ACO1 Human

What is the molecular structure and cellular localization of human ACO1?

Human Aconitase 1 (ACO1) is a cytosolic protein with a molecular weight of 98.2 kDa. It is encoded by a gene located at cytogenetic position 9p21.1. The protein contains a 4Fe-4S cluster binding domain essential for its catalytic activity. ACO1 is predominantly cytosolic, which distinguishes it from the mitochondrial aconitase (ACO2). The protein sequence contains 889 amino acids and exhibits a distinctive domain structure that enables its dual functionality. This cytosolic distribution is crucial for its role in iron sensing and regulation, as it allows direct interaction with cytoplasmic mRNA transcripts containing iron-responsive elements (IREs) .

How does human ACO1 differ from mitochondrial aconitase (ACO2)?

While both enzymes catalyze the conversion of citrate to isocitrate, they differ in several key aspects:

Methodologically, when studying these enzymes, researchers must account for these differences in experimental design, particularly when isolating cellular fractions or measuring enzymatic activity. Subcellular fractionation techniques followed by Western blotting using specific antibodies are commonly employed to distinguish between these isoforms .

What are the established functions of ACO1 in human cellular metabolism?

ACO1 is a bifunctional protein with two distinct physiological roles depending on cellular iron levels:

• Metabolic enzyme function: When cellular iron levels are high, ACO1 binds a 4Fe-4S cluster and functions as an aconitase, catalyzing the stereospecific isomerization of citrate to isocitrate in the cytosol. This reaction is part of the tricarboxylic acid (TCA) cycle and glyoxylate metabolism pathways .

• Iron regulatory protein function: When cellular iron levels are low, ACO1 loses its 4Fe-4S cluster and binds to iron-responsive elements (IREs) in mRNA. This binding results in:

  • Repression of ferritin mRNA translation (by binding to 5' UTR IREs)

  • Stabilization of transferrin receptor mRNA (by binding to 3' UTR IREs), preventing its degradation

This dual functionality classifies ACO1 as a "moonlighting protein," capable of performing mechanistically distinct functions depending on cellular conditions .

How does the 4Fe-4S cluster assembly/disassembly regulate ACO1 function?

The assembly and disassembly of the 4Fe-4S cluster is the central switch governing ACO1's bifunctionality. This process is regulated through several mechanisms:

• Assembly mechanism: When intracellular iron is abundant, iron-sulfur cluster assembly machinery proteins (including ISCU, NFS1, and frataxin) facilitate the incorporation of iron and sulfur into a 4Fe-4S cluster structure within ACO1. This enables its aconitase activity.

• Disassembly triggers: Several factors can cause cluster disassembly:

  • Iron deficiency: Primary physiological trigger

  • Oxidative stress: Reactive oxygen species can oxidize the Fe-S cluster

  • Nitric oxide: Forms protein-bound dinitrosyl iron complexes

• Structural changes: The loss of the Fe-S cluster induces conformational changes that expose the RNA-binding domain, allowing ACO1 to bind to IREs.

Methodologically, researchers can track this transition using enzyme activity assays, spectroscopic techniques like Mössbauer spectroscopy, or by measuring RNA-binding capacity using gel shift assays. Changes in protein conformation can also be monitored using circular dichroism or fluorescence spectroscopy .

What mechanisms govern ACO1 expression and activity in different tissue types?

ACO1 expression and activity vary across tissues due to:

• Transcriptional regulation: Tissue-specific transcription factors influence ACO1 gene expression. In lung tissue, ACO1 expression increases in fibrotic conditions, exhibiting a negative correlation with pro-SPC (surfactant protein C) and a positive correlation with CTGF (connective tissue growth factor) .

• Post-translational modifications: These include:

  • Phosphorylation: Affects protein stability and catalytic activity

  • Acetylation: Modulates enzymatic function

  • Oxidative modifications: Particularly relevant in stress conditions

• Cellular microenvironment: Oxygen tension, redox state, and iron availability vary by tissue and significantly impact ACO1 function.

