ADI1 Antibody

What is ADI1 and what role does it play in cancer research?

ADI1 (Acireductone Dioxygenase 1) is an enzyme involved in the methionine salvage pathway with a molecular mass of approximately 26 kDa. It has emerged as a significant target in cancer research due to its potential tumor suppressive role.

ADI1 catalyzes two different reactions between oxygen and acireductone 1,2-dihydroxy-3-keto-5-methylthiopentene (DHK-MTPene) depending on the metal bound in the active site:

• Fe-containing acireductone dioxygenase (Fe-ARD) produces formate and 2-keto-4-methylthiobutyrate (KMTB), the alpha-ketoacid precursor of methionine in the methionine recycle pathway

• Ni-containing acireductone dioxygenase (Ni-ARD) produces methylthiopropionate, carbon monoxide, and formate

Research has shown that ADI1 is downregulated in various cancers including prostate cancer and hepatocellular carcinoma (HCC). Higher ADI1 levels have been associated with favorable postoperative recurrence-free survival in HCC patients, supporting its role as a tumor suppressor .

What tissue expression patterns does ADI1 demonstrate?

ADI1 expression has been detected in multiple human tissues, with varying abundance:

In the prostate specifically, in situ hybridization experiments on human benign prostatic hyperplasia (BPH) tissues have shown that ADI1 mRNA is primarily expressed in epithelial cells, with little or no expression in stromal cells .

What types of ADI1 antibodies are available for research applications?

Several types of ADI1 antibodies are available for research applications:

These antibodies have been validated for various applications including Western blotting (WB), immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), and immunofluorescence (IF) .

How should I design experiments to study ADI1 expression in cancer tissues?

When designing experiments to study ADI1 expression in cancer tissues, consider the following methodological approach:

• Sample selection:

  • Include both tumorous and adjacent non-cancerous tissues from the same patients for paired analysis

  • Stratify samples by tumor grade/stage to evaluate correlation with disease progression

  • Include diverse tumor types if studying expression across cancer types

• Expression analysis techniques:

  • mRNA level: RT-qPCR, RNA-seq, or in situ hybridization

  • Protein level: Western blotting, immunohistochemistry on tissue microarrays

  • Subcellular localization: Immunofluorescence or fractionation followed by Western blotting

• Controls and normalization:

  • Use multiple housekeeping genes/proteins for normalization (e.g., β-actin for Western blots)

  • Include positive control tissues known to express high ADI1 levels (e.g., liver, kidney)

  • Include negative control tissues with low ADI1 expression (e.g., brain)

• Quantification methods:

  • For IHC: Use established scoring methods like the Grizzle method (scale 1-4) as described in literature

  • For Western blotting: Perform densitometry analysis normalized to loading controls

  • For RT-qPCR: Calculate relative expression using ΔΔCt method

Research has shown that ADI1 expression is frequently downregulated in tumors compared to adjacent normal tissues, with significant correlation to clinical outcomes .

What approaches can validate ADI1 antibody specificity for research applications?

Validating ADI1 antibody specificity is crucial for reliable research outcomes. A comprehensive validation should include:

• Western blot validation:

  • Verify single band of expected molecular weight (~26 kDa for ADI1)

  • Include positive control lysates from tissues known to express high ADI1 levels

  • Include negative controls (knockdown/knockout cells or low-expressing tissues)

  • Test cross-reactivity with recombinant ADI1 protein

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide before application

  • Specific signals should be blocked by the peptide

• Genetic validation:

  • Test antibody in ADI1 knockdown/knockout cells created using RNAi or CRISPR

  • Signal should be reduced/absent in these samples

• Orthogonal validation:

  • Compare results using multiple antibodies targeting different epitopes

  • Compare protein detection with mRNA expression data

  • Use tagged ADI1 constructs and detect with both anti-tag and anti-ADI1 antibodies

• Application-specific validation:

  • For IHC: Include appropriate isotype controls

  • For IF: Include secondary antibody-only controls

  • For IP: Include IgG controls

The literature describes generation of specific ADI1 antibodies by immunizing rabbits with His-tag-ADI1 fusion proteins, followed by affinity purification, with validation by Western blotting at 1:500 dilution .

How can I investigate the enzymatic activity of ADI1 in experimental systems?

Investigating ADI1 enzymatic activity requires specialized approaches to measure its aci-reductone dioxygenase (ARD) function:

• Recombinant protein activity assay:

  • Express and purify recombinant ADI1 from bacterial systems (E. coli)

  • Measure aci-reductone decay using spectrophotometric methods

  • Compare activity to control lysates lacking ADI1

  • Research has shown that bacterial lysates containing recombinant ADI1 produced a five-fold increase in aci-reductone decay over controls

• Metal dependency analysis:

  • Test activity with different metal ions (Fe²⁺ vs Ni²⁺)

  • Chelate endogenous metals and reconstitute with specific metals

  • Monitor different reaction products based on bound metal

• Site-directed mutagenesis approach:

  • Generate point mutations at key metal-binding residues (e.g., E94A, H133A)

  • These mutations selectively disrupt catalytic steps, affecting the methionine salvage pathway

  • Compare enzymatic activity between wild-type and mutant proteins

• Functional complementation:

  • Use yeast strains with disrupted ARD homolog (YMR009w)

  • Test growth restoration with human ADI1 expression

  • This approach demonstrated that human ADI1 can function in methionine metabolism in yeast

• Metabolite measurements:

  • Quantify metabolites in the methionine salvage pathway

  • Measure S-adenosylmethionine (SAMe) levels, which increase with ADI1 activity

  • Use liquid chromatography-mass spectrometry (LC-MS) for precise quantification

How does ADI1 contribute to tumor suppression through epigenetic mechanisms?

