ATG4B Human

What is the primary function of ATG4B in human autophagy?

ATG4B serves two essential functions in autophagy. First, it acts as a processing enzyme that cleaves the C-terminal amino acid of pro-LC3/GABARAP family proteins to expose a glycine residue, enabling their subsequent conjugation to phosphatidylethanolamine (PE) and membrane insertion. Second, ATG4B functions as a deconjugating enzyme that removes PE from lipidated LC3/GABARAP, releasing them back to the cytosol for reuse . This dual functionality makes ATG4B a key regulator of autophagosome formation, as proper LC3/GABARAP processing is essential for autophagosome membrane expansion and completion .

How does ATG4B differ from other ATG4 isoforms in humans?

Humans express four ATG4 isoforms (ATG4A, ATG4B, ATG4C, and ATG4D), but ATG4B demonstrates the broadest substrate specificity and highest catalytic activity. While ATG4B efficiently processes all LC3/GABARAP family proteins (MAP1LC3A, MAP1LC3B, MAP1LC3C, GABARAPL1, GABARAPL2, and GABARAP), ATG4A primarily processes GABARAP subfamily members with limited activity toward LC3 proteins . ATG4C and ATG4D exhibit significantly lower proteolytic activity. Knockout studies have shown that ATG4B depletion causes severe but incomplete autophagy defects, with residual activity attributed to compensatory functions of other ATG4 isoforms . This suggests an evolutionary mechanism ensuring autophagy pathway robustness, with ATG4B as the primary protease and other isoforms providing backup functionality.

What are the most effective approaches for manipulating ATG4B activity in experimental systems?

Several strategies can be employed to manipulate ATG4B activity for experimental studies:

• Genetic manipulation: CRISPR-Cas9 knockout or knockdown via siRNA/shRNA provides loss-of-function approaches. For ATG4B knockout validation, researchers should confirm protein absence via western blotting, verify introduced mutations through genomic sequencing, and assess functional effects on autophagy through LC3 processing assays . When interpreting results, consider potential compensation by other ATG4 isoforms.

• Dominant-negative mutants: Overexpression of catalytically inactive ATG4B (C74A/S) acts as a potent inhibitor of autophagy by competing with endogenous ATG4B for substrates. Electron microscopy studies have shown this approach leads to accumulation of isolation membranes and incomplete autophagosomes, demonstrating the importance of ATG4B in autophagosome completion .

• Redox-insensitive mutants: Mutations at regulatory cysteines (C292S/C361S) create ATG4B variants that remain active under oxidative conditions, enabling studies of redox regulation .

• Chemical modulators: Small-molecule inhibitors like NSC185058 allow temporal control of ATG4B inhibition without genetic manipulation, though specificity should be carefully validated .

What assays are available to measure ATG4B activity in vitro and in cells?

Multiple complementary assays can assess ATG4B activity:

• Biochemical assays: Purified recombinant ATG4B can be tested with fluorogenic substrates based on LC3 sequences, where cleavage generates measurable fluorescence. FRET-based substrates provide another sensitive method for detecting activity .

• Western blotting: Monitoring the ratio of LC3-I (cytosolic form) to LC3-II (PE-conjugated form), where increased ATG4B activity typically results in higher LC3-I levels due to enhanced delipidation. This approach works with endogenous proteins but isn't real-time .

• GFP-LC3 processing: Fluorescence microscopy can track ATG4B-mediated changes in GFP-LC3 distribution between cytosol and membranes in living cells. The dominant-negative ATG4B-C74A mutant causes accumulation of isolation membranes positive for GFP-Atg5, confirming the technique's specificity .

• Redox state analysis: Non-reducing SDS-PAGE can preserve disulfide bonds to assess ATG4B oxidation state, providing insights into its regulation under oxidative conditions .

How is ATG4B activity regulated by oxidative stress?

ATG4B activity is tightly regulated by cellular redox conditions through reversible oxidation of specific cysteine residues. Research has identified Cys292 and Cys361 as critical regulatory sites that form intramolecular disulfide bonds in response to reactive oxygen species (ROS) . When cells experience oxidative stress, these cysteines become oxidized, triggering conformational changes that inhibit ATG4B's protease activity. This regulation involves both direct effects on the catalytic site and formation of disulfide-linked oligomers that further suppress activity .

