ADAL Human

What is ADAL human protein and what is its primary function?

ADAL (Adenosine Deaminase-Like) is a nucleoside deaminase that plays a significant role in purine metabolism. It primarily catalyzes the hydrolytic deamination of adenosine or similar substrates. More specifically, ADAL catalyzes the hydrolysis of free cytosolic methylated adenosine nucleotide N(6)-methyl-AMP (N6-mAMP) to produce inositol monophosphate (IMP) and methylamine. This enzymatic activity is crucial for the catabolism of cytosolic N6-mAMP, which is derived from the degradation of mRNA containing N6-methylated adenine (m6A) . The enzyme has evolved to process specific nucleotide substrates, contributing to the complex network of nucleotide processing in human cells. Understanding this function is fundamental to researching RNA modification pathways and epitranscriptomic regulation mechanisms.

What is the molecular structure and composition of recombinant ADAL human protein?

Recombinant ADAL Human protein produced in E. coli is a single, non-glycosylated polypeptide chain containing 292 amino acids. The protein consists of amino acids 1-267 of the native sequence with a molecular mass of approximately 32.7 kDa . It is typically fused to a 25 amino acid His-tag at the N-terminus to facilitate purification using chromatographic techniques . The amino acid sequence of the recombinant form includes the His-tag followed by the functional protein sequence. The full amino acid sequence is:

MGSSHHHHHH SSGLVPRGSH MGSEFMIEAE EQQPCKTDFY SELPKVELHA HLNGSISSHT MKKLIAQKPD LKIHDQMTVI DKGKKRTLEE CFQMFQTIHQ LTSSPEDILM VTKDVIKEFA DDGVKYLELR STPRRENATG MTKKTYVESI LEGIKQSKQE NLDIDVRYLI AVDRRGGPLV AKETVKLAEE FFLSTEGTVL GLDLSGDPTV GQAKDFLEPL LEAKKAGLKL ALHLSEIPNQ KKETQILLDL LPDRIGHGTF LNSGEGGSLD LVDFVRQHRI PLGKAWSFRS SR .

This structure provides researchers with a stable protein suitable for various biochemical and enzymatic assays.

How is ADAL protein stored and what are the optimal conditions for maintaining its stability?

For optimal stability of ADAL human protein, researchers should follow specific storage protocols. Short-term storage (2-4 weeks) can be conducted at 4°C if the entire vial will be used within this period. For longer periods, the protein should be stored frozen at -20°C . When planning experiments that will span several weeks or months, it is recommended to add a carrier protein such as 0.1% human serum albumin (HSA) or bovine serum albumin (BSA) to enhance stability during long-term storage .

Multiple freeze-thaw cycles significantly reduce protein activity and should be strictly avoided. The standard formulation for ADAL includes 20mM Tris-HCl buffer (pH 8.0), 10% glycerol, and 0.4M Urea at a concentration of 1mg/ml . This formulation helps maintain the protein in a stable conformation while preventing aggregation. For experiments requiring different buffer conditions, researchers should perform buffer exchange gradually to prevent protein denaturation. Regular quality control testing is recommended to ensure the protein maintains >80% purity and activity throughout the research timeline.

What experimental protocols are most effective for assessing ADAL enzymatic activity in vitro?

Effective assessment of ADAL enzymatic activity requires carefully designed assay conditions that account for its specific substrate preferences. The most reliable approach involves monitoring the conversion of N6-methyl-AMP to IMP and methylamine through either spectrophotometric or chromatographic methods . For spectrophotometric assays, researchers can exploit the difference in absorption spectra between the substrate and products at 265nm wavelength.

A typical reaction mixture should contain:

• Purified recombinant ADAL (0.1-1μg)

• N6-methyl-AMP substrate (50-200μM)

• Reaction buffer (typically 50mM Tris-HCl, pH 7.5, 1mM DTT, 2mM MgCl₂)

• Total reaction volume: 100-200μL

The reaction is typically initiated by adding the enzyme and monitored over time at 37°C. For more precise quantification, HPLC or LC-MS analysis allows direct measurement of substrate depletion and product formation. Measuring enzyme kinetics requires determining initial velocities at various substrate concentrations to calculate Km and Vmax values through Michaelis-Menten kinetics analysis.

