{"gene":"SHARPIN","run_date":"2026-06-10T07:46:31","timeline":{"discoveries":[{"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 of SHARPIN mediates SHANK binding while the N-terminal half mediates homomultimerization.","method":"Yeast two-hybrid, co-immunoprecipitation from brain and heterologous cells, immunostaining/colocalization","journal":"Molecular and cellular neurosciences","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP from brain tissue plus heterologous cells, domain mapping, and colocalization; independently consistent across multiple methods in one study","pmids":["11178875"],"is_preprint":false},{"year":2011,"finding":"SHARPIN is a component of the linear ubiquitin chain assembly complex (LUBAC) together with HOIP (RNF31) and HOIL-1L (RBCK1); binding to HOIP stimulates formation of linear (Met1-linked) ubiquitin chains in vitro and in vivo, leading to linear ubiquitination of NEMO and activation of NF-κB signaling.","method":"Co-immunoprecipitation, in vitro ubiquitination assay, NF-κB reporter assay, SHARPIN-deficient mouse embryonic fibroblasts/B cells/macrophages","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro reconstitution of linear ubiquitin chain formation, reciprocal Co-IP, loss-of-function genetics; independently replicated in two simultaneous Nature papers (PMID 21455181 and 21455180)","pmids":["21455181","21455180"],"is_preprint":false},{"year":2011,"finding":"SHARPIN deficiency sensitizes cells to TNF-α-induced apoptosis via FADD- and caspase-8-dependent pathways, demonstrating that SHARPIN inhibits apoptosis independently of NF-κB activation.","method":"TNF-α stimulation of SHARPIN-deficient MEFs and primary cells; caspase-8/FADD genetic epistasis; cell death assays","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — loss-of-function genetics with defined pathway placement (FADD/caspase-8 dependence), replicated across multiple cell types and confirmed in vivo","pmids":["21455181"],"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, preventing the inactive-to-active conformational switch.","method":"RNAi screen, direct binding assay, fibroblasts/leukocytes/keratinocytes from SHARPIN-deficient mice, rescue by SHARPIN re-expression, integrin activity assays","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct binding demonstrated, loss-of-function with rescue across multiple primary cell types, mechanistic pathway placement (talin/kindlin competition)","pmids":["21947080"],"is_preprint":false},{"year":2011,"finding":"SHARPIN negatively associates with TRAF2-mediated NF-κB activation; mass spectrometry identified TRAF2 as a SHARPIN co-immunoprecipitated partner, and overexpression studies show SHARPIN can negatively modulate NF-κB via TRAF2 interaction.","method":"Co-immunoprecipitation, mass spectrometry, NF-κB luciferase reporter assay","journal":"PloS one","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP/MS identification plus reporter assay, single lab, no mechanistic follow-up mutagenesis","pmids":["21829440"],"is_preprint":false},{"year":2011,"finding":"SHARPIN interacts with NEMO (IKBKG) in macrophages; SHARPIN deficiency impairs NEMO-dependent signaling (p105 and ERK phosphorylation, p65 nuclear localization) downstream of TLR2, and this interaction is abrogated by the panr2 (L153P) NEMO mutation.","method":"Systems biology, co-immunoprecipitation, transcriptome analysis of SHARPIN-deficient macrophages","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP confirmed interaction, SHARPIN-deficient cell phenotyping with pathway-level readouts, single lab","pmids":["21709223"],"is_preprint":false},{"year":2011,"finding":"SHARPIN deficiency in keratinocytes leads to apoptosis via the intrinsic mitochondrial pathway (caspase-9 and caspase-3 activation, Bax/Bcl2 shift, mitochondrial membrane potential loss) rather than the extrinsic caspase-8 pathway.","method":"Annexin-V/PI FACS, transmission electron microscopy, caspase activity assays, western blotting in SHARPIN-deficient mouse skin","journal":"Journal of dermatological science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple complementary in vitro and in vivo assays, single lab, no rescue experiment","pmids":["21620685"],"is_preprint":false},{"year":2012,"finding":"The N-terminal portion of SHARPIN adopts a pleckstrin homology (PH) superfold that functions as a dimerization module rather than a ligand-binding domain; crystal structure determined to 2.0–2.6 Å resolution.","method":"X-ray crystallography (crystal structure of N-terminal SHARPIN domain)","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with functional validation of dimerization; single lab but rigorous structural method","pmids":["22549881"],"is_preprint":false},{"year":2013,"finding":"SHARPIN localizes to the trailing edge (uropod) of migrating lymphocytes and directly interacts with LFA-1 (αLβ2 integrin), maintaining LFA-1 in an inactive state to enable uropod detachment and control lymphocyte migration velocity.","method":"Live imaging, SHARPIN-deficient lymphocyte migration assays on ICAM-1, direct binding assay, rescue by SHARPIN re-expression, integrin activation measurement","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct binding plus localization by imaging plus loss-of-function with rescue, multiple orthogonal methods","pmids":["24210817"],"is_preprint":false},{"year":2014,"finding":"RIP1 (RIPK1) kinase activity is required for the inflammatory pathology in SHARPIN-deficient (cpdm) mice; kinase-dead Ripk1(K45A) completely protected against all cpdm-related pathology, placing RIPK1 kinase downstream of SHARPIN loss in TNF-driven inflammation.","method":"Genetic epistasis: Ripk1(K45A) knock-in mice crossed with cpdm mice; in vivo and in vitro necroptosis assays","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic epistasis with kinase-dead allele, complete phenotypic rescue, in vivo and in vitro validation","pmids":["24821972"],"is_preprint":false},{"year":2014,"finding":"Skin inflammation in SHARPIN-deficient mice is driven primarily by TNFR1 (but not TNFR2), and is dependent on TRADD- and FADD-mediated keratinocyte apoptosis; epidermis-restricted ablation of FADD combined with RIPK3 deficiency fully prevented skin inflammation.","method":"Genetic epistasis: compound knockout mice (Tnfr1-/-, Ripk3-/-, Casp8+/-, Fadd-/- keratinocyte-specific) crossed with Sharpin-deficient mice","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple complementary genetic epistasis experiments across two independent studies (PMIDs 25443632 and 25443631), consistent conclusions","pmids":["25443632","25443631"],"is_preprint":false},{"year":2015,"finding":"SHARPIN regulates TCR signaling in T cells; SHARPIN is conjugated with K63-linked ubiquitin chains, which inhibits the association of TCRζ with ZAP70, thereby affecting regulatory T cell generation and maintaining immunological homeostasis.","method":"Co-immunoprecipitation, ubiquitination assays, SHARPIN-deficient T cell signaling analysis, Treg transfer experiments","journal":"Nature immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with ubiquitination characterization and defined functional consequence (ZAP70 association), single lab","pmids":["26829767"],"is_preprint":false},{"year":2015,"finding":"SHARPIN is required for optimal NLRP3 inflammasome activation; SHARPIN-deficient macrophages show defects in canonical and non-canonical NLRP3 inflammasome activation, associated with impaired NF-κB and MAPK pathway priming.","method":"Loss-of-function analysis using Sharpin(cpdm) macrophages, NLRP3 inflammasome activation assays","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — defined cellular phenotype in loss-of-function system with pathway readouts, single lab","pmids":["25637014"],"is_preprint":false},{"year":2015,"finding":"SHARPIN binds integrin α-subunit cytoplasmic tails and LUBAC component RNF31 in a mutually exclusive manner via its ubiquitin-like (UBL) domain; residues V267 and L276 are required for both interactions, while F263 and I272 are specifically required for RNF31 but not integrin binding.","method":"Binding assays with SHARPIN mutants, competition assays with integrin α-tail and RNF31, NF-κB reporter assay, integrin activity assay","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — site-directed mutagenesis mapping two distinct binding surfaces, competition in vitro, functional validation in cells; single lab","pmids":["26600301"],"is_preprint":false},{"year":2016,"finding":"SHARPIN directly binds caspase-1 in a LUBAC-independent manner and disrupts p20/p10 dimer formation (the final step of caspase-1 processing), thereby inhibiting caspase-1 enzyme activation and maturation of IL-1β/IL-18.","method":"Co-immunoprecipitation of SHARPIN with caspase-1, caspase-1 processing assay, Sharpin-deficient mouse sepsis model, caspase-1 inhibitor rescue","journal":"The American journal of pathology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding plus mechanistic step (p20/p10 dimerization) plus in vivo genetic validation; single lab","pmids":["26968342"],"is_preprint":false},{"year":2016,"finding":"The NZF domain of SHARPIN (but not that of HOIL-1L) is critical for protection from programmed cell death by enhancing LUBAC recruitment to the activated TNFR complex; SHARPIN NZF binds K63-linked ubiquitin chains (in addition to linear chains), and this K63-binding activity is involved in LUBAC recruitment.","method":"Intercross of HOIL-1L and SHARPIN knockout mice, NZF domain mutants, TNFR complex recruitment assays, cell death assays","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain mutagenesis with functional rescue, defined mechanistic step (LUBAC-to-TNFR complex recruitment), single lab","pmids":["26976635"],"is_preprint":false},{"year":2016,"finding":"Immune dysregulation in SHARPIN-deficient mice is dependent on CYLD deubiquitinase; loss of SHARPIN impairs phosphorylation of CYLD at serine 418 (which normally inhibits CYLD activity), leading to enhanced CYLD-mediated removal of ubiquitin from RIPK1 and increased RIPK1 recruitment to death-signaling Complex II upon TNF stimulation.","method":"Genetic epistasis (Sharpin/Cyld double knockout), TNFR complex immunoprecipitation, CYLD phosphorylation assays, conditional Cyld deletion","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with full phenotypic rescue, biochemical pathway dissection (CYLD phosphorylation and RIPK1 recruitment), multiple orthogonal approaches in one study","pmids":["34887354"],"is_preprint":false},{"year":2016,"finding":"SHARPIN regulates mammary gland stromal function; mice with stromal-specific (S100a4-Cre) deletion of Sharpin have reduced ductal outgrowth and defects in collagen fibre assembly, contraction and degradation, while transplanted SHARPIN-null mammary epithelial cells in wild-type stroma develop normally.","method":"Conditional knockout (S100a4-Cre), mammary gland transplantation, collagen contraction/degradation assays, ECM stiffness measurement","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional knockout with cell-type specificity established by transplantation, multiple in vitro collagen assays; single lab","pmids":["27974362"],"is_preprint":false},{"year":2017,"finding":"SHARPIN