Affinage

ISCA2

Iron-sulfur cluster assembly 2 homolog, mitochondrial · UniProt Q86U28

Length
154 aa
Mass
16.5 kDa
Annotated
2026-06-10
25 papers in source corpus 16 papers cited in narrative 16 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 6/6 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

ISCA2 is a mitochondrial iron-sulfur cluster (ISC) assembly factor that acts late in the maturation pathway, functioning within an ISCA1-ISCA2-IBA57 subsystem dedicated specifically to the synthesis and delivery of [4Fe-4S] clusters to mitochondrial apo-proteins such as aconitase, respiratory complex I, and lipoic acid synthase, but not to [2Fe-2S] proteins (PMID:22323289, PMID:21987576). Mechanistically, [2Fe-2S]-loaded GLRX5 donates clusters to ISCA2, which together with ISCA1 undergoes reductive [2Fe-2S] fusion into a [4Fe-4S] cluster using electrons supplied by ferredoxin FDX2 and its reductase FDXR, a reaction facilitated by IBA57 (PMID:32817474). ISCA2 binds its cluster through three conserved cysteines and forms a [2Fe-2S]-bridged heterodimer with IBA57 in which cluster occupancy is required for complex assembly and which can reactivate apo-aconitase in vitro (PMID:30269484, PMID:10805735). ISCA1 then orchestrates downstream transfer by bridging ISCA2 and NFU1 in a transient ternary complex that hands off the [4Fe-4S] cluster to NFU1 for delivery to client proteins, while ISCA2 can also serve directly as a cluster donor to lipoyl synthase (PMID:33711344, PMID:33562493). Loss of ISCA2 selectively abolishes [4Fe-4S] enzyme activities, collapses mitochondrial respiration and membrane potential, and causes mtDNA depletion (PMID:29297947), with downstream consequences including ROS-driven IRP1 activation that suppresses ALAS2/heme synthesis in erythroid cells (PMID:35714932) and IRE-dependent blockade of HIF-2α translation in clear cell renal carcinoma (PMID:36097192). A homoallelic ISCA2 missense mutation (p.Gly77Ser) causes a mitochondrial disease characterized by destabilization of the entire late ISC subsystem (PMID:25539947).

Mechanistic history

Synthesis pass · year-by-year structured walk · 15 steps
  1. 2000 High

    Established where the ISCA2 ortholog resides and which residues matter, defining it as a mitochondrial protein whose conserved cysteines are functionally essential.

    Evidence Leader-sequence deletion analysis, cysteine mutagenesis, and mitochondrial fractionation in yeast

    PMID:10805735

    Open questions at the time
    • IMS vs matrix localization debated relative to later matrix assignments
    • did not define the molecular reaction the cysteines support
    • no human protein studied
  2. 2007 Medium

    Began defining client specificity by showing Isa proteins are needed for biotin synthase activity beyond simple cluster assembly.

    Evidence Genetic depletion and desthiobiotin-to-biotin conversion assays with BIO2 overexpression control in yeast

    PMID:17259550

    Open questions at the time
    • mechanism beyond cluster assembly not resolved
    • did not distinguish [2Fe-2S] vs [4Fe-4S] client classes
  3. 2010 Medium

    Placed ISCA2 relative to the monothiol glutaredoxin by showing Grx5 physically interacts with Isa1/Isa2 and Isa2 acts downstream or parallel.

    Evidence BiFC interaction and multi-copy suppressor/enzyme assays in fission yeast

    PMID:20085751

    Open questions at the time
    • BiFC does not establish direct stoichiometric binding
    • directionality of cluster flow not biochemically demonstrated here
  4. 2011 High

    Defined the [4Fe-4S]-specific role: Isa1-Isa2 form an iron-binding complex required for [4Fe-4S] but not [2Fe-2S] protein maturation, with epistasis to IBA57.

