Affinage

CIAO3

Cytosolic iron-sulfur assembly component 3 · UniProt Q9H6Q4

Length
476 aa
Mass
53.0 kDa
Annotated
2026-06-09
33 papers in source corpus 12 papers cited in narrative 12 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 5/5 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

CIAO3 (IOP1/NARFL; yeast ortholog Nar1) is an essential component of the cytosolic iron-sulfur (Fe-S) protein assembly (CIA) machinery, functioning as a metallocluster-bearing factor that connects early and late steps of cytosolic Fe-S protein maturation (PMID:18270200, PMID:21367862). It is selectively required for cytosolic but not mitochondrial Fe-S enzyme biogenesis: its depletion reduces cytosolic aconitase and xanthine oxidase activities while sparing mitochondrial aconitase, and triggers the iron-starvation response characteristic of cytosolic Fe-S cluster loss (PMID:18270200, PMID:21367862). The protein holds two Fe-S clusters at conserved N- and C-terminal cysteine motifs, both essential for function, with assembly of the buried C-terminal cluster dependent on the surface-exposed N-terminal motif (PMID:19385603); biophysical characterization defines these as [4Fe-4S] cofactors that are oxygen-sensitive, linking in vitro cluster lability to CIA phenotypes in vivo (PMID:41257193, PMID:25490157). CIAO3 operates as an external module of the MMS19-dependent CIA pathway, binding the core complex via a direct CIAO1 interaction (PMID:23585563), and forms a stable [4Fe-4S]-bound ternary complex with CIA2A and CIAO1 (PMID:32222833). Genetic ablation is lethal in mice, with embryonic lethality before E10.5 and lethality upon acute global knockout in adults (PMID:21367862), and endothelial-specific knockout causes acute lung injury driven by TNF-α-mediated immune activation (PMID:41187899).

Mechanistic history

Synthesis pass · year-by-year structured walk · 9 steps
  1. 2007 Medium

    The first functional readout linked CIAO3 to a regulatory role over the hypoxia response, establishing a cellular consequence of its loss before its biochemical role was defined.

    Evidence siRNA knockdown in mammalian cells with HRE reporter and target-gene expression assays

    PMID:16956324

    Open questions at the time
    • Mechanism connecting IOP1 to HIF-1alpha mRNA regulation not defined
    • Does not establish whether this effect is downstream of Fe-S assembly defects
  2. 2008 High

    Established the defining functional specificity of CIAO3 — that it is required for cytosolic but not mitochondrial Fe-S protein biogenesis — resolving which branch of Fe-S metabolism it serves.

    Evidence siRNA knockdown in HeLa/Hep3B with cytosolic vs mitochondrial enzyme activity assays and siRNA-resistant rescue

    PMID:18270200

    Open questions at the time
    • Did not define the molecular step CIAO3 performs within the CIA pathway
    • Direct partners not identified
  3. 2009 High

    Identified a physical interaction with the Fe-S scaffold ISCA1 and characterized the redox/cluster properties of the Nar1 ortholog, beginning to define CIAO3 as a metallocluster-binding factor.

    Evidence Co-IP, subcellular fractionation, and knockdown enzyme assays (ISCA1); site-directed mutagenesis with in vivo 55Fe radiolabeling in yeast (Nar1 two-cluster architecture)

    PMID:19385603 PMID:19864422

    Open questions at the time
    • ISCA1 interaction is single-lab Co-IP
    • Cluster identity quantified later, not in initial radiolabeling work
    • Functional consequence of two-cluster cooperativity for client maturation unclear
  4. 2011 High

    Demonstrated CIAO3 is essential in a mammalian organism, confirming the cytosolic-specific Fe-S role in vivo and elevating the gene from cell-line phenotype to organismal requirement.

    Evidence Constitutive and conditional mouse knockout with aconitase activity and viability assays

    PMID:21367862

    Open questions at the time
    • Tissue-level consequences of loss not dissected
    • Did not address molecular mechanism of cluster transfer
  5. 2013 Medium

    Placed CIAO3 within the architecture of the human CIA machinery as an external module distinct from the MMS19-MIP18-CIAO1 core, with CIAO1 as its direct binding partner.