interacts with PRMT5 (protein arginine methyltransferase 5) in a LUBAC-independent manner and increases PRMT5 methyltransferase activity; activated PRMT5 controls SOX10 and MITF expression via arginine dimethylation and inhibition of the transcriptional corepressor SKI.","method":"Co-immunoprecipitation, PRMT5 methyltransferase activity assay, SKI arginine dimethylation assay, SOX10/MITF expression analysis, SHARPIN knockdown/overexpression in melanoma cells","journal":"The Journal of clinical investigation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct protein interaction confirmed by Co-IP, in vitro enzymatic activity assay, defined downstream targets; single lab","pmids":["29227283"],"is_preprint":false},{"year":2017,"finding":"SHARPIN promotes p53 polyubiquitination and degradation in an MDM2-dependent manner; SHARPIN associates with MDM2, prolongs MDM2 protein stability, and facilitates p53 poly-ubiquitination.","method":"Co-immunoprecipitation, ubiquitination assay, p53 stability assay, SHARPIN depletion with rescue by p53 knockdown in breast cancer cell lines","journal":"Neoplasia","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP showing SHARPIN-MDM2 complex, ubiquitination assay, functional epistasis with p53 knockdown; single lab","pmids":["28063307"],"is_preprint":false},{"year":2017,"finding":"SHARPIN stabilizes estrogen receptor α (ERα) protein by inhibiting ERα poly-ubiquitination and facilitating ERα mono-ubiquitination at K302/303 sites; SHARPIN interacts with ERα in both cytosol and nucleus.","method":"Co-immunoprecipitation, ubiquitination assays, ERE luciferase reporter, SHARPIN knockdown/overexpression, ERα stability assays","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct Co-IP, specific ubiquitination site mapping, functional ERE reporter; single lab","pmids":["29100376"],"is_preprint":false},{"year":2017,"finding":"SHARPIN interacts with ARP2/3 complex and promotes lamellipodium formation in a LUBAC-independent manner; the ARP2/3-binding site on SHARPIN was identified and an ARP2/3-binding-deficient SHARPIN mutant failed to support lamellipodia.","method":"Mass spectrometry interactome (Sharpin interactome), co-immunoprecipitation, site-directed mutagenesis, lamellipodium formation assay","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — interactome MS plus Co-IP, domain mapping with loss-of-function mutant and functional readout; single lab","pmids":["28775156"],"is_preprint":false},{"year":2017,"finding":"SHARPIN promotes Versican expression synergistically with Wnt/β-catenin pathway activation to drive hepatocellular carcinoma invasion.","method":"Stable SHARPIN-overexpressing cell lines, microarray analysis, Wnt/β-catenin reporter assay, invasion assays, Versican knockdown, in vivo tumor xenograft","journal":"Oncogenesis","confidence":"Low","confidence_rationale":"Tier 3 / Weak — correlative microarray with functional validation but mechanism of synergy with Wnt not molecularly defined; single lab","pmids":["27941932"],"is_preprint":false},{"year":2017,"finding":"A novel SHARPIN-PRMT5-H3R2me1 axis regulates lung cancer cell invasion; SHARPIN-PRMT5 complex catalyzes monomethylation of histone H3 arginine 2 (H3R2me1) at metastasis-related gene loci, which is linked to H3K4me3 via MLL complex components ASH2 and WDR5.","method":"Co-immunoprecipitation, histone methylation assays, chromatin immunoprecipitation, SHARPIN knockdown in lung cancer cells","journal":"Oncotarget","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Co-IP and ChIP data but limited mechanistic dissection; single lab, no in vitro reconstitution","pmids":["28903384"],"is_preprint":false},{"year":2019,"finding":"SHARPIN suppresses β1-integrin activation by forming a complex with kindlin-1 and the integrin β1 cytoplasmic tail; SHARPIN directly binds the β1 CT and the sharpin-kindlin-1 complex inhibits talin head domain binding to the β1 CT.","method":"Biochemical binding assays (GST pulldown, co-immunoprecipitation), cell-based β1-integrin activation assay, talin competition assay","journal":"Cell communication and signaling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding demonstrated biochemically with defined molecular competition, functional validation in cells; single lab","pmids":["31429758"],"is_preprint":false},{"year":2019,"finding":"SHARPIN interacts with αIIb cytoplasmic tail of integrin αIIbβ3 in human platelets and participates in LUBAC-mediated Met1 linear ubiquitination of NEMO upon platelet stimulation; SHARPIN knockdown in megakaryocytes/platelets increases basal and agonist-induced fibrinogen binding and reduces Met1 ubiquitination and NF-κB activation.","method":"Co-immunoprecipitation and pull-down assays, super-resolution microscopy, SHARPIN knockdown in iPS-derived megakaryocytes/platelets, ubiquitination assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, defined functional consequences in platelet-lineage cells with knockdown, multiple orthogonal methods; single lab","pmids":["30804189"],"is_preprint":false},{"year":2019,"finding":"SHARPIN promotes melanoma migration and invasion by upregulating Rap1 GTPase expression and activating downstream p38 and JNK/c-Jun pathways; Rap1 activator/inhibitor treatments partially rescued invasion phenotypes.","method":"SHARPIN knockdown/overexpression in melanoma cells, invasion/migration assays, Rap1 pharmacological modulation, in vivo metastasis models","journal":"The Journal of investigative dermatology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — pathway placement by pharmacological rescue, no direct binding between SHARPIN and Rap1 demonstrated; single lab","pmids":["31401046"],"is_preprint":false},{"year":2019,"finding":"SHARPIN inhibits YAP protein in esophageal squamous cell carcinoma; SHARPIN associates with YAP and promotes YAP K48-dependent poly-ubiquitination and proteasomal degradation, thereby suppressing Hippo pathway target genes (CTGF, CYR61).","method":"Co-immunoprecipitation, ubiquitination assay, YAP protein stability assay, SHARPIN/YAP knockdown rescue experiments, TEAD reporter","journal":"Neoplasia","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct interaction by Co-IP with defined ubiquitination type, functional epistasis by double knockdown rescue; single lab","pmids":["31884247"],"is_preprint":false},{"year":2019,"finding":"SHARPIN interacts with Eya1 (Eyes absent 1) and enhances Eya1 function as a coactivator for Six transcription factors; this interaction is required for normal zebrafish ear and branchial arch development.","method":"GST pulldown, co-immunoprecipitation, Six/Eya coactivation assay, zebrafish morpholino knockdown","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding confirmed by GST pulldown and Co-IP, functional transcriptional assay, in vivo morpholino knockdown phenotype; single lab","pmids":["20956555"],"is_preprint":false},{"year":2020,"finding":"SHARPIN phosphorylation at serine 165 is required for optimal NF-κB activation; a phosphorylation-resistant S165A mutant shows impaired linear ubiquitination of NEMO and reduced NF-κB activation in response to TNFα.","method":"Mass spectrometry identification of phosphorylation site, phosphorylation-resistant mutant analysis, NEMO ubiquitination assay, NF-κB reporter assay","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS-identified phosphorylation validated by mutant analysis with defined functional readout (NEMO ubiquitination, NF-κB); single lab","pmids":["33392484"],"is_preprint":false},{"year":2020,"finding":"SHARPIN stabilizes β-catenin protein by competing with the E3 ubiquitin ligase β-TrCP1 for β-catenin binding, thereby reducing β-catenin K48-linked ubiquitination and proteasomal degradation in a linear ubiquitination-independent manner.","method":"Co-immunoprecipitation, ubiquitination assay, competition binding assay (SHARPIN vs β-TrCP1), SHARPIN knockdown/overexpression with β-catenin protein stability readout, in vivo xenograft","journal":"Gastric cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding by Co-IP, competition assay, ubiquitination assay showing mechanism; single lab","pmids":["33159601"],"is_preprint":false},{"year":2021,"finding":"SHARPIN directly interacts with HMGB1 and enhances HMGB1 expression; this SHARPIN-HMGB1 interaction was demonstrated by GST pull-down and Co-IP, and SHARPIN promotes M1-like macrophage polarization via HMGB1.","method":"GST pull-down, co-immunoprecipitation, SHARPIN knockdown with HMGB1 overexpression rescue, macrophage polarization assays","journal":"Metabolic brain disease","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, direct binding shown but mechanism of HMGB1 regulation by SHARPIN not fully resolved","pmids":["38805141"],"is_preprint":false},{"year":2021,"finding":"SHARPIN promotes ubiquitination and proteasomal degradation of von Hippel-Lindau protein (pVHL) through interaction involving the α and β domains of pVHL and the UBL domain of SHARPIN, leading to sustained HIF-2α activation.","method":"Co-immunoprecipitation (domain mapping), ubiquitination assay, pVHL stability assay, SHARPIN knockdown in ccRCC cell lines and xenograft","journal":"Cancer science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain-mapped direct interaction, ubiquitination assay with defined domains, functional rescue; single lab","pmids":["34339558"],"is_preprint":false},{"year":2022,"finding":"The N-terminal UBL domain of SHARPIN (residues 1-127) does not participate in LUBAC formation but enhances the structural stability of the central UBL domain (residues 128-309) and strengthens binding of the UBL domain to the HOIP UBA domain.","method":"Size exclusion chromatography, circular dichroism, thermal/urea unfolding, biosensor binding assays (N-terminal UBL domain vs central UBL domain constructs)","journal":"Protein expression and purification","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — biophysical characterization with multiple structural/biochemical methods; single lab, no mutagenesis","pmids":["34965468"],"is_preprint":false},{"year":2022,"finding":"SHARPIN S146 phosphorylation (induced by ERK1/2 and reversed by PP2A) is required for SHARPIN-ARP2/3 interaction and lamellipodia formation; S146A mutant SHARPIN fails to rescue 3D cancer cell invasion and in vivo metastasis in zebrafish.","method":"Mass spectrometry phosphoproteomics, in vitro kinase assay, CRISPR/Cas9 knockout with S146A mutant rescue, 3D invasion assay, zebrafish metastasis model","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS-identified phosphorylation validated by in vitro kinase assay, CRISPR rescue with phospho-mutant, in vivo metastasis model; single lab","pmids":["36148554"],"is_preprint":false},{"year":2022,"finding":"Platelet-specific SHARPIN deletion increases αIIbβ3 colocalization with talin and fibrinogen binding capacity, reduces NF-κB activation and linear ubiquitination upon platelet stimulation, and diminishes inflammation in murine colitis and peritonitis models.","method":"Platelet-specific conditional knockout (PF4-Cre, GPIbα-Cre crossed with Sharpin fl/fl), super-resolution microscopy, fibrinogen binding assay, ubiquitination assay, in vivo colitis/peritonitis models","journal":"Blood