ATG3 (Autophagy-Related Protein 3) antibodies are immunological tools designed to detect and study ATG3, a critical enzyme in the autophagy pathway. These antibodies are primarily used in research applications such as Western blotting (WB), enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), and flow cytometry . ATG3 functions as an E2-like conjugating enzyme essential for the lipidation of microtubule-associated protein 1 light chain 3 (LC3) with phosphatidylethanolamine (PE), a step required for autophagosome formation and autophagy progression .
ATG3 is a cytoplasmic protein expressed ubiquitously, with elevated levels in the heart, skeletal muscle, kidney, liver, and placenta . Its role extends beyond autophagy to cellular processes such as apoptosis, nutrient recycling, and pathogen clearance .
Catalyzes LC3-PE conjugation (LC3-II formation), enabling autophagosome membrane expansion .
Interacts with ATG12∼ATG5-ATG16L1 complex during autophagosome formation .
Regulates cell viability, death, and drug resistance in cancer .
ATG3 antibodies are utilized to investigate autophagy mechanisms and disease associations.
Non-Small Cell Lung Cancer (NSCLC): ATG3 upregulation promotes autophagy-mediated cell survival, while miR-204-5p and miR-16 suppress ATG3 to inhibit proliferation .
Hepatocellular Carcinoma (HCC): High ATG3 expression correlates with sorafenib resistance and tumor growth via autophagy activation .
Leukemia: Low ATG3 levels in acute myeloid leukemia (AML) reduce autophagy, increasing sensitivity to proteasome inhibitors .
Mycobacterial Infections: ATG3 inhibition by miR-155 facilitates Mycobacterium tuberculosis survival in dendritic cells .
Ischemia-Reperfusion Injury: ATG3 protects against liver damage but exacerbates myocardial injury .
A hydrophobic pocket in ATG12 binds residues Asp156, Met157, Tyr160, and Glu161 of ATG3 .
Mutations (e.g., D156A/M157A) disrupt ATG3-ATG12 binding, impairing LC3-II formation and autophagy .
Oxidation of ATG3 (e.g., during aging) inhibits autophagy by disrupting LC3 thioester bond formation .
ATG3, also known as autophagy-related protein 3, is a critical component of the autophagy pathway, essential for cellular homeostasis and the degradation of damaged organelles and proteins. ATG3 functions primarily as an E2-like enzyme that catalyzes the formation of the ATG8-phosphatidylethanolamine (ATG8-PE) conjugate, which represents a critical step in the autophagy pathway . The conjugation process involves the initial cleavage of the C-terminal arginine residue of ATG8 by ATG4 (a cysteine protease), exposing a glycine residue that is subsequently activated by ATG7. The activated glycine is then transferred to ATG3, where conjugation to phosphatidylethanolamine occurs . This process is enhanced by the presence of ATG12-ATG5 conjugate, which functions similarly to an E3 enzyme, highlighting the intricate regulatory mechanisms involved in autophagy .
Research-grade ATG3 antibodies are available in multiple formats to accommodate various experimental approaches. The primary types include mouse monoclonal antibodies such as the A-3 antibody that detects ATG3 protein from multiple species including mouse, rat, and human. These antibodies are available in both non-conjugated forms and various conjugated formats . The conjugated forms include:
Agarose-conjugated antibodies for immunoprecipitation applications
Horseradish peroxidase (HRP) conjugates for enhanced detection sensitivity
Fluorescent conjugates including phycoerythrin (PE) and fluorescein isothiocyanate (FITC)
Multiple Alexa Fluor conjugates for advanced fluorescence microscopy applications
These various formats enable researchers to select the most appropriate antibody configuration based on their specific experimental requirements.
