Iron-regulated assembly of the cytosolic iron–sulfur cluster biogenesis machinery

Fan, Xiaorui; Barshop, William D.; Vashisht, Ajay A.; Pandey, Vijaya; Leal, Stephanie; Rayatpisheh, Shima; Jami‐Alahmadi, Yasaman; Sha, Jihui; Wohlschlegel, James A.

doi:10.1016/j.jbc.2022.102094

Cited by 8 publications

(7 citation statements)

References 37 publications

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“…This further confirms the significant role of NARFL in the cytoplasmic iron-sulfur protein assembly pathway 38 . In a study by Fan XR et al 38 it was discovered that the NARFL-S161I mutant was unable to bind to the functional CIA complex. It was speculated that this mechanism could be related to the development of diffuse pulmonary arteriovenous malformation, a condition associated with this specific mutation, which was first identified by the researchers.…”

Section: Discussionsupporting

confidence: 76%

The Mechanism of Vascular Endothelial Dysfunction Induced by Ferroptosis Mediated by NARFL Knockout

Hu,

Luo,

et al. 2024

Preprint

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BACKGROUNDNuclear prelamin A recognition factor-like (NARFL) plays a crucial role in cytosolic iron-sulfur protein assembly (CIA) and protects cells against oxidative stress. In our previous study, we identified a novel homozygous mutation in NARFL that led to decreased expression in a consanguineous family with diffuse pulmonary arteriovenous malformations (DPAVMs) secondary to pulmonary hypertension. Additionally, we observed that narfl deletion in zebrafish resulted in larvae lethality, subintestinal vessel malformation, and increased oxidative stress. In this study, we aimed to further investigate the function of NARFL and elucidate the pathological manifestations of NARFL deficiency in zebrafish models, cellular models, mouse models, and clinical samples, focusing on the underlying molecular mechanisms.METHODSWe observed the behavioral and phenotypic abnormalities in zebrafish caused by narfl deletion and investigated the mechanism behind vascular morphological abnormalities. Furthermore, we constructedNARFLgene knockout stable cell lines in human pulmonary microvascular endothelial cells (HPMEC) to examine the morphological and functional changes in endothelial cells caused by NARFL deletion. We studied the effects of NARFL deletion on ferroptosis and its potential rescue using a ferroptosis inhibitor. To investigate the function of the human NARFL homolog Ciao3 gene in vascular development, we created a mouse model with a knockout of theCiao3gene. Finally, we compared the distribution of tagSNPs of NARFL using the SNaPshot method between cases and controls to confirm the role of the Ciao3 gene in endothelial dysfunction.RESULTSNarfl deletion in zebrafish resulted in larvae lethality, vascular malformation with abnormal blood flow, abnormal blood-brain barrier (BBB) structure, and brain neuron lesions. Fluorescence probe detection showed increased iron, enhanced oxidative stress, lipid peroxidation, and decreased mitochondrial respiration in response to narfl deficiency, which could be partially alleviated by the use of the ferroptosis inhibitor Ferrostatin-1. We observed downregulation of the iron-sulfur protein cyp2p8 expression in blood vessels of narfl-deficient zebrafish through qRT-PCR and WISH experiments. In HPMEC cells, NARFL deficiency resulted in decreased proliferation, abnormal mitochondrial morphology, increased levels of iron and oxidative stress, and decreased mitochondrial respiration. Functional experiments on endothelial cells revealed decreased tube formation ability and enhanced permeability in response to NARFL deficiency. WB experiments showed downregulation of GPX4, SLC7A11, and Ferritin, while TFR1 and IRP1 were upregulated. Downregulation of NARFL also affected the expression of the iron-sulfur protein CYP2J2. Co-IP results indicated that NARFL deletion led to incompatibility among the CIA system-associated proteins. In mice, Ciao3 deletion in the embryonic stage resulted in embryonic death, vascular dysplasia, impaired differentiation of endothelial progenitor cells, and abnormalities in the expression of ferroptosis-related proteins. Reduction of Ciao3 impaired vascular function and decreased ring formation ability in adult heterozygous mice.NARFLpolymorphisms rs11248948, rs2071952, and rs611289 were identified as susceptible sites for epilepsy, while rs11792680 was associated with susceptibility to pulmonary hypertension, epilepsy, and neurodegenerative diseases.CONCLUSIONNARFL knockout disrupts its interaction with CIA system-related proteins, leading to decreased aconitase activity, increased IRP1 activity, endothelial cell ferroptosis pathway abnormalities, enhanced ferroptosis and oxidative stress, and ultimately vascular endothelial dysfunction. This dysfunction is responsible for the death of embryos innarfl-/-zebrafish andCiao3-/-mice, as well as the susceptibility to pulmonary hypertension, epilepsy, and neurodegenerative diseases.What Is New?Elucidation of the mechanism behind NARFL knockout-induced death through dynamic visualization experimentsin vivoand mechanism and function experimentsin vitro:The study explored the function of NARFL, as it is known as a “knockout lethal” protein. Bothin vivoandin vitroexperiments have confirmed that NARFL acts as the “transmitter” of cytoplasmic iron-sulfur clusters. Its absence prevents interaction with associated proteins of the CIA system, leading to reduced cisaconitase activity, enhanced IRP1 activity, ferroptosis of endothelial cells, and increased oxidative stress, eventually resulting in cell death.Providing new research ideas for the study of cytoplasmic iron-sulfur proteins: Most current studies focus on the function of mitochondrial iron-sulfur proteins and their relationship with iron death. However, research on extramitochondrial iron-sulfur proteins is relatively limited. This study provides data support and research ideas for understanding the function of extramitochondrial iron-sulfur proteins by exploring the pathological mechanism of NARFL and the mediation of iron-sulfur protein maturation.What Are the Clinical Implications?From rare diseases to common diseases: Through the investigation of the lethal mechanism of NARFL knockout and the study ofNARFLgene polymorphisms associated with vascular endothelial dysfunction diseases, we propose the hypothesis that NARFL may be a susceptibility gene for these diseases. This study provides data support for the hypothesis and contributes to our understanding of the role of NARFL in vascular endothelial dysfunction diseases.