advances","confidence":"High","confidence_rationale":"Tier 2 / Strong — cell-type-specific conditional knockout, super-resolution imaging, multiple in vitro and in vivo readouts; same lab as earlier platelet paper but orthogonal conditional approach","pmids":["34991155"],"is_preprint":false},{"year":2022,"finding":"SHARPIN inhibits ferroptosis in cholangiocarcinoma via the p53/SLC7A11/GPX4 signaling pathway; SHARPIN silencing inhibits p53 ubiquitination and degradation, leading to p53 accumulation and downregulation of SLC7A11, GPX4, SOD-1, and SOD-2.","method":"siRNA knockdown and lentiviral overexpression, western blot (p53, SLC7A11, GPX4 levels), ubiquitination assay, ROS measurement, mitochondrial staining","journal":"Cancer science","confidence":"Low","confidence_rationale":"Tier 3 / Weak — correlation of SHARPIN with p53 ubiquitination inferred from protein levels without direct mechanistic dissection; single lab","pmids":["35968603"],"is_preprint":false},{"year":2023,"finding":"HOIL-1L and SHARPIN each form homo-dimers through their LTM (Leu-Thr-Met) motifs; crystal structures of the dimeric LTM motifs of both proteins reveal a shared dimerization mode, and the polyglucosan body myopathy-associated HOIL-1L A18P mutation disrupts LTM structural folding and dimer formation.","method":"Crystal structure determination of HOIL-1L and SHARPIN LTM homo-dimers, site-directed mutagenesis (A18P), dimerization assays","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structures with mutagenesis validation of disease-relevant mutation; single lab but rigorous structural method","pmids":["37976837"],"is_preprint":false},{"year":2014,"finding":"SIPL1 (SHARPIN) promotes PTEN polyubiquitination via K63-linked (not K48-linked) polyubiquitin chains using its UBL domain; this ubiquitination promotes SIPL1-PTEN complex formation.","method":"Co-immunoprecipitation with ubiquitin mutants (K48R, K63R, K0), UBL domain deletion mutant, ubiquitination assays","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic ubiquitin linkage mutant analysis with domain mapping; single lab","pmids":["25152374"],"is_preprint":false},{"year":2017,"finding":"A rare missense variant in SHARPIN (G186R/rs572750141) causes aberrant cellular localization of the variant protein and attenuated NF-κB activation; molecular dynamics simulations and Co-IP showed significantly reduced binding between G186R SHARPIN and HOIP.","method":"Functional complementation assay, NF-κB activation assay in variant-expressing cells, co-immunoprecipitation, molecular dynamics simulation, subcellular localization imaging","journal":"Molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple functional assays on a specific variant, Co-IP for binding, imaging for localization; single lab","pmids":["31216982"],"is_preprint":false},{"year":2024,"finding":"Human biallelic SHARPIN loss causes attenuated canonical NF-κB responses and propensity for TNF superfamily member-induced cell death in fibroblasts and B cells; anti-TNF therapy led to complete clinical and transcriptomic resolution of autoinflammation in an affected individual.","method":"Patient fibroblasts and B cells: NF-κB signaling assays, cell death assays; anti-TNF clinical intervention with transcriptomic resolution","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — human loss-of-function with defined cellular mechanistic readouts (NF-κB attenuation, cell death) and therapeutic intervention confirming TNF dependence; rare human disease genetics provides high orthogonal support","pmids":["38609546"],"is_preprint":false},{"year":2024,"finding":"SHARPIN NZF domains of both HOIL-1L and SHARPIN synergistically regulate LUBAC function; SHARPIN NZF binds K63-linked ubiquitin chains (in addition to linear chains) and predominantly regulates cell death protection independently of ubiquitin chain type, while HOIL-1L NZF binding to linear chains is required for full NF-κB activation and cooperates in cell death protection.","method":"NZF ubiquitin-binding assays, LUBAC function assays (NF-κB activation, cell death protection), compound NZF mutant analysis, small-molecule inhibitor screen","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic NZF mutant analysis with defined functional readouts, ubiquitin chain selectivity assays; single lab","pmids":["39528476"],"is_preprint":false}],"current_model":"SHARPIN is a multifunctional adaptor protein that operates both as an essential subunit of the LUBAC E3 ligase complex (with HOIP and HOIL-1L), where it stimulates Met1 linear ubiquitin chain formation on NEMO and other substrates to activate canonical NF-κB signaling and suppress RIPK1/caspase-8-dependent apoptosis, and independently of LUBAC as a direct inhibitor of β1- and αIIbβ3-integrin activation (by binding integrin α-subunit cytoplasmic tails and blocking talin/kindlin recruitment), a suppressor of caspase-1 processing (by disrupting p20/p10 dimer formation), a regulator of TCR proximal signaling (via K63-ubiquitin-dependent inhibition of ZAP70 recruitment), an enhancer of PRMT5 methyltransferase activity (affecting melanoma gene expression via SKI/SOX10/MITF), and a promoter of ARP2/3-dependent lamellipodium formation (via ERK-regulated S146 phosphorylation); its loss in mice and humans leads to TNF-driven, RIPK1-kinase-dependent and CYLD-deubiquitinase-dependent cell death, autoinflammation, and immunodeficiency."},"narrative":{"mechanistic_narrative":"SHARPIN is a multifunctional adaptor that operates as an essential accessory subunit of the linear ubiquitin chain assembly complex (LUBAC) and, separately, as a LUBAC-independent regulator of integrin activation and cytoskeletal dynamics [PMID:21455181, PMID:21455180, PMID:21947080]. Within LUBAC, SHARPIN binds HOIP (RNF31) and HOIL-1L (RBCK1) and stimulates formation of Met1-linked linear ubiquitin chains on NEMO to activate canonical NF-κB signaling [PMID:21455181, PMID:21455180]; this requires its central UBL domain interaction with the HOIP UBA domain (strengthened by an accessory N-terminal UBL domain), its NZF domain (which binds both linear and K63-linked chains to recruit LUBAC to the activated TNFR complex), and phosphorylation at S165 [PMID:34965468, PMID:26976635, PMID:39528476, PMID:33392484]. Beyond promoting inflammation-resolving signaling, SHARPIN restrains TNF-driven cell death: its loss sensitizes cells to FADD/caspase-8-dependent apoptosis and, in mice, drives RIPK1-kinase-dependent, TNFR1/TRADD/FADD-mediated keratinocyte death and CYLD-deubiquitinase-dependent RIPK1 recruitment to death-signaling complex II [PMID:21455181, PMID:24821972, PMID:25443632, PMID:25443631, PMID:34887354]. Independently of LUBAC, SHARPIN binds integrin α-subunit cytoplasmic tails (β1, αIIbβ3, αLβ2/LFA-1) through the same UBL surface used for RNF31, holding integrins inactive by blocking talin/kindlin recruitment and thereby controlling cell adhesion, lymphocyte uropod detachment, and platelet activation [PMID:21947080, PMID:26600301, PMID:31429758, PMID:24210817, PMID:30804189, PMID:34991155]. SHARPIN also promotes ARP2/3-dependent lamellipodium formation via ERK-controlled S146 phosphorylation [PMID:28775156, PMID:36148554], and was first identified as a SHANK-interacting postsynaptic density protein [PMID:11178875]. Biallelic SHARPIN loss in humans causes attenuated NF-κB responses, TNF-superfamily-induced cell death, and autoinflammation that resolves on anti-TNF therapy [PMID:38609546].","teleology":[{"year":2001,"claim":"Established the first molecular identity and localization of SHARPIN by showing it is a postsynaptic density protein that binds SHANK and self-associates, defining its modular architecture before any signaling role was known.","evidence":"Yeast two-hybrid, reciprocal Co-IP from brain and heterologous cells, colocalization, and domain mapping","pmids":["11178875"],"confidence":"High","gaps":["Functional consequence of SHANK binding at synapses not defined","No link to later ubiquitin/integrin roles established here"]},{"year":2011,"claim":"Defined SHARPIN's central mechanism by showing it is a LUBAC subunit that stimulates HOIP-dependent linear ubiquitination of NEMO to activate NF-κB, explaining the autoinflammatory phenotype of SHARPIN loss.","evidence":"Co-IP, in vitro linear ubiquitination reconstitution, NF-κB reporter, SHARPIN-deficient primary cells; two simultaneous Nature papers","pmids":["21455181","21455180"],"confidence":"High","gaps":["Did not resolve which SHARPIN domains mediate HOIP binding versus chain stimulation","Substrates beyond NEMO not enumerated"]},{"year":2011,"claim":"Separated SHARPIN's anti-apoptotic function from its NF-κB role by showing its loss sensitizes cells to TNF-induced FADD/caspase-8-dependent death independently of transcriptional activation.","evidence":"TNF-α stimulation of SHARPIN-deficient cells with FADD/caspase-8 genetic epistasis","pmids":["21455181"],"confidence":"High","gaps":["Upstream complex composition driving death not fully mapped here","Relationship to necroptosis not yet addressed"]},{"year":2011,"claim":"Revealed a LUBAC-independent function: SHARPIN is an endogenous integrin inhibitor that binds α-subunit tails and blocks the talin/kindlin-driven activation switch, linking it to cell adhesion control.","evidence":"RNAi screen, direct binding assay, SHARPIN-deficient primary cells with rescue, integrin activity assays","pmids":["21947080"],"confidence":"High","gaps":["Molecular details of how binding excludes talin/kindlin not yet resolved","Domain responsible for integrin binding not mapped here"]},{"year":2012,"claim":"Provided structural insight by showing the N-terminal region adopts a PH superfold serving as a dimerization module rather than a ligand-binding domain.","evidence":"X-ray crystallography (2.0–2.6 Å) of the N-terminal SHARPIN domain","pmids":["22549881"],"confidence":"High","gaps":["Functional role of dimerization in LUBAC or integrin contexts not tested","No structure of central UBL or NZF domains in this study"]},{"year":2013,"claim":"Extended integrin regulation to motility by showing SHARPIN localizes to the migrating-lymphocyte uropod and keeps LFA-1 inactive to permit trailing-edge detachment.","evidence":"Live imaging, migration assays on ICAM-1, direct binding, rescue, integrin activation measurement","pmids":["24210817"],"confidence":"High","gaps":["How SHARPIN is targeted to the uropod not defined","Interplay with LUBAC at the rear edge not addressed"]},{"year":2014,"claim":"Placed RIPK1 kinase activity downstream of SHARPIN loss in vivo, showing kinase-dead RIPK1 fully rescues cpdm pathology and implicating necroptotic/death signaling in the disease.","evidence":"Genetic epistasis with Ripk1(K45A) knock-in crossed to cpdm mice; necroptosis assays","pmids":["24821972"],"confidence":"High","gaps":["Biochemical basis of RIPK1 hyperactivation upon SHARPIN loss not yet defined here","Cell-type drivers not pinpointed"]},{"year":2014,"claim":"Dissected the cell-death axis driving cpdm skin inflammation, showing TNFR1- and TRADD/FADD-mediated keratinocyte apoptosis (with RIPK3) is the trigger.","evidence":"Compound knockout genetic epistasis crossed to Sharpin-deficient mice; two independent studies","pmids":["25443632","25443631"],"confidence":"High","gaps":["Contribution