ADI1's tumor suppressive role involves complex epigenetic mechanisms through the methionine salvage pathway:

• Promotion of MTA cycle:

  • ADI1 expression accelerates the 5'-methylthioadenosine (MTA) cycle

  • This leads to increased S-adenosylmethionine (SAMe) levels

• Altered DNA methylation profiles:

  • Elevated SAMe acts as a methyl donor for DNA methyltransferases

  • This alters genome-wide promoter methylation profiles

  • Research has shown ADI1 overexpression significantly changes methylation status of cancer-related gene promoters

• Repression of oncogenic genes:

  • ADI1-mediated hypermethylation suppresses expression of oncogenic genes

  • Caveolin-1 (CAV1), a growth-promoting protein in HCC, is markedly decreased upon ADI1 overexpression

  • This repression is mediated by increased methylation of the CAV1 promoter

• Regulation of non-coding RNAs:

  • ADI1 affects methylation of genes encoding various non-coding RNAs:

    • Antisense non-coding RNAs

    • Long non-coding RNAs (lncRNAs)

    • MicroRNAs (miRNAs)

  • This results in significant changes in their expression levels

• Growth suppression consequences:

  • The cumulative effect of these methylation changes leads to:

    • Reduced cell proliferation

    • Increased apoptosis

    • Suppressed tumor growth in xenograft models

Research using site-directed mutagenesis has shown that the enzymatic activity of ADI1 in the MTA cycle (dependent on E94 residue) is crucial for this tumor suppressive function, while other functions (dependent on H133) may have different effects .

How can I distinguish between enzymatic and non-enzymatic functions of ADI1?

Distinguishing between enzymatic and non-enzymatic functions of ADI1 requires sophisticated experimental approaches:

• Site-directed mutagenesis strategy:

  • Generate point mutations at key metal-binding residues:

    • E94A mutation: Disrupts specific catalytic step but keeps protein in MTA cycle

    • H133A mutation: Disrupts function, pushing metabolism away from MTA cycle

  • Compare functional outcomes between wild-type and mutant proteins

• Functional separation experiments:

  • Perform cell-based and xenograft experiments with cells expressing different ADI1 variants

  • Compare growth suppression, apoptosis induction, and other phenotypes

  • Surprisingly, research showed that point mutations that disrupt ADI1 enzymatic activity did not affect its ability to induce apoptosis in prostate cancer cells, suggesting these activities may be independent

• Protein-protein interaction studies:

  • Investigate physical interactions with other proteins (e.g., MT1-MMP)

  • Use co-immunoprecipitation, yeast two-hybrid screening, or proximity ligation assays

  • Research identified ADI1 interaction with MT1-MMP's cytoplasmic tail, suggesting non-enzymatic functions

• Pathway-specific metabolite analysis:

  • Measure changes in metabolites specific to the methionine salvage pathway

  • Compare these changes between wild-type and enzymatically inactive mutants

  • Correlate metabolite levels with biological outcomes

• Subcellular localization studies:

  • Investigate if enzymatic vs. non-enzymatic functions correlate with different localizations

  • Research has shown ADI1 can localize to both cytosolic and nuclear compartments

How should I interpret discrepancies between ADI1 protein and mRNA expression in cancer samples?

Interpreting discrepancies between ADI1 protein and mRNA expression requires consideration of multiple factors:

• Post-transcriptional regulation:

  • Investigate microRNA-mediated regulation of ADI1 mRNA

  • Examine mRNA stability using actinomycin D chase experiments

  • Consider RNA-binding proteins that might affect translation efficiency

• Post-translational modifications and protein stability:

  • Analyze protein half-life using cycloheximide chase assays

  • Investigate ubiquitination and proteasomal degradation

  • Examine effects of cancer-related stress conditions on protein stability

• Technical considerations:

  • Evaluate sensitivity and specificity of detection methods

  • For Western blot: Ensure complete protein extraction, appropriate antibody dilution (1:500 recommended for ADI1)

  • For IHC: Optimize antigen retrieval and scoring systems (e.g., Grizzle method)

• Sample heterogeneity:

  • Consider tumor heterogeneity and contamination with stromal cells

  • Research has shown ADI1 is primarily expressed in epithelial cells, with little expression in stromal cells

  • Use microdissection techniques for more precise analysis

• Clinical correlation analysis:

  • Determine whether protein or mRNA levels better correlate with clinical outcomes

  • Research has shown patients with higher ADI1 T/N (tumor/normal) ratio have better prognosis

  • TCGA database analysis revealed better prognosis in patients with higher ADI1 mRNA levels

When evaluating prognostic value, research suggests examining ADI1 expression as a ratio between tumorous and non-tumorous tissues from the same patient, rather than absolute expression levels, which showed more consistent correlation with patient outcomes .

