{"gene":"ISCA2","run_date":"2026-06-10T01:55:23","timeline":{"discoveries":[{"year":2012,"finding":"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.","method":"RNA interference knockdown in HeLa cells with enzymatic activity assays for [4Fe-4S] and [2Fe-2S] proteins, mitochondrial morphology analysis by electron microscopy","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean RNAi KD with multiple specific functional readouts, replicated across three proteins, consistent with yeast data from multiple labs","pmids":["22323289"],"is_preprint":false},{"year":2000,"finding":"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.","method":"Deletion analysis and site-directed mutagenesis of leader sequences and conserved cysteines, mitochondrial fractionation, growth complementation assays in yeast deletion mutants","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct localization by fractionation linked to functional consequence via mutagenesis, replicated with multiple mutant alleles","pmids":["10805735"],"is_preprint":false},{"year":2011,"finding":"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.","method":"Comprehensive in vivo analysis in S. cerevisiae including co-immunoprecipitation, iron-binding assays, targeting of bacterial ferredoxins to yeast mitochondria, epistasis with iba57 deletion","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal in vivo methods, replicated functional specificity for [4Fe-4S] vs [2Fe-2S], consistent with mammalian data","pmids":["21987576"],"is_preprint":false},{"year":2020,"finding":"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.","method":"In vitro reconstitution of mitochondrial [4Fe-4S] aconitase maturation without artificial reductants, using purified components; electron transfer assays with FDX1 vs FDX2 comparison","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — full biochemical reconstitution without artificial reductants, multiple component substitutions tested, mechanistic electron transfer pathway defined","pmids":["32817474"],"is_preprint":false},{"year":2018,"finding":"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.","method":"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":"Journal of the American Chemical Society","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstitution with mutagenesis and functional validation (aconitase reactivation), multiple orthogonal methods in one study","pmids":["30269484"],"is_preprint":false},{"year":2019,"finding":"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.","method":"Small-angle X-ray scattering (SAXS), bioinformatics-driven docking, structural modeling","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — low-resolution structural approach (SAXS, no atomic resolution), single lab, but functional validation of disease mutation at interface","pmids":["31831856"],"is_preprint":false},{"year":2021,"finding":"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.","method":"NMR-based interaction studies, characterization of binary and ternary complexes, cluster transfer assays","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structural/interaction data with mechanistic cluster transfer demonstrated, single lab but multiple orthogonal NMR approaches","pmids":["33711344"],"is_preprint":false},{"year":2021,"finding":"[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.","method":"In vitro reconstitution of human LIAS with purified cluster donor proteins, LC-MS activity assay, EPR spectroscopy","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with functional assay, single lab, ISCA2 as donor confirmed by LC-MS product measurement","pmids":["33562493"],"is_preprint":false},{"year":2010,"finding":"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.","method":"Bimolecular fluorescence complementation (BiFC) for in vivo interaction, multi-copy suppressor screen, growth and enzyme activity assays in S. pombe deletion mutants","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct in vivo interaction by BiFC plus epistasis by suppressor assay, single lab","pmids":["20085751"],"is_preprint":false},{"year":2007,"finding":"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.","method":"Yeast genetic depletion/deletion, desthiobiotin-to-biotin conversion assays, BIO2 overexpression complementation, Fe/S cluster assembly monitored on Bio2","journal":"Eukaryotic cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean genetic KD with specific enzymatic readout and overexpression negative control, single lab","pmids":["17259550"],"is_preprint":false},{"year":2014,"finding":"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.","method":"Patient fibroblast