    Evidence Co-IP, in vivo iron-binding, ferredoxin targeting, and iba57 epistasis in S. cerevisiae

    PMID:21987576

    Open questions at the time
    • in vitro cluster fusion chemistry not shown
    • electron donor unidentified at this stage
  5. 2011 Medium

    Demonstrated functional conservation of the ISCA system across eukaryotes and that depletion causes ROS rather than iron accumulation.

    Evidence RNAi in T. brucei with enzymatic assays and heterologous rescue by human ISCA proteins

    PMID:21790804

    Open questions at the time
    • human protein behavior inferred from rescue rather than direct assay
    • ROS source not mechanistically dissected
  6. 2012 High

    Confirmed in human cells that ISCA2 is specifically required for [4Fe-4S] protein maturation and acts late in the ISC pathway, with severe mitochondrial morphology defects upon loss.

    Evidence RNAi in HeLa cells with [4Fe-4S]/[2Fe-2S] enzyme assays and EM

    PMID:22323289

    Open questions at the time
    • did not resolve the biochemical step ISCA2 catalyzes
    • cluster donors and acceptors not yet defined
  7. 2018 High

    Resolved the ISCA2-IBA57 interface chemistry: a shared [2Fe-2S] cluster (ligated by three ISCA2 cysteines) is obligatory for heterodimer formation and the complex can reactivate apo-aconitase, defining a GLRX5→ISCA2→IBA57 transfer route.

    Evidence In vitro reconstitution, spectroscopy, cysteine mutagenesis, and aconitase reactivation

    PMID:30269484

    Open questions at the time
    • how [2Fe-2S] becomes [4Fe-4S] not addressed here
    • physiological electron source not tested
  8. 2018 Medium

    Quantified the cellular consequences of ISCA2 loss in patient and knockdown cells: selective [4Fe-4S] failure plus collapse of membrane potential, respiration, ATP, and mtDNA.

    Evidence siRNA and patient fibroblasts with respirometry, membrane potential, and mtDNA assays

    PMID:29297947

    Open questions at the time
    • single lab
    • causal chain from Fe-S loss to mtDNA depletion not mechanistically traced
  9. 2019 Medium

    Provided a low-resolution architecture of the [2Fe-2S] ISCA2-IBA57 complex and linked a disease mutation to the interaction interface.

    Evidence SAXS and bioinformatics docking with disease-mutation interface analysis

    PMID:31831856

    Open questions at the time
    • no atomic-resolution structure
    • model dependent on docking assumptions
  10. 2020 High

    Defined the physiological reaction mechanism: ISCA1-ISCA2 performs reductive [2Fe-2S] fusion to [4Fe-4S] using FDX2/FDXR electrons and IBA57 facilitation, with GLRX5 as donor.

    Evidence Full in vitro reconstitution of [4Fe-4S] aconitase maturation without artificial reductants, FDX1 vs FDX2 comparison

    PMID:32817474

    Open questions at the time
    • stoichiometry and intermediate states of fusion not fully resolved
    • downstream client handoff not addressed here
  11. 2021 High

    Established the downstream delivery logic: ISCA1 bridges ISCA2 and NFU1 in a transient ternary complex to transfer the assembled [4Fe-4S] cluster onward, while ISCA2 and NFU1 do not interact directly.

    Evidence NMR interaction studies and cluster transfer assays of binary/ternary complexes

    PMID:33711344

    Open questions at the time
    • client-selection rules for NFU1 not defined
    • single lab
  12. 2021 Medium

    Identified ISCA2 as a direct cluster donor to lipoyl synthase, broadening its acceptor repertoire.

    Evidence In vitro reconstitution of human LIAS with LC-MS turnover and EPR

    PMID:33562493

    Open questions at the time
    • in vivo relevance of direct ISCA2→LIAS donation vs NFU1 route not resolved
    • single lab
  13. 2022 Medium

    Connected ISCA2 loss to systemic iron/heme signaling by showing ROS-driven IRP1 activation suppresses ALAS2 translation and blocks erythroid heme synthesis.

