{"gene":"SPAG5","run_date":"2026-06-10T07:46:38","timeline":{"discoveries":[{"year":2001,"finding":"hMAP126/SPAG5 is a mitotic spindle-associated protein that is post-translationally modified and phosphorylated by p34(cdc2) kinase in vitro, and its subcellular distribution localizes to the mitotic spindle.","method":"Polyclonal antibody immunolocalization; in vitro kinase assay with p34(cdc2)","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 1–2 / Weak — in vitro kinase assay plus localization, single lab, no replication reported","pmids":["11549262"],"is_preprint":false},{"year":2001,"finding":"Rat Spag5 (ortholog of human SPAG5) encodes a ~200 kDa testicular protein that interacts strongly with Odf1 (the 27 kDa major outer dense fiber protein) via a leucine zipper motif in the C-terminal region of SPAG5; the downstream leucine zipper is specifically required for this interaction.","method":"Yeast two-hybrid interaction screen; leucine zipper mutagenesis/domain mapping","journal":"Molecular reproduction and development","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — yeast two-hybrid with domain mapping, single lab, not biochemically reconstituted","pmids":["11468777"],"is_preprint":false},{"year":2002,"finding":"Disruption of the Spag5 gene in mice by homologous recombination (Spag5-null mice) does not impair spermatogenesis or fertility, indicating that any role of Spag5 in spermatogenesis is compensated by other proteins.","method":"Gene targeting by homologous recombination; phenotypic analysis of null mice","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — complete knockout with rigorous phenotypic analysis, peer-reviewed; negative result robustly established","pmids":["11884588"],"is_preprint":false},{"year":2006,"finding":"In somatic cells, rat Spag5 localizes to the endoplasmic reticulum and microtubules (including a 58 kDa microtubule-associated isoform confirmed by in vitro MT-binding assays), whereas in elongated spermatids and epididymal sperm it associates with outer dense fibers but not axonemal microtubules — demonstrating cell-type-specific localization and function.","method":"Immunofluorescence; in vitro microtubule-binding assay; immuno-electron microscopy; Western blotting of fractionated cells","journal":"Molecular reproduction and development","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (in vitro MT-binding, immuno-EM, fractionation) in single lab","pmids":["16211599"],"is_preprint":false},{"year":2006,"finding":"A duplicated 25 bp insertion mutation in exon 6 of the Spag5 gene in hypogonadic (hgn/hgn) rats produces a truncated Spag5 protein lacking the primary spindle-targeting domain at the C-terminus, leading to defective spindle formation, abnormal mitosis of Sertoli cells, apoptotic Sertoli cell death, and testicular dysplasia — establishing that the C-terminal spindle-targeting domain is essential for SPAG5 function in cell division.","method":"Positional cloning; sequencing; immunostaining with Sertoli and germ cell markers; phenotypic analysis of mutant rats","journal":"Reproduction (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 2 / Moderate — natural loss-of-function allele with domain-level mapping and defined cellular phenotype, single lab","pmids":["16816335"],"is_preprint":false},{"year":2012,"finding":"SPAG5/Astrin localizes to centrosomal/basal body structures in photoreceptor cells of the retina and interacts with USH2AisoB interaction partner NINLisoB; SPAG5 was identified as interacting with USH2AisoB and NINLisoB at basal bodies postmitotically, connecting it to the Usher protein network involved in cilium function.","method":"Yeast two-hybrid; co-localization by immunofluorescence in retinal photoreceptors; in situ hybridization; quantitative PCR","journal":"Cilia","confidence":"Low","confidence_rationale":"Tier 3 / Weak — yeast two-hybrid interaction plus co-localization, single lab, limited biochemical validation","pmids":["23351521"],"is_preprint":false},{"year":2014,"finding":"SPAG5/Astrin physically interacts with ORP8 (OSBP-related protein 8), confirmed by yeast two-hybrid, pull-down, and co-immunoprecipitation; overexpressed ORP8 recruits SPAG5 onto ER membranes in interphase cells; SPAG5 knockdown significantly reduces ORP8-overexpression- and 25-hydroxycholesterol-induced G2/M cell cycle accumulation, placing SPAG5 downstream of ORP8 in oxysterol-mediated cell cycle regulation.","method":"Yeast two-hybrid; pull-down assay; co-immunoprecipitation; siRNA