TRIM25 ubiquitinates and degrades p62/SQSTM1 to suppress autophagy

Abstract

Autophagy is a conserved catabolic process in which organelles, macromolecules and pathogens are degraded via lysosomes. Sequestosome 1 (SQSTM1), also known as p62, the first autophagy receptor identified, binds to ubiquitin on targets and LC3 on phagophores, mediating the selective autophagy of ubiquitinated substrates. To identify the potential interacting partners for p62, Flag-tagged p62 was transfected into HEK293T cells and used as bait to Co-immunoprecipitate (Co-IP) with proteins that form a complex with p62. More materials and methods are provided in the Supplementary Materials and Methods. The proteins were then identified via mass spectrometry analysis. Two E3 ubiquitin ligases, TRIM25 and ITCH, were identified as the highest confidence hits in a list of identified proteins. Gene Ontology (GO) analysis revealed that p62-interacting proteins were enriched in the ubiquitin-dependent protein degradation, protein folding, oxidative phosphorylation, autophagy, etc., signalling pathways. Validation assays were then performed to test the E3 ubiquitin ligases for p62 identified in this study. Both endogenously and ectopically expressed p62 formed a complex with the E3 ubiquitin ligases TRIM25 and ITCH. GST pull-down assays revealed that recombinant TRIM25 and ITCH directly interact with p62. As detected by fluorescence microscopy analysis, mCherry-tagged TRIM25 and GFP-tagged p62 were colocalized mainly in the cytoplasm of HeLa cells. Ubiquitination assays were performed to determine whether TRIM25 and ITCH are merely interacting partners or true E3 ubiquitin ligases for p62. One potential explanation for this phenomenon is that ITCH requires assistance from a specific protein or undergoes a particular modification to activate its ability to ubiquitinate p62. Alternatively, it is conceivable that p62 needs to be modified to be ubiquitinated by ITCH. Exogenously expressed p62 was efficiently ubiquitinated by TRIM25 but not by ITCH. These results suggest that TRIM25 is an E3 ubiquitin ligase for p62, whereas ITCH is only an interacting partner. A reconstituted E. coli ubiquitination system, which has been used in several of our studies, was included in this study. The p62 proteins recovered from the E. coli ubiquitination system were subjected to mass spectrometry analysis, and fifteen Lys (K) residues of p62 were identified. As shown in Supplementary Figure S1D, K7 and K189 were validated as the two major sites for the TRIM25-mediated ubiquitination of p62 in mammalian cells. The mutant bearing simultaneous K-to-R substitutions (K7/189R) at the two Lys residues almost completely abolished the TRIM25-mediated ubiquitination of p62. Four shRNAs targeting TRIM25 were designed and tested in HeLa and Caski cells, and shTRIM25-1 and shTRIM25-2 were selected for further study. TRIM25 knockdown reduced p62 ubiquitination, which was effectively reversed by exogenously expressed TRIM25 in both HeLa and Caski cells. In HEK293T cells, the TRIM25-mediated reduction in p62 protein expression was blocked by treatment with the autophagy inhibitor bafilomycin (BAF) but not by treatment with the proteasome inhibitor bortezomib (BTZ). Upon undergoing ubiquitination, p62 enhances its interaction with LC3 through its LC3-interacting region (LIR) domain. This interaction enables p62 to be

1. Introduction

Autophagy is a conserved catabolic process in which organelles, macromolecules and pathogens are degraded via lysosomes. Sequestosome 1 (SQSTM1), also known as p62, the first autophagy receptor identified, binds to ubiquitin on targets and LC3 on phagophores, mediating the selective autophagy of ubiquitinated substrates.

To identify the potential interacting partners for p62, Flag-tagged p62 was transfected into HEK293T cells and used as bait to Co-immunoprecipitate (Co-IP) with proteins that form a complex with p62. More materials and methods are provided in the Supplementary Materials and Methods. The proteins were then identified via mass spectrometry analysis. Two E3 ubiquitin ligases, TRIM25 and ITCH, were identified as the highest confidence hits in a list of identified proteins. Gene Ontology (GO) analysis revealed that p62-interacting proteins were enriched in the ubiquitin-dependent protein degradation, protein folding, oxidative phosphorylation, autophagy, etc., signalling pathways.