    Evidence In vivo and in vitro Co-IP, knockdown, and overexpression experiments

    PMID:23585563

    Open questions at the time
    • Stoichiometry and dynamics of association not quantified
    • How CIAO3 transfers clusters to the core not shown
  6. 2014 Medium

    Refined cofactor identity by redox titration and connected CIAO3 loss to oxidative stress protection, broadening the functional context of the gene.

    Evidence EPR-monitored redox titration of yeast Nar1; yeast lifespan and paraquat assays with SOD2 rescue

    PMID:24486555 PMID:25490157

    Open questions at the time
    • Link between Fe-S function and oxidative stress phenotype mechanistically indirect
    • SOD2 rescue does not localize the defect
  7. 2020 High

    Defined a stable [4Fe-4S]-bound ternary complex of CIAO3 with CIA2A and CIAO1, providing a concrete molecular assembly that could mediate cluster handoff.

    Evidence SEC-MALS, UV-vis, EPR spectroscopy, and site-directed mutagenesis on reconstituted complex

    PMID:32222833

    Open questions at the time
    • Directionality of cluster transfer within complex not established
    • Structural basis of complex assembly not resolved
  8. 2025 High

    Resolved the full cofactor complement and oxygen sensitivity of the Nar1 ortholog, and revealed an endothelial pathophysiology for CIAO3 loss via TNF-α-driven immune activation.

    Evidence UV-vis, EPR, Mössbauer, native MS with in vitro reconstitution (cofactors); endothelial conditional KO mouse with scRNA-seq and TNF-α inhibition (lung injury)

    PMID:41187899 PMID:41257193

    Open questions at the time
    • The [2Fe-2S] cluster site is undetermined
    • Mechanistic link between endothelial Fe-S deficiency and cytokine storm not defined
    • In vitro cofactor states in E. coli may not match cellular state
  9. 2026 Medium

    Proposed a bipartite binding mechanism by which CIAO3 docks onto the CIA targeting complex through Cia1, positioning its cluster donor site for transfer to a Cia2 acceptor.

    Evidence Biochemical reconstitution, quantitative interaction assays, and AlphaFold modeling (preprint)

    PMID:41846961

    Open questions at the time
    • Preprint, not yet peer-reviewed
    • Cluster transfer event itself not directly observed
    • Structural models not experimentally validated

Open questions

Synthesis pass · forward-looking unresolved questions
  • It remains unresolved how CIAO3 mechanistically couples cluster receipt, its multi-cluster redox states, and directional handoff to client apoproteins, and how cytosolic Fe-S deficiency in specific tissues produces the observed immune and hypoxia-response phenotypes.
  • No experimental structure of the CIAO3-CTC complex
  • Directionality and trigger of cluster transfer not demonstrated
  • Causal chain from Fe-S defect to TNF-α-mediated lung injury unmapped

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0140104 molecular carrier activity 3 GO:0140096 catalytic activity, acting on a protein 2
Localization
GO:0005829 cytosol 2
Pathway
R-HSA-1430728 Metabolism 2 R-HSA-392499 Metabolism of proteins 2
Partners
CIA2ACIAO1ISCA1MIP18MMS19
Complex memberships
CIA targeting complex (CTC)CIAO3-CIA2A-CIAO1 ternary complex

