ADPRHL1 (ARH2) is a catalytically inactive member of the ADP-ribosylglycohydrolase family, expressed almost exclusively in striated muscle and most strongly in the heart. It retains the all-alpha ADP-ribosylhydrolase fold of its active paralogues ADPRH (ARH1, an ADP-ribosylarginine hydrolase) and ADPRS (ARH3, a serine/poly(ADP-ribose) hydrolase), but has lost the aspartate pair that coordinates the catalytic magnesium ions and the serines that grip the adenosine-ribose of the substrate, and recombinant ARH2 shows no hydrolase activity in the assays that detect ARH1 and ARH3 activity. Its function instead depends on the vestigial substrate cleft: the two adjacent serines that bind adenosine-ribose in ADPRH are replaced in ADPRHL1 by a di-arginine motif (Arg271-Arg272), and deleting one to four residues from that loop abolishes ventricular myofibril assembly in Xenopus without touching any catalytic residue. Loss of ADPRHL1 blocks striated myofibril assembly and outgrowth of the cardiac ventricle in frog embryos, and knockout in human embryonic stem cell-derived cardiomyocytes disorganises sarcomeres, reduces beta-1 integrin, paxillin and phosphorylated FAK, and impairs focal adhesion formation, calcium transients and electrical conduction through excessive activation of the ROCK-myosin II pathway. Epitope-tagged protein localises to stripes flanking the Z-disc, consistent with the sarcomeric and myofibrillar localisation recorded for the protein. No ligand, substrate or binding partner has been identified, so the molecular activity underlying these roles remains undefined.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000287 magnesium ion binding | IEA GO_REF:0000002 | REMOVE | Summary: This is the ancestral catalytic magnesium site transferred onto a protein in which that site has been dismantled. The row comes from interpro2go, which maps IPR012108 "ADP-ribosylarginine hydrolase" to GO:0000287, GO:0003875 and GO:0051725; ADPRHL1 matches IPR012108 because the entry's single member signature, PIRSF016939, does not separate the active ARH1 proteins from the inactive ARH2 ones. Of the 1282 proteins in IPR012108, 11 are reviewed Swiss-Prot entries, and 7 of those 11 are the entries Swiss-Prot itself names "Inactive ADP-ribosyltransferase ARH2" and annotates with a CAUTION that they lack the metal-binding residues. So a majority of the entry's own curated members are curated as catalytically dead, and the entry still supplies the metal-binding term. UniProt states the loss directly on this accession, and a residue census confirms it against a matched control: of ADPRH's six UniProt-annotated Mg(2+) ligands, ADPRHL1 keeps the coordinating oxygen at only three and loses it at three. Written as ADPRH position to ADPRHL1 residue, the survivors are Ser54 to Ser56, Asp55 to Asp57 and Asp302 to Glu304, and the losses are Asp56 to Asn58, Asp304 to Ala306 and Ser305 to Ala307. The decisive one is Asp56 to Asn58, because aspartate-to-asparagine at a vicinal catalytic aspartate is the very substitution used to abolish this family's activity experimentally - PMID:17075046 kills ARH3's reaction by replacing Asp77 and Asp78 with asparagine. The comparison is controlled rather than asserted: Dictyostelium ADPRH, a genuine ARH1 at 48.4% identity to human ADPRH - the same distance as ADPRHL1's 42.6-47.7% - retains 13 of 20 ligand sites and all six Mg(2+) donors, while every ADPRHL1 orthologue retains 6-7 sites and 2-3 donors. Retention is therefore not tracking sequence distance. One counter-consideration is recorded rather than suppressed: PMID:27217161's model notes that Asp100 of ADPRHL1 could provide an alternative coordination site for metals. That is a hedged modelling suggestion, presented in the source as an obstacle to substrate docking rather than as a functional site, and never tested - it does not support the annotation, whose provenance is specifically the ancestral catalytic site. Reason: Removed on the term's own provenance rather than by importing the hydrolase result. Magnesium binding as such has never been assayed on ARH2, and three of ADPRH's six Mg(2+) donor positions do survive, so the honest statement is not that ADPRHL1 binds no metal. All positions in this paragraph are given in ADPRH numbering, with the ADPRHL1 residue after the arrow: retained are Ser54 to Ser56, Asp55 to Asp57 and Asp302 to Glu304; lost are Asp56 to Asn58, Asp304 to Ala306 and Ser305 to Ala307. That reconciles with PMID:27217161, which reports three of the four critical aspartates lost with only Asp55 conserved - its verbatim sentence is quoted