{"gene":"SEPHS2","run_date":"2026-06-10T07:46:30","timeline":{"discoveries":[{"year":1997,"finding":"Mouse fetal SPS2 (SEPHS2) encodes a selenophosphate synthetase; the cysteine-substituted mutant (Sec→Cys) retains selenophosphate synthetase activity as measured by selenide-dependent AMP formation from ATP, demonstrating that the active-site selenocysteine/cysteine is catalytically essential.","method":"Baculovirus-insect cell overexpression, purification of Sec→Cys mutant protein, in vitro enzymatic assay (AMP formation from ATP)","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic reconstitution with purified protein and active-site mutagenesis in a single rigorous study","pmids":["9012797"],"is_preprint":false},{"year":1999,"finding":"Mouse SPS2 (SEPHS2) catalyzes selenophosphate synthesis; the Cys-substituted form complements the E. coli selD mutant and has measurable kinetic parameters (Km for ATP = 0.75 mM, Vmax = 9.23 nmol/min/mg). Substitution of Cys with Ala, Ser, or Thr abolishes complementation activity, establishing that a hydroxyl- or thiol-bearing residue at the active site is strictly required.","method":"In vivo complementation assay (E. coli selD mutant), purification of SPS2-CYS, in vitro enzymatic activity measurement, site-directed mutagenesis","journal":"Molecules and cells","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vivo complementation combined with in vitro kinetic assay and multiple active-site substitution mutants in one study","pmids":["10515607"],"is_preprint":false},{"year":2004,"finding":"Human SPS2 (SEPHS2) specifically functions in a selenite assimilation pathway, whereas paralog SPS1 depends on a selenocysteine-recycling (lyase) pathway; this substrate-specificity difference was established by differential complementation of an E. coli selD mutant with selenite vs. L-selenocysteine as selenium source.","method":"In vivo complementation assay in E. coli selD mutant with selenite or L-selenocysteine media; cloning from human lung adenocarcinoma cDNA library; TGA→TGT (Sec→Cys) mutation to allow expression","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic complementation with two distinct substrates distinguishing SPS1 vs SPS2, replicated from prior mouse work, but functional assay indirect (formate dehydrogenase H activity)","pmids":["15534230"],"is_preprint":false},{"year":2017,"finding":"Human SEPHS2 physically interacts with selenocysteine synthase SEPSECS and with paralog SEPHS1 within the selenocysteine biosynthesis machinery in mammalian cells; SEPHS2, SEPHS1, SEPSECS, and SECp43 also form homo- and hetero-oligomers.","method":"Bioluminescence resonance energy transfer (BRET) assay in mammalian cells; co-immunoprecipitation confirmation of SEPHS2–SEPSECS and SEPHS2–SEPHS1 interactions","journal":"Biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two orthogonal methods (BRET + Co-IP) in single lab confirming interactions","pmids":["28414460"],"is_preprint":false},{"year":2025,"finding":"METTL5-mediated 18S rRNA m6A methylation promotes SEPHS2 translation efficiency; depletion of METTL5 reduces SEPHS2 protein levels, decreases selenoprotein synthesis, elevates ROS, and induces apoptosis in multiple myeloma cells, placing SEPHS2 downstream of METTL5 in a translation-selenoprotein-ROS axis.","method":"METTL5 knockdown/knockout in MM cell lines and orthotopic xenograft model; polysome profiling/translation efficiency measurement; ROS assay; apoptosis assay; SEPHS2 rescue experiments","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with mechanistic rescue in vitro and in vivo, single lab","pmids":["40750759"],"is_preprint":false},{"year":2026,"finding":"SEPHS2 loss suppresses oxidative phosphorylation (OXPHOS) and redirects glucose metabolism toward gluconeogenesis and the pentose phosphate pathway (PPP) via a selenoprotein biosynthesis-independent mechanism: SEPHS2 knockout elevates intracellular NAD+ levels, activating deacetylase SIRT2, which promotes deacetylation-dependent stabilization of the gluconeogenic enzyme PCK1. SEPHS2 loss also promotes lung metastasis and sensitizes tumors to the PPP inhibitor 6-aminonicotinamide.","method":"OXPHOS-focused genetic screen; SEPHS2 KO cell lines; metabolic flux analysis; NAD+/NADH measurement; SIRT2 activity assay; PCK1 acetylation/stability assay; in vivo xenograft metastasis model; pharmacological inhibitor treatment","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal metabolic and biochemical assays in single lab with in vivo validation, but not yet independently replicated","pmids":["42035418"],"is_preprint":false},{"year":2024,"finding":"SEPHS2 was identified as a direct binding partner (target protein) of the kinase inhibitor dasatinib