Evidence

Reading pass · 12 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2007 IOP1 (CIAO3/NARFL) knockdown using siRNA in mammalian cells increases HIF-1alpha protein levels under both normoxic and hypoxic conditions and augments hypoxia-induced HRE reporter gene and endogenous HIF-1alpha target gene expression; mechanistically, IOP1 knockdown up-regulates HIF-1alpha mRNA levels, indicating IOP1 negatively regulates HIF-1alpha at the transcriptional level. siRNA knockdown in mammalian cells, HRE reporter gene assay, endogenous target gene expression analysis The Biochemical journal Medium 16956324
2008 IOP1 (CIAO3/NARFL) is required for cytosolic but not mitochondrial iron-sulfur (Fe-S) protein biogenesis in mammalian cells; siRNA knockdown of IOP1 in HeLa and Hep3B cells decreases cytosolic aconitase activity and xanthine oxidase activity (both cytosolic Fe-S proteins), but not mitochondrial aconitase activity. Knockdown also increases transferrin receptor 1 mRNA and decreases ferritin heavy chain protein levels, consistent with conversion of cytosolic aconitase to iron regulatory protein 1 upon Fe-S cluster loss. siRNA knockdown in HeLa and Hep3B cells, enzymatic activity assays for cytosolic and mitochondrial aconitase and xanthine oxidase, rescue with siRNA-resistant IOP1 construct The Journal of biological chemistry High 18270200
2009 Human IscA1 (ISCA1) physically interacts with IOP1 (CIAO3/NARFL) in the cytosolic iron-sulfur cluster assembly pathway; IscA1 is observed in both cytosolic and mitochondrial fractions and its siRNA knockdown decreases activity of mitochondrial Fe-S enzymes (succinate dehydrogenase, mitochondrial aconitase) as well as cytosolic aconitase. Co-immunoprecipitation, subcellular fractionation, siRNA knockdown, enzymatic activity assays The Journal of biological chemistry Medium 19864422
2009 Yeast CIA protein Nar1 (ortholog of CIAO3/IOP1) holds two Fe-S clusters at conserved N- and C-terminal cysteine motifs, both essential for Nar1 function and cell viability; the C-terminal Fe-S cluster is stably buried within Nar1, while the N-terminal cluster is surface-exposed; assembly of the C-terminal cluster depends on the N-terminal motif, indicating sequential or cooperative cluster assembly. Systematic site-directed mutagenesis of cysteine residues combined with in vivo 55Fe radiolabeling confirmed these roles. Site-directed mutagenesis, in vivo 55Fe radiolabeling in yeast, in vitro biochemistry, molecular modeling Biochemistry High 19385603
2011 Knockout of Iop1 (CIAO3/NARFL) in mice results in embryonic lethality before embryonic day 10.5; acute inducible global knockout in adult mice leads to lethality and significantly diminished cytosolic aconitase activity in liver extracts; inducible knockout in mouse embryonic fibroblasts causes diminished cytosolic but not mitochondrial aconitase activity and loss of cell viability, demonstrating an essential role for IOP1 in cytosolic Fe-S cluster assembly in mammals. Conditional and constitutive mouse knockout, enzymatic activity assays for cytosolic and mitochondrial aconitase, cell viability assays The Journal of biological chemistry High 21367862
2013 IOP1 (CIAO3/NARFL) is an external component of the human cytosolic iron-sulfur cluster assembly (CIA) machinery; MMS19, MIP18, and CIAO1 form a tight 'core' CIA complex, while IOP1 associates with this complex both in vivo and in vitro but behaves differently—knockdown of IOP1 does not affect levels of core components, whereas knockdown of any core component down-regulates all others. CIAO1 directly binds IOP1. MIP18 bridges MMS19 and CIAO1. IOP1 functions in the MMS19-dependent CIA pathway. Co-immunoprecipitation in vivo and in vitro, siRNA knockdown, overexpression experiments The Journal of biological chemistry Medium 23585563
