ACTMAP, formerly C19orf54, is a cytoplasmic cysteine protease that carries out the final proteolytic step of actin N-terminal maturation. Most proteins lose their initiator methionine co-translationally, but cytoplasmic beta- and gamma-actin instead retain it and have it N-terminally acetylated; ACTMAP subsequently excises the residue intact, as N-alpha-acetyl-methionine, after translation, exposing the Asp2 (or Glu2) that N-alpha-acetyltransferase NAA80 then acetylates to give mature actin. In muscle alpha-actins, whose initiator methionine is removed canonically, the equivalent substrate is the acetylated cysteine that becomes residue 1; this activity is established for the mouse enzyme and inferred for the human one by similarity. Catalysis depends on Cys132 inside a peptidase C39-like region structurally related to bacterial cysteine proteases: substituting this residue abolishes cleavage of immature actin, and covalent tryptoline butynamide probes that react with it cause immature actin to accumulate in human cells. A proline-rich disordered N-terminal region binds profilin 1 and profilin 2, the carriers of monomeric actin. Mice lacking ACTMAP are viable but build their cytoskeleton from immature actin in every tissue and develop a skeletal-muscle phenotype comprising shortened sarcomeric actin filaments, reduced muscle function and progressively centralized myonuclei.
Definition: Catalysis of the hydrolysis of the peptide bond between the N-alpha-acetylated N-terminal residue of a protein and the second residue, releasing an N-acetyl-amino acid such as N-acetyl-L-methionine or N-acetyl-L-cysteine and generating a new protein N-terminus. Unlike aminopeptidase activities, the alpha-amino group of the substrate's N-terminal residue is blocked by acetylation, so the released product is a substituted amino acid.
Justification: ACTMAP catalyses this reaction and GO has no term for it, so both of the terms GOA currently uses are wrong in different ways. GO:0004239 initiator methionyl aminopeptidase activity is defined as release of N-terminal initiator methionine and cross-references only EC 3.4.11.18, the canonical co-translational metal-dependent MetAP reaction, whereas ACTMAP releases N-acetyl-L-methionine and UniProt assigns it EC 3.4.11.-, deliberately not 3.4.11.18. GO:0070005 cysteine-type aminopeptidase activity gets the mechanism right but, like GO:0004239, descends from GO:0008238 exopeptidase activity, whose definition requires "a free N-terminal amino group" that an N-alpha-acetylated N-terminus does not have. GO:0008242 omega peptidase activity is the correct parent branch: it is not under exopeptidase activity, it is defined for "cleavage of non-standard peptide bonds releasing substituted amino acids", and it cross-references EC 3.4.19.1 acylaminoacyl-peptidase, the EC subclass for release of an N-acetyl amino acid from a polypeptide. The ontology already contains the pattern to copy: GO:0016920 pyroglutamyl-peptidase activity releases a single N-terminal residue whose alpha-amino group is blocked and is placed under GO:0008242 and GO:0008234 cysteine-type peptidase activity, and explicitly not under GO:0004177 aminopeptidase activity, so a dual-parented term of the same shape would fit ACTMAP exactly. Because the term should be substrate-agnostic it would also serve any other enzyme that removes an N-acetylated N-terminal residue. Adding it would let GO:0004239 revert to meaning only the METAP family, which is what 70 of the 74 gene products carrying it in five model organisms actually are.
