ADI1 Human

What is the molecular identity and primary function of ADI1?

ADI1, officially known as acireductone dioxygenase 1 (also 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase), belongs to the aci-reductone dioxygenase family of metal-binding enzymes. This enzyme plays a crucial role in the methionine salvage pathway, which recycles methionine from methylthioadenosine. ADI1 is also known by several synonyms including SIPL, FE-ARD, ARD, MTND, HMFT1638, MTCBP1, APL1, and NI-ARD .

The primary function of ADI1 varies depending on its cellular localization. While its canonical role involves methionine recycling in the cytoplasm, research indicates it may also regulate mRNA processing when localized in the nucleus, suggesting context-dependent functionality . The NCBI Gene ID for ADI1 is 55256, and it is located on human chromosome 2 .

How does ADI1 contribute to methionine metabolism?

ADI1 functions as a key enzyme in the methionine salvage pathway, which is essential for recycling methionine from 5'-methylthioadenosine (MTA). In this pathway, ADI1 catalyzes a critical step involving the conversion of acireductone intermediates. The cycle ultimately leads to the regeneration of S-adenosylmethionine (SAMe), a universal methyl donor in cellular processes .

Experimental evidence from studies manipulating ADI1 expression demonstrates that this protein can accelerate the MTA cycle, increasing SAMe levels within cells. This mechanism has significant downstream effects on cellular methylation processes, potentially altering gene expression profiles through promoter methylation changes . The metal binding properties of ADI1 are crucial for this enzymatic function, as different metal cofactors can direct the reaction toward different products.

What are the structural characteristics of the ADI1 protein?

Human ADI1 protein consists of 179 amino acids in its native form. When produced recombinantly (for example, in E. coli systems), it appears as a single, non-glycosylated polypeptide chain with a molecular mass of approximately 25.6kDa . Critical to its function are the metal-binding sites, particularly residues E94 and H133, which have been identified as essential for catalytic activity .

The protein contains specific domains characteristic of the aci-reductone dioxygenase family. Crystallographic and biochemical analyses reveal that ADI1 can bind different metal ions, which influences its catalytic outcomes. This metal-dependent functionality allows ADI1 to perform different reactions depending on which metal is bound at the active site . Recombinant production typically yields ADI1 with a molecular mass of 25.6kDa, though this may vary when fusion tags are added for purification purposes .

What is the tissue distribution pattern of ADI1 expression?

ADI1 shows variable expression across different human tissues, with particularly notable expression patterns in the brain. According to data from the Allen Brain Atlas, ADI1 exhibits tissue-specific expression in both adult human and mouse brain tissues . The expression pattern suggests potential specialized functions in neural tissues beyond its canonical metabolic role.

Multiple datasets including the Allen Brain Atlas Adult Human Brain Tissue Gene Expression Profiles and Allen Brain Atlas Adult Mouse Brain Tissue Gene Expression Profiles provide tissue-specific expression data that researchers can analyze to understand the relative abundance of ADI1 across different brain regions . Such differential expression may correlate with region-specific metabolic requirements or specialized functions in neural tissues.

What methods are recommended for quantifying ADI1 expression in experimental systems?

For accurate quantification of ADI1 expression, quantitative PCR (qPCR) represents a highly validated approach. Bio-Rad's PrimePCR assay (qHsaCED0047535) for human ADI1 has demonstrated excellent performance characteristics with 91% efficiency, an R² value of 0.9995, and 100% specificity . This assay targets a 117 bp amplicon in an exonic region of chromosome 2 (position 3502506-3502652).

When designing ADI1 expression experiments, researchers should consider:

The validated qPCR assay exhibits a cDNA Cq value of 21.78 in reference RNA samples, indicating relatively abundant expression that facilitates reliable detection in most experimental systems .

How does subcellular localization impact ADI1 function?

ADI1 demonstrates distinct functions depending on its subcellular localization. While primarily recognized for its role in methionine metabolism in the cytoplasm, evidence suggests that nuclear localization enables ADI1 to participate in mRNA processing events . This functional versatility highlights the importance of considering compartmentalization when investigating ADI1 activities.

Experimental approaches to study ADI1 localization include subcellular fractionation followed by western blotting, immunofluorescence microscopy, and the expression of fluorescently tagged ADI1 constructs. Each method provides complementary data on the protein's distribution pattern. Researchers should be aware that forcing ADI1 expression in specific compartments (through fusion with localization signals) can reveal location-dependent functions that may be obscured in total expression studies .

What is the evidence supporting ADI1's tumor suppressor activity?

