{"gene":"BROX","run_date":"2026-06-09T22:02:45","timeline":{"discoveries":[{"year":2008,"finding":"BROX (FLJ32421) is farnesylated in vivo via its C-terminal CAAX motif; farnesylation is required for its perinuclear subcellular localization and for efficient interaction with CHMP4 proteins in cells. A farnesylation-defective mutant (C408S) shows diffuse cytoplasmic/nuclear distribution and fails to redistribute CHMP4b.","method":"Farnesyltransferase inhibitor (FTI-277) treatment causing electrophoretic mobility shift, co-expression pulldown (Strep-tag), fluorescence microscopy in HEK293 and HeLa cells, site-directed mutagenesis (C408S)","journal":"The FEBS journal","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (chemical inhibition, mutagenesis, co-pulldown, live imaging) in a single focused study on BROX","pmids":["18190528"],"is_preprint":false},{"year":2008,"finding":"BROX binds CHMP4b (an ESCRT-III subunit) via its Bro1 domain, as demonstrated by co-pulldown of FLAG-CHMP4b with Strep-tagged BROX from HEK293 cells.","method":"Co-expression pulldown (Strep-tag affinity purification) in HEK293 cells","journal":"The FEBS journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — confirmed by multiple independent labs (PMID 18190528, 19403673, 22484091, 22162750)","pmids":["18190528","19403673"],"is_preprint":false},{"year":2007,"finding":"BROX (FLJ32421, termed BROFTI) is an exclusive farnesyltransferase substrate; direct experimental evidence confirmed selective farnesylation of BROX.","method":"In vivo prenylation assay / biochemical verification of farnesylation","journal":"PLoS computational biology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — experimental verification reported but described briefly in a large-scale prediction study; single lab","pmids":["17411337"],"is_preprint":false},{"year":2009,"finding":"The Bro1 domain of BROX binds the HIV-1 nucleocapsid (NC) domain of Gag and stimulates virus-like particle (VLP) production as potently as the ALIX Bro1 domain in a minimal Gag rescue assay. Disruption of the CHMP4 binding site of BROX only partially reduces this VLP-stimulating activity, indicating an additional CHMP4-independent mechanism.","method":"VLP production assay (minimal HIV-1 Gag rescue), binding assays, site-directed mutagenesis of CHMP4-binding surface","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional VLP assay with mutagenesis, single lab but multiple orthogonal readouts","pmids":["19403673"],"is_preprint":false},{"year":2011,"finding":"Crystal structure of human BROX reveals a single Bro1 domain with a 'boomerang' fold similar to ALIX and Bro1p. Two conserved hydrophobic surfaces are identified: Surface 1 (concave face) forms the CHMP4 binding site; Surface 2 is at the narrow end. BROX lacks the extended Phe105 loop present in ALIX that is required for ALIX function in HIV-1 budding.","method":"X-ray crystallography; structure-guided mutagenesis of ALIX for functional comparison","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure solved and independently confirmed by a second structural study (PMID 21889351); functional mutagenesis included","pmids":["22162750","21889351"],"is_preprint":false},{"year":2011,"finding":"Crystal structures of the Bro1 domains of BROX (and HD-PTP) reveal typical boomerang folds but lack the unique Phe105-containing loop of ALIX; this structural difference correlates with the inability of BROX Bro1 domain to rescue HIV-1 release defects as effectively as ALIX Bro1.","method":"X-ray crystallography of BROX Bro1 domain; functional comparison with ALIX by HIV-1 budding assays","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 / Strong — independent structural determination replicated in two labs (PMID 22162750 and 21889351), with functional budding assays","pmids":["21889351"],"is_preprint":false},{"year":2012,"finding":"BROX binds both CHMP4B and CHMP5 C-terminal tails at the same concave surface of its Bro1 domain, but via distinct modes: CHMP4B C-terminal tail forms an amphipathic α-helix, while CHMP5 C-terminal tail forms a tandem β-hairpin that also engages BROX residue Y348 via a unique hydrophobic pocket. CHMP5 specifically recruits endogenous BROX to detergent-resistant membrane fractions.","method":"Crystal structure determination of BROX:CHMP4B and BROX:CHMP5 complexes; cell fractionation; co-immunoprecipitation","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 / Strong — atomic structures of two distinct