Filaggrin is an epidermal protein of the S100 fused-type protein (SFTP) family, made as the ~400 kDa polyprotein precursor profilaggrin, which is the principal constituent of keratohyalin granules in the stratum granulosum. Profilaggrin has a two-part architecture whose halves become functionally separate proteins: an N-terminal S100 "A" domain carrying two EF-hands (one canonical, one S100-type pseudo-EF-hand) followed by a "B" domain with a nuclear localisation signal, and a tandem array of 10-12 copies of an intrinsically disordered, histidine- and arginine-rich ~324-residue filaggrin repeat. During terminal differentiation profilaggrin is dephosphorylated and proteolytically cleaved, releasing the N-terminal AB fragment, which binds calcium and partner proteins and translocates to the keratinocyte nucleus, and free filaggrin monomers. The repeat array drives assembly of keratohyalin granules as liquid-liquid phase-separated biomolecular condensates, and the released monomers bind keratin intermediate filaments along their rod domains through charge-complementary "ionic zipper" contacts, condensing them into macrofibrillar bundles and compacting the cell as it flattens into a squame. A fraction of filaggrin is covalently isodipeptide cross-linked by transglutaminases into the cornified envelope. At the granular-to-corneum transition an intracellular acidification dissolves the histidine-rich condensates; filaggrin is then citrullinated and completely degraded by caspase-14 and bleomycin hydrolase to free amino acids, urocanic acid and pyrrolidone carboxylic acid, which constitute natural moisturising factor and maintain stratum corneum hydration and acidity. Filaggrin is expressed chiefly in the granular layer of skin and other stratified epithelia. Loss-of-function variants, carried by up to 10% of people of European ancestry, cause ichthyosis vulgaris and are the strongest known genetic risk factor for atopic dermatitis and the associated atopic march of asthma and allergic sensitisation.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0001533 cornified envelope | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic propagation at PANTHER node PTN002503122 within PTHR22571 (S100-fused Epidermal Structural Protein). The donor set resolves to a tight, architecturally coherent SFTP clade: human FLG itself (P20930), human hornerin (Q86YZ3) and mouse Hrnr (MGI:3046938), plus mouse Flg2 (MGI:3645678). Filaggrin's presence in the cornified envelope is directly demonstrated on the target itself: proteinase-K digestion and sequencing of 145 cross-linked peptides from purified human foreskin cornified envelopes recovered isodipeptide cross-links involving filaggrin [PMID:7543090]. Reason: The node placement is sound - all donors are S100-fused epidermal barrier proteins sharing the S100 A-domain plus low-complexity repeat architecture, and cornified envelope incorporation is a shared property of the clade. FLG appearing in its own WITH/FROM is the expected marker that experimental grounding exists on the target, not circularity. The IBA agrees with the independent IDA from PMID:7543090. Supporting Evidence: PMID:7543090 In addition, cross-links involving loricrin and keratins 1, 2e, and 10 or filaggrin were recovered in both levels. The data establish for the first time that these several proteins are indeed cross-linked protein components of the CE structure. |
| GO:0036457 keratohyalin granule | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic propagation from PTN002503122, donors human FLG (self), human hornerin (Q86YZ3) and mouse Hrnr (MGI:3046938). The keratohyalin granule is the defining location of profilaggrin and is directly documented for the human protein [PMID:1429717], and the modern mechanism - that filaggrin is what assembles these granules, by liquid-liquid phase separation - is established in [PMID:32165560]. Reason: Correct and appropriately specific. The `is_active_in` qualifier is apt: the granule is not merely where filaggrin is found, it is a condensate that filaggrin itself scaffolds. Supporting Evidence: PMID:1429717 It is synthesized as a large precursor protein, profilaggrin, that consists of multiple filaggrin units and is localized in keratohyalin granules. PMID:32165560 Here, we found that filaggrin assembles KGs through liquid-liquid phase separation. |
