{"gene":"SHARPIN","run_date":"2026-04-28T20:42:07","timeline":{"discoveries":[{"year":2011,"finding":"SHARPIN functions as a novel component of the linear ubiquitin chain assembly complex (LUBAC) by binding to the LUBAC subunit HOIP (RNF31), thereby stimulating the formation of linear ubiquitin chains in vitro and in vivo, promoting linear ubiquitination of NEMO and subsequent NF-κB activation, and protecting against TNF-α-induced apoptosis via FADD- and caspase-8-dependent pathways.","method":"Co-immunoprecipitation, in vitro ubiquitination assay, genetic KO (cpdm mice), MEF and B cell functional assays","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1-2 — replicated independently in two simultaneous Nature papers with in vitro assays and genetic models","pmids":["21455181","21455180"],"is_preprint":false},{"year":2011,"finding":"SHARPIN is an endogenous inhibitor of β1-integrin activation; it directly binds to a conserved cytoplasmic region of integrin α-subunits and inhibits recruitment of talin and kindlin to the integrin, thereby preventing the switch from inactive to active integrin conformations.","method":"RNAi screen, direct binding assay, rescue experiments in SHARPIN-deficient fibroblasts/leukocytes/keratinocytes, re-expression of SHARPIN","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (RNAi screen, binding assay, genetic KO rescue) in one study, replicated in multiple cell types","pmids":["21947080"],"is_preprint":false},{"year":2001,"finding":"SHARPIN directly interacts with the ankyrin repeats of Shank family proteins and is enriched in the postsynaptic density (PSD) of excitatory synapses; the C-terminal half mediates Shank interaction while the N-terminal half mediates SHARPIN homomultimerization.","method":"Yeast two-hybrid, co-immunoprecipitation in heterologous cells and brain, immunostaining","journal":"Molecular and cellular neurosciences","confidence":"High","confidence_rationale":"Tier 2 — reciprocal co-IP and direct binding assay with domain mapping, foundational paper","pmids":["11178875"],"is_preprint":false},{"year":2012,"finding":"The N-terminal portion of SHARPIN adopts a pleckstrin homology (PH) superfold that acts as a dimerization module rather than a canonical ligand-recognition domain, extending the functional applications of this structural fold.","method":"Crystal structure determination of N-terminal SHARPIN fragment","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with functional validation of dimerization","pmids":["22549881"],"is_preprint":false},{"year":2014,"finding":"RIPK1 kinase activity is required for the inflammatory pathology in SHARPIN-deficient (cpdm) mice; crossing with kinase-dead Ripk1(K45A) mice fully protected against all cpdm-related pathology, placing RIPK1 kinase activity downstream of SHARPIN in TNF-driven inflammatory cell death.","method":"Genetic epistasis — Ripk1(K45A) knockin crossed with Sharpincpdm mice, in vitro and in vivo necroptosis assays","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 — clean genetic epistasis with rigorous in vivo phenotypic readout","pmids":["24821972"],"is_preprint":false},{"year":2014,"finding":"TNFR1 (but not TNFR2) signaling is the primary driver of multi-organ inflammation in Sharpin-deficient mice; RIPK3 deficiency combined with Caspase-8 heterozygosity almost completely suppressed the phenotype, placing necroptosis and apoptosis downstream of TNFR1 in SHARPIN-deficient inflammation.","method":"Genetic epistasis — Tnfr1/Tnfr2/Ripk3/Casp8/Mlkl deletions crossed with Sharpincpdm mice","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal genetic crosses with defined phenotypic readouts","pmids":["25443632"],"is_preprint":false},{"year":2014,"finding":"TRADD- and FADD-dependent keratinocyte apoptosis (not necroptosis) is the primary driver of skin inflammation in Sharpin-deficient mice; epidermis-restricted ablation of FADD combined with RIPK3 deficiency fully prevented skin inflammation.","method":"Genetic epistasis — tissue-specific KO (Krt14-Cre), FADD/RIPK3 double KO, primary keratinocyte apoptosis assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 — tissue-specific genetic dissection with defined cellular phenotype","pmids":["25443631"],"is_preprint":false},{"year":2011,"finding":"SHARPIN interacts with NEMO (confirmed by co-immunoprecipitation) and regulates NEMO-dependent signaling including p105 and ERK phosphorylation and p65 nuclear localization in a manner similar to the hypomorphic panr2 NEMO mutation, identifying SHARPIN as an essential adaptor in TLR2-induced NF-κB signaling in macrophages.","method":"Systems biology analysis, co-immunoprecipitation, transcriptomic analysis of cpdm macrophages","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — co-IP plus systems-level confirmation with genetic model","pmids":["21709223"],"is_preprint":false},{"year":2013,"finding":"SHARPIN localizes to the trailing edges (uropods) of chemokine-activated migrating lymphocytes, directly interacts with LFA-1 (lymphocyte-function-associated antigen-1), and inhibits intermediate and high-affinity forms of LFA-1, thereby regulating uropod detachment and migration velocity.","method":"Live cell imaging, direct binding assay, SHARPIN-deficient lymphocyte functional assays, rescue by re-expression","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 — direct binding, localization with functional consequence, and rescue experiment","pmids":["24210817"],"is_preprint":false},{"year":2016,"finding":"SHARPIN conjugated with K63-linked ubiquitin chains inhibits the association of TCRζ with the signaling kinase Zap70, affecting generation of regulatory T cells; SHARPIN deficiency results in enhanced proximal TCR signaling without affecting NF-κB activation.","method":"Co-immunoprecipitation, Treg transfer rescue experiments in SHARPIN-deficient mice, signaling assays","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 — mechanistic co-IP with K63-ubiquitin, functional rescue in vivo","pmids":["26829767"],"is_preprint":false},{"year":2016,"finding":"SHARPIN directly binds caspase-1 and disrupts p20/p10 dimer formation (the last step of caspase-1 processing), thereby inhibiting caspase-1 enzyme activation and maturation of IL-1β/IL-18 in a LUBAC-independent manner.","method":"Co-immunoprecipitation, biochemical dimerization assay, genetic KO (Casp1/Casp11 deletion in Sharpin-deficient background), in vivo sepsis model","journal":"The American journal of pathology","confidence":"High","confidence_rationale":"Tier 1-2 — direct binding assay plus mechanistic biochemical dissection plus genetic validation","pmids":["26968342"],"is_preprint":false},{"year":2015,"finding":"SHARPIN is required for optimal NLRP3 inflammasome activation by both canonical and non-canonical stimuli, and SHARPIN-deficient macrophages show dramatic defects in NF-κB and MAPK pathway activation, suggesting SHARPIN acts in transcriptional priming of NLRP3.","method":"Inflammasome activation assays in Sharpincpdm macrophages, IL-1β/caspase-1 cleavage assays","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 — clean KO with defined cellular phenotype, single lab","pmids":["25637014"],"is_preprint":false},{"year":2015,"finding":"SHARPIN's integrin inhibition function and its LUBAC-activating function are mutually exclusive; the integrin α-subunit tail competes with RNF31 (HOIP) for binding to the same ubiquitin-like (UBL) domain of SHARPIN, with two residues (V267, L276) required for both interactions.","method":"Domain mapping, in vitro competition binding assay, site-directed mutagenesis, NF-κB reporter assay","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1 — in vitro competition assay plus mutagenesis defining binding interface","pmids":["26600301"],"is_preprint":false},{"year":2016,"finding":"The NZF domain of SHARPIN (but not HOIL-1L) is critical for protection from programmed cell death by enhancing recruitment of LUBAC to the activated TNFR complex; this function depends on the SHARPIN NZF domain's ability to bind K63-linked ubiquitin chains.","method":"Intercrossing of HOIL-1L and SHARPIN KO mice, domain mutagenesis, TNFR complex recruitment assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis combined with domain mutagenesis and complex recruitment assay","pmids":["26976635"],"is_preprint":false},{"year":2017,"finding":"SHARPIN interacts with PRMT5 in a LUBAC-independent manner and increases PRMT5 multiprotein complex assembly and methyltransferase activity; activated PRMT5 controls expression of SOX10 and MITF transcription factors by arginine dimethylation and inhibition of the transcriptional corepressor SKI.","method":"Co-immunoprecipitation, in vitro methyltransferase activity assay, arginine dimethylation analysis, genetic rescue","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro enzymatic assay plus co-IP with mechanistic follow-through","pmids":["29227283"],"is_preprint":false},{"year":2017,"finding":"SHARPIN facilitates p53 poly-ubiquitination and degradation in an MDM2-dependent manner; SHARPIN associates with MDM2 (by co-immunoprecipitation) and prolongs MDM2 protein stability, thereby reducing p53 protein levels and target gene expression in breast cancer cells.","method":"Co-immunoprecipitation, ubiquitination assay, protein stability assay, RNA-seq, siRNA knockdown","journal":"Neoplasia","confidence":"Medium","confidence_rationale":"Tier 2 — co-IP and ubiquitination assay, single lab, moderate mechanistic depth","pmids":["28063307"],"is_preprint":false},{"year":2017,"finding":"SHARPIN stabilizes estrogen receptor α (ERα) by inhibiting ERα poly-ubiquitination while facilitating mono-ubiquitination at K302/303 sites; SHARPIN localizes in the cytosol and interacts with ERα in both cytosol and nucleus, promoting ERα-dependent transcription and breast cancer cell proliferation.","method":"Co-immunoprecipitation, ubiquitination assay, luciferase reporter, site-directed mutagenesis, siRNA knockdown","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 — ubiquitination site mapping plus co-IP, single lab","pmids":["29100376"],"is_preprint":false},{"year":2017,"finding":"SHARPIN interacts with PRMT5 to promote monomethylation of histone H3 at arginine 2 (H3R2me1) at metastasis-related gene loci, linking this mark to H3K4me3 via an MLL complex (ASH2/WDR5) to activate metastasis-related gene expression in lung cancer cells.","method":"Co-immunoprecipitation, histone methylation assay, chromatin immunoprecipitation","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 — co-IP with histone modification assay, single lab","pmids":["28903384"],"is_preprint":false},{"year":2016,"finding":"SHARPIN regulates mammary ductal outgrowth in a stromal-cell-intrinsic manner; SHARPIN-deficient stromal fibroblasts display defects in collagen fibre assembly, contraction, and degradation, leading to reduced ECM stiffness adjacent to invading ducts.","method":"Cell-type-specific KO (S100a4-Cre), mammary epithelial transplantation, collagen contraction/degradation in vitro assays","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 — tissue-specific deletion with transplantation experiment and in vitro functional assays","pmids":["27974362"],"is_preprint":false},{"year":2014,"finding":"SIPL1 (SHARPIN) promotes PTEN polyubiquitination via K63-linked polyubiquitin chains using its ubiquitin-like (UBL) domain; this ubiquitination promotes and stabilizes the SIPL1/PTEN complex.","method":"Ubiquitination assay with ubiquitin K48R and K63R mutants, co-immunoprecipitation, UBL domain mutagenesis","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 — ubiquitin chain-type dissection with mutagenesis, single lab","pmids":["25152374"],"is_preprint":false},{"year":2017,"finding":"SHARPIN interacts with the Arp2/3 complex and promotes lamellipodium formation in a LUBAC-independent manner; the Arp2/3-binding site on SHARPIN was mapped and an Arp2/3-binding-deficient mutant abrogated lamellipodium formation.","method":"Mass spectrometry interactome (Sharpin interactome), co-immunoprecipitation, domain mapping, Arp2/3-binding-deficient mutant rescue assay","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 — systematic interactome plus domain mutagenesis with functional readout","pmids":["28775156"],"is_preprint":false},{"year":2020,"finding":"SHARPIN serine 165 (S165) phosphorylation is constitutively present in lymphoblastoid cells and is further induced by TCR stimulation; a phosphorylation-resistant SHARPIN S165A mutant shows impaired linear ubiquitination of NEMO and reduced NF-κB activation in response to TNFα.","method":"Mass spectrometry phosphorylation analysis, phosphorylation-resistant mutant, NF-κB reporter assay, linear ubiquitination assay","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 — site-specific mutagenesis with functional assay, single lab","pmids":["33392484"],"is_preprint":false},{"year":2022,"finding":"SHARPIN S146 phosphorylation (sensitive to ERK1/2 inhibition and PP2A reactivation) mediates ARP2/3 complex interaction and lamellipodia formation, and is required for 3D cancer cell invasion and in vivo metastasis; S146A mutant SHARPIN fails to rescue invasion in SHARPIN-KO cancer cells.","method":"Mass spectrometry, in vitro kinase assay, CRISPR/Cas9 KO, S146A mutant rescue, zebrafish metastasis model","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro kinase assay plus mutagenesis plus in vivo model","pmids":["36148554"],"is_preprint":false},{"year":2021,"finding":"SHARPIN-deficient mouse phenotype (dermatitis, disrupted splenic architecture, loss of Peyer's patches) is fully dependent on CYLD deubiquitinase; absence of SHARPIN impairs CYLD phosphorylation at serine 418 (which normally inhibits CYLD), leading to enhanced RIPK1 recruitment to death-signaling Complex II following TNF stimulation.","method":"Genetic epistasis (Sharpin/Cyld double KO), conditional Cyld deletion (LysM-Cre, Cx3cr1-Cre), RIPK1 complex II immunoprecipitation, CYLD phosphorylation analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — clean genetic epistasis with mechanistic biochemical dissection of RIPK1 complex II","pmids":["34887354"],"is_preprint":false},{"year":2019,"finding":"SHARPIN interacts with integrin αIIb in human platelets (pull-down and co-immunoprecipitation) and co-immunoprecipitates with HOIP and HOIL-1 (LUBAC components); SHARPIN knockdown in megakaryocytes/platelets increases αIIbβ3-fibrinogen binding and reduces Met1 linear ubiquitination and NF-κB (RelA) activation.","method":"Pull-down, co-immunoprecipitation, SHARPIN knockdown in iPSC-derived megakaryocytes/platelets, flow cytometry, linear ubiquitination assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — reciprocal co-IP and functional KD with multiple cellular readouts","pmids":["30804189"],"is_preprint":false},{"year":2019,"finding":"SHARPIN suppresses β1-integrin activation by complexing with both the integrin β1 cytoplasmic tail and kindlin-1; kindlin-1 enhances the SHARPIN–β1 tail interaction, and the complex restricts talin head domain binding to the β1 tail.","method":"Biochemical binding assays, co-immunoprecipitation, talin competition assay in CHO cells","journal":"Cell communication and signaling","confidence":"Medium","confidence_rationale":"Tier 2 — direct binding and competition assay, single lab","pmids":["31429758"],"is_preprint":false},{"year":2020,"finding":"SHARPIN stabilizes β-catenin by competing with the E3 ubiquitin ligase β-TrCP1 for β-catenin binding, thereby decreasing β-catenin ubiquitination and preventing its proteasomal degradation in a linear ubiquitination-independent manner.","method":"Co-immunoprecipitation, ubiquitination assay, competition binding assay, in vitro and in vivo functional assays","journal":"Gastric cancer","confidence":"Medium","confidence_rationale":"Tier 2 — co-IP with competition assay and ubiquitination assay, single lab","pmids":["33159601"],"is_preprint":false},{"year":2021,"finding":"SHARPIN promotes ubiquitination and proteasomal degradation of von Hippel-Lindau protein (pVHL) using its ubiquitin-like domain; SHARPIN interaction requires the α and β domains of pVHL, and this leads to sustained HIF-2α activation in clear cell renal cell carcinoma.","method":"Co-immunoprecipitation with domain mapping, ubiquitination assay, siRNA knockdown, xenograft model","journal":"Cancer science","confidence":"Medium","confidence_rationale":"Tier 2 — co-IP with domain dissection and ubiquitination assay, single lab","pmids":["34339558"],"is_preprint":false},{"year":2019,"finding":"SHARPIN interacts with YAP protein and promotes YAP K48-linked poly-ubiquitination and degradation, thereby inhibiting YAP/TEAD target gene expression (CTGF, CYR61) and functioning as an endogenous inhibitor of YAP in esophageal squamous cell carcinoma.","method":"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, rescue experiment","journal":"Neoplasia","confidence":"Medium","confidence_rationale":"Tier 3 — single co-IP plus ubiquitination assay, single lab","pmids":["31884247"],"is_preprint":false},{"year":2011,"finding":"SHARPIN deficiency in keratinocytes triggers mitochondria-dependent (intrinsic) apoptosis characterized by mitochondrial membrane depolarization, shift in BCL2/BAX ratio, and activation of caspases 9 and 3 (but not 8), without involvement of the extrinsic caspase-8 pathway.","method":"FACS (Annexin V/PI), transmission electron microscopy, caspase activity assays, Western blot for BCL2/BAX","journal":"Journal of dermatological science","confidence":"Medium","confidence_rationale":"Tier 2 — multiple complementary assays defining the apoptotic pathway, single lab","pmids":["21620685"],"is_preprint":false},{"year":2023,"finding":"HOIL-1L and SHARPIN form homo-dimers through their LTM motifs; crystal structures of the dimeric LTM motifs reveal the molecular mechanism of dimerization and a shared mode for homo- and hetero-dimer formation; the polyglucosan body myopathy-associated HOIL-1L A18P mutation disrupts HOIL-1L LTM structural folding and dimer formation.","method":"Crystal structure determination of LTM motif dimers, disease mutation analysis, biochemical validation","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1 — crystal structures with functional and disease mutation validation","pmids":["37976837"],"is_preprint":false},{"year":2024,"finding":"The NZF domains of HOIL-1L and SHARPIN cooperatively regulate LUBAC function; HOIL-1L NZF preferentially binds linear ubiquitin chains while SHARPIN NZF binds K63-linked chains in addition to linear chains; simultaneous loss of both NZF ubiquitin-binding activities profoundly impairs NF-κB activation and cell death protection.","method":"NZF domain mutagenesis, ubiquitin chain-binding assays, NF-κB activation assays, cell death protection assays, compound screen","journal":"Cell death & disease","confidence":"High","confidence_rationale":"Tier 1-2 — domain mutagenesis with in vitro binding and multiple functional assays","pmids":["39528476"],"is_preprint":false},{"year":2024,"finding":"Biallelic SHARPIN loss in humans causes attenuated canonical NF-κB responses and propensity for cell death mediated by TNF superfamily members in fibroblasts and B cells; anti-TNF therapy completely resolved autoinflammation, confirming TNF-driven pathology.","method":"Patient-derived fibroblast and B cell assays, NF-κB signaling assays, anti-TNF treatment with transcriptomic resolution","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 — human genetic validation of LUBAC/NF-κB mechanism with functional cell assays and treatment response","pmids":["38609546"],"is_preprint":false},{"year":2021,"finding":"SHARPIN directly interacted with HMGB1 (demonstrated by GST pull-down and co-immunoprecipitation) and enhanced HMGB1 expression, promoting M1-like macrophage polarization and neuroinflammation in sevoflurane-induced neurotoxicity.","method":"GST pull-down, co-immunoprecipitation, SHARPIN siRNA knockdown, macrophage polarization assays","journal":"Metabolic brain disease","confidence":"Low","confidence_rationale":"Tier 3 — single co-IP/pulldown, single lab, limited mechanistic follow-up","pmids":["38805141"],"is_preprint":false},{"year":2010,"finding":"SHARPIN was identified as a novel interaction partner of Eya1 (Eyes Absent 1); SHARPIN (as Sipl1) and Rbck1 enhance the function of Eya proteins as coactivators for Six transcription factors, and morpholino knockdown of a Sipl1 ortholog in zebrafish produces a BOR syndrome-like phenotype with ear and branchial arch defects.","method":"GST pulldown, co-immunoprecipitation, coactivator transcriptional assay, zebrafish morpholino knockdown","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 — direct binding assay plus functional coactivator assay plus in vivo model, but in a developmental context not primary for mammalian SHARPIN focus","pmids":["20956555"],"is_preprint":false},{"year":2019,"finding":"Platelet-specific deletion of SHARPIN results in increased colocalization of αIIbβ3 with talin (by super-resolution microscopy), increased fibrinogen binding in response to ADP, and reduced NF-κB activation and linear ubiquitination of substrates; SHARPIN-null platelets show reduced inflammation in colitis and peritonitis models.","method":"Platelet-specific conditional KO (PF4-Cre/GPIbα-Cre), super-resolution microscopy, flow cytometry, in vivo inflammation models","journal":"Blood advances","confidence":"High","confidence_rationale":"Tier 2 — cell-type-specific KO with multiple orthogonal readouts","pmids":["34991155"],"is_preprint":false}],"current_model":"SHARPIN is a multifunctional adaptor protein that acts as an accessory subunit of the LUBAC E3 ligase complex (with HOIP/RNF31 and HOIL-1L/RBCK1) to catalyze linear (Met1-linked) ubiquitin chain formation on substrates including NEMO, thereby activating canonical NF-κB signaling and protecting cells from TNF-induced apoptosis and necroptosis; independently of LUBAC, SHARPIN inhibits β1- and β2-integrin activation by directly binding integrin α-subunit cytoplasmic tails and blocking talin/kindlin recruitment, inhibits caspase-1 by disrupting its p20/p10 dimer formation, suppresses TCR signaling by preventing ZAP70 association with TCRζ via K63-ubiquitin conjugation, promotes lamellipodium formation through ERK-dependent S146 phosphorylation-mediated Arp2/3 complex interaction, and regulates the stability of multiple proteins (p53 via MDM2, ERα, β-catenin, pVHL) through ubiquitination-dependent and -independent mechanisms."