{"gene":"CYRIB","run_date":"2026-06-09T22:57:19","timeline":{"discoveries":[{"year":2018,"finding":"FAM49B (CYRI-B) directly interacts with the active (GTP-bound) form of the small GTPase Rac1, and genetic disruption of this interaction compromises FAM49B function in suppressing T cell activation, PAK phosphorylation, and actin assembly.","method":"Co-immunoprecipitation, CRISPR-based genetic disruption of FAM49B-Rac interaction, CD69 induction assay, PAK phosphorylation assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal interaction confirmed by Co-IP, functional consequence of interaction disruption demonstrated by mutagenesis, replicated by independent labs in subsequent papers","pmids":["29632189"],"is_preprint":false},{"year":2018,"finding":"CYRI-B binds activated Rac1 via its DUF1394 domain (shared with CYFIP), inhibits Scar/WAVE-induced actin polymerization at the cell edge, and thereby restricts lamellipodia size and duration. CYRI-depleted cells display broad, stable lamellipodia enriched in Scar/WAVE, while CYRI overexpression suppresses Scar/WAVE recruitment.","method":"Protein pulldown/Co-IP for Rac1 binding, optogenetic Rac1 activation, live-cell imaging of protrusion dynamics, CYRI depletion and overexpression with Scar/WAVE localization readout","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (binding assay, optogenetics, live imaging, overexpression/depletion), replicated by independent labs","pmids":["30250061"],"is_preprint":false},{"year":2017,"finding":"FAM49B localizes to mitochondria and regulates mitochondrial fission; silencing FAM49B in pancreatic cancer cells increases mitochondrial fission and mitochondrial ROS generation.","method":"Mitochondrial fractionation/localization, ROS measurement, siRNA knockdown, proliferation and invasion assays","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — direct localization by fractionation with functional consequence (fission, ROS) shown, single lab with multiple readouts","pmids":["29059164"],"is_preprint":false},{"year":2019,"finding":"CYRI-B (FAM49B) binds RAC1 through its conserved DUF1394 domain and negatively regulates RAC1 signaling, thereby attenuating macropinocytosis, phagocytosis, and cell migration to confer host resistance to Salmonella infection. The bacterial effector SopE (a RAC1 activator) selectively targets CYRI to dampen its function.","method":"In vivo genome-wide mutagenesis screen, protein-protein binding assays for RAC1 interaction, macropinocytosis assay, phagocytosis assay, cell migration assay, bacterial infection models","journal":"Nature microbiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal functional assays, in vivo screen, mechanistic dissection of RAC1 binding domain, independently consistent with other labs' findings","pmids":["31285585"],"is_preprint":false},{"year":2020,"finding":"Crystal structure of whale shark CYRI-B reveals it comprises three distinct α-helical subdomains and is highly structurally related to the DUF1394-containing domain of CYFIP proteins, establishing the structural basis for Rac1 binding.","method":"X-ray crystallography","journal":"Acta crystallographica. Section D, Structural biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure solved by X-ray crystallography, first structure of any CYRI family member; single study but Tier 1 method","pmids":["33021503"],"is_preprint":false},{"year":2020,"finding":"Crystal structures of CYRI-B (lacking N-terminal helix) alone and in complex with active Rac1(Q61L) reveal: (1) a unique Rac1-effector interface in the N-terminal subdomain of CYRI-B, (2) conformational changes in a C-terminal 'Ratchet' subdomain upon Rac1 binding, and (3) that CYRI-A and CYRI-B can form autoinhibited homo- or heterodimers, providing an additional layer of regulation. CYRI-B directly competes with Scar/WAVE complex for Rac1 binding.","method":"X-ray crystallography of CYRI-BΔN and CYRI-BΔN:Rac1Q61L complex, structure-guided mutagenesis, competition binding assays","journal":"Structure (London, England : 1993)","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structures of both apo and Rac1-bound forms with multiple orthogonal validations in one rigorous study","pmids":["33217330"],"is_preprint":false},{"year":2021,"finding":"CYRI-A (the paralogue of CYRI-B) is transiently recruited to nascent macropinosomes in a PI3K- and RAC1-dependent manner, preceding RAB5A recruitment. CYRI-A regulates macropinosome formation and α5β1 integrin internalization; depletion of both CYRI-A and CYRI-B enhances surface integrin expression, migration, invasion, and anchorage-independent growth.","method":"Live-cell imaging of CYRI-A recruitment, PI3K and RAC1 inhibition, siRNA depletion, integrin surface expression by flow cytometry, invasion assays, 3D growth assays","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (live imaging, pharmacological inhibition, knockdown with multiple phenotypic readouts), single lab but highly rigorous","pmids":["34165494"],"is_preprint":false},{"year":2019,"finding":"Mass spectrometry-based unbiased screen for interactors of active Rac1 confirmed CYRI interaction with GTP-bound Rac1, supporting CYRI as a new class of Rac1 interactors.","method":"Mass spectrometry interactome screen with active Rac1 mutant as bait","journal":"Communicative & integrative biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — unbiased MS screen confirms Rac1 interaction, but single lab and not independently replicated in this paper","pmids":["31413787"],"is_preprint":false},{"year":2022,"finding":"miR-22 directly targets the 3′-UTR of FAM49B (confirmed by dual luciferase reporter assay), reducing FAM49B expression. FAM49B inhibits TRAF6/IKK signaling and downstream pro-inflammatory responses in hepatic ischemia/reperfusion injury in a Rac1-dependent manner (interference of Rac1 reversed FAM49B inhibition effects).","method":"Dual luciferase reporter assay for miR-22 targeting, miR-22 inhibition experiments, IKK phosphorylation measurement, in vivo Rac1 interference","journal":"Molecular immunology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — direct miR-22 targeting confirmed by reporter assay, Rac1 epistasis experiment performed, single lab","pmids":["35131594"],"is_preprint":false},{"year":2023,"finding":"LANCL1 stabilizes FAM49B protein by blocking FAM49B interaction with the E3 ubiquitin ligase TRIM21, thereby protecting FAM49B from ubiquitin-proteasome degradation. The LANCL1-FAM49B axis suppresses Rac1-NADPH oxidase-driven ROS production.","method":"Mass spectrometry (FAM49B identified as LANCL1 binding partner), co-immunoprecipitation for FAM49B-TRIM21 interaction, ubiquitination assay, ROS measurement, proteasome inhibitor experiments","journal":"Hepatology (Baltimore, Md.