| GO:0061436 establishment of skin barrier | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic propagation from PTN002503122, donors human FLG (self), human FLG2 (Q5D862), human hornerin (Q86YZ3) and mouse Flg2 (MGI:3645678). Barrier establishment is the shared, defining biology of the SFTP clade, and for FLG it is the single best-evidenced process: loss-of-function alleles cause ichthyosis vulgaris and are the strongest genetic risk factor for atopic dermatitis [PMID:19386895], and phase-separation dynamics of filaggrin condensates drive squame formation and barrier establishment [PMID:32165560]. Reason: Node placement is well supported and the term is the correct level of granularity for this gene. Concordant with the independent IEP annotation from PMID:23403047. Supporting Evidence: PMID:19386895 Loss of profilaggrin or filaggrin leads to a poorly formed stratum corneum (ichthyosis), which is also prone to water loss (xerosis). PMID:32165560 Thus, the pH shift appears to function specifically in altering the material properties of histidine-rich KGs, which in turn promote chromatin compaction, enucleation, and skin barrier establishment. |
| GO:0005198 structural molecule activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping of IPR003303 (Filaggrin repeat) to the root structural term. The signature matched is the tandem repeat region, and the directly demonstrated activity of that region is not architectural but a binding activity: filaggrin associates specifically with intermediate filaments and aligns them into macrofibrils, with roughly two filaggrin molecules per three-chain filament building block [PMID:6170061], and the histidine-rich filaggrins were shown in vitro to aggregate keratin filaments specifically into bundles [PMID:6195345]. The mechanism is a charge-complementary "ionic zipper" between cationic beta-turns of the repeat and the packed rod domains of the filament [PMID:7687298], and it is so sequence-degenerate that a 20-26 residue synthetic peptide is as effective as the full-length protein. In vitro the repeat binds type III vimentin/desmin filaments as well as type I/II keratin; in vivo the partner is keratin. Reason: This is not a claim that filaggrin lacks structural molecule activity - it plainly has one, and this review ACCEPTs the IDA to GO:0030280 (structural constituent of skin epidermis) for the cornified-envelope pool. GO:0030280 is_a GO:0005198, so the bare root-level IEA is redundant with a child term that is already asserted from direct evidence on the human protein, and it is the less informative of the two. Separately, the signature that generated this IEA is IPR003303 (Filaggrin repeat), and the directly demonstrated activity of that repeat region is a binding activity rather than an architectural one: it associates specifically with pre-formed intermediate filaments and condenses them into macrofibrils [PMID:6170061, PMID:6195345, PMID:7687298], and the repeat pool is subsequently deiminated and proteolysed to free amino acids [PMID:8417356] rather than retained in the structure it organises. The durable fix is therefore at the mapping level - IPR003303 is better mapped to GO:1990254 keratin filament binding - which is what is proposed as the replacement here. An independent OpenScientist analysis is consistent with this, finding every repeat region intrinsically disordered with low helix and sheet propensity and cationic only at acidic skin pH. Proposed replacements: keratin filament binding Supporting Evidence: PMID:6170061 We describe a class of cationic structural proteins that associate specifically with intermediate filaments (IF) but not with other types of cytoskeletal proteins. PMID:6170061 Stoichiometric analyses suggest that two molecules of filaggrin bind to each three-chain building block of the IF, possibly by ionic interactions with the coiled-coil alpha-helical regions of the IF. PMID:6195345 secondly by their ability in vitro to aggregate keratin filaments specifically into bundles PMID:7687298 Filaggrins of mammalian epidermis represent archetypical examples of intermediate filament-associated proteins that can bind large numbers of intermediate filaments in vitro (and keratin filaments in vivo) into macrofibrils. PMID:19386895 which binds to and condenses the keratin cytoskeleton and thereby contributes to the cell compaction process that is required for squame biogenesis file:human/FLG/FLG-hypotheses/mf-keratin-binding-vs-structural/openscientist.md The immediate molecular function of the mature ~324-aa filaggrin repeat unit is best described as **intermediate-filament / keratin binding that drives keratin bundling** (GO:0019215 / GO:1990254), *not* a generic **structural molecule activity** (GO:0005198). |