in this row's supported_by below, and nothing in this paragraph is a quotation. It uses a different denominator and a stricter criterion: its four are Asp55, Asp56, Asp302 and Asp304, and it scores Asp302 to Glu304 as lost because the residue is no longer an aspartate, whereas the donor test used here scores it retained because a glutamate still contributes a carboxylate oxygen. Both statements describe the same alignment. What is refuted is the only thing that put this term here: the row exists solely as interpro2go's mapping of IPR012108, that mapping describes the catalytic Mg(2+) centre of the active ARH1 members, and that centre is demonstrably dismantled - UniProt's own CAUTION records the metal-binding residues as absent, and the losses include the vicinal Asp56, the position whose aspartate-to-asparagine substitution abolishes the paralogue's activity. There is no independent support for the term anywhere in the clade: all seven reviewed ARH2 orthologues hold it by the same single IEA route. With its sole basis refuted and no measurement on either side, REMOVE rather than MARK_AS_OVER_ANNOTATED - though the latter would also have been defensible, and the caveat in the summary about ADPRHL1 Asp100, the one position in this row's reasoning cited in ADPRHL1 rather than ADPRH numbering, is why. Propagation Review Root cause: PROPAGATION BAD Failure modes: PSEUDO OR SUBACTIVITY LOSS WRONG ORTHOLOG OR PARALOG Sources checked: InterPro:IPR012108 · ADP-ribosylarginine hydrolase (InterPro family, member signature PIRSF016939) SUPPORTS SOURCE BUT NOT TARGET The signature is sound for the ARH1 members that justify it and unsound for the ARH2 members that outnumber them: 7 of the entry's 11 reviewed members are "Inactive ADP-ribosyltransferase ARH2" entries carrying a lack-of-catalytic-residues CAUTION. The three other InterPro signatures ADPRHL1 matches - IPR050792, IPR005502 and IPR036705 - map to nothing in interpro2go, which is the internal control showing this is one over-reaching entry rather than a careless pipeline. Supporting Evidence: file:human/ADPRHL1/ADPRHL1-uniprot.txt lacks the metal-binding and substrate-binding residues PMID:27217161 Three of the four critical aspartates of ADPRH have been lost in mammalian ADPRHL1 (hADPRH D56, D302, D304 lost, only D55 is conserved), suggesting that ADPRHL1 cation binding may be compromised. PMID:36497109 ARH2 (so far) does not exhibit any enzymatic activities [70], consistent with differences in primary sequences from critical residues found in ARH1 (e.g., 54-SDDT-57, 302-DSDS-305) file:human/ADPRHL1/ADPRHL1-bioinformatics/RESULTS.md Retention therefore is not tracking sequence distance here. |
| GO:0003875 ADP-ribosylarginine-[protein] hydrolase activity | IEA GO_REF:0000120 | REMOVE | Summary: The activity this term names has been measured on ARH2 and was not found. Ono et al. assayed recombinant ARH2 alongside ARH1 and ARH3 and report it inactive; the Moss laboratory's family review states the negative across the whole assay panel and, specifically for the reaction this term describes, that ARH2 does not appear to be responsible for hydrolysis of the ADP-ribose-acceptor linkage. UniProt records the same on this entry. Two independent automatic routes deliver the term anyway and neither examines a residue. The first is interpro2go via IPR012108. The second is the ARBA rule cited in this row's own WITH/FROM: ARBA00088955 has a single condition set, FunFam id 1.10.4080.10:FF:000002 AND taxon Mammalia, and this entry's cross-reference reads "FunFam; 1.10.4080.10:FF:000002; ADP-ribosylarginine hydrolase isoform X1". That FunFam contains both the active ARH1 proteins and the inactive ARH2 ones (ARH3 sits in FF:000001), so the rule is keyed on a structural family whose name asserts an activity that half its members have lost. The rule's taxon clause makes a testable prediction and GOA confirms it: the five mammalian ARH2 orthologues receive GO:0003875 via GO_REF:0000120 while Xenopus and zebrafish receive it via GO_REF:0000002 instead. Across all seven reviewed ARH2 orthologues, GO:0003875, GO:0000287 and GO:0051725 are held 21 times and every single one is IEA - no experimental, ISS or IBA support exists anywhere in the clade. PAINT, by contrast, gets this right: the GO:0003875 IBA reaches 20 gene products from node PTN009030515 and all 20 are ADPRH orthologues, with no ARH2 among them. The specificity question was also asked in the other direction and answered no: no ARH2 orthologue carries any ARH3-type term (GO:0140290, GO:0004649, GO:0140292 all return zero), and the