Parent term: omega peptidase activity
Mappings:
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: Correct, and a faithful mapping. UniProt's own SUBCELLULAR LOCATION line reads "Cytoplasm" with ECO:0000305|PubMed:36173861, and GO_REF:0000044 maps the SL-0086 Cytoplasm keyword to GO:0005737, so the pipeline has added nothing that UniProt does not already assert. The location is also what the biology requires: the substrate is newly synthesised, still-unpolymerised cytoplasmic actin, and the mapped interaction partners are the cytosolic profilins. Reason: Kept as general rather than refined to GO:0005829 cytosol. Both primary papers describe ACTMAP as cytosolic, but the Science full text is not available to check whether a fractionation or imaging experiment underpins that, and the descriptive word "cytosolic" in the JACS introduction is not itself a localisation measurement. Refining on that basis would be a specificity upgrade resting on prose, so the question is raised in suggested_questions instead. Supporting Evidence: file:human/ACTMAP/ACTMAP-uniprot.txt cleavage of immature acetylated N-terminal actin, thereby contributing |
| GO:0004239 initiator methionyl aminopeptidase activity | IEA GO_REF:0000107 | MODIFY | Summary: Same term problem as the IDA row below, reached by a different route, so it is treated the same way. Additionally, this row carries no evidence of its own. Its WITH/FROM is UniProtKB:J3QPC3, which resolves to ACTMP_MOUSE, mouse Actmap, 361 aa, Swiss-Prot reviewed, with the requested accession returning itself (so not a dead or merged entry). That donor does hold its own IDA for the term, but from PMID:36173861, the same publication that supports the human IDA sitting beside this row, because Haahr et al. assayed the human enzyme and a mouse knockout in one study. Three of the donor's four annotations to GO:0004239 (GO_REF:0000024 ISS, GO_REF:0000107 IEA, GO_REF:0000119 ISO) are themselves projections from human Q5BKX5, so the human and mouse records feed each other. Reason: Modified rather than removed: the donor is the genuine 1:1 ortholog and the propagation machinery worked correctly, so this is not a propagation error. What is wrong is the term, for the reasons given on the IDA row. The reciprocal circularity is recorded so that this row is not read as a second, independent line of support for an activity that in fact rests on one paper. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: CIRCULAR PROPAGATION Sources checked: UniProtKB:J3QPC3 Β· Actmap (mouse, ACTMP_MOUSE, 361 aa, Swiss-Prot reviewed) CIRCULAR OR REDUNDANT Holds its own IDA for GO:0004239, but from PMID:36173861, the same paper behind the human IDA; its ISS, ISO and IEA annotations to the term all cite human Q5BKX5. Proposed replacements: cysteine-type aminopeptidase activity Supporting Evidence: file:human/ACTMAP/ACTMAP-bioinformatics/RESULTS.md | GO:0004239 | `UniProtKB:J3QPC3` | ISS (GO_REF:0000024, UniProt), IEA (GO_REF:0000107, Ensembl), ISO (GO_REF:0000119, GO_Central), IDA (PMID:36173861, UniProt) | GO_REF:0000024, GO_REF:0000107, GO_REF:0000119 | |
| GO:0016485 protein processing | IEA GO_REF:0000107 | ACCEPT | Summary: The term is right and the ortholog projection is mechanically sound, so the row is kept. Its evidential contribution, however, is zero: the donor UniProtKB:J3QPC3 (mouse Actmap) holds its own IDA for GO:0016485 from PMID:36173861, which is the same publication supporting the human IDA row, and the donor's other two annotations to the term (GO_REF:0000107 IEA, GO_REF:0000119 ISO) both cite human Q5BKX5. So this is a reciprocal loop, not corroboration. Reason: Accepted because the term correctly describes ACTMAP's core biological role and the donor is the true ortholog. The propagation_review records the circularity so the row is not mistaken for independent support; there is no defect in the term itself and no basis for removal. Propagation Review Root cause: EVIDENCE CIRCULAR OR REDUNDANT Failure modes: CIRCULAR PROPAGATION Sources checked: UniProtKB:J3QPC3 Β· Actmap (mouse, ACTMP_MOUSE, 361 aa, Swiss-Prot reviewed) CIRCULAR OR REDUNDANT Its IDA for GO:0016485 is from PMID:36173861, the same paper as the human IDA; its IEA and ISO annotations to the term cite human Q5BKX5. Supporting Evidence: file:human/ACTMAP/ACTMAP-bioinformatics/RESULTS.md | GO:0016485 | `UniProtKB:J3QPC3` | IEA (GO_REF:0000107, Ensembl), ISO (GO_REF:0000119, GO_Central), IDA (PMID:36173861, UniProt) | GO_REF:0000107, GO_REF:0000119 | |
