{"gene":"MPST","run_date":"2026-06-10T02:59:51","timeline":{"discoveries":[{"year":2014,"finding":"Human TUM1/MPST exists as two splice isoforms with distinct subcellular localizations: TUM1-Iso1 is exclusively cytosolic, while TUM1-Iso2 localizes to both cytosol and mitochondria. Both isoforms catalyze conversion of 3-mercaptopyruvate to pyruvate and protein-bound persulfide with similar kinetics. TUM1 physically interacts with the L-cysteine desulfurase NFS1 and the rhodanese-like protein MOCS3, implicating it in molybdenum cofactor biosynthesis and cytosolic tRNA thiolation.","method":"Purification and kinetic characterization of recombinant isoforms; cellular localization by fluorescence microscopy; in vitro pulldown and in vivo split-EGFP interaction assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro enzymatic assay with purified proteins, direct subcellular localization imaging, and two orthogonal interaction methods (in vitro pulldown + split-EGFP in cells)","pmids":["25336638"],"is_preprint":false},{"year":2023,"finding":"Human TUM1/MPST is required for molybdenum cofactor (Moco) biosynthesis and cytosolic tRNA thiomodification in human cells, as demonstrated by reduced sulfite oxidase activity and decreased sulfur-modified tRNA levels in TUM1 CRISPR knockout HEK293 cells. TUM1 knockout also reduces H2S production and impairs cellular bioenergetics.","method":"CRISPR/Cas9 knockout in HEK293 cells; spectrophotometric sulfite oxidase activity assay; LC quantification of sulfur-modified tRNA; H2S measurement","journal":"Biomolecules","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean CRISPR KO with multiple orthogonal functional readouts in a single lab","pmids":["36671528"],"is_preprint":false},{"year":2006,"finding":"A nonsense mutation (Tyr85Stop) in human MPST produces a severely truncated protein that lacks enzymatic activity, as shown by in vitro heterologous expression and measurement of erythrocyte MPST activity, establishing that this residue is required for enzymatic function.","method":"Screening of 50 individuals; in vitro heterologous expression; transient transfection assay; erythrocyte MPST activity measurement","journal":"Toxicology letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro expression with activity assay plus in vivo erythrocyte activity, single lab","pmids":["16545926"],"is_preprint":false},{"year":2015,"finding":"MPST, but not CSE, is the primary source of H2S in coronary artery; rat coronary artery homogenates produce H2S via the MPST pathway using 3-mercaptopyruvate as substrate. MPST-derived H2S mediates coronary vasoconstriction in the presence of NO and vasorelaxation in its absence, acting through direct chemical reaction between H2S and NO.","method":"In vitro H2S production assay from coronary artery homogenates; ex vivo vasoreactivity experiments with 3-mercaptopyruvate; CSE knockout mouse in vivo coronary vasoreactivity; in vitro H2S–NO reaction assay","journal":"American journal of physiology. Heart and circulatory physiology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro enzymatic assay, ex vivo organ bath pharmacology, genetic (CSE KO) controls, and in vitro chemical mechanism, multiple orthogonal methods","pmids":["26519030"],"is_preprint":false},{"year":2022,"finding":"MPST directly interacts with AKT and reduces AKT phosphorylation in intestinal epithelial cells. MPST deficiency increases AKT-dependent apoptosis; overexpression of AKT rescues apoptosis caused by MPST loss, while AKT inhibition worsens it, placing MPST upstream of AKT in an anti-apoptotic pathway.","method":"Co-immunoprecipitation (direct interaction with AKT); phosphorylation assay; RNA-seq; siRNA knockdown and overexpression in HT29 cells; AKT overexpression rescue and AKT inhibitor epistasis; in vivo DSS colitis mouse model","journal":"Redox biology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP for interaction, epistasis by OE/inhibition, single lab with multiple approaches","pmids":["36126419"],"is_preprint":false},{"year":2022,"finding":"MPST deletion in mice causes impaired mitochondrial protein import by activating HIF1α and downregulating TIM/TOM translocase complex subunits, leading to suppression of the TCA cycle, oxidative phosphorylation, and fatty acid oxidation, and resulting in enhanced fat accumulation on high-fat diet. Sulfide donor administration reverses these changes.","method":"Mpst knockout mouse (HFD model); transcriptional and metabolic analysis; Western blot for TIM/TOM subunits; metabolic flux/respiratory measurements; sulfide donor pharmacological rescue","journal":"The Journal of experimental medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO mouse with defined molecular phenotype (TIM/TOM downregulation, metabolic measurements), pharmacological rescue, single lab","pmids":["35616614"],"is_preprint":false},{"year":2023,"finding":"Genetic double knockout of CTH and MPST in mice leads to paradoxical enhanced