of non-keratinocyte compartments not resolved","Apoptosis vs necroptosis balance across tissues left open"]},{"year":2014,"claim":"Identified a ubiquitin-ligase-like activity by showing SIPL1/SHARPIN promotes K63-linked PTEN polyubiquitination via its UBL domain, expanding its substrate repertoire.","evidence":"Co-IP with ubiquitin linkage mutants and UBL deletion constructs, ubiquitination assays","pmids":["25152374"],"confidence":"Medium","gaps":["No catalytic mechanism defined for SHARPIN itself","Single lab, no in vitro reconstitution"]},{"year":2015,"claim":"Connected SHARPIN to adaptive immunity by showing K63-ubiquitinated SHARPIN inhibits TCRζ–ZAP70 association, regulating Treg generation and homeostasis.","evidence":"Co-IP, ubiquitination assays, SHARPIN-deficient T cell signaling, Treg transfer","pmids":["26829767"],"confidence":"Medium","gaps":["Ligase generating SHARPIN K63 chains not identified","Single lab"]},{"year":2016,"claim":"Mapped competing binding surfaces, showing the UBL domain engages integrin α-tails and RNF31 mutually exclusively, providing a molecular switch between SHARPIN's LUBAC and integrin functions.","evidence":"Site-directed mutagenesis (V267/L276 shared; F263/I272 RNF31-specific), competition and functional assays","pmids":["26600301"],"confidence":"Medium","gaps":["Cellular signal controlling partner choice unknown","Single lab"]},{"year":2016,"claim":"Defined the SHARPIN NZF domain as the key element for cell-death protection through K63/linear chain binding that recruits LUBAC to the activated TNFR complex, distinguishing it from HOIL-1L NZF.","evidence":"HOIL-1L/SHARPIN double-knockout intercross, NZF mutants, TNFR recruitment and death assays","pmids":["26976635"],"confidence":"Medium","gaps":["Structural basis of dual chain recognition not resolved here","Single lab"]},{"year":2016,"claim":"Identified caspase-1 as a LUBAC-independent target, with SHARPIN disrupting p20/p10 dimerization to limit IL-1β/IL-18 maturation.","evidence":"Co-IP, caspase-1 processing assay, sepsis model, caspase-1 inhibitor rescue","pmids":["26968342"],"confidence":"Medium","gaps":["Reconciliation with reports of impaired inflammasome priming in SHARPIN-deficient macrophages not addressed","Single lab"]},{"year":2016,"claim":"Placed CYLD as a required effector of SHARPIN-loss pathology, showing reduced inhibitory CYLD-S418 phosphorylation enhances RIPK1 deubiquitination and complex-II recruitment.","evidence":"Sharpin/Cyld double-knockout epistasis, TNFR complex IP, CYLD phosphorylation assays, conditional Cyld deletion","pmids":["34887354"],"confidence":"High","gaps":["Kinase controlling CYLD-S418 phosphorylation not identified","How SHARPIN loss alters this phosphorylation mechanistically left open"]},{"year":2017,"claim":"Identified a LUBAC-independent enzymatic-enhancer role: SHARPIN binds PRMT5 and stimulates its methyltransferase activity to control SOX10/MITF via SKI, linking SHARPIN to melanoma gene expression.","evidence":"Co-IP, PRMT5 activity assay, SKI dimethylation assay, knockdown/overexpression in melanoma cells","pmids":["29227283"],"confidence":"Medium","gaps":["Structural basis of PRMT5 activation unknown","Single lab"]},{"year":2017,"claim":"Defined a cytoskeletal effector role by showing SHARPIN binds the ARP2/3 complex to drive lamellipodium formation independently of LUBAC.","evidence":"Interactome MS, Co-IP, ARP2/3-binding-deficient mutant, lamellipodium assay","pmids":["28775156"],"confidence":"Medium","gaps":["Regulation of the SHARPIN–ARP2/3 interaction not yet defined here","Single lab"]},{"year":2017,"claim":"Established a tissue-context function in stroma, showing stromal SHARPIN controls collagen assembly, contraction and degradation required for mammary ductal outgrowth.","evidence":"Stromal-specific (S100a4-Cre) knockout, mammary transplantation, collagen and ECM stiffness assays","pmids":["27974362"],"confidence":"Medium","gaps":["Molecular mediator of collagen-handling defect not identified","Single lab"]},{"year":2019,"claim":"Resolved the integrin-inhibition mechanism, showing SHARPIN forms a complex with kindlin-1 on the β1 tail that blocks talin head binding.","evidence":"GST pulldown, Co-IP, talin competition, cell-based β1 activation assay","pmids":["31429758"],"confidence":"Medium","gaps":["Apparent tension with earlier model of SHARPIN blocking kindlin recruitment not reconciled","Single lab"]},{"year":2019,"claim":"Extended integrin regulation and LUBAC signaling to platelets, showing SHARPIN binds the αIIb tail and contributes to agonist-induced Met1 ubiquitination of NEMO, restraining fibrinogen binding.","evidence":"Co-IP/pulldown, super-resolution microscopy, knockdown in iPS-derived megakaryocytes/platelets, ubiquitination assays","pmids":["30804189"],"confidence":"Medium","gaps":["In vivo platelet phenotype not yet established in this study","Single lab"]},{"year":2020,"claim":"Identified S165 phosphorylation as a positive regulatory mark required for efficient linear ubiquitination of NEMO and NF-κB activation.","evidence":"MS phosphosite identification, S165A mutant analysis, NEMO ubiquitination and NF-κB assays","pmids":["33392484"],"confidence":"Medium","gaps":["Kinase responsible for S165 phosphorylation not identified","Single lab"]},{"year":2022,"claim":"Confirmed the platelet role in vivo, showing platelet-specific SHARPIN deletion increases αIIbβ3/talin colocalization and fibrinogen binding while reducing NF-κB activation and inflammation.","evidence":"Platelet-specific conditional knockout, super-resolution imaging, fibrinogen and ubiquitination assays, colitis/peritonitis models","pmids":["34991155"],"confidence":"High","gaps":["Relative contributions of integrin vs LUBAC functions to the inflammation phenotype not separated","Same lab lineage as prior platelet work"]},{"year":2022,"claim":"Defined the regulatory logic of SHARPIN's cytoskeletal function, showing ERK1/2-driven (PP2A-reversed) S146 phosphorylation gates ARP2/3 binding, lamellipodia, and metastasis.","evidence":"Phosphoproteomics, in vitro kinase assay, CRISPR knockout with S146A rescue, 3D invasion and zebrafish metastasis","pmids":["36148554"],"confidence":"Medium","gaps":["Structural mechanism of phospho-dependent ARP2/3 engagement unknown","Single lab"]},{"year":2022,"claim":"Refined the LUBAC-architecture model, showing the N-terminal UBL domain does not join LUBAC but stabilizes the central UBL domain and strengthens its binding to the HOIP UBA domain.","evidence":"Size exclusion chromatography, circular dichroism, unfolding assays, biosensor binding","pmids":["34965468"],"confidence":"Medium","gaps":["No mutagenesis to test the stabilization model in cells","Single lab"]},{"year":2023,"claim":"Provided structural detail of SHARPIN self-association, showing LTM-motif homodimerization shared with HOIL-1L, with a disease-associated HOIL-1L mutation disrupting the fold.","evidence":"Crystal structures of SHARPIN and HOIL-1L LTM homodimers with A18P mutagenesis","pmids":["37976837"],"confidence":"High","gaps":["Functional consequence of SHARPIN LTM dimerization in LUBAC not tested","Single lab"]},{"year":2024,"claim":"Established SHARPIN as a human disease gene, showing biallelic loss attenuates NF-κB, sensitizes cells to TNF-superfamily death, and causes autoinflammation reversible by anti-TNF therapy.","evidence":"Patient fibroblasts/B cells NF-κB and death assays; anti-TNF clinical intervention with transcriptomic resolution","pmids":["38609546"],"confidence":"High","gaps":["Full spectrum of human phenotypes not yet defined","Genotype–phenotype relationships across variants not established"]},{"year":2024,"claim":"Refined the division of labor between LUBAC NZF domains, showing SHARPIN NZF protects against cell death largely independent of chain type while HOIL-1L NZF linear-chain binding drives full NF-κB activation.","evidence":"NZF ubiquitin-binding assays, compound NZF mutants, NF-κB and death readouts, inhibitor screen","pmids":["39528476"],"confidence":"Medium","gaps":["Structural basis of SHARPIN NZF chain-type promiscuity not resolved","Single lab"]},{"year":null,"claim":"How SHARPIN's many LUBAC-independent activities (integrin inhibition, caspase-1 suppression, PRMT5 activation, ARP2/3-driven motility, and multiple substrate ubiquitination/stabilization events) are coordinated and spatially partitioned within a single cell remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No unifying framework linking competing UBL-domain engagements in vivo","Signals switching SHARPIN between LUBAC and non-LUBAC pools unknown","Most cancer-context substrate findings rest on single labs without reconstitution"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,3,21]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[3,14,18,41]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1,38]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[3,8,25]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[20,21]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[20]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[1,2,11,12,40]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[2,9,10,16]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[1,38]},{"term_id":"R-HSA-1474244","term_label":"Extracellular matrix organization","supporting_discovery_ids":[3,17,24]},{"term_id":"R-HSA-109582","term_label":"Hemostasis","supporting_discovery_ids":[25,35]}],"complexes":["LUBAC"],"partners":["RNF31","RBCK1","IKBKG","SHANK","PRMT5","ITGB1","ITGA2B","ACTR2/3"],"other_free_text":[]}},"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":236,"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":161,"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":112,"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. : 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the C-terminal half of SHARPIN mediates SHANK binding while the N-terminal half mediates homomultimerization.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation from brain and heterologous cells, immunostaining/colocalization\",\n      \"journal\": \"Molecular and cellular neurosciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP from brain tissue plus heterologous cells, domain mapping, and colocalization; independently consistent across multiple methods in one study\",\n      \"pmids\": [\"11178875\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"SHARPIN is a component of the linear ubiquitin chain assembly complex (LUBAC) together with HOIP (RNF31) and HOIL-1L (RBCK1); binding to HOIP stimulates formation of linear (Met1-linked) ubiquitin chains in vitro and in vivo, leading to linear ubiquitination of NEMO and activation of NF-κB signaling.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ubiquitination assay, NF-κB reporter assay, SHARPIN-deficient mouse embryonic fibroblasts/B cells/macrophages\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro reconstitution of linear ubiquitin chain formation, reciprocal Co-IP, loss-of-function genetics; independently replicated in two simultaneous Nature papers (PMID 21455181 and 21455180)\",\n      \"pmids\": [\"21455181\", \"21455180\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"SHARPIN deficiency sensitizes cells to TNF-α-induced apoptosis via FADD- and caspase-8-dependent pathways, demonstrating that SHARPIN inhibits apoptosis independently of NF-κB activation.