Top-Nots: Candidate NOT Annotations from Existing Reviews

MATURE PIPELINE

Species: human, mouse, yeast, SCHPO, DROME, ANOGA, ACET2, BACSU, DESVH, ECOLI, METEA, METTP, PSEAE, PSEPK, SALTY, CANGA, CLOCL, ARATH, worm

Top-Nots: Candidate NOT Annotations from Existing Reviews

Overview

NOT annotations (negated annotations) in GO explicitly state that a gene product does NOT have a particular function, localization, or involvement in a process. They are valuable because they:

  1. Prevent propagation of incorrect IBA/IEA annotations to other species
  2. Document experimentally-tested negative results
  3. Correct systematic errors from domain-based prediction pipelines
  4. Provide essential context for computational analyses

This project mines existing AI gene reviews to find the strongest candidates for formal NOT annotations - cases where the reviewer found strong evidence that the gene definitively does NOT have the annotated function, not merely that evidence is insufficient.

Key distinction: A REMOVE action means "this annotation should be removed" - but only a subset of these warrant a NOT annotation. The best NOT candidates are cases where:
- There is positive experimental evidence the gene lacks the function
- The gene has a domain that predicts an activity it demonstrably lacks (pseudo-enzyme pattern)
- The misannotation is likely to recur via automated pipelines without an explicit NOT

Methodology

Candidates were identified by scanning all *-ai-review.yaml files for action: REMOVE or action: MARK_AS_OVER_ANNOTATED annotations whose review summaries contain multiple strong negative-evidence keywords (e.g., "does not have", "lacks catalytic", "pseudoenzyme", "no detectable activity", "domain lacks", "misannotation", "does not catalyze", "no intrinsic").

A keyword-based scoring system ranks candidates by confidence of negative evidence. Score >= 6 indicates very high confidence; score 4-5 indicates strong candidates; score 3 indicates moderate candidates worth manual review.

Statistics

Tier 1: Highest Confidence NOT Candidates (score >= 6)

These are the strongest cases where a domain predicts an activity that is demonstrably absent.

Species Gene UniProt Incorrect Term Evidence Score Pattern
DROME CG6051 Q9VB70 GO:0004438 phosphatidylinositol-3-phosphate phosphatase activity IEA 10 FYVE domain binds PI3P but has no phosphatase activity
DROME CG6051 Q9VB70 GO:0052629 phosphatidylinositol-3,5-bisphosphate 3-phosphatase activity IEA 9 Same gene, second phosphatase term
human MEX3B Q6ZN04 GO:0006468 protein phosphorylation ISS 9 RNA-binding/E3 ligase, NOT a kinase
ANOGA PGRPS1 Q7QFK2 GO:0008745 N-acetylmuramoyl-L-alanine amidase activity IBA 7 Non-catalytic PGRP, lacks zinc-binding residues
BACSU spoVAD P40869 GO:0016746 acyltransferase activity IEA 7 Thiolase-like fold but no enzymatic activity
SCHPO Epe1 O94603 GO:0016491 oxidoreductase activity IEA 7 JmjC pseudo-enzyme, degenerate active site
human AKTIP Q9H8T0 GO:0061631 ubiquitin conjugating enzyme activity IBA 7 UEV domain lacks catalytic cysteine
human CHRAC1 Q9NRG0 GO:0003887 DNA-directed DNA polymerase activity IEA 7 Histone-fold structural protein, no catalytic activity
human PLD5 Q8N7P1 GO:0003824 catalytic activity IEA 7 Pseudoenzyme, lacks conserved HKD motifs
yeast YDJ1 P25491 GO:0005524 ATP binding IEA 7 DnaJ/HSP40 stimulates Hsp70 ATPase but does not bind ATP itself
ACET2 cipB Q01866 GO:0004553 hydrolase activity, hydrolyzing O-glycosyl compounds IEA 6 Dockerin domain =/= hydrolase
SCHPO Epe1 O94603 GO:0051213 dioxygenase activity IEA 6 JmjC pseudo-enzyme
human CREB1 P16220 GO:0006468 protein phosphorylation IDA 6 Is phosphorylated BY kinases, not itself a kinase
human DNAJA4 Q8WW22 GO:0005524 ATP binding IEA 6 DnaJ/HSP40, same pattern as YDJ1
human GMFB P60983 GO:0006468 protein phosphorylation TAS 6 Glia maturation factor, not a kinase
human ILF3 Q12906 GO:0006468 protein phosphorylation IDA 6 dsRNA-binding protein, not a kinase
human MEX3B Q6ZN04 GO:0046777 protein autophosphorylation ISS 6 Same gene, second phosphorylation term
human PRRT1 Q99946 GO:0006468 protein phosphorylation ISS 6 SynDIG4, regulates phosphorylation of GRIA1 but is not a kinase
human RASA1 P20936 GO:0003924 GTPase activity TAS 6 Stimulates Ras GTPase, has no intrinsic GTPase activity
human SURF1 Q15526 GO:1902600 proton transmembrane transport IEA 6 Assembly factor, not itself a proton transporter
human SURF1 Q15526 GO:0004129 cytochrome-c oxidase activity IEA 6 Assembly factor for CIV, does not have CcO activity itself
yeast ATP10 P18496 GO:0051082 unfolded protein binding IPI 6 Assembly factor for ATP synthase, not a chaperone