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS-identified interaction confirmed by Co-IP, ubiquitination mechanism demonstrated; single lab with orthogonal methods","pmids":["37540188"],"is_preprint":false},{"year":2022,"finding":"Knockdown of CYRI-B in gastric cancer cells activates the Rac1-STAT3 pathway, promoting migration, invasion, and EMT. Collagen type I reduces CYRI-B expression via the collagen receptor DDR1.","method":"CYRI-B siRNA knockdown, western blotting for Rac1-STAT3 pathway activation, migration and invasion assays, DDR1 collagen receptor blocking experiments, in vivo xenograft","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — pathway placement by KD with defined molecular readouts, DDR1-mediated regulation identified; single lab","pmids":["36584745"],"is_preprint":false},{"year":2023,"finding":"CYRI-B is a negative regulator of CEACAM3-mediated phagocytosis: CYRI-B knockout in HL-60 cells enhances Rac-GTP loading and PAK phosphorylation downstream of CEACAM3, resulting in increased phagocytosis of bacteria. Complementation with CYRI-B reverts the knockout phenotype.","method":"Genome-wide CRISPR/Cas9 screen, clonal CYRI-B knockout generation, Rac-GTP loading assay, PAK phosphorylation western blot, phagocytosis assay with fluorescent bacteria, genetic complementation","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — genome-wide screen plus clonal KO with multiple molecular readouts and genetic complementation rescue, single lab but highly rigorous","pmids":["37264948"],"is_preprint":false},{"year":2024,"finding":"CYRI-B mediates macropinocytic uptake of lysophosphatidic acid receptor 1 (LPAR1); loss of CYRI-B in pancreatic cancer cells impairs LPAR1 internalization, reducing chemotactic responses to lysophosphatidic acid and inhibiting metastasis. In early disease, CYRI-B loss leads to enhanced ERK and JNK-induced proliferation in precancerous lesions.","method":"Cyrib gene deletion in mouse KRAS/p53-driven pancreatic cancer model, receptor internalization assay, chemotaxis assay, ERK/JNK phosphorylation, in vivo tumor progression analysis","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo genetic deletion with multiple mechanistic readouts (receptor trafficking, kinase activation, chemotaxis), single lab but multiple orthogonal methods","pmids":["38712822"],"is_preprint":false},{"year":2024,"finding":"Fam49b dampens TCR signal strength in thymocytes to prevent excessive negative selection; Fam49b-KO mice show excessive negative selection of DP thymocytes, impaired survival of SP thymocytes, and significant reductions in CD4 and CD8 SP thymocytes and peripheral T cells. Large proportions of TCRγδ+ and CD8αα+TCRαβ+ gut intraepithelial T lymphocytes were absent in Fam49b-KO mice.","method":"Novel Fam49b-KO mouse generation, flow cytometric analysis of thymic subsets, TCR signaling assays in primary cells","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean in vivo KO mouse model with defined developmental phenotypes and multiple cell subset analyses; consistent with in vitro T cell data from prior work","pmids":["39158947"],"is_preprint":false},{"year":2024,"finding":"In platelets, FAM49b negatively regulates lamellipodia formation and migration: Fam49b-/- platelets spread faster with larger areas on fibrinogen, are more prone to polarization and migration, and these phenotypes depend on functional WAVE complex (double Cyfip1/Fam49b KO eliminates lamellipodia, phenocopying Cyfip1 KO alone).","method":"Platelet-specific Fam49b-/-, Cyfip1-/-, and Cyfip1/Fam49b-/- mouse models, platelet spreading assay on fibrinogen, migration assay, structured micropatterns","journal":"Cells","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic models including double KO epistasis, multiple platelet functional readouts establishing pathway dependence on WAVE complex","pmids":["38391912"],"is_preprint":false},{"year":2024,"finding":"In Drosophila, CYRI acts as a molecular brake on the Rac-WRC-Arp2/3 pathway: loss of CYRI accelerates epidermal wound healing and enhances lamellipodial spreading in macrophages; CYRI also limits invasive border cell cluster migration by controlling cluster cohesion.","method":"Drosophila CYRI loss-of-function genetics, wound healing assay, live imaging of macrophage lamellipodia, border cell migration assay","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic loss-of-function in Drosophila with multiple in vivo phenotypic readouts; confirms conservation of CYRI function as WRC inhibitor","pmids":["39453414"],"is_preprint":false},{"year":2025,"finding":"FAM49B is cleaved by the cysteine protease AEP (asparagine endopeptidase) at residues N169 and N170, generating two fragments FAM49B(1-169) and FAM49B(171-324). While full-length FAM49B inhibits VSMC migration, the fragments bind Rac1 and increase its activity, inducing actin polymerization and promoting VSMC migration and atherosclerotic plaque formation.","method":"Mass spectrometry fragment identification, AEP cleavage site mutagenesis (N169/N170), AEP knockout mouse, adenoviral overexpression of full-length vs. fragment FAM49B in VSMCs, Rac1 activity assay, VSMC migration assay, in vivo atherosclerosis models","journal":"Arteriosclerosis, thrombosis, and vascular biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — cleavage site identified by MS and mutagenesis, functional distinction between full-length and fragments confirmed in multiple assays and in vivo models","pmids":["40567229"],"is_preprint":false},{"year":2025,"finding":"CYRI-B loss leads to accelerated focal adhesion maturation, formation of excessively large focal adhesions, accumulation of polymerized actin in stress fibres that acts as a barrier to microtubule targeting, and depletion of the integrin internalization mediator ERC1 from adhesion sites. This reveals a connection between CYRI-B-controlled lamellipodia dynamics and microtubule-mediated adhesion turnover.","method":"CYRI-B siRNA depletion, BioID proximity screen with paxillin as bait, focal adhesion morphometry, microtubule and actin imaging, ERC1 localization assay","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — BioID screen plus multiple orthogonal imaging and functional assays; mechanistic connection between CYRI-B, actin, microtubules, and adhesion turnover established","pmids":["41277545"],"is_preprint":false},{"year":2025,"finding":"FAM49B interacts with NEK9 and promotes NEK9-Thr210 phosphorylation; FAM49B knockdown reduces NEK9 phosphorylation and enhances K48-linked ubiquitination and degradation of c-Myc, suppressing CRC proliferation and migration.","method":"Co-immunoprecipitation of FAM49B-NEK9 interaction, western blot for NEK9-Thr210 phosphorylation, c-Myc ubiquitination assay (K48-specific), FAM49B knockdown with NEK9 rescue experiments, cell cycle analysis","journal":"BioFactors (Oxford, England)","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP interaction confirmed, downstream epistasis (NEK9 KD in FAM49B-OE cells) performed; single lab","pmids":["39780509"],"is_preprint":false},{"year":2021,"finding":"FAM49B interacts with ELAVL1 protein; endogenous FAM49B co-immunoprecipitates with ELAVL1 in breast cancer cells, and FAM49B positively regulates Rab10 and TLR4 expression by stabilizing ELAVL1 protein, thereby activating the ELAVL1/Rab10/TLR4/NF-κB signaling axis.","method":"Co-immunoprecipitation of FAM49B-ELAVL1, FAM49B knockdown with Rab10/TLR4 western blot, microarray analysis, xenograft model","journal":"Cancer cell international","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP interaction with downstream pathway effects; single lab with multiple readouts but limited mechanistic depth on how FAM49B stabilizes ELAVL1","pmids":["34645466"],"is_preprint":false},{"year":2025,"finding":"Downregulation of FAM49B in microglia (as modeled in human and murine microglial cells) leads to alterations in cytoskeletal maintenance, migration, surface adherence, energy homeostasis, autophagy, and increased microglial activation/inflammatory response.","method":"FAM49B knockdown in human and murine microglia, cytoskeletal assays, migration assay, metabolic assays, inflammatory cytokine measurement","journal":"npj aging","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — multiple cellular functional readouts in loss-of-function experiments; single lab, no independent replication","pmids":["41419490"],"is_preprint":false}],"current_model":"CYRI-B (FAM49B) is an evolutionarily conserved, GTP-Rac1-binding protein that competes with the Scar/WAVE complex for active Rac1 via its DUF1394 domain (structurally resolved by X-ray crystallography), thereby acting as a local inhibitor of Arp2/3-driven actin polymerization to restrict lamellipodia size and duration, regulate macropinocytosis and integrin trafficking, suppress phagocytosis, modulate focal adhesion maturation and turnover, and dampen T cell receptor and other Rac1-driven signaling cascades; its activity is further regulated by dimerization-based autoinhibition, miR-22-mediated transcriptional suppression, TRIM21-mediated ubiquitin-proteasome degradation (counteracted by LANCL1), and AEP-mediated proteolytic cleavage that converts the intact inhibitory protein into Rac1-activating fragments."