in live cells, discovered using the POST-IT proximity-tagging technology.","method":"POST-IT (Pup-On-target for Small molecule Target Identification Technology) non-diffusive proximity tagging in live cells; validated in live zebrafish embryos","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — novel target-ID method applied in single preprint study, no independent replication or mechanistic follow-up of SEPHS2–dasatinib interaction","pmids":["bio_10.1101_2024.09.06.611731"],"is_preprint":true}],"current_model":"SEPHS2 (SPS2) is a selenophosphate synthetase that uses an active-site selenocysteine (or cysteine in mutant forms) to catalyze selenophosphate synthesis from selenide and ATP, preferentially utilizing a selenite-assimilation pathway; it physically interacts with SEPHS1 and SEPSECS within the selenocysteine biosynthesis complex; its translation is promoted by METTL5-dependent rRNA methylation, coupling ribosome activity to selenoprotein synthesis and ROS control; and, independently of selenoprotein biosynthesis, SEPHS2 regulates cellular NAD+ levels to control SIRT2-mediated PCK1 stabilization, thereby linking selenium metabolism to oxidative phosphorylation and gluconeogenesis."},"narrative":{"mechanistic_narrative":"SEPHS2 is a selenophosphate synthetase that supplies the activated selenium donor for selenocysteine biosynthesis, coupling selenium assimilation to selenoprotein production and redox homeostasis [PMID:9012797, PMID:40750759]. The enzyme catalyzes selenide-dependent synthesis of selenophosphate from ATP through a catalytically essential active-site selenocysteine, which can be substituted by cysteine but not by alanine, serine, or threonine without loss of activity, establishing a strict requirement for a thiol/selenol nucleophile [PMID:9012797, PMID:10515607]. Functionally, SEPHS2 specifically serves a selenite-assimilation route, distinguishing it from its paralog SEPHS1, which operates through a selenocysteine-recycling pathway [PMID:15534230]. Within cells SEPHS2 physically associates with the selenocysteine biosynthesis machinery, interacting with SEPSECS and SEPHS1 and forming higher-order oligomers with these factors and SECp43 [PMID:28414460]. Its output is gated at the translational level: METTL5-dependent 18S rRNA m6A methylation promotes SEPHS2 translation, and loss of this input lowers SEPHS2 protein, reduces selenoprotein synthesis, raises ROS, and triggers apoptosis [PMID:40750759]. Independently of selenoprotein biosynthesis, SEPHS2 also constrains a metabolic axis in which its loss elevates intracellular NAD+, activating SIRT2 to deacetylate and stabilize the gluconeogenic enzyme PCK1, thereby suppressing oxidative phosphorylation and redirecting glucose flux toward gluconeogenesis and the pentose phosphate pathway with consequences for metastasis and PPP-inhibitor sensitivity [PMID:42035418].","teleology":[{"year":1997,"claim":"Established that SEPHS2 is a selenophosphate synthetase and that its active-site residue is catalytically essential, defining the enzyme's core chemistry.","evidence":"Baculovirus expression and purification of the Sec→Cys mutant with an in vitro selenide-dependent AMP-formation assay","pmids":["9012797"],"confidence":"High","gaps":["Activity measured on the Sec→Cys mutant rather than the native selenocysteine enzyme","No structural model of the active site"]},{"year":1999,"claim":"Defined kinetic parameters and the strict chemical requirement at the active site by showing only thiol/hydroxyl-bearing residues support catalysis.","evidence":"E. coli selD complementation plus in vitro kinetics and Cys→Ala/Ser/Thr substitution mutants","pmids":["10515607"],"confidence":"High","gaps":["Complementation uses a bacterial surrogate system","Does not address regulation in mammalian cells"]},{"year":2004,"claim":"Resolved how SEPHS2 differs functionally from its paralog SEPHS1 by showing SEPHS2 supports a selenite-assimilation pathway.","evidence":"Differential E. coli selD complementation with selenite versus L-selenocysteine as selenium source","pmids":["15534230"],"confidence":"Medium","gaps":["Functional readout was indirect (formate dehydrogenase H activity)","Used Sec→Cys mutant for expression"]},{"year":2017,"claim":"Placed SEPHS2 within a physical selenocysteine biosynthesis complex, connecting its enzymatic output to downstream Sec synthesis machinery.","evidence":"BRET in mammalian cells with co-immunoprecipitation confirmation of SEPHS2–SEPSECS and SEPHS2–SEPHS1 interactions","pmids":["28414460"],"confidence":"Medium","gaps":["Interaction stoichiometry and complex architecture undefined","Single-lab interaction data"]},{"year":2025,"claim":"Identified a translational control input by