endothelium-dependent vasorelaxation and reduced blood pressure, associated with compensatory upregulation of eNOS and soluble guanylate cyclase (sGC) α1 and β1 subunits, revealing that chronic H2S deficiency adaptively upregulates the NO/cGMP pathway.","method":"Double Cth/Mpst knockout mouse; aortic ring relaxation assay; blood pressure telemetry; Western blot for eNOS and sGC subunits; NOS inhibitor pharmacology","journal":"Frontiers in pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic double KO with multiple vascular functional readouts and molecular quantification, single lab","pmids":["36860295"],"is_preprint":false},{"year":2012,"finding":"Crystal structure of yeast Tum1 (MPST ortholog) at 1.90 Å resolution reveals two rhodanese-like domains (RLDs) but only one conserved active-site cysteine in the C-terminal RLD. An unidentified electron density near the active site suggests a potential cofactor in the sulfur transfer mechanism.","method":"X-ray crystallography at 1.90 Å resolution","journal":"Protein and peptide letters","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — crystal structure determined but functional validation of active-site cysteine not performed in this study; single paper","pmids":["22587783"],"is_preprint":false},{"year":2017,"finding":"Yeast Tum1 (ortholog of mammalian MPST) is involved in regulating sterol ester content; tum1Δ strains accumulate significantly higher sterol esters. This function is independent of the tRNA thiolation pathway (overexpression of thiolated tRNAs or deletion of UBA4 did not affect sterol ester content), indicating a distinct lipid metabolic role for Tum1.","method":"Yeast gene deletion (tum1Δ); lipid quantification; genetic epistasis with tRNA thiolation pathway mutants (uba4Δ); tRNA overexpression","journal":"BMC microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean deletion strain with lipid quantification and epistasis analysis, single lab","pmids":["28830344"],"is_preprint":false},{"year":2021,"finding":"Inhibition of 3-MPST with siRNA-mediated knockdown in primary rat astrocytes significantly increases acrylonitrile-induced cytotoxicity, while overexpression of 3-MPST attenuates it, demonstrating that the CBS/3-MPST-H2S pathway protects astrocytes against acrylonitrile toxicity.","method":"siRNA knockdown and overexpression of 3-MPST in primary rat astrocytes; cell viability assay; H2S content measurement; in vivo rat AN exposure model","journal":"Toxicology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — siRNA KD and OE with defined cytotoxicity readout, corroborated by in vivo measurements, single lab","pmids":["33486070"],"is_preprint":false},{"year":2025,"finding":"RNAi-mediated knockdown of MPST in neonatal patient-derived airway epithelial cells (nTAECs) during air-liquid interface differentiation reduces ciliated cell numbers at mid-differentiation, suppresses mitochondrial respiration without compensatory glycolysis increase, and induces early transcriptomic shifts in mitochondrial metabolic and epithelial differentiation programs, establishing MPST as required for mitochondrial metabolic integrity during airway epithelial development.","method":"RNAi knockdown in neonatal patient-derived tracheal airway epithelial cells (3D ALI model); RNA-seq; metabolic flux analysis (Seahorse); immunofluorescence for ciliated cell markers","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — multiple orthogonal methods (transcriptomics, metabolic flux, cell differentiation readout) but preprint, single lab, no replication","pmids":["41509323"],"is_preprint":true}],"current_model":"MPST (TUM1) is a mitochondrial/cytosolic sulfurtransferase that catalyzes conversion of 3-mercaptopyruvate to pyruvate and protein-bound persulfide, generating H2S; it exists as two splice isoforms with distinct compartmentalization, interacts with NFS1 and MOCS3 to contribute sulfur for molybdenum cofactor biosynthesis and cytosolic tRNA thiolation, serves as the primary H2S source in coronary artery where H2S modulates vasoreactivity via NO, directly binds AKT to suppress its phosphorylation and protect intestinal epithelial cells from apoptosis, and maintains mitochondrial protein import and oxidative metabolism in adipose and airway epithelial tissues."