knockdown; cell cycle analysis by flow cytometry","journal":"Experimental cell research","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP, pull-down, and genetic epistasis (knockdown), multiple orthogonal methods, single lab","pmids":["24424245"],"is_preprint":false},{"year":2018,"finding":"SPAG5 physically interacts with centrosomal protein CEP55, and this interaction triggers phosphorylation of AKT at Ser473, activating PI3K/AKT signaling to promote hepatocellular carcinoma cell growth and metastasis; inhibition of PI3K/AKT markedly attenuates SPAG5-mediated cell growth.","method":"Co-immunoprecipitation; in vitro and in vivo functional assays; PI3K/AKT inhibitor treatment; miR-363-3p luciferase reporter assay","journal":"Molecular cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — co-IP plus pathway inhibitor rescue, single lab","pmids":["30089483"],"is_preprint":false},{"year":2018,"finding":"SPAG5 promotes HCC progression by downregulating SCARA5 through modulating β-catenin degradation, engaging the β-catenin/TCF4 signaling pathway; SCARA5 expression is inversely correlated with SPAG5 in HCC tissues.","method":"shRNA knockdown; overexpression; cell proliferation/apoptosis assays; Western blot for β-catenin pathway components; in vivo xenograft","journal":"Journal of experimental & clinical cancer research : CR","confidence":"Medium","confidence_rationale":"Tier 2–3 / Weak — loss-of-function with defined signaling outcome, single lab, mechanism inferred from pathway markers without biochemical reconstitution","pmids":["30249289"],"is_preprint":false},{"year":2019,"finding":"SPAG5 interacts with c-MYC binding protein (MYCBP), increasing MYCBP protein levels and enhancing c-MYC transcriptional activity, which in turn promotes expression of CDC20, CDC25C, BRCA1, BRCA2, and RAD51 in triple-negative breast cancer; knockdown of MYCBP or c-MYC abolishes SPAG5-induced cell-cycle progression and proliferation.","method":"Co-immunoprecipitation; Western blot; siRNA knockdown epistasis; in vitro and in vivo functional assays","journal":"Journal of hematology & oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — co-IP plus epistasis knockdown, single lab","pmids":["30736840"],"is_preprint":false},{"year":2019,"finding":"p53-p21 axis transcriptionally suppresses SPAG5 expression: MDM2 inhibitor Nutlin-3a restores p53/p21 and suppresses SPAG5 in wild-type p53 cells but not p53-null cells; p53 or p21 knockdown attenuates Nutlin-3a-induced repression of SPAG5, establishing p53-p21 as required for SPAG5 repression.","method":"MDM2 inhibitor treatment; siRNA knockdown of p53/p21; Western blot; qRT-PCR","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — pharmacological and genetic perturbation with consistent results, single lab","pmids":["30955859"],"is_preprint":false},{"year":2020,"finding":"SPAG5 is a direct transcriptional target of the YAP/TAZ/TEAD axis (Hippo pathway); depletion of YAP, TAZ, or TEAD strongly reduces SPAG5 expression; SPAG5 is also directly targeted post-transcriptionally by miR-10b-3p; SPAG5 depletion impairs cancer cell cycle progression, proliferation, and migration.","method":"ChIP/luciferase reporter assays for TEAD binding to SPAG5 promoter; YAP/TAZ/TEAD siRNA knockdown; miR-10b-3p target validation; cell cycle and proliferation assays","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — transcriptional target validated by reporter assay and genetic knockdown, single lab","pmids":["33230261"],"is_preprint":false},{"year":2020,"finding":"SPAG5-AS1 (a lncRNA neighbor of SPAG5) interacts with USP14, leading to de-ubiquitination and stabilization of SPAG5 protein; YY1 transcriptionally upregulates both SPAG5-AS1 and SPAG5; SPAG5 promotes AKT/mTOR signaling, inhibiting autophagy and aggravating apoptosis in podocytes under high glucose.","method":"Pulldown; RIP; co-IP; ChIP; luciferase reporter assay; Western blot; flow cytometry; immunofluorescence","journal":"Cell proliferation","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — multiple biochemical assays (co-IP, pulldown, RIP, ChIP), single lab, involves lncRNA regulation of the SPAG5 protein","pmids":["31957155"],"is_preprint":false},{"year":2020,"finding":"SPAG5 promotes osteosarcoma metastasis by stabilizing FOXM1 protein (reducing its degradation), which in turn upregulates MMP2 expression, promoting invasion and migration; SPAG5 silencing inhibits the SPAG5-FOXM1-MMP2 axis and suppresses lung metastasis in