AK3L1 Human

What is AK3L1/AK4 and what are its key structural characteristics?

AK3L1 (also known as AK3, AK3L2, or AK4) is a member of the adenylate kinase family localized to the mitochondrial matrix . The protein regulates adenine and guanine nucleotide compositions within cells by catalyzing the reversible transfer of phosphate groups among nucleotides .

The human AK3L1/AK4 protein contains 223 amino acids in its core sequence (Ala 2-Tyr 223) with a predicted molecular weight of approximately 25.3 kDa for the untagged native protein . Recombinant versions may include fusion tags that alter the molecular weight:

• His-tagged version: 259 amino acids, 29.3 kDa

• His-GST-tagged version: calculated 53.0 kDa, observed 46 kDa

How does AK3L1/AK4 differ from other adenylate kinase family members?

AK3L1/AK4 represents a unique member of the adenylate kinase family with several distinguishing characteristics:

• Unlike other family members, AK3L1/AK4 appears to be enzymatically inactive despite high sequence homology with other adenylate kinases .

• While most adenylate kinases work primarily with ATP/AMP, AK3L1/AK4 may be active with GTP .

• AK3L1/AK4 is specifically localized to the mitochondrial matrix, indicating a specialized role in mitochondrial nucleotide metabolism .

• Expression patterns of AK3L1/AK4 are tissue-specific and developmentally regulated, suggesting specialized functions in different tissues .

What expression systems are commonly used for recombinant AK3L1/AK4 production?

Recombinant human AK3L1/AK4 can be produced in multiple expression systems, each with specific advantages:

• E. coli expression system: Yields non-glycosylated, single polypeptide chain protein. Commonly used for high-yield production of functional protein .

• Baculovirus-insect cell expression system: Provides eukaryotic post-translational modifications that may better reflect the native human protein state .

The choice of expression system depends on research requirements:

• For structural studies or basic biochemical characterization, E. coli-expressed protein may be sufficient

• For functional studies requiring proper folding and modifications, insect cell-expressed protein may be preferable

What challenges exist in studying AK3L1/AK4 enzymatic activity?

Studying AK3L1/AK4 enzymatic activity presents several methodological challenges:

• Apparent enzymatic inactivity: Despite structural similarity to other adenylate kinases, AK3L1/AK4 reportedly lacks conventional enzymatic activity . This necessitates specialized approaches to detect potential alternative activities.

• Substrate specificity: While adenylate kinases typically utilize ATP/AMP, AK3L1/AK4 may interact with GTP instead , requiring modified assay conditions.

• Mitochondrial localization: As a mitochondrial matrix protein , studying AK3L1/AK4 in its native environment requires careful subcellular fractionation or sophisticated imaging techniques.

• Protein stability: Recombinant protein requires specific storage conditions (4°C for short-term, -20°C with glycerol for long-term storage) to maintain structural integrity .

What is the current understanding of AK3L1/AK4's role in human disease?

AK3L1/AK4 has been implicated in several pathological processes:

• Bipolar disorder: Convergent functional genomics has identified AK3L1 as a candidate gene in bipolar disorder pathophysiology. Integration of genome-wide association data with functional genomics from both human and animal models suggests AK3L1 involvement in neuropsychiatric conditions .

• Cancer: AK3L1 has appeared in gene expression analyses of mammary tumor models, suggesting potential involvement in cancer-related processes .

• Metabolic disorders: Given its mitochondrial localization and role in nucleotide metabolism, AK3L1/AK4 may impact conditions involving mitochondrial dysfunction or nucleotide imbalance.

How can researchers approach contradictory findings regarding AK3L1/AK4 function?

When facing conflicting data about AK3L1/AK4 function, researchers should:

What are optimal purification strategies for obtaining functionally active AK3L1/AK4?

Based on published protocols, the following purification strategy is recommended:

• Expression system selection: Choose between E. coli (higher yield) or baculovirus-insect cells (better folding) based on specific research needs.

• Affinity purification: Utilize appropriate tag-based purification (His-tag or GST-tag) as the initial capture step .

• Buffer optimization: Maintain protein stability with appropriate buffer conditions:

  • For His-tagged protein: 20mM Tris-HCl pH-8, 2mM DTT, 20% glycerol

  • For His-GST-tagged protein: 20mM Tris, 500mM NaCl, pH 8.0

• Purity assessment: Confirm protein purity (>90% for research applications) via SDS-PAGE .

• Storage conditions: Store at 4°C if using within 2-4 weeks, or at -20°C with a carrier protein (0.1% HSA or BSA) for longer periods. Avoid multiple freeze-thaw cycles .

What techniques are most effective for detecting endogenous AK3L1/AK4 in biological samples?

Several complementary techniques can effectively detect endogenous AK3L1/AK4:

• ELISA: For quantitative detection in serum, plasma, or other biological fluids. Available assays have a sensitivity of 0.146ng/mL and detection range of 0.312-20ng/mL .

• Western blotting: For semi-quantitative protein expression analysis in tissue or cell lysates.

• qPCR: For mRNA expression analysis when protein detection is challenging.

