Recombinant Chicken Iron-sulfur cluster assembly 1 homolog, mitochondrial (ISCA1)

What is ISCA1 and what is its primary function in cellular metabolism?

ISCA1 (Iron-sulfur cluster assembly 1 homolog) is a member of the LYR family of proteins that contains a conserved tripeptide 'LYR' motif near the N-terminus. Its primary function is essential for the assembly of [4Fe-4S] clusters in key metabolic and respiratory enzymes. ISCA1 plays a crucial role in mitochondrial iron-sulfur (Fe-S) protein biogenesis, which is vital for cellular respiration and metabolic processes . The protein forms a stable complex in vivo with cysteine desulfurase (ISCS), which generates the inorganic sulfur required for Fe-S protein biogenesis . Defects in ISCA1 can severely impair both mitochondrial and cytosolic iron homeostasis, affecting multiple cellular functions dependent on iron-sulfur cluster-containing proteins .

Experimental evidence demonstrates that suppression of ISCA1 leads to inactivation of mitochondrial and cytosolic aconitases, activation of iron-responsive element-binding activity of iron regulatory protein 1 (IRP1), increased levels of iron regulatory protein 2 (IRP2), and abnormal punctate ferric iron accumulations in cells . These findings collectively indicate that ISCA1 is a critical component in the biogenesis of Fe-S clusters and the maintenance of cellular iron homeostasis.

What is the subcellular localization pattern of ISCA1?

ISCA1 demonstrates a dual localization pattern in mammalian cells. While it predominantly localizes to the mitochondrial compartment, as expected for a protein involved in mitochondrial Fe-S cluster assembly, it has also been detected in the nucleus of mammalian cells . This dual localization pattern is similar to that observed for ISCS, with which ISCA1 forms a stable complex .

The presence of ISCA1 in both mitochondria and nucleus suggests it may play roles in Fe-S cluster assembly in both compartments. The mitochondrial localization is consistent with its role in the biogenesis of mitochondrial [4Fe-4S] proteins, while its nuclear presence might indicate involvement in the assembly or repair of nuclear Fe-S proteins. The mitochondrial import of ISCA1 is facilitated by an uncleaved presequence, and mutations in this region (such as the p.V10G mutation) can severely affect both the mitochondrial import and stability of the protein .

How does ISCA1 deficiency affect cellular iron homeostasis?

ISCA1 deficiency disrupts both Fe-S cluster biogenesis and iron-sensing and regulation in human cells. The mechanism involves several key components of cellular iron homeostasis:

• Aconitase Activity: Suppression of ISCA1 inactivates both mitochondrial and cytosolic aconitases, which are Fe-S cluster-containing enzymes essential for the TCA cycle and cellular metabolism .

• Iron Regulatory Proteins (IRPs): ISCA1 depletion increases the iron-responsive element (IRE)-binding activity of both IRP1 and IRP2. Additionally, IRP2 protein levels increase approximately 2.5-fold in ISCA1 knock-down cells, suggesting cytosolic iron deficiency .

• Ferritin and Transferrin Receptor (TfR): Decreased ferritin protein levels and increased TfR protein levels are observed in ISCA1-depleted cells, consistent with translational repression of ferritin and stabilization of TfR mRNA by increased IRP binding .

• Iron Distribution: A characteristic pattern of iron accumulation emerges, with iron accumulating in mitochondria while the cytosol becomes functionally iron-depleted. This pattern of "mitochondrial iron overload with cytosolic iron depletion" is typical of defects in Fe-S cluster biogenesis .

This complex pattern of dysregulation demonstrates that ISCA1 is essential for maintaining proper iron distribution between mitochondrial and cytosolic compartments, and its absence leads to a cascade of effects that disrupt cellular iron homeostasis.

What are the pathological consequences of ISCA1 mutations?

Mutations in ISCA1 can lead to severe clinical manifestations, particularly affecting high-energy demanding tissues such as the nervous system. ISCA1 mutations have been associated with Multiple Mitochondrial Dysfunction Syndrome (MMDS), a group of severe autosomal recessive diseases characterized by:

• Infantile-onset mitochondrial encephalopathy: Patients typically present with severe early-onset leukodystrophy .

• Respiratory chain defects: Multiple defects in respiratory chain complexes (particularly complexes I, II, and IV) are observed, leading to impaired cellular respiration and energy production .