ATG3 expression patterns show notable variability across different tissue types, reflecting its diverse roles in cellular processes. Expression analysis has revealed that ATG3 is widely expressed throughout the body, with particularly significant levels detected in several key organs and tissues . The highest expression levels are observed in:
This widespread expression pattern underscores the fundamental importance of ATG3 in maintaining physiological processes across multiple organ systems. The varied expression levels likely reflect tissue-specific requirements for autophagy processes, which may correlate with metabolic demands, turnover rates of cellular components, or specialized functions of particular tissues.
ATG3 antibodies have been validated for multiple research applications, each providing unique insights into ATG3 biology. The primary validated applications include:
Western blotting (WB): For detecting and quantifying ATG3 protein levels in tissue or cellular lysates
Immunoprecipitation (IP): For isolating ATG3 protein complexes to study protein-protein interactions
Immunofluorescence (IF): For visualizing the subcellular localization of ATG3 in fixed cells
Enzyme-linked immunosorbent assay (ELISA): For quantitative detection of ATG3 in solution
These complementary approaches allow researchers to study ATG3 from multiple perspectives, including expression levels, interaction partners, and subcellular distribution. The antibodies have been validated across multiple species including human, mouse, and rat samples, enabling comparative studies across these model systems .
ATG3 antibodies serve as valuable tools for investigating the dynamics of autophagosome formation. Research has demonstrated that ATG3 localizes to the pre-autophagosomal structure (PAS) during autophagy induction . To visualize this process, researchers have developed experimental approaches that include:
Generation of fluorescently tagged ATG3 constructs (e.g., ATG3-GFP) for live-cell imaging
Immunofluorescence staining with anti-ATG3 antibodies to detect endogenous ATG3 localization
Co-localization studies with other autophagy markers to track progression of autophagosome formation
For the generation of fluorescently tagged constructs, the ATG3 open reading frame with flanking regions can be amplified by polymerase chain reaction and cloned into appropriate expression vectors. GFP-fused ATG3 (ATG3-GFP) constructs can be generated by insertion of restriction sites (such as BamHI) using site-directed mutagenesis, followed by ligation of the GFP sequence into the digested plasmids . These constructs allow dynamic tracking of ATG3 during autophagosome formation in real-time experiments.
The interaction between ATG3 and the ATG12-ATG5 conjugate (functioning as an E3-like enzyme) represents a critical regulatory step in autophagy. Multiple methodological approaches have been validated to study this interaction:
Size-exclusion chromatography: This technique effectively demonstrates co-elution of ATG3 fragments with the E3 complex (ATG12~ATG5-ATG16N), providing evidence of physical interaction .
Isothermal titration calorimetry (ITC): This approach enables quantitative measurement of binding affinity between ATG3 and ATG12~ATG5-ATG16N. Studies have determined a dissociation constant (KD) of approximately 41-94 nM for these interactions, indicating strong binding .
Co-immunoprecipitation assays: Using anti-ATG3 antibodies, researchers can precipitate ATG3 from cell lysates and detect co-precipitating ATG12~ATG5 complexes by Western blotting. This approach confirms the interaction at endogenous protein levels .
Mutational analysis: Generating alanine mutants of full-length ATG3 and measuring their affinities to ATG12~ATG5-ATG16N using ITC helps identify critical residues involved in the interaction. For example, mutations D156A, M157A, Y160A, and E161A in ATG3 significantly weaken the interaction with ATG12~ATG5 .
These complementary approaches provide comprehensive insights into the nature, strength, and structural determinants of ATG3-ATG12-ATG5 interactions.