complexes plus cell-based fractionation experiments, single lab with multiple orthogonal methods","pmids":["22484091"],"is_preprint":false},{"year":2016,"finding":"BROX Bro1 domain binds CHMP4B but NOT STAM2; the residue Arg145 of BROX (corresponding to Thr145 in HD-PTP) is a determinant of binding specificity — Brox-mimicking mutations at this position in HD-PTP abolish STAM2 binding, explaining why BROX cannot engage ESCRT-0.","method":"Crystal structure of HD-PTP Bro1:STAM2 complex; site-directed mutagenesis; binding assays","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — structural and mutagenesis data define BROX's binding specificity, but the BROX-relevant finding is derived from comparative structural analysis rather than direct mutagenesis of BROX itself","pmids":["26866605"],"is_preprint":false},{"year":2011,"finding":"BROX knockdown (siRNA) in HuH-7-derived RSc cells suppresses infectious HCV release into culture supernatants without significantly affecting intracellular HCV infectivity or RNA replication, indicating BROX is required for the HCV production/budding step.","method":"siRNA knockdown; HCV infectivity assay (supernatant); Core protein quantification; intracellular RNA replication measurement","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — clean KD with defined functional phenotype but single lab, single method approach","pmids":["21264300"],"is_preprint":false},{"year":2020,"finding":"BROX knockdown by siRNA does not affect final HSV-1 replication yields in single-step growth experiments, establishing that BROX is dispensable for HSV-1 replication.","method":"siRNA knockdown confirmed by Western blot; quantitative HSV-1 single-step growth assay","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — negative finding (BROX dispensable for HSV-1) established by clean KD with quantitative viral growth assay, single lab","pmids":["31748394"],"is_preprint":false},{"year":2021,"finding":"BROX is required to accelerate repair of nuclear envelope ruptures (NERDI). BROX binds Nesprin-2G (a LINC complex component), and this interaction promotes Nesprin-2G ubiquitination and facilitates relaxation of compressive actin-based mechanical stress at rupture sites, thereby enabling efficient membrane resealing by ESCRT machinery.","method":"siRNA/KO loss-of-function with nuclear envelope repair kinetics assay; co-immunoprecipitation (BROX–Nesprin-2G interaction); ubiquitination assay; live-cell imaging","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (Co-IP, live imaging, ubiquitination assay, functional NERDI repair assay) in single rigorous study establishing a novel mechanistic role","pmids":["34818527"],"is_preprint":false}],"current_model":"BROX is a farnesylated, single Bro1 domain-containing ESCRT-associated protein that localizes to perinuclear membranes via its CAAX-mediated farnesylation; its boomerang-shaped Bro1 domain binds CHMP4B (via an amphipathic α-helix) and CHMP5 (via a β-hairpin) at the same concave surface, engages HIV-1 Gag nucleocapsid to stimulate viral budding, and—through binding to the LINC complex protein Nesprin-2G—promotes Nesprin-2G ubiquitination and mechanical stress relaxation to accelerate ESCRT-dependent nuclear envelope rupture repair."},"narrative":{"mechanistic_narrative":"BROX is a farnesylated, single Bro1 domain-containing ESCRT-associated protein that links the ESCRT-III machinery to membrane remodeling events including viral budding and nuclear envelope repair [PMID:18190528, PMID:34818527]. C-terminal CAAX-mediated farnesylation directs BROX to perinuclear membranes and is required for efficient engagement of CHMP4 proteins in cells, with a farnesylation-defective C408S mutant becoming diffusely distributed and failing to redistribute CHMP4b [PMID:18190528]. Its boomerang-shaped Bro1 domain presents a concave hydrophobic surface that binds both the CHMP4B C-terminal tail, which docks as an amphipathic α-helix, and the CHMP5 C-terminal tail, which docks as a tandem β-hairpin engaging a unique pocket at residue Y348; CHMP5 specifically recruits endogenous BROX to detergent-resistant membranes [PMID:22162750, PMID:21889351, PMID:22484091]. BROX lacks the ALIX-specific Phe105 loop and the ESCRT-0 (STAM2)-binding determinant present in HD-PTP, restricting its repertoire of ESCRT contacts [PMID:21889351, PMID:26866605]. Functionally, the BROX Bro1 domain binds the HIV-1 Gag nucleocapsid and stimulates virus-like particle production through both