immunohistochemistry, dipstick enzyme assays for complex I, quantitative PCR for ISCA1/IBA57 expression, transmission electron microscopy","journal":"Journal of medical genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple functional readouts in patient-derived cells, single lab, mechanistic interpretation supported by prior biochemical work","pmids":["25539947"],"is_preprint":false},{"year":2018,"finding":"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.","method":"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","journal":"Human mutation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal functional readouts in both KD and patient cells, single lab","pmids":["29297947"],"is_preprint":false},{"year":2022,"finding":"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.","method":"siRNA knockdown in K562 cells, respiratory chain activity assays, ROS measurement, IRP1/ACO1 activity assays, ALAS2 expression analysis, heme quantification, differentiation assays","journal":"Biochimica et biophysica acta. Molecular cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal assays defining a signaling pathway from ISCA2 loss to heme deficiency, single lab","pmids":["35714932"],"is_preprint":false},{"year":2022,"finding":"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.","method":"High-throughput compound screen, siRNA knockdown, HIF protein quantification, IRE reporter assays, ferroptosis markers, xenograft mouse model","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological and genetic inhibition with multiple mechanistic readouts including in vivo validation, single lab","pmids":["36097192"],"is_preprint":false},{"year":2023,"finding":"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.","method":"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","journal":"Free radical biology & medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding assays with functional consequence in cell and mouse models, single lab","pmids":["37225108"],"is_preprint":false},{"year":2011,"finding":"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.","method":"RNAi knockdown in T. brucei procyclic form, enzymatic activity assays, heterologous rescue with human ISCA proteins, ROS and iron measurements","journal":"Molecular microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KD with specific enzymatic readouts and cross-species complementation demonstrating functional conservation, single lab","pmids":["21790804"],"is_preprint":false}],"current_model":"ISCA2 is a mitochondrial matrix protein that functions late in the iron-sulfur cluster (ISC) assembly pathway as part of an ISCA1-ISCA2-IBA57 subsystem specifically dedicated to synthesizing and inserting [4Fe-4S] clusters into mitochondrial apo-proteins: [2Fe-2S] clusters are transferred from GLRX5 to ISCA1-ISCA2, where FDX2/FDXR-driven reductive fusion generates [4Fe-4S] clusters in an IBA57-dependent manner; ISCA1 then orchestrates transfer of the [4Fe-4S] cluster to NFU1 via a transient ternary complex for delivery to specific client proteins, while the [2Fe-2S] ISCA2-IBA57 heterodimer (with conserved cysteines as cluster ligands) represents a key intermediate capable of reactivating apo-aconitase; loss of ISCA2 selectively abolishes [4Fe-4S] protein activities, impairs mitochondrial respiration and membrane potential, causes mtDNA depletion, and downstream consequences include ROS-mediated IRP1 activation, suppression of ALAS2/heme synthesis, and HIF-2α translational repression via IRE blockade."},"narrative":{"mechanistic_narrative":"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].","teleology":[{"year":2000,"claim":"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","pmids":["10805735"],"confidence":"High","gaps":["IMS vs matrix localization debated relative to later matrix assignments","did not define the molecular reaction the cysteines support","no human protein studied"]},{"year":2007,"claim":"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","pmids":["17259550"],"confidence":"Medium","gaps":["mechanism beyond cluster assembly not resolved","did not distinguish [2Fe-2S] vs [4Fe-4S] client classes"]},{"year":2010,"claim":"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","pmids":["20085751"],"confidence":"Medium","gaps":["BiFC does not establish direct stoichiometric binding","directionality of cluster flow not biochemically demonstrated here"]},{"year":2011,"claim":"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","pmids":["21987576"],"confidence":"High","gaps":["in vitro cluster fusion chemistry not shown","electron donor unidentified at this stage"]},{"year":2011,"claim":"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","pmids":["21790804"],"confidence":"Medium","gaps":["human