Abstract Despite ferritin's critical role in regulating cellular and systemic iron levels, our understanding of the structure and assembly mechanism of isoferritins, discovered over eight decades ago, remains limited. Unveiling how the composition and molecular architecture of hetero‐oligomeric ferritins confer distinct functionality to isoferritins is essential to understanding how the structural intricacies of H and L subunits influence their interactions with cellular machinery. In this study, ferritin heteropolymers with specific H to L subunit ratios were synthesized using a uniquely engineered plasmid design, followed by high‐resolution cryo‐electron microscopy analysis and deep learning‐based amino acid modeling. Our structural examination revealed unique architectural features during the self‐assembly mechanism of heteropolymer ferritins and demonstrated a significant preference for H‐L heterodimer formation over H‐H or L‐L homodimers. Unexpectedly, while dimers seem essential building blocks in the protein self‐assembly process, the overall mechanism of ferritin self‐assembly is observed to proceed randomly through diverse pathways. The physiological significance of these findings is discussed including how ferritin microheterogeneity could represent a tissue‐specific adaptation process that imparts distinctive tissue‐specific functions to isoferritins.
more »
« less
Ferritin microheterogeneity, subunit composition, functional, and physiological implications
Abstract Ferritin is a ubiquitous intracellular iron storage protein that plays a crucial role in iron homeostasis. Animal tissue ferritins consist of multiple isoforms (or isoferritins) with different proportions of H and L subunits that contribute to their structural and compositional heterogeneity, and thus physiological functions. Using size exclusion and anion exchange chromatography, capillary isoelectric focusing (cIEF), and SDS-capillary gel electrophoresis (SDS-CGE), we reveal for the first time a significant variation in ferritin subunit composition and isoelectric points, in both recombinant and native ferritins extracted from animal organs. Our results indicate that subunits composition is the main determinant of the mean pI of recombinant ferritin heteropolymers, and that ferritin microheterogeneity is a common property of both natural and recombinant proteins and appears to be an intrinsic feature of the cellular machinery during ferritin expression, regulation, post-translational modifications, and post-subunits assembly. The functional significance and physiological implications of ferritin heterogeneity in terms of iron metabolism, response to oxidative stress, tissue-specific functions, and pathological processes are discussed.
more »
« less
- Award ID(s):
- 1934666
- PAR ID:
- 10474972
- Publisher / Repository:
- Nature Portfolio Scientific Reports (Sci Rep) ISSN 2045-2322 (online)
- Date Published:
- Journal Name:
- Scientific Reports
- Volume:
- 13
- Issue:
- 1
- ISSN:
- 2045-2322
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Abstract Mammalian ferritins are predominantly heteropolymeric species consisting of 2 structurally similar, but functionally and genetically distinct subunit types, called H (Heavy) and L (Light). The two subunits co‐assemble in different H and L ratios to form 24‐mer shell‐like protein nanocages where thousands of iron atoms can be mineralized inside a hollow cavity. Here, we use differential scanning calorimetry (DSC) to study ferritin stability and understand how various combinations of H and L subunits confer aspects of protein structure–function relationships. Using a recently engineered plasmid design that enables the synthesis of complex ferritin nanostructures with specific H to L subunit ratios, we show that homopolymer L and heteropolymer L‐rich ferritins have a remarkable hyperthermostability (Tm = 115 ± 1°C) compared to their H‐ferritin homologues (Tm = 93 ± 1°C). Our data reveal a significant linear correlation between protein thermal stability and the number of L subunits present on the ferritin shell. A strong and unexpected iron‐induced protein thermal destabilization effect (ΔTmup to 20°C) is observed. To our knowledge, this is the first report of recombinant human homo‐ and hetero‐polymer ferritins that exhibit surprisingly high dissociation temperatures, the highest among all known ferritin species, including many known hyperthermophilic proteins and enzymes. This extreme thermostability of our L and L‐rich ferritins may have great potential for biotechnological applications.more » « less
-
Ferritin is a 24-mer protein nanocage that stores iron and regulates intracellular iron homeostasis. The nuclear receptor coactivator-4 (NCOA4) binds specifically to ferritin H subunits and facilitates the autophagic trafficking of ferritin to the lysosome for degradation and iron release. Using isothermal titration calorimetry (ITC), we studied the thermodynamics of the interactions between ferritin and the soluble fragment of NCOA4 (residues 383–522), focusing on the effects of the recently identified Fe–S cluster bound to NCOA4, ferritin subunit composition, and ferritin-iron loading. Our findings show that in the presence of the Fe–S cluster, the binding is driven by a more favorable enthalpy change and a decrease in entropy change, indicating a key role for the Fe–S cluster in the structural organization and stability of the complex. The ferritin iron core further enhances this association, increasing binding enthalpy and stabilizing the NCOA4-ferritin complex. The ferritin subunit composition primarily affects binding stoichiometry of the reaction based on the number of H subunits in the ferritin H/L oligomer. Our results demonstrate that both the Fe–S cluster and the ferritin iron core significantly affect the binding thermodynamics of the NCOA4-ferritin interactions, suggesting regulatory roles for the Fe–S cluster and ferritin iron content in ferritinophagy.more » « less
-
Ferritin is a protein that regulates the iron ions in humans by storing them in the form of iron oxides. Despite extensive efforts to understand the ferritin iron oxide structures, it is still not clear how ferritin proteins with a distinct light (L) and heavy (H) chain subunit ratio impact the biomineralization process. In situ graphene liquid cell-transmission electron microscopy (GLC-TEM) provides an indispensable platform to study the atomic structure of ferritin mineral cores in their native liquid environment. In this study, we report differences in the iron oxide formation in human spleen ferritins (HSFs) and human heart ferritins (HHFs) using in situ GLC-TEM. Scanning transmission electron microscopy (STEM) along with selected area electron diffraction (SAED) of the mineral core and electron energy loss spectroscopy (EELS) analyses enabled the visualization of morphologies, crystal structures and the chemistry of iron oxide cores in HSFs and HHFs. Our study revealed the presence of metastable ferrihydrite (5Fe 2 O 3 ·9H 2 O) as a dominant phase in hydrated HSFs and HHFs, while a stable hematite (α-Fe 2 O 3 ) phase predominated in non-hydrated HSFs and HHFs. In addition, a higher Fe 3+ /Fe 2+ ratio was found in HHFs in comparison with HSFs. This study provides new understanding on iron-oxide phases that exist in hydrated ferritin proteins from different human organs. Such new insights are needed to map ferritin biomineralization pathways and possible correlations with various iron-related disorders in humans.more » « less
-
Abstract The interaction between nuclear receptor coactivator 4 (NCOA4) and the iron storage protein ferritin is a crucial component of cellular iron homeostasis. The binding of NCOA4 to the FTH1 subunits of ferritin initiates ferritinophagy—a ferritin-specific autophagic pathway leading to the release of the iron stored inside ferritin. The dysregulation of NCOA4 is associated with several diseases, including neurodegenerative disorders and cancer, highlighting the NCOA4-ferritin interface as a prime target for drug development. Here, we present the cryo-EM structure of the NCOA4-FTH1 interface, resolving 16 amino acids of NCOA4 that are crucial for the interaction. The characterization of mutants, designed to modulate the NCOA4–FTH1 interaction, is used to validate the significance of the different features of the binding site. Our results explain the role of the large solvent-exposed hydrophobic patch found on the surface of FTH1 and pave the way for the rational development of ferritinophagy modulators.more » « less
An official website of the United States government