| GO:0070005 cysteine-type aminopeptidase activity | IDA PMID:36173861 Actin maturation requires the ACTMAP/C19orf54 protease. | ACCEPT | Summary: The best-founded molecular-function row on the record, and the only one that captures ACTMAP's mechanism. Both halves of the term are supported. Single N-terminal residue release: UniProt records four Rhea reactions in which exactly one N-terminal residue is hydrolysed off actin. Cysteine nucleophile: UniProt annotates ACT_SITE 132 inside a peptidase C39-like region (124-244) and the residue was tested, C132A being catalytically inactive and failing to cleave immature actin (ECO:0000269|PubMed:36173861). Four years later an independent laboratory reached the same conclusion by covalent chemistry: tryptoline butynamide stereoprobes react with WT but not C132A ACTMAP, two other cysteines (C119, C271) remain reactive, and the probe destabilises WT but not C132A protein. The activity claim therefore rests on a tested catalytic residue rather than on a domain name. On the unusual assigner, what is verified is that FlyBase holds two ISS annotations on the fly ortholog CG33108 (Q9VCE8) - GO:0070005 and GO:0016485, both GO_REF:0000024 with WITH/FROM UniProtKB:Q5BKX5 - so FlyBase was curating the fly gene from this human protein; that the human IDA was created to serve as the ISS source is a plausible inference from that, not an established fact. What is not in doubt is the consequence: this is the only row in GOA that records the cysteine mechanism, since UniProt annotated only GO:0004239. Reason: Accepted as the core molecular function. One caveat is recorded rather than acted on: GO:0070005 is a descendant of GO:0008238 exopeptidase activity, whose definition requires "a free N-terminal amino group", and ACTMAP's substrate N-terminus is N-alpha-acetylated. That is an argument for creating the missing omega-peptidase-style child (see proposed_new_terms), not for demoting the best available experimentally grounded term. Substrates are recorded machine-readably via has_input extensions to ACTB and ACTG1, which the term itself cannot express. Supporting Evidence: file:human/ACTMAP/ACTMAP-uniprot.txt /note="Peptidase C39-like" file:human/ACTMAP/ACTMAP-uniprot.txt C->A: Catalytically inactive, disrupts N-terminal PMID:42159598 AlphaFold predictions revealed that ACTMAP has structural similarity to bacterial cysteine proteases with C132 representing the catalytic nucleophile. PMID:42159598 we confirmed the stereoselective reactivity of WX-02-570 with recombinant WT-ACTMAP, but not a C132A mutant, both in HEK293T cells and in cell lysates by gel-ABPP PMID:42159598 Other representative cysteine mutants of ACTMAP (C119A and C271A) retained reactivity with WX-02-570 |
| GO:0004239 initiator methionyl aminopeptidase activity | IDA PMID:36173861 Actin maturation requires the ACTMAP/C19orf54 protease. | MODIFY | Summary: The activity is real but this is the wrong term for it, on three grounds that were computed rather than assumed. First, the term means canonical methionine aminopeptidase: its definition is "Catalysis of the release of N-terminal initiator methionine from peptides" and its only EC cross-reference is 3.4.11.18. ACTMAP does not release methionine; UniProt's own four Rhea reactions have it releasing N-acetyl-L-methionine or N-acetyl-L-cysteine, and UniProt deliberately assigns EC 3.4.11.-, not 3.4.11.18. The 2022 paper's central point is that actin "defies this rule". Second, the whole aminopeptidase branch is definitionally strained here: GO:0004239 descends from GO:0008238 exopeptidase activity, whose definition requires "a free N-terminal amino group", which an N-alpha-acetylated substrate does not have. GO already models the exact analogue correctly, in that GO:0016920 pyroglutamyl-peptidase activity releases a single N-terminal residue whose alpha-amino group is blocked and sits under GO:0008242 omega peptidase activity plus GO:0008234 cysteine-type peptidase activity, and not under GO:0004177 aminopeptidase activity. This second ground does not by itself discriminate GO:0004239 from the GO:0070005 proposed in its place, since both are descendants of GO:0008238; it