},"narrative":{"mechanistic_narrative":"CYRI-B (FAM49B) is an evolutionarily conserved regulator of actin-based cell behaviors that acts as a local brake on Rac1-driven cytoskeletal dynamics [PMID:30250061, PMID:39453414]. It directly binds active, GTP-loaded Rac1 through its DUF1394 domain, the same fold shared with the CYFIP subunit of the Scar/WAVE complex, and competes with Scar/WAVE for Rac1, thereby limiting Arp2/3-driven actin polymerization at the cell edge to restrict lamellipodia size and duration [PMID:30250061, PMID:33217330]. Crystal structures of CYRI-B alone and bound to Rac1(Q61L) define a unique Rac1-effector interface, a conformational 'Ratchet' subdomain that moves upon binding, and an autoinhibitory homo-/heterodimerization mode with its paralogue CYRI-A [PMID:33021503, PMID:33217330]. Through this Rac1-restraining activity CYRI-B governs macropinocytosis and receptor trafficking — controlling internalization of α5β1 integrin and the chemoattractant receptor LPAR1 [PMID:34165494, PMID:38712822] — and suppresses phagocytosis downstream of CEACAM3 by limiting Rac-GTP loading and PAK phosphorylation [PMID:37264948]. It also shapes focal adhesion maturation and microtubule-mediated adhesion turnover via control of polymerized actin and ERC1 localization [PMID:41277545], and dampens Rac1-dependent signaling outputs including T cell receptor signaling, where loss in mice causes excessive thymocyte negative selection and depletion of peripheral and gut T cell subsets [PMID:29632189, PMID:39158947]. CYRI-B abundance and activity are themselves regulated: by miR-22-mediated suppression of its 3′-UTR [PMID:35131594], by TRIM21-mediated ubiquitin-proteasome degradation that LANCL1 counteracts [PMID:37540188], and by AEP cleavage at N169/N170 that converts the intact inhibitor into Rac1-activating fragments promoting smooth-muscle migration and atherosclerosis [PMID:40567229]. Across cancer contexts CYRI-B loss generally de-represses Rac1 and downstream pathways such as Rac1-STAT3 to enhance migration, invasion and EMT [PMID:36584745, PMID:38712822].","teleology":[{"year":2017,"claim":"Established the first cellular role and localization for FAM49B by placing it at mitochondria as a restraint on mitochondrial fission and ROS, before its Rac1-centered function was known.","evidence":"Mitochondrial fractionation, siRNA knockdown, ROS and proliferation/invasion assays in pancreatic cancer cells","pmids":["29059164"],"confidence":"Medium","gaps":["Does not connect mitochondrial localization to Rac1 binding","Single lab, no reciprocal localization validation","Mechanism linking FAM49B to the fission machinery unresolved"]},{"year":2018,"claim":"Defined FAM49B/CYRI-B as a direct binder of active Rac1 that suppresses Rac1-driven actin assembly and signaling, answering what its molecular partner and activity are.","evidence":"Co-IP, CRISPR disruption of the FAM49B-Rac interaction, PAK phosphorylation and CD69 induction assays; independently, pulldown plus optogenetic Rac1 activation and live imaging of lamellipodia","pmids":["29632189","30250061"],"confidence":"High","gaps":["Did not resolve the structural interface","Did not establish how CYRI-B competes with Scar/WAVE at atomic detail"]},{"year":2019,"claim":"Localized CYRI-B's Rac1 binding to the DUF1394 domain and showed its restraint of macropinocytosis, phagocytosis and migration confers host defense, while an unbiased screen confirmed CYRI as a new class of active-Rac1 interactor.","evidence":"In vivo mutagenesis screen, domain-mapped binding assays, macropinocytosis/phagocytosis/migration assays, Salmonella infection models; separate mass-spectrometry active-Rac1 interactome","pmids":["31285585","31413787"],"confidence":"High","gaps":["MS screen single lab, not independently replicated within the study","How SopE selectively neutralizes CYRI not structurally defined"]},{"year":2020,"claim":"Provided the structural basis for CYRI-Rac1 recognition and revealed a dimerization-based autoinhibition layer, explaining how CYRI-B competes with Scar/WAVE.","evidence":"X-ray crystallography of whale shark CYRI-B and of CYRI-BΔN apo and Rac1(Q61L)-bound forms, structure-guided mutagenesis, competition binding assays","pmids":["33021503","33217330"],"confidence":"High","gaps":["Full-length protein including N-terminal helix not crystallized","Regulation of the dimer-to-monomer transition in cells not established"]},{"year":2021,"claim":"Connected CYRI proteins to receptor trafficking, showing PI3K/RAC1-dependent recruitment to nascent macropinosomes and control of integrin internalization, linking the actin brake to membrane uptake.","evidence":"Live-cell imaging of CYRI-A recruitment, PI3K/RAC1 inhibition, dual CYRI-A/B depletion, flow cytometry of surface integrin, invasion and 3D growth assays","pmids":["34165494"],"confidence":"High","gaps":["Relative contributions of CYRI-A versus CYRI-B not fully separated","Cargo selectivity of CYRI-dependent macropinocytosis incomplete"]},{"year":2022,"claim":"Identified upstream and downstream control nodes: miR-22 transcriptional suppression of FAM49B and its Rac1-dependent inhibition of TRAF6/IKK inflammatory signaling, and collagen/DDR1-mediated suppression feeding into Rac1-STAT3 in cancer.","evidence":"Dual luciferase reporter for miR-22 targeting, IKK phosphorylation and in vivo Rac1 interference in hepatic I/R; siRNA knockdown, Rac1-STAT3 western blots, DDR1 blocking and xenograft in gastric cancer","pmids":["35131594","36584745"],"confidence":"Medium","gaps":["Both single-lab studies","Direct biochemical link between FAM49B and TRAF6/IKK not shown","How DDR1 signaling lowers CYRI-B expression unresolved"]},{"year":2023,"claim":"Showed CYRI-B protein levels are set by a TRIM21 ubiquitin-degradation axis opposed by LANCL1, and confirmed its role as a negative regulator of CEACAM3-driven phagocytosis via Rac-GTP/PAK.","evidence":"MS, Co-IP of FAM49B-TRIM21, ubiquitination and proteasome-inhibitor assays, ROS measurement; genome-wide CRISPR screen, clonal CYRI-B KO, Rac-GTP/PAK readouts and complementation rescue","pmids":["37540188","37264948"],"confidence":"High","gaps":["LANCL1/TRIM21 axis single lab","TRIM21 ubiquitination site on FAM49B not mapped"]},{"year":2024,"claim":"In vivo genetics consolidated CYRI-B as a physiological Rac1 brake across tissues — receptor (LPAR1) trafficking and metastasis control in pancreatic cancer, TCR signal tuning in thymocyte selection, and WAVE-dependent lamellipodia restraint in platelets and Drosophila.","evidence":"Cyrib deletion in KRAS/p53 pancreatic model with receptor internalization/chemotaxis/ERK-JNK readouts; Fam49b-KO mice thymic flow cytometry; platelet Cyfip1/Fam49b single and double KO epistasis; Drosophila loss-of-function wound healing, macrophage and border cell assays","pmids":["38712822","39158947","38391912","39453414"],"confidence":"High","gaps":["Stage-specific dual role of CYRI-B in early proliferation versus late metastasis incompletely mechanistically defined","Cell-type-specific contributions of dimerization/autoinhibition in vivo not tested"]},{"year":2025,"claim":"Revealed proteolytic conversion of CYRI-B function and expanded its mechanistic reach: AEP cleavage produces Rac1-activating fragments, and