Protein complexes and neighborhoods driving autophagy

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.

Keywords: Autophagy; affinity purification; mass spectrometry; protein-protein interactions; proximity labeling; quantitative proteomics.

Figure 1.

Schematic representation of autophagosome biogenesis and maturation. MTORC1 inhibits autophagy via its inhibitory phosphorylations on ULK1 and ATG13 under nutrient-rich conditions. Under stress, autophagy is activated via the formation of an active tetrameric ULK1 initiation complex to promote autophagosome nucleation. PtdIns3K complex I catalyzes the production of PtdIns3Ps, which contributes to phagophore nucleation and omegasome formation. PtdIns3P-binding proteins like ZFYVE1/DFCP1 and WIPIs decorate the omegasomes. WIPI2 interaction with ATG16L1 mediates the recruitment of the ATG12–ATG5-ATG16L1 complex for the conjugation of LC3-I to PE and phagophore expansion and maturation. Additionally, lipid sources from ATG9 vesicles, ATG2A/B recruited by WIPIs and from cell membranes collectively help in expanding the phagophore membrane. Double-membraned autophagosomes fuse with lysosomes to form autolysosomes and their content is degraded by lysosomal hydrolases

Figure 2.

Classification of protein-protein interactions based on stability and binding affinity. Affinity is inversely proportional to the dissociation constant K_d. Based on stability and binding affinity, PPIs can be classified into quasi-permanent/obligate and transient/non-obligate interactions. In contrast to permanent complexes, transient complexes are dynamic with proteins associating and dissociating. Transient complexes can be subclassified as strong and weak based on affinity and temporal profile of interactions. Moreover, PPIs can be classified based on composition as homo-oligomers with identical proteins interacting and hetero-oligomers with non-identical chains interacting. Often, interactions change due to physiological conditions rather reflecting a continuous than a “static” classification

Figure 3.

AP-MS approaches. (A) AP of endogenous proteins is a method to identify bait interactors using bait-specific antibodies, which are coupled to beads. (B) Single-tagged bait proteins are expressed in cells and affinity purified using a tag-reactive antibody. (C) The TAP-tag consists of two biochemical tags used for purification. In the classical TAP, the first tag is the ZZ domain of protein-A followed by a TEV protease cleavage site and a second tag. This method involves two consecutive steps of purification

Figure 4.

Proximity labeling enzymes to study protein neighborhoods. Proximity labeling ligases can be classified into three types based on their activity: biotin ligases, PTM ligases and peroxidases. Biotin ligases (BioID, BioID2, AirID and BASU) catalyze the conversion of biotin to a reactive biotin intermediate, which labels lysine residues of proximal proteins. In presence of H₂O₂, peroxidases (APEX2 and HRP) convert biotin phenol to biotin-phenoxyl radical, which labels electron-rich amino acid residues such as Tyr. PTM ligases add peptide/protein tags to proximal proteins

Figure 5.

PL-MS approaches. (A) Biotin ligases and peroxidases generate reactive biotin/biotin-phenol intermediates, respectively, which tag proximal proteins. (B) Split variants of biotin ligases and peroxidases are used to identify proximal proteins, while two bait proteins interact to form a complex. Interactions of proteins bring the N/C terminal fragments of the ligases in close proximity allowing the formation of an active PL holo-enzyme. After cell lysis, biotinylated proteins are enriched using classical streptavidin-based enrichment prior to bottom-up LC-MS/MS analysis