showing METTL5-driven rRNA methylation promotes SEPHS2 production, linking ribosome modification to selenoprotein synthesis and ROS control.","evidence":"METTL5 knockdown/knockout with polysome profiling, ROS/apoptosis assays, and SEPHS2 rescue in myeloma cells and xenografts","pmids":["40750759"],"confidence":"Medium","gaps":["Whether SEPHS2 is a direct versus indirect translational target not fully resolved","Generality beyond multiple myeloma untested"]},{"year":2026,"claim":"Uncovered a selenoprotein-independent role for SEPHS2 in metabolic control via an NAD+–SIRT2–PCK1 axis governing OXPHOS and glucose routing.","evidence":"OXPHOS screen, SEPHS2 KO lines, metabolic flux, NAD+ measurement, SIRT2 activity and PCK1 acetylation/stability assays, plus xenograft metastasis and PPP-inhibitor treatment","pmids":["42035418"],"confidence":"Medium","gaps":["Mechanism linking SEPHS2 loss to NAD+ elevation not defined","Not independently replicated"]},{"year":null,"claim":"How the catalytic selenophosphate-synthetase function mechanistically connects to the selenoprotein-independent NAD+/metabolic role remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural mechanism bridging enzymatic and metabolic functions","The molecular event by which SEPHS2 sets NAD+ levels is unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,1]},{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[0,1]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0]}],"localization":[],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[3,4]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[5]}],"complexes":["selenocysteine biosynthesis complex"],"partners":["SEPHS1","SEPSECS","SECP43"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q99611","full_name":"Selenide, water dikinase 2","aliases":["Selenium donor protein 2","Selenophosphate synthase 2"],"length_aa":448,"mass_kda":47.3,"function":"Selenophosphate synthase that generates the selenium donor for selenocysteine biosynthesis by catalyzing formation of selenophosphate from selenide and ATP","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q99611/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/SEPHS2","classification":"Common Essential","n_dependent_lines":832,"n_total_lines":1208,"dependency_fraction":0.6887417218543046},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/SEPHS2","total_profiled":1310},"omim":[{"mim_id":"619597","title":"tRNA SELENOCYSTEINE 1-ASSOCIATED PROTEIN 1; TRNAU1AP","url":"https://www.omim.org/entry/619597"},{"mim_id":"606218","title":"SELENOPHOSPHATE SYNTHETASE 2; SEPHS2","url":"https://www.omim.org/entry/606218"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"liver","ntpm":377.1}],"url":"https://www.proteinatlas.org/search/SEPHS2"},"hgnc":{"alias_symbol":["SPS2","SPS2b"],"prev_symbol":[]},"alphafold":{"accession":"Q99611","domains":[],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q99611","model_url":"","pae_url":"","plddt_mean":null},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SEPHS2","jax_strain_url":"https://www.jax.org/strain/search?query=SEPHS2"},"sequence":{"accession":"Q99611","fasta_url":"https://rest.uniprot.org/uniprotkb/Q99611.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q99611/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q99611"}},"corpus_meta":[{"pmid":"15534230","id":"PMC_15534230","title":"Selenophosphate synthetase genes from lung adenocarcinoma cells: Sps1 for recycling L-selenocysteine and Sps2 for selenite assimilation.","date":"2004","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/15534230","citation_count":63,"is_preprint":false},{"pmid":"9012797","id":"PMC_9012797","title":"Fetal mouse selenophosphate synthetase 2 (SPS2): characterization of the cysteine mutant form overproduced in a baculovirus-insect cell system.","date":"1997","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/9012797","citation_count":47,"is_preprint":false},{"pmid":"31695102","id":"PMC_31695102","title":"Structural analysis of human SEPHS2 protein, a selenocysteine machinery component, over-expressed in triple negative breast cancer.","date":"2019","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/31695102","citation_count":26,"is_preprint":false},{"pmid":"38539903","id":"PMC_38539903","title":"Se Alleviated Pb-Caused Neurotoxicity in Chickens: SPS2-GPx1-GSH-IL-2/IL-17-NO Pathway, Selenoprotein Suppression, Oxidative Stress, and Inflammatory Injury.","date":"2024","source":"Antioxidants (Basel, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/38539903","citation_count":22,"is_preprint":false},{"pmid":"3302678","id":"PMC_3302678","title":"Increased copy number of the 5' end of the SPS2 gene inhibits sporulation of Saccharomyces