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Expression and characterization of the ferritin binding domain of Nuclear Receptor Coactivator-4 (NCOA4)

Magdalena Gryzik1, Ayush Srivastava1, Giovanna Longhi2

  • 1Molecular Biology Laboratory, Department of Molecular and Translational Medicine (DMMT), University of Brescia, Viale Europa 11, Brescia, Italy.

Biochimica Et Biophysica Acta. General Subjects
|July 30, 2017
PubMed

Insights

Nuclear Receptor Coactivator-4 (NCOA4) binds H-ferritin with high specificity and affinity, forming stable complexes. This interaction is crucial for understanding ferritinophagy and cellular iron regulation.

Area of Science:

  • Cellular Biology
  • Biochemistry
  • Molecular Mechanisms

Background:

  • Ferritinophagy regulates cellular iron homeostasis through autophagic degradation of ferritin.
  • Nuclear Receptor Coactivator-4 (NCOA4) acts as a selective cargo receptor, mediating ferritin recognition and targeting for degradation.
  • Understanding the NCOA4-ferritin interaction is key to elucidating ferritinophagy mechanisms.

Purpose of the Study:

  • To biochemically characterize the interaction between the ferritin-binding domain of NCOA4 and H-ferritin.
  • To determine the specificity and affinity of the NCOA4 fragment for different ferritin forms.
  • To investigate the influence of metal ions on the NCOA4-ferritin complex formation.

Main Methods:

  • Cloning, expression, and purification of the NCOA4(383-522) fragment in E. coli.
  • Circular Dichroism (CD) spectroscopy to assess secondary structure.
  • Electrophoretic mobility shift assays (EMSA) and ELISA to study ferritin binding.
  • Biochemical analysis of metal ion effects on complex formation.

Main Results:

  • The NCOA4(383-522) fragment was expressed as a soluble, dimeric form with low secondary structure.
  • NCOA4 fragment demonstrated high-affinity (nM range) and specific binding to H-ferritin, but not to L-ferritin or a specific H-ferritin mutant.
  • H-ferritin bound up to 24 NCOA4 fragments, forming stable, insoluble complexes.
  • Fe(II) partially inhibited the binding among tested divalent metal ions.

Conclusions:

  • The NCOA4 ferritin-binding domain exhibits high specificity for H-ferritin.
  • The formation of stable H-ferritin-NCOA4 complexes is iron-dependent and crucial for ferritinophagy.
  • These findings provide insights into the molecular basis of ferritinophagy and iron regulation.
Keywords:
FerritinFerritinophagyIron metabolismNCOA4

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