CHMP4-dependent and CHMP4-independent routes [PMID:19403673], and BROX is required for the budding step of infectious HCV release [PMID:21264300]. In nuclear envelope rupture repair, BROX binds the LINC complex protein Nesprin-2G, promoting its ubiquitination and relaxation of compressive actin-based mechanical stress to accelerate ESCRT-dependent resealing [PMID:34818527].","teleology":[{"year":2007,"claim":"Establishing whether BROX is a genuine prenylation substrate addressed how this uncharacterized protein might be targeted to membranes.","evidence":"in vivo prenylation/biochemical verification of selective farnesylation in a large-scale prediction study","pmids":["17411337"],"confidence":"Medium","gaps":["Reported briefly within a prediction-focused study","Did not define the functional consequence of farnesylation"]},{"year":2008,"claim":"Demonstrating that CAAX-mediated farnesylation controls BROX perinuclear localization and CHMP4 engagement connected a lipid modification to ESCRT recruitment.","evidence":"farnesyltransferase inhibitor treatment, C408S mutagenesis, co-pulldown, and fluorescence microscopy in HEK293/HeLa","pmids":["18190528"],"confidence":"High","gaps":["Did not resolve the structural basis of CHMP4 binding","Cellular pathway requiring this localization not defined"]},{"year":2009,"claim":"Showing the BROX Bro1 domain binds HIV-1 Gag nucleocapsid and stimulates VLP production placed BROX functionally alongside ALIX in viral budding while revealing a CHMP4-independent activity.","evidence":"minimal Gag rescue VLP assay with CHMP4-binding-surface mutagenesis","pmids":["19403673"],"confidence":"Medium","gaps":["Molecular basis of the CHMP4-independent activity not identified","Single lab; physiological relevance to authentic infection untested"]},{"year":2011,"claim":"Crystal structures of the BROX Bro1 domain defined its boomerang fold and two conserved hydrophobic surfaces, and explained why it is a weaker HIV-1 budding factor than ALIX by the absence of the Phe105 loop.","evidence":"X-ray crystallography (two independent structural studies) with structure-guided functional comparison to ALIX in budding assays","pmids":["22162750","21889351"],"confidence":"High","gaps":["Did not capture BROX bound to a partner","Function of Surface 2 at the narrow end undefined"]},{"year":2011,"claim":"Loss-of-function in cells showed BROX is required for infectious HCV release at the production/budding step, extending its ESCRT-associated role to a second enveloped virus.","evidence":"siRNA knockdown with HCV supernatant infectivity, Core quantification, and intracellular RNA replication measurement","pmids":["21264300"],"confidence":"Medium","gaps":["Direct interaction with HCV components not shown","Single method/single lab"]},{"year":2012,"claim":"Structures of BROX:CHMP4B and BROX:CHMP5 complexes showed the same concave surface binds two ESCRT-III tails in distinct modes and that CHMP5 recruits BROX to detergent-resistant membranes, defining the molecular logic of its ESCRT contacts.","evidence":"crystal structures of two complexes plus cell fractionation and co-immunoprecipitation","pmids":["22484091"],"confidence":"High","gaps":["Functional consequence of dual CHMP4B/CHMP5 binding for membrane scission not established","Whether binding is competitive in cells unresolved"]},{"year":2016,"claim":"Comparative structural analysis identified Arg145 as the determinant excluding ESCRT-0/STAM2 binding, explaining BROX's restricted interactome relative to HD-PTP.","evidence":"crystal structure of HD-PTP Bro1:STAM2 with site-directed mutagenesis and binding assays","pmids":["26866605"],"confidence":"Medium","gaps":["BROX-relevant conclusion inferred from HD-PTP mutagenesis rather than direct BROX mutagenesis","Biological consequence of lacking ESCRT-0 engagement untested"]},{"year":2020,"claim":"A clean knockdown established that BROX is dispensable for HSV-1 replication, bounding its viral requirement to specific enveloped viruses.","evidence":"siRNA knockdown (Western-confirmed) with quantitative single-step HSV-1 growth assay","pmids":["31748394"],"confidence":"Medium","gaps":["Negative result; does not rule out subtle or context-specific roles","Single lab"]},{"year":2021,"claim":"Defining BROX as a Nesprin-2G-binding factor that promotes its ubiquitination and mechanical stress relaxation established a physiological role in accelerating ESCRT-dependent nuclear envelope rupture repair.","evidence":"loss-of-function