new partners (NEK9, ELAVL1) and adhesion/microtubule control diversify its outputs.","evidence":"AEP cleavage-site MS/mutagenesis, AEP-KO mouse, fragment overexpression and Rac1 activity/VSMC migration/atherosclerosis assays; BioID-paxillin focal adhesion screen with imaging; Co-IP of NEK9 and ELAVL1 with downstream c-Myc/Rab10-TLR4 readouts; microglial knockdown phenotyping","pmids":["40567229","41277545","39780509","34645466","41419490"],"confidence":"High","gaps":["NEK9 and ELAVL1 interactions are single-lab Co-IP with limited mechanistic depth","Whether AEP-fragment switch operates outside vascular smooth muscle unknown","Microglial phenotypes not mechanistically tied to Rac1 or WAVE competition"]},{"year":null,"claim":"How CYRI-B integrates its multiple regulatory inputs (dimerization autoinhibition, miR-22, TRIM21/LANCL1, AEP cleavage) into a coherent threshold that determines when and where Rac1 is locally restrained remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model relating CYRI-B abundance, conformation and localization to lamellipodial output","Structural basis of the AEP-generated activating fragments' Rac1 binding not solved","Reconciliation of mitochondrial localization with the cytoskeletal Rac1 function unaddressed"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[1,3,5]},{"term_id":"GO:0140313","term_label":"molecular sequestering activity","supporting_discovery_ids":[1,5]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[1,14]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[1,6]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[1,17]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[2]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,3]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[3,6,12]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[11,13]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[3,6]}],"complexes":[],"partners":["RAC1","TRIM21","LANCL1","NEK9","ELAVL1","CYRI-A"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9NUQ9","full_name":"CYFIP-related Rac1 interactor B","aliases":["L1"],"length_aa":324,"mass_kda":36.7,"function":"Negatively regulates RAC1 signaling and RAC1-driven cytoskeletal remodeling (PubMed:30250061, PubMed:31285585). Regulates chemotaxis, cell migration and epithelial polarization by controlling the polarity, plasticity, duration and extent of protrusions. Limits Rac1 mediated activation of the Scar/WAVE complex, focuses protrusion signals and regulates pseudopod complexity by inhibiting Scar/WAVE-induced actin polymerization (PubMed:30250061). Protects against Salmonella bacterial infection. Attenuates processes such as macropinocytosis, phagocytosis and cell migration and restrict sopE-mediated bacterial entry (PubMed:31285585). Also restricts infection mediated by Mycobacterium tuberculosis and Listeria monocytogenes (By similarity). Involved in the regulation of mitochondrial dynamics and oxidative stress (PubMed:29059164)","subcellular_location":"Membrane; Mitochondrion","url":"https://www.uniprot.org/uniprotkb/Q9NUQ9/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CYRIB","classification":"Not Classified","n_dependent_lines":9,"n_total_lines":1208,"dependency_fraction":0.0074503311258278145},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CYRIB","total_profiled":1310},"omim":[{"mim_id":"621448","title":"CYFIP-RELATED RAC1 INTERACTOR A; CYRIA","url":"https://www.omim.org/entry/621448"},{"mim_id":"617978","title":"CYFIP-RELATED RAC1 INTERACTOR B; CYRIB","url":"https://www.omim.org/entry/617978"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"bone marrow","ntpm":187.0}],"url":"https://www.proteinatlas.org/search/CYRIB"},"hgnc":{"alias_symbol":["BM-009","CYRI-B","CYRI"],"prev_symbol":["FAM49B"]},"alphafold":{"accession":"Q9NUQ9","domains":[{"cath_id":"-","chopping":"31-130_149-194","consensus_level":"medium","plddt":93.8384,"start":31,"end":194},{"cath_id":"1.20.1050","chopping":"216-324","consensus_level":"medium","plddt":94.8171,"start":216,"end":324}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NUQ9","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NUQ9-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NUQ9-F1-predicted_aligned_error_v6.png","plddt_mean":91.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CYRIB","jax_strain_url":"https://www.jax.org/strain/search?query=CYRIB"},"sequence":{"accession":"Q9NUQ9","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9NUQ9.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9NUQ9/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NUQ9"}},"corpus_meta":[{"pmid":"29632189","id":"PMC_29632189","title":"Genome-wide CRISPR screen identifies FAM49B as a key regulator of actin dynamics and T cell activation.","date":"2018","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/29632189","citation_count":96,"is_preprint":false},{"pmid":"30250061","id":"PMC_30250061","title":"Fam49/CYRI interacts with Rac1 and locally suppresses protrusions.","date":"2018","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/30250061","citation_count":70,"is_preprint":false},{"pmid":"29059164","id":"PMC_29059164","title":"FAM49B, a novel regulator of mitochondrial function and integrity that suppresses tumor metastasis.","date":"2017","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/29059164","citation_count":58,"is_preprint":false},{"pmid":"31285585","id":"PMC_31285585","title":"CYRI/FAM49B negatively regulates RAC1-driven cytoskeletal remodelling and protects against bacterial infection.","date":"2019","source":"Nature microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/31285585","citation_count":44,"is_preprint":false},{"pmid":"32071545","id":"PMC_32071545","title":"TASP1 Promotes Gallbladder Cancer Cell Proliferation and Metastasis by Up-regulating FAM49B via PI3K/AKT Pathway.","date":"2020","source":"International journal of biological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/32071545","citation_count":31,"is_preprint":false},{"pmid":"35131594","id":"PMC_35131594","title":"FAM49B, restrained by miR-22, relieved hepatic ischemia/reperfusion injury by inhibiting TRAF6/IKK signaling pathway in a Rac1-dependent manner.","date":"2022","source":"Molecular immunology","url":"https://pubmed.ncbi.nlm.nih.gov/35131594","citation_count":28,"is_preprint":false},{"pmid":"34165494","id":"PMC_34165494","title":"CYRI-A limits invasive migration through macropinosome formation and integrin uptake regulation.","date":"2021","source":"The Journal of cell 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Md.)","url":"https://pubmed.ncbi.nlm.nih.gov/37540188","citation_count":17,"is_preprint":false},{"pmid":"33021503","id":"PMC_33021503","title":"Structure of CYRI-B (FAM49B), a key regulator of cellular actin assembly.","date":"2020","source":"Acta crystallographica. 