The physiological significance of this regulation has been demonstrated using site-directed mutagenesis of Cys292 and Cys361 to serine, creating redox-insensitive ATG4B variants. Cells expressing these mutants exhibit enhanced autophagic flux under basal conditions and resistance to oxidative stress-induced autophagy inhibition . This mechanism provides a direct link between cellular redox status and autophagy regulation, positioning ATG4B as a redox sensor that adjusts autophagic activity according to cellular ROS levels.

What is the catalytic mechanism of ATG4B and how do mutations affect its function?

ATG4B is a cysteine protease whose catalytic activity depends on a triad consisting of Cys74, His280, and Asp278 . The mechanism involves:

• The catalytic cysteine (Cys74) performs a nucleophilic attack on the carbonyl carbon of the peptide bond.

• The histidine (His280) acts as a general base, deprotonating the cysteine to enhance its nucleophilicity.

• The aspartate (Asp278) properly orients the histidine and stabilizes its protonated form.

Mutation of the catalytic cysteine (C74A or C74S) completely abolishes protease activity without affecting substrate binding, creating a dominant-negative inhibitor that has been widely used in research . This mutant binds to LC3/GABARAP substrates but cannot process them, effectively sequestering them from the endogenous ATG4B.

Structural studies have revealed that ATG4B undergoes significant conformational changes upon substrate binding, with a regulatory loop moving to expose the catalytic site. The enzyme recognizes specific features in LC3/GABARAP proteins, explaining its substrate specificity and enabling its precise role in autophagy regulation .

What happens to autophagosome formation when ATG4B function is compromised?

Disruption of ATG4B function through genetic knockout or dominant-negative approaches reveals its critical role in autophagosome biogenesis:

• Defective LC3 processing: ATG4B deficiency leads to accumulation of unprocessed pro-LC3 forms and reduced LC3-II formation, indicating impaired priming function .

• Incomplete autophagosome formation: Electron microscopy studies of cells expressing dominant-negative ATG4B-C74A show accumulation of isolation membranes and incompletely formed autophagosomes. The ratio of open structures to total autophagic structures is significantly higher than in control cells, suggesting impaired membrane closure .

• Altered membrane dynamics: ATG4B-C74A expression affects autophagosomal membrane length, with closed autophagic membranes appearing shorter than in control cells, indicating defects in membrane expansion .

• Impaired cargo degradation: Despite these defects, expressing pre-primed LC3B in ATG4-deficient cells can partially rescue autophagic degradation of cargo receptors like SQSTM1/p62, suggesting that while ATG4B is crucial for efficient autophagy, some compensatory mechanisms exist .

These findings collectively demonstrate that ATG4B plays essential roles in both initiating autophagosome formation through LC3/GABARAP priming and in later stages of autophagosome completion, potentially through its delipidation activity.

How do the priming and delipidation functions of ATG4B differentially impact autophagy?

The dual functions of ATG4B—priming and delipidation—have distinct implications for autophagy progression:

Experimental approaches to distinguish these functions include using separation-of-function mutants, where specific mutations selectively affect one activity, or employing pre-primed LC3/GABARAP constructs to bypass the requirement for priming while maintaining sensitivity to delipidation .

How can ATG4B be targeted therapeutically in disease contexts?

ATG4B represents a promising therapeutic target in multiple disease contexts due to its key regulatory role in autophagy:

• Cancer applications: In many cancers, ATG4B expression is upregulated, supporting enhanced autophagic capacity that helps cancer cells survive stress conditions. Small-molecule inhibitors of ATG4B can sensitize resistant cancer cells to chemotherapy by blocking this pro-survival mechanism .

• Neurodegenerative diseases: In conditions characterized by protein aggregation like Alzheimer's and Parkinson's diseases, enhancing ATG4B priming activity while maintaining appropriate delipidation rates may promote clearance of toxic protein aggregates .

• Strategic approaches: Rather than simply inhibiting or activating ATG4B globally, targeted approaches may include:

  • Isoform-specific modulators that target ATG4B while sparing other ATG4 family members

  • Compounds that selectively affect either priming or delipidation functions

  • Agents that prevent oxidative inhibition of ATG4B under stress conditions by protecting Cys292/Cys361 from oxidation

• Delivery considerations: For CNS applications, blood-brain barrier penetration is crucial. For cancer applications, tumor-specific delivery systems may enhance efficacy while reducing systemic effects on normal tissues.