BGC project: catalytic/cofactor terms on non-catalytic subunits & pseudoenzymes

NOT candidates from the biosynthetic-gene-cluster reviews (BGC.md). EryCII is a heme-less
cytochrome-P450 homologue; PqsB and the act chain-length factor are condensing-enzyme folds
with no active site (the catalytic residues are in their partner subunit).

Species Gene UniProt Incorrect Term Evidence Score Pattern
SACEN eryCII A4F7P2 GO:0020037 heme binding IEA 9 P450 homologue lacks the conserved heme-ligating Cys; apo structure (PDB 2YJN); UniProt "lacks the heme-binding sites"
SACEN eryCII A4F7P2 GO:0004497 monooxygenase activity IEA 9 No heme → no P450 monooxygenase chemistry; functions as a GT activator
SACEN eryCII A4F7P2 GO:0005506 iron ion binding IEA 8 No heme iron; depends on the absent heme cofactor
PSEAE pqsB Q9I4X2 GO:0016746 acyltransferase activity IEA 7 FabH/KAS-III fold but no active site (Cys-129/His-269 are in PqsC); contributes_to only
STRCO actI-ORF2 Q02062 GO:0016746 acyltransferase activity IEA 7 Type II PKS chain-length factor; "does not have an active site"

Tier 2: Strong NOT Candidates (score 4-5)

Pseudo-enzymes and Wrong Catalytic Activity

Species Gene UniProt Incorrect Term Evidence Score
ACET2 cipA Q06851 GO:0004553 hydrolase activity, hydrolyzing O-glycosyl compounds IEA 5
ANOGA PGRPLD A7UTR2 GO:0008745 N-acetylmuramoyl-L-alanine amidase activity IEA 5
BACSU fliY P24073 GO:0003774 cytoskeletal motor activity IEA 5
DESVH DVU_3336 Q725T9 GO:0004673 protein histidine kinase activity IEA 5
DESVH DVU_3336 Q725T9 GO:0016301 kinase activity IEA 5
DESVH rpoN Q72BK7 GO:0016779 nucleotidyltransferase activity IEA 5
DESVH rpoN Q72BK7 GO:0016740 transferase activity IEA 5
DROME CG6051 Q9VB70 GO:0004721 phosphoprotein phosphatase activity IEA 5
DROME CG6051 Q9VB70 GO:0046856 phosphatidylinositol dephosphorylation IEA 5
DROME Ccs A1Z850 GO:0016491 oxidoreductase activity IEA 5
SALTY slrP Q8ZQQ2 GO:0051082 unfolded protein binding IPI 5
human ADM2 Q7Z4H4 GO:0006468 protein phosphorylation IDA 5
human AIP O00170 GO:0003755 peptidyl-prolyl cis-trans isomerase activity IEA 5
human BIRC6 Q9NR09 GO:0006468 protein phosphorylation TAS 5
human CHRAC1 Q9NRG0 GO:0016740 transferase activity IEA 5
human CHRAC1 Q9NRG0 GO:0003887 DNA-directed DNA polymerase activity NAS 5
human CTBP1 Q13363 GO:0006468 protein phosphorylation TAS 5
human DNAJA2 O60884 GO:0005524 ATP binding IEA 5
human EGFR P00533 GO:0004709 MAP kinase kinase kinase activity NAS 5
human IDH3B O43837 GO:0016616 oxidoreductase activity, acting on CH-OH donors IEA 5
human LIPE Q05469 GO:0006468 protein phosphorylation TAS 5
human MORC3 Q14149 GO:0018105 peptidyl-serine phosphorylation IDA 5
human PICK1 Q9NRD5 GO:0006468 protein phosphorylation ISS 5
human PIWIL1 Q96J94 GO:0003729 mRNA binding IEA 5
human RARA P10276 GO:0006468 protein phosphorylation IMP 5
human RUNX3 Q13761 GO:0006468 protein phosphorylation IDA 5
human SLC3A2 P08195 GO:0005975 carbohydrate metabolic process IEA 5
mouse Dnaja3 Q99M87 GO:0005524 ATP binding IEA 5
yeast HST1 P50111 GO:0004864 protein phosphatase inhibitor activity IMP 5