The structural basis for ATG3 recognition by ATG12 involves specific regions and residues that facilitate their interaction. Research has identified ATG3's flexible region (ATG3 FR) as the primary site for ATG12 binding, with particular importance of a region termed RIA12 . The interaction demonstrates several key features:
The β-strand region of ATG3 RIA12 (residues 154-157) forms critical contacts with ATG12
Key residues in this interaction include Asp156, Met157, Tyr160, and Glu161 of ATG3
A cluster of eight consecutive negatively charged residues (144-151) in ATG3 contributes significantly to binding affinity
The M pocket on ATG12 accommodates ATG3 Met157, similar to how the L site on LC3 accommodates residues in LIR interactions
When compared to other autophagy protein interactions, such as the p62 LIR-LC3B complex, the ATG3 RIA12-ATG12 interaction shows both similarities and distinct differences:
Similarities:
The backbone residues of the tetrad of p62 LIR can be superposed on those of the β-strand region of ATG3 RIA12
Both interactions involve electrostatic interactions between acidic residues and basic patches on their binding partners
Differences:
These structural insights establish ATG3 RIA12 as a unique linear sequence specifically evolved to bind ATG12, distinct from other known interaction motifs in the autophagy pathway.
Mutations in the ATG3 RIA12 region have profound effects on LC3 lipidation and consequently on autophagy progression. Functional studies have established clear correlations between the binding affinity of ATG3 mutants to ATG12~ATG5 and their ability to support LC3-II formation .
The most critical mutations and their effects include:
Point mutations D156A and M157A severely impair LC3-II formation, consistent with their dramatic reduction in binding affinity to ATG12~ATG5
The double mutant D156A/M157A completely abolishes detectable LC3-II formation
Mutation of the eight consecutive negatively charged residues (144-151) to alanine (8×Ala mutation) causes severe reduction in LC3-II levels, confirming the importance of these residues in cellular contexts
These mutations specifically impair the ATG3-ATG12 interaction without affecting other aspects of ATG3 function. For example, the D156A/M157A double mutant retains the ability to interact with ATG7 (the E1-like enzyme) and can form a thioester conjugate with LC3, demonstrating that the defect is specifically in the E2-E3 interaction required for efficient LC3 lipidation .
These findings establish that the ATG3 RIA12-ATG12 interaction is essential for LC3-II production and consequently for normal autophagy progression, providing important mechanistic insights into the regulation of autophagosome formation.
The interactions of ATG3 with ATG12 and ATG7 involve distinct regions and mechanisms, reflecting their different functions in the autophagy pathway:
ATG3-ATG12 interaction:
Primarily mediated by the flexible region (FR) of ATG3, specifically the RIA12 region
Critical residues include Asp156, Met157, Tyr160, and Glu161
A cluster of eight consecutive negatively charged residues (144-151) provides additional binding energy
ATG3-ATG7 interaction:
Interestingly, the ATG3-ATG7 interaction appears to be enhanced when ATG3 is conjugated with LC3. This is evidenced by reduced co-precipitation of ATG7 with the C264A mutant of ATG3, which cannot form the LC3~ATG3 intermediate. This observation suggests that the LC3~ATG3 intermediate has a higher affinity for ATG7 than unconjugated ATG3 .
These distinct interaction mechanisms ensure the proper sequential progression of the autophagy conjugation cascade, with ATG3 first interacting with ATG7 to receive LC3, followed by interaction with ATG12~ATG5 to facilitate LC3 transfer to phosphatidylethanolamine.
When using ATG3 antibodies in experimental settings, appropriate controls are essential to ensure the validity and reliability of results. Recommended controls include:
For Western blotting:
Positive control: Lysates from tissues with known high ATG3 expression (kidney, liver, heart, skeletal muscle)
Negative control: ATG3 knockout cell lysates or siRNA-treated samples
Loading control: Probing for housekeeping proteins such as α-tubulin
Specificity control: Pre-absorption with recombinant ATG3 protein
For immunoprecipitation:
For immunofluorescence:
Peptide competition: Pre-incubation of antibody with immunizing peptide
Secondary antibody-only control: Omission of primary antibody
Knockdown validation: siRNA-treated or knockout cells
For functional studies:
These controls help confirm antibody specificity, validate experimental protocols, and provide benchmarks for interpreting experimental results.