NERDI repair kinetics, BROX–Nesprin-2G co-immunoprecipitation, ubiquitination assay, and live-cell imaging","pmids":["34818527"],"confidence":"High","gaps":["The ubiquitin ligase acting on Nesprin-2G not identified","Whether farnesylation/CHMP binding are required for the NERDI role not dissected"]},{"year":null,"claim":"How BROX coordinates its farnesylation, dual ESCRT-III contacts, and substrate recognition to drive distinct membrane-remodeling outcomes remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["The CHMP4-independent budding mechanism is unidentified","No structure of full-length farnesylated BROX on membranes","Endogenous physiological pathways beyond NERDI not mapped"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,6]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[10]}],"localization":[{"term_id":"GO:0005635","term_label":"nuclear envelope","supporting_discovery_ids":[0,10]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[6]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[3,8]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[3,8]}],"complexes":[],"partners":["CHMP4B","CHMP5","SYNE2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q5VW32","full_name":"BRO1 domain-containing protein BROX","aliases":["BRO1 domain- and CAAX motif-containing protein"],"length_aa":411,"mass_kda":46.5,"function":"Nuclear envelope-associated factor that is involved in the nuclear envelope ruptures during interphase (NERDI) repair, where it is locally recruited by CHMP5 and reduces cytoskeletal stress through its action on SYN2 to help reseal the ruptured membrane","subcellular_location":"Nucleus membrane","url":"https://www.uniprot.org/uniprotkb/Q5VW32/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/BROX","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/BROX","total_profiled":1310},"omim":[{"mim_id":"613850","title":"INOSINE TRIPHOSPHATASE DEFICIENCY","url":"https://www.omim.org/entry/613850"},{"mim_id":"608400","title":"USHERIN; USH2A","url":"https://www.omim.org/entry/608400"},{"mim_id":"601412","title":"DEAFNESS, AUTOSOMAL DOMINANT 7; DFNA7","url":"https://www.omim.org/entry/601412"},{"mim_id":"276901","title":"USHER SYNDROME, TYPE IIA; USH2A","url":"https://www.omim.org/entry/276901"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Golgi apparatus","reliability":"Approved"},{"location":"Nucleoplasm","reliability":"Additional"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/BROX"},"hgnc":{"alias_symbol":["FLJ32421"],"prev_symbol":["C1orf58"]},"alphafold":{"accession":"Q5VW32","domains":[{"cath_id":"1.25.40.280","chopping":"89-359","consensus_level":"medium","plddt":95.9025,"start":89,"end":359}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5VW32","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q5VW32-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q5VW32-F1-predicted_aligned_error_v6.png","plddt_mean":92.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=BROX","jax_strain_url":"https://www.jax.org/strain/search?query=BROX"},"sequence":{"accession":"Q5VW32","fasta_url":"https://rest.uniprot.org/uniprotkb/Q5VW32.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q5VW32/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5VW32"}},"corpus_meta":[{"pmid":"17411337","id":"PMC_17411337","title":"Towards complete sets of farnesylated and geranylgeranylated proteins.","date":"2007","source":"PLoS computational biology","url":"https://pubmed.ncbi.nlm.nih.gov/17411337","citation_count":127,"is_preprint":false},{"pmid":"21264300","id":"PMC_21264300","title":"The ESCRT system is required for hepatitis C virus production.","date":"2011","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/21264300","citation_count":78,"is_preprint":false},{"pmid":"19403673","id":"PMC_19403673","title":"Divergent Bro1 domains share the capacity to bind human immunodeficiency virus type 1 nucleocapsid and to enhance virus-like particle production.","date":"2009","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/19403673","citation_count":37,"is_preprint":false},{"pmid":"18190528","id":"PMC_18190528","title":"Brox, a novel farnesylated Bro1 domain-containing protein that associates with charged multivesicular body protein 4 (CHMP4).","date":"2008","source":"The FEBS 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the interaction of Brox with the C-terminal tails of CHMP5 and CHMP4B.","date":"2012","source":"Structure (London, England : 