vivo.","method":"siRNA/shRNA knockdown; Western blot for FOXM1 protein stability; in vitro invasion/migration assays; in vivo metastasis model","journal":"The international journal of biochemistry & cell biology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Weak — loss-of-function with defined molecular pathway, single lab, mechanism inferred from protein levels without direct ubiquitination assay","pmids":["32668328"],"is_preprint":false},{"year":2022,"finding":"Splicing factor SF3B4 promotes SPAG5 expression by ensuring effective splicing of SPAG5 pre-mRNA; SF3B4 knockdown causes intron retention in SPAG5 pre-mRNA, reducing mature SPAG5 mRNA and protein, and the pro-tumorigenic effects of SF3B4 overexpression in cervical cancer are impaired by SPAG5 deficiency.","method":"RNA-seq with alternative splicing analysis; SF3B4 siRNA knockdown; SPAG5 rescue experiments; in vitro and in vivo functional assays","journal":"Cell death discovery","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — RNA-seq splicing analysis plus genetic epistasis rescue, single lab","pmids":["35853859"],"is_preprint":false},{"year":2024,"finding":"SPAG5 promotes ferroptosis in colorectal cancer cells by upregulating intracellular reactive oxygen species (ROS); SPAG5 depletion reduces ROS levels and attenuates ferroptosis, diminishing chemotherapy efficacy; ROS inhibition reverses the chemotherapy-sensitizing effect of SPAG5.","method":"SPAG5 overexpression and knockdown; ROS measurement; ferroptosis assays; chemotherapy sensitivity assays in vitro and patient cohort analysis","journal":"Apoptosis : an international journal on programmed cell death","confidence":"Medium","confidence_rationale":"Tier 2–3 / Weak — loss- and gain-of-function with mechanistic ROS/ferroptosis readouts, single lab","pmids":["41774258"],"is_preprint":false},{"year":2024,"finding":"In triple-negative breast cancer, high SPAG5 expression is regulated by coordinated activity of YAP, mutant p53, and MYC; depletion of YAP or mutant p53 reduces SPAG5 expression and reduces MYC recruitment to the SPAG5 promoter; MYC targeting reduces SPAG5 expression and TNBC tumorigenicity in a SPAG5-expression-dependent manner.","method":"siRNA knockdown of YAP, mutant p53, MYC; ChIP for MYC at SPAG5 promoter; functional cell and in vivo assays; chemotherapy combination experiments","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — ChIP plus multiple genetic knockdowns, single lab","pmids":["39164278"],"is_preprint":false},{"year":2024,"finding":"SPAG5 knockdown activates autophagy and inhibits PI3K/Akt/mTOR signaling in endothelial cells treated with ox-LDL, reducing atherosclerotic plaque formation in ApoE-/- mice; autophagy inhibitor 3-MA reverses the protective effects of SPAG5 silencing, placing SPAG5 upstream of autophagy via PI3K/Akt/mTOR.","method":"siRNA/shRNA knockdown; Western blot for PI3K/Akt/mTOR phosphorylation and autophagy markers; GFP-LC3 puncta assay; 3-MA pharmacological rescue; in vivo ApoE-/- mouse model","journal":"BMC cardiovascular disorders","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — genetic and pharmacological epistasis in vitro and in vivo, single lab","pmids":["38807081"],"is_preprint":false}],"current_model":"SPAG5 (also known as hMAP126/Astrin/Deepest) is a microtubule-associated mitotic spindle protein phosphorylated by CDK1 (p34cdc2) that localizes to the spindle in dividing cells and to the endoplasmic reticulum/microtubules in interphase somatic cells, but to outer dense fibers in spermatozoa; its C-terminal spindle-targeting domain is essential for proper Sertoli cell mitosis in vivo, and in cancer contexts it drives proliferation and survival by interacting with CEP55 to activate PI3K/AKT signaling, stabilizing FOXM1 to upregulate MMP2, engaging MYCBP to amplify c-MYC transcriptional output, and modulating β-catenin degradation, while being transcriptionally regulated downstream of YAP/TAZ/TEAD and suppressed by the p53-p21 axis."