For broader substrate specificity studies, researchers should test ADAL activity against various substrates including different N6-substituted purine nucleoside monophosphates and O6-substituted compounds, as ADAL has demonstrated capability to catalyze the removal of different alkyl groups from these compounds in vitro .

How can researchers differentiate between ADAL and classical adenosine deaminase (ADA) in experimental settings?

Differentiating between ADAL and classical adenosine deaminase (ADA) in experimental settings requires attention to their distinct substrate specificities, inhibition profiles, and biochemical properties. While both enzymes belong to the deaminase family, they have evolved to catalyze reactions with different substrates and exhibit distinct kinetic parameters.

The key differences that researchers should exploit include:

• Substrate specificity: ADAL preferentially catalyzes the deamination of N6-methyl-AMP and related methylated nucleotides, while classical ADA primarily acts on adenosine and 2'-deoxyadenosine . A comparative substrate panel assay using both methylated and non-methylated substrates can effectively distinguish between these enzymes.

• Inhibition profile: ADAL and ADA show different sensitivities to known deaminase inhibitors. For instance, pentostatin (deoxycoformycin) and EHNA (erythro-9-(2-hydroxy-3-nonyl)adenine) strongly inhibit ADA but have variable effects on ADAL. A well-designed inhibitor panel can provide clear discrimination.

• Molecular weight differences: ADAL has a molecular mass of approximately 32.7 kDa, which differs from ADA's molecular weight . Western blot analysis using specific antibodies against each protein can clearly differentiate between them.

• Catalytic efficiency: Measuring kcat/Km values for both enzymes across a range of substrates reveals distinct catalytic efficiency profiles that serve as enzymatic fingerprints for identification purposes.

For definitive differentiation in complex biological samples, immunological methods using specific antibodies against unique epitopes of each protein, coupled with mass spectrometry analysis of reaction products, provide the most reliable discrimination between these related but functionally distinct enzymes.

What role does ADAL play in RNA modification pathways and epitranscriptomic regulation?

ADAL plays a crucial role in RNA modification pathways, particularly in the processing of N6-methylated adenine (m6A) containing RNA degradation products. The enzyme specifically catalyzes the hydrolysis of N6-methyl-AMP (N6-mAMP), which is derived from the degradation of mRNA containing m6A modifications . This function positions ADAL as an important component in the epitranscriptomic regulatory network.

The m6A RNA modification is the most prevalent internal modification in eukaryotic mRNA and plays critical roles in mRNA splicing, export, stability, and translation. When m6A-containing mRNAs undergo degradation, they generate N6-mAMP, which requires further processing by ADAL to produce IMP and methylamine. This process ensures the proper recycling of nucleotides and prevents the accumulation of potentially harmful methylated nucleotides in the cytosol.

Research suggests that ADAL's activity may influence several aspects of cellular function:

• RNA metabolism: By processing degradation products of m6A-modified RNAs, ADAL contributes to nucleotide recycling pathways essential for cellular homeostasis.

• Epitranscriptomic regulation: The dynamic interplay between m6A writers (methyltransferases), readers (proteins that recognize m6A), erasers (demethylases), and processors like ADAL creates a complex regulatory network that influences gene expression patterns.

• Cellular stress response: Preliminary studies indicate that ADAL activity may be modulated under various stress conditions, suggesting its involvement in adaptive cellular responses.

Future research directions include investigating how ADAL activity coordinates with other enzymes involved in RNA modification and degradation, and determining whether ADAL dysfunction contributes to diseases associated with aberrant RNA processing.

What are the optimal expression systems for producing high-quality recombinant ADAL for research purposes?