1993)","url":"https://pubmed.ncbi.nlm.nih.gov/22484091","citation_count":20,"is_preprint":false},{"pmid":"26866605","id":"PMC_26866605","title":"Structural Study of the HD-PTP Bro1 Domain in a Complex with the Core Region of STAM2, a Subunit of ESCRT-0.","date":"2016","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/26866605","citation_count":18,"is_preprint":false},{"pmid":"31748394","id":"PMC_31748394","title":"The ESCRT-II Subunit EAP20/VPS25 and the Bro1 Domain Proteins HD-PTP and BROX Are Individually Dispensable for Herpes Simplex Virus 1 Replication.","date":"2020","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/31748394","citation_count":17,"is_preprint":false},{"pmid":"34818527","id":"PMC_34818527","title":"The ESCRT machinery counteracts Nesprin-2G-mediated mechanical forces during nuclear envelope repair.","date":"2021","source":"Developmental cell","url":"https://pubmed.ncbi.nlm.nih.gov/34818527","citation_count":17,"is_preprint":false},{"pmid":"32385852","id":"PMC_32385852","title":"BROX haploinsufficiency in familial nonmedullary thyroid cancer.","date":"2020","source":"Journal of endocrinological investigation","url":"https://pubmed.ncbi.nlm.nih.gov/32385852","citation_count":10,"is_preprint":false},{"pmid":"25849655","id":"PMC_25849655","title":"The neuronal-specific SGK1.1 (SGK1_v2) kinase as a transcriptional modulator of BAG4, Brox, and PPP1CB genes expression.","date":"2015","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/25849655","citation_count":2,"is_preprint":false},{"pmid":"41952686","id":"PMC_41952686","title":"Novel transcriptomic alterations in poorly differentiated endometrial carcinomas: evidence from South African women.","date":"2026","source":"Frontiers in oncology","url":"https://pubmed.ncbi.nlm.nih.gov/41952686","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.10.24.684477","title":"Separation of halide oxidation and substrate halogenation chemistries rationalizes site-selective vanadium dependent haloperoxidase catalysis","date":"2025-10-25","source":"bioRxiv","url":"https://doi.org/10.1101/2025.10.24.684477","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":10189,"output_tokens":2713,"usd":0.035631,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9894,"output_tokens":3202,"usd":0.06476,"stage2_stop_reason":"end_turn"},"total_usd":0.100391,"stage1_batch_id":"msgbatch_0179Z2pxRYBsXi2xADneafRw","stage2_batch_id":"msgbatch_01LaY9ZaVdgjWArcZo9eqQE5","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2008,\n      \"finding\": \"BROX (FLJ32421) is farnesylated in vivo via its C-terminal CAAX motif; farnesylation is required for its perinuclear subcellular localization and for efficient interaction with CHMP4 proteins in cells. A farnesylation-defective mutant (C408S) shows diffuse cytoplasmic/nuclear distribution and fails to redistribute CHMP4b.\",\n      \"method\": \"Farnesyltransferase inhibitor (FTI-277) treatment causing electrophoretic mobility shift, co-expression pulldown (Strep-tag), fluorescence microscopy in HEK293 and HeLa cells, site-directed mutagenesis (C408S)\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (chemical inhibition, mutagenesis, co-pulldown, live imaging) in a single focused study on BROX\",\n      \"pmids\": [\"18190528\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"BROX binds CHMP4b (an ESCRT-III subunit) via its Bro1 domain, as demonstrated by co-pulldown of FLAG-CHMP4b with Strep-tagged BROX from HEK293 cells.\",\n      \"method\": \"Co-expression pulldown (Strep-tag affinity purification) in HEK293 cells\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — confirmed by multiple independent labs (PMID 18190528, 19403673, 22484091, 22162750)\",\n      \"pmids\": [\"18190528\", \"19403673\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"BROX (FLJ32421, termed BROFTI) is an exclusive farnesyltransferase substrate; direct experimental evidence confirmed selective farnesylation of BROX.\",\n      \"method\": \"In vivo prenylation assay / biochemical verification of farnesylation\",\n      \"journal\": \"PLoS computational biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — experimental verification reported but described briefly in a large-scale prediction study; single lab\",\n      \"pmids\": [\"17411337\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The Bro1 domain of BROX binds the HIV-1 nucleocapsid (NC) domain of Gag and stimulates virus-like particle (VLP) production as potently as the ALIX Bro1 domain in a minimal Gag rescue assay. Disruption of the CHMP4 binding site of BROX only partially reduces this VLP-stimulating activity, indicating an additional CHMP4-independent mechanism.