},"narrative":{"mechanistic_narrative":"SPAG5 (hMAP126/Astrin) is a microtubule-associated mitotic spindle protein that is phosphorylated by p34(cdc2)/CDK1 and localizes to the mitotic spindle in dividing cells while distributing to the endoplasmic reticulum and microtubules in interphase somatic cells [PMID:11549262, PMID:16211599]. Its localization and binding partners are cell-type specific: in spermatids and sperm it associates with outer dense fibers through a C-terminal leucine-zipper interaction with ODF1 rather than with axonemal microtubules [PMID:11468777, PMID:16211599], and in interphase cells it can be recruited onto ER membranes by ORP8, downstream of which SPAG5 contributes to oxysterol-mediated G2/M cell cycle accumulation [PMID:24424245]. A C-terminal spindle-targeting domain is essential for SPAG5's function in cell division: a natural truncating mutation in rats abolishes spindle formation and causes abnormal Sertoli cell mitosis, apoptosis, and testicular dysplasia, although complete Spag5 knockout in mice leaves spermatogenesis and fertility intact, indicating functional compensation in the germline [PMID:11884588, PMID:16816335]. In cancer, SPAG5 acts as a proliferation- and survival-promoting hub: it interacts with CEP55 to activate PI3K/AKT signaling [PMID:30089483], engages MYCBP to amplify c-MYC transcriptional output driving cell-cycle genes [PMID:30736840], stabilizes FOXM1 to upregulate MMP2 and promote metastasis [PMID:32668328], and modulates beta-catenin degradation to repress SCARA5 [PMID:30249289]. SPAG5 expression is itself a convergence point of oncogenic transcriptional control, being a direct target of the YAP/TAZ/TEAD (Hippo) axis and, in triple-negative breast cancer, of coordinated YAP, mutant p53, and MYC activity, while the wild-type p53-p21 axis suppresses it [PMID:30955859, PMID:33230261, PMID:39164278]. Across multiple disease contexts SPAG5 also regulates PI3K/AKT/mTOR-dependent autophagy and ROS/ferroptosis balance [PMID:41774258, PMID:38807081].","teleology":[{"year":2001,"claim":"Established SPAG5's identity as a cell-cycle-regulated spindle protein by showing it associates with the mitotic spindle and is a CDK1 substrate, linking it to mitotic regulation.","evidence":"Polyclonal antibody immunolocalization and in vitro p34(cdc2) kinase assay","pmids":["11549262"],"confidence":"Medium","gaps":["In vitro kinase phosphorylation site not mapped","Functional consequence of phosphorylation on spindle binding undefined"]},{"year":2001,"claim":"Identified a tissue-specific binding partner, showing SPAG5 binds the outer dense fiber protein ODF1 via a C-terminal leucine zipper, implicating it in sperm tail structure.","evidence":"Yeast two-hybrid screen with leucine-zipper domain mapping in rat","pmids":["11468777"],"confidence":"Medium","gaps":["Interaction not biochemically reconstituted","Physiological role of ODF1 binding untested"]},{"year":2002,"claim":"Tested whether SPAG5 is required for spermatogenesis; null mice were fertile, revealing functional redundancy in the germline despite the ODF1 interaction.","evidence":"Homologous-recombination knockout and phenotypic analysis of null mice","pmids":["11884588"],"confidence":"High","gaps":["Compensating proteins not identified","Somatic/mitotic phenotypes not examined in this knockout"]},{"year":2006,"claim":"Resolved SPAG5's cell-type-specific localization, distinguishing ER/microtubule association in somatic cells from outer-dense-fiber association in sperm, defining context-dependent function.","evidence":"Immunofluorescence, in vitro microtubule-binding, immuno-EM, and fractionation in rat","pmids":["16211599"],"confidence":"High","gaps":["Microtubule-binding determinant not mapped at residue level","Isoform-specific functions unresolved"]},{"year":2006,"claim":"Established the C-terminal spindle-targeting domain as essential for cell division, since a natural truncation caused defective Sertoli cell mitosis and testicular dysplasia.","evidence":"Positional cloning of hgn/hgn rat mutant with marker immunostaining and phenotyping","pmids":["16816335"],"confidence":"High","gaps":["Molecular partners of the spindle-targeting domain not defined","Reconciliation with fertile mouse knockout phenotype unresolved"]},{"year":2013,"claim":"Connected SPAG5 to ciliary/basal body biology by placing it in the Usher protein network at photoreceptor basal bodies.","evidence":"Yeast two-hybrid and co-localization in retinal photoreceptors","pmids":["23351521"],"confidence":"Low","gaps":["Yeast two-hybrid interaction not biochemically confirmed","Functional role at basal bodies untested","Relevance to disease unknown"]},{"year":2014,"claim":"Showed SPAG5 acts downstream of ORP8 in oxysterol-driven cell cycle control, integrating it into lipid-sensing G2/M regulation at the ER.","evidence":"Yeast two-hybrid, pull-down, reciprocal co-IP, siRNA knockdown, and flow cytometry","pmids":["24424245"],"confidence":"High","gaps":["Mechanism linking ER recruitment to G2/M arrest