ISCU Human

What is the biochemical function of ISCU in human cells?

ISCU serves as a scaffold protein that plays a crucial role in the assembly of iron-sulfur (Fe-S) clusters, which are essential prosthetic groups for numerous proteins involved in electron transport, enzymatic catalysis, and cellular iron sensing. In humans, ISCU functions within the mitochondrial Fe-S cluster assembly machinery, working cooperatively with other proteins including the cysteine desulfurase complex (NIA)2, frataxin (FXN), and ferredoxin 2 (FDX2) .

The protein participates in a multi-step process involving:

• Acquisition of sulfur from cysteine via the cysteine desulfurase NFS1

• Coordination of iron atoms

• Assembly of the iron-sulfur cluster on the scaffold

• Transfer of the assembled cluster to recipient proteins

Methodologically, researchers investigate ISCU function through enzymatic assays measuring both cysteine desulfurase activity and Fe-S cluster assembly rates, often using different reductants like dithiothreitol (DTT) or the physiologically relevant reduced ferredoxin 2 (rdFDX2) .

What structural conformations does human ISCU adopt?

Human ISCU exhibits a remarkable conformational equilibrium that appears functionally significant. Based on nuclear magnetic resonance (NMR) studies, wild-type ISCU exists in two interconverting states:

• Structured state (S): Represents approximately 30% of molecules

• Dynamically disordered state (D): Represents approximately 70% of molecules

This conformational equilibrium is physiologically relevant, as evidenced by variants like ISCU(M108I) and ISCU(D39V) that populate only the structured state and display altered functional properties .

To study these conformational states, researchers typically employ:

• Two-dimensional 1H–15N TROSY-HSQC NMR experiments with 15N-labeled proteins

• Circular dichroism (CD) spectroscopy to assess secondary structure content

• Chemical shift perturbation analysis to identify structural changes upon mutation

How does aberrant ISCU splicing contribute to human disease?

Hereditary myopathy with lactic acidosis (HML) is caused by an intronic mutation in the ISCU gene that leads to incorrect splicing. The improperly spliced transcripts contain a 100 or 86 bp intron sequence that encodes a non-functional ISCU protein .

The methodological approach to studying ISCU-related myopathy typically involves:

• Analysis of tissue-specific splicing patterns using RT-PCR and qPCR

• Transgenic mouse models expressing human HML-mutated ISCU

• Comparison of transcript levels across different tissues and muscle fiber types

• Investigation of splicing factors (e.g., SRSF3) that regulate tissue-specific ISCU splicing

• Cell culture models with overexpression or knockdown of candidate splicing regulators

Research has demonstrated that skeletal muscle contains the highest levels of incorrectly spliced ISCU transcripts compared to other tissues, with slow-twitch muscles showing particularly elevated levels .

How do specific ISCU variants differ in their biochemical properties?

ISCU variants exhibit significant differences in structure, function, and protein-protein interactions. The table below summarizes key differences between wild-type ISCU and two well-studied variants:

The methodological approach to characterizing these differences includes:

• NMR titration experiments to study protein-protein interactions

• Enzymatic assays with different reductants to assess functional properties

• Protein stability analyses

• Backbone chemical shift assignments through 3D NMR experiments (HNCA, HNCO, HNCACB, CBCA(CO)NH)

Notably, the ISCU(M108I) variant has been shown to bypass the frataxin requirement in Fe-S cluster assembly when the physiological reductant rdFDX2 is used, suggesting potential alternative pathways for therapeutic intervention .

What methodological challenges exist when studying ISCU protein-protein interactions?

Investigating ISCU interactions with its protein partners presents several technical challenges:

• Complex formation dynamics: NMR titration experiments reveal that when ISCU(M108I) is incorporated into the [NIA–ISCU–FXN]2 complex, the addition of rdFDX2 causes displacement of FXN. This dynamic nature of protein interactions requires carefully designed experiments that can capture transient states .

• Signal assignment difficulties: Some residues, including those in the "99LPPVK103" loop of ISCU, are not observable in NMR experiments due to internal dynamics, creating "blind spots" in interaction mapping .

• Reductant-dependent interactions: The choice of reductant (DTT versus rdFDX2) significantly affects experimental outcomes, with physiological reductants sometimes yielding different results than commonly used laboratory reagents .

Methodological solutions include:

• Combined use of multiple biophysical techniques (NMR, CD, enzymatic assays)

• Careful experimental design with appropriate controls

• Isotope labeling strategies to distinguish signals from different proteins in complexes

• Chemical shift perturbation analysis using the equation: ΔδNH = √[(ΔδH)2 + (ΔδN/5)2]

What mechanisms explain the tissue-specific symptoms of ISCU-related myopathy?

The striking tissue specificity of hereditary myopathy with lactic acidosis (HML) appears related to differential splicing regulation across tissues. Research methodologies addressing this question include:

• Comparative tissue analysis: Studies confirm that skeletal muscle contains the highest levels of incorrectly spliced ISCU transcripts compared to heart, brain, liver, and kidney tissues .

• Muscle fiber type specificity: Within skeletal muscle, slow-twitch muscles (e.g., soleus) exhibit significantly higher levels of incorrectly spliced ISCU compared to gastrocnemius and quadriceps .