The production of high-quality recombinant ADAL protein requires careful selection of expression systems based on experimental requirements. Based on current research practices, Escherichia coli remains the predominant expression system for ADAL production . The bacterial expression offers several advantages including cost-effectiveness, high protein yield, and simplified purification protocols.

For E. coli-based expression, the following optimized conditions have yielded consistently high-quality ADAL protein:

• Expression vector selection: pET-based vectors with T7 promoter systems provide tight regulation and high expression levels. The inclusion of an N-terminal His-tag facilitates efficient purification while minimally affecting protein function .

• Host strain optimization: BL21(DE3) derivatives, particularly Rosetta or Arctic Express strains, can enhance proper folding of ADAL, especially when expression is conducted at lower temperatures (16-18°C).

• Induction parameters: Optimal expression typically involves induction with 0.1-0.5mM IPTG at mid-log phase (OD600 = 0.6-0.8) followed by overnight expression at 18°C, which balances yield with proper folding.

For applications requiring post-translational modifications or higher structural integrity, eukaryotic expression systems should be considered. Insect cell expression using baculovirus vectors provides a compromise between yield and proper folding, while mammalian cell expression (typically HEK293 or CHO cells) offers the most native-like protein structure but with lower yields.

The purification strategy should include:

• Initial capture using Ni-NTA affinity chromatography

• Intermediate purification using ion exchange chromatography

• Polishing step using size exclusion chromatography to remove aggregates

Final protein purity should exceed 80% for standard applications and 95% for structural studies, with typical yields ranging from 5-15mg per liter of bacterial culture .

How can researchers design experiments to investigate ADAL's substrate specificity beyond its canonical functions?

Investigating ADAL's substrate specificity beyond its canonical functions requires a systematic experimental approach that probes the enzyme's ability to recognize and process structurally diverse substrates. Researchers should design a comprehensive substrate screening strategy that encompasses both hypothesis-driven and unbiased approaches.

A multi-tiered experimental design should include:

• Structural analog screening: Test a panel of systematically modified nucleotide analogs that vary in:

  • Base modifications (various N6-substitutions beyond methyl groups)

  • Sugar modifications (ribose vs. deoxyribose, 2'-O-modifications)

  • Phosphate modifications (mono-, di-, and triphosphates)

• High-throughput activity assays: Utilize enzyme-coupled assays that detect ammonia release or substrate consumption to rapidly screen potential substrates. This can be performed in 96-well format with colorimetric or fluorometric readouts.

• Reaction product characterization: For positive hits from initial screening, confirm actual deamination using LC-MS/MS analysis to identify specific reaction products and rule out false positives from coupled assays.

• Competition assays: Determine relative substrate preferences by measuring ADAL activity against the canonical substrate (N6-methyl-AMP) in the presence of increasing concentrations of test substrates.

• Structure-activity relationship analysis: Correlate chemical features of substrates with enzymatic parameters to identify key molecular determinants of recognition.

For each potential substrate, researchers should determine full kinetic parameters (Km, Vmax, kcat) and compare catalytic efficiency (kcat/Km) across substrates to establish a hierarchy of substrate preference. This approach can reveal unexpected activities of ADAL on previously unrecognized substrates, potentially uncovering novel physiological roles beyond its established function in processing methylated nucleotides .

What are the current challenges and limitations in ADAL research, and what methodological advances might address them?

Current ADAL research faces several significant challenges that limit our comprehensive understanding of this enzyme's function and biological significance. These challenges span from technical difficulties in protein production to gaps in understanding its physiological roles.

Key Current Challenges:

• Structural characterization limitations: Despite availability of recombinant protein, high-resolution structural data for ADAL remains limited, hampering structure-based functional studies and rational inhibitor design.

• Physiological substrate uncertainty: While in vitro studies have established N6-methyl-AMP as a substrate, the full spectrum of physiologically relevant substrates in different cellular contexts remains unclear.

• Limited tools for studying endogenous ADAL: There is a scarcity of highly specific antibodies and chemical probes for studying endogenous ADAL activity and localization in cellular contexts.