},"narrative":{"teleology":[{"year":2001,"claim":"Before SHARPIN's signaling roles were known, its initial identification as a Shank-binding postsynaptic density protein established it as a scaffolding adaptor capable of homomultimerization, setting the stage for understanding its broader interaction repertoire.","evidence":"Yeast two-hybrid and co-immunoprecipitation from brain lysates with domain mapping","pmids":["11178875"],"confidence":"High","gaps":["Functional significance of SHARPIN at excitatory synapses remains undefined","Whether synaptic SHARPIN affects Shank-dependent signaling is untested"]},{"year":2010,"claim":"Before LUBAC function was linked to SHARPIN, its interaction with Eya1 and enhancement of Six-dependent transcription revealed a role in developmental transcriptional coactivation, broadening the protein's functional scope beyond scaffolding.","evidence":"GST pulldown, coactivator assay, zebrafish morpholino knockdown producing BOR-like phenotype","pmids":["20956555"],"confidence":"Medium","gaps":["The mammalian developmental relevance of the SHARPIN–Eya1 axis is not confirmed by knockout","Whether this function is LUBAC-dependent is unknown"]},{"year":2011,"claim":"The central mechanistic advance was the simultaneous discovery by two groups that SHARPIN is a bona fide LUBAC subunit: it binds HOIP, stimulates linear ubiquitin chain formation on NEMO, activates NF-κB, and protects cells from TNF-induced apoptosis, explaining the chronic proliferative dermatitis (cpdm) mouse phenotype.","evidence":"Co-immunoprecipitation, in vitro ubiquitination, cpdm mouse genetic model, MEF and B cell assays (two independent Nature papers)","pmids":["21455181","21455180","21709223"],"confidence":"High","gaps":["Structural basis of SHARPIN–HOIP interaction at atomic resolution was not yet defined","Relative contributions of SHARPIN versus HOIL-1L NZF domains to LUBAC recruitment were unclear"]},{"year":2011,"claim":"In parallel with the LUBAC discovery, SHARPIN was found to directly bind integrin α-subunit cytoplasmic tails and inhibit β1-integrin activation by preventing talin and kindlin recruitment, establishing a LUBAC-independent function.","evidence":"RNAi screen, direct binding assay, rescue in SHARPIN-deficient fibroblasts/leukocytes/keratinocytes","pmids":["21947080"],"confidence":"High","gaps":["Whether integrin inhibition contributes to the cpdm phenotype was not separated from NF-κB defects","Structural mechanism of α-tail binding was not resolved"]},{"year":2012,"claim":"Crystallography of the N-terminal domain revealed a PH superfold that functions as a dimerization module rather than a ligand-binding domain, providing structural basis for SHARPIN self-association.","evidence":"Crystal structure of N-terminal SHARPIN fragment with functional dimerization validation","pmids":["22549881"],"confidence":"High","gaps":["How dimerization affects LUBAC assembly or integrin inhibition was not tested","Full-length SHARPIN structure remained unavailable"]},{"year":2014,"claim":"Genetic epistasis experiments resolved the cell death pathways downstream of SHARPIN loss: TNFR1 (not TNFR2) is the primary driver, RIPK1 kinase activity is essential, and both apoptosis (via TRADD/FADD in keratinocytes) and necroptosis contribute tissue-specifically to the cpdm phenotype.","evidence":"Intercross of cpdm mice with Ripk1(K45A), Tnfr1-KO, Tnfr2-KO, Ripk3-KO, Casp8-het, tissue-specific FADD deletion","pmids":["24821972","25443632","25443631"],"confidence":"High","gaps":["Which LUBAC substrates beyond NEMO are critical for cell death protection was not defined","Non-TNF cytokine contributions were not fully excluded"]},{"year":2015,"claim":"Binding competition experiments showed that the integrin α-tail and HOIP bind the same UBL domain surface on SHARPIN, making integrin inhibition and LUBAC activation mutually exclusive — revealing a molecular switch governing SHARPIN's dual functions.","evidence":"In vitro competition binding assay, V267/L276 mutagenesis, NF-κB reporter assay","pmids":["26600301"],"confidence":"High","gaps":["How the switch is regulated in vivo (post-translational modifications, localization) is unknown","Whether the competition is relevant in all cell types is untested"]},{"year":2016,"claim":"SHARPIN was shown to have LUBAC-independent roles in immune regulation: it inhibits caspase-1 by disrupting p20/p10 dimerization, and its K63-ubiquitin conjugation suppresses TCRζ–ZAP70 association, affecting regulatory T cell generation.","evidence":"Co-immunoprecipitation with dimerization assay and genetic validation (caspase-1); co-IP with K63-Ub and Treg transfer rescue (TCR signaling)","pmids":["26968342","26829767"],"confidence":"High","gaps":["The E3 ligase catalyzing K63-ubiquitination of SHARPIN in T cells is unidentified","Whether caspase-1 inhibition is relevant in vivo beyond the sepsis model is unclear"]},{"year":2016,"claim":"The SHARPIN NZF domain was shown to be specifically required for LUBAC recruitment to the TNFR complex via K63-linked ubiquitin binding, distinguishing it functionally from the HOIL-1L NZF domain.","evidence":"NZF mutagenesis, TNFR complex recruitment assay, HOIL-1L/SHARPIN double KO intercross","pmids":["26976635"],"confidence":"High","gaps":["Whether NZF-mediated recruitment is regulated by post-translational modifications is unknown"]},{"year":2017,"claim":"SHARPIN was found to interact with the Arp2/3 complex in a LUBAC-independent manner to promote lamellipodium formation, and separately to enhance PRMT5 methyltransferase activity controlling SOX10/MITF expression and metastasis-related gene transcription, expanding its roles to cytoskeletal dynamics and epigenetic regulation.","evidence":"Mass spectrometry interactome, Arp2/3-binding-deficient mutant (lamellipodium); co-IP with in vitro methyltransferase and ChIP assays (PRMT5)","pmids":["28775156","29227283","28903384"],"confidence":"High","gaps":["The kinase phosphorylating S146 to regulate Arp2/3 binding was not yet identified","Whether PRMT5 activation by SHARPIN occurs in non-cancer contexts is untested"]},{"year":2017,"claim":"Multiple studies established SHARPIN as a regulator of protein stability through ubiquitination-dependent mechanisms: it promotes MDM2-dependent p53 degradation and stabilizes ERα by switching its ubiquitination from poly- to mono-ubiquitination.","evidence":"Co-immunoprecipitation, ubiquitination assays, protein stability assays in breast cancer cells","pmids":["28063307","29100376"],"confidence":"Medium","gaps":["Whether SHARPIN acts as a direct E3 ligase or purely as an adaptor for these substrates is unresolved","Single-lab findings for each substrate"]},{"year":2019,"claim":"Platelet-specific studies confirmed SHARPIN's dual function in an important physiological context: it inhibits αIIbβ3-integrin activation (increasing talin–integrin colocalization upon deletion) and supports LUBAC-dependent linear ubiquitination and NF-κB activation in platelets.","evidence":"Platelet-specific conditional KO (PF4-Cre, GPIbα-Cre), super-resolution microscopy, flow cytometry, in vivo inflammation models","pmids":["30804189","34991155"],"confidence":"High","gaps":["Whether platelet SHARPIN loss affects thrombosis risk in vivo needs further study","The relative importance of integrin versus NF-κB functions in platelet biology is not separated"]},{"year":2021,"claim":"The full cpdm phenotype was shown to require the deubiquitinase CYLD: SHARPIN loss impairs IKK-mediated CYLD phosphorylation at S418, unleashing CYLD activity that deubiquitinates RIPK1 and promotes its recruitment to death-inducing Complex II.","evidence":"Sharpin/Cyld double KO, tissue-specific Cyld deletion, RIPK1 Complex II immunoprecipitation, CYLD phosphorylation analysis","pmids":["34887354"],"confidence":"High","gaps":["Whether CYLD directly deubiquitinates linear chains on NEMO or acts on other substrates is not resolved","Whether pharmacological CYLD inhibition could be therapeutic is untested"]},{"year":2022,"claim":"ERK1/2-dependent phosphorylation of SHARPIN at S146 was identified as the signal controlling Arp2/3 interaction, lamellipodia formation, and cancer cell invasion/metastasis, revealing a phospho-switch for SHARPIN's cytoskeletal function.","evidence":"In vitro kinase assay, mass spectrometry, CRISPR KO with S146A mutant rescue, zebrafish metastasis model","pmids":["36148554"],"confidence":"High","gaps":["The PP2A holoenzyme subunit targeting pS146 is not identified","Whether S146 phosphorylation affects other SHARPIN functions is unknown"]},{"year":2023,"claim":"Crystal structures of SHARPIN LTM motif dimers revealed the molecular basis for LUBAC subunit homo- and hetero-dimerization, and linked a disease-associated HOIL-1L mutation (A18P) to disrupted dimer formation.","evidence":"Crystal structure determination of LTM dimers with disease mutation structural analysis","pmids":["37976837"],"confidence":"High","gaps":["Full LUBAC holocomplex structure is still unavailable","How LTM dimerization regulates catalytic activity quantitatively is not defined"]},{"year":2024,"claim":"Biallelic SHARPIN loss was identified in human patients with autoinflammation, confirming the mouse-to-human translational relevance: patient cells show attenuated NF-κB and TNF-driven cell death, and anti-TNF therapy fully resolved disease, establishing SHARPIN deficiency as a human Mendelian autoinflammatory condition.","evidence":"Patient-derived fibroblast and B cell functional assays, NF-κB signaling, anti-TNF treatment with transcriptomic validation","pmids":["38609546"],"confidence":"High","gaps":["The full clinical spectrum of human SHARPIN deficiency beyond autoinflammation is not characterized","Whether integrin dysregulation contributes to the human phenotype is unknown"]},{"year":2024,"claim":"Cooperative regulation by HOIL-1L and SHARPIN NZF domains was demonstrated: HOIL-1L NZF preferentially binds linear chains while SHARPIN NZF binds both K63 and linear chains, and simultaneous loss profoundly impairs NF-κB and cell death protection, refining the model of accessory subunit function within LUBAC.","evidence":"NZF domain mutagenesis, ubiquitin chain-binding assays, NF-κB and cell death functional assays","pmids":["39528476"],"confidence":"High","gaps":["How NZF ubiquitin-type