| GO:0005509 calcium ion binding | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping of IPR002048 (EF-hand domain), and in this case the electronic call is correct and understates the available evidence. Calcium binding by the two N-terminal EF-hands was demonstrated biochemically in vitro [PMID:8417356], and a 2.2 A crystal structure of the human profilaggrin N-terminal S100 domain was solved with bound calcium ions [PMID:25760235, PDB 4PCW]. UniProt places Ca(2+) binding residues at 62, 64, 66, 68 and 73 - i.e. in the canonical EF-hand 2 only; EF-hand 1 is the S100-type pseudo-EF-hand. Calcium dependence is functionally meaningful: the A and B domains together bind annexin II and keratin intermediate filaments in a calcium-dependent manner [PMID:32893105]. Reason: Directly demonstrated, with structural proof. Scope note for downstream users: this is a property of profilaggrin and of the cleaved N-terminal AB fragment, not of the mature filaggrin repeat monomer, which contains no EF-hand. It is a regulatory activity rather than the barrier-forming one. An evidence upgrade from IEA to IDA citing PMID:25760235 would be justified. Supporting Evidence: PMID:8417356 It contains two alpha-helical regions, termed EF-hands, that bind calcium in vitro. This is the first example of functional calcium-binding domains fused to a structural protein. PMID:32893105 Profilaggrin B domain cooperates with the S100 domain to bind annexin II and keratin intermediate filaments in a calcium-dependent manner using exposed cationic surface. file:human/FLG/FLG-hypotheses/function-hypothesis-go-0005509/openscientist.md Calcium binding is a genuine, direct molecular function of the gene product, localized to the **N-terminal profilaggrin S100 "fused-type" domain**, which contains two EF-hand motifs. |
| GO:0046914 transition metal ion binding | IEA GO_REF:0000002 | REMOVE | Summary: InterPro2GO mapping of IPR034325 (S-100 domain). Soluble S100 family members often carry a secondary Zn(2+)/Cu(2+) site at the dimer interface, and the mapping propagates that expectation to every S100-domain protein. No such site has been demonstrated for filaggrin. The dedicated 2.2 A structural study of exactly this domain reports only calcium ions bound and discusses no transition metal [PMID:25760235], and UniProt P20930 annotates five Ca(2+) binding residues and no transition-metal site at all. Reason: A signature-level over-propagation with no experimental support in this protein and with the most direct available evidence - a calcium-bound crystal structure of the very domain in question - silent on transition metals. This is exactly the category of demonstrably unwarranted electronic mapping that should be removed rather than retained as a hedge; the genuine metal-binding activity of the domain is already captured by GO:0005509. An independent OpenScientist audit adds three convergent lines: the deposited 4PCW coordinates contain eight calcium atoms and no transition-metal atoms; residues 1-91 carry only two histidines and no cysteines, lacking the His3Asp/His4-6 or Cys-cluster chemistry that characterised metal-binding S100 proteins use, and those two histidines are 8-15 A from any calcium site and cannot cluster into a motif; and the genuinely Zn-binding S100 proteins (S100A7, S100A8, S100A9, S100B) instead carry the specific child term GO:0008270 with experimental evidence. Because the mapping is at the signature level, all six human S100 fused-type proteins carrying IPR034325 (FLG, FLG2, HRNR, RPTN, TCHH, CRNN) have inherited the same unsupported claim. The honest caveat is that no study has directly assayed this domain for Zn(2+)/Cu(2+) binding, so the case rests on structure, sequence and comparative annotation rather than a direct negative experiment; a confirmatory assay is listed under suggested_experiments. Supporting Evidence: PMID:25760235 To explore its molecular function, we determined a 2.2 Γ
-resolution crystal structure of the N-terminal fused-type S100 domain of human profilaggrin with bound calcium ions. file:human/FLG/FLG-hypotheses/function-hypothesis-go-0046914/openscientist.md a HETATM audit of the deposited coordinates finds **8 calcium atoms and zero transition-metal atoms** file:human/FLG/FLG-hypotheses/function-hypothesis-go-0046914/openscientist.md The IEA annotation of FLG to **transition metal ion binding (GO:0046914)** is an **over-annotation** β a family-level InterPro2GO carry-over rather than a member-specific, experimentally supported molecular function of the filaggrin gene product. |