Wrong Binding, Localization, and Process (score 4-5)

Species Gene UniProt Incorrect Term Evidence Score
ANOGA PGRPS1 Q7QFK2 GO:0008270 zinc ion binding IEA 4
ANOGA PGRPS1 Q7QFK2 GO:0009253 peptidoglycan catabolic process IEA 4
CLOCL cbpA P38058 GO:0030245 cellulose catabolic process IEA 4
CLOCL hbpA Q9RGE7 GO:0000272 polysaccharide catabolic process IEA 4
DESVH DVU_3335 Q725U0 GO:0006355 regulation of DNA-templated transcription IEA 4
DESVH DVU_3336 Q725T9 GO:0000155 phosphorelay sensor kinase activity IEA 4
DESVH DVU_3336 Q725T9 GO:0016740 transferase activity IEA 4
DESVH DVU_3336 Q725T9 GO:0034220 monoatomic ion transmembrane transport IEA 4
DESVH kdpC Q725T8 GO:0005524 ATP binding IEA 4
DESVH kdpC Q725T8 GO:0016787 hydrolase activity IEA 4
DROME Ccs A1Z850 GO:0019430 removal of superoxide radicals IEA 4
DROME Ccs A1Z850 GO:0016209 antioxidant activity IEA 4
ECOLI DnaJ P08622 GO:0005524 ATP binding IEA 4
SCHPO Epe1 O94603 GO:0046872 metal ion binding IEA 4
SCHPO pmp20 O14313 GO:0008379 thioredoxin peroxidase activity IEA 4
human AIP O00170 GO:0003755 peptidyl-prolyl cis-trans isomerase activity IDA 4
human APBB1 O00213 GO:0006915 apoptotic process IEA 4
human BCL2 P10415 GO:0000209 protein polyubiquitination IDA 4
human BECN1 Q14457 GO:0006915 apoptotic process IEA 4
human C1QBP Q07021 GO:0006915 apoptotic process IEA 4
human CDC25B P30305 GO:0006468 protein phosphorylation IDA 4
human CDK1 P06493 GO:0006915 apoptotic process IEA 4
human CDK1 P06493 GO:0016579 protein deubiquitination TAS 4
human CFTR P13569 GO:0016853 isomerase activity IEA 4
human CHMP3 Q9Y3E7 GO:0140678 molecular function inhibitor activity EXP 4
human CHRAC1 Q9NRG0 GO:0071897 DNA biosynthetic process IEA 4
human CPT1C Q8TCG5 GO:0016740 transferase activity IEA 4
human GMFG O60234 GO:0006468 protein phosphorylation TAS 4
human GRPEL1 Q9HAV7 GO:0051082 unfolded protein binding IBA 4
human HCST Q9UBK5 GO:0006468 protein phosphorylation IGI 4
human HSPB6 O14558 GO:0005212 structural constituent of eye lens IEA 4
human HSPG2 P98160 GO:0005509 calcium ion binding IEA 4
human IL7R P16871 GO:0003823 antigen binding TAS 4
human KCTD11 Q693B1 GO:0016740 transferase activity IEA 4
human MORC3 Q14149 GO:0006468 protein phosphorylation IDA 4
human PDGFA P04085 GO:0038083 peptidyl-tyrosine autophosphorylation NAS 4
human PEX14 O75381 GO:0034614 cellular response to reactive oxygen species IDA 4
human PIWIL1 Q96J94 GO:0003729 mRNA binding ISS 4
human PPP3CB P16298 GO:0006468 protein phosphorylation ISS 4
human SCG5 P05408 GO:0005634 nucleus IEA 4
human SIRT1 Q96EB6 GO:0004857 enzyme inhibitor activity IEA 4
human SLC14A1 Q13336 GO:0005372 water transmembrane transporter activity IEA 4
human SPR P35270 GO:0008106 alcohol dehydrogenase (NADP+) activity TAS 4
human TMEM67 Q5HYA8 GO:0051082 unfolded protein binding IPI 4
mouse Pld4 Q497R3 GO:0004630 phospholipase D activity IEA 4

Tier 3: Moderate Candidates (score 3, selected highlights)

These need manual review but include notable patterns. Many of these are better treated as simple removals rather than formal NOT annotations — a NOT annotation requires positive evidence that the function is absent, not merely insufficient evidence that it is present.