2014 EPR-monitored redox titration of yeast Nar1 (ortholog of CIAO3) determined the midpoint potentials of its two Fe-S cofactors in their detectable redox states, confirming the identity and quantifying the cofactors. EPR-monitored redox titration with chemical reductants/oxidants and redox mediators Journal of visualized experiments : JoVE Medium 25490157
2014 Nar1 deficiency in yeast (ortholog of CIAO3) results in shortened replicative lifespan and sensitivity to the oxidative stress agent paraquat; increased expression of mitochondrial superoxide dismutase (Sod2) rescues both the shortened lifespan and paraquat sensitivity, indicating that Nar1 promotes protection against oxidative stress through a pathway involving mitochondrial ROS management. Yeast genetics, lifespan assays, paraquat sensitivity assays, overexpression of SOD2 Mechanisms of ageing and development Medium 24486555
2020 CIAO3 forms a stable ternary complex with CIA2A and CIAO1 that is bound to a [4Fe-4S] cluster; this was demonstrated by size exclusion chromatography coupled with multiangle light scattering, UV-vis absorption, and EPR spectroscopies. Site-directed mutagenesis data suggest a structural role for the C-terminal [4Fe-4S] cluster of CIAO3. Size exclusion chromatography-multiangle light scattering (SEC-MALS), UV-vis absorption spectroscopy, EPR spectroscopy, site-directed mutagenesis Journal of biological inorganic chemistry : JBIC High 32222833
2025 Ciao3 conditional knockout in endothelial cells (EC-CKO) of mice causes acute lung injury (ALI) associated with elevated cytokine levels (cytokine storm), immune activation, and abnormal cell-cell communication between endothelial cells and immune cells; TNF-α inhibition reverses ALI development. scRNA-seq identified monocytes and macrophages as central effectors, and CypA, FN1, and THBS signaling pathways as the main changed pathways. Conditional knockout mouse model (EC-CKO), cytokine measurement, bulk RNA-seq, single-cell RNA-seq, TNF-α inhibition experiment, immunohistochemistry Life sciences Medium 41187899
2025 Yeast Nar1 (ortholog of CIAO3) binds a [4Fe-4S] cluster at site 1 and an unexpected [2Fe-2S] cluster at an unknown site when recombinantly produced in E. coli; Fe-S reconstitution installs a second [4Fe-4S] cluster at site 2, yielding protein with up to three Fe-S cofactors. Two of the Fe-S clusters are rapidly destroyed by molecular oxygen, linking Nar1 oxygen sensitivity in vitro to CIA pathway phenotypes observed in vivo. The [2Fe-2S] cluster is proposed to occupy a cavity equivalent to the [2Fe]H cofactor in [FeFe]-hydrogenases. UV-vis spectroscopy, EPR spectroscopy, Mössbauer spectroscopy, native mass spectrometry, in vitro Fe-S reconstitution Chemical science High 41257193
2026 Nar1 (CIAO3/IOP1) binds the CIA targeting complex (CTC) through two distinct interfaces: a primary electrostatic interface anchoring Nar1 to a conserved acidic surface on the Cia1 subunit, and a secondary interface involving Nar1's divergent targeting complex recognition peptide at the Cia1-Cia2 interface. Computational structural models position Nar1's putative Fe-S cluster donor site adjacent to a proposed acceptor site on Cia2, suggesting that this bipartite binding mechanism positions Nar1 for Fe-S cluster transfer to the CTC. Biochemical reconstitution, quantitative protein-protein interaction assays, AlphaFold structural modeling bioRxivpreprint Medium 41846961