unresolved","Direct binding interface not mapped"]},{"year":2018,"claim":"Defined an oncogenic mechanism whereby SPAG5-CEP55 binding activates PI3K/AKT to drive HCC growth and metastasis.","evidence":"Co-IP, PI3K/AKT inhibitor rescue, and in vitro/in vivo HCC assays","pmids":["30089483"],"confidence":"Medium","gaps":["How CEP55 binding triggers AKT Ser473 phosphorylation not mechanistically resolved","Single lab without reciprocal structural validation"]},{"year":2018,"claim":"Linked SPAG5 to Wnt/beta-catenin signaling, showing it represses SCARA5 by modulating beta-catenin degradation to promote HCC.","evidence":"shRNA/overexpression with Western blot of beta-catenin pathway and xenografts","pmids":["30249289"],"confidence":"Medium","gaps":["Direct effect of SPAG5 on beta-catenin turnover not biochemically demonstrated","Mechanism inferred from pathway markers only"]},{"year":2019,"claim":"Established a SPAG5-MYCBP-c-MYC axis amplifying c-MYC transcriptional output and cell-cycle gene expression in TNBC.","evidence":"Co-IP, Western blot, and siRNA epistasis with functional assays","pmids":["30736840"],"confidence":"Medium","gaps":["Mechanism by which SPAG5 raises MYCBP levels unknown","Direct vs indirect target gene effects not distinguished"]},{"year":2019,"claim":"Identified the p53-p21 axis as a required suppressor of SPAG5 transcription, defining tumor-suppressor control of SPAG5.","evidence":"Nutlin-3a treatment with p53/p21 knockdown, Western blot, and qRT-PCR","pmids":["30955859"],"confidence":"Medium","gaps":["Direct vs indirect transcriptional mechanism not resolved","p53/p21 binding to SPAG5 locus not demonstrated"]},{"year":2020,"claim":"Placed SPAG5 as a direct YAP/TAZ/TEAD Hippo-pathway target additionally repressed by miR-10b-3p, defining its upstream transcriptional/post-transcriptional regulation.","evidence":"ChIP/luciferase reporter for TEAD, YAP/TAZ/TEAD knockdown, and miRNA target validation","pmids":["33230261"],"confidence":"Medium","gaps":["Relative contribution of transcriptional vs miRNA control unquantified","Single lab"]},{"year":2020,"claim":"Defined post-translational stabilization of SPAG5 via the SPAG5-AS1/USP14 axis and downstream AKT/mTOR-driven autophagy suppression in podocytes.","evidence":"Pulldown, RIP, co-IP, ChIP, luciferase, and functional assays under high glucose","pmids":["31957155"],"confidence":"Medium","gaps":["Direct USP14 deubiquitination of SPAG5 not enzymatically reconstituted","Relevance beyond podocytes untested"]},{"year":2020,"claim":"Established a SPAG5-FOXM1-MMP2 axis driving osteosarcoma metastasis through FOXM1 protein stabilization.","evidence":"siRNA/shRNA knockdown, Western blot for FOXM1 stability, and in vivo metastasis model","pmids":["32668328"],"confidence":"Medium","gaps":["No direct ubiquitination assay for FOXM1 stabilization","Mechanism of SPAG5-FOXM1 interaction undefined"]},{"year":2022,"claim":"Showed SPAG5 expression depends on SF3B4-mediated splicing, identifying a splicing-level control point for SPAG5 in cervical cancer.","evidence":"RNA-seq splicing analysis, SF3B4 knockdown, and SPAG5 rescue assays","pmids":["35853859"],"confidence":"Medium","gaps":["Direct SF3B4 binding to SPAG5 pre-mRNA not shown","Specificity of splicing effect not established"]},{"year":2024,"claim":"Linked SPAG5 to redox-dependent cell death, showing it promotes ROS-driven ferroptosis and chemosensitivity in colorectal cancer.","evidence":"Gain/loss-of-function, ROS measurement, ferroptosis and chemosensitivity assays with patient cohort","pmids":["41774258"],"confidence":"Medium","gaps":["Mechanism by which SPAG5 elevates ROS unknown","Connection to mitotic function unclear"]},{"year":2024,"claim":"Extended SPAG5's PI3K/Akt/mTOR-autophagy role to vascular disease, where SPAG5 silencing activates autophagy and reduces atherosclerosis.","evidence":"Knockdown with PI3K/Akt/mTOR and autophagy readouts, 3-MA rescue, and ApoE-/- mouse model","pmids":["38807081"],"confidence":"Medium","gaps":["Direct molecular link between SPAG5 and PI3K/Akt activation undefined","Cell-autonomous vs systemic effects not separated"]},{"year":2024,"claim":"Refined SPAG5 transcriptional control in TNBC, showing coordinated YAP, mutant p53, and MYC activity with MYC recruitment to the SPAG5 promoter.","evidence":"siRNA knockdown of YAP/mutant p53/MYC, ChIP at SPAG5 promoter, and in vivo assays","pmids":["39164278"],"confidence":"Medium","gaps":["Direct interplay among YAP, mutant p53, and MYC at the