Abstract
ABSTRACT Autophagy summarizes evolutionarily conserved, intracellular degradation processes targeting cytoplasmic material for lysosomal degradation. These encompass constitutive processes as well as stress responses, which are often found dysregulated in diseases. Autophagy pathways help in the clearance of damaged organelles, protein aggregates and macromolecules, mediating their recycling and maintaining cellular homeostasis. Protein-protein interaction networks contribute to autophagosome biogenesis, substrate loading, vesicular trafficking and fusion, protein translocations across membranes and degradation in lysosomes. Hypothesis-free proteomic approaches tremendously helped in the functional characterization of protein-protein interactions to uncover molecular mechanisms regulating autophagy. In this review, we elaborate on the importance of understanding protein-protein-interactions of varying affinities and on the strengths of mass spectrometry-based proteomic approaches to study these, generating new mechanistic insights into autophagy regulation. We discuss in detail affinity purification approaches and recent developments in proximity labeling coupled to mass spectrometry, which uncovered molecular principles of autophagy mechanisms. Abbreviations: AMPK: AMP-activated protein kinase; AP-MS: affinity purification-mass spectrometry; APEX2: ascorbate peroxidase-2; ATG: autophagy related; BioID: proximity-dependent biotin identification; ER: endoplasmic reticulum; GFP: green fluorescent protein; iTRAQ: isobaric tag for relative and absolute quantification; MS: mass spectrometry; PCA: protein-fragment complementation assay; PL-MS: proximity labeling-mass spectrometry; PtdIns3P: phosphatidylinositol-3-phosphate; PTM: posttranslational modification; PUP-IT: pupylation-based interaction tagging; RFP: red fluorescent protein; SILAC: stable isotope labeling by amino acids in cell culture; TAP: tandem affinity purification; TMT: tandem mass tag.
Highlights
The majority of proteins are degraded via two pathways: the ubiquitin proteasome system and autophagy
We discuss in detail affinity purification approaches and recent developments in proximity labeling coupled to mass spectrometry, which uncovered molecular principles of autophagy mechanisms
Autophagy has been broadly cate gorized into three different subtypes: macroautophagy, micro autophagy and chaperone-mediated autophagy, which differ in the mode of transport and delivery of substrates for lyso somal degradation
Summary
The majority of proteins are degraded via two pathways: the ubiquitin proteasome system and autophagy. The authors expressed 3x HA-tagged TMEM192, a lysosomal membrane protein, in HEK293T cells and per formed anti-HA AP to enrich lysosomes prior MS-based identification and quantification They identified a change in the localization of a protein called NUFIP1 (nuclear FMR1 interacting protein 1), which shifted from the nucleus toward lysosomes/autophagosomes during starvation-induced autop hagy and suggested that NUFIP1 might act as a potential receptor for ribophagy [100]. Pupylation-based interaction tagging (PUP-IT) is a method that was developed to identify PPIs at membranes This approach involves the expression of a bait protein fused to the bacterial Pup ligase PafA and co-expression of a Pup (prokaryotic ubiquitin-like protein) variant with a C-terminal Gly-Gly-Glu sequence. ATG101 interacts only with ATG13 to [152]
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