• Lipoic acid metabolism impairment: Severe defects in lipoic acid synthesis affect pyruvate dehydrogenase and α-ketoglutarate dehydrogenase complexes, further compromising cellular metabolism .

• Clinical features: Non-ketotic hyperglycinemia, myopathy, lactic acidosis, and often early death characterize the clinical picture .

For example, a homozygous missense mutation in ISCA1 (c.29T>G; p.V10G) identified in a patient resulted in a dramatic reduction of ISCA1 protein levels. This mutation, located in the uncleaved presequence, severely affected both mitochondrial import and stability of ISCA1, leading to impaired biogenesis of mitochondrial [4Fe-4S] proteins and the clinical manifestations described above .

What is the role of ISCA1 in cancer biology?

Recent research has identified ISCA1 as a potential prognostic marker for various cancers, particularly bladder cancer (BLCA). Several key findings highlight its role in cancer biology:

• Immune correlation: ISCA1 gene expression is positively related to four immune signatures (chemokine, immunostimulator, MHC, and receptor) in BLCA .

• Prognostic significance: High levels of ISCA1 expression are associated with poorer prognosis in BLCA patients, suggesting that ISCA1 is a risk factor in this cancer type .

• Immune checkpoint correlation: ISCA1 expression is positively linked with various immune checkpoints, including CTLA4, PDCD1, CD86, and CD274 in BLCA .

• Immune cell correlation: There is a significant positive correlation between ISCA1 expression and 20 different immune cell scores in BLCA, indicating a potential role in tumor immunology .

These findings suggest that ISCA1 could serve as both a prognostic biomarker and potentially a therapeutic target in cancer treatment, particularly in BLCA. The strong association with immune markers also points to a possible role in modulating tumor immune responses, although the exact mechanisms require further investigation.

What experimental approaches are optimal for studying ISCA1 function in vitro?

For comprehensive investigation of ISCA1 function in vitro, researchers should consider a multi-faceted experimental approach:

• Recombinant Protein Expression and Purification:

  • Express recombinant ISCA1 in E. coli using pET-based vectors with appropriate tags (His-tag, GST) for purification

  • Optimize expression conditions (temperature, IPTG concentration, induction time) to maintain proper folding

  • Purify under anaerobic conditions to preserve integrity of iron-sulfur clusters

  • Verify protein quality using SDS-PAGE, western blotting, and mass spectrometry

• Protein-Protein Interaction Studies:

  • Co-immunoprecipitation to identify ISCA1 binding partners

  • Pull-down assays using tagged ISCA1 to confirm direct interactions

  • Bioluminescence resonance energy transfer (BRET) or fluorescence resonance energy transfer (FRET) for studying interactions in living cells

  • Cross-linking coupled with mass spectrometry to map interaction surfaces

• Enzymatic Assays for Fe-S Cluster Assembly:

  • UV-visible spectroscopy to monitor Fe-S cluster formation

  • Circular dichroism (CD) spectroscopy to assess secondary structure and Fe-S cluster binding

  • Electron paramagnetic resonance (EPR) spectroscopy to characterize Fe-S cluster properties

  • Activity assays measuring reconstitution of Fe-S proteins (e.g., aconitase)

• Cell-Free Fe-S Cluster Assembly Systems:

  • Reconstitute Fe-S cluster assembly using purified components (ISCA1, ISCS, ferrous iron, reducing agents)

  • Monitor kinetics of cluster formation using spectroscopic techniques

  • Assess the impact of mutations on assembly efficiency

These methods provide complementary approaches to dissect ISCA1 function, from structural characteristics to protein interactions and enzymatic activities in the Fe-S cluster assembly pathway.

How can RNA interference techniques be optimized to study ISCA1 function in cellular models?

RNA interference (RNAi) techniques provide powerful tools for studying ISCA1 function in cellular models. Based on the studies described in the search results, the following optimization strategies can be implemented:

• siRNA Design and Delivery:

  • Design multiple siRNA sequences targeting different regions of ISCA1 mRNA to ensure specificity

  • Include appropriate negative controls (scrambled siRNA) and positive controls (siRNA targeting known genes)

  • Optimize transfection conditions based on cell type (lipofection for HeLa cells, electroporation for hard-to-transfect cells)

  • Consider stable knockdown using shRNA for long-term studies

• Validation of Knockdown Efficiency:

  • Western blotting to quantify ISCA1 protein reduction (aim for >80% reduction as achieved in the referenced studies)