    Evidence siRNA in K562 cells with IRP1/ACO1 activity, ALAS2, heme, and differentiation assays

    PMID:35714932

    Open questions at the time
    • single cell model
    • direct vs ROS-mediated IRP1 effects not fully separated
  14. 2022 Medium

    Revealed a therapeutic vulnerability: ISCA2 inhibition blocks IRE-dependent HIF-α translation and triggers ferroptosis in renal carcinoma in vitro and in vivo.

    Evidence Compound screen, siRNA, IRE reporters, ferroptosis markers, and ccRCC xenografts

    PMID:36097192

    Open questions at the time
    • selectivity of inhibitor for ISCA2 not exhaustively defined
    • single lab
  15. 2023 Medium

    Identified copper as an inhibitor of ISCA2-dependent Fe-S assembly, linking the protein to copper toxicity in Wilson's disease models.

    Evidence In vitro copper-binding assays and Fe-S activity in copper-loaded cells and ATP7A/B models

    PMID:37225108

    Open questions at the time
    • physiological copper occupancy of ISCA2 not quantified
    • single lab

Open questions

Synthesis pass · forward-looking unresolved questions
  • How client specificity is encoded — which apo-proteins receive clusters directly from ISCA2 versus via NFU1, and how the system is regulated by metal competition in vivo — remains open.
  • no atomic-resolution structure of the active [4Fe-4S] ISCA1-ISCA2 species
  • in vivo hierarchy of direct vs NFU1-mediated client delivery undefined
  • physiological copper regulation of ISCA2 unquantified

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:0005739 mitochondrion 3
Pathway
R-HSA-1430728 Metabolism 3 R-HSA-1852241 Organelle biogenesis and maintenance 2
Partners
FDX2FDXRGLRX5IBA57ISCA1LIASNFU1
Complex memberships
ISCA1-ISCA2-IBA57 late ISC subsystemISCA1-ISCA2-NFU1 transient ternary complexISCA2-IBA57 [2Fe-2S] heterodimer