Species Gene UniProt Incorrect Term Evidence Pattern
ACET2 celC A3DJ77 GO:0008422 beta-glucosidase activity IEA Xylanase, not a glucosidase
ACET2 xynZ P10478 GO:0033905 xylan endo-1,3-beta-xylosidase activity IDA Wrong xylanase specificity
DROME Ccs A1Z850 GO:0004784 superoxide dismutase activity IEA Copper chaperone for SOD1, not SOD itself
DESVH fliA Q726C4 GO:0003899 DNA-directed RNA polymerase activity IEA Sigma factor, not the catalytic subunit
METEA mxaI P14775 GO:0004022 alcohol dehydrogenase (NAD+) activity IEA Beta subunit, catalysis is in alpha
SCHPO Epe1 O94603 GO:0032452 histone demethylase activity IBA Core pseudo-enzyme case
SCHPO Epe1 O94603 GO:0032454 histone H3K9 demethylase activity IDA Even IDA is contested
SCHPO pmp20 O14313 GO:0004601 peroxidase activity IEA Distinct from thioredoxin peroxidase
SCHPO sou1 Q9Y6Z9 GO:0050085 mannitol 2-dehydrogenase (NADP+) activity IEA Different substrate specificity
human AGRN O00468 GO:0005200 structural constituent of cytoskeleton TAS Extracellular proteoglycan, not cytoskeletal
human APEX1 P27695 GO:0033892 deoxyribonuclease (pyrimidine dimer) activity IDA AP endonuclease, not pyrimidine dimer nuclease
human APEX1 P27695 GO:0004844 uracil DNA N-glycosylase activity TAS Misattributed activity
human ATF2 P15336 GO:0018107 peptidyl-threonine phosphorylation IDA Is phosphorylated, not a kinase
human ATP6V0C P27449 GO:0046933 proton-transporting ATP synthase activity, rotational TAS V-ATPase, not F-ATP synthase
human CAPG P40121 GO:0051014 actin filament severing IBA Caps filaments, does not sever
human CFTR P13569 GO:0006695 cholesterol biosynthetic process IEA Chloride channel, not cholesterol synthesis
human CTLA4 P16410 GO:0050853 B cell receptor signaling pathway IBA T cell inhibitory receptor
human DCN P07585 GO:0003723 RNA binding HDA HTP artifact; decorin is extracellular
human IL7R P16871 GO:0003823 antigen binding TAS Cytokine receptor, not antigen-binding
human PSMD1 Q99461 GO:0016887 ATPase activity IBA Structural lid subunit, not the ATPase ring
mouse Ang2 P21258 GO:0006412 translation IEA Angiogenin, ribonuclease activity, not a ribosome
worm pgl-1 Q9XTR1 GO:0003677 DNA binding IEA P granule component, RNA-binding

Emerging Patterns

1. Pseudo-enzyme Pattern (Highest value for NOT annotations)

Proteins with enzyme-family domains that have lost catalytic activity. These are the strongest NOT candidates because:
- Automated pipelines will repeatedly re-annotate them
- The negative evidence is biochemically definitive
- NOT annotations prevent IBA propagation

Examples: Epe1 (JmjC pseudo-demethylase), PLD5 (pseudo-phospholipase D), AKTIP (pseudo-E2), AIP (pseudo-PPIase), CG6051 (pseudo-phosphatase), CPT1C (pseudo-transferase), Pld4 (pseudo-PLD)

2. Domain =/= Function Pattern

Domains that serve structural/binding roles but are annotated with the catalytic activity of the domain family.
- Dockerin domains annotated as hydrolases (cipB, cipA, celX)
- Sensor domains annotated as kinases (DVU_3336)
- DnaJ domains annotated as ATP-binding (YDJ1, DNAJA2, DNAJA4, Dnaja3, DnaJ)
- PGRP domains annotated as amidases (PGRPS1, PGRPLD)

3. "Is Phosphorylated" =/= "Does Phosphorylation" Pattern (Over-annotation — removals, not NOTs)

A systematic class of over-annotation where proteins annotated to GO:0006468 (protein phosphorylation) are actually kinase SUBSTRATES, and the cited paper only demonstrates their phosphorylation BY kinases, not that they are kinases. This is especially common with TAS and IDA evidence codes.

