Browsing by Author "Irimia-Dominguez, Jose"

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    Cryo-EM structures and functional characterization of homo- and heteropolymers of human ferritin variants
    (Nature, 2020-11-26) Irimia-Dominguez, Jose; Sun, Chen; Li, Kunpeng; Muhoberac, Barry B.; Hallinan, Grace I.; Garringer, Holly J.; Ghetti, Bernardino; Jiang, Wen; Vidal, Ruben; Pathology and Laboratory Medicine, School of Medicine
    The role of abnormal brain iron metabolism in neurodegenerative diseases is still insufficiently understood. Here, we investigate the molecular basis of the neurodegenerative disease hereditary ferritinopathy (HF), in which dysregulation of brain iron homeostasis is the primary cause of neurodegeneration. We mutagenized ferritin's three-fold pores (3FPs), i.e. the main entry route for iron, to investigate ferritin's iron management when iron must traverse the protein shell through the disrupted four-fold pores (4FPs) generated by mutations in the ferritin light chain (FtL) gene in HF. We assessed the structure and properties of ferritins using cryo-electron microscopy and a range of functional analyses in vitro. Loss of 3FP function did not alter ferritin structure but led to a decrease in protein solubility and iron storage. Abnormal 4FPs acted as alternate routes for iron entry and exit in the absence of functional 3FPs, further reducing ferritin iron-storage capacity. Importantly, even a small number of MtFtL subunits significantly compromises ferritin solubility and function, providing a rationale for the presence of ferritin aggregates in cell types expressing different levels of FtLs in patients with HF. These findings led us to discuss whether modifying pores could be used as a pharmacological target in HF.
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    Sub-3 Å Apoferritin Structure Determined With Full Range of Phase Shifts Using A Single Position Of Volta Phase Plate
    (Elsevier, 2019-05-01) Li, Kunpeng; Sun, Chen; Klose, Thomas; Irimia-Dominguez, Jose; Vago, Frank S.; Vidal, Ruben; Jiang, Wen; Pathology and Laboratory Medicine, School of Medicine
    Volta Phase Plate (VPP) has become an invaluable tool for cryo-EM structural determination of small protein complexes by increasing image contrast. Currently, the standard protocol of VPP usage periodically changes the VPP position to a fresh spot during data collection. Such a protocol was to target the phase shifts to a relatively narrow range (around 90°) based on the observations of increased phase shifts and image blur associated with more images taken with a single VPP position. Here, we report a 2.87 Å resolution structure of apoferritin reconstructed from a dataset collected using only a single position of VPP. The reconstruction resolution and map density features are nearly identical to the reconstruction from the control dataset collected with periodic change of VPP positions. Further experiments have verified that similar results, including a 2.5 Å resolution structure, could be obtained with a full range of phase shifts, different spots of variable phase shift increasing rates, and at different ages of the VPP post-installation. Furthermore, we have found that the phase shifts at low resolutions, probably related to the finite size of the Volta spots, could not be correctly modeled by current CTF model using a constant phase shift at all frequencies. In dataset III, severe beam tilt issue was identified but could be computationally corrected with iterative refinements. The observations in this study may provide new insights into further improvement of both the efficiency and robustness of VPP, and to help turn VPP into a plug-and-play device for high-resolution cryo-EM.
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    Systemic and cerebral iron homeostasis in ferritin knock-out mice
    (PLoS, 2015-01-28) Li, Wei; Garringer, Holly J.; Goodwin, Charles B.; Richine, Briana; Acton, Anthony; VanDuyn, Natalia; Muhoberac, Barry B.; Irimia-Dominguez, Jose; Chan, Rebecca J.; Peacock, Munro; Nass, Richard; Ghetti, Bernardino; Vidal, Ruben; Department of Pathology and Laboratory Medicine, IU School of Medicine
    Ferritin, a 24-mer heteropolymer of heavy (H) and light (L) subunits, is the main cellular iron storage protein and plays a pivotal role in iron homeostasis by modulating free iron levels thus reducing radical-mediated damage. The H subunit has ferroxidase activity (converting Fe(II) to Fe(III)), while the L subunit promotes iron nucleation and increases ferritin stability. Previous studies on the H gene (Fth) in mice have shown that complete inactivation of Fth is lethal during embryonic development, without ability to compensate by the L subunit. In humans, homozygous loss of the L gene (FTL) is associated with generalized seizure and atypical restless leg syndrome, while mutations in FTL cause a form of neurodegeneration with brain iron accumulation. Here we generated mice with genetic ablation of the Fth and Ftl genes. As previously reported, homozygous loss of the Fth allele on a wild-type Ftl background was embryonic lethal, whereas knock-out of the Ftl allele (Ftl-/-) led to a significant decrease in the percentage of Ftl-/- newborn mice. Analysis of Ftl-/- mice revealed systemic and brain iron dyshomeostasis, without any noticeable signs of neurodegeneration. Our findings indicate that expression of the H subunit can rescue the loss of the L subunit and that H ferritin homopolymers have the capacity to sequester iron in vivo. We also observed that a single allele expressing the H subunit is not sufficient for survival when both alleles encoding the L subunit are absent, suggesting the need of some degree of complementation between the subunits as well as a dosage effect.