is an argument for the new term in proposed_new_terms, which the first and third grounds are not. Third, the term is now doing double duty: across five model organisms it is carried by 74 gene products, of which only 4 are ACTMAP-family and 70 are METAP1/METAP1D/METAP2, yeast MAP1/MAP2, RNPEPL1 and fly MAP1A/MAP1B/und, so one term covers both the metal-dependent co-translational MetAPs that remove unmodified methionine and the cysteine-nucleophile ACTMAP family that removes the acetylated residue post-translationally. Reason: Modified, not removed, and explicitly not a criticism of the curator. GO:0004239 was obsoleted in 2015 and reinstated on 2023-03-14 with its present narrow definition (QuickGO carries the comment "This term was reinstated from obsolete"); this annotation is dated 2023-03-31, seventeen days later. The curator picked the closest term available at the time, and the underlying problem is that the ontology has no term for release of an N-alpha-acetylated N-terminal residue. GO:0070005 is proposed as the replacement because it is the best currently existing term: it is already on this record with the same IDA support, it retains single-N-terminal-residue release and the cysteine mechanism, and it drops the false assertion of EC 3.4.11.18 MetAP activity. Only the first and third grounds above discriminate the two terms and so motivate this MODIFY; the second applies equally to both and motivates the term request instead. What it loses is the residue identity, which is exactly what the proposed new term would restore. Replacing the term here also removes the incorrect implication that ACTMAP belongs to the METAP family, and the same correction applies family-wide: all six reviewed members of PTHR28631 now carry GO:0004239, the five non-human ones (mouse, bovine, Xenopus, zebrafish, Drosophila) having inherited it from this human annotation. Proposed replacements: cysteine-type aminopeptidase activity Supporting Evidence: file:human/ACTMAP/ACTMAP-uniprot.txt EC=3.4.11.- {ECO:0000269|PubMed:36173861} file:human/ACTMAP/ACTMAP-uniprot.txt Reaction=N-terminal N(alpha)-acetyl-L-methionyl-L-aspartyl-[protein] + PMID:36173861 Protein synthesis generally starts with a methionine that is removed during translation. However, cytoplasmic actin defies this rule because its synthesis involves noncanonical excision of the acetylated methionine by an unidentified enzyme after translation. file:human/ACTMAP/ACTMAP-bioinformatics/RESULTS.md - `GO:0004239` (initiator methionyl aminopeptidase activity) is under `GO:0008238`: **True** file:human/ACTMAP/ACTMAP-bioinformatics/RESULTS.md - `GO:0008242` omega peptidase activity is under `GO:0008238`: **False** file:human/ACTMAP/ACTMAP-bioinformatics/RESULTS.md - of these, **4** are ACTMAP-family (PANTHER PTHR28631) and **70** are not Knowledge gap: GO has no term for hydrolytic release of an N-alpha-acetylated N-terminal residue from a protein, which is the reaction ACTMAP catalyses, so every term currently available to it is either wrong about the substrate or silent about it. OPEN ONTOLOGY RESIDUAL_SUBGAP Resolve: Create the term proposed in proposed_new_terms and re-annotate the six reviewed ACTMAP-family gene products that carry GO:0004239. "- `GO:0008242` omega peptidase activity is under `GO:0008238`: **False**" β file:human/ACTMAP/ACTMAP-bioinformatics/RESULTS.md |
| GO:0016485 protein processing | IDA PMID:36173861 Actin maturation requires the ACTMAP/C19orf54 protease. | ACCEPT | Summary: Correct and core. ACTMAP's only known biological role is a protein-maturation cleavage event, which is precisely what GO:0016485 denotes: "Any protein maturation process achieved by the cleavage of a peptide bond or bonds within a protein." Removing the acetylated initiator residue is the step that converts immature actin into a substrate for NAA80 and hence into mature actin, and genetic loss of ACTMAP in human cells and in mice leaves actin unprocessed. Reason: Accepted as the core biological process, with has_input extensions to ACTB and ACTG1 so that the substrate is recorded machine-readably. The term itself is silent about the substrate, and the obvious repair - adding GO:0030047 actin modification, which the pathway's next enzyme NAA80 carries by IDA from three papers - is wrong on GO's own structure: GO:0016485 sits under