• Incomplete understanding of regulation: The mechanisms controlling ADAL expression, activity, and turnover in different tissues and under various physiological conditions remain poorly characterized.

Methodological Advances to Address These Challenges:

• Cryo-EM and advanced crystallography: Application of these techniques to ADAL and its complexes with substrates/inhibitors would provide critical structural insights to understand substrate recognition and catalytic mechanisms.

• Metabolomics approaches: Untargeted metabolomics in ADAL knockout/knockdown systems could reveal accumulated substrates and diminished products, helping identify novel physiological substrates.

• CRISPR-based tools: Development of ADAL knockout and knock-in cell lines with fluorescent or affinity tags would enable precise tracking of ADAL localization and interactions.

• Activity-based protein profiling: Design of activity-based probes specific to ADAL would allow monitoring of enzymatic activity in complex biological samples.

• Single-molecule enzymology: Application of these techniques could provide insights into ADAL's catalytic cycle and potential processivity on polymer substrates.

• Tissue-specific conditional knockout models: Development of such animal models would help elucidate ADAL's role in different physiological and pathological contexts.

Addressing these challenges through methodological innovations will significantly advance our understanding of ADAL's biological functions and potentially reveal new therapeutic opportunities targeting this enzyme.

How does ADAL function integrate with other enzymatic pathways in nucleotide metabolism?

ADAL's function is intricately connected with multiple enzymatic pathways in nucleotide metabolism, positioning it as a key player in the broader landscape of cellular nucleotide processing and recycling. Its role in deaminating N6-methyl-AMP links RNA modification pathways with purine salvage networks, creating important metabolic intersections.

The integration of ADAL with other enzymatic pathways occurs at several critical junctions:

• RNA degradation and nucleotide salvage: When m6A-modified RNAs are degraded, they generate N6-methyl-AMP, which ADAL processes to produce IMP . This IMP can then enter the purine salvage pathway, where it can be converted to AMP or GMP through the action of adenylosuccinate synthetase/lyase or IMP dehydrogenase/GMP synthetase, respectively. This process ensures efficient recycling of valuable nucleotide components.

• Epitranscriptomic regulation: ADAL functions downstream of the m6A modification machinery, which includes writers (METTL3/METTL14 complex), readers (YTHDF proteins), and erasers (FTO, ALKBH5). The activity of ADAL in processing methylated nucleotides may indirectly influence the availability of methyl donors (e.g., S-adenosylmethionine) for RNA modification, creating a potential feedback loop.

• Adenosine metabolism: While distinct from classical adenosine deaminase (ADA), ADAL likely participates in specialized branches of adenosine-derived molecule metabolism, particularly those involving methylated derivatives. This relationship potentially connects ADAL to pathways involving S-adenosylhomocysteine and the methionine cycle.

• Purine biosynthesis regulation: By contributing to the pool of IMP through its deaminase activity, ADAL potentially influences the balance between de novo purine synthesis and salvage pathways, particularly under conditions where methylated nucleotides are abundant.

This integrated view of ADAL function highlights its potential importance in maintaining nucleotide homeostasis and suggests that its dysregulation might have ripple effects across multiple metabolic pathways.

What are the implications of ADAL dysfunction in human diseases and potential therapeutic applications?

While direct evidence linking ADAL dysfunction to specific human diseases remains limited, emerging research suggests potential implications in several pathological conditions. The enzyme's role in processing methylated nucleotides positions it at a critical junction of RNA modification and nucleotide metabolism pathways, both of which have been implicated in various diseases.

Potential Disease Associations:

• Cancer biology: Alterations in RNA modification pathways, particularly m6A, have been linked to various cancers. As ADAL processes the degradation products of m6A-modified RNAs, its dysfunction might contribute to imbalances in methylated nucleotide pools, potentially influencing cancer cell metabolism and proliferation. Changes in ADAL expression or activity could potentially serve as biomarkers or therapeutic targets in specific cancer types.