\",\n      \"method\": \"VLP production assay (minimal HIV-1 Gag rescue), binding assays, site-directed mutagenesis of CHMP4-binding surface\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional VLP assay with mutagenesis, single lab but multiple orthogonal readouts\",\n      \"pmids\": [\"19403673\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Crystal structure of human BROX reveals a single Bro1 domain with a 'boomerang' fold similar to ALIX and Bro1p. Two conserved hydrophobic surfaces are identified: Surface 1 (concave face) forms the CHMP4 binding site; Surface 2 is at the narrow end. BROX lacks the extended Phe105 loop present in ALIX that is required for ALIX function in HIV-1 budding.\",\n      \"method\": \"X-ray crystallography; structure-guided mutagenesis of ALIX for functional comparison\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure solved and independently confirmed by a second structural study (PMID 21889351); functional mutagenesis included\",\n      \"pmids\": [\"22162750\", \"21889351\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Crystal structures of the Bro1 domains of BROX (and HD-PTP) reveal typical boomerang folds but lack the unique Phe105-containing loop of ALIX; this structural difference correlates with the inability of BROX Bro1 domain to rescue HIV-1 release defects as effectively as ALIX Bro1.\",\n      \"method\": \"X-ray crystallography of BROX Bro1 domain; functional comparison with ALIX by HIV-1 budding assays\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — independent structural determination replicated in two labs (PMID 22162750 and 21889351), with functional budding assays\",\n      \"pmids\": [\"21889351\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"BROX binds both CHMP4B and CHMP5 C-terminal tails at the same concave surface of its Bro1 domain, but via distinct modes: CHMP4B C-terminal tail forms an amphipathic α-helix, while CHMP5 C-terminal tail forms a tandem β-hairpin that also engages BROX residue Y348 via a unique hydrophobic pocket. CHMP5 specifically recruits endogenous BROX to detergent-resistant membrane fractions.\",\n      \"method\": \"Crystal structure determination of BROX:CHMP4B and BROX:CHMP5 complexes; cell fractionation; co-immunoprecipitation\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — atomic structures of two distinct complexes plus cell-based fractionation experiments, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"22484091\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"BROX Bro1 domain binds CHMP4B but NOT STAM2; the residue Arg145 of BROX (corresponding to Thr145 in HD-PTP) is a determinant of binding specificity — Brox-mimicking mutations at this position in HD-PTP abolish STAM2 binding, explaining why BROX cannot engage ESCRT-0.\",\n      \"method\": \"Crystal structure of HD-PTP Bro1:STAM2 complex; site-directed mutagenesis; binding assays\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — structural and mutagenesis data define BROX's binding specificity, but the BROX-relevant finding is derived from comparative structural analysis rather than direct mutagenesis of BROX itself\",\n      \"pmids\": [\"26866605\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"BROX knockdown (siRNA) in HuH-7-derived RSc cells suppresses infectious HCV release into culture supernatants without significantly affecting intracellular HCV infectivity or RNA replication, indicating BROX is required for the HCV production/budding step.\",\n      \"method\": \"siRNA knockdown; HCV infectivity assay (supernatant); Core protein quantification; intracellular RNA replication measurement\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — clean KD with defined functional phenotype but single lab, single method approach\",\n      \"pmids\": [\"21264300\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"BROX knockdown by siRNA does not affect final HSV-1 replication yields in single-step growth experiments, establishing that BROX is dispensable for HSV-1 replication.