Several lines of evidence support ADI1's role as a tumor suppressor, particularly in hepatocellular carcinoma (HCC). Significant reduction of ADI1 protein and mRNA levels has been observed in HCC tissues compared to normal liver tissues . Critically, higher ADI1 expression levels correlate with favorable postoperative recurrence-free survival in HCC patients, suggesting prognostic value .

Experimental manipulation of ADI1 levels in HCC cells reveals a consistent negative correlation between ADI1 expression and cell proliferation. Both cell-based assays and xenograft models demonstrate that overexpression of wild-type ADI1 suppresses tumor growth. Conversely, knockdown of ADI1 promotes cell proliferation, further supporting its growth-inhibitory function . These findings align with previous implications of ADI1 as a tumor suppressor in prostate cancer, suggesting a broader role across multiple cancer types.

What molecular mechanisms underlie ADI1's growth suppression effects?

The growth suppression effect of ADI1 operates through its enzymatic activity in the methionine salvage pathway. Research using ADI1 mutants with alterations at metal-binding sites (E94A and H133A) demonstrates that the tumor-suppressive function depends on accelerating the methylthioadenosine (MTA) cycle rather than simply disrupting it .

A key downstream effect of enhanced ADI1 activity is increased S-adenosylmethionine (SAMe) production, which serves as a universal methyl donor. Elevated SAMe levels alter the methylation status of key regulatory genes. For example, caveolin-1 (CAV1), a growth-promoting protein in HCC, is markedly decreased upon ADI1 overexpression due to increased promoter methylation .

Genome-wide methylation analysis reveals that ADI1 overexpression significantly alters promoter methylation profiles across numerous cancer-related genes, including:

• Protein-coding genes like CAV1

• Antisense non-coding RNAs

• Long non-coding RNAs

• MicroRNA genes

These epigenetic changes result in altered expression patterns that collectively contribute to HCC growth suppression .

What is ADI1's role in viral infections, particularly hepatitis C?

ADI1 (also known as MTCBP-1 or Sip-L in some literature) has been identified as capable of supporting hepatitis C virus (HCV) replication in otherwise non-permissive cell lines . Experimental evidence indicates that mouse hepatoma cells coexpressing human CD81 and ADI1/Sip-L supported both HCV infection and replication, suggesting ADI1 creates a more permissive cellular environment for the virus .

Interestingly, overexpression of human ADI1/Sip-L in 293 cells enhances cell entry of HCV but does not appear to significantly impact viral replication after entry . This selective effect on viral entry suggests a specific mechanism involving ADI1 in early viral infection stages, possibly through interactions with cellular receptors or membrane components involved in viral internalization.

Beyond HCV, ADI1 has also been associated with Klebsiella infections, though the mechanistic details of this association require further investigation . The dual role of ADI1 in both tumor suppression and viral facilitation highlights the complexity of its biological functions and the need for context-specific experimental designs.

How can researchers effectively manipulate ADI1 expression and activity in experimental systems?

Researchers have several validated approaches for manipulating ADI1 expression and activity:

Overexpression Systems:

• Plasmid-based expression of wild-type ADI1 has been successfully implemented in multiple cell lines

• Recombinant ADI1 can be produced in E. coli as a single, non-glycosylated polypeptide (179 amino acids) with a molecular mass of 25.6kDa

• Expression constructs often include an N-terminal His-tag to facilitate purification

Mutagenesis Approaches:

• Site-directed mutagenesis targeting metal-binding sites (E94A and H133A) has proven effective for studying catalytic mechanisms

• These mutations selectively disrupt different catalytic steps, allowing researchers to determine whether ADI1 effects depend on staying in or leaving the MTA cycle

Protein Handling Considerations:
For recombinant ADI1 protein:

• Formulation in 20mM Tris-HCl buffer (pH 8.0) with 0.15M NaCl, 10% glycerol, and 1mM DTT maintains stability

• Storage at 4°C is suitable if the entire preparation will be used within 2-4 weeks

• For longer storage, freezing at -20°C with addition of carrier protein (0.1% HSA or BSA) is recommended

• Multiple freeze-thaw cycles should be avoided to maintain protein integrity

What cell and animal models are most appropriate for ADI1 research?