  • qRT-PCR to confirm reduction at the mRNA level

  • Monitor expression of other Fe-S cluster assembly components (IBA57, NFU1, IND1, MIA40) to ensure specificity

• Rescue Experiments:

  • Design RNAi-resistant versions of ISCA1 (using synonymous mutations) for complementation studies

  • Express wild-type and mutant versions (e.g., ISCA1 V10G) to assess functional consequences

  • Quantify the degree of phenotypic rescue to determine relative function of mutant proteins

• Phenotypic Assays Following Knockdown:

  • Cell proliferation assays to assess growth defects

  • Respiratory chain complex activities (RCCI, RCCII, RCCIV)

  • Steady-state levels of Fe-S proteins by western blotting

  • Medium acidification as an indicator of respiratory defects

  • Iron regulatory protein (IRP) binding assays using electrophoretic mobility shift assays

This systematic approach to RNAi studies, combined with comprehensive phenotypic analyses, allows for detailed characterization of ISCA1 function and the consequences of its deficiency in cellular models.

What are the structural and functional differences between human and chicken ISCA1 homologs?

While the search results don't provide specific information about chicken ISCA1, I can outline an approach for comparative analysis of human and chicken ISCA1 homologs:

Structural Comparisons:

• Sequence Analysis:

  • Perform sequence alignment to identify conserved domains, particularly the LYR motif near the N-terminus

  • Compare mitochondrial targeting sequences and their predicted cleavage sites

  • Identify species-specific sequence variations that might affect function

• Structural Prediction and Modeling:

  • Generate homology models based on available crystal structures

  • Compare predicted secondary and tertiary structures

  • Analyze surface electrostatics and potential interaction interfaces

Functional Comparisons:

• Expression Patterns:

  • Compare tissue-specific expression profiles in human and chicken tissues

  • Analyze developmental expression patterns

• Protein Interactions:

  • Identify conserved and species-specific interaction partners

  • Compare binding affinities with key partners (e.g., ISCS homologs)

• Complementation Studies:

  • Test whether chicken ISCA1 can complement human ISCA1 deficiency in cellular models

  • Assess whether human ISCA1 can rescue phenotypes in chicken cell models

• Biochemical Properties:

  • Compare Fe-S cluster binding properties

  • Assess stability and activity under various conditions (pH, temperature, oxidative stress)

This comparative approach would provide valuable insights into conserved functions and species-specific adaptations of ISCA1, potentially revealing evolutionary aspects of Fe-S cluster assembly mechanisms.

How does the ISCA1 p.V10G mutation affect protein function at the molecular level?

The ISCA1 p.V10G mutation, identified in a patient with infantile-onset leukodystrophy, has profound effects on protein function at the molecular level. The search results provide detailed insights into these effects:

These molecular defects collectively explain the severe clinical phenotype observed in patients with the ISCA1 p.V10G mutation, demonstrating how a single amino acid change in the mitochondrial targeting sequence can have cascading effects on protein function and cellular metabolism.

What methodologies are most effective for monitoring Fe-S cluster assembly in ISCA1-dependent pathways?

Monitoring Fe-S cluster assembly in ISCA1-dependent pathways requires a combination of biochemical, spectroscopic, and cellular approaches:

• Activity Assays of Fe-S Enzymes:

  • Aconitase activity assays: Both mitochondrial and cytosolic aconitases are sensitive indicators of [4Fe-4S] cluster assembly

  • Respiratory chain complex activities: Measuring activities of RCCI and RCCII, which contain multiple Fe-S clusters

  • Lipoic acid-dependent enzyme activities: Pyruvate dehydrogenase and α-ketoglutarate dehydrogenase as indicators of Fe-S cluster-dependent lipoic acid synthesis

• Protein-Based Indicators:

  • Western blotting of Fe-S proteins to monitor steady-state levels (SDHB, NDUFS1, etc.)

  • Iron regulatory protein (IRP) binding assays using [α-32P]CTP-labeled IRE probes and electrophoretic mobility shift assays

  • Quantification of IRP2 protein levels as an indicator of cytosolic iron status

• Iron Homeostasis Parameters:

  • Ferritin and transferrin receptor protein levels as indicators of cellular iron regulation

  • Iron staining techniques to visualize subcellular iron distribution and identify abnormal accumulations

  • Quantification of labile iron pool using fluorescent probes

• Spectroscopic Methods:

  • UV-visible spectroscopy to monitor characteristic absorbance of different types of Fe-S clusters

  • Electron paramagnetic resonance (EPR) spectroscopy for detailed characterization of Fe-S cluster types

  • Mössbauer spectroscopy to analyze iron states in different cellular compartments

• Real-time Monitoring Approaches:

  • Fluorescent protein fusions to track ISCA1 localization and dynamics

  • FRET-based sensors for monitoring Fe-S cluster transfer between proteins

  • Live-cell imaging of Fe-S protein assembly using specifically designed probes

These complementary approaches provide a comprehensive toolkit for monitoring Fe-S cluster assembly in ISCA1-dependent pathways, allowing researchers to detect defects at multiple levels from protein stability to enzymatic activity and iron homeostasis.