| GO:0005515 protein binding | IPI PMID:23629652 Kallikrein-related peptidase 5 functions in proteolytic proc... | KEEP AS NON CORE | Summary: IntAct-curated interaction with KLK5 (Q9Y337). The relationship is enzyme-substrate: KLK5 was identified as a profilaggrin-processing protease, the two co-localise in the stratum granulosum, and KLK5 knockdown raises profilaggrin levels [PMID:23629652]. Filaggrin is the substrate here, not the actor. Reason: The interaction is real and correctly curated, but bare `protein binding` conveys no functional information about FLG and an enzyme-substrate contact is not a molecular function of the substrate. Retained as a documented interaction, not as a core function. Where FLG does have informative, pocket-mediated partners - annexin II, stratifin/14-3-3 sigma and HSP27 binding the hydrophobic pockets of the N-terminal S100 dimer [PMID:25760235] - those would be the interactions worth capturing. Supporting Evidence: PMID:23629652 KLK5 knockdown in normal cultured human epidermal keratinocytes resulted in higher levels of profilaggrin, indicating that KLK5 potentially functions in profilaggrin cleavage. PMID:23629652 we reveal that KLK5 and profilaggrin co-localize in the stratum granulosum in human epidermis PMID:25760235 The profilaggrin S100 domain formed a stable dimer, which contained two hydrophobic pockets that provide a molecular interface for protein interactions. |
| GO:0036464 cytoplasmic ribonucleoprotein granule | IDA GO_REF:0000052 | MODIFY | Summary: This is a mapping artefact rather than a finding. The Human Protein Atlas reports FLG localised to "cytoplasmic bodies" in HaCaT keratinocytes (Enhanced reliability, antibodies HPA027505 and HPA030188); GO_REF:0000052 maps the HPA "cytoplasmic bodies" class onto GO:0036464. In a keratinocyte line, FLG-positive cytoplasmic puncta are keratohyalin-granule-like condensates - HaCaT is the very cell line in which filaggrin condensate assembly was characterised [PMID:32165560] - not ribonucleoprotein granules. Reason: GO:0036464 requires ribonucleoprotein content, and filaggrin has no known RNA-binding activity nor any reported association with P-bodies or stress granules. The observed structure is well characterised and has its own term: GO:0036457 keratohyalin granule, which is already independently supported for this gene by IDA and IBA. The underlying imaging observation is sound; only the term choice is wrong. Proposed replacements: keratohyalin granule Supporting Evidence: PMID:32165560 Here, we found that filaggrin assembles KGs through liquid-liquid phase separation. PMID:1429717 It is synthesized as a large precursor protein, profilaggrin, that consists of multiple filaggrin units and is localized in keratohyalin granules. |
| GO:0070268 cornification | IDA PMID:7543090 The proteins elafin, filaggrin, keratin intermediate filamen... | ACCEPT | Summary: Filaggrin was recovered as an isodipeptide cross-linked component of cornified envelopes purified from human foreskin epidermis, establishing its participation in the terminal cornification programme [PMID:7543090]. This is complemented by the demonstration that filaggrin condensate dynamics drive chromatin compaction, enucleation and squame formation [PMID:32165560]. Reason: Core biological process for this gene, directly evidenced, and at the right level of specificity. Supporting Evidence: PMID:7543090 In addition, cross-links involving loricrin and keratins 1, 2e, and 10 or filaggrin were recovered in both levels. The data establish for the first time that these several proteins are indeed cross-linked protein components of the CE structure. PMID:32165560 Thus, the pH shift appears to function specifically in altering the material properties of histidine-rich KGs, which in turn promote chromatin compaction, enucleation, and skin barrier establishment. |
| GO:0031012 extracellular matrix | HDA PMID:28344315 Proteomic characterization of human multiple myeloma bone ma... | REMOVE | Summary: Derived from a shotgun proteomic survey of decellularised extracellular matrix from multiple myeloma bone marrow [PMID:28344315]. Filaggrin is an intracellular protein of the epidermal granular layer with no secretory signal peptide, and it is not expressed in bone marrow; UniProt records expression in skin and a set of stratified epithelia, not haematopoietic tissue. Reason: Detection of a large, abundant skin protein in a bone-marrow proteome is the signature of keratinocyte/skin-derived sample contamination, a well-known confounder of shotgun proteomics, rather than evidence of extracellular-matrix localisation. The claim is also biologically incoherent: nothing in the filaggrin literature places it outside the cell, and its terminal fate is complete intracellular proteolysis to free amino acids [PMID:8417356]. This is an unsupported high-throughput inference, not a curator's reading of a functional experiment. Supporting Evidence: PMID:8417356 Later, filaggrin itself is degraded to free amino acids that participate in maintenance of epidermal flexibility. |