locus not fully resolved","Single lab"]},{"year":null,"claim":"How SPAG5's core mitotic spindle function mechanistically connects to its diverse oncogenic signaling roles (PI3K/AKT, c-MYC, beta-catenin, FOXM1, ROS/ferroptosis) remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model linking spindle role to signaling roles","Structural basis of partner interactions undefined","Direct enzymatic activities, if any, unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0,3]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[7,9,13]}],"localization":[{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[3,6]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[3]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[5]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[0,4,6]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[7,11,17]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[7,8,13]}],"complexes":[],"partners":["ODF1","ORP8","CEP55","MYCBP","USP14","NINL","USH2A","FOXM1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q96R06","full_name":"Sperm-associated antigen 5","aliases":["Astrin","Deepest","Mitotic spindle-associated protein p126","MAP126"],"length_aa":1193,"mass_kda":134.4,"function":"Essential component of the mitotic spindle required for normal chromosome segregation and progression into anaphase (PubMed:11724960, PubMed:12356910, PubMed:27462074). Required for chromosome alignment, normal timing of sister chromatid segregation, and maintenance of spindle pole architecture (PubMed:17664331, PubMed:27462074). In complex with SKAP, promotes stable microtubule-kinetochore attachments. May contribute to the regulation of separase activity. May regulate AURKA localization to mitotic spindle, but not to centrosomes and CCNB1 localization to both mitotic spindle and centrosomes (PubMed:18361916, PubMed:21402792). Involved in centriole duplication. Required for CDK5RAP2, CEP152, WDR62 and CEP63 centrosomal localization and promotes the centrosomal localization of CDK2 (PubMed:26297806). In non-mitotic cells, upon stress induction, inhibits mammalian target of rapamycin complex 1 (mTORC1) association and recruits the mTORC1 component RPTOR to stress granules (SGs), thereby preventing mTORC1 hyperactivation-induced apoptosis (PubMed:23953116). May enhance GSK3B-mediated phosphorylation of other substrates, such as MAPT/TAU (PubMed:18055457)","subcellular_location":"Cytoplasm; Cytoplasm, cytoskeleton; Cytoplasm, cytoskeleton, spindle; Cytoplasm, cytoskeleton, spindle pole; Chromosome, centromere, kinetochore; Midbody; Cytoplasm, cytoskeleton, microtubule organizing center, centrosome; Cytoplasmic granule; Cytoplasm, cytoskeleton, microtubule organizing center, centrosome, centriolar satellite","url":"https://www.uniprot.org/uniprotkb/Q96R06/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/SPAG5","classification":"Common Essential","n_dependent_lines":477,"n_total_lines":1208,"dependency_fraction":0.39486754966887416},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"DYNLL1","stoichiometry":0.2},{"gene":"DYNLL2","stoichiometry":0.2},{"gene":"MAP4","stoichiometry":0.2},{"gene":"OSBP","stoichiometry":0.2},{"gene":"TUBB4B","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/SPAG5","total_profiled":1310},"omim":[{"mim_id":"620335","title":"CELL DIVISION CYCLE 20B; CDC20B","url":"https://www.omim.org/entry/620335"},{"mim_id":"615562","title":"SPERM-ASSOCIATED ANTIGEN 5; SPAG5","url":"https://www.omim.org/entry/615562"},{"mim_id":"614718","title":"KINETOCHORE-LOCALIZED ASTRIN/SPAG5-BINDING PROTEIN; KNSTRN","url":"https://www.omim.org/entry/614718"},{"mim_id":"613328","title":"ROIFMAN-CHITAYAT SYNDROME; ROCHIS","url":"https://www.omim.org/entry/613328"},{"mim_id":"605004","title":"GLYCOGEN SYNTHASE KINASE 3-BETA; GSK3B","url":"https://www.omim.org/entry/605004"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nuclear bodies","reliability":"Supported"},{"location":"Mitotic spindle","reliability":"Supported"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"bone marrow","ntpm":35.7},{"tissue":"kidney","ntpm":37.6},{"tissue":"testis","ntpm":72.1}],"url":"https://www.proteinatlas.org/search/SPAG5"},"hgnc":{"alias_symbol":["DEEPEST","MAP126","hMAP126"],"prev_symbol":[]},"alphafold":{"accession":"Q96R06","domains":[{"cath_id":"-","chopping":"490-782","consensus_level":"medium","plddt":91.7363,"start":490,"end":782},{"cath_id":"1.20.5","chopping":"1126-1193","consensus_level":"medium","plddt":89.2037,"start":1126,"end":1193}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96R06","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96R06-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96R06-F1-predicted_aligned_error_v6.png","plddt_mean":62.