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sciences","url":"https://pubmed.ncbi.nlm.nih.gov/36499755","citation_count":8,"is_preprint":false},{"pmid":"38712822","id":"PMC_38712822","title":"CYRI-B-mediated macropinocytosis drives metastasis via lysophosphatidic acid receptor uptake.","date":"2024","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/38712822","citation_count":8,"is_preprint":false},{"pmid":"31413787","id":"PMC_31413787","title":"CYRI/ Fam49 Proteins Represent a New Class of Rac1 Interactors.","date":"2019","source":"Communicative & integrative biology","url":"https://pubmed.ncbi.nlm.nih.gov/31413787","citation_count":8,"is_preprint":false},{"pmid":"29653078","id":"PMC_29653078","title":"Characterization of CyrI, the hydroxylase involved in the last step of cylindrospermopsin biosynthesis: Binding studies, site-directed mutagenesis and stereoselectivity.","date":"2018","source":"Archives of biochemistry and biophysics","url":"https://pubmed.ncbi.nlm.nih.gov/29653078","citation_count":7,"is_preprint":false},{"pmid":"36584745","id":"PMC_36584745","title":"Downregulation of CYRI-B promotes migration, invasion and EMT by activating the Rac1-STAT3 pathway in gastric cancer.","date":"2022","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/36584745","citation_count":6,"is_preprint":false},{"pmid":"39453414","id":"PMC_39453414","title":"CYRI controls epidermal wound closure and cohesion of invasive border cell cluster in Drosophila.","date":"2024","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/39453414","citation_count":6,"is_preprint":false},{"pmid":"36892409","id":"PMC_36892409","title":"CYRI proteins: controllers of actin dynamics in the cellular 'eat vs walk' decision.","date":"2023","source":"Biochemical Society transactions","url":"https://pubmed.ncbi.nlm.nih.gov/36892409","citation_count":5,"is_preprint":false},{"pmid":"35205432","id":"PMC_35205432","title":"Characterization and Functional Study of FAM49B Reveals Its Effect on Cell Proliferation in HEK293T Cells.","date":"2022","source":"Genes","url":"https://pubmed.ncbi.nlm.nih.gov/35205432","citation_count":3,"is_preprint":false},{"pmid":"38391912","id":"PMC_38391912","title":"Differential Role of the RAC1-Binding Proteins FAM49b (CYRI-B) and CYFIP1 in Platelets.","date":"2024","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/38391912","citation_count":3,"is_preprint":false},{"pmid":"39780509","id":"PMC_39780509","title":"FAM49B drives colorectal cancer progression by stabilizing c-Myc through NEK9 phosphorylation.","date":"2025","source":"BioFactors (Oxford, England)","url":"https://pubmed.ncbi.nlm.nih.gov/39780509","citation_count":2,"is_preprint":false},{"pmid":"37264948","id":"PMC_37264948","title":"A genome-wide genetic screen identifies CYRI-B as a negative regulator of CEACAM3-mediated phagocytosis.","date":"2023","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/37264948","citation_count":2,"is_preprint":false},{"pmid":"39158947","id":"PMC_39158947","title":"Fam49b dampens TCR signal strength to regulate survival of positively selected thymocytes and peripheral T cells.","date":"2024","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/39158947","citation_count":2,"is_preprint":false},{"pmid":"41246334","id":"PMC_41246334","title":"Single-cell and spatial transcriptomics integration reveals FAM49B promotes tumor-associated macrophages polarization in colorectal cancer via the MK pathway.","date":"2025","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/41246334","citation_count":1,"is_preprint":false},{"pmid":"40567229","id":"PMC_40567229","title":"FAM49B Fragmentation by Asparagine Endopeptidase Promotes Vascular Smooth Muscle Cell Migration in Atherogenesis.","date":"2025","source":"Arteriosclerosis, thrombosis, and vascular biology","url":"https://pubmed.ncbi.nlm.nih.gov/40567229","citation_count":0,"is_preprint":false},{"pmid":"41419490","id":"PMC_41419490","title":"Age-related nigral downregulation of the Parkinson's risk factor FAM49B primes human microglia for inflammaging.","date":"2025","source":"npj aging","url":"https://pubmed.ncbi.nlm.nih.gov/41419490","citation_count":0,"is_preprint":false},{"pmid":"41113986","id":"PMC_41113986","title":"FAM49B suppresses ovarian cancer cell growth through regulating MAPK signaling.","date":"2025","source":"American journal of cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/41113986","citation_count":0,"is_preprint":false},{"pmid":"41277545","id":"PMC_41277545","title":"CYRI-B loss promotes enlarged mature focal adhesions and restricts microtubule and ERC1 access to the cell leading edge.","date":"2025","source":"Journal of cell 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driving.","date":"2021","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/34402856","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.04.22.646545","title":"Transcription-based identification of uncharacterized genes in the human immune response","date":"2025-04-24","source":"bioRxiv","url":"https://doi.org/10.1101/2025.04.22.646545","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":18136,"output_tokens":5525,"usd":0.068641,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":13967,"output_tokens":4255,"usd":0.088105,"stage2_stop_reason":"end_turn"},"total_usd":0.156746,"stage1_batch_id":"msgbatch_01ACAxJQabDsht39JXeWG22d","stage2_batch_id":"msgbatch_01QEU22A9U5sbPTgsMzTmvSf","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2018,\n      \"finding\": \"FAM49B (CYRI-B) directly interacts with the active (GTP-bound) form of the small GTPase Rac1, and genetic disruption of this interaction compromises FAM49B function in suppressing T cell activation, PAK phosphorylation, and actin assembly.\",\n      \"method\": \"Co-immunoprecipitation, CRISPR-based genetic disruption of FAM49B-Rac interaction, CD69 induction assay, PAK phosphorylation assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal interaction confirmed by Co-IP, functional consequence of interaction disruption demonstrated by mutagenesis, replicated by independent labs in subsequent papers\",\n      \"pmids\": [\"29632189\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CYRI-B binds activated Rac1 via its DUF1394 domain (shared with CYFIP), inhibits Scar/WAVE-induced actin polymerization at the cell edge, and thereby restricts lamellipodia size and duration. CYRI-depleted cells display broad, stable lamellipodia enriched in Scar/WAVE, while CYRI overexpression suppresses Scar/WAVE recruitment.\",\n      \"method\": \"Protein pulldown/Co-IP for Rac1 binding, optogenetic Rac1 activation, live-cell imaging of protrusion dynamics, CYRI depletion and overexpression with Scar/WAVE localization readout\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (binding assay, optogenetics, live imaging, overexpression/depletion), replicated by independent labs\",\n      \"pmids\": [\"30250061\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"FAM49B localizes to mitochondria and regulates mitochondrial fission; silencing FAM49B in pancreatic cancer cells increases mitochondrial fission and mitochondrial ROS generation.\",\n      \"method\": \"Mitochondrial fractionation/localization, ROS measurement, siRNA knockdown, proliferation and invasion assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — direct localization by fractionation with functional consequence (fission, ROS) shown, single lab with multiple readouts\",\n      \"pmids\": [\"29059164\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CYRI-B (FAM49B) binds RAC1 through its conserved DUF1394 domain and negatively regulates RAC1 signaling, thereby attenuating macropinocytosis, phagocytosis, and cell migration to confer host resistance to Salmonella infection. The bacterial effector SopE (a RAC1 activator) selectively targets CYRI to dampen its function.\",\n      \"method\": \"In vivo genome-wide mutagenesis screen, protein-protein binding assays for RAC1 interaction, macropinocytosis assay, phagocytosis assay, cell migration assay, bacterial infection models\",\n      \"journal\": \"Nature microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal functional assays, in vivo screen, mechanistic dissection of RAC1 binding domain, independently consistent with other labs' findings\",\n      \"pmids\": [\"31285585\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Crystal structure of whale shark CYRI-B reveals it comprises three distinct α-helical subdomains and is highly structurally related to the DUF1394-containing domain of CYFIP proteins, establishing the structural basis for Rac1 binding.