GO:0006508 proteolysis and NOT under GO:0036211 protein modification process, while GO:0030047 sits under GO:0036211 and NOT under GO:0006508. The two branches are disjoint, so a proteolytic event does not belong in the modification branch, and NAA80's precedent is an acetyl transfer that does not extend to peptide-bond hydrolysis. The extension records the same substrate fact with no branch violation and without the unwanted GO:0030036 actin-cytoskeleton-organization ancestry that GO:0030047 would confer. Supporting Evidence: file:human/ACTMAP/ACTMAP-bioinformatics/RESULTS.md | GO:0016485 | protein processing | True | False | False | True | file:human/ACTMAP/ACTMAP-bioinformatics/RESULTS.md | GO:0030047 | actin modification | False | True | True | False | PMID:42159598 protein identified in a haploid genetic screen to function as a protease that post-translationally cleaves the N-terminally acetylated methionine from Ξ²- and Ξ³-actin, which are then reacetylated by N-acetyltransferase NAA80 to generate mature actins. PMID:36173861 Thus, ACTMAP encodes the missing factor required for the synthesis of mature actin and regulates specific actin-dependent traits in vivo. |
| GO:0005522 profilin binding | IPI PMID:36173861 Actin maturation requires the ACTMAP/C19orf54 protease. | NEW | Summary: Proposed new annotation, and the only interaction worth annotating for this gene. GOA currently records no interaction term at all. UniProt's SUBUNIT section states, with ECO:0000269|PubMed:36173861, that ACTMAP interacts via its N-terminus with PFN2 isoforms IIa and IIb and with PFN1, and its DOMAIN section maps the binding surface to the N-terminal proline-rich disordered region, so this is a region-level mapping rather than a bare pull-down. An independent laboratory recovered PFN1 and PFN2 in ACTMAP immunoprecipitates and refers to them as established ACTMAP interactors. GO:0005522 profilin binding is specific and informative where GO:0005515 would not be, and profilin is biologically the right partner class, being the carrier of monomeric G-actin, the form ACTMAP must act on. Reason: Annotates the binding event only. UniProt frames the functional consequence as "the interactions may facilitate efficient cleavage of the acetylated N-terminus of immature actin", which is a hypothesis, so no substrate-delivery or adaptor function is claimed here. Two caveats are recorded rather than glossed. The JACS observation is that the profilin signal falls when ACTMAP is liganded, and those authors explicitly note the effect may be indirect because ACTMAP itself is degraded; the sentence is cited only for the fact that profilins are recovered with ACTMAP, which is what it asserts. Separately, none of the 220 IntAct records for ACTMAP involve profilin, and 212 of the 216 ACTMAP-side records are against isoform Q5BKX5-3, which lacks the catalytic cysteine, so the profilin interaction rests on the primary paper rather than on the high-throughput interaction corpus, and none of that corpus is proposed for annotation. Supporting Evidence: file:human/ACTMAP/ACTMAP-uniprot.txt Interacts (via N-terminus) with PFN2 isoforms IIa and IIb; the file:human/ACTMAP/ACTMAP-uniprot.txt interactions may facilitate efficient cleavage of the acetylated N- file:human/ACTMAP/ACTMAP-uniprot.txt The N-terminal proline-rich disordered region contributes to PMID:42159598 These IP-MS experiments also revealed stereoselective reductions in the enrichment of established ACTMAP-interacting proteins, such as profilin 1 and 2 (PFN1/2) |
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Download this section (compressed HTML)Q: Should GO create a term for release of an N-alpha-acetylated N-terminal residue, parented under GO:0008242 omega peptidase activity and GO:0008234 cysteine-type peptidase activity in the same way as GO:0016920 pyroglutamyl-peptidase activity? At present GO:0004239 initiator methionyl aminopeptidase activity, whose sole EC cross-reference is 3.4.11.18, is carried by 74 gene products across five model organisms, 70 of them METAP-family and 4 of them ACTMAP-family; that five-taxon count understates the family, because asking each reviewed PTHR28631 member directly gives six of six carrying the term. One new term would separate the two families and would fix all six reviewed ACTMAP-family records together.