How does ISCA1 interact with the cellular iron-sensing machinery?

ISCA1 plays a critical role in cellular iron homeostasis through its interactions with the iron-sensing machinery. The search results reveal several key mechanisms:

• Iron Regulatory Protein 1 (IRP1) Regulation:

  • IRP1 functions as cytosolic aconitase when it contains an intact [4Fe-4S] cluster

  • ISCA1 is essential for assembly of this cluster; ISCA1 deficiency leads to loss of the cluster

  • When the Fe-S cluster is lost, IRP1 converts to its RNA-binding form

  • This conversion allows IRP1 to bind iron-responsive elements (IREs) in target mRNAs

• Iron Regulatory Protein 2 (IRP2) Regulation:

  • ISCA1 deficiency leads to approximately 2.5-fold increase in IRP2 protein levels

  • This increase is statistically significant and consistent with cytosolic iron deficiency

  • IRP2 stabilization occurs because the iron-dependent degradation of IRP2 is impaired in iron-deficient conditions

• IRE-Binding Activity Modulation:

  • Extracts from ISCA1-depleted cells show increased IRE-binding activity of both IRP1 and IRP2

  • This is demonstrated using electrophoretic mobility shift assays with labeled IRE probes

  • The increased binding activity affects translation of IRE-containing mRNAs

• Downstream Effects on Iron-Regulated Proteins:

  • Ferritin: Translation is repressed due to increased IRP binding to 5'-UTR IREs

  • Transferrin receptor (TfR): mRNA is stabilized and translation is promoted due to IRP binding to 3'-UTR IREs

  • These changes alter cellular iron uptake and storage patterns

These interactions create a feedback loop where ISCA1 deficiency leads to impaired Fe-S cluster assembly, which then activates the iron-sensing machinery, resulting in a cellular iron starvation response despite potential mitochondrial iron overload.

What experimental approaches can be used to investigate ISCA1's role in mitochondrial iron accumulation?

Investigating ISCA1's role in mitochondrial iron accumulation requires a multi-faceted approach combining imaging, biochemical, and genetic techniques:

• Iron Visualization Techniques:

  • Perls' Prussian blue staining with DAB enhancement to visualize ferric iron accumulations in cells

  • Fluorescent iron probes (e.g., rhodamine B-[(1,10-phenanthrolin-5-yl)aminocarbonyl]benzyl ester) for live-cell imaging

  • Transmission electron microscopy with electron-dense iron staining

  • Synchrotron X-ray fluorescence microscopy for high-resolution mapping of elemental iron

• Subcellular Fractionation and Iron Quantification:

  • Isolation of pure mitochondrial fractions using differential centrifugation

  • Atomic absorption spectroscopy or inductively coupled plasma mass spectrometry (ICP-MS) to quantify iron content in mitochondrial versus cytosolic fractions

  • Ferrozine-based colorimetric assays for iron quantification

• Mitochondrial Iron Import/Export Studies:

  • Radio-labeled iron uptake assays in isolated mitochondria

  • Analysis of mitochondrial iron transporters (mitoferrin-1/2) expression and activity

  • Assessment of iron export mechanisms and their regulation

• Genetic Manipulation Approaches:

  • ISCA1 knockdown using RNA interference to induce the phenotype

  • Rescue experiments with wild-type and mutant ISCA1 to assess reversibility

  • Generation of cell lines with fluorescently tagged mitochondria and iron sensors

• Integration with Iron Homeostasis Parameters:

  • Measurement of labile iron pool in different cellular compartments

  • Assessment of iron-sulfur protein activities as indicators of functional iron utilization

  • Analysis of iron-responsive gene expression patterns

These methods allow for comprehensive characterization of mitochondrial iron accumulation in ISCA1-deficient cells, providing insights into both the mechanisms and consequences of altered iron distribution in mitochondrial disorders.