| GO:0001533 cornified envelope | IDA PMID:7543090 The proteins elafin, filaggrin, keratin intermediate filamen... | ACCEPT | Summary: Direct evidence: cornified envelopes purified from human foreskin epidermis were digested with proteinase K and 145 unique cross-linked peptides were sequenced, recovering isodipeptide cross-links involving filaggrin at both the outer and middle levels of the envelope [PMID:7543090]. Reason: Directly demonstrated on the human protein, and the covalent nature of the association makes this an unusually solid localisation claim. Supporting Evidence: PMID:7543090 In addition, cross-links involving loricrin and keratins 1, 2e, and 10 or filaggrin were recovered in both levels. PMID:7543090 The recovery of cross-links involving keratins further suggests that the subjacent cytoplasmic keratin intermediate filament-filaggrin network is anchored to the developing CE during these events. |
| GO:0030280 structural constituent of skin epidermis | IDA PMID:7543090 The proteins elafin, filaggrin, keratin intermediate filamen... | ACCEPT | Summary: This is the one molecular function of filaggrin that is genuinely structural rather than a binding activity. A fraction of filaggrin is covalently isodipeptide cross-linked by transglutaminases into the cornified envelope, becoming a permanent constituent of that structure [PMID:7543090], and the keratin filament-filaggrin network is anchored to the developing envelope. Reason: Correctly specific for an epidermal structure, and directly demonstrated by peptide sequencing of cross-linked envelope material. Preferable to the bare GO:0005198 IEA, which this review proposes to repoint to the repeat's binding activity. Note the division of labour within the protein: this term describes the cross-linked cornified-envelope pool, while the bulk of filaggrin acts transiently on keratin and is then degraded. Accepted as a real but secondary molecular function, not as the gene's primary activity. Supporting Evidence: PMID:7543090 The data establish for the first time that these several proteins are indeed cross-linked protein components of the CE structure. PMID:7543090 The recovery of cross-links involving keratins further suggests that the subjacent cytoplasmic keratin intermediate filament-filaggrin network is anchored to the developing CE during these events. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-6810357 | KEEP AS NON CORE | Summary: Reactome pathway assertion from "Fillagrin and keratin intermediate filaments polymerize forming a network". Profilaggrin and filaggrin are cytoplasmic throughout their working life, so the compartment is not wrong, but `cytosol` is far less informative than the granule and envelope terms that describe where filaggrin actually acts. Reason: True but uninformative. The functionally meaningful locations are the keratohyalin granule (GO:0036457) and the cornified envelope (GO:0001533); much of the cytosolic pool is in fact phase-separated into condensates rather than freely dissolved [PMID:32165560]. Supporting Evidence: PMID:32165560 Here, we found that filaggrin assembles KGs through liquid-liquid phase separation. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-6810937 | KEEP AS NON CORE | Summary: Reactome pathway assertion from "Lamellar bodies bind the early cornified envelope". Same compartment call as the other Reactome cytosol rows. Reason: Correct compartment but uninformative relative to GO:0036457 and GO:0001533. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-6811539 | KEEP AS NON CORE | Summary: Reactome pathway assertion from "Reinforcement of the Cornified Envelope". Same compartment call as the other Reactome cytosol rows. Reason: Correct compartment but uninformative relative to GO:0036457 and GO:0001533. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-6814298 | KEEP AS NON CORE | Summary: Reactome pathway assertion from "Late envelope proteins bind cornified envelope:CDSN". Same compartment call as the other Reactome cytosol rows. Reason: Correct compartment but uninformative relative to GO:0036457 and GO:0001533. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-6814387 | KEEP AS NON CORE | Summary: Reactome pathway assertion from "CASP14 cleaves filaggrin". Caspase-14 cleavage of the filaggrin repeat unit is a genuine, experimentally mapped step in filaggrin catabolism [PMID:21654840], and it occurs in the cytoplasm of cornifying cells. Reason: Correct compartment but uninformative as a location. The underlying event matters biologically - it is how filaggrin is converted into natural moisturising factor - but that is a process, not this cellular-component annotation. Supporting Evidence: PMID:21654840 We found that caspase-14 cleaves the FLG repeat unit and identified two caspase-14 cleavage sites. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-6814734 | KEEP AS NON CORE | Summary: Reactome pathway assertion from "CDSN binds the cornified envelope". Same compartment call as the other Reactome cytosol rows. Reason: Correct compartment but uninformative relative to GO:0036457 and GO:0001533. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-8849797 | KEEP AS NON CORE | Summary: Reactome pathway assertion from "Membrane proteases cleave Profilaggrin producing Filaggrin". Same compartment call as the other Reactome cytosol rows. Reason: Correct compartment but uninformative relative to GO:0036457 and GO:0001533. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-8934819 | KEEP AS NON CORE | Summary: Reactome pathway assertion from "Cytoplasmic proteases cleave Profilaggrin producing Filaggrin". Same compartment call as the other Reactome cytosol rows. Reason: Correct compartment but uninformative relative to GO:0036457 and GO:0001533. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-8942224 | KEEP AS NON CORE | Summary: Reactome pathway assertion from "Filaggrin binds Keratin tonofilament:Desmosome". The underlying reaction is the keratin-binding activity of filaggrin, directly demonstrated in [PMID:6170061] and [PMID:7687298]; the derived annotation here is only the cytosolic compartment. Reason: Correct compartment but uninformative as a location. The informative content of this Reactome reaction is the molecular function, which this review captures by repointing GO:0005198 to GO:1990254 keratin filament binding. Supporting Evidence: PMID:6170061 They interact in vitro with the IF several different types of cells to form large fibers or macrofibrils in which many IF are highly aligned in parallel arrays. |
| GO:0036457 keratohyalin granule | IDA PMID:12850301 Analysis of epidermal-type transglutaminase (transglutaminas... | ACCEPT | Summary: Annotation made by a UniProt curator from the full text of an immunohistochemical study of transglutaminase 3 in human epidermis and cultured keratinocytes, in which profilaggrin was co-examined as a differentiation marker [PMID:12850301]. The cached record here is abstract-only, so the granule imaging the curator saw is not visible to this review. Reason: The claim is independently and abundantly established for this protein - keratohyalin granule localisation of profilaggrin is its defining cell-biological property [PMID:1429717, PMID:32165560] - and the paper's author list includes Presland, Dale and Fleckman, the group that characterised human profilaggrin. There is no basis to second-guess an experimental annotation whose full text has not been read, and the conclusion is correct regardless. Supporting Evidence: PMID:12850301 In cultured keratinocytes, TGase 3 was expressed in differentiating cells coincident with profilaggrin and keratin 10 expressions. PMID:1429717 It is synthesized as a large precursor protein, profilaggrin, that consists of multiple filaggrin units and is localized in keratohyalin granules. |
| GO:0036457 keratohyalin granule | IDA PMID:1429717 Characterization of the human epidermal profilaggrin gene. G... | ACCEPT | Summary: The foundational characterisation of the human profilaggrin gene and protein, which established both the keratohyalin-granule localisation and the N-terminal S100-like domain [PMID:1429717]. UniProt cites the same paper for its subcellular location statement. Reason: Directly evidenced, specific, and the defining location of the protein. Supporting Evidence: PMID:1429717 It is synthesized as a large precursor protein, profilaggrin, that consists of multiple filaggrin units and is localized in keratohyalin granules. |
| GO:0005634 nucleus | HDA PMID:21630459 Proteomic characterization of the human sperm nucleus. | KEEP AS NON CORE | Summary: The cited evidence is a shotgun proteomic catalogue of purified human sperm nuclei [PMID:21630459], which is a weak basis for this claim in a keratinocyte protein and is prone to the same contamination confound as the bone-marrow ECM dataset. The term itself, however, is independently correct for FLG on much better evidence: the cleaved profilaggrin N-terminal domain carries a functional bipartite nuclear localisation signal in its B domain [PMID:12230510] and is seen by immuno-electron microscopy in the nuclei of transition-layer keratinocytes [PMID:9800950]. Reason: Retained because the localisation is real, not