selectivity is determined structurally is unresolved","Whether NZF functions can be pharmacologically targeted is untested"]},{"year":null,"claim":"Key unresolved questions include the full-length atomic structure of SHARPIN, the in vivo regulatory logic governing the UBL domain switch between HOIP and integrin binding, whether SHARPIN's LUBAC-independent functions (caspase-1 inhibition, PRMT5 activation, Arp2/3 interaction) are interdependent or independently regulated, and the complete clinical spectrum of human SHARPIN deficiency.","evidence":"","pmids":[],"confidence":"Low","gaps":["Full-length SHARPIN structure not available","In vivo regulation of the HOIP–integrin binding switch is undefined","Interplay among LUBAC-independent functions is untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,7,12,13]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[1,8,20,22,25]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[10,14,26,27]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[16]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[1,8,24]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,7,9,11,32]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,7,21,26]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[4,5,6,23,29,32]},{"term_id":"R-HSA-1500931","term_label":"Cell-Cell communication","supporting_discovery_ids":[1,8,24,25]}],"complexes":["LUBAC"],"partners":["RNF31","RBCK1","IKBKG","PRMT5","CASP1","CYLD","SHANK1","ACTR2"],"other_free_text":[]},"mechanistic_narrative":"SHARPIN is a multifunctional adaptor protein that serves as an accessory subunit of the linear ubiquitin chain assembly complex (LUBAC), where it binds HOIP/RNF31 via its ubiquitin-like (UBL) domain and facilitates linear (Met1-linked) ubiquitination of NEMO, thereby activating canonical NF-κB signaling and protecting cells from TNF-induced apoptosis and necroptosis; the inflammatory pathology caused by SHARPIN deficiency is driven by TNFR1 signaling, RIPK1 kinase activity, and the deubiquitinase CYLD, and biallelic SHARPIN loss in humans causes autoinflammation that is fully resolved by anti-TNF therapy [PMID:21455181, PMID:21455180, PMID:24821972, PMID:25443632, PMID:34887354, PMID:38609546]. Independent of LUBAC, SHARPIN directly binds integrin α-subunit cytoplasmic tails through its UBL domain — competing with HOIP for the same binding surface — to inhibit integrin activation by blocking talin and kindlin recruitment, a function demonstrated for β1-integrins, β2-integrins (LFA-1), and αIIbβ3 in platelets [PMID:21947080, PMID:24210817, PMID:30804189, PMID:26600301]. SHARPIN also acts outside the LUBAC–integrin axis: it inhibits caspase-1 by disrupting p20/p10 dimerization, suppresses proximal TCR signaling via K63-ubiquitin-dependent exclusion of ZAP70 from TCRζ, promotes lamellipodium formation through ERK-dependent S146 phosphorylation and Arp2/3 complex interaction, enhances PRMT5 methyltransferase activity, and modulates the stability of p53 (via MDM2), β-catenin, and pVHL through ubiquitination-dependent mechanisms [PMID:26968342, PMID:26829767, PMID:36148554, PMID:29227283, PMID:28063307, PMID:33159601, PMID:34339558]. Biallelic loss-of-function mutations in SHARPIN cause a human autoinflammatory disease characterized by attenuated NF-κB responses and TNF superfamily–driven cell death [PMID:38609546]."},"prefetch_data":{"uniprot":{"accession":"Q9H0F6","full_name":"Sharpin","aliases":["Shank-associated RH domain-interacting protein","Shank-interacting protein-like 1","hSIPL1"],"length_aa":387,"mass_kda":39.9,"function":"Component of the LUBAC complex which conjugates linear polyubiquitin chains in a head-to-tail manner to substrates and plays a key role in NF-kappa-B activation and regulation of inflammation (PubMed:21455173, PubMed:21455180, PubMed:21455181). LUBAC conjugates linear polyubiquitin to IKBKG and RIPK1 and is involved in activation of the canonical NF-kappa-B and the JNK signaling pathways (PubMed:21455173, PubMed:21455180, PubMed:21455181). Linear ubiquitination mediated by the LUBAC complex interferes with TNF-induced cell death and thereby prevents inflammation (PubMed:21455173, PubMed:21455180, PubMed:21455181). LUBAC is recruited to the TNF-R1 signaling complex (TNF-RSC) following polyubiquitination of TNF-RSC components by BIRC2 and/or BIRC3 and to conjugate linear polyubiquitin to IKBKG and possibly other components contributing to the stability of the complex (PubMed:21455173, PubMed:21455180, PubMed:21455181). The LUBAC complex is also involved in innate immunity by conjugating linear polyubiquitin chains at the surface of bacteria invading the cytosol to form the ubiquitin coat surrounding bacteria (PubMed:28481331). LUBAC is not able to initiate formation of the bacterial ubiquitin coat, and can only promote formation of linear polyubiquitins on pre-existing ubiquitin (PubMed:28481331). The bacterial ubiquitin coat acts as an 'eat-me' signal for xenophagy and promotes NF-kappa-B activation (PubMed:28481331). Together with OTULIN, the LUBAC complex regulates the canonical Wnt signaling during angiogenesis (PubMed:23708998)","subcellular_location":"Cytoplasm, cytosol; Synapse","url":"https://www.uniprot.org/uniprotkb/Q9H0F6/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SHARPIN","classification":"Not Classified","n_dependent_lines":50,"n_total_lines":1208,"dependency_fraction":0.041390728476821195},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/SHARPIN","total_profiled":1310},"omim":[{"mim_id":"620795","title":"AUTOINFLAMMATION WITH EPISODIC FEVER AND IMMUNE DYSREGULATION; AIFID","url":"https://www.omim.org/entry/620795"},{"mim_id":"620632","title":"IMMUNODEFICIENCY 115 WITH AUTOINFLAMMATION; IMD115","url":"https://www.omim.org/entry/620632"},{"mim_id":"612487","title":"RING FINGER PROTEIN 31; RNF31","url":"https://www.omim.org/entry/612487"},{"mim_id":"611885","title":"SHANK-ASSOCIATED RH DOMAIN INTERACTOR; SHARPIN","url":"https://www.omim.org/entry/611885"},{"mim_id":"610924","title":"RANBP-TYPE AND C3HC4-TYPE ZINC FINGER-CONTAINING 1; RBCK1","url":"https://www.omim.org/entry/610924"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Plasma membrane","reliability":"Approved"},{"location":"Golgi apparatus","reliability":"Additional"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/SHARPIN"},"hgnc":{"alias_symbol":["DKFZP434N1923","SIPL1"],"prev_symbol":[]},"alphafold":{"accession":"Q9H0F6","domains":[{"cath_id":"2.30.29.30","chopping":"21-124","consensus_level":"high","plddt":88.1258,"start":21,"end":124},{"cath_id":"3.10.20.90","chopping":"213-300","consensus_level":"high","plddt":90.4263,"start":213,"end":300},{"cath_id":"1.10.287","chopping":"170-208","consensus_level":"high","plddt":94.8485,"start":170,"end":208}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H0F6","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H0F6-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H0F6-F1-predicted_aligned_error_v6.png","plddt_mean":74.25},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SHARPIN","jax_strain_url":"https://www.jax.org/strain/search?query=SHARPIN"},"sequence":{"accession":"Q9H0F6","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9H0F6.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9H0F6/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H0F6"}},"corpus_meta":[{"pmid":"21455181","id":"PMC_21455181","title":"SHARPIN 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mice.","date":"2014","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/25443632","citation_count":234,"is_preprint":false},{"pmid":"17538631","id":"PMC_17538631","title":"Spontaneous mutations in the mouse Sharpin gene result in multiorgan inflammation, immune system dysregulation and dermatitis.","date":"2007","source":"Genes and immunity","url":"https://pubmed.ncbi.nlm.nih.gov/17538631","citation_count":182,"is_preprint":false},{"pmid":"21947080","id":"PMC_21947080","title":"SHARPIN is an endogenous inhibitor of β1-integrin activation.","date":"2011","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/21947080","citation_count":165,"is_preprint":false},{"pmid":"25443631","id":"PMC_25443631","title":"Sharpin prevents skin inflammation by inhibiting TNFR1-induced keratinocyte apoptosis.","date":"2014","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/25443631","citation_count":160,"is_preprint":false},{"pmid":"11178875","id":"PMC_11178875","title":"Sharpin, a novel postsynaptic density protein that directly interacts with the shank family of proteins.","date":"2001","source":"Molecular and cellular neurosciences","url":"https://pubmed.ncbi.nlm.nih.gov/11178875","citation_count":126,"is_preprint":false},{"pmid":"35968603","id":"PMC_35968603","title":"SHARPIN promotes cell proliferation of cholangiocarcinoma and inhibits ferroptosis via p53/SLC7A11/GPX4 signaling.","date":"2022","source":"Cancer science","url":"https://pubmed.ncbi.nlm.nih.gov/35968603","citation_count":111,"is_preprint":false},{"pmid":"26216893","id":"PMC_26216893","title":"The Inflammatory Caspases-1 and -11 Mediate the Pathogenesis of Dermatitis in Sharpin-Deficient Mice.","date":"2015","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/26216893","citation_count":64,"is_preprint":false},{"pmid":"25550157","id":"PMC_25550157","title":"SHARPIN overexpression induces tumorigenesis in human prostate cancer LNCaP, DU145 and PC-3 cells via NF-κB/ERK/Akt signaling pathway.","date":"2015","source":"Medical oncology (Northwood, London, England)","url":"https://pubmed.ncbi.nlm.nih.gov/25550157","citation_count":63,"is_preprint":false},{"pmid":"21709223","id":"PMC_21709223","title":"Systems analysis identifies an essential role for SHANK-associated RH domain-interacting protein (SHARPIN) in macrophage Toll-like receptor 2 (TLR2) responses.","date":"2011","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/21709223","citation_count":61,"is_preprint":false},{"pmid":"22452937","id":"PMC_22452937","title":"SHARPIN is a key regulator of immune and inflammatory responses.","date":"2012","source":"Journal of cellular and molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/22452937","citation_count":55,"is_preprint":false},{"pmid":"20179993","id":"PMC_20179993","title":"Newly identified tumor-associated role of human Sharpin.","date":"2010","source":"Molecular and cellular biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/20179993","citation_count":55,"is_preprint":false},{"pmid":"26829767","id":"PMC_26829767","title":"SHARPIN controls regulatory T cells by negatively modulating the T cell antigen receptor complex.","date":"2016","source":"Nature immunology","url":"https://pubmed.ncbi.nlm.nih.gov/26829767","citation_count":53,"is_preprint":false},{"pmid":"25637014","id":"PMC_25637014","title":"Cutting edge: SHARPIN is required for optimal NLRP3 inflammasome activation.","date":"2015","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/25637014","citation_count":52,"is_preprint":false},{"pmid":"24465642","id":"PMC_24465642","title":"Chronic