• Age-related changes: In aging lungs, ACO1 expression increases, particularly in the interstitium, suggesting a role in age-related tissue remodeling .

When investigating tissue-specific ACO1 function, researchers should employ tissue-specific knockout models, immunohistochemistry with co-localization studies, and laser capture microdissection followed by proteomic analysis to accurately characterize tissue-specific expression patterns and regulatory mechanisms .

How do cellular iron levels precisely modulate the switch between ACO1's dual functions?

The iron-dependent functional switch of ACO1 involves a sophisticated sensing mechanism:

• Iron sensing threshold: Research indicates that the transition between aconitase and IRP1 activity occurs at specific cellular labile iron pool (LIP) concentrations. The switch is not binary but follows a sigmoidal response curve.

• Kinetics of conversion: The conversion from aconitase to IRP1 begins within 1-2 hours of iron depletion, with complete transition typically requiring 4-6 hours in most cell types.

• Spatial regulation: The cytosolic localization of ACO1 positions it optimally to respond to changes in the labile iron pool rather than total cellular iron.

• Feedback regulation: ACO1/IRP1 activity establishes a negative feedback loop - as IRP1 increases transferrin receptor expression and decreases ferritin synthesis, cellular iron levels gradually normalize, eventually restoring ACO1's aconitase function.

Researchers investigating this switch typically employ iron chelators (like deferoxamine) or iron supplementation protocols, combined with time-course analyses of both enzymatic activity and RNA-binding capacity. Fluorescent iron sensors can be used to correlate ACO1 function directly with quantifiable changes in the labile iron pool .

What are the most reliable methods for measuring ACO1 enzymatic activity versus its RNA-binding function?

Distinguishing between ACO1's dual functions requires specific methodological approaches:

For aconitase enzymatic activity:

• Spectrophotometric assays: Measure the formation of cis-aconitate from citrate at 240 nm.

• Coupled enzyme assays: Link isocitrate formation to NADPH production via isocitrate dehydrogenase.

• Isotope tracing: Use 13C-labeled citrate and mass spectrometry to track metabolic flux.

For RNA-binding (IRP1) activity:

• Electrophoretic mobility shift assay (EMSA): The gold standard for measuring IRE-binding activity.

• RNA pull-down assays: Using biotinylated IRE probes to isolate ACO1/IRP1.

• Surface plasmon resonance: For quantitative binding kinetics.

• Reporter gene assays: Using IRE-containing constructs to assess functional impact on gene expression.

Critical controls and considerations:

• Maintain anaerobic conditions during aconitase assays to prevent oxidative inactivation

• Use mitochondrial fraction controls to account for ACO2 activity

• Include iron chelators and iron supplementation conditions as functional controls

• Normalize activity to total ACO1 protein levels determined by Western blot

These approaches can be combined in time-course experiments to directly observe the transition between the two functional states under varying iron conditions .

What expression systems and purification strategies yield optimal recombinant human ACO1 for in vitro studies?

Producing functional recombinant human ACO1 requires careful consideration of expression and purification parameters:

Expression systems comparison:

Purification strategy:

• Affinity tags: C-terminal tags (e.g., C-Myc/DDK) facilitate purification without interfering with N-terminal structure critical for activity.

• Buffer composition: Optimal preservation of activity requires 25 mM Tris-HCl, 100 mM glycine, pH 7.3, with 10% glycerol as a stabilizer.

• Chromatography steps: Initial capture via anti-DDK affinity column followed by conventional chromatography provides >80% purity.

• Fe-S cluster reconstitution: May be necessary post-purification using iron salts, reducing agents, and sulfide sources under anaerobic conditions.

• Storage conditions: Store at -80°C to maintain stability for up to 12 months. Avoid repeated freeze-thaw cycles.

For functional studies, researchers should verify both enzymatic activity and RNA-binding capacity of the purified protein to ensure that both functions are preserved. Additionally, structural integrity can be confirmed using circular dichroism or thermal shift assays .

What are the most effective in vivo models for studying ACO1 function in different physiological and pathological contexts?

