ETHE1: experimental and database evidence

Human enzymology and original access

• PMID:23144459, original article, accessible PMC text. Actual Experimental Procedures, Results, Tables 1–2 and Figures 2–6 captions were read. The human preparation has a reported N-terminal 11-residue deletion and C-terminal His6 tag and was expressed in E. coli. Plasma emission spectroscopy measures iron content. Oxygen consumption, GSSH kinetics and HPLC sulfite detection establish persulfide dioxygenase chemistry; the confirming mass-spectrometric result is explicitly data not shown. GSH alone was not a substrate, and its activating effect was unresolved. The preparation is monomeric by gel filtration. Figure 6 is a model based on an Arabidopsis structure, not a human crystal. No figure pixels or supplementary material were inspected.
• PMID:25596185, original article, PMC text, structure 4CHL. Operative protein-production, activity, crystallography and solution-mass Methods/Results and main captions were read. The resolved polymer is GPVD plus human canonical residues 21–254 after tag cleavage, a deliberately engineered deletion. Figure 3A measures GSSH-dependent oxygen consumption. The crystal resolves one iron per protomer, coordinated by His79, His135 and Asp154; GSSH was manually docked. The solution preparation is predominantly dimeric. The Figure 3C caption calls an approximately 51.56-kDa species monomeric, whereas the Results and orthogonal measurements describe dimers; this wording discrepancy does not establish an obligate native state. No figure pixels or supplements were inspected.
• PMID:19136963, original article, actual author-upload access route. Selected main Results, Methods and Figure 4 caption were read. Printed pages 203–204 distinguish mouse knockout tissue, human HeLa overexpression and affinity-purified mature human ETHE1 expressed in E. coli. Figure 4c reports oxygen consumption by the human recombinant enzyme. The main processing passage reports peptide mapping supporting Arg7–Val8 cleavage in ETHE1-HA; its supplementary figures were not inspected. The exact recombinant deletion boundary is not independently reconstructed. Earlier iron-per-dimer wording is not generalized over later measurements. No figure pixels were inspected.
• PMID:14732903, original article, PMC text. The full original was accessible; the localization/import Methods, Results, Figure 6 caption and relevant Discussion were read. Native human fibroblast mitochondrial protease protection and soluble-versus-membrane separation support matrix location (Figure 6c–d). Full human ETHE1 cDNA with a C-terminal HA tag was separately tested in HeLa and COS7 cells. A P21-start comparator is an engineered construct; the import experiment only bounded the cleavage length, rather than establishing the later residue-8 mature start. The antibody description gives both a peptide-position statement and an inconsistent C-terminal description; no new epitope mapping is inferred. No figure pixels or supplementary data were inspected.
• PMID:12398897, original article, PubMed. The identifier and abstract were verified, but original Methods, Results and figures remained unavailable after bounded publisher/repository attempts. The abstract reports HSCO nuclear–cytoplasmic shuttling and RelA export; assay constructs and conditions were not established. The later mitochondrial papers are not a reading of this original, and no tag, fragment or overexpression-artifact verdict follows from the access limit. Its reference retains full_text_unavailable: true.

Localization and the antibody-dependent nuclear observation

The human localization study PMID:14732903(https://doi.org/10.1086/381653) distinguishes mitochondrial import from suborganellar location. Its Methods, Cell Fractionation and Biochemical Assays; Mitochondrial Targeting; ETHE1 Cloning, and Results, In Vivo Import, Protease-Protection Test and Suborganellar Localization; Figure 6 caption were read. Native human fibroblast ETHE1 was protected in intact mitochondria and enriched in the soluble mitochondrial fraction. Full-length human HA-tagged constructs in HeLa and COS-7 cells provide separate import/imaging evidence. The engineered deletion of the first 20 residues was a comparator, not an observed natural splice product or the measured cleavage site. The Discussion reports failure to detect nuclear staining, including after leptomycin B under the reported conditions. No figure pixels or supplements from this paper were examined here.

Current HPA ETHE1 subcellular data contain an important distinction. HPA028360 and HPA029028 have enhanced mitochondrial assignments in A-431, U-251MG and U2OS cells. HPA029029 has supported antibody-level assignments to mitochondria and nucleoplasm in those three lines. All three selected images were inspected: the first two chiefly show extranuclear punctate/reticular signal, whereas the third has diffuse central/nuclear green signal together with stronger extranuclear structures. That selected HPA029029 image lacks a visible blue nuclear overlay. These observations are not a quantitative colocalization measurement or an independent antibody-specificity experiment. The aggregate HPA conclusion is mainly mitochondrial with enhanced reliability; it does not list nuclear localization as an aggregate main location.

The entire 86-residue HPA029029 antigen is shared by the displayed 254-residue sequence and all captured RefSeq alternatives, including the predicted XP record. Therefore this antibody cannot identify which product contributes the nuclear signal. This positive but antibody-dependent evidence supports retaining a bounded non-core nuclear context, while leaving the disagreement with the 2004 experiment unresolved. It does not establish predominant nuclear residence, nucleocytoplasmic shuttling or a nuclear biochemical activity. The older HSCO study PMID:12398897 remains unavailable at operative-original level in this review; it is not dismissed as an artifact.