cerevisiae.","date":"1987","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/3302678","citation_count":21,"is_preprint":false},{"pmid":"28414460","id":"PMC_28414460","title":"Analysis of Novel Interactions between Components of the Selenocysteine Biosynthesis Pathway, SEPHS1, SEPHS2, SEPSECS, and SECp43.","date":"2017","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/28414460","citation_count":18,"is_preprint":false},{"pmid":"36144391","id":"PMC_36144391","title":"Whole-Genome Sequence Analysis of an Endophytic Fungus Alternaria sp. SPS-2 and Its Biosynthetic Potential of Bioactive Secondary Metabolites.","date":"2022","source":"Microorganisms","url":"https://pubmed.ncbi.nlm.nih.gov/36144391","citation_count":11,"is_preprint":false},{"pmid":"10515607","id":"PMC_10515607","title":"Fetal mouse selenophosphate synthetase 2 (SPS2): biological activities of mutant forms in Escherichia coli.","date":"1999","source":"Molecules and cells","url":"https://pubmed.ncbi.nlm.nih.gov/10515607","citation_count":8,"is_preprint":false},{"pmid":"40750759","id":"PMC_40750759","title":"METTL5 regulates SEPHS2-mediated selenoprotein synthesis to promote multiple myeloma survival and progression.","date":"2025","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/40750759","citation_count":5,"is_preprint":false},{"pmid":"41988776","id":"PMC_41988776","title":"Evaluating SEPHS2 expression and glutathione peroxidase as biomarkers in recurrent pregnancy loss: a new insight.","date":"2026","source":"Biomarkers : biochemical indicators of exposure, response, and susceptibility to chemicals","url":"https://pubmed.ncbi.nlm.nih.gov/41988776","citation_count":0,"is_preprint":false},{"pmid":"42035418","id":"PMC_42035418","title":"SEPHS2 loss reprograms cancer metabolism from oxidative phosphorylation to gluconeogenesis via PCK1 stabilization.","date":"2026","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/42035418","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.09.06.611731","title":"Target protein identification in live cells and organisms with a non-diffusive proximity tagging system","date":"2024-09-08","source":"bioRxiv","url":"https://doi.org/10.1101/2024.09.06.611731","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8300,"output_tokens":2031,"usd":0.027682,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9029,"output_tokens":2399,"usd":0.05256,"stage2_stop_reason":"end_turn"},"total_usd":0.080242,"stage1_batch_id":"msgbatch_013LWZZT6MWaqB9qyTSeQBmU","stage2_batch_id":"msgbatch_0194ZzQaTGNpruoNJBbEMbt3","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1997,\n      \"finding\": \"Mouse fetal SPS2 (SEPHS2) encodes a selenophosphate synthetase; the cysteine-substituted mutant (Sec→Cys) retains selenophosphate synthetase activity as measured by selenide-dependent AMP formation from ATP, demonstrating that the active-site selenocysteine/cysteine is catalytically essential.\",\n      \"method\": \"Baculovirus-insect cell overexpression, purification of Sec→Cys mutant protein, in vitro enzymatic assay (AMP formation from ATP)\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic reconstitution with purified protein and active-site mutagenesis in a single rigorous study\",\n      \"pmids\": [\"9012797\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Mouse SPS2 (SEPHS2) catalyzes selenophosphate synthesis; the Cys-substituted form complements the E. coli selD mutant and has measurable kinetic parameters (Km for ATP = 0.75 mM, Vmax = 9.23 nmol/min/mg). Substitution of Cys with Ala, Ser, or Thr abolishes complementation activity, establishing that a hydroxyl- or thiol-bearing residue at the active site is strictly required.\",\n      \"method\": \"In vivo complementation assay (E. coli selD mutant), purification of SPS2-CYS, in vitro enzymatic activity measurement, site-directed mutagenesis\",\n      \"journal\": \"Molecules and cells\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vivo complementation combined with in vitro kinetic assay and multiple active-site substitution mutants in one study\",\n      \"pmids\": [\"10515607\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Human SPS2 (SEPHS2) specifically functions in a selenite assimilation pathway, whereas paralog SPS1 depends on a selenocysteine-recycling (lyase) pathway; this substrate-specificity difference was established by differential complementation of an E. coli selD mutant with selenite vs. L-selenocysteine as selenium source.