Suggested experts: GO ontology editors, MEROPS and peptidase classification curators
Q: Does GO intend "covalent modification" in GO:0030047 actin modification to include hydrolytic removal of a residue, or only additive modifications? The question decides whether a substrate-specific process term is available to ACTMAP at all. On GO's present structure the answer looks like no: GO:0016485 protein processing sits under GO:0006508 proteolysis and not under GO:0036211 protein modification process, while GO:0030047 sits under GO:0036211 and not under GO:0006508, so the two branches are disjoint and a proteolytic event has no place in the modification branch. Both existing users of the term are consistent with that reading, since NAA80's three IDAs are acetyl transfer and the term's only child is actin ubiquitination. This review therefore records ACTMAP's substrate with has_input extensions rather than a process term. A secondary question follows if the answer is yes: GO:0030047 also entails GO:0030036 actin cytoskeleton organization, which an enzyme that merely modifies an actin molecule has not earned.
Suggested experts: GO ontology editors, cytoskeleton curators
Q: Does UniProt's SUBCELLULAR LOCATION for ACTMAP rest on a fractionation or imaging experiment that would support Cytosol rather than the current Cytoplasm? Both primary papers describe ACTMAP as cytosolic in prose, and the substrate, profilin-bound unpolymerised actin, is cytosolic, but the current annotation is ECO:0000305 curator inference and the Science full text was not available for checking.
Suggested experts: UniProt curators
Q: Should PAN-GO annotate ACTMAP? UniProt's DR line records 0 GO annotations based on evolutionary models for Q5BKX5, yet PTHR28631 is a single-subfamily family (PTHR28631:SF1 ACTIN MATURATION PROTEASE, 1109 proteins, 845 proteomes, 2999 taxa) with one reviewed member per species and IDA-grade evidence on the human protein. There is no paralog to confound a transfer, so this looks like a coverage gap rather than a deliberate exclusion.
Suggested experts: PAN-GO and PAINT curators
Q: Will MGI curate mouse Actmap? It still carries root ND annotations in all three aspects (GO:0003674, GO:0008150, GO:0005575 via GO_REF:0000015), so the knockout phenotype reported in the same paper that named the gene, namely shortened sarcomeric actin filaments, reduced muscle function and centralized myonuclei, is unrepresented in GO, and the human record has nothing to receive by orthology beyond the UniProt annotations.
Suggested experts: MGI curators
Q: Does human ACTMAP cleave the acetylated cysteine of muscle alpha-actins directly? UniProt annotates this activity for human by similarity from mouse (ECO:0000250 UniProtKB:J3QPC3) while the human demonstration covers beta- and gamma-actin, so the human enzyme's activity on ACTA1, ACTC1 and ACTA2 is inferred rather than shown.
Suggested experts: muscle cell biologists, UniProt curators
Experiment: Compare full-length ACTMAP with an N-terminally truncated form lacking the proline-rich region (residues 1-70) in a reconstituted cleavage assay on purified immature beta-actin, with and without profilin 1 or profilin 2, measuring cleavage kinetics by mass spectrometry of the released N-acetyl-methionine. Re-express the same constructs in ACTMAP-knockout human cells and quantify immature beta-actin with the N-terminus-specific antibody. UniProt currently states the functional role only as a possibility.
Hypothesis: Profilin binding by the proline-rich disordered N-terminus is functionally required for efficient cleavage of immature actin, rather than being an incidental interaction.
Type: biochemical reconstitution and cellular rescue
Experiment: Present purified ACTMAP with matched substrates differing only in N-terminal acetylation state (acetyl-Met-Asp-Asp-Asp versus Met-Asp-Asp-Asp, and acetyl-Cys-Asp-Asp versus Cys-Asp-Asp) and measure release of the first residue. A strict requirement for acetylation would settle the EC assignment as 3.4.19-type rather than 3.4.11-type, and would decide the ontology placement question directly instead of by analogy to pyroglutamyl-peptidase.
Hypothesis: ACTMAP is an omega-type peptidase whose activity requires the substrate N-terminus to be N-alpha-acetylated, and it cannot act on a free N-terminal methionine.