},"narrative":{"mechanistic_narrative":"MPST (TUM1) is a rhodanese-family 3-mercaptopyruvate sulfurtransferase that converts 3-mercaptopyruvate to pyruvate and a protein-bound persulfide, providing a major enzymatic source of H2S and reactive sulfur in mammalian cells [PMID:25336638, PMID:26519030]. It is expressed as two splice isoforms with distinct compartmentalization—a cytosolic-only form and a form distributed between cytosol and mitochondria—both of which are catalytically active [PMID:25336638]. Through physical interaction with the cysteine desulfurase NFS1 and the rhodanese-like protein MOCS3, MPST channels sulfur into molybdenum cofactor biosynthesis and cytosolic tRNA thiomodification, and its loss reduces sulfite oxidase activity, sulfur-modified tRNA levels, H2S output, and cellular bioenergetics [PMID:25336638, PMID:36671528]. The persulfide/H2S it generates supports mitochondrial function: MPST loss activates HIF1α, downregulates TIM/TOM import translocase subunits, and suppresses the TCA cycle, oxidative phosphorylation, and fatty acid oxidation, a defect reversible by sulfide donors [PMID:35616614]. MPST-derived H2S is the principal sulfide source in coronary artery, where it modulates vasoreactivity through direct chemical reaction with NO [PMID:26519030], and chronic combined H2S deficiency adaptively upregulates the NO/cGMP pathway [PMID:36860295]. MPST also directly binds AKT to suppress its phosphorylation, protecting intestinal epithelial cells from apoptosis [PMID:36126419]. A nonsense mutation (Tyr85Stop) abolishes enzymatic activity, defining residues required for catalysis [PMID:16545926].","teleology":[{"year":2006,"claim":"Established that specific protein sequence is required for MPST catalytic activity by showing a truncating mutation abolishes function, linking genotype to enzymatic loss.","evidence":"Heterologous expression of a Tyr85Stop variant plus erythrocyte MPST activity measurement","pmids":["16545926"],"confidence":"Medium","gaps":["No structure-function mapping of which catalytic step the truncation eliminates","Clinical phenotype of carriers not defined"]},{"year":2012,"claim":"Resolved the structural architecture of the MPST ortholog, showing two rhodanese-like domains with a single active-site cysteine in the C-terminal domain that mediates sulfur transfer.","evidence":"X-ray crystallography of yeast Tum1 at 1.90 Å","pmids":["22587783"],"confidence":"Medium","gaps":["Functional validation of the active-site cysteine not performed","Identity of unassigned electron density near the active site unknown"]},{"year":2014,"claim":"Defined MPST isoform compartmentalization and its physical partners, establishing it as a dual cytosolic/mitochondrial sulfurtransferase feeding sulfur-trafficking complexes.","evidence":"Recombinant isoform kinetics, fluorescence localization, in vitro pulldown and split-EGFP interaction with NFS1 and MOCS3","pmids":["25336638"],"confidence":"High","gaps":["Stoichiometry and regulation of the NFS1/MOCS3 sulfur-relay not resolved","Functional difference between the two isoforms beyond localization unclear"]},{"year":2015,"claim":"Identified MPST as the dominant H2S source in coronary artery and clarified that its product modulates vascular tone via direct reaction with NO rather than a single fixed effect.","evidence":"H2S production assays from arterial homogenates, ex vivo vasoreactivity, CSE knockout controls, in vitro H2S–NO reaction","pmids":["26519030"],"confidence":"High","gaps":["Molecular identity of the H2S–NO reaction product in tissue not defined","Receptor/channel targets downstream of the H2S–NO interaction not mapped"]},{"year":2022,"claim":"Connected MPST to mitochondrial protein import and oxidative metabolism, showing sulfide supply maintains TIM/TOM translocase expression and energy metabolism.","evidence":"Mpst knockout mouse on high-fat diet, TIM/TOM Western blots, respiratory/flux measurements, sulfide donor rescue","pmids":["35616614"],"confidence":"Medium","gaps":["Mechanism by which sulfide controls HIF1α/TIM-TOM expression not defined","Direct persulfidation targets in import machinery not identified"]},{"year":2022,"claim":"Placed MPST upstream of AKT in an anti-apoptotic pathway by direct binding that suppresses AKT phosphorylation in intestinal epithelium.","evidence":"Co-IP, phosphorylation assays, siRNA/overexpression and AKT epistasis in HT29 cells, DSS colitis mouse model","pmids":["36126419"],"confidence":"Medium","gaps":["Single Co-IP without reciprocal/structural validation of direct binding","Whether suppression depends on persulfidation of AKT not established"]},{"year":2023,"claim":"Confirmed in human cells that MPST is required for molybdenum cofactor biosynthesis and tRNA thiolation, validating its sulfur-donor role beyond interaction data.","evidence":"CRISPR knockout in HEK293, sulfite oxidase activity, LC quantification of thio-modified tRNA, H2S measurement","pmids":["36671528"],"confidence":"Medium","gaps":["Relative contribution of MPST versus alternative sulfur donors not quantified","Single-lab, single cell line"]},{"year":2023,"claim":"Revealed adaptive crosstalk between H2S and NO signaling, where chronic combined H2S deficiency upregulates eNOS and sGC to enhance vasorelaxation.","evidence":"Cth/Mpst double knockout mouse, aortic ring relaxation, blood pressure telemetry, eNOS/sGC Western blots, NOS inhibitor pharmacology","pmids":["36860295"],"confidence":"Medium","gaps":["MPST-specific contribution not separable from CTH in the double knockout","Signaling mechanism driving NO/cGMP upregulation not defined"]},{"year":2025,"claim":"Extended MPST's