\",\n      \"method\": \"In vivo complementation assay in E. coli selD mutant with selenite or L-selenocysteine media; cloning from human lung adenocarcinoma cDNA library; TGA→TGT (Sec→Cys) mutation to allow expression\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic complementation with two distinct substrates distinguishing SPS1 vs SPS2, replicated from prior mouse work, but functional assay indirect (formate dehydrogenase H activity)\",\n      \"pmids\": [\"15534230\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Human SEPHS2 physically interacts with selenocysteine synthase SEPSECS and with paralog SEPHS1 within the selenocysteine biosynthesis machinery in mammalian cells; SEPHS2, SEPHS1, SEPSECS, and SECp43 also form homo- and hetero-oligomers.\",\n      \"method\": \"Bioluminescence resonance energy transfer (BRET) assay in mammalian cells; co-immunoprecipitation confirmation of SEPHS2–SEPSECS and SEPHS2–SEPHS1 interactions\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two orthogonal methods (BRET + Co-IP) in single lab confirming interactions\",\n      \"pmids\": [\"28414460\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"METTL5-mediated 18S rRNA m6A methylation promotes SEPHS2 translation efficiency; depletion of METTL5 reduces SEPHS2 protein levels, decreases selenoprotein synthesis, elevates ROS, and induces apoptosis in multiple myeloma cells, placing SEPHS2 downstream of METTL5 in a translation-selenoprotein-ROS axis.\",\n      \"method\": \"METTL5 knockdown/knockout in MM cell lines and orthotopic xenograft model; polysome profiling/translation efficiency measurement; ROS assay; apoptosis assay; SEPHS2 rescue experiments\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with mechanistic rescue in vitro and in vivo, single lab\",\n      \"pmids\": [\"40750759\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"SEPHS2 loss suppresses oxidative phosphorylation (OXPHOS) and redirects glucose metabolism toward gluconeogenesis and the pentose phosphate pathway (PPP) via a selenoprotein biosynthesis-independent mechanism: SEPHS2 knockout elevates intracellular NAD+ levels, activating deacetylase SIRT2, which promotes deacetylation-dependent stabilization of the gluconeogenic enzyme PCK1. SEPHS2 loss also promotes lung metastasis and sensitizes tumors to the PPP inhibitor 6-aminonicotinamide.\",\n      \"method\": \"OXPHOS-focused genetic screen; SEPHS2 KO cell lines; metabolic flux analysis; NAD+/NADH measurement; SIRT2 activity assay; PCK1 acetylation/stability assay; in vivo xenograft metastasis model; pharmacological inhibitor treatment\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal metabolic and biochemical assays in single lab with in vivo validation, but not yet independently replicated\",\n      \"pmids\": [\"42035418\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"SEPHS2 was identified as a direct binding partner (target protein) of the kinase inhibitor dasatinib in live cells, discovered using the POST-IT proximity-tagging technology.\",\n      \"method\": \"POST-IT (Pup-On-target for Small molecule Target Identification Technology) non-diffusive proximity tagging in live cells; validated in live zebrafish embryos\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — novel target-ID method applied in single preprint study, no independent replication or mechanistic follow-up of SEPHS2–dasatinib interaction\",\n      \"pmids\": [\"bio_10.1101_2024.09.06.611731\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"SEPHS2 (SPS2) is a selenophosphate synthetase that uses an active-site selenocysteine (or cysteine in mutant forms) to catalyze selenophosphate synthesis from selenide and ATP, preferentially utilizing a selenite-assimilation pathway; it physically interacts with SEPHS1 and SEPSECS within the selenocysteine biosynthesis complex; its translation is promoted by METTL5-dependent rRNA methylation, coupling ribosome activity to selenoprotein synthesis and ROS control; and, independently of selenoprotein biosynthesis, SEPHS2 regulates cellular NAD+ levels to control SIRT2-mediated PCK1 stabilization, thereby linking selenium metabolism to oxidative phosphorylation and gluconeogenesis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SEPHS2 is a selenophosphate synthetase that supplies the activated selenium donor for selenocysteine biosynthesis, coupling selenium assimilation to selenoprotein production and redox homeostasis [#0, #4]. The enzyme catalyzes selenide-dependent synthesis of selenophosphate from ATP through a catalytically essential active-site selenocysteine, which can be substituted by cysteine but not by alanine, serine, or threonine without loss of activity, establishing a strict requirement for a thiol/selenol nucleophile [#0, #1]. Functionally, SEPHS2 specifically serves a selenite-assimilation route, distinguishing it from its paralog SEPHS1, which operates through a selenocysteine-recycling pathway [#2]. Within cells SEPHS2 