• Splicing factor identification: The splicing factor serine/arginine-rich splicing factor 3 (SRSF3) has been identified as a potential regulator of ISCU splicing, with higher expression in soleus muscle correlating with increased aberrant splicing .

• Experimental validation: Overexpression of SRSF3 in human myoblasts increases levels of incorrectly spliced ISCU, while knockdown results in decreased levels, confirming a regulatory role .

This research highlights the importance of considering tissue-specific regulatory mechanisms when studying diseases associated with aberrant splicing.

What are the recommended protocols for expressing and purifying human ISCU?

Research-grade human ISCU protein preparation requires specific methodological considerations:

• Expression system selection: E. coli BL21(DE3) or similar strains are commonly used for recombinant expression.

• Isotope labeling strategy: For NMR studies, growth in minimal media with 15N-ammonium chloride and/or 13C-glucose enables production of labeled protein. This is essential for structural studies and investigating protein-protein interactions .

• Purification approach: A multi-step protocol typically involves:

  • Affinity chromatography (e.g., His-tag purification)

  • Ion exchange chromatography

  • Size exclusion chromatography

• Buffer optimization: Buffers containing 20 mM HEPES (pH 7.6), 150 mM NaCl, and reducing agents like 2 mM TCEP have been successfully used for maintaining ISCU stability during NMR studies .

• Mutagenesis methods: Site-directed mutagenesis techniques such as Polymerase Incomplete Primer Extension (PIPE) are effective for generating variants like M108I and D39V .

• Quality control measures: Verification of purity by SDS-PAGE and confirmation of proper folding by CD spectroscopy or NMR are essential before proceeding with functional studies.

How can researchers effectively study the conformational dynamics of ISCU?

ISCU's conformational equilibrium between structured and disordered states presents both challenges and opportunities for structural biology. Advanced methodological approaches include:

• NMR spectroscopy techniques:

  • Two-dimensional (2D) 1H–15N HSQC or TROSY-HSQC experiments

  • Three-dimensional (3D) experiments (HNCA, HNCO, HNCACB, CBCA(CO)NH)

  • Non-uniform sampling (NUS) with processing using algorithms like NESTA for efficient data collection

• Chemical shift analysis:

  • Backbone assignments enable mapping of structural changes

  • Chemical shift perturbation analysis helps identify interaction interfaces

  • Comparison of shift differences between variants provides structural insights

• Complementary techniques:

  • Circular dichroism spectroscopy for secondary structure assessment

  • Hydrogen-deuterium exchange mass spectrometry for dynamics

  • Molecular dynamics simulations to model conformational transitions

Research shows that residues exhibiting the largest chemical shift differences between ISCU variants (Δδ NH > 1 ppm) include G38, V40, K42, Q44, I45, F58, K59, G62, C63, A66, I67, and C106–A110, many of which map to the ISCU–NFS1 interface in the complex structure .

What strategies can address data contradictions in ISCU research?

Researchers may encounter contradictory results when studying ISCU, necessitating careful methodological approaches:

How can structural and functional data on ISCU inform therapeutic development?

Translating basic ISCU research into therapeutic strategies requires integration of structural, biochemical, and cellular approaches:

• Splicing modulation strategies: Given that HML results from aberrant splicing, approaches to correct splicing could be therapeutic. This may involve targeting splicing factors like SRSF3 that regulate ISCU splicing .

• Structure-guided drug design: Detailed structural information, particularly at protein-protein interfaces, can guide the development of small molecules that modulate ISCU function or protein interactions.

• Variant-inspired bypass pathways: The observation that ISCU(M108I) can bypass the frataxin requirement suggests potential alternative pathways that could be therapeutically exploited .

• Methodological considerations:

  • Target validation through multiple experimental approaches

  • Development of cellular and animal models that recapitulate tissue-specific effects

  • High-throughput screening assays that reflect physiologically relevant interactions

  • Interdisciplinary integration of structural biology, biochemistry, and genetics

• Regulatory considerations: Researchers should be aware of Title IX requirements combined with New York Human Rights Law and New York Education Law 129-B when developing human subject research protocols .

What novel technologies are advancing ISCU research methodologies?

Several cutting-edge approaches are enhancing our ability to study ISCU:

• Advanced NMR techniques:

  • Non-uniform sampling approaches that significantly reduce experiment time

  • NESTA and NMRPipe processing methods for improved spectral quality

  • Automated assignment tools like the PINE server for efficient data analysis

• Integrative structural biology:

  • Combining NMR data with cryo-electron microscopy

  • Computational modeling and molecular dynamics simulations

  • Mass spectrometry-based structural approaches

• Gene editing technologies:

  • CRISPR-Cas9 for generating cellular models with specific ISCU mutations

  • Base editing for precise correction of disease-causing mutations

• Single-cell technologies:

  • Analysis of splicing patterns at the single-cell level

  • Investigation of cell-to-cell variation in ISCU expression and function

• Academic planning tools:

  • Schedule Builder and Degree Works systems to organize research time efficiently

  • Progress Survey tools to track experimental outcomes and identify challenges early