residue census scores ADPRHL1 at 6-7 of ARH3's 17 sites against 14-17 for the ARH3 clade itself. ADPRHL1 is not a misfiled serine hydrolase; it is not a hydrolase. Reason: A catalytic activity that was directly assayed on this protein and not detected, arriving from two fold-based pipelines neither of which inspects the catalytic residues. The field's own caveat - that the negative was obtained with model substrates - is why no replacement hydrolase term is proposed rather than why the term is kept: GO cannot express "might hydrolyse something unidentified", and inventing a general hydrolase term to hold that hedge would be an over-annotation of the opposite sign. Propagation Review Root cause: PROPAGATION BAD Failure modes: PSEUDO OR SUBACTIVITY LOSS WRONG ORTHOLOG OR PARALOG Sources checked: ARBA:ARBA00088955 · ARBA rule, FunFam 1.10.4080.10:FF:000002 AND taxon Mammalia gives GO:0003875 SOURCE BAD The rule's whole condition set is a CATH FunFam membership plus a taxon. The FunFam is named "ADP-ribosylarginine hydrolase isoform X1" and contains ADPRH and ADPRHL1 alike, so the rule grants a catalytic molecular function on the strength of a family name. Its Mammalia clause is visible in GOA as the split between GO_REF:0000120 for the five mammalian ARH2 orthologues and GO_REF:0000002 for the frog and zebrafish ones. InterPro:IPR012108 · ADP-ribosylarginine hydrolase (InterPro family, member signature PIRSF016939) SUPPORTS SOURCE BUT NOT TARGET Same entry as on the GO:0000287 and GO:0051725 rows; 7 of its 11 reviewed members are Swiss-Prot "Inactive ADP-ribosyltransferase ARH2" entries. Supporting Evidence: PMID:17075046 The rate of O-acetyl-ADP-ribose hydrolysis by recombinant ARH3 was 250-fold that observed with ARH1; ARH2 and poly(ADP-ribose) glycohydrolase were inactive. PMID:36497109 ARH2 does not appear to be responsible for hydrolysis of ADP-ribose-acceptor linkage. PMID:32726316 The sequence changes suggest binding of ADP-ribosylated protein substrates and cation-mediated catalysis are both abolished in Adprhl1 and biochemical assays have confirmed the lack of any comparable enzymatic activity file:human/ADPRHL1/ADPRHL1-uniprot.txt showing no activity against O-acetyl-ADP-ribose |
| GO:0030017 sarcomere | IEA GO_REF:0000044 | ACCEPT | Summary: The automatic mapping of UniProt SubCell SL-0313 (Sarcomere) to GO:0030017. Its input is this entry's SUBCELLULAR LOCATION line, "Cytoplasm, myofibril, sarcomere", which carries ECO:0000250|UniProtKB:Q6AZR2 - so this row and the ISS row below are the same underlying observation arriving by two routes rather than two independent findings, and they are given the same verdict. The observation itself is the Xenopus work: an N-terminally epitope-tagged Adprhl1 localises to stripes flanking the Z-disc. Two caveats are recorded rather than glossed. It is over-expressed tagged protein, and the same study reports that its peptide antibody was not sensitive enough to detect endogenous Adprhl1 in situ, so the endogenous distribution has not been imaged in any species. And no human localisation experiment exists at all. Kept because the inference is nonetheless well founded - a 74.6%-identical 1:1 ortholog, a heart- and skeletal-muscle-restricted expression pattern, and human knockout cardiomyocytes in which sarcomere arrangement is disturbed - and because a location term that is very likely true should not be deleted on the strength of an assay caveat. Reason: A true but indirectly evidenced compartment call. Same datum as the ISS row, reached through UniProt's SubCell vocabulary rather than through the curator's ortholog transfer. Supporting Evidence: file:human/ADPRHL1/ADPRHL1-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm, myofibril, sarcomere PMID:27217161 Recombinant Adprhl1 can localize to stripes adjacent to the Z-disc |
| GO:0051725 protein de-ADP-ribosylation | IEA GO_REF:0000002 | REMOVE | Summary: The biological process that the absent hydrolase activity would carry out, transferred from the same IPR012108 signature and falling with it. The direction of this term was checked explicitly rather than assumed, because this family is exactly where reaction direction goes wrong: GO:0051725 is the removal of ADP-ribose, which is the correct direction for an ARH-family protein, so this is not the error of annotating a protein as performing the modification it removes. It fails for a different reason - ADPRHL1 has never been shown to remove ADP-ribose from anything, and was assayed for exactly that and found inactive. Nothing else in the literature supports the process either. The