protein behavior inferred from rescue rather than direct assay","ROS source not mechanistically dissected"]},{"year":2012,"claim":"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","pmids":["22323289"],"confidence":"High","gaps":["did not resolve the biochemical step ISCA2 catalyzes","cluster donors and acceptors not yet defined"]},{"year":2018,"claim":"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","pmids":["30269484"],"confidence":"High","gaps":["how [2Fe-2S] becomes [4Fe-4S] not addressed here","physiological electron source not tested"]},{"year":2018,"claim":"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","pmids":["29297947"],"confidence":"Medium","gaps":["single lab","causal chain from Fe-S loss to mtDNA depletion not mechanistically traced"]},{"year":2019,"claim":"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","pmids":["31831856"],"confidence":"Medium","gaps":["no atomic-resolution structure","model dependent on docking assumptions"]},{"year":2020,"claim":"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","pmids":["32817474"],"confidence":"High","gaps":["stoichiometry and intermediate states of fusion not fully resolved","downstream client handoff not addressed here"]},{"year":2021,"claim":"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","pmids":["33711344"],"confidence":"High","gaps":["client-selection rules for NFU1 not defined","single lab"]},{"year":2021,"claim":"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","pmids":["33562493"],"confidence":"Medium","gaps":["in vivo relevance of direct ISCA2→LIAS donation vs NFU1 route not resolved","single lab"]},{"year":2022,"claim":"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","pmids":["35714932"],"confidence":"Medium","gaps":["single cell model","direct vs ROS-mediated IRP1 effects not fully separated"]},{"year":2022,"claim":"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","pmids":["36097192"],"confidence":"Medium","gaps":["selectivity of inhibitor for ISCA2 not exhaustively defined","single lab"]},{"year":2023,"claim":"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","pmids":["37225108"],"confidence":"Medium","gaps":["physiological copper occupancy of ISCA2 not quantified","single lab"]},{"year":null,"claim":"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.","evidence":"","pmids":[],"confidence":"Medium","gaps":["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":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[4,7]},{"term_id":"GO:0140104","term_label":"molecular carrier activity","supporting_discovery_ids":[3,6,7]}],"localization":[{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[0,1,11]}],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[0,3,11]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[3,6]}],"complexes":["ISCA1-ISCA2-IBA57 late ISC subsystem","ISCA2-IBA57 [2Fe-2S] heterodimer","ISCA1-ISCA2-NFU1 transient ternary complex"],"partners":["ISCA1","IBA57","GLRX5","NFU1","FDX2","FDXR","LIAS"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q86U28","full_name":"Iron-sulfur cluster assembly 2 homolog, mitochondrial","aliases":["HESB-like domain-containing protein 1"],"length_aa":154,"mass_kda":16.5,"function":"Involved in the maturation of mitochondrial 4Fe-4S proteins functioning late in the iron-sulfur cluster assembly pathway. May be involved in the binding of an intermediate of Fe/S cluster assembly","subcellular_location":"Mitochondrion","url":"https://www.uniprot.org/uniprotkb/Q86U28/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/ISCA2","classification":"Common Essential","n_dependent_lines":921,"n_total_lines":1208,"dependency_fraction":0.7624172185430463},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/ISCA2","total_profiled":1310},"omim":[{"mim_id":"616370","title":"MULTIPLE MITOCHONDRIAL DYSFUNCTIONS SYNDROME 4; MMDS4","url":"https://www.omim.org/entry/616370"},{"mim_id":"615330","title":"MULTIPLE MITOCHONDRIAL DYSFUNCTIONS SYNDROME 3; MMDS3","url":"https://www.omim.org/entry/615330"},{"mim_id":"615317","title":"IRON-SULFUR CLUSTER ASSEMBLY 2; ISCA2","url":"https://www.omim.org/entry/615317"},{"mim_id":"615316","title":"IRON-SULFUR CLUSTER ASSEMBLY FACTOR IBA57; IBA57","url":"https://www.omim.org/entry/615316"},{"mim_id":"611006","title":"IRON-SULFUR CLUSTER ASSEMBLY 1; ISCA1","url":"https://www.omim.org/entry/611006"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/ISCA2"},"hgnc":{"alias_symbol":["ISA2"],"prev_symbol":["HBLD1"]},"alphafold":{"accession":"Q86U28","domains":[{"cath_id":"2.60.300.12","chopping":"52-153","consensus_level":"high","plddt":90.576,"start":52,"end":153}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q86U28","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q86U28-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q86U28-F1-predicted_aligned_error_v6.png","plddt_mean":77.