What are common challenges with ADI1 antibody applications and how can they be addressed?

Several challenges may arise when working with ADI1 antibodies. Here are methodological solutions for common issues:

• High background in immunohistochemistry:

  • Optimize antibody dilution (start with manufacturer's recommendation)

  • Extend blocking time (use 5-7% BSA or normal serum from secondary antibody species)

  • Include 0.1-0.3% Triton X-100 in wash buffer

  • Perform antigen retrieval optimization

  • Use more specific detection systems like polymer-based detection

• Weak or absent signal in Western blotting:

  • Verify ADI1 expression in your sample type (consider positive controls like liver or kidney tissue)

  • Optimize protein extraction (use 1% SDS lysis buffer as described in literature)

  • Increase protein loading (30μg recommended)

  • Reduce transfer time for small proteins like ADI1 (~26 kDa)

  • Use enhanced chemiluminescence for detection

• Non-specific bands in immunoblotting:

  • Increase blocking time and concentration

  • Optimize antibody dilution (1:500 dilution recommended for ADI1 antibodies)

  • Include 0.1% Tween-20 in wash buffer

  • Consider using freshly prepared samples to avoid degradation products

  • Validate with peptide competition assays

• Poor reproducibility across experiments:

  • Implement stringent sample handling protocols

  • Standardize tissue processing methods

  • Use the same antibody lot when possible

  • Include consistent positive and negative controls

  • Develop robust normalization strategies

• Cross-reactivity concerns:

  • Validate using knockdown/knockout samples

  • Test antibodies on multiple cell lines with varying ADI1 expression

  • Confirm specificity using recombinant ADI1 protein

  • Consider using antibodies targeting different epitopes for confirmation

How can I optimize protocols for studying ADI1 in relation to its role in the methionine salvage pathway?

Optimizing protocols for studying ADI1's role in the methionine salvage pathway requires specialized approaches:

• Cell system optimization:

  • Use cell lines with manipulated ADI1 expression:

    • Stable overexpression of wild-type ADI1

    • Expression of enzymatically inactive mutants (E94A, H133A)

    • Knockdown using shRNA (multiple clones recommended)

  • Select appropriate control cells with similar background

• Metabolite measurement:

  • Optimize extraction methods for methionine cycle metabolites

  • Use liquid chromatography-mass spectrometry (LC-MS) for precise quantification

  • Include internal standards for normalization

  • Measure key metabolites including SAMe, MTA, and MTOB

• Methylation analysis protocols:

  • For genome-wide methylation studies:

    • Use bisulfite sequencing or methylation arrays

    • Focus on promoter regions of cancer-related genes

  • For targeted methylation analysis:

    • Use methylation-specific PCR for genes of interest (e.g., CAV1)

    • Perform bisulfite pyrosequencing for quantitative analysis

• Functional readouts:

  • Measure cell proliferation using multiple methods:

    • Cell counting

    • MTT/MTS assays

    • BrdU incorporation

  • Assess apoptosis using:

    • TUNEL assay (as described in literature)

    • Annexin V/PI staining

    • Caspase activity assays

• In vivo model considerations:

  • Design xenograft experiments with cells expressing:

    • Wild-type ADI1

    • E94A mutant (on-pathway)

    • H133A mutant (off-pathway)

  • Measure tumor growth kinetics

  • Perform IHC analysis of tumor tissues for downstream targets like CAV1

What stability considerations should be addressed when working with ADI1 antibodies in research protocols?

Stability considerations are crucial for maintaining antibody performance and ensuring reliable results:

• Storage and handling:

  • Store antibodies at recommended temperature (-20°C long-term)

  • Ship at 4°C for short-term transport

  • Avoid repeated freeze-thaw cycles by preparing small aliquots

  • Many ADI1 antibodies are supplied in PBS with 50% glycerol and 0.02% sodium azide

• Buffer composition:

  • For long-term stability, verify buffer components:

    • PBS pH 7.4 is commonly used

    • 50% glycerol prevents freezing damage

    • 0.02% sodium azide prevents microbial growth

  • For working solutions, dilute in appropriate buffer with stabilizers like BSA

• Stability testing:

  • Periodically test antibody performance against reference samples

  • Include positive controls in each experiment to monitor sensitivity

  • Consider industry standards for antibody stability testing

  • Research on anti-drug antibodies suggests stability for several years in undiluted human biological matrix

• Sample preparation stability:

  • Process tissues consistently to avoid variability

  • For cell lysates, use protease inhibitor cocktails

  • For Western blotting, maintain consistent protein denaturation (95°C for 10 min)

  • Process samples immediately or store appropriately to prevent degradation

• Assay-specific considerations:

  • For IHC: Optimize fixation time and antigen retrieval methods

  • For IF: Minimize exposure to light to prevent photobleaching

  • For ELISA: Prepare fresh reagents for coating and detection

  • For WB: Use fresh transfer buffers and ECL substrates