\",\n      \"method\": \"TNF-α stimulation of SHARPIN-deficient MEFs and primary cells; caspase-8/FADD genetic epistasis; cell death assays\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — loss-of-function genetics with defined pathway placement (FADD/caspase-8 dependence), replicated across multiple cell types and confirmed in vivo\",\n      \"pmids\": [\"21455181\"],\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, preventing the inactive-to-active conformational switch.\",\n      \"method\": \"RNAi screen, direct binding assay, fibroblasts/leukocytes/keratinocytes from SHARPIN-deficient mice, rescue by SHARPIN re-expression, integrin activity assays\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct binding demonstrated, loss-of-function with rescue across multiple primary cell types, mechanistic pathway placement (talin/kindlin competition)\",\n      \"pmids\": [\"21947080\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"SHARPIN negatively associates with TRAF2-mediated NF-κB activation; mass spectrometry identified TRAF2 as a SHARPIN co-immunoprecipitated partner, and overexpression studies show SHARPIN can negatively modulate NF-κB via TRAF2 interaction.\",\n      \"method\": \"Co-immunoprecipitation, mass spectrometry, NF-κB luciferase reporter assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP/MS identification plus reporter assay, single lab, no mechanistic follow-up mutagenesis\",\n      \"pmids\": [\"21829440\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"SHARPIN interacts with NEMO (IKBKG) in macrophages; SHARPIN deficiency impairs NEMO-dependent signaling (p105 and ERK phosphorylation, p65 nuclear localization) downstream of TLR2, and this interaction is abrogated by the panr2 (L153P) NEMO mutation.\",\n      \"method\": \"Systems biology, co-immunoprecipitation, transcriptome analysis of SHARPIN-deficient macrophages\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP confirmed interaction, SHARPIN-deficient cell phenotyping with pathway-level readouts, single lab\",\n      \"pmids\": [\"21709223\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"SHARPIN deficiency in keratinocytes leads to apoptosis via the intrinsic mitochondrial pathway (caspase-9 and caspase-3 activation, Bax/Bcl2 shift, mitochondrial membrane potential loss) rather than the extrinsic caspase-8 pathway.\",\n      \"method\": \"Annexin-V/PI FACS, transmission electron microscopy, caspase activity assays, western blotting in SHARPIN-deficient mouse skin\",\n      \"journal\": \"Journal of dermatological science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple complementary in vitro and in vivo assays, single lab, no rescue experiment\",\n      \"pmids\": [\"21620685\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The N-terminal portion of SHARPIN adopts a pleckstrin homology (PH) superfold that functions as a dimerization module rather than a ligand-binding domain; crystal structure determined to 2.0–2.6 Å resolution.\",\n      \"method\": \"X-ray crystallography (crystal structure of N-terminal SHARPIN domain)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with functional validation of dimerization; single lab but rigorous structural method\",\n      \"pmids\": [\"22549881\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"SHARPIN localizes to the trailing edge (uropod) of migrating lymphocytes and directly interacts with LFA-1 (αLβ2 integrin), maintaining LFA-1 in an inactive state to enable uropod detachment and control lymphocyte migration velocity.\",\n      \"method\": \"Live imaging, SHARPIN-deficient lymphocyte migration assays on ICAM-1, direct binding assay, rescue by SHARPIN re-expression, integrin activation measurement\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct binding plus localization by imaging plus loss-of-function with rescue, multiple orthogonal methods\",\n      \"pmids\": [\"24210817\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"RIP1 (RIPK1) kinase activity is required for the inflammatory pathology in SHARPIN-deficient (cpdm) mice; kinase-dead Ripk1(K45A) completely protected against all cpdm-related pathology, placing RIPK1 kinase downstream of SHARPIN loss in TNF-driven inflammation.\",\n      \"method\": \"Genetic epistasis: Ripk1(K45A) knock-in mice crossed with cpdm mice; in vivo and in vitro necroptosis assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic epistasis with kinase-dead allele, complete phenotypic rescue, in vivo and in vitro validation\",\n      \"pmids\": [\"24821972\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Skin inflammation in SHARPIN-deficient mice is driven primarily by TNFR1 (but not TNFR2), and is dependent on TRADD- and FADD-mediated keratinocyte apoptosis; epidermis-restricted ablation of FADD combined with RIPK3 deficiency fully prevented skin inflammation.\",\n      \"method\": \"Genetic epistasis: compound knockout mice (Tnfr1-/-, Ripk3-/-, Casp8+/-, Fadd-/- keratinocyte-specific) crossed with Sharpin-deficient mice\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple complementary genetic epistasis experiments across two independent studies (PMIDs 25443632 and 25443631), consistent conclusions\",\n      \"pmids\": [\"25443632\", \"25443631\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"SHARPIN regulates TCR signaling in T cells; SHARPIN is conjugated with K63-linked ubiquitin chains, which inhibits the association of TCRζ with ZAP70, thereby affecting regulatory T cell generation and maintaining immunological homeostasis.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, SHARPIN-deficient T cell signaling analysis, Treg transfer experiments\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with ubiquitination characterization and defined functional consequence (ZAP70 association), single lab\",\n      \"pmids\": [\"26829767\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"SHARPIN is required for optimal NLRP3 inflammasome activation; SHARPIN-deficient macrophages show defects in canonical and non-canonical NLRP3 inflammasome activation, associated with impaired NF-κB and MAPK pathway priming.\",\n      \"method\": \"Loss-of-function analysis using Sharpin(cpdm) macrophages, NLRP3 inflammasome activation assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — defined cellular phenotype in loss-of-function system with pathway readouts, single lab\",\n      \"pmids\": [\"25637014\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"SHARPIN binds integrin α-subunit cytoplasmic tails and LUBAC component RNF31 in a mutually exclusive manner via its ubiquitin-like (UBL) domain; residues V267 and L276 are required for both interactions, while F263 and I272 are specifically required for RNF31 but not integrin binding.\",\n      \"method\": \"Binding assays with SHARPIN mutants, competition assays with integrin α-tail and RNF31, NF-κB reporter assay, integrin activity assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — site-directed mutagenesis mapping two distinct binding surfaces, competition in vitro, functional validation in cells; single lab\",\n      \"pmids\": [\"26600301\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SHARPIN directly binds caspase-1 in a LUBAC-independent manner and disrupts p20/p10 dimer formation (the final step of caspase-1 processing), thereby inhibiting caspase-1 enzyme activation and maturation of IL-1β/IL-18.\",\n      \"method\": \"Co-immunoprecipitation of SHARPIN with caspase-1, caspase-1 processing assay, Sharpin-deficient mouse sepsis model, caspase-1 inhibitor rescue\",\n      \"journal\": \"The American journal of pathology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding plus mechanistic step (p20/p10 dimerization) plus in vivo genetic validation; single lab\",\n      \"pmids\": [\"26968342\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The NZF domain of SHARPIN (but not that of HOIL-1L) is critical for protection from programmed cell death by enhancing LUBAC recruitment to the activated TNFR complex; SHARPIN NZF binds K63-linked ubiquitin chains (in addition to linear chains), and this K63-binding activity is involved in LUBAC recruitment.\",\n      \"method\": \"Intercross of HOIL-1L and SHARPIN knockout mice, NZF domain mutants, TNFR complex recruitment assays, cell death assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain mutagenesis with functional rescue, defined mechanistic step (LUBAC-to-TNFR complex recruitment), single lab\",\n      \"pmids\": [\"26976635\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Immune dysregulation in SHARPIN-deficient mice is dependent on CYLD deubiquitinase; loss of SHARPIN impairs phosphorylation of CYLD at serine 418 (which normally inhibits CYLD activity), leading to enhanced CYLD-mediated removal of ubiquitin from RIPK1 and increased RIPK1 recruitment to death-signaling Complex II upon TNF stimulation.\",\n      \"method\": \"Genetic epistasis (Sharpin/Cyld double knockout), TNFR complex immunoprecipitation, CYLD phosphorylation assays, conditional Cyld deletion\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with full phenotypic rescue, biochemical pathway dissection (CYLD phosphorylation and RIPK1 recruitment), multiple orthogonal approaches in one study\",\n      \"pmids\": [\"34887354\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SHARPIN regulates mammary gland stromal function; mice with stromal-specific (S100a4-Cre) deletion of Sharpin have reduced ductal outgrowth and defects in collagen fibre assembly, contraction and degradation, while transplanted SHARPIN-null mammary epithelial cells in wild-type stroma develop normally.\",\n      \"method\": \"Conditional knockout (S100a4-Cre), mammary gland transplantation, collagen contraction/degradation assays, ECM stiffness measurement\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional knockout with cell-type specificity established by transplantation, multiple in vitro collagen assays; single lab\",\n      \"pmids\": [\"27974362\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"SHARPIN interacts with PRMT5 (protein arginine methyltransferase 5) in a LUBAC-independent manner and increases PRMT5 methyltransferase activity; activated PRMT5 controls SOX10 and MITF expression via arginine dimethylation and inhibition of the transcriptional corepressor SKI.\",\n      \"method\": \"Co-immunoprecipitation, PRMT5 methyltransferase activity assay, SKI arginine dimethylation assay, SOX10/MITF expression analysis, SHARPIN knockdown/overexpression in melanoma cells\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct protein interaction confirmed by Co-IP, in vitro enzymatic activity assay, defined downstream targets; single lab\",\n      \"pmids\": [\"29227283\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"SHARPIN promotes p53 polyubiquitination and degradation in an MDM2-dependent manner; SHARPIN associates with MDM2, prolongs MDM2 protein stability, and facilitates p53 poly-ubiquitination.