How can ISCA1 mutations be effectively screened in patients with suspected mitochondrial disorders?

Effective screening for ISCA1 mutations in patients with suspected mitochondrial disorders requires a comprehensive diagnostic approach:

• Clinical Indicators for ISCA1 Testing:

  • Early-onset leukodystrophy

  • Multiple respiratory chain complex deficiencies

  • Impaired lipoic acid metabolism

  • Infantile-onset encephalopathy

  • Non-ketotic hyperglycinemia

  • Lactic acidosis

• Genetic Testing Approaches:

  • Targeted Sequencing: MitoExome sequencing focusing on mitochondrial and nuclear genes involved in mitochondrial function

  • Whole Exome Sequencing (WES): For comprehensive coverage of all coding regions

  • Whole Genome Sequencing (WGS): To identify non-coding and regulatory mutations

  • Mitochondrial DNA analysis to rule out mtDNA-based disorders

• Biochemical Screening:

  • Activity assays of respiratory chain complexes (particularly RCCI, RCCII, and RCCIV)

  • Lipoylation status of proteins using western blot analysis with anti-lipoic acid antibodies

  • Aconitase activity assays (mitochondrial and cytosolic)

  • Lactate/pyruvate ratio in blood and cerebrospinal fluid

• Protein-Level Analysis:

  • Western blotting to assess ISCA1 protein levels

  • Analysis of other Fe-S assembly components to identify related defects

  • Immunofluorescence studies to assess ISCA1 localization

• Functional Validation of Variants:

  • Complementation studies in patient fibroblasts or ISCA1-depleted cell lines

  • Assessment of mitochondrial import using fluorescently tagged ISCA1 variants

  • Protein stability analyses of mutant ISCA1 proteins

This multi-level screening approach allows for efficient identification of ISCA1 mutations and their functional classification, facilitating accurate diagnosis and potential therapeutic interventions for patients with ISCA1-related mitochondrial disorders.

What is the potential of ISCA1 as a prognostic marker in cancer?

ISCA1 shows significant promise as a prognostic marker in cancer, particularly in bladder cancer (BLCA). The search results provide several lines of evidence supporting this potential:

• Survival Correlation:

  • Kaplan-Meier curve analysis shows that patients with high ISCA1 expression have worse survival outcomes compared to those with low ISCA1 expression in BLCA

  • This correlation indicates that ISCA1 expression levels could serve as a risk stratification marker

• Immune Correlation Profiles:

  • ISCA1 gene expression is positively related to four key immune signatures (chemokine, immunostimulator, MHC, and receptor) in BLCA

  • There is a significant positive correlation between ISCA1 expression and multiple tumor-related immune cell populations

  • These correlations suggest ISCA1 may play a role in modulating tumor immune responses

• Immune Checkpoint Association:

  • ISCA1 shows substantial positive correlations with important immune checkpoint molecules:

    • CTLA4 (Cytotoxic T-Lymphocyte Associated Protein 4)

    • PDCD1 (Programmed Cell Death 1)

    • CD86 (B7-2)

    • CD274 (PD-L1)

  • This association with immune checkpoints suggests potential relevance for immunotherapy response prediction

• Prognostic Model Development:

  • An immune risk scoring model incorporating ISCA1 showed better performance compared to the Tumor Immune Dysfunction and Exclusion (TIDE) model

  • This indicates that ISCA1-based models could improve current approaches to cancer prognosis

• Genetic and Epigenetic Correlations:

  • Analysis of SNV (Single Nucleotide Variation), CNV (Copy Number Variation), and methylation patterns in relation to ISCA1 expression provides additional layers for prognostic assessment

These findings collectively suggest that ISCA1 could serve as a valuable prognostic marker for cancer, particularly BLCA. Its integration into multi-parameter prognostic models could enhance risk stratification and potentially guide treatment decisions, especially regarding immunotherapy approaches.

How can advanced imaging techniques be optimized to study ISCA1-dependent iron distribution in cells?