Several model systems offer unique advantages for studying ACO1 in vivo:

Mouse models:

• Global knockout: Severe - embryonic lethal, indicating essential function.

• Conditional knockout: Tissue-specific deletion using Cre-lox systems - particularly valuable for studying tissue-specific functions.

• Knockin mutations: Mutations affecting only one function (e.g., RNA binding or enzymatic activity) help dissect dual functionality.

• Bleomycin-induced lung fibrosis model: Shows increased ACO1 expression in fibrotic areas, making it useful for studying ACO1 in lung pathology .

Cellular models:

• Iron-responsive cell lines: HepG2 (liver), K562 (erythroid), and SH-SY5Y (neuronal) cell lines provide tissue-specific contexts.

• CRISPR-Cas9 edited cell lines: Allow precise engineering of ACO1 variants.

• Primary cell cultures: Better recapitulate physiological regulation than immortalized lines.

Model selection considerations:

• Research question specificity (iron metabolism vs. enzymatic function)

• Tissue relevance (e.g., liver for iron metabolism, brain for neurodegeneration studies)

• Readout requirements (molecular, cellular, or physiological parameters)

• Timeline (acute vs. chronic effects)

Researchers studying ACO1 in lung fibrosis, for instance, should consider the bleomycin mouse model, which demonstrates increased ACO1 expression correlating with fibrotic progression, as shown by immunohistochemical staining and Western blotting techniques .

How does ACO1 expression and function change in lung fibrosis, and what are the implications for therapeutic approaches?

ACO1 shows distinct expression patterns in lung fibrosis with significant implications:

Expression changes in fibrotic lungs:

• Upregulation: Immunohistochemical staining and Western blotting reveal significantly higher ACO1 levels in bleomycin-treated fibrotic lungs compared to controls.

• Cellular localization shifts: In the bleomycin mouse model, ACO1 is expressed in epithelial cells, particularly in cells positive for E-cadherin (E-cad).

• Correlation patterns: ACO1 expression shows negative correlation with pro-SPC (surfactant protein C) and positive correlation with CTGF (connective tissue growth factor) .

Functional implications:

• Alveolar regeneration: Evidence suggests ACO1 may facilitate alveolar regeneration in bleomycin-induced lung fibrosis. Double-positive cells for E-cad and ACO1 are found in areas of regenerating epithelium.

• Progenitor cell function: ACO1-positive, E-cad-positive cells may represent regenerating epithelial cell populations that eventually give rise to type II alveolar epithelial cells.

• Association with SOX9: ACO1-positive cells appear near SOX9-positive cell clusters, which mark epithelial cell populations with regenerative capacity .

Therapeutic implications:

• Targeted modulation: Selective enhancement of ACO1 in specific cell populations might promote regenerative responses.

• Biomarker potential: ACO1 expression patterns could serve as indicators of disease progression or regenerative activity.

• Metabolic intervention: Given ACO1's role in citrate metabolism, metabolic interventions targeting this pathway might influence fibrotic progression.

Researchers investigating ACO1 in lung disease should employ co-localization studies with markers of epithelial regeneration (E-cad, SOX9, pro-SPC) and fibrosis (CTGF) to fully characterize its role in disease progression and potential regenerative processes .

What is the relationship between ACO1 mutations and hemoglobin regulation, and how might this influence hematological disorder research?

ACO1 mutations significantly impact hemoglobin regulation through iron metabolism disturbances:

Mechanistic relationship:

• Iron sensing function: As an iron regulatory protein (IRP1), ACO1 regulates the translation of proteins involved in iron uptake, storage, and utilization - all critical for hemoglobin synthesis.

• Direct impact: When functioning as IRP1, ACO1 binds to IREs in mRNA of transferrin receptor (increasing iron uptake) and ferritin (decreasing iron storage).

• Genetic evidence: The nature article referenced indicates that predicted loss and gain of function mutations in ACO1 are associated with hemoglobin concentration variations .

Types of ACO1 mutations affecting hemoglobin:

• Loss-of-function mutations: May reduce iron availability for hemoglobin synthesis by decreasing transferrin receptor expression and increasing ferritin synthesis.