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ISCA2","jax_strain_url":"https://www.jax.org/strain/search?query=ISCA2"},"sequence":{"accession":"Q86U28","fasta_url":"https://rest.uniprot.org/uniprotkb/Q86U28.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q86U28/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q86U28"}},"corpus_meta":[{"pmid":"22323289","id":"PMC_22323289","title":"The human mitochondrial ISCA1, ISCA2, and IBA57 proteins are required for [4Fe-4S] protein maturation.","date":"2012","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/22323289","citation_count":172,"is_preprint":false},{"pmid":"10805735","id":"PMC_10805735","title":"Role of Saccharomyces cerevisiae ISA1 and ISA2 in iron homeostasis.","date":"2000","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/10805735","citation_count":148,"is_preprint":false},{"pmid":"21987576","id":"PMC_21987576","title":"Specialized function of yeast Isa1 and Isa2 proteins in the maturation of mitochondrial [4Fe-4S] proteins.","date":"2011","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/21987576","citation_count":135,"is_preprint":false},{"pmid":"25539947","id":"PMC_25539947","title":"ISCA2 mutation causes infantile neurodegenerative mitochondrial disorder.","date":"2014","source":"Journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/25539947","citation_count":80,"is_preprint":false},{"pmid":"32817474","id":"PMC_32817474","title":"Mitochondrial [4Fe-4S] protein assembly involves reductive [2Fe-2S] cluster fusion on ISCA1-ISCA2 by electron flow from ferredoxin FDX2.","date":"2020","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/32817474","citation_count":71,"is_preprint":false},{"pmid":"20085751","id":"PMC_20085751","title":"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.","date":"2010","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/20085751","citation_count":53,"is_preprint":false},{"pmid":"36097192","id":"PMC_36097192","title":"ISCA2 inhibition decreases HIF and induces ferroptosis in clear cell renal 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medicine","url":"https://pubmed.ncbi.nlm.nih.gov/37225108","citation_count":34,"is_preprint":false},{"pmid":"21790804","id":"PMC_21790804","title":"Stage-specific requirement for Isa1 and Isa2 proteins in the mitochondrion of Trypanosoma brucei and heterologous rescue by human and Blastocystis orthologues.","date":"2011","source":"Molecular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/21790804","citation_count":33,"is_preprint":false},{"pmid":"29297947","id":"PMC_29297947","title":"Loss-of-function mutations in ISCA2 disrupt 4Fe-4S cluster machinery and cause a fatal leukodystrophy with hyperglycinemia and mtDNA depletion.","date":"2018","source":"Human mutation","url":"https://pubmed.ncbi.nlm.nih.gov/29297947","citation_count":24,"is_preprint":false},{"pmid":"12966069","id":"PMC_12966069","title":"A HEAT-repeats containing protein, IaiH, stabilizes the iron-sulfur cluster bound to the cyanobacterial IscA homologue, IscA2.","date":"2003","source":"Journal of biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12966069","citation_count":24,"is_preprint":false},{"pmid":"29122497","id":"PMC_29122497","title":"Further delineation of the phenotypic spectrum of ISCA2 defect: A report of ten new cases.","date":"2017","source":"European journal of paediatric neurology : EJPN : official journal of the European Paediatric Neurology Society","url":"https://pubmed.ncbi.nlm.nih.gov/29122497","citation_count":23,"is_preprint":false},{"pmid":"33562493","id":"PMC_33562493","title":"Characterization and Reconstitution of Human Lipoyl Synthase (LIAS) Supports ISCA2 and ISCU as Primary Cluster Donors and an Ordered Mechanism of Cluster Assembly.","date":"2021","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/33562493","citation_count":22,"is_preprint":false},{"pmid":"31831856","id":"PMC_31831856","title":"Structural properties of [2Fe-2S] ISCA2-IBA57: a complex of the mitochondrial iron-sulfur cluster assembly 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Molecular cell research","url":"https://pubmed.ncbi.nlm.nih.gov/35714932","citation_count":8,"is_preprint":false},{"pmid":"40595605","id":"PMC_40595605","title":"Amylopectin branch trimming and biosynthesis elucidated by the rice isoamylase ISA1-ISA2 heterocomplex.","date":"2025","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/40595605","citation_count":4,"is_preprint":false},{"pmid":"36613634","id":"PMC_36613634","title":"Foliar