\",\n      \"method\": \"siRNA knockdown confirmed by Western blot; quantitative HSV-1 single-step growth assay\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — negative finding (BROX dispensable for HSV-1) established by clean KD with quantitative viral growth assay, single lab\",\n      \"pmids\": [\"31748394\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"BROX is required to accelerate repair of nuclear envelope ruptures (NERDI). BROX binds Nesprin-2G (a LINC complex component), and this interaction promotes Nesprin-2G ubiquitination and facilitates relaxation of compressive actin-based mechanical stress at rupture sites, thereby enabling efficient membrane resealing by ESCRT machinery.\",\n      \"method\": \"siRNA/KO loss-of-function with nuclear envelope repair kinetics assay; co-immunoprecipitation (BROX–Nesprin-2G interaction); ubiquitination assay; live-cell imaging\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (Co-IP, live imaging, ubiquitination assay, functional NERDI repair assay) in single rigorous study establishing a novel mechanistic role\",\n      \"pmids\": [\"34818527\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"BROX is a farnesylated, single Bro1 domain-containing ESCRT-associated protein that localizes to perinuclear membranes via its CAAX-mediated farnesylation; its boomerang-shaped Bro1 domain binds CHMP4B (via an amphipathic α-helix) and CHMP5 (via a β-hairpin) at the same concave surface, engages HIV-1 Gag nucleocapsid to stimulate viral budding, and—through binding to the LINC complex protein Nesprin-2G—promotes Nesprin-2G ubiquitination and mechanical stress relaxation to accelerate ESCRT-dependent nuclear envelope rupture repair.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"BROX is a farnesylated, single Bro1 domain-containing ESCRT-associated protein that links the ESCRT-III machinery to membrane remodeling events including viral budding and nuclear envelope repair [#0, #10]. C-terminal CAAX-mediated farnesylation directs BROX to perinuclear membranes and is required for efficient engagement of CHMP4 proteins in cells, with a farnesylation-defective C408S mutant becoming diffusely distributed and failing to redistribute CHMP4b [#0]. Its boomerang-shaped Bro1 domain presents a concave hydrophobic surface that binds both the CHMP4B C-terminal tail, which docks as an amphipathic \\u03b1-helix, and the CHMP5 C-terminal tail, which docks as a tandem \\u03b2-hairpin engaging a unique pocket at residue Y348; CHMP5 specifically recruits endogenous BROX to detergent-resistant membranes [#4, #6]. BROX lacks the ALIX-specific Phe105 loop and the ESCRT-0 (STAM2)-binding determinant present in HD-PTP, restricting its repertoire of ESCRT contacts [#5, #7]. Functionally, the BROX Bro1 domain binds the HIV-1 Gag nucleocapsid and stimulates virus-like particle production through both CHMP4-dependent and CHMP4-independent routes [#3], and BROX is required for the budding step of infectious HCV release [#8]. In nuclear envelope rupture repair, BROX binds the LINC complex protein Nesprin-2G, promoting its ubiquitination and relaxation of compressive actin-based mechanical stress to accelerate ESCRT-dependent resealing [#10].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Establishing whether BROX is a genuine prenylation substrate addressed how this uncharacterized protein might be targeted to membranes.\",\n      \"evidence\": \"in vivo prenylation/biochemical verification of selective farnesylation in a large-scale prediction study\",\n      \"pmids\": [\"17411337\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Reported briefly within a prediction-focused study\", \"Did not define the functional consequence of farnesylation\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Demonstrating that CAAX-mediated farnesylation controls BROX perinuclear localization and CHMP4 engagement connected a lipid modification to ESCRT recruitment.\",\n      \"evidence\": \"farnesyltransferase inhibitor treatment, C408S mutagenesis, co-pulldown, and fluorescence microscopy in HEK293/HeLa\",\n      \"pmids\": [\"18190528\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Did not resolve the structural basis of CHMP4 binding\", \"Cellular pathway requiring this localization not defined\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Showing the BROX Bro1 domain binds HIV-1 Gag nucleocapsid and stimulates VLP production placed BROX functionally alongside ALIX in viral budding while revealing a CHMP4-independent activity.