ATG4B recombinant protein: a core substrate of UBE3C-mediated ubiquitination in the autophagy regulatory network

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This article focuses on the molecular characteristics and autophagy regulatory functions of ATG4B, systematically elaborating its core role as the only specific cysteine protease of the Atg8 family in LC3 precursor cleavage and LC3-II delipidation, analyzing the molecular mechanism by which UBE3C modifies ATG4B Lys119 via K33-branched ubiquitin chains to regulate its activity without inducing degradation, and exploring the fine regulation of the autophagy pathway by ATG4B ubiquitination under normal and starvation conditions.

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ATG4B Recombinant Protein: A Core Substrate of UBE3C-Mediated Ubiquitination in the Autophagy Regulatory Network

Summary: This article focuses on the molecular characteristics and autophagy regulatory functions of ATG4B, systematically elaborating its core role as the only specific cysteine protease of the Atg8 family in LC3 precursor cleavage and LC3-II delipidation, analyzing the molecular mechanism by which UBE3C modifies ATG4B at Lys119 via K33-branched ubiquitin chains to regulate its activity without inducing degradation, and discussing the fine-tuned regulation of the autophagy pathway by ATG4B ubiquitination under normal and starvation conditions.

I. The Core Position of ATG4B in the Autophagy Cascade.

Autophagy is a conserved degradation system for proteins and organelles, regulated by multiple autophagy-related genes. ATG4B is a core protein essential for cleaving pro-MAP1LC3/LC3 or deconjugating lipidated LC3-II to promote autophagosome formation. Atg4 is the only specific cysteine protease of Atg8 family proteins and plays a key role in the autophagy cascade. There are four Atg4 homologs in mammalian cells, among which ATG4B has the strongest enzymatic activity and the broadest spectrum for all substrates (such as MAP1LC3/LC3 and the GABARAP subfamily, hereinafter referred to as LC3). This enzymatic activity advantage and broad substrate spectrum make ATG4B an irreplaceable core protease in the autophagy regulatory network.

During autophagy biogenesis, ATG4B cleaves the precursor form of LC3 to generate LC3-I, exposing the active glycine residue that conjugates with phosphatidylethanolamine on the membrane to form LC3-II, thereby allowing expansion of the phagophore membrane. Once autophagosome biosynthesis is complete, ATG4B also induces delipidation of LC3-II from the membrane, regenerating LC3-I to participate in new autophagosome formation. This bidirectional catalytic function of “cleavage-delipidation” enables ATG4B to exert regulatory roles at both critical nodes of autophagosome formation initiation and termination.

II. The Post-Translational Modification Regulatory Network of ATG4B.

The protein stability and activity of ATG4B can be regulated by various post-translational modifications. ULK1 phosphorylates ATG4B at Ser316 to inhibit ATG4B activity and LC3 processing. In glioblastoma, the kinase STK26/MST4 phosphorylates ATG4B at Ser383 to activate ATG4B activity and increase the autophagy pathway. In hepatocellular carcinoma cells, AKT phosphorylation of ATG4B can inhibit mitochondrial activity and enhance the warfarin effect. The DDIT4/REDD1-TXNIP pro-oxidant complex regulates reactive oxygen species generation and then controls stress-induced autophagy and maintains motility by regulating ATG4B activity. In addition, Cys292 and Cys361 are key sites for regulating the oxidized form and oligomer formation of ATG4B, and reversible oxidative modification can regulate the protease activity of human ATG4B, thereby affecting autophagy.

Once ATG4B is nitrosylated at Cys189 and Cys292, its enzymatic activity is also reduced, thereby mediating neurotoxicity. When ATG4B is O-GlcNAcylated, it can promote autophagy by increasing its hydroxylase activity. Furthermore, ubiquitination mediated by the E3 ubiquitin ligase RNF5 can impair ATG4B stability, thereby affecting autophagy levels and sensitivity to bacterial infection. However, whether ATG4B can be ubiquitinated by other E3 ligases under normal or starvation conditions, and whether ATG4B ubiquitination leads to protein degradation, still need further elucidation. This unresolved scientific question is precisely the core proposition that UBE3C regulation of ATG4B research aims to answer.