Detecting ATG3-ATG12-ATG5 interactions requires careful optimization of experimental conditions. Based on published protocols, the following recommendations can improve detection sensitivity and specificity:
Cell preparation:
Immunoprecipitation protocol:
Western blotting detection:
Alternative approaches:
These optimized conditions enhance the detection of physiologically relevant ATG3-ATG12-ATG5 interactions and provide quantitative data for comparative analyses.
Validating ATG3 antibody specificity is crucial for generating reliable experimental data. Multiple complementary approaches are recommended:
Genetic validation:
Molecular weight verification:
ATG3 should appear at the expected molecular weight (~36-40 kDa)
Detection of potential post-translational modifications or isoforms should be consistent with literature
Cross-reactivity assessment:
Functional validation:
Peptide competition:
Pre-incubation of antibody with immunizing peptide or recombinant ATG3 should abolish signal
These validation approaches ensure that observed signals genuinely represent ATG3 and not non-specific interactions, providing a solid foundation for subsequent experimental analyses.
Studying ATG3-mediated LC3 lipidation presents several technical challenges that researchers should anticipate and address:
Basal autophagy variation:
LC3-I versus LC3-II discrimination:
Transient nature of interactions:
Interpreting mutant effects:
Quantification challenges:
Autophagy flux versus accumulation:
Addressing these pitfalls through careful experimental design and appropriate controls ensures more reliable and interpretable data when studying ATG3-mediated LC3 lipidation.
Optimizing immunofluorescence protocols for ATG3 detection requires consideration of cell type-specific factors and technical parameters:
Fixation method selection:
Paraformaldehyde (4%) works well for most applications but may mask some epitopes
Methanol fixation can improve detection of some ATG3 epitopes but may disrupt membrane structures
Test both methods to determine optimal preservation of ATG3 signal
Permeabilization optimization:
Triton X-100 (0.1-0.5%) for general permeabilization
Saponin (0.1%) for milder permeabilization that better preserves membrane structures
Digitonin (10-50 μg/ml) for selective plasma membrane permeabilization
Antibody concentration titration:
Start with manufacturer's recommended dilution
Test serial dilutions to determine optimal signal-to-noise ratio
Include secondary antibody-only controls to assess background
Signal amplification strategies:
Tyramide signal amplification for low-abundance detection
Fluorescently-conjugated primary antibodies to reduce background
Consider super-resolution microscopy techniques for detailed localization studies
Co-localization markers:
Cell type-specific considerations:
Neuronal cells: Longer fixation times may be needed
Adipocytes: Additional permeabilization steps may be required
Primary cells: May require gentler fixation than cell lines
These optimizations should be systematically tested and validated to establish reliable protocols for ATG3 detection across different experimental systems.
ATG3 undergoes various conformational changes during autophagy, and distinguishing between these states requires specialized approaches:
Conformation-specific antibodies:
Develop or obtain antibodies that recognize specific conformational epitopes
Validate specificity using mutants locked in particular conformations
Fluorescence resonance energy transfer (FRET):
Design ATG3 constructs tagged with FRET donor and acceptor pairs
Monitor conformational changes through FRET efficiency alterations
Use sites flanking known flexible regions or interaction domains
Limited proteolysis approaches:
Different conformational states often show altered susceptibility to proteolytic digestion
Compare digestion patterns between active and inactive states
Combine with mass spectrometry for detailed mapping
Structural biology techniques:
X-ray crystallography of ATG3 in different states (free, bound to ATG7, bound to ATG12~ATG5)
Cryo-electron microscopy for larger complexes
NMR spectroscopy for dynamic conformational analyses
Site-specific crosslinking:
Introduce photoreactive amino acids at strategic positions
Crosslink under different conditions to capture transient states
Identify crosslinked partners by mass spectrometry
Proximity labeling approaches:
Fusion of ATG3 with enzymes like BioID or APEX2
Map proximal proteins under different autophagy conditions
Identify changing interaction landscapes during conformational shifts
These approaches provide complementary information about ATG3 conformational dynamics, offering insights into the molecular mechanisms underlying its functions in the autophagy pathway.