GO_REF:0000054 describes LIFEdb GFP-fusion localization, not HPA. Its current resource URL redirects to a general proteomics page, and the exact ETHE1 clone images were not recovered. The broad GO cytoplasm term includes organelles. Refining it to matrix here rests on the independent native-human experiment above, not on a claim that the historical GFP-fusion experiment itself resolved matrix or excluded soluble cytosol. The separately imported experimental HSCO cytoplasm assertion is retained as non-core rather than replaced by matrix; its operative original remains unread. Mitochondrial HPA images likewise do not resolve the matrix; their refinement uses the independent fractionation result.

Proteomics and protein-group limits

PMID:34800366(https://doi.org/10.1016/j.cmet.2021.11.001), PMC original and supplements: actual Results and STAR Methods covering fractionation, subtractive/spatial proteomics, importomics, carbonate/sonication and protein-group identification were read. Exact ETHE1 rows were inspected in Supplementary Tables S1–S3 (mmc2.xlsx–mmc4.xlsx). The group is O95571/M0QXB5/M0QY80/M0QX80, with O95571/M0QXB5 as majority identifiers. It has subtractive Class 1, spatial Cluster 1 and TOMM40 Class 1 support; the multiple-cell-line experiment includes HEK, HeLa, Huh7 and U2OS. TOMM70 depletion did not produce the same positive dependence. These are protein-group measurements, not separate localization assays for each product.

The independent matrix refinement uses the native-human experiment in PMID:14732903. MitoCoP's peripheral classification is not rewritten as soluble-matrix evidence; preparation and assay context remain distinct. No MitoCoP figure pixels were inspected.

Interaction screens

PMID:32296183(https://doi.org/10.1038/s41586-020-2188-x), PMC original and the archived HuRI data were examined at target-record level. The main text describes nine Y2H screens, quadruplicate pairwise retesting and sequence confirmation. The exact ETHE1 records comprise human ORFs tested in yeast, with three method records per pair (array, pooling and validated two-hybrid). Orthogonal MAPPIT/GPCA validation of a study-wide subset is not attributed to each of these pairs.

The ETHE1 ORF is 2869. The archived Q9H8Y8-1 record has the same accession stem as the imported Q9H8Y8 partner; the imported identifier is unchanged. Exact sequence equivalence of archived clones to every current natural product is not established. The four generic interactions remain supported non-core associations. Self-association is likewise retained without choosing a universal monomer/dimer state from differing recombinant preparations.

Phylogenetic placement, rules and reaction mappings

The actual PANTHER PTHR43084 tree places human O95571 at PTN002534467. The actual family MSA ungaps to the exact 254-residue sequence. The mitochondrial IBD at PTN000940093 and the glutathione-metabolism and sulfur-dioxygenase IBDs at PTN002454072 all lie on this target path; no target-path NOT record was found in the captured current IBD source. The target's own experimentally grounded appearance in WITH is legitimate. Arabidopsis AT1G53580/Q9C8L4 identity, placement and current experimental source records were traced, but its primary papers were not independently read in this lane.

Current ARBA00026962 matches ETHE1 through condition set 370 of 380 (zero-based): FunFam 3.60.15.10:FF:000013 plus Eukaryota. ARBA00090275 has the same matching conjunction in its sole condition set, mapping sulfur dioxygenase activity. These are actual current-rule matches, not assumptions about a historical rule version. Their functional conclusions are corroborated by the human experiments below.

IPR044528 is the persulfide-dioxygenase-like MBL-fold domain, with an actual target match and mappings to sulfur dioxygenase activity and glutathione metabolism. Membership in this specific domain is not used to assign generic MBL hydrolase, beta-lactamase or glyoxalase chemistry.

Current GO:0050313, EC 1.13.11.18 and the official UniProt O95571 reaction record agree on oxidation of S-sulfanylglutathione with oxygen and water to sulfite, glutathione and protons. Direct Rhea download routes returned HTTP 403; the RHEA:12981 cross-reference and equation were verified through UniProt and the independent EC/GO records, without pretending the blocked Rhea body was read. ETHE1 directly transforms a glutathione conjugate, supporting glutathione metabolism; this is not evidence of glutathione synthesis, free-glutathione oxidation or sulfate production by ETHE1.

Reactome location and the metal-composition query

The actual human R-HSA-1614605 chain is event 1614605 → catalyst 1614523 → complex 1614521 → protein 1614628 → O95571. Protein 1614628 itself has the matrix compartment and modeled residues 8–254, so the localization does not depend only on the event's mixed compartment list. This agrees with human fractionation evidence.

However, complex 1614521 explicitly contains two matrix zinc entities 880067, rather than merely having an outdated display name. The human biochemical and structural findings instead support one iron per protomer. A separate, unreported composition query is proposed for Reactome; the matrix annotation remains valid. The event also represents SQRDL-bound persulfide, so it should not be presented as the exact GSSH assay setup. No source cache is changed, and no alternative-metal capability is universally excluded.