proliferative dermatitis in Sharpin null mice: development of an autoinflammatory disease in the absence of B and T lymphocytes and IL4/IL13 signaling.","date":"2014","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/24465642","citation_count":48,"is_preprint":false},{"pmid":"24210817","id":"PMC_24210817","title":"SHARPIN regulates uropod detachment in migrating lymphocytes.","date":"2013","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/24210817","citation_count":48,"is_preprint":false},{"pmid":"20811394","id":"PMC_20811394","title":"Inhibition of NF-κB signaling retards eosinophilic dermatitis in SHARPIN-deficient mice.","date":"2010","source":"The Journal of investigative 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regulates the development of clear cell renal cell carcinoma by promoting von Hippel-Lindau protein ubiquitination and degradation.","date":"2021","source":"Cancer science","url":"https://pubmed.ncbi.nlm.nih.gov/34339558","citation_count":9,"is_preprint":false},{"pmid":"21829440","id":"PMC_21829440","title":"SHARPIN negatively associates with TRAF2-mediated NFκB activation.","date":"2011","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/21829440","citation_count":9,"is_preprint":false},{"pmid":"34991155","id":"PMC_34991155","title":"Platelet SHARPIN regulates platelet adhesion and inflammatory responses through associations with αIIbβ3 and LUBAC.","date":"2022","source":"Blood advances","url":"https://pubmed.ncbi.nlm.nih.gov/34991155","citation_count":8,"is_preprint":false},{"pmid":"29248549","id":"PMC_29248549","title":"A role of SIPL1/SHARPIN in promoting resistance to hormone therapy in breast cancer.","date":"2017","source":"Biochimica et biophysica acta. 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Section F, Structural biology and crystallization communications","url":"https://pubmed.ncbi.nlm.nih.gov/22750873","citation_count":2,"is_preprint":false},{"pmid":"37976837","id":"PMC_37976837","title":"Mechanistic insights into the homo-dimerization of HOIL-1L and SHARPIN.","date":"2023","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/37976837","citation_count":1,"is_preprint":false},{"pmid":"39528476","id":"PMC_39528476","title":"Synergistic involvement of the NZF domains of the LUBAC accessory subunits HOIL-1L and SHARPIN in the regulation of LUBAC function.","date":"2024","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/39528476","citation_count":1,"is_preprint":false},{"pmid":"35798064","id":"PMC_35798064","title":"Overexpression of SHARPIN promotes tumor progression in ovarian cancer.","date":"2022","source":"Experimental and molecular pathology","url":"https://pubmed.ncbi.nlm.nih.gov/35798064","citation_count":1,"is_preprint":false},{"pmid":"24506958","id":"PMC_24506958","title":"[Effects of SIPL1 screened by suppression subtractive hybridization (SSH) on biological function and drug resistance of renal cell carcinoma cells].","date":"2013","source":"Zhonghua zhong liu za zhi [Chinese journal of oncology]","url":"https://pubmed.ncbi.nlm.nih.gov/24506958","citation_count":1,"is_preprint":false},{"pmid":"38156288","id":"PMC_38156288","title":"Mind bomb 2 limits inflammatory dermatitis in Sharpin mutant mice independently of cell death.","date":"2023","source":"PNAS nexus","url":"https://pubmed.ncbi.nlm.nih.gov/38156288","citation_count":0,"is_preprint":false},{"pmid":"38805141","id":"PMC_38805141","title":"SHARPIN contributes to sevoflurane-induced neonatal neurotoxicity through up-regulating HMGB1 to repress M2 like-macrophage polarization.","date":"2024","source":"Metabolic brain disease","url":"https://pubmed.ncbi.nlm.nih.gov/38805141","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.10.01.24314738","title":"Identification of novel associations of candidate loci with Alzheimer’s disease by leveraging the shared genetic basis with hippocampal volume","date":"2024-10-02","source":"bioRxiv","url":"https://doi.org/10.1101/2024.10.01.24314738","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.02.11.25322052","title":"Genetic Variants that Modulate Alzheimer’s Disease Risk Deregulate Protein-Protein Correlations in the Gyrus Temporalis Medius","date":"2025-02-14","source":"bioRxiv","url":"https://doi.org/10.1101/2025.02.11.25322052","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.03.10.25323307","title":"Genetic dissection of hippocampal sclerosis of ageing using magnetic resonance imaging surrogates","date":"2025-03-11","source":"bioRxiv","url":"https://doi.org/10.1101/2025.03.10.25323307","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.05.09.593462","title":"Construction of a breast cancer predictive nomogram based on diverse cell death methods and reveal tumor microenvironment characterization","date":"2024-05-14","source":"bioRxiv","url":"https://doi.org/10.1101/2024.05.09.593462","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":46206,"output_tokens":7927,"usd":0.128762},"stage2":{"model":"claude-opus-4-6","input_tokens":11907,"output_tokens":4570,"usd":0.260678},"total_usd":0.38944,"stage1_batch_id":"msgbatch_011rkcQdVV1RaFq9XhcG6df6","stage2_batch_id":"msgbatch_01RzgrJafdF4EWHhj5mNi6dh","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2011,\n      \"finding\": \"SHARPIN functions as a novel component of the linear ubiquitin chain assembly complex (LUBAC) by binding to the LUBAC subunit HOIP (RNF31), thereby stimulating the formation of linear ubiquitin chains in vitro and in vivo, promoting linear ubiquitination of NEMO and subsequent NF-κB activation, and protecting against TNF-α-induced apoptosis via FADD- and caspase-8-dependent pathways.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ubiquitination assay, genetic KO (cpdm mice), MEF and B cell functional assays\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — replicated independently in two simultaneous Nature papers with in vitro assays and genetic models\",\n      \"pmids\": [\"21455181\", \"21455180\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"SHARPIN is an endogenous inhibitor of β1-integrin activation; it directly binds to a conserved cytoplasmic region of integrin α-subunits and inhibits recruitment of talin and kindlin to the integrin, thereby preventing the switch from inactive to active integrin conformations.\",\n      \"method\": \"RNAi screen, direct binding assay, rescue experiments in SHARPIN-deficient fibroblasts/leukocytes/keratinocytes, re-expression of SHARPIN\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (RNAi screen, binding assay, genetic KO rescue) in one study, replicated in multiple cell types\",\n      \"pmids\": [\"21947080\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"SHARPIN directly interacts with the ankyrin repeats of Shank family proteins and is enriched in the postsynaptic density (PSD) of excitatory synapses; the C-terminal half mediates Shank interaction while the N-terminal half mediates SHARPIN homomultimerization.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation in heterologous cells and brain, immunostaining\",\n      \"journal\": \"Molecular and cellular neurosciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP and direct binding assay with domain mapping, foundational paper\",\n      \"pmids\": [\"11178875\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The N-terminal portion of SHARPIN adopts a pleckstrin homology (PH) superfold that acts as a dimerization module rather than a canonical ligand-recognition domain, extending the functional applications of this structural fold.\",\n      \"method\": \"Crystal structure determination of N-terminal SHARPIN fragment\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with functional validation of dimerization\",\n      \"pmids\": [\"22549881\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"RIPK1 kinase activity is required for the inflammatory pathology in SHARPIN-deficient (cpdm) mice; crossing with kinase-dead Ripk1(K45A) mice fully protected against all cpdm-related pathology, placing RIPK1 kinase activity downstream of SHARPIN in TNF-driven inflammatory cell death.\",\n      \"method\": \"Genetic epistasis — Ripk1(K45A) knockin crossed with Sharpincpdm mice, in vitro and in vivo necroptosis assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean genetic epistasis with rigorous in vivo phenotypic readout\",\n      \"pmids\": [\"24821972\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"TNFR1 (but not TNFR2) signaling is the primary driver of multi-organ inflammation in Sharpin-deficient mice; RIPK3 deficiency combined with Caspase-8 heterozygosity almost completely suppressed the phenotype, placing necroptosis and apoptosis downstream of TNFR1 in SHARPIN-deficient inflammation.\",\n      \"method\": \"Genetic epistasis — Tnfr1/Tnfr2/Ripk3/Casp8/Mlkl deletions crossed with Sharpincpdm mice\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal genetic crosses with defined phenotypic readouts\",\n      \"pmids\": [\"25443632\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"TRADD- and FADD-dependent keratinocyte apoptosis (not necroptosis) is the primary driver of skin inflammation in Sharpin-deficient mice; epidermis-restricted ablation of FADD combined with RIPK3 deficiency fully prevented skin inflammation.\",\n      \"method\": \"Genetic epistasis — tissue-specific KO (Krt14-Cre), FADD/RIPK3 double KO, primary keratinocyte apoptosis assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — tissue-specific genetic dissection with defined cellular phenotype\",\n      \"pmids\": [\"25443631\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"SHARPIN interacts with NEMO (confirmed by co-immunoprecipitation) and regulates NEMO-dependent signaling including p105 and ERK phosphorylation and p65 nuclear localization in a manner similar to the hypomorphic panr2 NEMO mutation, identifying SHARPIN as an essential adaptor in TLR2-induced NF-κB signaling in macrophages.\",\n      \"method\": \"Systems biology analysis, co-immunoprecipitation, transcriptomic analysis of cpdm macrophages\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — co-IP plus systems-level confirmation with genetic model\",\n      \"pmids\": [\"21709223\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"SHARPIN localizes to the trailing edges (uropods) of chemokine-activated migrating lymphocytes, directly interacts with LFA-1 (lymphocyte-function-associated antigen-1), and inhibits intermediate and high-affinity forms of LFA-1, thereby regulating uropod detachment and migration velocity.\",\n      \"method\": \"Live cell imaging, direct binding assay, SHARPIN-deficient lymphocyte functional assays, rescue by re-expression\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct binding, localization with functional consequence, and rescue experiment\",\n      \"pmids\": [\"24210817\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SHARPIN conjugated with K63-linked ubiquitin chains inhibits the association of TCRζ with the signaling kinase Zap70, affecting generation of regulatory T cells; SHARPIN deficiency results in enhanced proximal TCR signaling without affecting NF-κB activation.