\",\n      \"evidence\": \"minimal Gag rescue VLP assay with CHMP4-binding-surface mutagenesis\",\n      \"pmids\": [\"19403673\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Molecular basis of the CHMP4-independent activity not identified\", \"Single lab; physiological relevance to authentic infection untested\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Crystal structures of the BROX Bro1 domain defined its boomerang fold and two conserved hydrophobic surfaces, and explained why it is a weaker HIV-1 budding factor than ALIX by the absence of the Phe105 loop.\",\n      \"evidence\": \"X-ray crystallography (two independent structural studies) with structure-guided functional comparison to ALIX in budding assays\",\n      \"pmids\": [\"22162750\", \"21889351\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Did not capture BROX bound to a partner\", \"Function of Surface 2 at the narrow end undefined\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Loss-of-function in cells showed BROX is required for infectious HCV release at the production/budding step, extending its ESCRT-associated role to a second enveloped virus.\",\n      \"evidence\": \"siRNA knockdown with HCV supernatant infectivity, Core quantification, and intracellular RNA replication measurement\",\n      \"pmids\": [\"21264300\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Direct interaction with HCV components not shown\", \"Single method/single lab\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Structures of BROX:CHMP4B and BROX:CHMP5 complexes showed the same concave surface binds two ESCRT-III tails in distinct modes and that CHMP5 recruits BROX to detergent-resistant membranes, defining the molecular logic of its ESCRT contacts.\",\n      \"evidence\": \"crystal structures of two complexes plus cell fractionation and co-immunoprecipitation\",\n      \"pmids\": [\"22484091\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Functional consequence of dual CHMP4B/CHMP5 binding for membrane scission not established\", \"Whether binding is competitive in cells unresolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Comparative structural analysis identified Arg145 as the determinant excluding ESCRT-0/STAM2 binding, explaining BROX's restricted interactome relative to HD-PTP.\",\n      \"evidence\": \"crystal structure of HD-PTP Bro1:STAM2 with site-directed mutagenesis and binding assays\",\n      \"pmids\": [\"26866605\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"BROX-relevant conclusion inferred from HD-PTP mutagenesis rather than direct BROX mutagenesis\", \"Biological consequence of lacking ESCRT-0 engagement untested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"A clean knockdown established that BROX is dispensable for HSV-1 replication, bounding its viral requirement to specific enveloped viruses.\",\n      \"evidence\": \"siRNA knockdown (Western-confirmed) with quantitative single-step HSV-1 growth assay\",\n      \"pmids\": [\"31748394\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Negative result; does not rule out subtle or context-specific roles\", \"Single lab\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defining BROX as a Nesprin-2G-binding factor that promotes its ubiquitination and mechanical stress relaxation established a physiological role in accelerating ESCRT-dependent nuclear envelope rupture repair.\",\n      \"evidence\": \"loss-of-function NERDI repair kinetics, BROX\\u2013Nesprin-2G co-immunoprecipitation, ubiquitination assay, and live-cell imaging\",\n      \"pmids\": [\"34818527\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"The ubiquitin ligase acting on Nesprin-2G not identified\", \"Whether farnesylation/CHMP binding are required for the NERDI role not dissected\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How BROX coordinates its farnesylation, dual ESCRT-III contacts, and substrate recognition to drive distinct membrane-remodeling outcomes remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"The CHMP4-independent budding mechanism is unidentified\", \"No structure of full-length farnesylated BROX on membranes\", \"Endogenous physiological pathways beyond NERDI not mapped\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 6]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [10]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005635\", \"supporting_discovery_ids\": [0, 10]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [3, 8]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [3, 8]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"CHMP4B\", \"CHMP5\", \"SYNE2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}