• Neurological disorders: Given the importance of precise RNA regulation in neuronal function, ADAL dysfunction might contribute to neurological conditions. Accumulation of methylated nucleotides due to impaired ADAL activity could potentially disrupt neuronal metabolism and function.

• Metabolic disorders: As part of nucleotide metabolism pathways, ADAL dysfunction might influence broader metabolic networks, potentially contributing to inherited or acquired metabolic conditions.

Therapeutic Potential:

The therapeutic applications related to ADAL fall into several categories:

• ADAL modulators: Development of specific inhibitors or activators of ADAL could provide tools to manipulate methylated nucleotide metabolism in disease contexts where this pathway is dysregulated.

• Diagnostic applications: Assays measuring ADAL activity or expression might serve as biomarkers for conditions associated with altered methylated nucleotide metabolism.

• Enzyme replacement therapy: In theoretical cases of ADAL deficiency, recombinant enzyme administration could potentially address metabolic imbalances.

• Combination approaches: Targeting ADAL in combination with other enzymes involved in RNA modification pathways might provide synergistic effects in diseases characterized by epitranscriptomic dysregulation.

Future research should focus on comprehensive phenotypic characterization of ADAL deficiency models and systematic analysis of ADAL expression and activity across different disease states to better define its pathophysiological significance and therapeutic potential.

How can advanced computational approaches enhance ADAL research and predict novel functions?

Advanced computational approaches offer powerful tools to accelerate ADAL research, providing insights into structure-function relationships, predicting novel substrates, and uncovering potential regulatory mechanisms. The integration of computational methods with experimental validation creates a synergistic approach to exploring ADAL biology.

Key Computational Strategies:

• Structural bioinformatics and molecular modeling:

  • Homology modeling based on related deaminases can predict ADAL's three-dimensional structure

  • Molecular dynamics simulations can reveal conformational changes during substrate binding and catalysis

  • Virtual screening against the active site can identify potential novel substrates or inhibitors

  • QM/MM (quantum mechanics/molecular mechanics) methods can elucidate the detailed deamination mechanism

• Systems biology approaches:

  • Pathway analysis integrating transcriptomic, proteomic, and metabolomic data can place ADAL in broader functional networks

  • Flux balance analysis can predict metabolic consequences of ADAL perturbation

  • Gene co-expression network analysis can identify functional associations and potential regulatory relationships

• Machine learning applications:

  • Deep learning models trained on enzyme-substrate interaction data can predict novel ADAL substrates

  • Natural language processing of scientific literature can extract hidden relationships between ADAL and other biological processes

  • Classification algorithms applied to large-scale cancer genomics datasets can identify correlations between ADAL expression and disease phenotypes

• Evolutionary analysis:

  • Phylogenetic comparisons across species can reveal evolutionary conservation patterns and highlight functionally important regions

  • Analysis of positive selection signatures can identify adaptively evolved residues that may confer specialized functions

These computational approaches generate testable hypotheses that guide experimental design, creating an iterative discovery process. For example, computational prediction of novel substrates can be validated through in vitro enzymatic assays, while predicted structural features can guide mutagenesis studies to confirm functional importance of specific residues.

The integration of diverse computational methods creates a multidimensional view of ADAL function that complements and enhances traditional experimental approaches, ultimately accelerating discovery in this emerging area of nucleotide metabolism research.

What cutting-edge technologies are being applied to advance understanding of ADAL function and regulation?

The study of ADAL is being revolutionized by several cutting-edge technologies that provide unprecedented insights into its function, regulation, and biological significance. These technologies span multiple disciplines and offer complementary approaches to understanding this enzyme's role in nucleotide metabolism.

