner. Classical bulk stimuli such as nutrient starvation mainly induce degradation of ubiquitin-dependent soluble SARs and not of ubiquitin-independent, membrane-bound SARs. In contrast, treatment with the iron chelator deferiprone leads to the degradation of ubiquitin-dependent and -independent SARs linked to mitophagy and reticulophagy/ER-phagy. Our approach is automatable and supports large-scale screening assays paving the way to (pre)clinical applications and monitoring of specific autophagy flux.Abbreviation AMBRA1 autophagy and beclin 1 regulator 1; ATG autophagy related; BafA1 bafilomycin A1; BNIP1 BCL2 interacting protein 1; BNIP3 BCL2 interacting protein 3; BNIP3L/NIX BCL2 interacting protein 3-like; CALCOCO2/NDP52 calcium binding and coiled-coil domain 2; CCPG1 cell cycle progression 1; CV coefficients of variations; CCCP carbonyl cyanide m-chlorophenyl hydrazone; DFP deferiprone; ER endoplasmic reticulum; FKBP8 FKBP prolyl isomerase 8; GABARAPL GABA type A receptor associated protein like; LC liquid chromatography; LOD limit of detection; LOQ limit of quantification; MAP1LC3 microtubule associated protein 1 light chain 3; MS mass spectrometry; NCOA4 nuclear receptor coactivator 4; NBR1 NBR1 autophagy cargo receptor; NUFIP1 nuclear FMR1 interacting protein 1; OPTN optineurin; PHB2 prohibitin 2; PNPLA2/ATGL patatin like phospholipase domain containing 2; POI protein of interest; PTM posttranslational modification; PRM parallel reaction monitoring; RB1CC1/FIP200 RB1 inducible coiled-coil 1; RETREG1/FAM134B reticulophagy regulator 1; RPS6KB1 ribosomal protein S6 kinase B1; RTN3 reticulon 3; SARs selective autophagy receptors; SQSTM1/p62 sequestosome 1; STBD1 starch binding domain 1; TAX1BP1 Tax1 binding protein 1; TFEB transcription factor EB; TNIP1 TNFAIP3 interacting protein 1; TOLLIP toll interacting protein; ULK1 unc-51 like autophagy activating kinase 1; WBP2 WW domain binding protein 2; WDFY3/Alfy WD repeat and FYVE domain containing 3; WIPI2 WD repeat domain, phosphoinositide interacting 2.