Native processing, sequence records and engineered proteins

The displayed UniProt O95571 sequence has 254 residues and equals RefSeq NP_055112.2. Its annotated 1–7 transit segment and 8–254 processed chain describe maturation, not additional isoforms. The 2009 original reports Arg7–Val8 processing in its main text; the corresponding supplementary measurements were not inspected here.

These are deterministic comparisons of the official captured sequences, not assays or inferred UniProt isoform identifiers. The natural 243-residue record has an internal deletion and is not the recombinant N-terminal Δ11 protein merely because the lengths coincide. Neither targeting, stability, processing nor catalytic activity of the alternative records is established by the Δ11 or Δ20 preparations. Missing a region that contains a ligand in the canonical structure is not by itself an inactivity verdict. Antigen sequence overlap cannot resolve which product produced a cellular signal.

Coupling the measured reaction to physiological processes

The executed reaction is supported by PMID:23144459(https://doi.org/10.1074/jbc.M112.407411), Experimental Procedures (enzyme assay and reaction-product analysis), Results (GSSH kinetics, Table 2 and Figure 5), and PMID:25596185(https://doi.org/10.1093/hmg/ddv007), Results (single-iron active site and Figure 3A) and Methods (protein preparation and persulfide dioxygenase assay). The former shows sulfite HPLC co-elution with an enzyme-free control; its confirmatory mass-spectrometric result is reported as data not shown. The latter independently measures GSSH-dependent oxygen consumption and resolves one iron per protomer; its substrate pose is manually docked. Neither claim requires an obligate dimer.

The current sulfur dioxygenase term describes the GSSH-to-sulfite-and-glutathione reaction. The same catalytic step directly changes a glutathione conjugate and supplies persulfide oxidation within sulfide catabolism, supporting the existing glutathione-metabolism and sulfide-oxidation contexts. These contexts do not attribute free-H2S oxidation or sulfate production to ETHE1. The matrix location comes independently from PMID:14732903(https://doi.org/10.1086/381653), Methods (proteinase K protection and suborganellar fractionation) and Results/Figure 6c–d, rather than from the enzyme preparation or antibody imaging alone.

Feedback clarification: curated records, TST and pathway coupling

The unchanged local UniProt record is now an explicit reference. CC:COFACTOR supports one Fe(2+) per subunit; CC:SUBUNIT preserves monomer/dimer preparation differences and the TST interaction; CC:SUBCELLULAR LOCATION retains the older cytoplasm and nucleus reports separately. Its reference title is derived from the record's ID, first AC and recommended full name. These curated statements do not establish biochemical equivalence among the shorter RefSeq products.

The actual PMID:19136963 author-uploaded original, main Results discussing structural association with rhodanese and Methods Cloning and expression of rhodaneseHis, provides positive human-cell association evidence. ETHE1-HA expressed in HeLa was recovered with anti-rhodanese; coexpressed ETHE1-HA and human rhodanese-His6 were reciprocally immunoprecipitated. The main text refers to Supplementary Figure 3b–c, but those panels were not inspected. The experiment does not establish purified binary binding, an unmodified endogenous complex or direct substrate channeling. The bacterial operon/fusion observation in the same passage is distinct from these human-cell experiments.

The 2009 main Discussion describes sulfite produced by ETHE1 being used downstream by rhodanese. PMID:23144459(https://pmc.ncbi.nlm.nih.gov/articles/PMC3531769/) likewise discusses a postulated downstream rhodanese role and alternative sulfur flows; its tested ETHE1 reaction uses GSSH and does not turn over thiosulfate. Consequently, the TST association is not evidence that TST is obligatorily immediately upstream of ETHE1, or that the co-immunoprecipitation experiment reconstitutes metabolite transfer. Native coupling remains a focused question.

For nuclear localization, GO_REF:0000052 itself supplies the imported mitochondrion annotation. The separate positive nuclear observation comes from the named HPA029029 antibody record and inspected image at the HPA portal, with the antibody/aggregate/product limitations stated above. It does not verify the inaccessible HSCO paper's shuttling mechanism.

Iron binding remains ACCEPT as part of the characterized catalytic mechanism. The one core explicitly describes iron coordination while retaining sulfur dioxygenase as its single molecular-function term; no second core or new annotation is needed. The full construct and native-fractionation limits remain in the source sections above, allowing the repeated row rationales to be shortened without changing their decisions.

Reproducible Reactome composition query

The following fields are transcribed from the captured official ContentService responses, not reconstructed from the event-summary cache. This remains an unreported, probable composition query, with the supported matrix annotation retained.

The cached source is preserved; the duplicated zinc components are not silently replaced. Independent human measurements and the curated cofactor record motivate checking composition, without excluding every possible metal condition or product.

Captured complex-response SHA256: c76165024547c563c253ed0c6d553081f306bba4a3e291525096ce8720485586.