\",\n      \"method\": \"Co-immunoprecipitation, Treg transfer rescue experiments in SHARPIN-deficient mice, signaling assays\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic co-IP with K63-ubiquitin, functional rescue in vivo\",\n      \"pmids\": [\"26829767\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SHARPIN directly binds caspase-1 and disrupts p20/p10 dimer formation (the last step of caspase-1 processing), thereby inhibiting caspase-1 enzyme activation and maturation of IL-1β/IL-18 in a LUBAC-independent manner.\",\n      \"method\": \"Co-immunoprecipitation, biochemical dimerization assay, genetic KO (Casp1/Casp11 deletion in Sharpin-deficient background), in vivo sepsis model\",\n      \"journal\": \"The American journal of pathology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — direct binding assay plus mechanistic biochemical dissection plus genetic validation\",\n      \"pmids\": [\"26968342\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"SHARPIN is required for optimal NLRP3 inflammasome activation by both canonical and non-canonical stimuli, and SHARPIN-deficient macrophages show dramatic defects in NF-κB and MAPK pathway activation, suggesting SHARPIN acts in transcriptional priming of NLRP3.\",\n      \"method\": \"Inflammasome activation assays in Sharpincpdm macrophages, IL-1β/caspase-1 cleavage assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with defined cellular phenotype, single lab\",\n      \"pmids\": [\"25637014\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"SHARPIN's integrin inhibition function and its LUBAC-activating function are mutually exclusive; the integrin α-subunit tail competes with RNF31 (HOIP) for binding to the same ubiquitin-like (UBL) domain of SHARPIN, with two residues (V267, L276) required for both interactions.\",\n      \"method\": \"Domain mapping, in vitro competition binding assay, site-directed mutagenesis, NF-κB reporter assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro competition assay plus mutagenesis defining binding interface\",\n      \"pmids\": [\"26600301\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The NZF domain of SHARPIN (but not HOIL-1L) is critical for protection from programmed cell death by enhancing recruitment of LUBAC to the activated TNFR complex; this function depends on the SHARPIN NZF domain's ability to bind K63-linked ubiquitin chains.\",\n      \"method\": \"Intercrossing of HOIL-1L and SHARPIN KO mice, domain mutagenesis, TNFR complex recruitment assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis combined with domain mutagenesis and complex recruitment assay\",\n      \"pmids\": [\"26976635\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"SHARPIN interacts with PRMT5 in a LUBAC-independent manner and increases PRMT5 multiprotein complex assembly and methyltransferase activity; activated PRMT5 controls expression of SOX10 and MITF transcription factors by arginine dimethylation and inhibition of the transcriptional corepressor SKI.\",\n      \"method\": \"Co-immunoprecipitation, in vitro methyltransferase activity assay, arginine dimethylation analysis, genetic rescue\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro enzymatic assay plus co-IP with mechanistic follow-through\",\n      \"pmids\": [\"29227283\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"SHARPIN facilitates p53 poly-ubiquitination and degradation in an MDM2-dependent manner; SHARPIN associates with MDM2 (by co-immunoprecipitation) and prolongs MDM2 protein stability, thereby reducing p53 protein levels and target gene expression in breast cancer cells.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, protein stability assay, RNA-seq, siRNA knockdown\",\n      \"journal\": \"Neoplasia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP and ubiquitination assay, single lab, moderate mechanistic depth\",\n      \"pmids\": [\"28063307\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"SHARPIN stabilizes estrogen receptor α (ERα) by inhibiting ERα poly-ubiquitination while facilitating mono-ubiquitination at K302/303 sites; SHARPIN localizes in the cytosol and interacts with ERα in both cytosol and nucleus, promoting ERα-dependent transcription and breast cancer cell proliferation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, luciferase reporter, site-directed mutagenesis, siRNA knockdown\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ubiquitination site mapping plus co-IP, single lab\",\n      \"pmids\": [\"29100376\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"SHARPIN interacts with PRMT5 to promote monomethylation of histone H3 at arginine 2 (H3R2me1) at metastasis-related gene loci, linking this mark to H3K4me3 via an MLL complex (ASH2/WDR5) to activate metastasis-related gene expression in lung cancer cells.\",\n      \"method\": \"Co-immunoprecipitation, histone methylation assay, chromatin immunoprecipitation\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP with histone modification assay, single lab\",\n      \"pmids\": [\"28903384\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SHARPIN regulates mammary ductal outgrowth in a stromal-cell-intrinsic manner; SHARPIN-deficient stromal fibroblasts display defects in collagen fibre assembly, contraction, and degradation, leading to reduced ECM stiffness adjacent to invading ducts.\",\n      \"method\": \"Cell-type-specific KO (S100a4-Cre), mammary epithelial transplantation, collagen contraction/degradation in vitro assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — tissue-specific deletion with transplantation experiment and in vitro functional assays\",\n      \"pmids\": [\"27974362\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"SIPL1 (SHARPIN) promotes PTEN polyubiquitination via K63-linked polyubiquitin chains using its ubiquitin-like (UBL) domain; this ubiquitination promotes and stabilizes the SIPL1/PTEN complex.\",\n      \"method\": \"Ubiquitination assay with ubiquitin K48R and K63R mutants, co-immunoprecipitation, UBL domain mutagenesis\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ubiquitin chain-type dissection with mutagenesis, single lab\",\n      \"pmids\": [\"25152374\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"SHARPIN interacts with the Arp2/3 complex and promotes lamellipodium formation in a LUBAC-independent manner; the Arp2/3-binding site on SHARPIN was mapped and an Arp2/3-binding-deficient mutant abrogated lamellipodium formation.\",\n      \"method\": \"Mass spectrometry interactome (Sharpin interactome), co-immunoprecipitation, domain mapping, Arp2/3-binding-deficient mutant rescue assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — systematic interactome plus domain mutagenesis with functional readout\",\n      \"pmids\": [\"28775156\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SHARPIN serine 165 (S165) phosphorylation is constitutively present in lymphoblastoid cells and is further induced by TCR stimulation; a phosphorylation-resistant SHARPIN S165A mutant shows impaired linear ubiquitination of NEMO and reduced NF-κB activation in response to TNFα.\",\n      \"method\": \"Mass spectrometry phosphorylation analysis, phosphorylation-resistant mutant, NF-κB reporter assay, linear ubiquitination assay\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — site-specific mutagenesis with functional assay, single lab\",\n      \"pmids\": [\"33392484\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SHARPIN S146 phosphorylation (sensitive to ERK1/2 inhibition and PP2A reactivation) mediates ARP2/3 complex interaction and lamellipodia formation, and is required for 3D cancer cell invasion and in vivo metastasis; S146A mutant SHARPIN fails to rescue invasion in SHARPIN-KO cancer cells.\",\n      \"method\": \"Mass spectrometry, in vitro kinase assay, CRISPR/Cas9 KO, S146A mutant rescue, zebrafish metastasis model\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro kinase assay plus mutagenesis plus in vivo model\",\n      \"pmids\": [\"36148554\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SHARPIN-deficient mouse phenotype (dermatitis, disrupted splenic architecture, loss of Peyer's patches) is fully dependent on CYLD deubiquitinase; absence of SHARPIN impairs CYLD phosphorylation at serine 418 (which normally inhibits CYLD), leading to enhanced RIPK1 recruitment to death-signaling Complex II following TNF stimulation.\",\n      \"method\": \"Genetic epistasis (Sharpin/Cyld double KO), conditional Cyld deletion (LysM-Cre, Cx3cr1-Cre), RIPK1 complex II immunoprecipitation, CYLD phosphorylation analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean genetic epistasis with mechanistic biochemical dissection of RIPK1 complex II\",\n      \"pmids\": [\"34887354\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"SHARPIN interacts with integrin αIIb in human platelets (pull-down and co-immunoprecipitation) and co-immunoprecipitates with HOIP and HOIL-1 (LUBAC components); SHARPIN knockdown in megakaryocytes/platelets increases αIIbβ3-fibrinogen binding and reduces Met1 linear ubiquitination and NF-κB (RelA) activation.\",\n      \"method\": \"Pull-down, co-immunoprecipitation, SHARPIN knockdown in iPSC-derived megakaryocytes/platelets, flow cytometry, linear ubiquitination assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP and functional KD with multiple cellular readouts\",\n      \"pmids\": [\"30804189\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"SHARPIN suppresses β1-integrin activation by complexing with both the integrin β1 cytoplasmic tail and kindlin-1; kindlin-1 enhances the SHARPIN–β1 tail interaction, and the complex restricts talin head domain binding to the β1 tail.\",\n      \"method\": \"Biochemical binding assays, co-immunoprecipitation, talin competition assay in CHO cells\",\n      \"journal\": \"Cell communication and signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct binding and competition assay, single lab\",\n      \"pmids\": [\"31429758\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SHARPIN stabilizes β-catenin by competing with the E3 ubiquitin ligase β-TrCP1 for β-catenin binding, thereby decreasing β-catenin ubiquitination and preventing its proteasomal degradation in a linear ubiquitination-independent manner.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, competition binding assay, in vitro and in vivo functional assays\",\n      \"journal\": \"Gastric cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP with competition assay and ubiquitination assay, single lab\",\n      \"pmids\": [\"33159601\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SHARPIN promotes ubiquitination and proteasomal degradation of von Hippel-Lindau protein (pVHL) using its ubiquitin-like domain; SHARPIN interaction requires the α and β domains of pVHL, and this leads to sustained HIF-2α activation in clear cell renal cell carcinoma.