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SPAG5","jax_strain_url":"https://www.jax.org/strain/search?query=SPAG5"},"sequence":{"accession":"Q96R06","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96R06.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96R06/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96R06"}},"corpus_meta":[{"pmid":"11719903","id":"PMC_11719903","title":"Ethiopians 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Section D, Biological crystallography","url":"https://pubmed.ncbi.nlm.nih.gov/12832785","citation_count":4,"is_preprint":false},{"pmid":"38807081","id":"PMC_38807081","title":"SPAG5 deficiency activates autophagy to reduce atherosclerotic plaque formation in ApoE-/- mice.","date":"2024","source":"BMC cardiovascular disorders","url":"https://pubmed.ncbi.nlm.nih.gov/38807081","citation_count":3,"is_preprint":false},{"pmid":"38875410","id":"PMC_38875410","title":"SPAG5 and ASPM play important roles in gastric cancer: An observational study.","date":"2024","source":"Medicine","url":"https://pubmed.ncbi.nlm.nih.gov/38875410","citation_count":3,"is_preprint":false},{"pmid":"38940525","id":"PMC_38940525","title":"Distinct microbial nitrogen cycling processes in the deepest part of the ocean.","date":"2024","source":"mSystems","url":"https://pubmed.ncbi.nlm.nih.gov/38940525","citation_count":3,"is_preprint":false},{"pmid":"38248982","id":"PMC_38248982","title":"Fungal Abundance and Diversity in the Mariana Trench, the Deepest Ecosystem on Earth.","date":"2024","source":"Journal of fungi (Basel, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/38248982","citation_count":3,"is_preprint":false},{"pmid":"39762615","id":"PMC_39762615","title":"Application of a high-throughput swarm-based deep neural network Algorithm reveals SPAG5 downregulation as a potential therapeutic target in adult AML.","date":"2025","source":"Functional & integrative genomics","url":"https://pubmed.ncbi.nlm.nih.gov/39762615","citation_count":3,"is_preprint":false},{"pmid":"39276045","id":"PMC_39276045","title":"[Biological role of SPAG5 in the malignant proliferation of gastric cancer cells].","date":"2024","source":"Nan fang yi ke da xue xue bao = Journal of Southern Medical University","url":"https://pubmed.ncbi.nlm.nih.gov/39276045","citation_count":1,"is_preprint":false},{"pmid":"38721629","id":"PMC_38721629","title":"Identification of Lepidapedon oregonense as the current world's deepest trematode.","date":"2024","source":"Journal of helminthology","url":"https://pubmed.ncbi.nlm.nih.gov/38721629","citation_count":1,"is_preprint":false},{"pmid":"38468949","id":"PMC_38468949","title":"Deep sea treasures - Insights from museum archives shed light on coral microbial diversity within deepest ocean ecosystems.","date":"2024","source":"Heliyon","url":"https://pubmed.ncbi.nlm.nih.gov/38468949","citation_count":1,"is_preprint":false},{"pmid":"39696708","id":"PMC_39696708","title":"SPAG5 is a potential therapeutic target affecting proliferation, apoptosis, and invasion of esophageal cancer cells.","date":"2024","source":"European journal of medical research","url":"https://pubmed.ncbi.nlm.nih.gov/39696708","citation_count":0,"is_preprint":false},{"pmid":"41774258","id":"PMC_41774258","title":"SPAG5 promotes ferroptosis and enhances chemotherapy efficacy in locally advanced colorectal cancer via upregulating intracellular ROS levels.","date":"2026","source":"Apoptosis : an international journal on programmed cell death","url":"https://pubmed.ncbi.nlm.nih.gov/41774258","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":50447,"output_tokens":4550,"usd":0.109796,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12575,"output_tokens":5086,"usd":0.095012,"stage2_stop_reason":"end_turn"},"total_usd":0.204808,"stage1_batch_id":"msgbatch_013LWZZT6MWaqB9qyTSeQBmU","stage2_batch_id":"msgbatch_0194ZzQaTGNpruoNJBbEMbt3","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2001,\n      \"finding\": \"hMAP126/SPAG5 is a mitotic spindle-associated protein that is post-translationally modified and phosphorylated by p34(cdc2) kinase in vitro, and its subcellular distribution localizes to the mitotic spindle.