Advanced imaging techniques offer powerful tools for studying ISCA1-dependent iron distribution in cells. Based on the search results and current scientific approaches, the following optimization strategies can be implemented:

• Enhanced Perls' Prussian Blue Staining:

  • Optimize DAB enhancement protocols for maximum sensitivity

  • Combine with fluorescent counterstains for subcellular compartments

  • Use digital image analysis for quantitative assessment of staining intensity

  • Apply spectral unmixing to distinguish true iron staining from artifacts

• Fluorescent Probes for Live-Cell Imaging:

  • Select iron-specific fluorescent probes (e.g., Phen Green SK, RhoNox-1)

  • Combine with mitochondria-specific dyes (e.g., MitoTracker) for colocalization studies

  • Implement ratiometric imaging approaches to account for probe concentration variations

  • Use fluorescence lifetime imaging microscopy (FLIM) to detect probe-iron interactions

• Super-Resolution Microscopy Approaches:

  • Apply structured illumination microscopy (SIM) for improved resolution of iron deposits

  • Use stimulated emission depletion (STED) microscopy for nanoscale visualization

  • Implement single-molecule localization microscopy (PALM/STORM) for precise mapping

  • Combine with expansion microscopy for physical enlargement of subcellular structures

• Correlative Light and Electron Microscopy (CLEM):

  • Identify regions of interest by fluorescence microscopy

  • Apply electron microscopy to the same regions for ultrastructural details

  • Use energy-dispersive X-ray spectroscopy (EDX) or electron energy loss spectroscopy (EELS) for elemental analysis

  • Implement cryo-preparation techniques to preserve native state

• Advanced Analytical Techniques:

  • X-ray fluorescence microscopy for quantitative elemental mapping

  • Secondary ion mass spectrometry (SIMS) imaging for isotope-specific detection

  • Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) for trace element analysis

  • Synchrotron radiation X-ray absorption spectroscopy for iron oxidation state determination

Optimizing these imaging approaches provides complementary views of iron distribution at different resolution scales, allowing for comprehensive characterization of how ISCA1 deficiency affects cellular iron homeostasis and compartmentalization.

What are the most promising therapeutic approaches for ISCA1-deficiency disorders?

Although the search results don't directly discuss therapeutic approaches for ISCA1-deficiency disorders, we can propose promising avenues based on our understanding of the pathophysiology:

These therapeutic strategies would need to be evaluated in appropriate model systems before clinical translation. The severe, early-onset nature of ISCA1 deficiency disorders suggests that early intervention, possibly even prenatally or neonatally, might be necessary for optimal outcomes.

How might high-throughput screening approaches be used to identify modulators of ISCA1 function?

High-throughput screening (HTS) approaches offer powerful tools for identifying modulators of ISCA1 function that could have both research and therapeutic applications:

• Cell-Based Screening Platforms:

  • Reporter Systems:

    • Generate cell lines with iron-responsive element (IRE)-controlled fluorescent reporters

    • Develop ISCA1-dependent enzyme activity reporters (e.g., aconitase-linked reporters)

    • Create transcriptional reporters for Fe-S cluster-dependent transcription factors

  • Phenotypic Screens:

    • Viability assays in ISCA1-deficient cells to identify rescue compounds

    • High-content imaging of mitochondrial function (membrane potential, morphology)

    • Multiplex assays for respiratory chain complex activities

• Biochemical Screening Approaches:

  • Direct ISCA1 Activity Assays:

    • Develop fluorescence-based Fe-S cluster transfer assays

    • Create high-throughput adaptations of Fe-S enzyme reconstitution assays

    • Implement thermal shift assays to identify ISCA1 stabilizers

  • Protein Interaction Screens:

    • AlphaScreen or FRET-based assays for ISCA1-ISCS interactions

    • Split-luciferase complementation assays for protein complex formation

    • Protein microarrays to identify novel ISCA1 interactors

• Genetic Screening Strategies:

  • CRISPR Screens:

    • Genome-wide CRISPR activation (CRISPRa) screens to identify enhancers of ISCA1 function

    • CRISPR interference (CRISPRi) screens to find synthetic lethal interactions

    • Focused CRISPR screens targeting iron metabolism genes

  • RNA Interference Libraries:

    • siRNA/shRNA screens targeting the mitochondrial proteome

    • Synthetic genetic array approaches in model organisms

• In Silico Screening Approaches:

  • Virtual screening of compound libraries against ISCA1 structural models

  • Molecular dynamics simulations to identify druggable pockets

  • Systems biology approaches to identify network-based intervention points

• Validation and Secondary Screening:

  • Dose-response confirmation in multiple cell types

  • Mechanistic validation using biochemical and cellular assays

  • Testing in patient-derived cells and animal models of ISCA1 deficiency

These HTS approaches would facilitate the identification of chemical or genetic modulators of ISCA1 function, potentially leading to research tools and therapeutic leads for ISCA1-related disorders.