closest is PMID:35816343, in which a germline p.Asp78Val allele expressed in prostate cells activates PARP1 and raises poly(ADP-ribose) levels while wild-type ADPRHL1 suppresses proliferation; that is consistent with wild-type ADPRHL1 restraining cellular ADP-ribosylation somehow, but it is ectopic expression of a single variant in cancer cell lines with no demonstration that ADPRHL1 acts on ADP-ribose itself, and it cannot support a de-ADP-ribosylation annotation. Note that Asp78 is not one of the ligand positions in the residue census, so the variant does not bear on the catalytic question either. Reason: A process term that exists solely as the downstream consequence of a catalytic activity this protein does not have. Removed on the same evidence as the GO:0003875 row, and from the same signature. Propagation Review Root cause: PROPAGATION BAD Failure modes: PSEUDO OR SUBACTIVITY LOSS WRONG ORTHOLOG OR PARALOG Sources checked: InterPro:IPR012108 · ADP-ribosylarginine hydrolase (InterPro family, member signature PIRSF016939) SUPPORTS SOURCE BUT NOT TARGET The third of the three terms interpro2go attaches to this entry. It reaches all seven reviewed ARH2 orthologues, in every case as the only evidence any of them has for the process. Supporting Evidence: PMID:36497109 ARH2 does not appear to be responsible for hydrolysis of ADP-ribose-acceptor linkage. PMID:17075046 The rate of O-acetyl-ADP-ribose hydrolysis by recombinant ARH3 was 250-fold that observed with ARH1; ARH2 and poly(ADP-ribose) glycohydrolase were inactive. file:human/ADPRHL1/ADPRHL1-uniprot.txt lacks the metal-binding and substrate-binding residues |
| GO:0003242 cardiac chamber ballooning | ISS GO_REF:0000024 | ACCEPT | Summary: A sound ortholog transfer. The WITH/FROM entity is correct for an ISS row - the sequence-similar protein, not a partner - and it resolves to ARHL1_XENLA, a reviewed Swiss-Prot entry for Xenopus laevis adprhl1, 354 aa, a 1:1 ortholog at 74.6% aligned identity (published as 75%). Its own annotation is IMP from PMID:27217161, where morpholino knockdown of all Adprhl1 variants prevents outgrowth of the ventricle while leaving cardiomyocyte number, electrical conduction and myofibrillar gene expression intact. A fully paginated reference-projection test on that PMID returns 3 annotations over 1 entity, so this is one curator reading one paper about one gene, not a bulk import and not a complex-level phenotype spread across a set. The mammalian evidence is thinner but not contradictory: mice lacking Adprhl1 exons 3-4 are normal, but that allele still produces the smaller ADPRHL1 species and so is not a null. ADPRHL1 is heart- and skeletal-muscle-enriched in human tissue and expressed in left ventricle myocardium, and human variants in the locus associate with ventricular conduction phenotypes, so nothing about the human gene argues against the transfer. Reason: Legitimate ISS from a reviewed 1:1 ortholog at 75% identity whose underlying evidence is a specific, single-gene loss-of-function experiment in the same organ. Supporting Evidence: PMID:27217161 Morpholino oligonucleotide-mediated knockdown of all Adprhl1 variants inhibits striated myofibril assembly and prevents outgrowth of the ventricle. PMID:36497109 ARH2 is a cytoplasmic protein expressed primarily in heart and skeletal muscle and may be involved in cardiac myofibril assembly and cardiac chamber outgrowth |
| GO:0030017 sarcomere | ISS GO_REF:0000024 | ACCEPT | Summary: The curator's ortholog transfer of the Xenopus localisation, and the origin of the SubCell IEA row above. Q6AZR2 holds GO:0030017 by IMP from PMID:27217161 - IMP rather than IDA because the localisation was obtained from transgenically over-expressed, N-terminally tagged protein. The same caveats therefore apply here as to the IEA row: it is tagged over-expressed protein, the endogenous protein has never been imaged in situ in any species, and there is no human localisation experiment. It is accepted because the ortholog relationship is close and unambiguous, because the compartment is consistent with everything else known about the protein - muscle-restricted expression, a knockout that disorganises sarcomeres in human cardiomyocytes, and a reported association of mouse ARH2 with a synemin, desmin and triadin-containing assembly - and because the two GO:0030017 rows describe one observation and must take one action. Reason: Correctly executed ISS from a reviewed 1:1 ortholog. The assay caveat is recorded as a knowledge gap rather than used to delete a compartment call that is very likely true. Supporting Evidence: PMID:27217161 Recombinant Adprhl1 can localize to stripes adjacent to the Z-disc PMID:27217161 immunocytochemistry of hearts using the same antibody was not sufficiently sensitive to detect endogenous Adprhl1 protein in situ |