\",\n      \"method\": \"Co-immunoprecipitation with domain mapping, ubiquitination assay, siRNA knockdown, xenograft model\",\n      \"journal\": \"Cancer science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP with domain dissection and ubiquitination assay, single lab\",\n      \"pmids\": [\"34339558\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"SHARPIN interacts with YAP protein and promotes YAP K48-linked poly-ubiquitination and degradation, thereby inhibiting YAP/TEAD target gene expression (CTGF, CYR61) and functioning as an endogenous inhibitor of YAP in esophageal squamous cell carcinoma.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, rescue experiment\",\n      \"journal\": \"Neoplasia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single co-IP plus ubiquitination assay, single lab\",\n      \"pmids\": [\"31884247\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"SHARPIN deficiency in keratinocytes triggers mitochondria-dependent (intrinsic) apoptosis characterized by mitochondrial membrane depolarization, shift in BCL2/BAX ratio, and activation of caspases 9 and 3 (but not 8), without involvement of the extrinsic caspase-8 pathway.\",\n      \"method\": \"FACS (Annexin V/PI), transmission electron microscopy, caspase activity assays, Western blot for BCL2/BAX\",\n      \"journal\": \"Journal of dermatological science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple complementary assays defining the apoptotic pathway, single lab\",\n      \"pmids\": [\"21620685\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"HOIL-1L and SHARPIN form homo-dimers through their LTM motifs; crystal structures of the dimeric LTM motifs reveal the molecular mechanism of dimerization and a shared mode for homo- and hetero-dimer formation; the polyglucosan body myopathy-associated HOIL-1L A18P mutation disrupts HOIL-1L LTM structural folding and dimer formation.\",\n      \"method\": \"Crystal structure determination of LTM motif dimers, disease mutation analysis, biochemical validation\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structures with functional and disease mutation validation\",\n      \"pmids\": [\"37976837\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The NZF domains of HOIL-1L and SHARPIN cooperatively regulate LUBAC function; HOIL-1L NZF preferentially binds linear ubiquitin chains while SHARPIN NZF binds K63-linked chains in addition to linear chains; simultaneous loss of both NZF ubiquitin-binding activities profoundly impairs NF-κB activation and cell death protection.\",\n      \"method\": \"NZF domain mutagenesis, ubiquitin chain-binding assays, NF-κB activation assays, cell death protection assays, compound screen\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — domain mutagenesis with in vitro binding and multiple functional assays\",\n      \"pmids\": [\"39528476\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Biallelic SHARPIN loss in humans causes attenuated canonical NF-κB responses and propensity for cell death mediated by TNF superfamily members in fibroblasts and B cells; anti-TNF therapy completely resolved autoinflammation, confirming TNF-driven pathology.\",\n      \"method\": \"Patient-derived fibroblast and B cell assays, NF-κB signaling assays, anti-TNF treatment with transcriptomic resolution\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — human genetic validation of LUBAC/NF-κB mechanism with functional cell assays and treatment response\",\n      \"pmids\": [\"38609546\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SHARPIN directly interacted with HMGB1 (demonstrated by GST pull-down and co-immunoprecipitation) and enhanced HMGB1 expression, promoting M1-like macrophage polarization and neuroinflammation in sevoflurane-induced neurotoxicity.\",\n      \"method\": \"GST pull-down, co-immunoprecipitation, SHARPIN siRNA knockdown, macrophage polarization assays\",\n      \"journal\": \"Metabolic brain disease\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single co-IP/pulldown, single lab, limited mechanistic follow-up\",\n      \"pmids\": [\"38805141\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"SHARPIN was identified as a novel interaction partner of Eya1 (Eyes Absent 1); SHARPIN (as Sipl1) and Rbck1 enhance the function of Eya proteins as coactivators for Six transcription factors, and morpholino knockdown of a Sipl1 ortholog in zebrafish produces a BOR syndrome-like phenotype with ear and branchial arch defects.\",\n      \"method\": \"GST pulldown, co-immunoprecipitation, coactivator transcriptional assay, zebrafish morpholino knockdown\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct binding assay plus functional coactivator assay plus in vivo model, but in a developmental context not primary for mammalian SHARPIN focus\",\n      \"pmids\": [\"20956555\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Platelet-specific deletion of SHARPIN results in increased colocalization of αIIbβ3 with talin (by super-resolution microscopy), increased fibrinogen binding in response to ADP, and reduced NF-κB activation and linear ubiquitination of substrates; SHARPIN-null platelets show reduced inflammation in colitis and peritonitis models.\",\n      \"method\": \"Platelet-specific conditional KO (PF4-Cre/GPIbα-Cre), super-resolution microscopy, flow cytometry, in vivo inflammation models\",\n      \"journal\": \"Blood advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — cell-type-specific KO with multiple orthogonal readouts\",\n      \"pmids\": [\"34991155\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SHARPIN is a multifunctional adaptor protein that acts as an accessory subunit of the LUBAC E3 ligase complex (with HOIP/RNF31 and HOIL-1L/RBCK1) to catalyze linear (Met1-linked) ubiquitin chain formation on substrates including NEMO, thereby activating canonical NF-κB signaling and protecting cells from TNF-induced apoptosis and necroptosis; independently of LUBAC, SHARPIN inhibits β1- and β2-integrin activation by directly binding integrin α-subunit cytoplasmic tails and blocking talin/kindlin recruitment, inhibits caspase-1 by disrupting its p20/p10 dimer formation, suppresses TCR signaling by preventing ZAP70 association with TCRζ via K63-ubiquitin conjugation, promotes lamellipodium formation through ERK-dependent S146 phosphorylation-mediated Arp2/3 complex interaction, and regulates the stability of multiple proteins (p53 via MDM2, ERα, β-catenin, pVHL) through ubiquitination-dependent and -independent mechanisms.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"SHARPIN is a multifunctional adaptor protein that serves as an accessory subunit of the linear ubiquitin chain assembly complex (LUBAC), where it binds HOIP/RNF31 via its ubiquitin-like (UBL) domain and facilitates linear (Met1-linked) ubiquitination of NEMO, thereby activating canonical NF-κB signaling and protecting cells from TNF-induced apoptosis and necroptosis; the inflammatory pathology caused by SHARPIN deficiency is driven by TNFR1 signaling, RIPK1 kinase activity, and the deubiquitinase CYLD, and biallelic SHARPIN loss in humans causes autoinflammation that is fully resolved by anti-TNF therapy [PMID:21455181, PMID:21455180, PMID:24821972, PMID:25443632, PMID:34887354, PMID:38609546]. Independent of LUBAC, SHARPIN directly binds integrin α-subunit cytoplasmic tails through its UBL domain — competing with HOIP for the same binding surface — to inhibit integrin activation by blocking talin and kindlin recruitment, a function demonstrated for β1-integrins, β2-integrins (LFA-1), and αIIbβ3 in platelets [PMID:21947080, PMID:24210817, PMID:30804189, PMID:26600301]. SHARPIN also acts outside the LUBAC–integrin axis: it inhibits caspase-1 by disrupting p20/p10 dimerization, suppresses proximal TCR signaling via K63-ubiquitin-dependent exclusion of ZAP70 from TCRζ, promotes lamellipodium formation through ERK-dependent S146 phosphorylation and Arp2/3 complex interaction, enhances PRMT5 methyltransferase activity, and modulates the stability of p53 (via MDM2), β-catenin, and pVHL through ubiquitination-dependent mechanisms [PMID:26968342, PMID:26829767, PMID:36148554, PMID:29227283, PMID:28063307, PMID:33159601, PMID:34339558]. Biallelic loss-of-function mutations in SHARPIN cause a human autoinflammatory disease characterized by attenuated NF-κB responses and TNF superfamily–driven cell death [PMID:38609546].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Before SHARPIN's signaling roles were known, its initial identification as a Shank-binding postsynaptic density protein established it as a scaffolding adaptor capable of homomultimerization, setting the stage for understanding its broader interaction repertoire.\",\n      \"evidence\": \"Yeast two-hybrid and co-immunoprecipitation from brain lysates with domain mapping\",\n      \"pmids\": [\"11178875\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional significance of SHARPIN at excitatory synapses remains undefined\", \"Whether synaptic SHARPIN affects Shank-dependent signaling is untested\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Before LUBAC function was linked to SHARPIN, its interaction with Eya1 and enhancement of Six-dependent transcription revealed a role in developmental transcriptional coactivation, broadening the protein's functional scope beyond scaffolding.\",\n      \"evidence\": \"GST pulldown, coactivator assay, zebrafish morpholino knockdown producing BOR-like phenotype\",\n      \"pmids\": [\"20956555\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The mammalian developmental relevance of the SHARPIN–Eya1 axis is not confirmed by knockout\", \"Whether this function is LUBAC-dependent is unknown\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"The central mechanistic advance was the simultaneous discovery by two groups that SHARPIN is a bona fide LUBAC subunit: it binds HOIP, stimulates linear ubiquitin chain formation on NEMO, activates NF-κB, and protects cells from TNF-induced apoptosis, explaining the chronic proliferative dermatitis (cpdm) mouse phenotype.\",\n      \"evidence\": \"Co-immunoprecipitation, in vitro ubiquitination, cpdm mouse genetic model, MEF and B cell assays (two independent Nature papers)\",\n      \"pmids\": [\"21455181\", \"21455180\", \"21709223\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of SHARPIN–HOIP interaction at atomic resolution was not yet defined\", \"Relative contributions of SHARPIN versus HOIL-1L NZF domains to LUBAC recruitment were unclear\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"In parallel with the LUBAC discovery, SHARPIN was found to directly bind integrin α-subunit cytoplasmic tails and inhibit β1-integrin activation by preventing talin and kindlin recruitment, establishing a LUBAC-independent function.