ATG16L2 Antibody

What is ATG16L2 and how does it differ from ATG16L1?

ATG16L2 is a novel isoform of mammalian ATG16L, consisting of similar domain structures as ATG16L1. It is a 619 amino-acid protein belonging to the WD repeat ATG16 family and contains 7 WD repeats. The protein has three structural regions: an N-terminal region homologous to the ATG5-binding region of ATG16L1, a putative coiled-coil domain in the middle region, and seven WD repeats at the C-terminal region .

While ATG16L1 and ATG16L2 share structural similarities, they differ significantly in function. The sequence homology between their N- and C-terminal regions is well conserved (32.1% and 43.0% amino acid identity, respectively), but the middle region shows relatively low homology (20.7% amino acid identity) . This difference in the middle region containing the coiled-coil domain is critical for their distinct functions in autophagy.

What are the known isoforms of ATG16L2?

There are three known isoforms of ATG16L2 with molecular weights of 69kDa, 56kDa, and 31kDa . Two alternative splicing isoforms have been specifically characterized:

• ATG16L2α (short isoform) - 602 amino acids, lacks exon 8 (63 bp)

• ATG16L2β (long isoform) - 623 amino acids, contains all 18 exons

The expression of these isoforms varies by tissue type, with ATG16L2β being dominant in most mouse tissues, in contrast to ATG16L1 which shows tissue-specific alternative splicing .

What are the recommended applications for ATG16L2 antibodies in research?

ATG16L2 antibodies can be utilized in multiple research applications:

Testing has confirmed reactivity with human and mouse samples . It's recommended to perform antibody titration for each specific experimental system to obtain optimal results.

How should ATG16L2 antibodies be optimized for immunohistochemistry in tissue samples?

For optimal immunohistochemistry results with ATG16L2 antibodies:

• Use heat-induced epitope retrieval (HIER) with pH 6 buffer for paraffin sections

• For immunofluorescence, use PFA/Triton X-100 for fixation and permeabilization

• Grade samples as low (<50%) or high (≥50%) according to the proportion of ATG16L2-positive cells for quantitative analysis

• Include appropriate positive controls (mouse spleen or testis tissue have shown positive results)

• Compare expression between normal and pathological tissues, as ATG16L2 expression differs significantly between them

When analyzing colorectal tissue specifically, note that strong cytoplasmic positivity has been observed in ganglion cells .

How does ATG16L2 differ functionally from ATG16L1 in autophagy?

Despite structural similarities, ATG16L2 and ATG16L1 exhibit distinct functional roles in autophagy:

• Complex Formation: Both ATG16L2 and ATG16L1 interact with ATG5 and self-oligomerize to form an ~800-kDa complex

• Subcellular Localization: ATG16L1 localizes to phagophores during autophagy, while ATG16L2 is predominantly cytosolic and not recruited to phagophores

• Autophagosome Formation: ATG16L2 cannot compensate for ATG16L1 in autophagosome formation, and knockdown of endogenous ATG16L2 does not affect autophagosome formation

• ATG5 Binding: Both proteins bind ATG5 with similar affinity (calculated EC50 values: 46.2±18.3 nM for ATG16L2 and 53.5±17.5 nM for ATG16L1)

• Rab33B Binding: ATG16L2 has significantly weaker Rab33B-binding affinity compared to ATG16L1

The functional differences are primarily attributed to the middle region containing the coiled-coil domain, which shows the lowest sequence homology between the two proteins .

What is the role of ATG16L2 in inflammasome regulation?

ATG16L2 plays a significant role in regulating inflammasome activity:

• ATG16L2 inhibits NLRP3 inflammasome activation through promoting ATG5-12-16L1 complex assembly and autophagy

• ATG16L2 deficiency attenuates LPS-induced autophagy flux in macrophages through mediating ATG5-12-16L1

• NLRP3 inflammasome activation is elevated in ATG16L2-deficient macrophages, which also display defects in mitochondrial integrity and respiration

• ATG16L2 knockout mice show increased susceptibility to DSS-induced intestinal damage, which can be ameliorated by inhibition of NLRP3

These findings suggest ATG16L2 could be a potential target for manipulating inflammatory responses in various disease contexts.

How does ATG16L2 expression correlate with cancer prognosis?

Research has revealed significant correlations between ATG16L2 expression and cancer outcomes:

These findings suggest ATG16L2 could serve as a valuable prognostic biomarker in colorectal cancer .

What is the evidence for ATG16L2's role in inflammatory diseases?

ATG16L2 has been implicated in various inflammatory conditions:

• Genome-wide association studies have identified ATG16L2 as independently associated with inflammatory diseases including Crohn's disease and systemic lupus erythematosus

• ATG16L2 contributes distinctly to autophagy and cellular ontogeny in myeloid, lymphoid, and epithelial lineages compared to ATG16L1

• In some scenarios, ATG16L2 may act as an endogenous dominant-negative inhibitor by competing with ATG16L1 for binding to ATG5, while simultaneously displacing ATG16L1 and leading to its proteasomal degradation, resulting in blocked autophagy

• ATG16L2 knockout mice display phenotypes distinct from ATG16L1 knockout mice, indicating that these homologs contribute differently to disease processes

The complex interplay between ATG16L1 and ATG16L2 appears critical for maintaining immune homeostasis, with dysregulation potentially contributing to inflammatory disease pathogenesis.