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, p53 stability assay, SHARPIN depletion with rescue by p53 knockdown in breast cancer cell lines\",\n      \"journal\": \"Neoplasia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP showing SHARPIN-MDM2 complex, ubiquitination assay, functional epistasis with p53 knockdown; single lab\",\n      \"pmids\": [\"28063307\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"SHARPIN stabilizes estrogen receptor α (ERα) protein by inhibiting ERα poly-ubiquitination and facilitating ERα mono-ubiquitination at K302/303 sites; SHARPIN interacts with ERα in both cytosol and nucleus.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, ERE luciferase reporter, SHARPIN knockdown/overexpression, ERα stability assays\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct Co-IP, specific ubiquitination site mapping, functional ERE reporter; single lab\",\n      \"pmids\": [\"29100376\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"SHARPIN interacts with ARP2/3 complex and promotes lamellipodium formation in a LUBAC-independent manner; the ARP2/3-binding site on SHARPIN was identified and an ARP2/3-binding-deficient SHARPIN mutant failed to support lamellipodia.\",\n      \"method\": \"Mass spectrometry interactome (Sharpin interactome), co-immunoprecipitation, site-directed mutagenesis, lamellipodium formation assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — interactome MS plus Co-IP, domain mapping with loss-of-function mutant and functional readout; single lab\",\n      \"pmids\": [\"28775156\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"SHARPIN promotes Versican expression synergistically with Wnt/β-catenin pathway activation to drive hepatocellular carcinoma invasion.\",\n      \"method\": \"Stable SHARPIN-overexpressing cell lines, microarray analysis, Wnt/β-catenin reporter assay, invasion assays, Versican knockdown, in vivo tumor xenograft\",\n      \"journal\": \"Oncogenesis\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — correlative microarray with functional validation but mechanism of synergy with Wnt not molecularly defined; single lab\",\n      \"pmids\": [\"27941932\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"A novel SHARPIN-PRMT5-H3R2me1 axis regulates lung cancer cell invasion; SHARPIN-PRMT5 complex catalyzes monomethylation of histone H3 arginine 2 (H3R2me1) at metastasis-related gene loci, which is linked to H3K4me3 via MLL complex components ASH2 and WDR5.\",\n      \"method\": \"Co-immunoprecipitation, histone methylation assays, chromatin immunoprecipitation, SHARPIN knockdown in lung cancer cells\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Co-IP and ChIP data but limited mechanistic dissection; single lab, no in vitro reconstitution\",\n      \"pmids\": [\"28903384\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"SHARPIN suppresses β1-integrin activation by forming a complex with kindlin-1 and the integrin β1 cytoplasmic tail; SHARPIN directly binds the β1 CT and the sharpin-kindlin-1 complex inhibits talin head domain binding to the β1 CT.\",\n      \"method\": \"Biochemical binding assays (GST pulldown, co-immunoprecipitation), cell-based β1-integrin activation assay, talin competition assay\",\n      \"journal\": \"Cell communication and signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding demonstrated biochemically with defined molecular competition, functional validation in cells; single lab\",\n      \"pmids\": [\"31429758\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"SHARPIN interacts with αIIb cytoplasmic tail of integrin αIIbβ3 in human platelets and participates in LUBAC-mediated Met1 linear ubiquitination of NEMO upon platelet stimulation; SHARPIN knockdown in megakaryocytes/platelets increases basal and agonist-induced fibrinogen binding and reduces Met1 ubiquitination and NF-κB activation.\",\n      \"method\": \"Co-immunoprecipitation and pull-down assays, super-resolution microscopy, SHARPIN knockdown in iPS-derived megakaryocytes/platelets, ubiquitination assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, defined functional consequences in platelet-lineage cells with knockdown, multiple orthogonal methods; single lab\",\n      \"pmids\": [\"30804189\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"SHARPIN promotes melanoma migration and invasion by upregulating Rap1 GTPase expression and activating downstream p38 and JNK/c-Jun pathways; Rap1 activator/inhibitor treatments partially rescued invasion phenotypes.\",\n      \"method\": \"SHARPIN knockdown/overexpression in melanoma cells, invasion/migration assays, Rap1 pharmacological modulation, in vivo metastasis models\",\n      \"journal\": \"The Journal of investigative dermatology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — pathway placement by pharmacological rescue, no direct binding between SHARPIN and Rap1 demonstrated; single lab\",\n      \"pmids\": [\"31401046\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"SHARPIN inhibits YAP protein in esophageal squamous cell carcinoma; SHARPIN associates with YAP and promotes YAP K48-dependent poly-ubiquitination and proteasomal degradation, thereby suppressing Hippo pathway target genes (CTGF, CYR61).\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, YAP protein stability assay, SHARPIN/YAP knockdown rescue experiments, TEAD reporter\",\n      \"journal\": \"Neoplasia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct interaction by Co-IP with defined ubiquitination type, functional epistasis by double knockdown rescue; single lab\",\n      \"pmids\": [\"31884247\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"SHARPIN interacts with Eya1 (Eyes absent 1) and enhances Eya1 function as a coactivator for Six transcription factors; this interaction is required for normal zebrafish ear and branchial arch development.\",\n      \"method\": \"GST pulldown, co-immunoprecipitation, Six/Eya coactivation assay, zebrafish morpholino knockdown\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding confirmed by GST pulldown and Co-IP, functional transcriptional assay, in vivo morpholino knockdown phenotype; single lab\",\n      \"pmids\": [\"20956555\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SHARPIN phosphorylation at serine 165 is required for optimal NF-κB activation; a phosphorylation-resistant S165A mutant shows impaired linear ubiquitination of NEMO and reduced NF-κB activation in response to TNFα.\",\n      \"method\": \"Mass spectrometry identification of phosphorylation site, phosphorylation-resistant mutant analysis, NEMO ubiquitination assay, NF-κB reporter assay\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS-identified phosphorylation validated by mutant analysis with defined functional readout (NEMO ubiquitination, NF-κB); single lab\",\n      \"pmids\": [\"33392484\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SHARPIN stabilizes β-catenin protein by competing with the E3 ubiquitin ligase β-TrCP1 for β-catenin binding, thereby reducing β-catenin K48-linked ubiquitination and proteasomal degradation in a linear ubiquitination-independent manner.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, competition binding assay (SHARPIN vs β-TrCP1), SHARPIN knockdown/overexpression with β-catenin protein stability readout, in vivo xenograft\",\n      \"journal\": \"Gastric cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding by Co-IP, competition assay, ubiquitination assay showing mechanism; single lab\",\n      \"pmids\": [\"33159601\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SHARPIN directly interacts with HMGB1 and enhances HMGB1 expression; this SHARPIN-HMGB1 interaction was demonstrated by GST pull-down and Co-IP, and SHARPIN promotes M1-like macrophage polarization via HMGB1.\",\n      \"method\": \"GST pull-down, co-immunoprecipitation, SHARPIN knockdown with HMGB1 overexpression rescue, macrophage polarization assays\",\n      \"journal\": \"Metabolic brain disease\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, direct binding shown but mechanism of HMGB1 regulation by SHARPIN not fully resolved\",\n      \"pmids\": [\"38805141\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SHARPIN promotes ubiquitination and proteasomal degradation of von Hippel-Lindau protein (pVHL) through interaction involving the α and β domains of pVHL and the UBL domain of SHARPIN, leading to sustained HIF-2α activation.\",\n      \"method\": \"Co-immunoprecipitation (domain mapping), ubiquitination assay, pVHL stability assay, SHARPIN knockdown in ccRCC cell lines and xenograft\",\n      \"journal\": \"Cancer science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain-mapped direct interaction, ubiquitination assay with defined domains, functional rescue; single lab\",\n      \"pmids\": [\"34339558\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"The N-terminal UBL domain of SHARPIN (residues 1-127) does not participate in LUBAC formation but enhances the structural stability of the central UBL domain (residues 128-309) and strengthens binding of the UBL domain to the HOIP UBA domain.\",\n      \"method\": \"Size exclusion chromatography, circular dichroism, thermal/urea unfolding, biosensor binding assays (N-terminal UBL domain vs central UBL domain constructs)\",\n      \"journal\": \"Protein expression and purification\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — biophysical characterization with multiple structural/biochemical methods; single lab, no mutagenesis\",\n      \"pmids\": [\"34965468\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SHARPIN S146 phosphorylation (induced by ERK1/2 and reversed by PP2A) is required for SHARPIN-ARP2/3 interaction and lamellipodia formation; S146A mutant SHARPIN fails to rescue 3D cancer cell invasion and in vivo metastasis in zebrafish.\",\n      \"method\": \"Mass spectrometry phosphoproteomics, in vitro kinase assay, CRISPR/Cas9 knockout with S146A mutant rescue, 3D invasion assay, zebrafish metastasis model\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS-identified phosphorylation validated by in vitro kinase assay, CRISPR rescue with phospho-mutant, in vivo metastasis model; single lab\",\n      \"pmids\": [\"36148554\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Platelet-specific SHARPIN deletion increases αIIbβ3 colocalization with talin and fibrinogen binding capacity, reduces NF-κB activation and linear ubiquitination upon platelet stimulation, and diminishes inflammation in murine colitis and peritonitis models.\",\n      \"method\": \"Platelet-specific conditional knockout (PF4-Cre, GPIbα-Cre crossed with Sharpin fl/fl), super-resolution microscopy, fibrinogen binding assay, ubiquitination assay, in vivo colitis/peritonitis models\",\n      \"journal\": \"Blood advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — cell-type-specific conditional knockout, super-resolution imaging, multiple in vitro and in vivo readouts; same lab as earlier platelet paper but orthogonal conditional approach\",\n      \"pmids\": [\"34991155\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SHARPIN inhibits ferroptosis in cholangiocarcinoma via the p53/SLC7A11/GPX4 signaling pathway; SHARPIN silencing inhibits p53 ubiquitination and degradation, leading to p53 accumulation and downregulation of SLC7A11, GPX4, SOD-1, and SOD-2.