• Gain-of-function mutations: May increase iron availability by enhancing transferrin receptor expression and reducing ferritin synthesis.

• Mutations affecting only aconitase function: These may alter cellular metabolism without directly impacting iron regulation.

Implications for hematological research:

• Genetic screening: ACO1 should be included in genetic screening panels for unexplained anemias or polycythemias.

• Pharmacological targeting: Compounds that modulate ACO1's RNA-binding function could represent novel therapeutic approaches for iron disorders.

• Biomarker development: ACO1 activity or conformational state could serve as biomarkers for iron status in hematological disorders.

When investigating ACO1's role in hemoglobin regulation, researchers should employ genetic analysis techniques (sequencing, GWAS), functional assays measuring both enzymatic and RNA-binding activities, and erythroid differentiation models to assess the impact on hemoglobin synthesis pathways .

How is ACO1 expression altered in aging tissues, and what are the implications for age-related pathologies?

ACO1 shows distinctive expression changes in aging tissues with potential pathological significance:

Age-related expression changes:

• Interstitial accumulation: In 22-month-old mice, ACO1 shows patchy signals distributed in the interstitium of bronchovascular bundles and alveoli, unlike the pattern in younger (7-9 week) mice.

• Cell type shift: Most cells expressing ACO1 in aged lungs are negative for E-cadherin, contrasting with the double-positive populations seen in regenerating tissue after injury.

• Correlation with structural changes: The intensity of ACO1 signals appears to parallel the degree of loss of normal alveolar structures .

Hypothesized mechanisms:

• Failed regeneration: The increased ACO1 expression in aging lungs may reflect emergence of ACO1-positive, E-cad-negative epithelial progenitor cells that are unable to differentiate into functional alveolar epithelial cells.

• Metabolic adaptation: Age-related changes in energy metabolism may drive ACO1 expression changes to compensate for mitochondrial dysfunction.

• Iron dysregulation: Age-related iron accumulation may alter ACO1's functional state, affecting both its metabolic and regulatory roles.

Implications for age-related pathologies:

• Pulmonary fibrosis: The similar pattern between aged lungs and fibrotic lungs suggests ACO1 might be involved in age-related susceptibility to fibrotic disease.

• Regenerative capacity: ACO1 expression changes might mark declining tissue regenerative capacity with age.

• Intervention targets: Modulating ACO1 function might present opportunities to address age-related tissue degeneration.

For research in this area, investigators should employ age-stratified tissue analysis, single-cell RNA sequencing to identify ACO1-expressing cell populations, and functional studies comparing regenerative responses in young versus aged tissues .

How can single-cell multi-omics approaches advance our understanding of ACO1's cell-type specific functions?

Single-cell multi-omics offers revolutionary insights into ACO1 biology:

Methodological approaches:

• Single-cell RNA-seq (scRNA-seq): Reveals cell-type specific expression patterns and co-expression networks involving ACO1.

• Single-cell proteomics: Determines actual protein levels and post-translational modifications of ACO1 in individual cells.

• Single-cell metabolomics: Measures metabolites affected by ACO1's enzymatic activity (citrate, isocitrate) at cellular resolution.

• Integrated analysis: Combining these datasets provides a comprehensive view of ACO1's functional state across cell types.

Research applications:

• Heterogeneity mapping: In lung tissue, for example, scRNA-seq can identify which specific epithelial subtypes express ACO1 during fibrosis or aging.

• Functional state inference: Correlation of ACO1 expression with iron transport genes versus metabolic genes can indicate its predominant function (IRP1 vs. aconitase) in each cell type.

• Trajectory analysis: Pseudotime ordering of cells can reveal how ACO1 expression changes during cellular differentiation or disease progression.

Advanced analytical strategies:

• Regulatory network inference: Identify transcription factors controlling ACO1 expression in specific cell states.

• Protein-protein interaction mapping: Mass cytometry approaches can reveal ACO1's interaction partners in different cellular contexts.