mitochondrial-metabolic requirement to airway epithelial development, where its loss impairs ciliated differentiation and respiration.","evidence":"RNAi knockdown in patient-derived airway epithelial 3D ALI cultures, RNA-seq, Seahorse flux, ciliated-cell immunofluorescence (preprint)","pmids":["41509323"],"confidence":"Medium","gaps":["Preprint, single lab, no replication","Whether the defect is sulfide-dependent and rescuable not tested"]},{"year":null,"claim":"How MPST partitions its persulfide output among Moco/tRNA sulfur trafficking, mitochondrial import maintenance, AKT regulation, and vascular signaling within a single cell remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying model connecting MPST's enzymatic persulfide chemistry to its distinct downstream phenotypes","Direct persulfidation targets across these pathways not catalogued"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,1,2,3]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[4]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[0,5]}],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[1,5]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,4,6]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[1]}],"complexes":[],"partners":["NFS1","MOCS3","AKT"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P25325","full_name":"3-mercaptopyruvate sulfurtransferase","aliases":[],"length_aa":297,"mass_kda":33.2,"function":"Transfer of a sulfur ion to cyanide or to other thiol compounds. Also has weak rhodanese activity. Detoxifies cyanide and is required for thiosulfate biosynthesis. Acts as an antioxidant. In combination with cysteine aminotransferase (CAT), contributes to the catabolism of cysteine and is an important producer of hydrogen sulfide in the brain, retina and vascular endothelial cells. Hydrogen sulfide H(2)S is an important synaptic modulator, signaling molecule, smooth muscle contractor and neuroprotectant. Its production by the 3MST/CAT pathway is regulated by calcium ions","subcellular_location":"Cytoplasm; Mitochondrion; Synapse, synaptosome","url":"https://www.uniprot.org/uniprotkb/P25325/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/MPST","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/MPST","total_profiled":1310},"omim":[{"mim_id":"602496","title":"MERCAPTOPYRUVATE SULFURTRANSFERASE; MPST","url":"https://www.omim.org/entry/602496"},{"mim_id":"249650","title":"MERCAPTOLACTATE-CYSTEINE DISULFIDURIA; MCDU","url":"https://www.omim.org/entry/249650"},{"mim_id":"180370","title":"THIOSULFATE SULFURTRANSFERASE; TST","url":"https://www.omim.org/entry/180370"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Mitochondria","reliability":"Approved"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"liver","ntpm":370.6}],"url":"https://www.proteinatlas.org/search/MPST"},"hgnc":{"alias_symbol":["MST","TST2","TUM1"],"prev_symbol":[]},"alphafold":{"accession":"P25325","domains":[{"cath_id":"3.40.250.10","chopping":"9-158","consensus_level":"high","plddt":97.0859,"start":9,"end":158},{"cath_id":"3.40.250.10","chopping":"160-286","consensus_level":"high","plddt":97.914,"start":160,"end":286}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P25325","model_url":"https://alphafold.ebi.ac.uk/files/AF-P25325-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P25325-F1-predicted_aligned_error_v6.png","plddt_mean":95.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MPST","jax_strain_url":"https://www.jax.org/strain/search?query=MPST"},"sequence":{"accession":"P25325","fasta_url":"https://rest.uniprot.org/uniprotkb/P25325.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P25325/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P25325"}},"corpus_meta":[{"pmid":"36126419","id":"PMC_36126419","title":"MPST deficiency promotes intestinal epithelial cell apoptosis and aggravates inflammatory bowel disease via AKT.","date":"2022","source":"Redox biology","url":"https://pubmed.ncbi.nlm.nih.gov/36126419","citation_count":92,"is_preprint":false},{"pmid":"25336638","id":"PMC_25336638","title":"Characterization and interaction studies of two isoforms of the dual localized 3-mercaptopyruvate sulfurtransferase TUM1 from humans.","date":"2014","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/25336638","citation_count":63,"is_preprint":false},{"pmid":"32139477","id":"PMC_32139477","title":"The Calcium Sensor CBL2 and Its Interacting Kinase CIPK6 Are Involved in Plant Sugar Homeostasis via Interacting with Tonoplast Sugar Transporter TST2.","date":"2020","source":"Plant physiology","url":"https://pubmed.ncbi.nlm.nih.gov/32139477","citation_count":54,"is_preprint":false},{"pmid":"26519030","id":"PMC_26519030","title":"MPST but not CSE is the primary regulator of hydrogen sulfide production and function in the coronary artery.","date":"2015","source":"American journal of physiology. Heart and circulatory physiology","url":"https://pubmed.ncbi.nlm.nih.gov/26519030","citation_count":41,"is_preprint":false},{"pmid":"8394175","id":"PMC_8394175","title":"Drosophila