Evidence

Reading pass · 16 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2012 ISCA2 (along with ISCA1 and IBA57) is specifically required for maturation of mitochondrial [4Fe-4S] proteins (aconitase, respiratory complex I, lipoic acid synthase) but not [2Fe-2S] proteins (ferrochelatase); RNAi depletion in HeLa cells caused massively swollen mitochondria devoid of cristae and loss of [4Fe-4S] enzyme activities, placing ISCA2 late in the ISC assembly pathway. RNA interference knockdown in HeLa cells with enzymatic activity assays for [4Fe-4S] and [2Fe-2S] proteins, mitochondrial morphology analysis by electron microscopy Molecular biology of the cell High 22323289
2000 Yeast Isa2p (ortholog of human ISCA2) localizes to the mitochondrial intermembrane space via a bipartite N-terminal leader sequence; both the mitochondrial import signal and the second IMS-targeting sequence are required for function. Three invariant cysteine residues in Isa2p are essential for function and likely involved in iron binding. Deletion analysis and site-directed mutagenesis of leader sequences and conserved cysteines, mitochondrial fractionation, growth complementation assays in yeast deletion mutants Molecular and cellular biology High 10805735
2011 Yeast Isa1 and Isa2 form a complex required specifically for maturation of mitochondrial [4Fe-4S] proteins but not [2Fe-2S] proteins or cytosolic [4Fe-4S] proteins; both proteins bind iron in vivo, and this iron is proposed to be used for de novo [4Fe-4S] cluster synthesis rather than as a donor for [2Fe-2S] cluster assembly on Isu1/Isu2. Comprehensive in vivo analysis in S. cerevisiae including co-immunoprecipitation, iron-binding assays, targeting of bacterial ferredoxins to yeast mitochondria, epistasis with iba57 deletion The Journal of biological chemistry High 21987576
2020 ISCA1-ISCA2 undergoes reductive [2Fe-2S] cluster fusion to form [4Fe-4S] clusters; this reaction requires electrons from mitochondrial ferredoxin FDX2 (not FDX1) and its reductase FDXR, and is facilitated by IBA57. [2Fe-2S]-GLRX5 serves as the cluster donor to ISCA1-ISCA2. This defines the physiologically relevant mechanism of late-acting ISC factors in mitochondrial [4Fe-4S] cluster synthesis. In vitro reconstitution of mitochondrial [4Fe-4S] aconitase maturation without artificial reductants, using purified components; electron transfer assays with FDX1 vs FDX2 comparison Proceedings of the National Academy of Sciences of the United States of America High 32817474
2018 IBA57 forms a heterodimeric complex with ISCA2 bridged by a [2Fe-2S] cluster; cluster binding is absolutely required for complex formation. The conserved cysteine of IBA57 and the three conserved cysteines of ISCA2 act as cluster ligands. The [2Fe-2S] ISCA2-IBA57 complex is resistant to oxidation and can reactivate apo-aconitase in vitro. A cluster transfer pathway GLRX5 → ISCA2 → IBA57 was defined. In vitro complex reconstitution, NMR/UV-Vis spectroscopy, site-directed mutagenesis of cysteine ligands, aconitase reactivation assay, exposure of [2Fe-2S] ISCA2 or [2Fe-2S] GLRX5 + apo ISCA2 to IBA57 Journal of the American Chemical Society High 30269484
2019 Low-resolution structural model of the [2Fe-2S]2+ ISCA2-IBA57 complex determined by SAXS and bioinformatics docking shows a dimer-of-dimers organization with ISCA2 providing the homodimerization core; the [2Fe-2S] cluster is located outside the ISCA2 core and is shared with IBA57. The pathogenic IBA57 Arg146Trp mutation disrupts the ISCA2-IBA57 interaction interface. Small-angle X-ray scattering (SAXS), bioinformatics-driven docking, structural modeling Scientific reports Medium 31831856
2021 ISCA1 acts as the key orchestrator of [4Fe-4S] protein maturation by interacting with both ISCA2 and NFU1; ISCA2 and NFU1 do not interact with each other directly. ISCA1 promotes formation of a transient ISCA1-ISCA2-NFU1 ternary complex and drives [4Fe-4S] cluster transfer from the ISCA1-ISCA2 assembly site to NFU1, which then delivers clusters to specific apo proteins. NMR-based interaction studies, characterization of binary and ternary complexes, cluster transfer assays Journal of molecular biology High 33711344
2021 [2Fe-2S]-cluster-bound forms of human ISCA2 (and ISCU) were found capable of reconstituting human lipoyl synthase (LIAS) and enabling complete product turnover, identifying ISCA2 as a primary cluster donor to LIAS in vitro. In vitro reconstitution of human LIAS with purified cluster donor proteins, LC-MS activity assay, EPR spectroscopy International journal of molecular sciences Medium 33562493