\",\n      \"method\": \"siRNA knockdown and lentiviral overexpression, western blot (p53, SLC7A11, GPX4 levels), ubiquitination assay, ROS measurement, mitochondrial staining\",\n      \"journal\": \"Cancer science\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — correlation of SHARPIN with p53 ubiquitination inferred from protein levels without direct mechanistic dissection; single lab\",\n      \"pmids\": [\"35968603\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"HOIL-1L and SHARPIN each form homo-dimers through their LTM (Leu-Thr-Met) motifs; crystal structures of the dimeric LTM motifs of both proteins reveal a shared dimerization mode, and the polyglucosan body myopathy-associated HOIL-1L A18P mutation disrupts LTM structural folding and dimer formation.\",\n      \"method\": \"Crystal structure determination of HOIL-1L and SHARPIN LTM homo-dimers, site-directed mutagenesis (A18P), dimerization assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structures with mutagenesis validation of disease-relevant mutation; single lab but rigorous structural method\",\n      \"pmids\": [\"37976837\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"SIPL1 (SHARPIN) promotes PTEN polyubiquitination via K63-linked (not K48-linked) polyubiquitin chains using its UBL domain; this ubiquitination promotes SIPL1-PTEN complex formation.\",\n      \"method\": \"Co-immunoprecipitation with ubiquitin mutants (K48R, K63R, K0), UBL domain deletion mutant, ubiquitination assays\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic ubiquitin linkage mutant analysis with domain mapping; single lab\",\n      \"pmids\": [\"25152374\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"A rare missense variant in SHARPIN (G186R/rs572750141) causes aberrant cellular localization of the variant protein and attenuated NF-κB activation; molecular dynamics simulations and Co-IP showed significantly reduced binding between G186R SHARPIN and HOIP.\",\n      \"method\": \"Functional complementation assay, NF-κB activation assay in variant-expressing cells, co-immunoprecipitation, molecular dynamics simulation, subcellular localization imaging\",\n      \"journal\": \"Molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple functional assays on a specific variant, Co-IP for binding, imaging for localization; single lab\",\n      \"pmids\": [\"31216982\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Human biallelic SHARPIN loss causes attenuated canonical NF-κB responses and propensity for TNF superfamily member-induced cell death in fibroblasts and B cells; anti-TNF therapy led to complete clinical and transcriptomic resolution of autoinflammation in an affected individual.\",\n      \"method\": \"Patient fibroblasts and B cells: NF-κB signaling assays, cell death assays; anti-TNF clinical intervention with transcriptomic resolution\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — human loss-of-function with defined cellular mechanistic readouts (NF-κB attenuation, cell death) and therapeutic intervention confirming TNF dependence; rare human disease genetics provides high orthogonal support\",\n      \"pmids\": [\"38609546\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"SHARPIN NZF domains of both HOIL-1L and SHARPIN synergistically regulate LUBAC function; SHARPIN NZF binds K63-linked ubiquitin chains (in addition to linear chains) and predominantly regulates cell death protection independently of ubiquitin chain type, while HOIL-1L NZF binding to linear chains is required for full NF-κB activation and cooperates in cell death protection.\",\n      \"method\": \"NZF ubiquitin-binding assays, LUBAC function assays (NF-κB activation, cell death protection), compound NZF mutant analysis, small-molecule inhibitor screen\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic NZF mutant analysis with defined functional readouts, ubiquitin chain selectivity assays; single lab\",\n      \"pmids\": [\"39528476\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SHARPIN is a multifunctional adaptor protein that operates both as an essential subunit of the LUBAC E3 ligase complex (with HOIP and HOIL-1L), where it stimulates Met1 linear ubiquitin chain formation on NEMO and other substrates to activate canonical NF-κB signaling and suppress RIPK1/caspase-8-dependent apoptosis, and independently of LUBAC as a direct inhibitor of β1- and αIIbβ3-integrin activation (by binding integrin α-subunit cytoplasmic tails and blocking talin/kindlin recruitment), a suppressor of caspase-1 processing (by disrupting p20/p10 dimer formation), a regulator of TCR proximal signaling (via K63-ubiquitin-dependent inhibition of ZAP70 recruitment), an enhancer of PRMT5 methyltransferase activity (affecting melanoma gene expression via SKI/SOX10/MITF), and a promoter of ARP2/3-dependent lamellipodium formation (via ERK-regulated S146 phosphorylation); its loss in mice and humans leads to TNF-driven, RIPK1-kinase-dependent and CYLD-deubiquitinase-dependent cell death, autoinflammation, and immunodeficiency.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SHARPIN is a multifunctional adaptor that operates as an essential accessory subunit of the linear ubiquitin chain assembly complex (LUBAC) and, separately, as a LUBAC-independent regulator of integrin activation and cytoskeletal dynamics [#1, #3]. Within LUBAC, SHARPIN binds HOIP (RNF31) and HOIL-1L (RBCK1) and stimulates formation of Met1-linked linear ubiquitin chains on NEMO to activate canonical NF-\\u03baB signaling [#1]; this requires its central UBL domain interaction with the HOIP UBA domain (strengthened by an accessory N-terminal UBL domain), its NZF domain (which binds both linear and K63-linked chains to recruit LUBAC to the activated TNFR complex), and phosphorylation at S165 [#33, #15, #41, #29]. Beyond promoting inflammation-resolving signaling, SHARPIN restrains TNF-driven cell death: its loss sensitizes cells to FADD/caspase-8-dependent apoptosis and, in mice, drives RIPK1-kinase-dependent, TNFR1/TRADD/FADD-mediated keratinocyte death and CYLD-deubiquitinase-dependent RIPK1 recruitment to death-signaling complex II [#2, #9, #10, #16]. Independently of LUBAC, SHARPIN binds integrin \\u03b1-subunit cytoplasmic tails (\\u03b21, \\u03b1IIb\\u03b23, \\u03b1L\\u03b22/LFA-1) through the same UBL surface used for RNF31, holding integrins inactive by blocking talin/kindlin recruitment and thereby controlling cell adhesion, lymphocyte uropod detachment, and platelet activation [#3, #13, #24, #8, #25, #35]. SHARPIN also promotes ARP2/3-dependent lamellipodium formation via ERK-controlled S146 phosphorylation [#21, #34], and was first identified as a SHANK-interacting postsynaptic density protein [#0]. Biallelic SHARPIN loss in humans causes attenuated NF-\\u03baB responses, TNF-superfamily-induced cell death, and autoinflammation that resolves on anti-TNF therapy [#40].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Established the first molecular identity and localization of SHARPIN by showing it is a postsynaptic density protein that binds SHANK and self-associates, defining its modular architecture before any signaling role was known.\",\n      \"evidence\": \"Yeast two-hybrid, reciprocal Co-IP from brain and heterologous cells, colocalization, and domain mapping\",\n      \"pmids\": [\"11178875\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of SHANK binding at synapses not defined\", \"No link to later ubiquitin/integrin roles established here\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Defined SHARPIN's central mechanism by showing it is a LUBAC subunit that stimulates HOIP-dependent linear ubiquitination of NEMO to activate NF-\\u03baB, explaining the autoinflammatory phenotype of SHARPIN loss.\",\n      \"evidence\": \"Co-IP, in vitro linear ubiquitination reconstitution, NF-\\u03baB reporter, SHARPIN-deficient primary cells; two simultaneous Nature papers\",\n      \"pmids\": [\"21455181\", \"21455180\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve which SHARPIN domains mediate HOIP binding versus chain stimulation\", \"Substrates beyond NEMO not enumerated\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Separated SHARPIN's anti-apoptotic function from its NF-\\u03baB role by showing its loss sensitizes cells to TNF-induced FADD/caspase-8-dependent death independently of transcriptional activation.\",\n      \"evidence\": \"TNF-\\u03b1 stimulation of SHARPIN-deficient cells with FADD/caspase-8 genetic epistasis\",\n      \"pmids\": [\"21455181\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream complex composition driving death not fully mapped here\", \"Relationship to necroptosis not yet addressed\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Revealed a LUBAC-independent function: SHARPIN is an endogenous integrin inhibitor that binds \\u03b1-subunit tails and blocks the talin/kindlin-driven activation switch, linking it to cell adhesion control.\",\n      \"evidence\": \"RNAi screen, direct binding assay, SHARPIN-deficient primary cells with rescue, integrin activity assays\",\n      \"pmids\": [\"21947080\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular details of how binding excludes talin/kindlin not yet resolved\", \"Domain responsible for integrin binding not mapped here\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Provided structural insight by showing the N-terminal region adopts a PH superfold serving as a dimerization module rather than a ligand-binding domain.\",\n      \"evidence\": \"X-ray crystallography (2.0\\u20132.6 \\u00c5) of the N-terminal SHARPIN domain\",\n      \"pmids\": [\"22549881\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional role of dimerization in LUBAC or integrin contexts not tested\", \"No structure of central UBL or NZF domains in this study\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Extended integrin regulation to motility by showing SHARPIN localizes to the migrating-lymphocyte uropod and keeps LFA-1 inactive to permit trailing-edge detachment.\",\n      \"evidence\": \"Live imaging, migration assays on ICAM-1, direct binding, rescue, integrin activation measurement\",\n      \"pmids\": [\"24210817\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How SHARPIN is targeted to the uropod not defined\", \"Interplay with LUBAC at the rear edge not addressed\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Placed RIPK1 kinase activity downstream of SHARPIN loss in vivo, showing kinase-dead RIPK1 fully rescues cpdm pathology and implicating necroptotic/death signaling in the disease.\",\n      \"evidence\": \"Genetic epistasis with Ripk1(K45A) knock-in crossed to cpdm mice; necroptosis assays\",\n      \"pmids\": [\"24821972\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Biochemical basis of RIPK1 hyperactivation upon SHARPIN loss not yet defined here\", \"Cell-type drivers not pinpointed\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Dissected the cell-death axis driving cpdm skin inflammation, showing TNFR1- and TRADD/FADD-mediated keratinocyte apoptosis (with RIPK3) is the trigger.