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Section: Discussionsupporting

confidence: 76%

The Mechanism of Vascular Endothelial Dysfunction Induced by Ferroptosis Mediated by NARFL Knockout

Hu,

Luo,

et al. 2024

Preprint

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“…NUBP2 deficiency in humans is not reported, but the ortholog Nbp35 seems to be essential in yeast. [30] NUBP2 is part of the Fe−S cluster biogenesis machinery, [31] of which several components were detected, including NUBP1 (P53384), GLRX3 (O76003), GLRX5 (Q86SX6), FDXR (P22570), FXN (Q16595), FDX1 (P10109), NFU1 (Q9UMS0), NFS1 (Q9Y697) and CIAO1 (O76071, all grey, Figure 3 B and Supporting Information Figure 8A). [ 30 , 32 ] These proteins were not regulated upon treatment with TRIP.…”

Section: Resultsmentioning

confidence: 99%

An Anticancer Rhenium Tricarbonyl Targets Fe−S Cluster Biogenesis in Ovarian Cancer Cells

et al. 2022

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Target identification remains a critical challenge in inorganic drug discovery to deconvolute potential polypharmacology. Herein, we describe an improved approach to prioritize candidate protein targets based on a combination of dose‐dependent chemoproteomics and treatment effects in living cancer cells for the rhenium tricarbonyl compound TRIP. Chemoproteomics revealed 89 distinct dose‐dependent targets with concentrations of competitive saturation between 0.1 and 32 μM despite the broad proteotoxic effects of TRIP. Target‐response networks revealed two highly probable targets of which the Fe−S cluster biogenesis factor NUBP2 was competitively saturated by free TRIP at nanomolar concentrations. Importantly, TRIP treatment led to a down‐regulation of Fe−S cluster containing proteins and upregulated ferritin. Fe−S cluster depletion was further verified by assessing mitochondrial bioenergetics. Consequently, TRIP emerges as a first‐in‐class modulator of the scaffold protein NUBP2, which disturbs Fe−S cluster biogenesis at sub‐cytotoxic concentrations in ovarian cancer cells.

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“…However, these resources are scarce and incomplete. Only recently (2021)(2022) have iron-related canonical pathways been added to the curated database from QIAGEN Ingenuity Pathway Analysis (IPA, Qiagen) (https://www. qiagen.com/ingenuity).…”

Section: Bioinformatics Approaches Tailored For the Analysis Of Iron ...mentioning

confidence: 99%

“…Additionally, a recently developed, targeted mass spectrometry approach provides better sensitivity and quantitation and is applicable when the interacting partners are known or suspected. This approach was used to confirm that the cytosolic Fe-S cluster assembly complex forms a metabolon consisting of scaffolding components, the targeting complex, and recipient apo-proteins ( Fan et al, 2022 ).…”

Section: High Throughput Approaches For Identifying Iron Trafficking ...mentioning

confidence: 99%

Iron-tracking strategies: Chaperones capture iron in the cytosolic labile iron pool

Philpott

Protchenko²,

Wang³

et al. 2023

Front. Mol. Biosci.

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Cells express hundreds of iron-dependent enzymes that rely on the iron cofactors heme, iron-sulfur clusters, and mono-or di-nuclear iron centers for activity. Cells require systems for both the assembly and the distribution of iron cofactors to their cognate enzymes. Proteins involved in the binding and trafficking of iron ions in the cytosol, called cytosolic iron chaperones, have been identified and characterized in mammalian cells. The first identified iron chaperone, poly C-binding protein 1 (PCBP1), has also been studied in mice using genetic models of conditional deletion in tissues specialized for iron handling. Studies of iron trafficking in mouse tissues have necessitated the development of new approaches, which have revealed new roles for PCBP1 in the management of cytosolic iron. These approaches can be applied to investigate use of other nutrient metals in mammals.

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Iron-regulated assembly of the cytosolic iron–sulfur cluster biogenesis machinery

Cited by 8 publications

References 37 publications

The Mechanism of Vascular Endothelial Dysfunction Induced by Ferroptosis Mediated by NARFL Knockout

The Mechanism of Vascular Endothelial Dysfunction Induced by Ferroptosis Mediated by NARFL Knockout

An Anticancer Rhenium Tricarbonyl Targets Fe−S Cluster Biogenesis in Ovarian Cancer Cells

Iron-tracking strategies: Chaperones capture iron in the cytosolic labile iron pool

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