2010 In fission yeast, Grx5 (monothiol glutaredoxin) physically interacts with Isa1 and Isa2 proteins in mitochondria, as demonstrated by bimolecular fluorescence complementation; overexpression of isa2+ suppressed growth defects of Δgrx5 mutant and partly restored Fe-S enzyme activities, placing Isa2 downstream of or parallel to Grx5 in the mitochondrial Fe-S assembly pathway. Bimolecular fluorescence complementation (BiFC) for in vivo interaction, multi-copy suppressor screen, growth and enzyme activity assays in S. pombe deletion mutants Biochemical and biophysical research communications Medium 20085751
2007 Yeast Isa2 (and Isa1) are required for in vivo catalytic activity of biotin synthase (Bio2) but not for de novo synthesis of its Fe/S clusters; depletion of Isa proteins reduced Bio2 protein levels, but overexpression of BIO2 did not rescue the desthiobiotin utilization defect, indicating the Isa proteins are essential for Bio2 function beyond cluster assembly. Yeast genetic depletion/deletion, desthiobiotin-to-biotin conversion assays, BIO2 overexpression complementation, Fe/S cluster assembly monitored on Bio2 Eukaryotic cell Medium 17259550
2014 A homoallelic missense mutation in ISCA2 (p.Gly77Ser) causes mitochondrial depletion, reduced complex I activity, and decreased ISCA1 and IBA57 expression in patient fibroblasts, demonstrating that ISCA2 loss of function disrupts the entire late ISC assembly subsystem. Patient fibroblast immunohistochemistry, dipstick enzyme assays for complex I, quantitative PCR for ISCA1/IBA57 expression, transmission electron microscopy Journal of medical genetics Medium 25539947
2018 Loss of ISCA2 in patient cells specifically impairs [4Fe-4S] protein function (aconitase, ETC complex II) but not [2Fe-2S] proteins; ISCA2 deficiency also diminishes mitochondrial membrane potential, respiration, ATP production, and causes mtDNA depletion. Cellular knockdown (siRNA) and patient-derived fibroblasts with Seahorse XF respirometry, mitochondrial membrane potential assays, enzymatic activity assays for [2Fe-2S] vs [4Fe-4S] proteins, mtDNA quantification Human mutation Medium 29297947
2022 ISCA2 knockdown in K562 erythroid cells impairs [4Fe-4S] cluster formation, reduces mitochondrial respiratory chain complex activities, causes ROS accumulation, which then inhibits cytoplasmic aconitase (ACO1/IRP1) Fe-S cluster, converting ACO1 to its IRP1 form; elevated IRP1 activity suppresses ALAS2 (key heme synthesis enzyme) translation via IRE, thereby inhibiting heme synthesis and erythroid differentiation. siRNA knockdown in K562 cells, respiratory chain activity assays, ROS measurement, IRP1/ACO1 activity assays, ALAS2 expression analysis, heme quantification, differentiation assays Biochimica et biophysica acta. Molecular cell research Medium 35714932
2022 ISCA2 inhibition (pharmacological or siRNA) in ccRCC cells decreases HIF-2α protein levels by blocking iron-responsive element (IRE)-dependent translation, and at higher concentrations also decreases HIF-1α; ISCA2 inhibition triggers the iron starvation response, leading to iron/metal overload and ferroptotic cell death. An orally available ISCA2 inhibitor reduced ccRCC xenograft growth in vivo with decreased HIF-α levels and increased lipid peroxidation. High-throughput compound screen, siRNA knockdown, HIF protein quantification, IRE reporter assays, ferroptosis markers, xenograft mouse model Oncogene Medium 36097192
2023 Human ISCA2 (along with ISCA1 and ISCU) has strong copper-binding activity; excess copper binding to these proteins inhibits iron-sulfur cluster assembly, providing a mechanism for copper-induced cytotoxicity and Fe-S enzyme deficiency in Wilson's disease models. In vitro copper-binding assays with purified proteins, Fe-S enzyme activity assays in copper-loaded cells, ATP7A/ATP7B knockout/knockdown cell and mouse models Free radical biology & medicine Medium 37225108
2011 TbIsa1 and TbIsa2 in Trypanosoma brucei are required for assembly of Fe-S clusters in mitochondrial aconitase, fumarase, and succinate dehydrogenase; human Isa (ISCA) orthologues partially rescue TbIsa1/2 knockdown, demonstrating functional conservation. ROS (but not iron) accumulates in TbIsa-depleted mitochondria. RNAi knockdown in T. brucei procyclic form, enzymatic activity assays, heterologous rescue with human ISCA proteins, ROS and iron measurements Molecular microbiology Medium 21790804