III. Identification and Mechanism of UBE3C as a Novel E3 Ligase for ATG4B.

A new study identified a novel E3 ligase for ATG4B, namely UBE3C, through mass spectrometry. UBE3C primarily assembles K33-branched ubiquitin chains at the Lys119 site of ATG4B without causing ATG4B degradation. This finding has important mechanistic significance——ubiquitination is typically regarded as a signal for protein degradation, whereas UBE3C-mediated K33-branched ubiquitin chains regulate ATG4B function in a non-degradative manner, expanding the understanding of the functional diversity of ubiquitination modifications.

Protein ubiquitination is catalyzed by a series of enzymes, mainly including E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin ligases. Polyubiquitin chains are typically linked to a lysine residue or the N-terminal methionine residue of the substrate through one of the seven lysine residues of ubiquitin or the N-terminal methionine residue of ubiquitin, which may be a fundamental degradation signal for proteins and may also alter protein localization, function, or activity. Lys48 chains target their substrates to the 26S proteasome for degradation, Lys63 chains function in the NF-κB signaling pathway, DNA repair, and receptor endocytosis, Lys29 chains can degrade proteins through lysosomes, Lys29/Lys33 can be assembled on AMP-activated protein kinase-related kinase family substrates, participating in cell polarity and proliferation, Lys6 may regulate DNA repair, Lys27 may participate in stress response pathways, and K11 and K48 chains participate in endoplasmic reticulum-associated degradation. The assembly of K33-branched ubiquitin chains on ATG4B represents a non-degradative regulatory ubiquitination modification.

E3 ligases play a key role in ubiquitination and can be divided into three structurally distinct types: E3s containing a domain homologous to the E6AP C-terminus, N-end rule E3s, and E3s with a Really Interesting New Gene/RING finger structure, including their derivatives U-Box and Plant Homeo-Domain. Studies have found that the human HECT E3 ligase UBE3C can mediate K29 and K48 polyubiquitin chains, with a preference for K48 linkages. Whether UBE3C can also assemble other Ub chains on substrates remains unclear, and this study is the first to confirm that UBE3C can assemble K33-branched ubiquitin chains on ATG4B, expanding the understanding of the substrate modification spectrum of UBE3C.

IV. Regulatory Effects of ATG4B Ubiquitination on the Autophagy Pathway.

Increased ATG4B ubiquitination caused by UBE3C overexpression inhibits the autophagy pathway under both normal and starvation conditions, possibly due to weakened ATG4B activity and ATG4B-LC3 interaction. Once the Lys119 site of ATG4B is mutated to arginine, this weakening can be reversed. More importantly, under starvation conditions, the interaction between ATG4B and UBE3C is significantly reduced, and consequently the K33-branched ubiquitin chains on ATG4B are removed. Therefore, starvation-induced autophagy may be partially inhibited due to increased levels of ATG4B ubiquitination. This finding reveals the dynamic regulatory characteristics of ATG4B ubiquitination modification——under nutrient-rich conditions, the interaction between UBE3C and ATG4B is enhanced, inhibiting ATG4B activity through K33 ubiquitin chains; under starvation conditions, their interaction is weakened, the ubiquitin chains are removed, and ATG4B activity is restored to initiate autophagy. Overall, this study reveals a novel modification mode of ATG4B, in which UBE3C regulates ATG4B activity through specific ubiquitination to control autophagy without causing ATG4B degradation.

V. Conclusion.

In ATG4B-related basic research and autophagy mechanism exploration, high-quality recombinant ATG4B protein is a core tool for dissecting its protease activity and ubiquitination modification regulatory mechanisms. The His tag facilitates purification through immobilized metal affinity chromatography and enables detection and capture via anti-His tag antibodies. To meet the needs of ATG4B function research and autophagy regulatory mechanism exploration, UIA provides ATG4B His Tag Protein, Human. This product is suitable for applications including enzymatic activity analysis of ATG4B in LC3 precursor cleavage and LC3-II delipidation, research on UBE3C-mediated ATG4B ubiquitination modification, mechanistic studies of ATG4B-LC3 interaction, and development of autophagy pathway regulatory strategies.

This article is reviewed and published by the technical expert team of UA

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