How can ATG16L2 function be effectively modulated in experimental systems?

Several approaches have been validated for modulating ATG16L2 function in research:

• Genetic Manipulation:

  • Complete knockout models: ATG16L2 knockout mice have been generated to study systemic effects

  • Cell-specific knockdown: siRNA targeting ATG16L2 has been used to assess its role in autophagy

  • Overexpression systems: FLAG-tagged ATG16L2 expression plasmids have been constructed and transfected into cell lines like RKO cells

• Chimeric Analysis:

  • Creating chimeric proteins between ATG16L1 and ATG16L2 has helped identify functional domains responsible for their distinct roles in autophagy

• In vivo Models:

  • Subcutaneous injection of ATG16L2-overexpressing cells into BALB/c nude mice has been used to assess tumor growth effects

  • DSS-induced colitis models in ATG16L2 knockout mice have revealed roles in intestinal inflammation

These approaches provide complementary insights into ATG16L2 function across different biological contexts.

What are the recommended protocols for analyzing ATG16L2 and ATG5 interactions?

To effectively study ATG16L2-ATG5 interactions:

• Co-immunoprecipitation:

  • Use 0.5-4.0 μg of ATG16L2 antibody for 1.0-3.0 mg of total protein lysate

  • Include controls for non-specific binding

  • Western blot using anti-ATG5 antibodies to detect interactions

• Direct Binding Assays:

  • Utilize purified components (T7-tagged ATG16L2 and GST-tagged ATG5)

  • Incubate beads coupled with T7-ATG16L2 with various concentrations of GST-ATG5

  • Detect bound proteins with anti-GST antibody

  • Calculate EC50 values to quantify binding affinity (reported EC50 for ATG16L2·ATG5 interaction: 46.2±18.3 nM)

• Cellular Assays:

  • Immunofluorescence co-localization studies

  • FRET-based interaction assays

  • Proximity ligation assays for detecting endogenous protein interactions

These methods have successfully demonstrated that ATG16L2 interacts with ATG5 with similar affinity to ATG16L1, despite functional differences in autophagy.

How can researchers address inconsistent ATG16L2 antibody detection in western blots?

When encountering variability in ATG16L2 detection:

• Isoform Consideration: Be aware that ATG16L2 has three isoforms with molecular weights of approximately 69kDa, 56kDa, and 31kDa . Ensure you're looking at the appropriate molecular weight range.

• Sample Preparation:

  • Use fresh tissue/cell lysates when possible

  • Include protease inhibitors in lysis buffers

  • Optimize protein extraction protocols for membrane-associated proteins

  • Consider non-denaturing conditions if detecting complexes

• Antibody Optimization:

  • Test different antibody dilutions (recommended range: 1:500-1:2000)

  • Increase incubation time at 4°C overnight

  • Optimize blocking conditions (BSA vs. milk)

  • Test different detection systems (HRP vs. fluorescent)

• Positive Controls:

  • Include known positive samples (mouse spleen or testis tissues have shown reliable detection)

  • Consider using overexpression systems alongside endogenous detection

• Detection Enhancement:

  • Use more sensitive ECL substrates for low abundance detection

  • Consider signal amplification systems for weakly expressed proteins

What approaches can resolve discrepancies between ATG16L2 mRNA and protein expression data?

When facing discrepancies between mRNA and protein expression:

• Post-transcriptional Regulation:

  • Analyze miRNA regulation of ATG16L2 through predictive algorithms and experimental validation

  • Assess mRNA stability through actinomycin D chase experiments

  • Evaluate alternative splicing patterns using PCR with isoform-specific primers

• Post-translational Modifications:

  • Investigate protein stability using cyclohexamide chase assays

  • Analyze ubiquitination status through immunoprecipitation followed by ubiquitin blotting

  • Examine proteasomal degradation using inhibitors like MG132

• Technical Considerations:

  • Ensure primers detect all relevant isoforms in qPCR

  • Verify antibody specificity through knockout/knockdown controls

  • Use multiple antibodies targeting different epitopes

  • Normalize data appropriately for both techniques

• Spatial and Temporal Factors:

  • Consider subcellular localization differences that may affect extraction efficiency

  • Account for differences in half-life between mRNA and protein

  • Analyze time-course data to capture dynamic regulation

Studies have shown that ATG16L2 expression can be lower in tumor tissues than normal tissues at both mRNA and protein levels , but regulatory mechanisms may vary across experimental systems.

What are the key unanswered questions regarding ATG16L2 function in autophagy regulation?

Several critical questions remain to be addressed:

• Molecular Mechanisms:

  • How does ATG16L2 promote ATG5-12-16L1 complex assembly despite not being recruited to phagophores?

  • What structural features in the middle region account for the functional differences between ATG16L1 and ATG16L2?