Based on published research, several model systems have proven valuable for ADI1 studies:

Cell Models:

• Hepatocellular carcinoma (HCC) cell lines have been extensively validated for ADI1 functional studies

• 293 cells have been used for viral interaction studies, particularly for HCV entry experiments

• Mouse hepatoma cells coexpressing human CD81 and ADI1 serve as models for viral infection studies

Validation Methods:
When establishing new model systems, researchers should confirm:

• Baseline ADI1 expression (using qPCR assays like qHsaCED0047535)

• Protein expression patterns via western blotting

• Enzymatic activity using appropriate biochemical assays

• Response to known ADI1 modulators

Xenograft Models:
Cells with manipulated ADI1 expression (either overexpression or knockdown) have been successfully used in xenograft experiments to assess the impact on tumor growth in vivo . These models allow for assessment of ADI1's effects on tumor development in a more physiologically relevant context than cell culture alone.

What biochemical assays are available for measuring ADI1 enzymatic activity?

ADI1 enzymatic activity can be assessed through several complementary approaches:

Direct Enzymatic Assays:

• Measurement of 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase activity

• Tracking conversion of acireductone substrates to corresponding products

• Spectrophotometric detection of reaction products

Pathway Analysis:

• Quantification of S-adenosylmethionine (SAMe) levels by HPLC or LC-MS/MS

• Assessment of methionine recycling efficiency using isotope-labeled precursors

• Measurement of 5'-methylthioadenosine (MTA) levels to assess pathway flux

Metal Dependence:
Given ADI1's nature as a metalloenzyme, activity assays should consider:

• Testing activity with different metal cofactors (Fe2+, Ni2+)

• Chelation experiments to confirm metal dependency

• Site-directed mutagenesis of metal-binding residues (E94, H133) as controls for specificity

These assays should be complemented with expression analysis to correlate enzymatic activity with protein levels, particularly when examining disease-related alterations in ADI1 function.

How does ADI1 influence epigenetic regulation and genome-wide methylation patterns?

ADI1 exerts significant effects on epigenetic regulation through its role in the MTA cycle and subsequent modulation of S-adenosylmethionine (SAMe) levels. As demonstrated in hepatocellular carcinoma models, ADI1 overexpression increases SAMe levels, which then serves as a methyl donor for DNA methyltransferases .

Genome-wide methylation analysis reveals that ADI1 overexpression significantly alters promoter methylation profiles across numerous genes. This effect extends beyond protein-coding genes to include regulatory elements and non-coding RNA genes. The altered methylation patterns result in significant changes in gene expression that collectively contribute to ADI1's phenotypic effects .

Key experimental observations include:

• Altered methylation status of the caveolin-1 (CAV1) promoter upon ADI1 overexpression

• Changes in methylation patterns of genes encoding antisense non-coding RNAs

• Methylation changes in long non-coding RNA genes

• Modified methylation of microRNA gene promoters

These findings suggest that ADI1's role extends beyond basic metabolism to include epigenetic regulation, positioning it as a potential link between metabolic pathways and gene expression control mechanisms.

What is the significance of ADI1's interaction with the mRNA processing machinery?

While ADI1 is primarily recognized for its enzymatic role in methionine salvage, evidence suggests it may also regulate mRNA processing in the nucleus . This dual functionality positions ADI1 at the intersection of metabolism and gene expression regulation, suggesting a potential regulatory feedback mechanism between these processes.

Research questions that remain to be fully addressed include:

• The specific mRNA processing steps influenced by ADI1

• Whether this function is dependent on or independent of its enzymatic activity

• The protein interaction partners of ADI1 in the nucleus

• How nuclear ADI1 levels are regulated in response to cellular conditions

Investigating these aspects requires specialized approaches such as RNA immunoprecipitation, mass spectrometry-based interaction studies, and transcriptome-wide analyses of splicing and other RNA processing events in the context of ADI1 manipulation.

How might ADI1 function be therapeutically targeted in cancer and viral infections?

The dual roles of ADI1 in tumor suppression and viral facilitation present both opportunities and challenges for therapeutic development. Several strategic approaches might be considered:

For Cancer Applications:

• Developing small molecules that enhance ADI1 enzymatic activity to promote its tumor-suppressive effects

• Targeting upstream regulators of ADI1 expression to increase its levels in cancer cells

• Mimicking downstream effects of ADI1 by directly modulating SAMe levels or targeting key ADI1-regulated genes like CAV1

For Viral Infections:

• Creating inhibitors that selectively block ADI1's interaction with viral components without affecting its metabolic functions

• Developing peptide-based approaches to disrupt the specific protein-protein interactions involved in ADI1-mediated viral entry

• Exploring combination approaches that maintain ADI1's beneficial effects while mitigating its viral-facilitating properties

A comprehensive understanding of the structural determinants of ADI1's different functions will be crucial for designing selective modulators. High-resolution structural studies, coupled with functional characterization of ADI1 variants, will provide essential insights for rational drug design approaches targeting this multifunctional protein.