Important caveat: Being a phosphorylation substrate does NOT definitively mean the protein lacks kinase activity — autophosphorylation exists, and some proteins are both substrates and kinases. These are therefore primarily over-annotation candidates (the evidence cited doesn't support the annotation), not necessarily true NOT candidates (where there is positive evidence the activity is absent).

However, none of these 19 genes have a kinase domain in InterPro, which strengthens the case. The nuance is:
- GO:0006468 (protein phosphorylation) is a process term — a gene product can be "involved in" phosphorylation via regulation without being a kinase. So a NOT for process involvement is a higher bar than a NOT for kinase MF.
- For a formal NOT on the MF (e.g. NOT GO:0004672 protein kinase activity), the absence of a kinase domain IS strong structural evidence and could justify NOTs for some of these.
- For the BP annotation GO:0006468, these are better treated as evidence-insufficient removals unless the gene has no plausible regulatory role in phosphorylation either.

Examples: CREB1, MEX3B, ATF2, RARA, RUNX3, ILF3, GMFB, ADM2, BIRC6, PICK1, PRRT1, CTBP1, LIPE, HCST, CDC25B, PPP3CB, GMFG, MORC3, PDGFA

This is the single largest category by count. Most should be simple removals, not formal NOTs. The exception: proteins with no kinase domain AND no plausible regulatory role in phosphorylation could warrant NOT annotations specifically on the MF term GO:0004672 (protein kinase activity), where structural absence of the kinase domain is positive evidence.

4. Assembly Factor =/= Complex Activity Pattern

Proteins required for assembly of multi-subunit complexes but annotated with the activity of the complex itself.
- SURF1: Assembly factor for Complex IV, annotated with cytochrome-c oxidase activity and proton transport
- ATP10: Assembly factor for ATP synthase, annotated with unfolded protein binding
- IDH3B: Regulatory subunit annotated with catalytic activity of the alpha subunit
- Ccs: Copper chaperone for SOD1, annotated with SOD activity

5. Upstream Regulator =/= Direct Activity

Genes annotated with the activity of their downstream targets.
- EGFR annotated as MAP3K (activates RAF, the actual MAP3K)
- RASA1 annotated with GTPase activity (stimulates Ras GTPase)

6. Non-catalytic Family Members

Family members that have lost the signature catalytic activity.
- PGRPS1, PGRPLD: PGRP family, lost amidase activity, retain binding
- Epe1: JmjC family, lost demethylase activity
- CG6051: Myotubularin family, lost phosphatase activity

Relationship to Other Projects

Next Steps

STATUS

Completed

In Progress

Last updated: 2026-03-06

NOTES

2026-03-06

Project Creation

Scanned 6,319 REMOVE/MARK_AS_OVER_ANNOTATED annotations across all gene reviews. Identified 22 very high-confidence (score >= 6) and 115 strong (score >= 4) candidates for formal NOT annotations across 19 species.

The most impactful pattern is pseudo-enzymes: proteins with enzyme-family domains that have demonstrably lost catalytic activity. These are particularly valuable NOT annotations because automated pipelines (InterPro2GO, IBA) will repeatedly re-annotate them without explicit NOT annotations to prevent propagation.

Key insight: The DnaJ/HSP40 ATP binding pattern (YDJ1, DNAJA2, DNAJA4, Dnaja3, DnaJ) represents a systematic class error worth addressing at the pipeline level - DnaJ proteins stimulate HSP70 ATPase but do not themselves bind ATP.

Expanded Search

Added keywords for "misannotation", "does not catalyze", "no intrinsic", "erroneous", "does not bind", "not itself", "false positive". This expanded the candidate pool from 53 to 250 at score >= 3.

Major new finding: the "is phosphorylated" =/= "does phosphorylation" pattern is the single largest category by count, with ~19 human genes incorrectly annotated to GO:0006468 protein phosphorylation because a paper described their phosphorylation by kinases. This is a systematic issue with TAS/IDA evidence codes.

Also identified the assembly factor pattern (SURF1, ATP10, IDH3B, Ccs) where proteins required for complex assembly get annotated with the complex's activity.