  • What is the significance of the lower Rab33B-binding affinity of ATG16L2 compared to ATG16L1?

• Regulatory Pathways:

  • Which upstream signaling pathways specifically regulate ATG16L2 expression and function?

  • How is the balance between ATG16L1 and ATG16L2 maintained in different cell types?

  • What environmental factors influence ATG16L2 expression and activity?

• Alternative Functions:

  • Does ATG16L2 regulate non-canonical autophagy pathways?

  • What role does ATG16L2 play in other cellular processes beyond autophagy?

  • How does ATG16L2 influence mitochondrial integrity and respiration?

• Therapeutic Potential:

  • Can modulation of ATG16L2 be exploited for therapeutic benefit in inflammatory or neoplastic conditions?

  • What are the consequences of long-term ATG16L2 modulation in vivo?

These questions represent important directions for future ATG16L2 research with potential implications for disease treatment.

How might emerging technologies advance our understanding of ATG16L2 biology?

Several cutting-edge approaches could significantly enhance ATG16L2 research:

• CRISPR-Based Techniques:

  • CRISPR activation/inhibition for precise temporal control of ATG16L2 expression

  • Base editing to introduce disease-associated variants

  • CRISPR screening to identify genetic modifiers of ATG16L2 function

• Advanced Imaging:

  • Super-resolution microscopy to visualize ATG16L2 complexes at nanoscale resolution

  • Live-cell imaging with fluorescently tagged ATG16L2 to track dynamics

  • Correlative light and electron microscopy to link ATG16L2 localization with ultrastructural features

• Proteomics Approaches:

  • Proximity labeling (BioID, APEX) to map the ATG16L2 interactome

  • Mass spectrometry to identify post-translational modifications

  • Thermal proteome profiling to assess ATG16L2 stability under different conditions

• Single-Cell Technologies:

  • Single-cell RNA-seq to identify cell-specific ATG16L2 expression patterns

  • Single-cell proteomics to correlate ATG16L2 with autophagy markers

  • Spatial transcriptomics to map ATG16L2 expression in tissue contexts

• Computational Approaches:

  • Molecular dynamics simulations to understand structural differences between ATG16L1 and ATG16L2

  • Machine learning to predict regulatory networks involving ATG16L2

  • Systems biology modeling of autophagy incorporating ATG16L2 functions

These technologies could resolve longstanding questions about ATG16L2's distinct contributions to cellular function in health and disease.

How does ATG16L2 function differ from other ATG family members in experimental systems?

Comparative analysis reveals distinct characteristics of ATG16L2:

• Complex Formation and Localization:

  • ATG16L2 forms an ~800-kDa complex similar to ATG16L1, but unlike ATG16L1, it is not recruited to phagophores

  • While most ATG proteins localize to autophagosomal structures during autophagy induction, ATG16L2 remains predominantly cytosolic

• Functional Redundancy:

  • Unlike many ATG proteins that show some functional redundancy, ATG16L2 cannot compensate for ATG16L1 function in autophagosome formation

  • Knockdown of endogenous ATG16L2 does not affect autophagosome formation, unlike most core ATG proteins

• Binding Partners:

  • ATG16L2 binds ATG5 with similar affinity to ATG16L1 but has significantly reduced Rab33B binding capacity

  • Evidence suggests ATG16L2 can form hetero-oligomers with ATG16L1, a feature not commonly observed among ATG proteins

• Disease Associations:

  • While many ATG proteins are broadly implicated in autophagy-related diseases, ATG16L2 shows specific associations with inflammatory conditions like Crohn's disease and systemic lupus erythematosus

  • ATG16L2's role in cancer appears distinct, with overexpression inhibiting cell proliferation

These differences highlight ATG16L2's unique position within the autophagy machinery.

What is the most effective experimental design to distinguish between ATG16L1 and ATG16L2 functions?

To effectively distinguish between ATG16L1 and ATG16L2 functions:

• Sequential Knockout/Knockdown Approach:

  • Generate single and double knockout/knockdown systems

  • Analyze autophagy markers (LC3-II formation, p62 degradation) in each condition

  • Perform rescue experiments with each protein to assess functional complementation

• Domain Swap Experiments:

  • Create chimeric proteins exchanging the N-terminal, middle (coiled-coil), and C-terminal regions

  • Assess which domains confer specific functions (previous research indicates the middle region is critical)

  • Evaluate localization, complex formation, and autophagy induction capacity

• Interaction Proteomics:

  • Perform comparative immunoprecipitation followed by mass spectrometry

  • Identify unique binding partners for each protein

  • Validate key interactions through secondary methods

• Transcriptional Profiling:

  • Compare gene expression changes following ATG16L1 vs. ATG16L2 manipulation

  • Identify unique transcriptional signatures associated with each protein

  • Validate key targets through functional studies

• Tissue-Specific Analysis:

  • Examine expression and function in different tissues (ATG16L1 and ATG16L2 show distinct tissue expression patterns)

  • Use conditional knockout models to assess tissue-specific roles

  • Evaluate disease phenotypes in tissue-specific knockout models