Source papers

Stage 0 corpus · 25 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2012 The human mitochondrial ISCA1, ISCA2, and IBA57 proteins are required for [4Fe-4S] protein maturation. Molecular biology of the cell 172 22323289
2000 Role of Saccharomyces cerevisiae ISA1 and ISA2 in iron homeostasis. Molecular and cellular biology 148 10805735
2011 Specialized function of yeast Isa1 and Isa2 proteins in the maturation of mitochondrial [4Fe-4S] proteins. The Journal of biological chemistry 135 21987576
2014 ISCA2 mutation causes infantile neurodegenerative mitochondrial disorder. Journal of medical genetics 80 25539947
2020 Mitochondrial [4Fe-4S] protein assembly involves reductive [2Fe-2S] cluster fusion on ISCA1-ISCA2 by electron flow from ferredoxin FDX2. Proceedings of the National Academy of Sciences of the United States of America 71 32817474
2010 Monothiol glutaredoxin Grx5 interacts with Fe-S scaffold proteins Isa1 and Isa2 and supports Fe-S assembly and DNA integrity in mitochondria of fission yeast. Biochemical and biophysical research communications 53 20085751
2022 ISCA2 inhibition decreases HIF and induces ferroptosis in clear cell renal carcinoma. Oncogene 50 36097192
2007 The ISC [corrected] proteins Isa1 and Isa2 are required for the function but not for the de novo synthesis of the Fe/S clusters of biotin synthase in Saccharomyces cerevisiae. Eukaryotic cell 47 17259550
2018 IBA57 Recruits ISCA2 to Form a [2Fe-2S] Cluster-Mediated Complex. Journal of the American Chemical Society 44 30269484
2023 Copper exerts cytotoxicity through inhibition of iron-sulfur cluster biogenesis on ISCA1/ISCA2/ISCU assembly proteins. Free radical biology & medicine 34 37225108
2011 Stage-specific requirement for Isa1 and Isa2 proteins in the mitochondrion of Trypanosoma brucei and heterologous rescue by human and Blastocystis orthologues. Molecular microbiology 33 21790804
2018 Loss-of-function mutations in ISCA2 disrupt 4Fe-4S cluster machinery and cause a fatal leukodystrophy with hyperglycinemia and mtDNA depletion. Human mutation 24 29297947
2003 A HEAT-repeats containing protein, IaiH, stabilizes the iron-sulfur cluster bound to the cyanobacterial IscA homologue, IscA2. Journal of biochemistry 24 12966069
2017 Further delineation of the phenotypic spectrum of ISCA2 defect: A report of ten new cases. European journal of paediatric neurology : EJPN : official journal of the European Paediatric Neurology Society 23 29122497
2021 Characterization and Reconstitution of Human Lipoyl Synthase (LIAS) Supports ISCA2 and ISCU as Primary Cluster Donors and an Ordered Mechanism of Cluster Assembly. International journal of molecular sciences 22 33562493
2019 Structural properties of [2Fe-2S] ISCA2-IBA57: a complex of the mitochondrial iron-sulfur cluster assembly machinery. Scientific reports 21 31831856
2018 Neonatal mitochondrial leukoencephalopathy with brain and spinal involvement and high lactate: expanding the phenotype of ISCA2 gene mutations. Metabolic brain disease 20 29359243
2017 Simultaneous silencing of isoamylases ISA1, ISA2 and ISA3 by multi-target RNAi in potato tubers leads to decreased starch content and an early sprouting phenotype. PloS one 19 28708852
2019 Multiple Mitochondrial Dysfunctions Syndrome 4 Due to ISCA2 Gene Defects: A Review. Child neurology open 16 31106229
2021 ISCA1 Orchestrates ISCA2 and NFU1 in the Maturation of Human Mitochondrial [4Fe-4S] Proteins. Journal of molecular biology 12 33711344
2019 A novel ISCA2 variant responsible for an early-onset neurodegenerative mitochondrial disorder: a case report of multiple mitochondrial dysfunctions syndrome 4. BMC neurology 10 31279336
2022 ISCA2 deficiency leads to heme synthesis defects and impaired erythroid differentiation in K562 cells by indirect ROS-mediated IRP1 activation. Biochimica et biophysica acta. Molecular cell research 8 35714932
2025 Amylopectin branch trimming and biosynthesis elucidated by the rice isoamylase ISA1-ISA2 heterocomplex. Nature communications 4 40595605
2022 Foliar Application of dsRNA Targeting Endogenous Potato (Solanum tuberosum) Isoamylase Genes ISA1, ISA2, and ISA3 Confers Transgenic Phenotype. International journal of molecular sciences 4 36613634
2024 A novel missense mutation in ISCA2 causes aberrant splicing and leads to multiple mitochondrial dysfunctions syndrome 4. Frontiers in psychiatry 2 39544370

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