physically associates with the selenocysteine biosynthesis machinery, interacting with SEPSECS and SEPHS1 and forming higher-order oligomers with these factors and SECp43 [#3]. Its output is gated at the translational level: METTL5-dependent 18S rRNA m6A methylation promotes SEPHS2 translation, and loss of this input lowers SEPHS2 protein, reduces selenoprotein synthesis, raises ROS, and triggers apoptosis [#4]. Independently of selenoprotein biosynthesis, SEPHS2 also constrains a metabolic axis in which its loss elevates intracellular NAD+, activating SIRT2 to deacetylate and stabilize the gluconeogenic enzyme PCK1, thereby suppressing oxidative phosphorylation and redirecting glucose flux toward gluconeogenesis and the pentose phosphate pathway with consequences for metastasis and PPP-inhibitor sensitivity [#5].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Established that SEPHS2 is a selenophosphate synthetase and that its active-site residue is catalytically essential, defining the enzyme's core chemistry.\",\n      \"evidence\": \"Baculovirus expression and purification of the Sec\\u2192Cys mutant with an in vitro selenide-dependent AMP-formation assay\",\n      \"pmids\": [\"9012797\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Activity measured on the Sec\\u2192Cys mutant rather than the native selenocysteine enzyme\", \"No structural model of the active site\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Defined kinetic parameters and the strict chemical requirement at the active site by showing only thiol/hydroxyl-bearing residues support catalysis.\",\n      \"evidence\": \"E. coli selD complementation plus in vitro kinetics and Cys\\u2192Ala/Ser/Thr substitution mutants\",\n      \"pmids\": [\"10515607\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Complementation uses a bacterial surrogate system\", \"Does not address regulation in mammalian cells\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Resolved how SEPHS2 differs functionally from its paralog SEPHS1 by showing SEPHS2 supports a selenite-assimilation pathway.\",\n      \"evidence\": \"Differential E. coli selD complementation with selenite versus L-selenocysteine as selenium source\",\n      \"pmids\": [\"15534230\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional readout was indirect (formate dehydrogenase H activity)\", \"Used Sec\\u2192Cys mutant for expression\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Placed SEPHS2 within a physical selenocysteine biosynthesis complex, connecting its enzymatic output to downstream Sec synthesis machinery.\",\n      \"evidence\": \"BRET in mammalian cells with co-immunoprecipitation confirmation of SEPHS2\\u2013SEPSECS and SEPHS2\\u2013SEPHS1 interactions\",\n      \"pmids\": [\"28414460\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Interaction stoichiometry and complex architecture undefined\", \"Single-lab interaction data\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified a translational control input by showing METTL5-driven rRNA methylation promotes SEPHS2 production, linking ribosome modification to selenoprotein synthesis and ROS control.\",\n      \"evidence\": \"METTL5 knockdown/knockout with polysome profiling, ROS/apoptosis assays, and SEPHS2 rescue in myeloma cells and xenografts\",\n      \"pmids\": [\"40750759\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether SEPHS2 is a direct versus indirect translational target not fully resolved\", \"Generality beyond multiple myeloma untested\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Uncovered a selenoprotein-independent role for SEPHS2 in metabolic control via an NAD+\\u2013SIRT2\\u2013PCK1 axis governing OXPHOS and glucose routing.\",\n      \"evidence\": \"OXPHOS screen, SEPHS2 KO lines, metabolic flux, NAD+ measurement, SIRT2 activity and PCK1 acetylation/stability assays, plus xenograft metastasis and PPP-inhibitor treatment\",\n      \"pmids\": [\"42035418\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking SEPHS2 loss to NAD+ elevation not defined\", \"Not independently replicated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the catalytic selenophosphate-synthetase function mechanistically connects to the selenoprotein-independent NAD+/metabolic role remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural mechanism bridging enzymatic and metabolic functions\", \"The molecular event by which SEPHS2 sets NAD+ levels is unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [3, 4]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"complexes\": [\"selenocysteine biosynthesis complex\"],\n    \"partners\": [\"SEPHS1\", \"SEPSECS\", \"SECp43\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}