Source papers

Stage 0 corpus · 33 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2017 Toxic PRn poly-dipeptides encoded by the C9orf72 repeat expansion block nuclear import and export. Proceedings of the National Academy of Sciences of the United States of America 185 28069952
1994 Nar-1 and Nar-2, Two Loci Required for Mla12-Specified Race-Specific Resistance to Powdery Mildew in Barley. The Plant cell 81 12244263
2008 A role for IOP1 in mammalian cytosolic iron-sulfur protein biogenesis. The Journal of biological chemistry 62 18270200
2006 Differential regulation of the Chlamydomonas Nar1 gene family by carbon and nitrogen. Protist 59 16905358
1993 The Ustilago maydis nar1 gene encoding nitrate reductase activity: sequence and transcriptional regulation. Gene 54 8370542
2000 Evolution of the family of pRN plasmids and their integrase-mediated insertion into the chromosome of the crenarchaeon Sulfolobus solfataricus. Journal of molecular biology 52 11054282
2008 Familial non-medullary thyroid carcinoma (FNMTC): analysis of fPTC/PRN, NMTC1, MNG1 and TCO susceptibility loci and identification of somatic BRAF and RAS mutations. Endocrine-related cancer 44 18310288
2009 Human ISCA1 interacts with IOP1/NARFL and functions in both cytosolic and mitochondrial iron-sulfur protein biogenesis. The Journal of biological chemistry 42 19864422
2009 Crucial role of conserved cysteine residues in the assembly of two iron-sulfur clusters on the CIA protein Nar1. Biochemistry 40 19385603
2013 IOP1 protein is an external component of the human cytosolic iron-sulfur cluster assembly (CIA) machinery and functions in the MMS19 protein-dependent CIA pathway. The Journal of biological chemistry 37 23585563
2007 IOP1, a novel hydrogenase-like protein that modulates hypoxia-inducible factor-1alpha activity. The Biochemical journal 37 16956324
2008 Evidence for the horizontal transfer of an integrase gene from a fusellovirus to a pRN-like plasmid within a single strain of Sulfolobus and the implications for plasmid survival. Microbiology (Reading, England) 36 18227242
1981 Gene roles in the prn cluster of Aspergillus nidulans. Current genetics 32 24189952
2011 Mouse knock-out of IOP1 protein reveals its essential role in mammalian cytosolic iron-sulfur protein biogenesis. The Journal of biological chemistry 31 21367862
2011 Studies on Prn variation in the mouse model and comparison with epidemiological data. PloS one 28 21464955
1978 Reduced expression of a distal gene of the prn gene cluster in deletion mutants of Aspergillus nidulans: genetic evidence for a dicistronic messenger in an eukaryote. Molecular & general genetics : MGG 25 355839
2020 Efficacy of a Bruton's Tyrosine Kinase Inhibitor (PRN-473) in the treatment of canine pemphigus foliaceus. Veterinary dermatology 24 31899567
1998 Sequence, exon-intron organization, transcription and mutational analysis of prnA, the gene encoding the transcriptional activator of the prn gene cluster in Aspergillus nidulans. Molecular microbiology 23 9622360
2020 CIAO3 protein forms a stable ternary complex with two key players of the human cytosolic iron-sulfur cluster assembly machinery. Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry 14 32222833
2004 Comparative sequence analysis of Bordetella bronchiseptica pertactin gene (prn) repeat region variants in swine vaccines and field isolates. Vaccine 13 15519707
2020 Effect of FHA and Prn on Bordetella pertussis colonization of mice is dependent on vaccine type and anatomical site. PloS one 11 32822419
2003 The new strategy for allele identification of the genes coding for pertussis toxin subunit S1 (ptx S1) and pertactin (prn) in Bordetella pertussis. Journal of microbiological methods 11 14607408
2020 Nitrate Respiration in Thermus thermophilus NAR1: from Horizontal Gene Transfer to Internal Evolution. Genes 9 33158244
2014 EPR monitored redox titration of the cofactors of Saccharomyces cerevisiae Nar1. Journal of visualized experiments : JoVE 8 25490157
2014 Nar1 deficiency results in shortened lifespan and sensitivity to paraquat that is rescued by increased expression of mitochondrial superoxide dismutase. Mechanisms of ageing and development 7 24486555
2007 Activation of Prn-p gene and stable transfection of Prn-p cDNA in leukemia MEL and neuroblastoma N2a cells increased production of PrP(C) but not prevented DNA fragmentation initiated by serum deprivation. Journal of cellular physiology 6 17186498
2022 Enhanced recovery after surgery multimodality pain regimen performs similar to PRN narcotics on outcomes and pain control after cardiac surgery: A quality improvement project. Journal of cardiac surgery 4 35352395
2024 Aflibercept 5+PRN with retinal laser photocoagulation is more effective than retinal laser photocoagulation alone and aflibercept 3+PRN with retinal laser photocoagulation in patients with high-risk proliferative diabetic retinopathy and diabetic macular edema: a 12-month clinical trial. Frontiers in endocrinology 3 38455651
2025 Ciao3 knockout causes acute lung injury through immune activation using single cell sequencing. Life sciences 1 41187899
2026 Nar1 binds the cytosolic iron sulfur cluster assembly targeting complex via a bipartite interaction interface. bioRxiv : the preprint server for biology 0 41846961
2025 Yeast [FeFe]-hydrogenase-like protein Nar1 binds a [2Fe-2S] cluster. Chemical science 0 41257193
2008 [Expression of prn gene of Bordetella bronchiseptica and development of a recombinant protein-based indirect ELISA for antibodies detection]. Wei sheng wu xue bao = Acta microbiologica Sinica 0 18479059
2007 [Analysis of chosen parameters of immuno response in mice immunized with whole-cell or acellular pertussis vaccines and challenged with B. pertussis strains harbouring different ptxS1/prn allele genes combinations]. Medycyna doswiadczalna i mikrobiologia 0 17929411

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