When developing ATG4B-targeted therapeutics, researchers must consider potential compensation by other ATG4 isoforms and assess effects on both basal and stress-induced autophagy pathways .

What are emerging technologies and future directions in ATG4B research?

The ATG4B research field continues to evolve with several exciting directions:

• Advanced imaging techniques: Super-resolution microscopy and single-molecule tracking now enable researchers to visualize ATG4B dynamics during autophagosome formation with unprecedented detail. Correlative light and electron microscopy (CLEM) allows precise correlation between fluorescence signals and ultrastructural features .

• Proteomics approaches: Comprehensive analysis of ATG4B interactors and substrates using techniques like BioID or APEX proximity labeling is uncovering novel functions beyond canonical LC3/GABARAP processing .

• Structural biology advances: Cryo-EM and advanced crystallography techniques are providing deeper insights into ATG4B structure in complex with substrates and regulatory partners, facilitating structure-based drug design efforts .

• Systems biology integration: Multi-omics approaches are helping to position ATG4B within the broader autophagy regulatory network, revealing unexpected connections with other cellular pathways and stress responses .

• Computational approaches: Molecular dynamics simulations can predict how specific mutations or modifications alter ATG4B structure and function, guiding experimental design and therapeutic development .

Future research should address outstanding questions including the spatial regulation of ATG4B activity at autophagosomal membranes, the precise timing of priming versus delipidation events, and the potential role of ATG4B in non-canonical autophagy pathways .

What are common pitfalls in ATG4B research and how can they be avoided?

Researchers studying ATG4B frequently encounter several methodological challenges:

• Distinguishing ATG4B effects from other ATG4 isoforms: When manipulating ATG4B expression, compensatory changes in other ATG4 isoforms may occur. Solution: Include comprehensive analysis of all ATG4 family members at both mRNA and protein levels, and consider generating multiple knockout models (single, double, triple) to assess functional redundancy .

• Interpreting autophagy phenotypes: Changes in LC3 lipidation or autophagosome number can result from either increased autophagy induction or blocked autophagosome-lysosome fusion. Solution: Include flux assays with lysosomal inhibitors like bafilomycin A1 to differentiate between these possibilities .

• Overexpression artifacts: Excessive overexpression of ATG4B or dominant-negative mutants may cause non-physiological effects. Solution: Use inducible expression systems, validate with endogenous protein manipulation, and include rescue experiments with physiological expression levels .

• Redox sensitivity in sample preparation: The oxidation state of ATG4B is easily altered during cell lysis and protein preparation. Solution: Include reducing or alkylating agents (DTT, NEM) in lysis buffers to preserve the in vivo redox state, and prepare samples under anaerobic conditions when studying oxidative modifications .

• Heterogeneity in cell populations: Individual cells may show varying levels of ATG4B activity and autophagy. Solution: Complement population-based assays (western blots) with single-cell techniques (immunofluorescence, flow cytometry) to capture this heterogeneity .

How should researchers design controls for ATG4B activity assays?

Proper controls are critical for reliable interpretation of ATG4B activity assays:

• Positive controls:

  • Recombinant wild-type ATG4B protein for in vitro assays

  • Starvation-induced autophagy to verify system responsiveness in cellular assays

  • ATG4B overexpression to demonstrate maximum processing capacity

• Negative controls:

  • Catalytically inactive ATG4B-C74A/S mutant to establish baseline in activity assays

  • Non-cleavable LC3 substrate (LC3-G120A) to verify specificity of cleavage

  • ATG4B knockout cells to establish complete loss-of-function baseline

• Specificity controls:

  • Testing multiple LC3/GABARAP family substrates to assess specificity

  • Including other ATG4 family proteins to determine isoform selectivity

  • Using ATG4B-specific inhibitors to confirm that observed effects are ATG4B-dependent

• Oxidation controls:

  • Include both reducing (DTT, GSH) and oxidizing (H₂O₂, GSSG) conditions when studying redox regulation

  • Use C292S/C361S ATG4B mutants resistant to oxidative inhibition as controls for redox studies

• Time course and concentration dependence:

  • Test multiple time points and enzyme/substrate concentrations to ensure measurements are made in the linear range

  • Include kinetic analyses rather than single time-point measurements when possible