awdK-pn, a homologue of the metastasis suppressor gene nm23, suppresses the Tum-1 haematopoietic oncogene.","date":"1993","source":"Nature genetics","url":"https://pubmed.ncbi.nlm.nih.gov/8394175","citation_count":37,"is_preprint":false},{"pmid":"35616614","id":"PMC_35616614","title":"MPST sulfurtransferase maintains mitochondrial protein import and cellular bioenergetics to attenuate obesity.","date":"2022","source":"The Journal of experimental medicine","url":"https://pubmed.ncbi.nlm.nih.gov/35616614","citation_count":36,"is_preprint":false},{"pmid":"38431528","id":"PMC_38431528","title":"Phosphorylation of sugar transporter TST2 by protein kinase CPK27 enhances drought tolerance in tomato.","date":"2024","source":"Plant physiology","url":"https://pubmed.ncbi.nlm.nih.gov/38431528","citation_count":30,"is_preprint":false},{"pmid":"16545926","id":"PMC_16545926","title":"Evidence for a functional genetic polymorphism of the human mercaptopyruvate sulfurtransferase (MPST), a cyanide detoxification enzyme.","date":"2006","source":"Toxicology letters","url":"https://pubmed.ncbi.nlm.nih.gov/16545926","citation_count":27,"is_preprint":false},{"pmid":"39611527","id":"PMC_39611527","title":"CBL1/CIPK23 phosphorylates tonoplast sugar transporter TST2 to enhance sugar accumulation in sweet orange (Citrus sinensis).","date":"2024","source":"Journal of integrative plant biology","url":"https://pubmed.ncbi.nlm.nih.gov/39611527","citation_count":18,"is_preprint":false},{"pmid":"18575748","id":"PMC_18575748","title":"Tum-1, a tumstatin fragment, gene delivery into hepatocellular carcinoma suppresses tumor growth through inhibiting angiogenesis.","date":"2008","source":"International journal of oncology","url":"https://pubmed.ncbi.nlm.nih.gov/18575748","citation_count":16,"is_preprint":false},{"pmid":"33486070","id":"PMC_33486070","title":"Acute acrylonitrile exposure inhibits endogenous H2S biosynthesis in rat brain and liver: The role of CBS/3-MPST-H2S pathway in its astrocytic toxicity.","date":"2021","source":"Toxicology","url":"https://pubmed.ncbi.nlm.nih.gov/33486070","citation_count":14,"is_preprint":false},{"pmid":"27915245","id":"PMC_27915245","title":"The yeast TUM1 affects production of hydrogen sulfide from cysteine treatment during fermentation.","date":"2016","source":"FEMS yeast research","url":"https://pubmed.ncbi.nlm.nih.gov/27915245","citation_count":14,"is_preprint":false},{"pmid":"2507641","id":"PMC_2507641","title":"Microplate selection technique (MPST). A new method for selecting mouse transfectants expressing human gene products.","date":"1989","source":"Journal of immunological methods","url":"https://pubmed.ncbi.nlm.nih.gov/2507641","citation_count":14,"is_preprint":false},{"pmid":"37640250","id":"PMC_37640250","title":"Structural and functional characterisation of Tst2, a novel TRPV1 inhibitory peptide from the Australian sea anemone Telmatactis stephensoni.","date":"2023","source":"Biochimica et biophysica acta. 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Both isoforms catalyze conversion of 3-mercaptopyruvate to pyruvate and protein-bound persulfide with similar kinetics. TUM1 physically interacts with the L-cysteine desulfurase NFS1 and the rhodanese-like protein MOCS3, implicating it in molybdenum cofactor biosynthesis and cytosolic tRNA thiolation.\",\n      \"method\": \"Purification and kinetic characterization of recombinant isoforms; cellular localization by fluorescence microscopy; in vitro pulldown and in vivo split-EGFP interaction assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro enzymatic assay with purified proteins, direct subcellular localization imaging, and two orthogonal interaction methods (in vitro pulldown + split-EGFP in cells)\",\n      \"pmids\": [\"25336638\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Human TUM1/MPST is required for molybdenum cofactor (Moco) biosynthesis and cytosolic tRNA thiomodification in human cells, as demonstrated by reduced sulfite oxidase activity and decreased sulfur-modified tRNA levels in TUM1 CRISPR knockout HEK293 cells. TUM1 knockout also reduces H2S production and impairs cellular bioenergetics.\",\n      \"method\": \"CRISPR/Cas9 knockout in HEK293 cells; spectrophotometric sulfite oxidase activity assay; LC quantification of sulfur-modified tRNA; H2S measurement\",\n      \"journal\": \"Biomolecules\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean CRISPR KO with multiple orthogonal functional readouts in a single lab\",\n      \"pmids\": [\"36671528\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"A nonsense mutation (Tyr85Stop) in human MPST produces a severely truncated protein that lacks enzymatic activity, as shown by in vitro heterologous expression and measurement of erythrocyte MPST activity, establishing that this residue is required for enzymatic function.