Type: enzyme substrate-specificity assay
Experiment: In human iPSC-derived skeletal myotubes, disrupt ACTMAP and measure the N-terminal processing state of ACTA1 separately from ACTB and ACTG1 by targeted mass spectrometry, then assay sarcomeric thin-filament length and contractile function. This tests directly whether the tissue restriction of the mouse phenotype follows from substrate identity, and would supply the human evidence the current record lacks.
Hypothesis: The muscle phenotype of ACTMAP loss is mediated by failure to process muscle alpha-actin, not by the beta- and gamma-actin defect that is present in all tissues.
Type: cellular genetics with targeted proteomics
Experiment: Exploit the observation that WX-02-623 both inhibits and destabilises ACTMAP: compare acute inhibition with an equipotent non-degrading analogue, and with the stereochemically matched inactive enantiomer WX-02-624, in a time course of immature beta-actin accumulation and of actin filament dynamics. The reported proteasome-dependent but neddylation-independent degradation is itself unexplained and would be addressed by the same series.
Hypothesis: Covalent Cys132 ligands can be used as acute chemical probes of actin maturation, separating the consequences of losing mature actin from the consequences of losing the ACTMAP protein.
Type: chemical biology with stereochemical controls
What is not known β curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: Nothing is known about how ACTMAP itself is regulated, and no function has been attached to its one mapped post-translational modification.
OPEN BIOLOGY RESIDUAL_SUBGAP
What is known: Ser316 is a confidently mapped phosphosite, reported independently by three large-scale mitotic phosphoproteomics studies, so the modification is not in question; its consequence for activity, localisation or stability is entirely unstudied, as is why the site should be mitotic.
What would resolve it: Phosphomimetic and non-phosphorylatable substitutions at Ser316 assayed for cleavage activity, profilin binding and protein stability across the cell cycle.
Provenance (the field's own admissions):
Gap: Why loss of ACTMAP produces a functional defect only in skeletal muscle, when immature actin accumulates in every tissue, is unexplained.
OPEN BIOLOGY BP_DARK
What is known: Both halves of the contrast are established in the knockout mouse: the cytoskeleton is built from immature actin across all tissues, yet the phenotype is restricted to shortened sarcomeric filaments, reduced muscle function and centralized nuclei.
What would resolve it: Determine whether the tissue restriction follows from substrate identity (muscle alpha-actin versus beta- and gamma-actin) or from the mechanical demands placed on thin-filament length.
Provenance (the field's own admissions):
Gap: There is no experimental structure of ACTMAP; the fold assignment and the identity of the catalytic nucleophile rest on AlphaFold models combined with mutagenesis, and no enzyme-substrate complex exists.
OPEN BIOLOGY MF_DARK
What is known: The functional conclusion is nonetheless secure, because C132A is catalytically dead and covalent probes engage WT but not C132A protein while two other cysteines stay reactive. What is missing is how the enzyme recognises an acetylated N-terminus, which no model predicts.
What would resolve it: A crystal or cryo-EM structure of ACTMAP, ideally with a covalent butynamide adduct or an N-terminal actin peptide bound.
Provenance (the field's own admissions):
Gap: How covalent liganding of Cys132 triggers proteasomal degradation of ACTMAP, and which ligase does it, is unknown.
OPEN BIOLOGY RESIDUAL_SUBGAP
What is known: The phenomenon is characterised: degradation is stereoselective, requires the catalytic cysteine, is blocked by MG-132 and is not blocked by the neddylation inhibitor MLN4924, and liganding promotes binding to HSP90AA1, HSP90AB1 and CCT2.
What would resolve it: Genetic screens for the mediators of stereoprobe-induced ACTMAP degradation, as the authors themselves propose.
Provenance (the field's own admissions):
Gap: The model organism database record for the orthologue is empty, so the in vivo phenotype cannot be reached through GO at all.
OPEN CURATION WHOLLY_DARK
What is known: Mouse Actmap carries 13 GO annotations, but three of them are root ND rows covering all three aspects, and PAN-GO reports zero evolutionary-model annotations for the human protein; the underlying phenotype data have been published since 2022.
What would resolve it: MGI curation of PMID:36173861, and PAN-GO coverage of PTHR28631.
Provenance (the field's own admissions):
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