\",\n      \"evidence\": \"RNAi screen, direct binding assay, rescue in SHARPIN-deficient fibroblasts/leukocytes/keratinocytes\",\n      \"pmids\": [\"21947080\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether integrin inhibition contributes to the cpdm phenotype was not separated from NF-κB defects\", \"Structural mechanism of α-tail binding was not resolved\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Crystallography of the N-terminal domain revealed a PH superfold that functions as a dimerization module rather than a ligand-binding domain, providing structural basis for SHARPIN self-association.\",\n      \"evidence\": \"Crystal structure of N-terminal SHARPIN fragment with functional dimerization validation\",\n      \"pmids\": [\"22549881\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How dimerization affects LUBAC assembly or integrin inhibition was not tested\", \"Full-length SHARPIN structure remained unavailable\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Genetic epistasis experiments resolved the cell death pathways downstream of SHARPIN loss: TNFR1 (not TNFR2) is the primary driver, RIPK1 kinase activity is essential, and both apoptosis (via TRADD/FADD in keratinocytes) and necroptosis contribute tissue-specifically to the cpdm phenotype.\",\n      \"evidence\": \"Intercross of cpdm mice with Ripk1(K45A), Tnfr1-KO, Tnfr2-KO, Ripk3-KO, Casp8-het, tissue-specific FADD deletion\",\n      \"pmids\": [\"24821972\", \"25443632\", \"25443631\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which LUBAC substrates beyond NEMO are critical for cell death protection was not defined\", \"Non-TNF cytokine contributions were not fully excluded\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Binding competition experiments showed that the integrin α-tail and HOIP bind the same UBL domain surface on SHARPIN, making integrin inhibition and LUBAC activation mutually exclusive — revealing a molecular switch governing SHARPIN's dual functions.\",\n      \"evidence\": \"In vitro competition binding assay, V267/L276 mutagenesis, NF-κB reporter assay\",\n      \"pmids\": [\"26600301\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How the switch is regulated in vivo (post-translational modifications, localization) is unknown\", \"Whether the competition is relevant in all cell types is untested\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"SHARPIN was shown to have LUBAC-independent roles in immune regulation: it inhibits caspase-1 by disrupting p20/p10 dimerization, and its K63-ubiquitin conjugation suppresses TCRζ–ZAP70 association, affecting regulatory T cell generation.\",\n      \"evidence\": \"Co-immunoprecipitation with dimerization assay and genetic validation (caspase-1); co-IP with K63-Ub and Treg transfer rescue (TCR signaling)\",\n      \"pmids\": [\"26968342\", \"26829767\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The E3 ligase catalyzing K63-ubiquitination of SHARPIN in T cells is unidentified\", \"Whether caspase-1 inhibition is relevant in vivo beyond the sepsis model is unclear\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"The SHARPIN NZF domain was shown to be specifically required for LUBAC recruitment to the TNFR complex via K63-linked ubiquitin binding, distinguishing it functionally from the HOIL-1L NZF domain.\",\n      \"evidence\": \"NZF mutagenesis, TNFR complex recruitment assay, HOIL-1L/SHARPIN double KO intercross\",\n      \"pmids\": [\"26976635\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether NZF-mediated recruitment is regulated by post-translational modifications is unknown\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"SHARPIN was found to interact with the Arp2/3 complex in a LUBAC-independent manner to promote lamellipodium formation, and separately to enhance PRMT5 methyltransferase activity controlling SOX10/MITF expression and metastasis-related gene transcription, expanding its roles to cytoskeletal dynamics and epigenetic regulation.\",\n      \"evidence\": \"Mass spectrometry interactome, Arp2/3-binding-deficient mutant (lamellipodium); co-IP with in vitro methyltransferase and ChIP assays (PRMT5)\",\n      \"pmids\": [\"28775156\", \"29227283\", \"28903384\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The kinase phosphorylating S146 to regulate Arp2/3 binding was not yet identified\", \"Whether PRMT5 activation by SHARPIN occurs in non-cancer contexts is untested\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Multiple studies established SHARPIN as a regulator of protein stability through ubiquitination-dependent mechanisms: it promotes MDM2-dependent p53 degradation and stabilizes ERα by switching its ubiquitination from poly- to mono-ubiquitination.\",\n      \"evidence\": \"Co-immunoprecipitation, ubiquitination assays, protein stability assays in breast cancer cells\",\n      \"pmids\": [\"28063307\", \"29100376\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether SHARPIN acts as a direct E3 ligase or purely as an adaptor for these substrates is unresolved\", \"Single-lab findings for each substrate\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Platelet-specific studies confirmed SHARPIN's dual function in an important physiological context: it inhibits αIIbβ3-integrin activation (increasing talin–integrin colocalization upon deletion) and supports LUBAC-dependent linear ubiquitination and NF-κB activation in platelets.\",\n      \"evidence\": \"Platelet-specific conditional KO (PF4-Cre, GPIbα-Cre), super-resolution microscopy, flow cytometry, in vivo inflammation models\",\n      \"pmids\": [\"30804189\", \"34991155\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether platelet SHARPIN loss affects thrombosis risk in vivo needs further study\", \"The relative importance of integrin versus NF-κB functions in platelet biology is not separated\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"The full cpdm phenotype was shown to require the deubiquitinase CYLD: SHARPIN loss impairs IKK-mediated CYLD phosphorylation at S418, unleashing CYLD activity that deubiquitinates RIPK1 and promotes its recruitment to death-inducing Complex II.\",\n      \"evidence\": \"Sharpin/Cyld double KO, tissue-specific Cyld deletion, RIPK1 Complex II immunoprecipitation, CYLD phosphorylation analysis\",\n      \"pmids\": [\"34887354\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether CYLD directly deubiquitinates linear chains on NEMO or acts on other substrates is not resolved\", \"Whether pharmacological CYLD inhibition could be therapeutic is untested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"ERK1/2-dependent phosphorylation of SHARPIN at S146 was identified as the signal controlling Arp2/3 interaction, lamellipodia formation, and cancer cell invasion/metastasis, revealing a phospho-switch for SHARPIN's cytoskeletal function.\",\n      \"evidence\": \"In vitro kinase assay, mass spectrometry, CRISPR KO with S146A mutant rescue, zebrafish metastasis model\",\n      \"pmids\": [\"36148554\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The PP2A holoenzyme subunit targeting pS146 is not identified\", \"Whether S146 phosphorylation affects other SHARPIN functions is unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Crystal structures of SHARPIN LTM motif dimers revealed the molecular basis for LUBAC subunit homo- and hetero-dimerization, and linked a disease-associated HOIL-1L mutation (A18P) to disrupted dimer formation.\",\n      \"evidence\": \"Crystal structure determination of LTM dimers with disease mutation structural analysis\",\n      \"pmids\": [\"37976837\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full LUBAC holocomplex structure is still unavailable\", \"How LTM dimerization regulates catalytic activity quantitatively is not defined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Biallelic SHARPIN loss was identified in human patients with autoinflammation, confirming the mouse-to-human translational relevance: patient cells show attenuated NF-κB and TNF-driven cell death, and anti-TNF therapy fully resolved disease, establishing SHARPIN deficiency as a human Mendelian autoinflammatory condition.\",\n      \"evidence\": \"Patient-derived fibroblast and B cell functional assays, NF-κB signaling, anti-TNF treatment with transcriptomic validation\",\n      \"pmids\": [\"38609546\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The full clinical spectrum of human SHARPIN deficiency beyond autoinflammation is not characterized\", \"Whether integrin dysregulation contributes to the human phenotype is unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Cooperative regulation by HOIL-1L and SHARPIN NZF domains was demonstrated: HOIL-1L NZF preferentially binds linear chains while SHARPIN NZF binds both K63 and linear chains, and simultaneous loss profoundly impairs NF-κB and cell death protection, refining the model of accessory subunit function within LUBAC.\",\n      \"evidence\": \"NZF domain mutagenesis, ubiquitin chain-binding assays, NF-κB and cell death functional assays\",\n      \"pmids\": [\"39528476\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How NZF ubiquitin-type selectivity is determined structurally is unresolved\", \"Whether NZF functions can be pharmacologically targeted is untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the full-length atomic structure of SHARPIN, the in vivo regulatory logic governing the UBL domain switch between HOIP and integrin binding, whether SHARPIN's LUBAC-independent functions (caspase-1 inhibition, PRMT5 activation, Arp2/3 interaction) are interdependent or independently regulated, and the complete clinical spectrum of human SHARPIN deficiency.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Full-length SHARPIN structure not available\", \"In vivo regulation of the HOIP–integrin binding switch is undefined\", \"Interplay among LUBAC-independent functions is untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 7, 12, 13]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [1, 8, 20, 22, 25]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [10, 14, 26, 27]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [16]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [1, 8, 24]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 7, 9, 11, 32]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 7, 21, 26]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [4, 5, 6, 23, 29, 32]},\n      {\"term_id\": \"R-HSA-1500931\", \"supporting_discovery_ids\": [1, 8, 24, 25]}\n    ],\n    \"complexes\": [\n      \"LUBAC\"\n    ],\n    \"partners\": [\n      \"RNF31\",\n      \"RBCK1\",\n      \"IKBKG\",\n      \"PRMT5\",\n      \"CASP1\",\n      \"CYLD\",\n      \"SHANK1\",\n      \"ACTR2\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}