\",\n      \"method\": \"Screening of 50 individuals; in vitro heterologous expression; transient transfection assay; erythrocyte MPST activity measurement\",\n      \"journal\": \"Toxicology letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro expression with activity assay plus in vivo erythrocyte activity, single lab\",\n      \"pmids\": [\"16545926\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"MPST, but not CSE, is the primary source of H2S in coronary artery; rat coronary artery homogenates produce H2S via the MPST pathway using 3-mercaptopyruvate as substrate. MPST-derived H2S mediates coronary vasoconstriction in the presence of NO and vasorelaxation in its absence, acting through direct chemical reaction between H2S and NO.\",\n      \"method\": \"In vitro H2S production assay from coronary artery homogenates; ex vivo vasoreactivity experiments with 3-mercaptopyruvate; CSE knockout mouse in vivo coronary vasoreactivity; in vitro H2S–NO reaction assay\",\n      \"journal\": \"American journal of physiology. Heart and circulatory physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro enzymatic assay, ex vivo organ bath pharmacology, genetic (CSE KO) controls, and in vitro chemical mechanism, multiple orthogonal methods\",\n      \"pmids\": [\"26519030\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"MPST directly interacts with AKT and reduces AKT phosphorylation in intestinal epithelial cells. MPST deficiency increases AKT-dependent apoptosis; overexpression of AKT rescues apoptosis caused by MPST loss, while AKT inhibition worsens it, placing MPST upstream of AKT in an anti-apoptotic pathway.\",\n      \"method\": \"Co-immunoprecipitation (direct interaction with AKT); phosphorylation assay; RNA-seq; siRNA knockdown and overexpression in HT29 cells; AKT overexpression rescue and AKT inhibitor epistasis; in vivo DSS colitis mouse model\",\n      \"journal\": \"Redox biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP for interaction, epistasis by OE/inhibition, single lab with multiple approaches\",\n      \"pmids\": [\"36126419\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"MPST deletion in mice causes impaired mitochondrial protein import by activating HIF1α and downregulating TIM/TOM translocase complex subunits, leading to suppression of the TCA cycle, oxidative phosphorylation, and fatty acid oxidation, and resulting in enhanced fat accumulation on high-fat diet. Sulfide donor administration reverses these changes.\",\n      \"method\": \"Mpst knockout mouse (HFD model); transcriptional and metabolic analysis; Western blot for TIM/TOM subunits; metabolic flux/respiratory measurements; sulfide donor pharmacological rescue\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO mouse with defined molecular phenotype (TIM/TOM downregulation, metabolic measurements), pharmacological rescue, single lab\",\n      \"pmids\": [\"35616614\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Genetic double knockout of CTH and MPST in mice leads to paradoxical enhanced endothelium-dependent vasorelaxation and reduced blood pressure, associated with compensatory upregulation of eNOS and soluble guanylate cyclase (sGC) α1 and β1 subunits, revealing that chronic H2S deficiency adaptively upregulates the NO/cGMP pathway.\",\n      \"method\": \"Double Cth/Mpst knockout mouse; aortic ring relaxation assay; blood pressure telemetry; Western blot for eNOS and sGC subunits; NOS inhibitor pharmacology\",\n      \"journal\": \"Frontiers in pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic double KO with multiple vascular functional readouts and molecular quantification, single lab\",\n      \"pmids\": [\"36860295\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Crystal structure of yeast Tum1 (MPST ortholog) at 1.90 Å resolution reveals two rhodanese-like domains (RLDs) but only one conserved active-site cysteine in the C-terminal RLD. An unidentified electron density near the active site suggests a potential cofactor in the sulfur transfer mechanism.\",\n      \"method\": \"X-ray crystallography at 1.90 Å resolution\",\n      \"journal\": \"Protein and peptide letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — crystal structure determined but functional validation of active-site cysteine not performed in this study; single paper\",\n      \"pmids\": [\"22587783\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Yeast Tum1 (ortholog of mammalian MPST) is involved in regulating sterol ester content; tum1Δ strains accumulate significantly higher sterol esters. This function is independent of the tRNA thiolation pathway (overexpression of thiolated tRNAs or deletion of UBA4 did not affect sterol ester content), indicating a distinct lipid metabolic role for Tum1.