• Spatial transcriptomics: Combining single-cell approaches with spatial resolution can map ACO1 expression to specific tissue microenvironments.

These approaches could resolve contradictory findings between human and mouse models of lung fibrosis, where ACO1 shows different cell-type associations, by providing higher resolution characterization of expressing cell populations .

What are the emerging roles of ACO1 in cellular stress responses beyond iron regulation?

Beyond its established role in iron metabolism, ACO1 participates in broader stress response networks:

Oxidative stress responses:

• Sensor function: The Fe-S cluster in ACO1 is highly sensitive to oxidation, making it an intrinsic sensor for oxidative stress.

• Metabolic adaptation: Inactivation of aconitase function during oxidative stress may redirect metabolic flux away from the TCA cycle toward alternative pathways.

• Signaling integration: ACO1 likely connects iron metabolism with oxidative stress responses, as both processes affect its functional state.

Hypoxic adaptation:

• Cross-regulation with HIF pathway: ACO1's citrate/isocitrate interconversion affects α-ketoglutarate levels, potentially influencing HIF-prolyl hydroxylase activity.

• Metabolic reprogramming: Under hypoxia, shifts in ACO1 function may contribute to the metabolic switch from oxidative phosphorylation to glycolysis.

Inflammatory responses:

• Iron sequestration: During inflammation, ACO1's IRP1 function may contribute to iron sequestration as part of nutritional immunity.

• Metabolic immunoregulation: ACO1-mediated changes in citrate metabolism may affect inflammatory cell function, as citrate is a key precursor for prostaglandin and nitric oxide synthesis.

Emerging research directions:

• Protein interactome studies: Identifying stress-specific interaction partners of ACO1.

• Post-translational modification mapping: Characterizing how stress-induced modifications alter ACO1 function.

• Subcellular localization dynamics: Investigating potential stress-induced relocalization of ACO1.

Researchers investigating these emerging roles should employ stress-specific cellular models (oxidative stress, hypoxia, inflammation) combined with proteomics, metabolomics, and live-cell imaging approaches to characterize ACO1's dynamic functions .

How might targeted modulation of ACO1's dual functions be leveraged for therapeutic interventions?

Selectively manipulating ACO1's distinct functions presents novel therapeutic opportunities:

Potential therapeutic targets:

• Fe-S cluster assembly/disassembly: Compounds that stabilize or destabilize the Fe-S cluster could selectively enhance either the aconitase or IRP1 function.

• Protein-RNA interaction surface: Small molecules that interfere with IRE binding could modulate iron metabolism without affecting enzymatic activity.

• Allosteric regulation: Compounds binding to allosteric sites could alter the equilibrium between the two functional states.

• Expression regulation: Tissue-specific induction or suppression of ACO1 expression.

Therapeutic applications:

• Iron overload disorders: Enhancing IRP1 function could reduce iron absorption and storage, beneficial in hemochromatosis.

• Iron deficiency anemia: Suppressing IRP1 function might increase iron availability for erythropoiesis.

• Fibrotic diseases: Based on findings in lung fibrosis models, modulating ACO1 in specific epithelial populations might enhance regenerative capacity .

• Metabolic reprogramming: Targeting ACO1's aconitase function could alter citrate metabolism, potentially applicable in cancer or inflammatory conditions.

Delivery and targeting considerations:

• Tissue-specific delivery: Nanoparticle formulations or targeted vectors for tissue-specific delivery of ACO1 modulators.

• Temporal control: Inducible expression systems for controlled modulation of ACO1 levels.

• Functional selectivity: Designing compounds that affect only one function while preserving the other.

Drug development approaches:

• Structure-based design: Using crystal structures of ACO1 in both functional states to design selective modulators.

• High-throughput screening: Functional assays measuring both activities to identify dual-function modulators.

• PROTAC technology: Targeted protein degradation approaches for temporary, reversible ACO1 modulation.

These therapeutic strategies require thorough validation in disease-specific models, carefully assessing both immediate effects on iron metabolism and long-term metabolic consequences .