Application of dsRNA Targeting Endogenous Potato (Solanum tuberosum) Isoamylase Genes ISA1, ISA2, and ISA3 Confers Transgenic Phenotype.","date":"2022","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/36613634","citation_count":4,"is_preprint":false},{"pmid":"39544370","id":"PMC_39544370","title":"A novel missense mutation in ISCA2 causes aberrant splicing and leads to multiple mitochondrial dysfunctions syndrome 4.","date":"2024","source":"Frontiers in psychiatry","url":"https://pubmed.ncbi.nlm.nih.gov/39544370","citation_count":2,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.06.23.661224","title":"LIKE EARLY STARVATION is involved in the regulation of starch initiation in potato (  <i>Solanum tuberosum</i>  cv. Désirée) tubers","date":"2025-06-24","source":"bioRxiv","url":"https://doi.org/10.1101/2025.06.23.661224","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":15440,"output_tokens":4485,"usd":0.056798,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12643,"output_tokens":4506,"usd":0.087932,"stage2_stop_reason":"end_turn"},"total_usd":0.14473,"stage1_batch_id":"msgbatch_01DUPACZi7AAWwuZiUjseQF4","stage2_batch_id":"msgbatch_018yt7chE3f48PEFtDwtj6AE","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2012,\n      \"finding\": \"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.\",\n      \"method\": \"RNA interference knockdown in HeLa cells with enzymatic activity assays for [4Fe-4S] and [2Fe-2S] proteins, mitochondrial morphology analysis by electron microscopy\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean RNAi KD with multiple specific functional readouts, replicated across three proteins, consistent with yeast data from multiple labs\",\n      \"pmids\": [\"22323289\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"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.\",\n      \"method\": \"Deletion analysis and site-directed mutagenesis of leader sequences and conserved cysteines, mitochondrial fractionation, growth complementation assays in yeast deletion mutants\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct localization by fractionation linked to functional consequence via mutagenesis, replicated with multiple mutant alleles\",\n      \"pmids\": [\"10805735\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"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.\",\n      \"method\": \"Comprehensive in vivo analysis in S. cerevisiae including co-immunoprecipitation, iron-binding assays, targeting of bacterial ferredoxins to yeast mitochondria, epistasis with iba57 deletion\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal in vivo methods, replicated functional specificity for [4Fe-4S] vs [2Fe-2S], consistent with mammalian data\",\n      \"pmids\": [\"21987576\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"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.\",\n      \"method\": \"In vitro reconstitution of mitochondrial [4Fe-4S] aconitase maturation without artificial reductants, using purified components; electron transfer assays with FDX1 vs FDX2 comparison\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — full biochemical reconstitution without artificial reductants, multiple component substitutions tested, mechanistic electron transfer pathway defined\",\n      \"pmids\": [\"32817474\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"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.\",\n      \"method\": \"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\",\n      \"journal\": \"Journal of the American Chemical Society\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstitution with mutagenesis and functional validation (aconitase reactivation), multiple orthogonal methods in one study\",\n      \"pmids\": [\"30269484\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"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.\",\n      \"method\": \"Small-angle X-ray scattering (SAXS), bioinformatics-driven docking, structural modeling\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — low-resolution structural approach (SAXS, no atomic resolution), single lab, but functional validation of disease mutation at interface\",\n      \"pmids\": [\"31831856\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"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.\",\n      \"method\": \"NMR-based interaction studies, characterization of binary and ternary complexes, cluster transfer assays\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structural/interaction data with mechanistic cluster transfer demonstrated, single lab but multiple orthogonal NMR approaches\",\n      \"pmids\": [\"33711344\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"[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.