ATE1 Antibody

What is ATE1 and what cellular processes does it regulate?

ATE1 is an enzyme that catalyzes the non-ribosomal, post-translational addition of the amino acid arginine to acceptor proteins. This process, known as arginylation, is critical for protein degradation via the ubiquitin pathway. ATE1 is important for the posttranslational conjugation of arginine to N-terminal aspartate-, glutamate-, and possibly cystine-containing substrates . Research has demonstrated ATE1's involvement in cardiovascular development, angiogenesis, adipogenesis, muscle contraction, neurodegeneration, neurite outgrowth during brain development, and cancer progression . Recent studies have identified ATE1 as a tumor suppressor in hepatocellular carcinoma (HCC), showing it can inhibit cancer progression by suppressing Wnt/β-catenin signaling .

What is the molecular mechanism through which ATE1 regulates cancer progression?

ATE1 inhibits liver cancer progression through a mechanism involving the regulation of Wnt/β-catenin signaling. Specifically, ATE1 accelerates the degradation of β-catenin by regulating the turnover of Regulator of G Protein Signaling 5 (RGS5) . RGS5 appears to be involved in regulating the activity of GSK3-β, a crucial component of the cytoplasmic destruction complex . Experimental evidence shows that treatment with a GSK inhibitor (CHIR99021) cooperates with ablation of ATE1 or RGS5 overexpression to promote Wnt/β-catenin signaling, while ATE1 overexpression or RGS5 knockdown does not reverse the effect of GSK inhibitor . This RGS5-mediated pathway represents the key mechanism through which ATE1 exerts its tumor-suppressive effects.

What criteria should be considered when selecting an ATE1 antibody for research?

When selecting an ATE1 antibody, researchers should consider:

• Target species reactivity: Verify the antibody has been validated for your species of interest. Most commercial ATE1 antibodies show reactivity with human, mouse, and rat samples .

• Application compatibility: Ensure the antibody is validated for your specific application (WB, IHC, ELISA) . For instance, some antibodies may be optimized for Western blot but not for immunohistochemistry.

• Immunogen sequence: Check which portion of ATE1 was used as the immunogen. Some antibodies target specific regions which may affect detection of splice variants .

• Molecular weight detection: ATE1 has a calculated molecular weight of 59 kDa, but the observed molecular weight typically ranges from 59-65 kDa . Confirm that the antibody detects proteins in this range.

• Validation data: Review available validation data showing detection in relevant cell lines or tissues such as HepG2 cells, mouse liver tissue, HeLa cells, or Jurkat cells .

What dilutions and protocols are recommended for Western blot detection of ATE1?

For optimal Western blot detection of ATE1:

For protein turnover studies, cells can be treated with cycloheximide (10 μg/mL) for various time points before Western blot analysis to assess ATE1 stability or its effect on substrate degradation .

What are the optimal conditions for immunohistochemical detection of ATE1?

For immunohistochemical detection of ATE1 in tissue sections:

How can I troubleshoot multiple bands or inconsistent results with ATE1 antibodies?

When encountering multiple bands or inconsistent results with ATE1 antibodies:

• Verify sample integrity: ATE1 can be subject to degradation. Ensure proper sample handling and include fresh protease inhibitors during preparation.

• Adjust blocking conditions: Optimize blocking solution (BSA vs. NFDM) and duration to reduce non-specific binding.

• Consider splice variants: ATE1 has at least two distinct isoforms due to alternative splicing, which may appear as separate bands on Western blots .

• Test antibody specificity: Include positive controls (like HepG2 cells or mouse liver tissue) and negative controls (knockdown or knockout samples if available) .

• Optimize antibody concentration: Titrate antibody dilution to find the optimal concentration that maximizes specific signal while minimizing background.

• Check subcellular localization: ATE1 can localize to both the cytoplasm and nucleus, so different extraction methods may yield different results .

What are the best positive controls for validating ATE1 antibody performance?

For validating ATE1 antibody performance, the following positive controls have been experimentally verified:

When establishing a new protocol, these samples provide reliable positive controls for antibody validation. For negative controls, consider using siRNA knockdown of ATE1 in a positive control cell line.

How should researchers interpret variations in ATE1 molecular weight across different experimental systems?

Variations in ATE1 molecular weight (observed range 59-65 kDa compared to calculated 59 kDa) across experimental systems may result from:

• Post-translational modifications: ATE1 itself may undergo modifications that alter its electrophoretic mobility.

• Splice variants: Alternative splicing results in at least two distinct isoforms with potentially different molecular weights .

• Species-specific differences: Human, mouse, and rat ATE1 may show slight variations in molecular weight.

• Technical variations: Different gel systems, running buffers, and protein markers can affect apparent molecular weight.

When comparing ATE1 across different experimental conditions, consistent sample preparation and electrophoresis conditions are essential for accurate interpretation. Always include appropriate molecular weight markers and consider running known positive controls alongside experimental samples.