1. CRISPR-Cas9 Genome Editing Technologies:
CRISPR-based approaches are enabling precise manipulation of ADAL in cellular and animal models. This includes:

• Gene knockout studies to assess loss-of-function phenotypes

• Knock-in of tagged versions for tracking endogenous protein

• Base editing to introduce specific mutations that alter activity without disrupting expression

• CRISPRi/CRISPRa systems for temporal control of ADAL expression

2. Advanced Structural Biology Techniques:

• Cryo-electron microscopy for high-resolution structural analysis without crystallization requirements

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map protein dynamics and conformational changes upon substrate binding

• Single-particle analysis to capture different conformational states during the catalytic cycle

3. Advanced Metabolomics and Chemical Biology:

• Untargeted metabolomics using high-resolution mass spectrometry to identify novel metabolites processed by ADAL

• Stable isotope tracing to map metabolic flux through ADAL-dependent pathways

• Activity-based protein profiling using chemical probes designed to capture ADAL in its active state

• Chemoproteomics approaches to identify protein-protein interactions involving ADAL

4. Single-Cell Technologies:

• Single-cell RNA sequencing to map ADAL expression across different cell types and states

• Single-cell proteomics to correlate ADAL protein levels with cellular phenotypes

• Spatial transcriptomics to understand ADAL expression patterns within tissues

5. Biophysical Methods:

• Surface plasmon resonance and microscale thermophoresis to measure binding affinities with potential substrates

• Nanopore sequencing to detect m6A modifications in RNA and correlate with ADAL activity

• Optical tweezers and other single-molecule techniques to observe enzyme kinetics at unprecedented resolution

The integration of these diverse technologies is creating a comprehensive understanding of ADAL's function and regulation. As these methods continue to evolve and become more accessible, we can anticipate accelerated progress in defining ADAL's broader biological significance and potential therapeutic applications.

How might genetic variation in ADAL affect enzyme function across different populations?

Genetic variation in ADAL may have significant implications for enzyme function across different populations, potentially contributing to individual and population-level differences in nucleotide metabolism and RNA processing. Understanding these variations is important for personalizing therapeutic approaches and interpreting population-specific disease associations.

Types of Genetic Variation and Functional Implications:

• Coding region variations:

  • Missense variants may alter substrate specificity, catalytic efficiency, or protein stability

  • Nonsense variants could lead to truncated proteins with compromised function

  • Frameshift mutations typically result in loss of function through nonsense-mediated decay

• Regulatory region variations:

  • Promoter variants may alter expression levels in specific tissues

  • Enhancer/silencer variations could modify context-dependent expression

  • 5'UTR variants might affect translation efficiency

  • 3'UTR variants could influence mRNA stability or miRNA targeting

Population Genetics Considerations:

Different human populations show distinct patterns of genetic variation due to evolutionary history, selection pressures, and demographic factors. For ADAL, population-specific variants might reflect adaptations to different environmental exposures or dietary patterns that influence nucleotide metabolism.

A comprehensive analysis of population genetics data reveals several key considerations:

• Allele frequency distributions: Some ADAL variants show significant frequency differences across populations, suggesting potential functional adaptations or genetic drift effects.

• Selection signatures: Analysis of selection metrics (Fst, iHS, etc.) could reveal whether certain ADAL variants have been under positive selection in specific populations.

• Linkage disequilibrium patterns: Population-specific LD blocks containing ADAL may influence how variants are inherited together and their cumulative functional impact.

Methodological Approaches:

To properly assess the impact of population genetic variation in ADAL:

• Computational predictions: Tools like SIFT, PolyPhen, and CADD can predict functional impacts of coding variants.

• Experimental validation: Site-directed mutagenesis and expression of population-specific variants can directly measure altered enzyme kinetics or substrate specificities.

• Population pharmacogenomics: Studies correlating ADAL variants with drug responses related to nucleotide metabolism could reveal clinically relevant associations.

• Phenome-wide association studies (PheWAS): Linking ADAL variants to diverse phenotypes could uncover unexpected functions across different populations.

Understanding population-specific ADAL variation has important implications for precision medicine approaches targeting nucleotide metabolism pathways and could reveal novel insights into the evolution of RNA modification systems across human populations.