\",\n      \"method\": \"In vitro reconstitution of human LIAS with purified cluster donor proteins, LC-MS activity assay, EPR spectroscopy\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with functional assay, single lab, ISCA2 as donor confirmed by LC-MS product measurement\",\n      \"pmids\": [\"33562493\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"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.\",\n      \"method\": \"Bimolecular fluorescence complementation (BiFC) for in vivo interaction, multi-copy suppressor screen, growth and enzyme activity assays in S. pombe deletion mutants\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct in vivo interaction by BiFC plus epistasis by suppressor assay, single lab\",\n      \"pmids\": [\"20085751\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"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.\",\n      \"method\": \"Yeast genetic depletion/deletion, desthiobiotin-to-biotin conversion assays, BIO2 overexpression complementation, Fe/S cluster assembly monitored on Bio2\",\n      \"journal\": \"Eukaryotic cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean genetic KD with specific enzymatic readout and overexpression negative control, single lab\",\n      \"pmids\": [\"17259550\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"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.\",\n      \"method\": \"Patient fibroblast immunohistochemistry, dipstick enzyme assays for complex I, quantitative PCR for ISCA1/IBA57 expression, transmission electron microscopy\",\n      \"journal\": \"Journal of medical genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple functional readouts in patient-derived cells, single lab, mechanistic interpretation supported by prior biochemical work\",\n      \"pmids\": [\"25539947\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"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.\",\n      \"method\": \"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\",\n      \"journal\": \"Human mutation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal functional readouts in both KD and patient cells, single lab\",\n      \"pmids\": [\"29297947\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"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.\",\n      \"method\": \"siRNA knockdown in K562 cells, respiratory chain activity assays, ROS measurement, IRP1/ACO1 activity assays, ALAS2 expression analysis, heme quantification, differentiation assays\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal assays defining a signaling pathway from ISCA2 loss to heme deficiency, single lab\",\n      \"pmids\": [\"35714932\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"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.\",\n      \"method\": \"High-throughput compound screen, siRNA knockdown, HIF protein quantification, IRE reporter assays, ferroptosis markers, xenograft mouse model\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological and genetic inhibition with multiple mechanistic readouts including in vivo validation, single lab\",\n      \"pmids\": [\"36097192\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"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.\",\n      \"method\": \"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\",\n      \"journal\": \"Free radical biology & medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding assays with functional consequence in cell and mouse models, single lab\",\n      \"pmids\": [\"37225108\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"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.\",\n      \"method\": \"RNAi knockdown in T. brucei procyclic form, enzymatic activity assays, heterologous rescue with human ISCA proteins, ROS and iron measurements\",\n      \"journal\": \"Molecular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KD with specific enzymatic readouts and cross-species complementation demonstrating functional conservation, single lab\",\n      \"pmids\": [\"21790804\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ISCA2 is a mitochondrial matrix protein that functions late in the iron-sulfur cluster (ISC) assembly pathway as part of an ISCA1-ISCA2-IBA57 subsystem specifically dedicated to synthesizing and inserting [4Fe-4S] clusters into mitochondrial apo-proteins: [2Fe-2S] clusters are transferred from GLRX5 to ISCA1-ISCA2, where FDX2/FDXR-driven reductive fusion generates [4Fe-4S] clusters in an IBA57-dependent manner; ISCA1 then orchestrates transfer of the [4Fe-4S] cluster to NFU1 via a transient ternary complex for delivery to specific client proteins, while the [2Fe-2S] ISCA2-IBA57 heterodimer (with conserved cysteines as cluster ligands) represents a key intermediate capable of reactivating apo-aconitase; loss of ISCA2 selectively abolishes [4Fe-4S] protein activities, impairs mitochondrial respiration and membrane potential, causes mtDNA depletion, and downstream consequences include ROS-mediated IRP1 activation, suppression of ALAS2/heme synthesis, and HIF-2α translational repression via IRE blockade.