Abstract
Nucleotide insertions in the ferritin light chain (FTL) polypeptide gene cause hereditary ferritinopathy, a neurodegenerative disease characterized by abnormal accumulation of ferritin and iron in the central nervous system. Here we describe for the first time the protein structure and iron storage function of the FTL mutant p.Phe167SerfsX26 (MT-FTL), which has a C terminus altered in sequence and extended in length. MT-FTL polypeptides assembled spontaneously into soluble, spherical 24-mers that were ultrastructurally indistinguishable from those of the wild type. Far-UV CD showed a decrease in α-helical content, and 8-anilino-1-naphthalenesulfonate fluorescence revealed the appearance of hydrophobic binding sites. Near-UV CD and proteolysis studies suggested little or no structural alteration outside of the C-terminal region. In contrast to wild type, MT-FTL homopolymers precipitated at much lower iron loading, had a diminished capacity to incorporate iron, and were less thermostable. However, precipitation was significantly reversed by addition of iron chelators both in vitro and in vivo. Our results reveal substantial protein conformational changes localized at the 4-fold pore of MT-FTL homopolymers and imply that the C terminus of the MT-FTL polypeptide plays an important role in ferritin solubility, stability, and iron management. We propose that the protrusion of some portion of the C terminus above the spherical shell allows it to cross-link with other mutant polypeptides through iron bridging, leading to enhanced mutant precipitation by iron. Our data suggest that hereditary ferritinopathy pathogenesis is likely to result from a combination of reduction in iron storage function and enhanced toxicity associated with iron-induced ferritin aggregates.
Highlights
Iron is an essential element needed for vital processes such as neuronal development, myelination, synthesis, and catabolism of neurotransmitters and electron transport, as well as heme and iron-sulfur cluster synthesis [1]
The results shown here suggest that the C terminus of the MT-ferritin light chain (FTL) polypeptide plays an important role in the solubility and stability of ferritin, and in iron management, and provide insights into the molecular mechanisms involved in the generation of ferritin inclusion bodies and the accumulation of iron observed in patients with hereditary ferritinopathy (HF)
In this work we investigate the protein structure and iron storage function of ferritin homopolymers formed from a light chain variant p.Phe167SerfsX26 that causes HF [9]
Summary
Cloning and Expression of Ferritin Polypeptides—cDNAs containing the sequence of human WT-FTL and human mutant FTL498 – 499InsTC were introduced into the pET28a(ϩ) expression vector (Novagen, EMD Chemicals Inc.). Iron Loading of Apoferritins—Freshly prepared ferrous ammonium sulfate (0.5– 4.5 mM) in 10 mM HCl was added to MT- and WT-FTL apoferritin homopolymers (1 M) in 0.1 M Hepes buffer (pH 7.4) at room temperature [16]. Thermolysin Treatment of WT- and MT-FTL Apoferritin Homopolymers—Proteolysis of recombinant MT- and WTFTL homopolymers was initiated by adding to 10 g of ferritin a 10-fold concentrated stock solution (36.5 units/mg) of thermolysin (Fluka) in Hepes (0.1 M) (pH 7.0), 10 mM CaCl2 to a final concentration of 0.2 mg/ml. Characterization of Detergent-insoluble MT-FTL Ferritin from Astrocyte Cultures—Cerebral cortical astrocytes cultures were homogenized in lysis buffer (3 ml of 50 mM Tris-HCl (pH 7.4), 1% SDS, 30 units/ml benzonase, 2 mM MgCl2) containing Complete protease inhibitor mixture (Roche Applied Science) and incubated for 15 min at room temperature. Washed with PBS, and further incubated for 1 h at room temperature with the secondary antibodies diluted in blocking solution
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