because the cited dataset establishes it - the supporting references added here, not the sperm-nucleus proteome, are what carry the claim. It is non-core and fragment-specific: only the cleaved N-terminal AB peptide enters the nucleus, transiently, at the granular-to-cornified transition; mature filaggrin repeats do not. Re-anchoring this annotation to PMID:9800950 or PMID:12230510 with an experimental evidence code would be an improvement. Supporting Evidence: PMID:9800950 Antibodies specific for the amino-terminal domains of profilaggrin showed localization in keratohyalin granules in the granular cells, but stained the nucleus in transition cells. PMID:12230510 The nuclear localization signals in human and mouse profilaggrin were shown to be functional by transfection of epithelial cells and depended on the absence of filaggrin sequences. |
| GO:0061436 establishment of skin barrier | IEP PMID:23403047 Defects of filaggrin-like proteins in both lesional and nonl... | ACCEPT | Summary: Expression-pattern evidence from an FLG-genotyped cohort of 73 adults with atopic dermatitis and 73 matched controls: FLG, hornerin and FLG2 were all reduced in atopic skin relative to controls, irrespective of FLG genotype, and more so in lesional than non-lesional skin [PMID:23403047]. Reason: Appropriate use of IEP for a correlation between expression level and barrier state, and the process term is correct and well corroborated by the genetics of loss-of-function alleles [PMID:19386895] and by the IBA. IEP is inherently weaker than the direct evidence, so this row supports rather than establishes the annotation. Supporting Evidence: PMID:23403047 Compared with control subjects, FLG, HRNR, and FLG2 were detected at significantly lower levels in the skin of patients with AD, irrespective of their FLG genotype. PMID:19386895 Loss of profilaggrin or filaggrin leads to a poorly formed stratum corneum (ichthyosis), which is also prone to water loss (xerosis). |
| GO:0030216 keratinocyte differentiation | TAS PMID:17704059 Role of ADAM-9 disintegrin-cysteine-rich domains in human ke... | KEEP AS NON CORE | Summary: The term is correct for FLG - filaggrin is both a marker and an effector of keratinocyte terminal differentiation, and its condensate dynamics were shown to govern that programme [PMID:32165560] - but the cited reference does not support it. PMID:17704059 is a study of ADAM-9 disintegrin-cysteine-rich domains in keratinocyte migration and beta1 integrin engagement; filaggrin is not its subject and the abstract does not mention it. Reason: Kept because the biology is right, marked non-core because GO:0030216 is a broad parent process relative to FLG's specific role, which is better captured by GO:0070268 cornification and GO:0061436 establishment of skin barrier. The reference should be replaced: PMID:32165560 is the appropriate primary support. Supporting Evidence: PMID:32165560 The dynamics of phase separation governed terminal differentiation and were disrupted by human skin barrier disease-associated mutations. |
| GO:0140693 molecular condensate scaffold activity | IDA PMID:32165560 Liquid-liquid phase separation drives skin barrier formation... | NEW | Summary: Proposed new molecular function, covering the activity by which filaggrin builds keratohyalin granules. Filaggrin and its paralogs are intrinsically disordered low-complexity repeat proteins that assemble keratohyalin granules by liquid-liquid phase separation [PMID:32165560]. The tandem repeat array is the functional unit, on two independent assays. In a transfection-localisation assay a single FLG repeat stayed diffuse in the cytoplasm while constructs of four or more repeats formed KG-like structures. In a separate quantitative measurement of critical concentration, wild-type filaggrin (n = 12) phase separated at ~2 uM whereas disease-associated variants of four or fewer repeats required ~130 to over 1500 uM - a roughly 65- to 750-fold rise. Four-repeat constructs therefore do form granules, but only at concentrations far above the wild-type threshold, which is why n = 4 appears in both results. The N-terminal S100 domain lowers the critical concentration further by dimerising while it is still attached. Endogenous granules behave as liquid condensates whose coalescence is limited by keratin filament bundles, and whose pH-triggered dissolution at the granular-to-corneum transition drives chromatin compaction and enucleation. Reason: GO:0140693 is defined as "binding and bringing together two or more macromolecules in contact, permitting those molecules to organize as a molecular condensate" - a precise fit for what filaggrin does, and