\",\n      \"method\": \"Yeast gene deletion (tum1Δ); lipid quantification; genetic epistasis with tRNA thiolation pathway mutants (uba4Δ); tRNA overexpression\",\n      \"journal\": \"BMC microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean deletion strain with lipid quantification and epistasis analysis, single lab\",\n      \"pmids\": [\"28830344\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Inhibition of 3-MPST with siRNA-mediated knockdown in primary rat astrocytes significantly increases acrylonitrile-induced cytotoxicity, while overexpression of 3-MPST attenuates it, demonstrating that the CBS/3-MPST-H2S pathway protects astrocytes against acrylonitrile toxicity.\",\n      \"method\": \"siRNA knockdown and overexpression of 3-MPST in primary rat astrocytes; cell viability assay; H2S content measurement; in vivo rat AN exposure model\",\n      \"journal\": \"Toxicology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — siRNA KD and OE with defined cytotoxicity readout, corroborated by in vivo measurements, single lab\",\n      \"pmids\": [\"33486070\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RNAi-mediated knockdown of MPST in neonatal patient-derived airway epithelial cells (nTAECs) during air-liquid interface differentiation reduces ciliated cell numbers at mid-differentiation, suppresses mitochondrial respiration without compensatory glycolysis increase, and induces early transcriptomic shifts in mitochondrial metabolic and epithelial differentiation programs, establishing MPST as required for mitochondrial metabolic integrity during airway epithelial development.\",\n      \"method\": \"RNAi knockdown in neonatal patient-derived tracheal airway epithelial cells (3D ALI model); RNA-seq; metabolic flux analysis (Seahorse); immunofluorescence for ciliated cell markers\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — multiple orthogonal methods (transcriptomics, metabolic flux, cell differentiation readout) but preprint, single lab, no replication\",\n      \"pmids\": [\"41509323\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"MPST (TUM1) is a mitochondrial/cytosolic sulfurtransferase that catalyzes conversion of 3-mercaptopyruvate to pyruvate and protein-bound persulfide, generating H2S; it exists as two splice isoforms with distinct compartmentalization, interacts with NFS1 and MOCS3 to contribute sulfur for molybdenum cofactor biosynthesis and cytosolic tRNA thiolation, serves as the primary H2S source in coronary artery where H2S modulates vasoreactivity via NO, directly binds AKT to suppress its phosphorylation and protect intestinal epithelial cells from apoptosis, and maintains mitochondrial protein import and oxidative metabolism in adipose and airway epithelial tissues.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MPST (TUM1) is a rhodanese-family 3-mercaptopyruvate sulfurtransferase that converts 3-mercaptopyruvate to pyruvate and a protein-bound persulfide, providing a major enzymatic source of H2S and reactive sulfur in mammalian cells [#0, #3]. It is expressed as two splice isoforms with distinct compartmentalization—a cytosolic-only form and a form distributed between cytosol and mitochondria—both of which are catalytically active [#0]. Through physical interaction with the cysteine desulfurase NFS1 and the rhodanese-like protein MOCS3, MPST channels sulfur into molybdenum cofactor biosynthesis and cytosolic tRNA thiomodification, and its loss reduces sulfite oxidase activity, sulfur-modified tRNA levels, H2S output, and cellular bioenergetics [#0, #1]. The persulfide/H2S it generates supports mitochondrial function: MPST loss activates HIF1α, downregulates TIM/TOM import translocase subunits, and suppresses the TCA cycle, oxidative phosphorylation, and fatty acid oxidation, a defect reversible by sulfide donors [#5]. MPST-derived H2S is the principal sulfide source in coronary artery, where it modulates vasoreactivity through direct chemical reaction with NO [#3], and chronic combined H2S deficiency adaptively upregulates the NO/cGMP pathway [#6]. MPST also directly binds AKT to suppress its phosphorylation, protecting intestinal epithelial cells from apoptosis [#4]. A nonsense mutation (Tyr85Stop) abolishes enzymatic activity, defining residues required for catalysis [#2].\",\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"Established that specific protein sequence is required for MPST catalytic activity by showing a truncating mutation abolishes function, linking genotype to enzymatic loss.\",\n      \"evidence\": \"Heterologous expression of a Tyr85Stop variant plus erythrocyte MPST activity measurement\",\n      \"pmids\": [\"16545926\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure-function mapping of which catalytic step the truncation eliminates\", \"Clinical phenotype of carriers not defined\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Resolved the structural architecture of the MPST ortholog, showing two rhodanese-like domains with a single active-site cysteine in the C-terminal domain that mediates sulfur transfer.