| GO:0055003 cardiac myofibril assembly | ISS GO_REF:0000024 | ACCEPT | Summary: The best-supported annotation on this gene, and the only one with independent human evidence behind it. The ISS is faithful to its donor: Q6AZR2 holds GO:0055003 by IMP from PMID:27217161, where knockdown blocks striated myofibril assembly and over-expression produces disarrayed, branching myofibrils with sarcomere division at the actin-Z-disc boundary. It is independently corroborated in human cells that GOA has not yet seen: CRISPR knockout of ADPRHL1 in H9 embryonic stem cell-derived cardiomyocytes leaves cardiac differentiation and myofibrillar gene expression intact but disturbs sarcomere arrangement. Accepted as it stands, with two additive recommendations recorded separately rather than by modifying this row - a more specific ventricular term that exists in GO but has been curated only onto TrEMBL copies of the Xenopus gene, and the human IMP evidence that should sit alongside this ISS. Why the human sarcomere result is left as a curation recommendation rather than proposed as its own row, when the same paper's focal adhesion result is proposed: the focal adhesion finding is the paper's central, quantified result, measured on four independent markers and closed by a pharmacological rescue, and it has no annotation in GOA at all. The sarcomere finding is a qualitative supplementary-figure observation with no quantification, and the process it would support is already annotated here and refined by the GO:0055005 row below - so a third overlapping myofibril term drawn from the weaker of the paper's two observations would add granularity the observation does not carry. GO:0045214 sarcomere organization was considered on that basis and declined. Reason: A faithful ortholog transfer of a specific loss-of-function phenotype, now also supported by a human knockout. Left unmodified because the row correctly reflects the term its cited donor holds; the refinement is proposed additively below. Supporting Evidence: PMID:27217161 Morpholino oligonucleotide-mediated knockdown of all Adprhl1 variants inhibits striated myofibril assembly and prevents outgrowth of the ventricle. PMID:37880701 the arrangement of sarcomeres was less regular |
| GO:0055005 ventricular cardiac myofibril assembly | ISS GO_REF:0000024 | NEW | Summary: Proposed addition, and it exists only because a reference-projection test on the 2020 CRISPR paper turned up a split donor. All three of ADPRHL1's ISS rows transfer from UniProtKB:Q6AZR2, the Swiss-Prot entry for Xenopus laevis adprhl1, which carries the 2016 morpholino paper's three IMP annotations. But Xenbase curated the 2020 CRISPR paper onto two TrEMBL accessions for the same frog gene, A0A8J0UG54 and A0A8J0UIM8 (adprhl1.L), which the UniProt ISS pipeline never consulted. Those entries hold GO:0055005 ventricular cardiac myofibril assembly, GO:0030041 actin filament polymerization and GO:0036342 post-anal tail morphogenesis by IMP, and none of the three has ever propagated to any mammal. GO:0055005 is an is_a descendant of the GO:0055003 that ADPRHL1 already holds, and the evidence for it is specific: CRISPR deletion of one to four residues from the Arg271-Arg272 loop at the centre of the vestigial ADP-ribosylhydrolase cleft causes loss of ventricular myofibril assembly, which is also the cleanest demonstration anywhere that ADPRHL1's function is cleft-dependent and catalysis-independent. Human relevance is direct - ADPRHL1 is expressed in left ventricle myocardium and its knockout disorganises sarcomeres in human cardiomyocytes. GO:0030041 was considered and declined for human: the Xenopus IMP is a morphological inference from myofibril phenotypes rather than a polymerization assay, and the human mechanism places any actin effect downstream of ROCK-myosin II. GO:0036342 is Xenopus-specific and not transferable. Reason: A more specific term than the existing GO:0055003 row, curated for the orthologous gene from a later paper, which failed to propagate because the two Xenopus papers were curated onto different accessions of the same gene. Added rather than substituted for GO:0055003, because that row correctly reflects the donor it cites. Supporting Evidence: PMID:32726316 Mutant alleles encode discrete loss of 1, 3 or 4 amino acids from a di-arginine (Arg271-Arg272) containing peptide loop at the centre of the ancestral ADP-ribosylhydrolase site. PMID:32726316 Thus despite lacking catalytic activity, it is the modified (adenosine-ribose) substrate binding cleft of Adprhl1 that fulfils an essential role during heart formation. PMID:37880701 the arrangement of sarcomeres was less regular |