\",\n      \"evidence\": \"Compound knockout genetic epistasis crossed to Sharpin-deficient mice; two independent studies\",\n      \"pmids\": [\"25443632\", \"25443631\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Contribution of non-keratinocyte compartments not resolved\", \"Apoptosis vs necroptosis balance across tissues left open\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identified a ubiquitin-ligase-like activity by showing SIPL1/SHARPIN promotes K63-linked PTEN polyubiquitination via its UBL domain, expanding its substrate repertoire.\",\n      \"evidence\": \"Co-IP with ubiquitin linkage mutants and UBL deletion constructs, ubiquitination assays\",\n      \"pmids\": [\"25152374\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No catalytic mechanism defined for SHARPIN itself\", \"Single lab, no in vitro reconstitution\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Connected SHARPIN to adaptive immunity by showing K63-ubiquitinated SHARPIN inhibits TCR\\u03b6\\u2013ZAP70 association, regulating Treg generation and homeostasis.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, SHARPIN-deficient T cell signaling, Treg transfer\",\n      \"pmids\": [\"26829767\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ligase generating SHARPIN K63 chains not identified\", \"Single lab\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Mapped competing binding surfaces, showing the UBL domain engages integrin \\u03b1-tails and RNF31 mutually exclusively, providing a molecular switch between SHARPIN's LUBAC and integrin functions.\",\n      \"evidence\": \"Site-directed mutagenesis (V267/L276 shared; F263/I272 RNF31-specific), competition and functional assays\",\n      \"pmids\": [\"26600301\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cellular signal controlling partner choice unknown\", \"Single lab\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Defined the SHARPIN NZF domain as the key element for cell-death protection through K63/linear chain binding that recruits LUBAC to the activated TNFR complex, distinguishing it from HOIL-1L NZF.\",\n      \"evidence\": \"HOIL-1L/SHARPIN double-knockout intercross, NZF mutants, TNFR recruitment and death assays\",\n      \"pmids\": [\"26976635\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of dual chain recognition not resolved here\", \"Single lab\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identified caspase-1 as a LUBAC-independent target, with SHARPIN disrupting p20/p10 dimerization to limit IL-1\\u03b2/IL-18 maturation.\",\n      \"evidence\": \"Co-IP, caspase-1 processing assay, sepsis model, caspase-1 inhibitor rescue\",\n      \"pmids\": [\"26968342\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Reconciliation with reports of impaired inflammasome priming in SHARPIN-deficient macrophages not addressed\", \"Single lab\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Placed CYLD as a required effector of SHARPIN-loss pathology, showing reduced inhibitory CYLD-S418 phosphorylation enhances RIPK1 deubiquitination and complex-II recruitment.\",\n      \"evidence\": \"Sharpin/Cyld double-knockout epistasis, TNFR complex IP, CYLD phosphorylation assays, conditional Cyld deletion\",\n      \"pmids\": [\"34887354\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase controlling CYLD-S418 phosphorylation not identified\", \"How SHARPIN loss alters this phosphorylation mechanistically left open\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identified a LUBAC-independent enzymatic-enhancer role: SHARPIN binds PRMT5 and stimulates its methyltransferase activity to control SOX10/MITF via SKI, linking SHARPIN to melanoma gene expression.\",\n      \"evidence\": \"Co-IP, PRMT5 activity assay, SKI dimethylation assay, knockdown/overexpression in melanoma cells\",\n      \"pmids\": [\"29227283\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of PRMT5 activation unknown\", \"Single lab\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Defined a cytoskeletal effector role by showing SHARPIN binds the ARP2/3 complex to drive lamellipodium formation independently of LUBAC.\",\n      \"evidence\": \"Interactome MS, Co-IP, ARP2/3-binding-deficient mutant, lamellipodium assay\",\n      \"pmids\": [\"28775156\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Regulation of the SHARPIN\\u2013ARP2/3 interaction not yet defined here\", \"Single lab\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Established a tissue-context function in stroma, showing stromal SHARPIN controls collagen assembly, contraction and degradation required for mammary ductal outgrowth.\",\n      \"evidence\": \"Stromal-specific (S100a4-Cre) knockout, mammary transplantation, collagen and ECM stiffness assays\",\n      \"pmids\": [\"27974362\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular mediator of collagen-handling defect not identified\", \"Single lab\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Resolved the integrin-inhibition mechanism, showing SHARPIN forms a complex with kindlin-1 on the \\u03b21 tail that blocks talin head binding.\",\n      \"evidence\": \"GST pulldown, Co-IP, talin competition, cell-based \\u03b21 activation assay\",\n      \"pmids\": [\"31429758\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Apparent tension with earlier model of SHARPIN blocking kindlin recruitment not reconciled\", \"Single lab\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Extended integrin regulation and LUBAC signaling to platelets, showing SHARPIN binds the \\u03b1IIb tail and contributes to agonist-induced Met1 ubiquitination of NEMO, restraining fibrinogen binding.\",\n      \"evidence\": \"Co-IP/pulldown, super-resolution microscopy, knockdown in iPS-derived megakaryocytes/platelets, ubiquitination assays\",\n      \"pmids\": [\"30804189\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo platelet phenotype not yet established in this study\", \"Single lab\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identified S165 phosphorylation as a positive regulatory mark required for efficient linear ubiquitination of NEMO and NF-\\u03baB activation.\",\n      \"evidence\": \"MS phosphosite identification, S165A mutant analysis, NEMO ubiquitination and NF-\\u03baB assays\",\n      \"pmids\": [\"33392484\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Kinase responsible for S165 phosphorylation not identified\", \"Single lab\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Confirmed the platelet role in vivo, showing platelet-specific SHARPIN deletion increases \\u03b1IIb\\u03b23/talin colocalization and fibrinogen binding while reducing NF-\\u03baB activation and inflammation.\",\n      \"evidence\": \"Platelet-specific conditional knockout, super-resolution imaging, fibrinogen and ubiquitination assays, colitis/peritonitis models\",\n      \"pmids\": [\"34991155\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contributions of integrin vs LUBAC functions to the inflammation phenotype not separated\", \"Same lab lineage as prior platelet work\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined the regulatory logic of SHARPIN's cytoskeletal function, showing ERK1/2-driven (PP2A-reversed) S146 phosphorylation gates ARP2/3 binding, lamellipodia, and metastasis.\",\n      \"evidence\": \"Phosphoproteomics, in vitro kinase assay, CRISPR knockout with S146A rescue, 3D invasion and zebrafish metastasis\",\n      \"pmids\": [\"36148554\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural mechanism of phospho-dependent ARP2/3 engagement unknown\", \"Single lab\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Refined the LUBAC-architecture model, showing the N-terminal UBL domain does not join LUBAC but stabilizes the central UBL domain and strengthens its binding to the HOIP UBA domain.\",\n      \"evidence\": \"Size exclusion chromatography, circular dichroism, unfolding assays, biosensor binding\",\n      \"pmids\": [\"34965468\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No mutagenesis to test the stabilization model in cells\", \"Single lab\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Provided structural detail of SHARPIN self-association, showing LTM-motif homodimerization shared with HOIL-1L, with a disease-associated HOIL-1L mutation disrupting the fold.\",\n      \"evidence\": \"Crystal structures of SHARPIN and HOIL-1L LTM homodimers with A18P mutagenesis\",\n      \"pmids\": [\"37976837\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of SHARPIN LTM dimerization in LUBAC not tested\", \"Single lab\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Established SHARPIN as a human disease gene, showing biallelic loss attenuates NF-\\u03baB, sensitizes cells to TNF-superfamily death, and causes autoinflammation reversible by anti-TNF therapy.\",\n      \"evidence\": \"Patient fibroblasts/B cells NF-\\u03baB and death assays; anti-TNF clinical intervention with transcriptomic resolution\",\n      \"pmids\": [\"38609546\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full spectrum of human phenotypes not yet defined\", \"Genotype\\u2013phenotype relationships across variants not established\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Refined the division of labor between LUBAC NZF domains, showing SHARPIN NZF protects against cell death largely independent of chain type while HOIL-1L NZF linear-chain binding drives full NF-\\u03baB activation.\",\n      \"evidence\": \"NZF ubiquitin-binding assays, compound NZF mutants, NF-\\u03baB and death readouts, inhibitor screen\",\n      \"pmids\": [\"39528476\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of SHARPIN NZF chain-type promiscuity not resolved\", \"Single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How SHARPIN's many LUBAC-independent activities (integrin inhibition, caspase-1 suppression, PRMT5 activation, ARP2/3-driven motility, and multiple substrate ubiquitination/stabilization events) are coordinated and spatially partitioned within a single cell remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No unifying framework linking competing UBL-domain engagements in vivo\", \"Signals switching SHARPIN between LUBAC and non-LUBAC pools unknown\", \"Most cancer-context substrate findings rest on single labs without reconstitution\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 3, 21]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [3, 14, 18, 41]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 38]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [3, 8, 25]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [20, 21]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [20]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [1, 2, 11, 12, 40]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [2, 9, 10, 16]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 38]},\n      {\"term_id\": \"R-HSA-1474244\", \"supporting_discovery_ids\": [3, 17, 24]},\n      {\"term_id\": \"R-HSA-109582\", \"supporting_discovery_ids\": [25, 35]}\n    ],\n    \"complexes\": [\"LUBAC\"],\n    \"partners\": [\"RNF31\", \"RBCK1\", \"IKBKG\", \"SHANK\", \"PRMT5\", \"ITGB1\", \"ITGA2B\", \"ACTR2/3\"]\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}