\",\n      \"method\": \"X-ray crystallography\",\n      \"journal\": \"Acta crystallographica. Section D, Structural biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure solved by X-ray crystallography, first structure of any CYRI family member; single study but Tier 1 method\",\n      \"pmids\": [\"33021503\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Crystal structures of CYRI-B (lacking N-terminal helix) alone and in complex with active Rac1(Q61L) reveal: (1) a unique Rac1-effector interface in the N-terminal subdomain of CYRI-B, (2) conformational changes in a C-terminal 'Ratchet' subdomain upon Rac1 binding, and (3) that CYRI-A and CYRI-B can form autoinhibited homo- or heterodimers, providing an additional layer of regulation. CYRI-B directly competes with Scar/WAVE complex for Rac1 binding.\",\n      \"method\": \"X-ray crystallography of CYRI-BΔN and CYRI-BΔN:Rac1Q61L complex, structure-guided mutagenesis, competition binding assays\",\n      \"journal\": \"Structure (London, England : 1993)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structures of both apo and Rac1-bound forms with multiple orthogonal validations in one rigorous study\",\n      \"pmids\": [\"33217330\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CYRI-A (the paralogue of CYRI-B) is transiently recruited to nascent macropinosomes in a PI3K- and RAC1-dependent manner, preceding RAB5A recruitment. CYRI-A regulates macropinosome formation and α5β1 integrin internalization; depletion of both CYRI-A and CYRI-B enhances surface integrin expression, migration, invasion, and anchorage-independent growth.\",\n      \"method\": \"Live-cell imaging of CYRI-A recruitment, PI3K and RAC1 inhibition, siRNA depletion, integrin surface expression by flow cytometry, invasion assays, 3D growth assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (live imaging, pharmacological inhibition, knockdown with multiple phenotypic readouts), single lab but highly rigorous\",\n      \"pmids\": [\"34165494\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Mass spectrometry-based unbiased screen for interactors of active Rac1 confirmed CYRI interaction with GTP-bound Rac1, supporting CYRI as a new class of Rac1 interactors.\",\n      \"method\": \"Mass spectrometry interactome screen with active Rac1 mutant as bait\",\n      \"journal\": \"Communicative & integrative biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — unbiased MS screen confirms Rac1 interaction, but single lab and not independently replicated in this paper\",\n      \"pmids\": [\"31413787\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"miR-22 directly targets the 3′-UTR of FAM49B (confirmed by dual luciferase reporter assay), reducing FAM49B expression. FAM49B inhibits TRAF6/IKK signaling and downstream pro-inflammatory responses in hepatic ischemia/reperfusion injury in a Rac1-dependent manner (interference of Rac1 reversed FAM49B inhibition effects).\",\n      \"method\": \"Dual luciferase reporter assay for miR-22 targeting, miR-22 inhibition experiments, IKK phosphorylation measurement, in vivo Rac1 interference\",\n      \"journal\": \"Molecular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — direct miR-22 targeting confirmed by reporter assay, Rac1 epistasis experiment performed, single lab\",\n      \"pmids\": [\"35131594\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"LANCL1 stabilizes FAM49B protein by blocking FAM49B interaction with the E3 ubiquitin ligase TRIM21, thereby protecting FAM49B from ubiquitin-proteasome degradation. The LANCL1-FAM49B axis suppresses Rac1-NADPH oxidase-driven ROS production.\",\n      \"method\": \"Mass spectrometry (FAM49B identified as LANCL1 binding partner), co-immunoprecipitation for FAM49B-TRIM21 interaction, ubiquitination assay, ROS measurement, proteasome inhibitor experiments\",\n      \"journal\": \"Hepatology (Baltimore, Md.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS-identified interaction confirmed by Co-IP, ubiquitination mechanism demonstrated; single lab with orthogonal methods\",\n      \"pmids\": [\"37540188\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Knockdown of CYRI-B in gastric cancer cells activates the Rac1-STAT3 pathway, promoting migration, invasion, and EMT. Collagen type I reduces CYRI-B expression via the collagen receptor DDR1.\",\n      \"method\": \"CYRI-B siRNA knockdown, western blotting for Rac1-STAT3 pathway activation, migration and invasion assays, DDR1 collagen receptor blocking experiments, in vivo xenograft\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — pathway placement by KD with defined molecular readouts, DDR1-mediated regulation identified; single lab\",\n      \"pmids\": [\"36584745\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CYRI-B is a negative regulator of CEACAM3-mediated phagocytosis: CYRI-B knockout in HL-60 cells enhances Rac-GTP loading and PAK phosphorylation downstream of CEACAM3, resulting in increased phagocytosis of bacteria. Complementation with CYRI-B reverts the knockout phenotype.\",\n      \"method\": \"Genome-wide CRISPR/Cas9 screen, clonal CYRI-B knockout generation, Rac-GTP loading assay, PAK phosphorylation western blot, phagocytosis assay with fluorescent bacteria, genetic complementation\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genome-wide screen plus clonal KO with multiple molecular readouts and genetic complementation rescue, single lab but highly rigorous\",\n      \"pmids\": [\"37264948\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CYRI-B mediates macropinocytic uptake of lysophosphatidic acid receptor 1 (LPAR1); loss of CYRI-B in pancreatic cancer cells impairs LPAR1 internalization, reducing chemotactic responses to lysophosphatidic acid and inhibiting metastasis. In early disease, CYRI-B loss leads to enhanced ERK and JNK-induced proliferation in precancerous lesions.\",\n      \"method\": \"Cyrib gene deletion in mouse KRAS/p53-driven pancreatic cancer model, receptor internalization assay, chemotaxis assay, ERK/JNK phosphorylation, in vivo tumor progression analysis\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo genetic deletion with multiple mechanistic readouts (receptor trafficking, kinase activation, chemotaxis), single lab but multiple orthogonal methods\",\n      \"pmids\": [\"38712822\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Fam49b dampens TCR signal strength in thymocytes to prevent excessive negative selection; Fam49b-KO mice show excessive negative selection of DP thymocytes, impaired survival of SP thymocytes, and significant reductions in CD4 and CD8 SP thymocytes and peripheral T cells. Large proportions of TCRγδ+ and CD8αα+TCRαβ+ gut intraepithelial T lymphocytes were absent in Fam49b-KO mice.\",\n      \"method\": \"Novel Fam49b-KO mouse generation, flow cytometric analysis of thymic subsets, TCR signaling assays in primary cells\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean in vivo KO mouse model with defined developmental phenotypes and multiple cell subset analyses; consistent with in vitro T cell data from prior work\",\n      \"pmids\": [\"39158947\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In platelets, FAM49b negatively regulates lamellipodia formation and migration: Fam49b-/- platelets spread faster with larger areas on fibrinogen, are more prone to polarization and migration, and these phenotypes depend on functional WAVE complex (double Cyfip1/Fam49b KO eliminates lamellipodia, phenocopying Cyfip1 KO alone).