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"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 [#0, #2]. 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 [#3]. 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 [#4, #1]. 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 [#6, #7]. Loss of ISCA2 selectively abolishes [4Fe-4S] enzyme activities, collapses mitochondrial respiration and membrane potential, and causes mtDNA depletion [#11], with downstream consequences including ROS-driven IRP1 activation that suppresses ALAS2/heme synthesis in erythroid cells [#12] and IRE-dependent blockade of HIF-2\\u03b1 translation in clear cell renal carcinoma [#13]. A homoallelic ISCA2 missense mutation (p.Gly77Ser) causes a mitochondrial disease characterized by destabilization of the entire late ISC subsystem [#10].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Established where the ISCA2 ortholog resides and which residues matter, defining it as a mitochondrial protein whose conserved cysteines are functionally essential.\",\n      \"evidence\": \"Leader-sequence deletion analysis, cysteine mutagenesis, and mitochondrial fractionation in yeast\",\n      \"pmids\": [\"10805735\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"IMS vs matrix localization debated relative to later matrix assignments\", \"did not define the molecular reaction the cysteines support\", \"no human protein studied\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Began defining client specificity by showing Isa proteins are needed for biotin synthase activity beyond simple cluster assembly.\",\n      \"evidence\": \"Genetic depletion and desthiobiotin-to-biotin conversion assays with BIO2 overexpression control in yeast\",\n      \"pmids\": [\"17259550\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"mechanism beyond cluster assembly not resolved\", \"did not distinguish [2Fe-2S] vs [4Fe-4S] client classes\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Placed ISCA2 relative to the monothiol glutaredoxin by showing Grx5 physically interacts with Isa1/Isa2 and Isa2 acts downstream or parallel.\",\n      \"evidence\": \"BiFC interaction and multi-copy suppressor/enzyme assays in fission yeast\",\n      \"pmids\": [\"20085751\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"BiFC does not establish direct stoichiometric binding\", \"directionality of cluster flow not biochemically demonstrated here\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"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.\",\n      \"evidence\": \"Co-IP, in vivo iron-binding, ferredoxin targeting, and iba57 epistasis in S. cerevisiae\",\n      \"pmids\": [\"21987576\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"in vitro cluster fusion chemistry not shown\", \"electron donor unidentified at this stage\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Demonstrated functional conservation of the ISCA system across eukaryotes and that depletion causes ROS rather than iron accumulation.\",\n      \"evidence\": \"RNAi in T. brucei with enzymatic assays and heterologous rescue by human ISCA proteins\",\n      \"pmids\": [\"21790804\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"human protein behavior inferred from rescue rather than direct assay\", \"ROS source not mechanistically dissected\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"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.\",\n      \"evidence\": \"RNAi in HeLa cells with [4Fe-4S]/[2Fe-2S] enzyme assays and EM\",\n      \"pmids\": [\"22323289\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"did not resolve the biochemical step ISCA2 catalyzes\", \"cluster donors and acceptors not yet defined\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"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\\u2192ISCA2\\u2192IBA57 transfer route.\",\n      \"evidence\": \"In vitro reconstitution, spectroscopy, cysteine mutagenesis, and aconitase reactivation\",\n      \"pmids\": [\"30269484\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"how [2Fe-2S] becomes [4Fe-4S] not addressed here\", \"physiological electron source not tested\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"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.