| GO:0051894 positive regulation of focal adhesion assembly | IMP PMID:37880701 The pseudoenzyme ADPRHL1 affects cardiac function by regulat... | NEW | Summary: Proposed addition from the only functional experiment ever performed on the human protein, which contributes nothing to GOA at present - a fully paginated reference-projection test on PMID:37880701 returns zero annotations over zero entities. CRISPR knockout of ADPRHL1 in human H9 embryonic stem cell-derived cardiomyocytes reduces beta-1 integrin, phosphorylated FAK, paxillin and talin, and impairs focal adhesion formation; the cells detach from the culture surface, and calcium transients and microelectrode-array conduction are perturbed. Inhibiting ROCK or myosin II restores focal adhesions and rescues the electrical and calcium phenotypes, placing the defect downstream of excessive ROCK-myosin II activity. The direction matters and is stated by the experiment: knockout decreases focal adhesion formation, so wild-type ADPRHL1 promotes it. The regulation term rather than the bare GO:0048041 is chosen deliberately - ADPRHL1 is not a structural component of focal adhesions and the paper's mechanism is regulatory and indirect, running through the ROCK-myosin II pathway. Evidence code is IMP: this is a genetic knockout scored by phenotype, not a direct assay of ADPRHL1 acting on a focal adhesion. No term is proposed for the calcium and conduction phenotypes, which the same experiments show are downstream of the adhesion defect and are rescued by relieving it. One caveat recorded rather than argued away: the paper's own model makes the focal adhesion defect downstream of de-repressed ROCK-myosin II, so a curator might reasonably prefer a Rho-kinase-pathway term over the focal adhesion one. This row stays on the phenotype that was actually measured, on four markers, and rescued - the pathway step is inferred from RNA-seq and inhibitor sensitivity, and no assay places ADPRHL1 on ROCK itself. Reason: Direct human loss-of-function evidence for a regulatory role in focal adhesion assembly, in the correct direction, currently absent from GOA entirely. Supporting Evidence: PMID:37880701 suggesting that ADPRHL1 deficiency disrupts the FAs in these cells PMID:37880701 the expression of Paxillin, a structural protein in FAs, in KO-CM was markedly reduced PMID:37880701 Our results demonstrated that ADPRHL1-deficient cardiomyocytes exhibited abnormal adhesion, calcium transients and electrophysiological activity. |
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Download this section (compressed HTML)Q: To InterPro and GO Central: IPR012108 "ADP-ribosylarginine hydrolase" maps to GO:0000287, GO:0003875 and GO:0051725, but a majority of its own reviewed members are pseudoenzymes. Of the entry's 1282 proteins, 11 are Swiss-Prot; 4 are named "ADP-ribosylhydrolase ARH1" and 7 are named "Inactive ADP-ribosyltransferase ARH2", each carrying a CAUTION that it lacks the metal-binding and substrate-binding residues. The result is 21 IEA annotations across seven ARH2 orthologues with no experimental, ISS or IBA support anywhere behind them. Either the entry should be split so that PIRSF016939's ARH1 and ARH2 branches are separate signatures, or the three GO mappings should be withdrawn from it. Note the contrast that shows this is one entry and not a systemic problem: the other three signatures ADPRHL1 matches - IPR050792, IPR005502 and IPR036705 - map to nothing at all, and PAINT independently reaches the correct answer, giving GO:0003875 to 20 ADPRH orthologues from node PTN009030515 and to no ARH2 protein.
Suggested experts: InterPro curators, GO Central
Q: To UniProt (ARBA): rule ARBA00088955 assigns GO:0003875 on a single condition set, FunFam id 1.10.4080.10:FF:000002 AND taxon Mammalia. That FunFam is named "ADP-ribosylarginine hydrolase isoform X1" and contains ADPRH and ADPRHL1 alike - P54922, Q8NDY3, Q8BGK2, Q5XIB3, Q3ZBM1, Q5RCJ0, Q6AZR2 and Q5XJB9 all match it, while ARH3 sits in FF:000001. The rule therefore grants a catalytic molecular function to five mammalian pseudoenzymes on the strength of a structural family's name, in entries whose own CAUTION line says the catalytic residues are absent. A negative taxon or a negative condition excluding the ARH2 orthologues would fix it.