\",\n      \"method\": \"Platelet-specific Fam49b-/-, Cyfip1-/-, and Cyfip1/Fam49b-/- mouse models, platelet spreading assay on fibrinogen, migration assay, structured micropatterns\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic models including double KO epistasis, multiple platelet functional readouts establishing pathway dependence on WAVE complex\",\n      \"pmids\": [\"38391912\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In Drosophila, CYRI acts as a molecular brake on the Rac-WRC-Arp2/3 pathway: loss of CYRI accelerates epidermal wound healing and enhances lamellipodial spreading in macrophages; CYRI also limits invasive border cell cluster migration by controlling cluster cohesion.\",\n      \"method\": \"Drosophila CYRI loss-of-function genetics, wound healing assay, live imaging of macrophage lamellipodia, border cell migration assay\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic loss-of-function in Drosophila with multiple in vivo phenotypic readouts; confirms conservation of CYRI function as WRC inhibitor\",\n      \"pmids\": [\"39453414\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"FAM49B is cleaved by the cysteine protease AEP (asparagine endopeptidase) at residues N169 and N170, generating two fragments FAM49B(1-169) and FAM49B(171-324). While full-length FAM49B inhibits VSMC migration, the fragments bind Rac1 and increase its activity, inducing actin polymerization and promoting VSMC migration and atherosclerotic plaque formation.\",\n      \"method\": \"Mass spectrometry fragment identification, AEP cleavage site mutagenesis (N169/N170), AEP knockout mouse, adenoviral overexpression of full-length vs. fragment FAM49B in VSMCs, Rac1 activity assay, VSMC migration assay, in vivo atherosclerosis models\",\n      \"journal\": \"Arteriosclerosis, thrombosis, and vascular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — cleavage site identified by MS and mutagenesis, functional distinction between full-length and fragments confirmed in multiple assays and in vivo models\",\n      \"pmids\": [\"40567229\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CYRI-B loss leads to accelerated focal adhesion maturation, formation of excessively large focal adhesions, accumulation of polymerized actin in stress fibres that acts as a barrier to microtubule targeting, and depletion of the integrin internalization mediator ERC1 from adhesion sites. This reveals a connection between CYRI-B-controlled lamellipodia dynamics and microtubule-mediated adhesion turnover.\",\n      \"method\": \"CYRI-B siRNA depletion, BioID proximity screen with paxillin as bait, focal adhesion morphometry, microtubule and actin imaging, ERC1 localization assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — BioID screen plus multiple orthogonal imaging and functional assays; mechanistic connection between CYRI-B, actin, microtubules, and adhesion turnover established\",\n      \"pmids\": [\"41277545\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"FAM49B interacts with NEK9 and promotes NEK9-Thr210 phosphorylation; FAM49B knockdown reduces NEK9 phosphorylation and enhances K48-linked ubiquitination and degradation of c-Myc, suppressing CRC proliferation and migration.\",\n      \"method\": \"Co-immunoprecipitation of FAM49B-NEK9 interaction, western blot for NEK9-Thr210 phosphorylation, c-Myc ubiquitination assay (K48-specific), FAM49B knockdown with NEK9 rescue experiments, cell cycle analysis\",\n      \"journal\": \"BioFactors (Oxford, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP interaction confirmed, downstream epistasis (NEK9 KD in FAM49B-OE cells) performed; single lab\",\n      \"pmids\": [\"39780509\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"FAM49B interacts with ELAVL1 protein; endogenous FAM49B co-immunoprecipitates with ELAVL1 in breast cancer cells, and FAM49B positively regulates Rab10 and TLR4 expression by stabilizing ELAVL1 protein, thereby activating the ELAVL1/Rab10/TLR4/NF-κB signaling axis.\",\n      \"method\": \"Co-immunoprecipitation of FAM49B-ELAVL1, FAM49B knockdown with Rab10/TLR4 western blot, microarray analysis, xenograft model\",\n      \"journal\": \"Cancer cell international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP interaction with downstream pathway effects; single lab with multiple readouts but limited mechanistic depth on how FAM49B stabilizes ELAVL1\",\n      \"pmids\": [\"34645466\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Downregulation of FAM49B in microglia (as modeled in human and murine microglial cells) leads to alterations in cytoskeletal maintenance, migration, surface adherence, energy homeostasis, autophagy, and increased microglial activation/inflammatory response.\",\n      \"method\": \"FAM49B knockdown in human and murine microglia, cytoskeletal assays, migration assay, metabolic assays, inflammatory cytokine measurement\",\n      \"journal\": \"npj aging\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — multiple cellular functional readouts in loss-of-function experiments; single lab, no independent replication\",\n      \"pmids\": [\"41419490\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CYRI-B (FAM49B) is an evolutionarily conserved, GTP-Rac1-binding protein that competes with the Scar/WAVE complex for active Rac1 via its DUF1394 domain (structurally resolved by X-ray crystallography), thereby acting as a local inhibitor of Arp2/3-driven actin polymerization to restrict lamellipodia size and duration, regulate macropinocytosis and integrin trafficking, suppress phagocytosis, modulate focal adhesion maturation and turnover, and dampen T cell receptor and other Rac1-driven signaling cascades; its activity is further regulated by dimerization-based autoinhibition, miR-22-mediated transcriptional suppression, TRIM21-mediated ubiquitin-proteasome degradation (counteracted by LANCL1), and AEP-mediated proteolytic cleavage that converts the intact inhibitory protein into Rac1-activating fragments.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CYRI-B (FAM49B) is an evolutionarily conserved regulator of actin-based cell behaviors that acts as a local brake on Rac1-driven cytoskeletal dynamics [#1, #15]. It directly binds active, GTP-loaded Rac1 through its DUF1394 domain, the same fold shared with the CYFIP subunit of the Scar/WAVE complex, and competes with Scar/WAVE for Rac1, thereby limiting Arp2/3-driven actin polymerization at the cell edge to restrict lamellipodia size and duration [#1, #5]. Crystal structures of CYRI-B alone and bound to Rac1(Q61L) define a unique Rac1-effector interface, a conformational 'Ratchet' subdomain that moves upon binding, and an autoinhibitory homo-/heterodimerization mode with its paralogue CYRI-A [#4, #5]. Through this Rac1-restraining activity CYRI-B governs macropinocytosis and receptor trafficking — controlling internalization of α5β1 integrin and the chemoattractant receptor LPAR1 [#6, #12] — and suppresses phagocytosis downstream of CEACAM3 by limiting Rac-GTP loading and PAK phosphorylation [#11]. It also shapes focal adhesion maturation and microtubule-mediated adhesion turnover via control of polymerized actin and ERC1 localization [#17], and dampens Rac1-dependent signaling outputs including T cell receptor signaling, where loss in mice causes excessive thymocyte negative selection and depletion of peripheral and gut T cell subsets [#0, #13]. CYRI-B abundance and activity are themselves regulated: by miR-22-mediated suppression of its 3′-UTR [#8], by TRIM21-mediated ubiquitin-proteasome degradation that LANCL1 counteracts [#9], and by AEP cleavage at N169/N170 that converts the intact inhibitor into Rac1-activating fragments promoting smooth-muscle migration and atherosclerosis [#16]. Across cancer contexts CYRI-B loss generally de-represses Rac1 and downstream pathways such as Rac1-STAT3 to enhance migration, invasion and EMT [#10, #12].\",\n  \"teleology\": [\n    {\n      \"year\": 2017,\n      \"claim\": \"Established the first cellular role and localization for FAM49B by placing it at mitochondria as a restraint on mitochondrial fission and ROS, before its Rac1-centered function was known.