\",\n      \"evidence\": \"siRNA and patient fibroblasts with respirometry, membrane potential, and mtDNA assays\",\n      \"pmids\": [\"29297947\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"single lab\", \"causal chain from Fe-S loss to mtDNA depletion not mechanistically traced\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Provided a low-resolution architecture of the [2Fe-2S] ISCA2-IBA57 complex and linked a disease mutation to the interaction interface.\",\n      \"evidence\": \"SAXS and bioinformatics docking with disease-mutation interface analysis\",\n      \"pmids\": [\"31831856\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"no atomic-resolution structure\", \"model dependent on docking assumptions\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"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.\",\n      \"evidence\": \"Full in vitro reconstitution of [4Fe-4S] aconitase maturation without artificial reductants, FDX1 vs FDX2 comparison\",\n      \"pmids\": [\"32817474\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"stoichiometry and intermediate states of fusion not fully resolved\", \"downstream client handoff not addressed here\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"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.\",\n      \"evidence\": \"NMR interaction studies and cluster transfer assays of binary/ternary complexes\",\n      \"pmids\": [\"33711344\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"client-selection rules for NFU1 not defined\", \"single lab\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identified ISCA2 as a direct cluster donor to lipoyl synthase, broadening its acceptor repertoire.\",\n      \"evidence\": \"In vitro reconstitution of human LIAS with LC-MS turnover and EPR\",\n      \"pmids\": [\"33562493\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"in vivo relevance of direct ISCA2\\u2192LIAS donation vs NFU1 route not resolved\", \"single lab\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Connected ISCA2 loss to systemic iron/heme signaling by showing ROS-driven IRP1 activation suppresses ALAS2 translation and blocks erythroid heme synthesis.\",\n      \"evidence\": \"siRNA in K562 cells with IRP1/ACO1 activity, ALAS2, heme, and differentiation assays\",\n      \"pmids\": [\"35714932\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"single cell model\", \"direct vs ROS-mediated IRP1 effects not fully separated\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Revealed a therapeutic vulnerability: ISCA2 inhibition blocks IRE-dependent HIF-\\u03b1 translation and triggers ferroptosis in renal carcinoma in vitro and in vivo.\",\n      \"evidence\": \"Compound screen, siRNA, IRE reporters, ferroptosis markers, and ccRCC xenografts\",\n      \"pmids\": [\"36097192\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"selectivity of inhibitor for ISCA2 not exhaustively defined\", \"single lab\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identified copper as an inhibitor of ISCA2-dependent Fe-S assembly, linking the protein to copper toxicity in Wilson's disease models.\",\n      \"evidence\": \"In vitro copper-binding assays and Fe-S activity in copper-loaded cells and ATP7A/B models\",\n      \"pmids\": [\"37225108\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"physiological copper occupancy of ISCA2 not quantified\", \"single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How client specificity is encoded \\u2014 which apo-proteins receive clusters directly from ISCA2 versus via NFU1, and how the system is regulated by metal competition in vivo \\u2014 remains open.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"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\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [4, 7]},\n      {\"term_id\": \"GO:0140104\", \"supporting_discovery_ids\": [3, 6, 7]},\n      {\"term_id\": \"GO:0005506\", \"supporting_discovery_ids\": [2, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [0, 1, 11]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [0, 3, 11]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [3, 6]}\n    ],\n    \"complexes\": [\n      \"ISCA1-ISCA2-IBA57 late ISC subsystem\",\n      \"ISCA2-IBA57 [2Fe-2S] heterodimer\",\n      \"ISCA1-ISCA2-NFU1 transient ternary complex\"\n    ],\n    \"partners\": [\n      \"ISCA1\",\n      \"IBA57\",\n      \"GLRX5\",\n      \"NFU1\",\n      \"FDX2\",\n      \"FDXR\",\n      \"LIAS\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}