Suggested experts: UniProt ARBA developers
Q: To UniProt (nomenclature): Q8NDY3's RecName is "Inactive ADP-ribosyltransferase ARH2", and the same name is used for all seven ARH2 orthologues. Transferase is the wrong reaction direction. The entry's own SIMILARITY line assigns it to the ADP-ribosylglycohydrolase family, its AltName is "[Protein ADP-ribosylarginine] hydrolase-like protein 1", and no ARH-family protein has been proposed to add ADP-ribose. Suggest "Inactive ADP-ribosylhydrolase ARH2", consistent with ADPRH_HUMAN "ADP-ribosylhydrolase ARH1" and ADPRS_HUMAN "ADP-ribosylhydrolase ARH3". This name is what GOA prints in the GENE NAME column of every ADPRHL1 annotation.
Suggested experts: UniProt nomenclature curators
Q: To GOA and Xenbase: the ISS pipeline and Xenbase are curating the same Xenopus laevis gene onto different accessions, and one whole paper is invisible as a result. All three of human ADPRHL1's ISS rows transfer from UniProtKB:Q6AZR2 (ARHL1_XENLA, Swiss-Prot), which carries the 2016 morpholino paper. Xenbase curated the 2020 CRISPR paper (PMID:32726316) onto A0A8J0UG54 and A0A8J0UIM8, TrEMBL entries for adprhl1.L, giving GO:0055005, GO:0030041 and GO:0036342 by IMP - none of which has reached any mammalian orthologue. Merging or cross-linking those accessions would let the later evidence propagate.
Suggested experts: GOA curators, Xenbase curators
Q: To GOA: PMID:37880701 is the only functional experiment ever performed on human ADPRHL1 - a CRISPR knockout in H9 embryonic stem cell-derived cardiomyocytes - and it contributes zero annotations. Beyond the focal adhesion term proposed here, it supports human IMP evidence for GO:0055003, which the gene currently holds only by ISS from frog.
Suggested experts: GOA curators
Q: To the ADPRH reviewers (paint/ADPRH, PR 2332): the two reviews were run independently and agree on every residue and mapped position across the five shared positions and five shared accessions. They differ on the class label at seven of those calls, because that review scores substitutions with hand-defined conservative groups while this one used BLOSUM62. The difference is entirely at aspartate-to-asparagine and serine-to-alanine, which BLOSUM62 scores positively. This review resolves the clash in favour of the ADPRH review by adding a third, mechanism-anchored test - whether the coordinating carboxylate or hydroxyl oxygen survives - which calls both substitutions lost. The anchor is that aspartate-to-asparagine at a vicinal catalytic aspartate is exactly the substitution PMID:17075046 uses to abolish ARH3 activity. Recorded here rather than silently absorbed.
Suggested experts: ai-gene-review PAINT campaign reviewers
Experiment: Identify what the Arg271-Arg272 cleft binds. Express wild-type ADPRHL1 and the loop-deletion alleles characterised in PMID:32726316 as tagged baits in human iPSC-derived cardiomyocytes and compare interactomes; a partner lost specifically by the loop deletions is a candidate for the molecular function that this review could not annotate.
Experiment: Re-test hydrolase activity against physiological substrates. The published negatives used O-acetyl-ADP-ribose and other model substrates; the family review flags that as a possible explanation for the null result. Assay recombinant ADPRHL1 against ADP-ribosylated proteins purified from cardiac muscle, and against phospho-ribosylated derivatives, which the 2020 paper raises as a candidate remaining ligand.
Experiment: Image endogenous ADPRHL1. Knock a fluorescent tag into the native ADPRHL1 locus in human iPSCs, differentiate to cardiomyocytes, and image against the knockout line as the specificity control. This would give the first localisation of the protein at native abundance in any species and the first in human, and would test whether the Z-disc-flanking distribution seen with over-expressed tagged Xenopus protein is genuine.
Experiment: Make a true mammalian null. The existing mouse allele deletes exons 3-4 and still produces the smaller ADPRHL1 species. A full knockout, verified by loss of both protein species on western blot, would test whether the Xenopus chamber-outgrowth requirement holds in a mammal and would validate or refute the three ISS rows in this review.
Experiment: Determine which ADPRHL1 protein species matters. The 40 kDa and 23 kDa forms differ in abundance across development and the mouse exon 3-4 allele separates them genetically. Define the 23 kDa species by mass spectrometry and test the two forms separately for rescue of the knockout phenotype in human cardiomyocytes.