the mechanism that unifies its granule biology with its keratin-organising role. GOA currently has no molecular function term describing this. It is distinct from the keratin-binding activity proposed for GO:0005198 and from the cornified-envelope structural role of GO:0030280, and it is the activity most directly perturbed by the FLG repeat-number variants that cause human disease. Supporting Evidence: PMID:32165560 Here, we found that filaggrin assembles KGs through liquid-liquid phase separation. PMID:32165560 Over a wide range of expression levels, disease-associated mutations with β€4 repeats exhibited a large increase (~130 to >1500 ΞΌM) in critical concentration required for phase separation (Fig. 2B and fig. S7, B to F). By contrast, wild-type filaggrin (n = 12) phase separated at ~2 ΞΌM. PMID:32165560 Filaggrin and its less-studied (often less-abundant) paralogs are intrinsically disordered repeat proteins with a low-complexity (LC) sequence. PMID:32165560 The S100 domain is known to dimerize (29), and when fused to filaggrin variants, it reduced the critical concentration for phase separation PMID:32165560 Our findings suggest a model whereby reciprocal density-dependent interactions between LC domains of terminal differentiationβspecific keratins and KGs structure the cytoplasm to form an elaborate, interwoven network of stabilized liquid-like KGs and keratin filament bundles. |
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Download this section (compressed HTML)Q: Is the keratin-bundling activity of the filaggrin repeat separable from its condensate-scaffolding activity, or are they the same physical interaction observed at two scales? A repeat variant that phase-separates normally but fails to bundle keratin (or the converse) would settle whether GO should record one molecular function or two.
Q: Should GO:0030280 (structural constituent of skin epidermis) be reserved for the cross-linked cornified-envelope pool of filaggrin, with the keratin-organising activity of the soluble pool annotated separately? At present a single molecular function term is doing both jobs for several epidermal proteins.
Q: Does the profilaggrin N-terminal AB fragment have a molecular function in the nucleus, or is nuclear translocation simply a disposal route? Its identified partners annexin II, 14-3-3 sigma and HSP27 are all cytoplasmic, and no nuclear target has been reported.
Q: Is there any experimental evidence that any S100 fused-type protein binds a transition metal? If not, the InterPro2GO mapping of IPR034325 to GO:0046914 may be propagating an unwarranted transition-metal claim across the whole SFTP family, not just FLG.
Experiment: Test transition-metal binding of the recombinant profilaggrin S100 domain directly by isothermal titration calorimetry and inductively coupled plasma mass spectrometry with Zn(2+) and Cu(2+), in the presence and absence of saturating Ca(2+). This would either supply the missing evidence for GO:0046914 or confirm its removal.
Hypothesis: The profilaggrin S100 domain does not bind transition metals, and GO:0046914 is an unwarranted InterPro2GO propagation.
Experiment: Engineer FLG repeat variants that decouple charge from phase behaviour - for example His-to-Gln substitutions that abolish pH-triggered dissolution while preserving net charge, and Arg-to-Lys substitutions that preserve charge while removing cation-pi capacity - and assay condensate formation and keratin bundling in parallel in HaCaT cells and in vitro with reconstituted K1/K10 filaments.
Hypothesis: Condensate scaffolding and keratin bundling are separable activities of the filaggrin repeat, driven by distinct sequence features.
Experiment: Quantify by targeted proteomics what fraction of filaggrin ends up cross-linked into the cornified envelope versus degraded to natural moisturising factor, across FLG genotypes. This would establish whether the structural (GO:0030280) and transient condensate/keratin-binding pools are comparable in size.
Hypothesis: The cornified-envelope cross-linked pool of filaggrin is a minor side branch relative to the pool degraded to natural moisturising factor.
Experiment: Reconstitute keratohyalin-granule-like condensates in vitro from purified filaggrin repeat protein and K1/K10 filaments and image by cryo-electron tomography, to test the ionic zipper model directly and determine whether keratin is excluded from, wets, or is embedded within the condensate.
Hypothesis: Filaggrin condenses keratin filaments by distributed electrostatic contacts along the filament rod domain, as predicted by the ionic zipper model.
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