How can researchers assess the functional relationship between ATE1 and its substrates like RGS5?

To investigate the functional relationship between ATE1 and substrates like RGS5:

• Protein turnover analysis: Treat cells with cycloheximide (10 μg/mL) and monitor RGS5 degradation kinetics in the presence or absence of ATE1 manipulation .

• Co-immunoprecipitation studies: Use ATE1 antibodies to pull down protein complexes and probe for RGS5 association.

• Double immunofluorescence: Perform dual staining for ATE1 and RGS5 to assess co-localization patterns.

• Pathway reporter assays: Implement Cignal Finder 10-Pathway Reporter Arrays to determine which signaling pathways are affected by ATE1 manipulation .

• Loss- and gain-of-function experiments: Combine ATE1 knockdown/overexpression with RGS5 manipulation to establish epistatic relationships.

These approaches can help elucidate whether ATE1's effects on pathways like Wnt/β-catenin signaling are mediated directly through RGS5 or involve additional mechanisms.

What experimental approaches can differentiate direct ATE1 substrate arginylation from indirect effects?

To differentiate direct substrate arginylation by ATE1 from indirect effects:

• In vitro arginylation assays: Using purified recombinant ATE1 and candidate substrates to assess direct enzymatic activity.

• Site-directed mutagenesis: Mutate putative arginylation sites (N-terminal Asp-, Glu-, or Cys-) in substrate proteins to prevent modification.

• Mass spectrometry: Identify arginylated residues in proteins following ATE1 overexpression or in cells treated with ATE1 inhibitors.

• Time-course experiments: Establish the temporal sequence of events following ATE1 manipulation to distinguish primary from secondary effects.

• Substrate specificity analysis: Compare the effects of ATE1 on multiple potential substrates to establish patterns of specificity.

These approaches can help researchers move beyond correlation to establish causation in ATE1-mediated effects on cellular processes.

How can ATE1 antibodies be utilized in studying cancer progression mechanisms?

ATE1 antibodies can be valuable tools for investigating cancer progression mechanisms:

• Prognostic biomarker analysis: Assess ATE1 expression in patient tumor samples via immunohistochemistry to correlate with clinical outcomes. Low ATE1 expression correlates with aggressive clinicopathologic features and poorer prognosis in HCC .

• Wnt/β-catenin pathway investigation: Use ATE1 antibodies alongside β-catenin and RGS5 antibodies to examine the complete signaling axis in cancer tissues.

• Therapeutic response prediction: Monitor changes in ATE1 expression following treatment with various cancer therapeutics, particularly those targeting the Wnt pathway.

• Cancer model validation: Verify ATE1 knockdown or overexpression in cancer models (cell lines, xenografts) to ensure experimental validity.

• Metastasis studies: Examine differential ATE1 expression between primary tumors and metastatic lesions, given ATE1's reported influence on cancer metastasis .

These applications leverage ATE1 antibodies beyond basic detection to provide insights into cancer biology and potential therapeutic approaches.

What roles might ATE1 play in cellular processes beyond protein degradation?

Beyond protein degradation, emerging research suggests ATE1 may influence:

• Transcriptional regulation: ATE1's nuclear localization suggests potential roles in gene expression control .

• Signal transduction: ATE1 affects the Wnt/β-catenin pathway through RGS5-mediated mechanisms, suggesting broader roles in cell signaling .

• Cellular stress responses: The N-end rule pathway, which involves ATE1, is implicated in cellular responses to various stressors.

• Development and differentiation: ATE1's roles in cardiovascular development and adipogenesis point to functions in cellular differentiation programs .

• Neurological functions: ATE1 has been implicated in neurodegeneration and neurite outgrowth during brain development .

Researchers investigating these areas should consider using multiple detection methods (antibody-based and functional assays) to comprehensively assess ATE1's diverse roles.

How might post-translational modifications affect ATE1 function and detection?

Post-translational modifications may impact ATE1 function and detection in several ways:

• Activity regulation: Modifications could alter ATE1's enzymatic activity without changing expression levels.

• Localization changes: PTMs might affect nuclear-cytoplasmic shuttling, altering ATE1's cellular distribution .

• Substrate specificity: Modifications could change ATE1's affinity for different substrates.

• Antibody epitope masking: PTMs near antibody recognition sites may affect detection efficiency.

• Protein stability: Modifications could alter ATE1's half-life and steady-state levels.

Researchers should be aware that antibodies raised against different epitopes may show variable detection efficiency depending on the modification state of ATE1 in their experimental system.

What statistical approaches are recommended for analyzing ATE1 expression data in clinical samples?

For analyzing ATE1 expression data in clinical samples, the following statistical approaches are recommended:

These approaches have been successfully applied in HCC studies to establish ATE1 as a prognostic biomarker. Researchers should consider significance thresholds (typically p < 0.05) and appropriate sample sizes for statistical power .