\",\n      \"evidence\": \"Mitochondrial fractionation, siRNA knockdown, ROS and proliferation/invasion assays in pancreatic cancer cells\",\n      \"pmids\": [\"29059164\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not connect mitochondrial localization to Rac1 binding\", \"Single lab, no reciprocal localization validation\", \"Mechanism linking FAM49B to the fission machinery unresolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defined FAM49B/CYRI-B as a direct binder of active Rac1 that suppresses Rac1-driven actin assembly and signaling, answering what its molecular partner and activity are.\",\n      \"evidence\": \"Co-IP, CRISPR disruption of the FAM49B-Rac interaction, PAK phosphorylation and CD69 induction assays; independently, pulldown plus optogenetic Rac1 activation and live imaging of lamellipodia\",\n      \"pmids\": [\"29632189\", \"30250061\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve the structural interface\", \"Did not establish how CYRI-B competes with Scar/WAVE at atomic detail\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Localized CYRI-B's Rac1 binding to the DUF1394 domain and showed its restraint of macropinocytosis, phagocytosis and migration confers host defense, while an unbiased screen confirmed CYRI as a new class of active-Rac1 interactor.\",\n      \"evidence\": \"In vivo mutagenesis screen, domain-mapped binding assays, macropinocytosis/phagocytosis/migration assays, Salmonella infection models; separate mass-spectrometry active-Rac1 interactome\",\n      \"pmids\": [\"31285585\", \"31413787\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"MS screen single lab, not independently replicated within the study\", \"How SopE selectively neutralizes CYRI not structurally defined\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Provided the structural basis for CYRI-Rac1 recognition and revealed a dimerization-based autoinhibition layer, explaining how CYRI-B competes with Scar/WAVE.\",\n      \"evidence\": \"X-ray crystallography of whale shark CYRI-B and of CYRI-BΔN apo and Rac1(Q61L)-bound forms, structure-guided mutagenesis, competition binding assays\",\n      \"pmids\": [\"33021503\", \"33217330\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length protein including N-terminal helix not crystallized\", \"Regulation of the dimer-to-monomer transition in cells not established\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Connected CYRI proteins to receptor trafficking, showing PI3K/RAC1-dependent recruitment to nascent macropinosomes and control of integrin internalization, linking the actin brake to membrane uptake.\",\n      \"evidence\": \"Live-cell imaging of CYRI-A recruitment, PI3K/RAC1 inhibition, dual CYRI-A/B depletion, flow cytometry of surface integrin, invasion and 3D growth assays\",\n      \"pmids\": [\"34165494\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contributions of CYRI-A versus CYRI-B not fully separated\", \"Cargo selectivity of CYRI-dependent macropinocytosis incomplete\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified upstream and downstream control nodes: miR-22 transcriptional suppression of FAM49B and its Rac1-dependent inhibition of TRAF6/IKK inflammatory signaling, and collagen/DDR1-mediated suppression feeding into Rac1-STAT3 in cancer.\",\n      \"evidence\": \"Dual luciferase reporter for miR-22 targeting, IKK phosphorylation and in vivo Rac1 interference in hepatic I/R; siRNA knockdown, Rac1-STAT3 western blots, DDR1 blocking and xenograft in gastric cancer\",\n      \"pmids\": [\"35131594\", \"36584745\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Both single-lab studies\", \"Direct biochemical link between FAM49B and TRAF6/IKK not shown\", \"How DDR1 signaling lowers CYRI-B expression unresolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Showed CYRI-B protein levels are set by a TRIM21 ubiquitin-degradation axis opposed by LANCL1, and confirmed its role as a negative regulator of CEACAM3-driven phagocytosis via Rac-GTP/PAK.\",\n      \"evidence\": \"MS, Co-IP of FAM49B-TRIM21, ubiquitination and proteasome-inhibitor assays, ROS measurement; genome-wide CRISPR screen, clonal CYRI-B KO, Rac-GTP/PAK readouts and complementation rescue\",\n      \"pmids\": [\"37540188\", \"37264948\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"LANCL1/TRIM21 axis single lab\", \"TRIM21 ubiquitination site on FAM49B not mapped\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"In vivo genetics consolidated CYRI-B as a physiological Rac1 brake across tissues — receptor (LPAR1) trafficking and metastasis control in pancreatic cancer, TCR signal tuning in thymocyte selection, and WAVE-dependent lamellipodia restraint in platelets and Drosophila.\",\n      \"evidence\": \"Cyrib deletion in KRAS/p53 pancreatic model with receptor internalization/chemotaxis/ERK-JNK readouts; Fam49b-KO mice thymic flow cytometry; platelet Cyfip1/Fam49b single and double KO epistasis; Drosophila loss-of-function wound healing, macrophage and border cell assays\",\n      \"pmids\": [\"38712822\", \"39158947\", \"38391912\", \"39453414\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stage-specific dual role of CYRI-B in early proliferation versus late metastasis incompletely mechanistically defined\", \"Cell-type-specific contributions of dimerization/autoinhibition in vivo not tested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Revealed proteolytic conversion of CYRI-B function and expanded its mechanistic reach: AEP cleavage produces Rac1-activating fragments, and new partners (NEK9, ELAVL1) and adhesion/microtubule control diversify its outputs.\",\n      \"evidence\": \"AEP cleavage-site MS/mutagenesis, AEP-KO mouse, fragment overexpression and Rac1 activity/VSMC migration/atherosclerosis assays; BioID-paxillin focal adhesion screen with imaging; Co-IP of NEK9 and ELAVL1 with downstream c-Myc/Rab10-TLR4 readouts; microglial knockdown phenotyping\",\n      \"pmids\": [\"40567229\", \"41277545\", \"39780509\", \"34645466\", \"41419490\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"NEK9 and ELAVL1 interactions are single-lab Co-IP with limited mechanistic depth\", \"Whether AEP-fragment switch operates outside vascular smooth muscle unknown\", \"Microglial phenotypes not mechanistically tied to Rac1 or WAVE competition\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How CYRI-B integrates its multiple regulatory inputs (dimerization autoinhibition, miR-22, TRIM21/LANCL1, AEP cleavage) into a coherent threshold that determines when and where Rac1 is locally restrained remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model relating CYRI-B abundance, conformation and localization to lamellipodial output\", \"Structural basis of the AEP-generated activating fragments' Rac1 binding not solved\", \"Reconciliation of mitochondrial localization with the cytoskeletal Rac1 function unaddressed\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1, 3, 5]},\n      {\"term_id\": \"GO:0140313\", \"supporting_discovery_ids\": [1, 5]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [1, 14]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [1, 6]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [1, 17]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 3]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [3, 6, 12]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [11, 13]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [3, 6]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"RAC1\", \"TRIM21\", \"LANCL1\", \"NEK9\", \"ELAVL1\", \"CYRI-A\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}