\",\n      \"evidence\": \"X-ray crystallography of yeast Tum1 at 1.90 Å\",\n      \"pmids\": [\"22587783\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional validation of the active-site cysteine not performed\", \"Identity of unassigned electron density near the active site unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Defined MPST isoform compartmentalization and its physical partners, establishing it as a dual cytosolic/mitochondrial sulfurtransferase feeding sulfur-trafficking complexes.\",\n      \"evidence\": \"Recombinant isoform kinetics, fluorescence localization, in vitro pulldown and split-EGFP interaction with NFS1 and MOCS3\",\n      \"pmids\": [\"25336638\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and regulation of the NFS1/MOCS3 sulfur-relay not resolved\", \"Functional difference between the two isoforms beyond localization unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identified MPST as the dominant H2S source in coronary artery and clarified that its product modulates vascular tone via direct reaction with NO rather than a single fixed effect.\",\n      \"evidence\": \"H2S production assays from arterial homogenates, ex vivo vasoreactivity, CSE knockout controls, in vitro H2S–NO reaction\",\n      \"pmids\": [\"26519030\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular identity of the H2S–NO reaction product in tissue not defined\", \"Receptor/channel targets downstream of the H2S–NO interaction not mapped\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Connected MPST to mitochondrial protein import and oxidative metabolism, showing sulfide supply maintains TIM/TOM translocase expression and energy metabolism.\",\n      \"evidence\": \"Mpst knockout mouse on high-fat diet, TIM/TOM Western blots, respiratory/flux measurements, sulfide donor rescue\",\n      \"pmids\": [\"35616614\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which sulfide controls HIF1α/TIM-TOM expression not defined\", \"Direct persulfidation targets in import machinery not identified\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Placed MPST upstream of AKT in an anti-apoptotic pathway by direct binding that suppresses AKT phosphorylation in intestinal epithelium.\",\n      \"evidence\": \"Co-IP, phosphorylation assays, siRNA/overexpression and AKT epistasis in HT29 cells, DSS colitis mouse model\",\n      \"pmids\": [\"36126419\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single Co-IP without reciprocal/structural validation of direct binding\", \"Whether suppression depends on persulfidation of AKT not established\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Confirmed in human cells that MPST is required for molybdenum cofactor biosynthesis and tRNA thiolation, validating its sulfur-donor role beyond interaction data.\",\n      \"evidence\": \"CRISPR knockout in HEK293, sulfite oxidase activity, LC quantification of thio-modified tRNA, H2S measurement\",\n      \"pmids\": [\"36671528\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relative contribution of MPST versus alternative sulfur donors not quantified\", \"Single-lab, single cell line\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Revealed adaptive crosstalk between H2S and NO signaling, where chronic combined H2S deficiency upregulates eNOS and sGC to enhance vasorelaxation.\",\n      \"evidence\": \"Cth/Mpst double knockout mouse, aortic ring relaxation, blood pressure telemetry, eNOS/sGC Western blots, NOS inhibitor pharmacology\",\n      \"pmids\": [\"36860295\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"MPST-specific contribution not separable from CTH in the double knockout\", \"Signaling mechanism driving NO/cGMP upregulation not defined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Extended MPST's mitochondrial-metabolic requirement to airway epithelial development, where its loss impairs ciliated differentiation and respiration.\",\n      \"evidence\": \"RNAi knockdown in patient-derived airway epithelial 3D ALI cultures, RNA-seq, Seahorse flux, ciliated-cell immunofluorescence (preprint)\",\n      \"pmids\": [\"41509323\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, single lab, no replication\", \"Whether the defect is sulfide-dependent and rescuable not tested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How MPST partitions its persulfide output among Moco/tRNA sulfur trafficking, mitochondrial import maintenance, AKT regulation, and vascular signaling within a single cell remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unifying model connecting MPST's enzymatic persulfide chemistry to its distinct downstream phenotypes\", \"Direct persulfidation targets across these pathways not catalogued\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 1, 2, 3]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [0, 5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [1, 5]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 4, 6]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"NFS1\", \"MOCS3\", \"AKT\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}