• Immunohistochemistry/Immunofluorescence: For visualizing subcellular localization and tissue distribution patterns.

When selecting detection methods, researchers should consider:

• Required sensitivity based on expected expression levels

• The need to distinguish AK3L1/AK4 from other adenylate kinase family members

• Whether protein or mRNA detection is more relevant to the research question

How should researchers design loss-of-function and gain-of-function experiments for AK3L1/AK4?

Loss-of-function approaches:

• CRISPR/Cas9 gene editing: For complete knockout studies in cell lines or model organisms .

• RNA interference: Using siRNA or shRNA for temporary knockdown when complete knockout is lethal or to study acute effects.

• Dominant-negative mutants: Based on structural information to interfere with potential protein-protein interactions.

Gain-of-function approaches:

• Overexpression systems: Using appropriate vectors with mitochondrial targeting sequences to ensure proper localization.

• Inducible expression: To control timing and magnitude of expression.

• Mutant expression: Introducing mutations that might confer novel activities based on structural comparisons with other adenylate kinases.

Critical considerations:

• Verify mitochondrial localization of modified proteins

• Confirm expression levels via multiple methods

• Include appropriate controls to account for non-specific effects

• Assess functional outcomes beyond just protein levels

What experimental approaches can resolve the apparent enzymatic inactivity of AK3L1/AK4?

To investigate AK3L1/AK4's apparent enzymatic inactivity despite structural similarity to active adenylate kinases, researchers should consider:

• Comprehensive substrate screening: Test beyond canonical adenylate kinase substrates, including various nucleotides and potential alternative phosphate acceptors.

• Assay condition optimization: Systematically vary pH, temperature, ion concentrations, and potential cofactors that might be required for activity.

• Structural biology approaches: Employ X-ray crystallography or cryo-EM to identify subtle structural differences that might explain inactivity.

• Protein interaction studies: Identify potential binding partners that might be required for in vivo activity but absent in purified systems.

• Comparative approach: Align AK3L1/AK4 sequence with active adenylate kinases to identify key differences, then create mutants that might restore activity.

How can researchers effectively study AK3L1/AK4's role in mitochondrial nucleotide homeostasis?

Studying AK3L1/AK4's role in mitochondrial nucleotide homeostasis requires specialized approaches:

• Subcellular fractionation: Develop protocols for isolating intact mitochondria while preserving nucleotide pools.

• Metabolomics: Employ liquid chromatography-mass spectrometry (LC-MS/MS) to accurately quantify mitochondrial nucleotide levels.

• Genetic manipulation: Compare nucleotide profiles in cells with normal, reduced, or elevated AK3L1/AK4 levels.

• Metabolic flux analysis: Use isotopically labeled precursors to track nucleotide synthesis and turnover rates.

• Functional correlation: Link changes in nucleotide levels to mitochondrial function parameters (membrane potential, ATP production, respiratory capacity).

• Stress response: Examine how AK3L1/AK4 affects nucleotide homeostasis under conditions like hypoxia or nutrient deprivation.

What comparative analyses between human AK3L1/AK4 and orthologues in model organisms are most informative?

When comparing human AK3L1/AK4 with orthologues in model organisms, researchers should focus on:

Researchers should:

• Verify conservation of key functional domains relevant to their specific research questions

• Consider species-specific differences in subcellular localization and expression patterns

• Validate findings across multiple model systems when possible

• Determine whether observed phenotypes in model organisms translate to human systems

How might AK3L1/AK4 contribute to cellular stress response pathways?

As a mitochondrial protein involved in nucleotide metabolism, AK3L1/AK4 likely intersects with multiple stress response pathways:

• Metabolic stress: AK3L1/AK4 may help maintain nucleotide balance during periods of metabolic fluctuation.

• Oxidative stress: Nucleotide metabolism interfaces with redox biology, suggesting potential roles in oxidative stress responses.

• Mitochondrial stress: AK3L1/AK4 might contribute to mitochondrial quality control mechanisms or bioenergetic adaptations.

• Disease contexts: Bipolar disorder and other conditions associated with AK3L1/AK4 often involve altered stress responses.

Researchers investigating these connections should consider measuring AK3L1/AK4 expression and activity under various stress conditions while monitoring relevant cellular outcomes.

What potential roles might AK3L1/AK4 play in mitochondrial-nuclear communication?

Given its mitochondrial localization and involvement in nucleotide metabolism, AK3L1/AK4 may contribute to mitochondrial-nuclear communication:

• Retrograde signaling: AK3L1/AK4-mediated changes in nucleotide pools might influence nuclear gene expression.

• Mitochondrial DNA maintenance: By regulating nucleotide availability, AK3L1/AK4 could impact mitochondrial DNA replication and repair.

• Energy status communication: As part of nucleotide metabolism pathways, AK3L1/AK4 might transmit information about mitochondrial energy status to the nucleus.

• Integration with transcriptional programs: AK3L1/AK4 activity may coordinate with nuclear transcription factors that respond to metabolic changes.

This emerging research area requires methods that can simultaneously monitor mitochondrial and nuclear events while manipulating AK3L1/AK4 function.