What is not known — curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: ADPRHL1 has no known molecular function. No ligand, no substrate, no verified protein partner and no measured biochemical activity of any kind has been reported. The genetics say the vestigial substrate cleft is what matters, but what that cleft binds is undetermined - the 2020 study lists it as an open question, asking whether Adprhl1 retains binding for a partially degraded ADP-ribose derivative such as phospho-ribose, or whether the cleft is itself a target for ADP-ribosylation.
OPEN BIOLOGY MF_DARK
What is known: Established: the protein exists and is detected by mass spectrometry (UniProt PE 1); it folds on the all-alpha ADP-ribosylglycohydrolase scaffold; it is inactive against O-acetyl-ADP-ribose and against the ADP-ribose-acceptor linkages that ARH1 and ARH3 cleave; three of six magnesium-coordinating positions and both adenosine-ribose serines are lost; and one to four residue deletions in the Arg271-Arg272 loop abolish ventricular myofibril assembly. Not established: any binding activity, any partner, and whether the loop acts as a binding surface, a protein-protein interface or something else. IntAct returns zero interactions for Q8NDY3, against 17 for ADPRH and 35 for ADPRS by the same query, so this is a real absence and not a failed lookup.
Significance: Every molecular function currently in GOA for this gene is removed by this review, so after it the gene has no MF annotation at all. That is the honest state: ADPRHL1 is an essential cardiac protein with a completely undefined activity, and the family review itself cautions that the in vitro negatives may reflect the use of model substrates rather than genuine inertness.
What would resolve it: Identify what the Arg271-Arg272 cleft binds - for example by photo-crosslinking or pull-down from cardiomyocyte extracts using wild-type versus loop-deleted ADPRHL1 as bait - and re-test hydrolase activity against physiological ADP-ribosylated cardiac substrates rather than the small-molecule model substrates used so far.
Provenance (the field's own admissions):
Gap: The subcellular localisation of endogenous ADPRHL1 has never been imaged, in any species. Every localisation statement in the record - UniProt's SUBCELLULAR LOCATION line, the SubCell IEA row and the ISS row - descends from a single Xenopus experiment using transgenically over-expressed, N-terminally epitope-tagged protein.
OPEN BIOLOGYCURATION CC_DARK
What is known: Established: over-expressed tagged Xenopus Adprhl1 localises to stripes flanking the Z-disc and a diffuse stripe at the H-zone; human ADPRHL1 is heart-, skeletal-muscle- and tongue-enriched and expressed in left ventricle myocardium; and human ADPRHL1 knockout disorganises sarcomeres. Not established: where the endogenous protein is, at native abundance, in any species - the antibody raised against the protein was not sensitive enough to detect it in situ - and nothing at all about the human protein's location.
Significance: Two of the seven GOA rows are this one observation, and it is the only positive evidence for the cellular component. If the tagged construct mislocalises, both rows are wrong together and there is no independent check anywhere in the record.
What would resolve it: Endogenous immunofluorescence or a knock-in fluorescent tag at the native locus in human iPSC-derived cardiomyocytes, with the ADPRHL1 knockout line as the specificity control.
Provenance (the field's own admissions):
Gap: Whether a mammal actually requires ADPRHL1 for heart development is untested. The only published mammalian allele deletes exons 3-4, and those animals are normal - but the allele still produces the smaller ADPRHL1 protein species, so it is not a null and cannot answer the question.
NARROWING BIOLOGY BP_DARK
What is known: Established: loss of all Adprhl1 variants in Xenopus prevents ventricle outgrowth; loss in human embryonic stem cell-derived cardiomyocytes impairs adhesion, calcium handling and conduction; and mice lacking exons 3-4 are viable and normal while retaining the small protein species, which localises the essential activity to the C-terminal portion. Not established: the phenotype of a true mammalian null. As of the 2022 family review no Arh2-deficient mouse had been reported; a knockout has since been described only in a preprint with no PubMed identifier, which this review does not rely on.
Significance: Three of the seven GOA rows are ISS transfers of Xenopus developmental phenotypes into human. They are sound as ortholog inferences, but no mammalian loss-of-function result yet tests them, and the one mammalian allele that exists is hypomorphic by construction.
What would resolve it: A conditional or full mouse Adprhl1 null in which loss of both the 40 kDa and the smaller protein species is confirmed by western blot, phenotyped for chamber morphogenesis and myofibril organisation.
Provenance (the field's own admissions):
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