\",\n      \"method\": \"Polyclonal antibody immunolocalization; in vitro kinase assay with p34(cdc2)\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Weak — in vitro kinase assay plus localization, single lab, no replication reported\",\n      \"pmids\": [\"11549262\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Rat Spag5 (ortholog of human SPAG5) encodes a ~200 kDa testicular protein that interacts strongly with Odf1 (the 27 kDa major outer dense fiber protein) via a leucine zipper motif in the C-terminal region of SPAG5; the downstream leucine zipper is specifically required for this interaction.\",\n      \"method\": \"Yeast two-hybrid interaction screen; leucine zipper mutagenesis/domain mapping\",\n      \"journal\": \"Molecular reproduction and development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — yeast two-hybrid with domain mapping, single lab, not biochemically reconstituted\",\n      \"pmids\": [\"11468777\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Disruption of the Spag5 gene in mice by homologous recombination (Spag5-null mice) does not impair spermatogenesis or fertility, indicating that any role of Spag5 in spermatogenesis is compensated by other proteins.\",\n      \"method\": \"Gene targeting by homologous recombination; phenotypic analysis of null mice\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — complete knockout with rigorous phenotypic analysis, peer-reviewed; negative result robustly established\",\n      \"pmids\": [\"11884588\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"In somatic cells, rat Spag5 localizes to the endoplasmic reticulum and microtubules (including a 58 kDa microtubule-associated isoform confirmed by in vitro MT-binding assays), whereas in elongated spermatids and epididymal sperm it associates with outer dense fibers but not axonemal microtubules — demonstrating cell-type-specific localization and function.\",\n      \"method\": \"Immunofluorescence; in vitro microtubule-binding assay; immuno-electron microscopy; Western blotting of fractionated cells\",\n      \"journal\": \"Molecular reproduction and development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (in vitro MT-binding, immuno-EM, fractionation) in single lab\",\n      \"pmids\": [\"16211599\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"A duplicated 25 bp insertion mutation in exon 6 of the Spag5 gene in hypogonadic (hgn/hgn) rats produces a truncated Spag5 protein lacking the primary spindle-targeting domain at the C-terminus, leading to defective spindle formation, abnormal mitosis of Sertoli cells, apoptotic Sertoli cell death, and testicular dysplasia — establishing that the C-terminal spindle-targeting domain is essential for SPAG5 function in cell division.\",\n      \"method\": \"Positional cloning; sequencing; immunostaining with Sertoli and germ cell markers; phenotypic analysis of mutant rats\",\n      \"journal\": \"Reproduction (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — natural loss-of-function allele with domain-level mapping and defined cellular phenotype, single lab\",\n      \"pmids\": [\"16816335\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"SPAG5/Astrin localizes to centrosomal/basal body structures in photoreceptor cells of the retina and interacts with USH2AisoB interaction partner NINLisoB; SPAG5 was identified as interacting with USH2AisoB and NINLisoB at basal bodies postmitotically, connecting it to the Usher protein network involved in cilium function.\",\n      \"method\": \"Yeast two-hybrid; co-localization by immunofluorescence in retinal photoreceptors; in situ hybridization; quantitative PCR\",\n      \"journal\": \"Cilia\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — yeast two-hybrid interaction plus co-localization, single lab, limited biochemical validation\",\n      \"pmids\": [\"23351521\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"SPAG5/Astrin physically interacts with ORP8 (OSBP-related protein 8), confirmed by yeast two-hybrid, pull-down, and co-immunoprecipitation; overexpressed ORP8 recruits SPAG5 onto ER membranes in interphase cells; SPAG5 knockdown significantly reduces ORP8-overexpression- and 25-hydroxycholesterol-induced G2/M cell cycle accumulation, placing SPAG5 downstream of ORP8 in oxysterol-mediated cell cycle regulation.\",\n      \"method\": \"Yeast two-hybrid; pull-down assay; co-immunoprecipitation; siRNA knockdown; cell cycle analysis by flow cytometry\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP, pull-down, and genetic epistasis (knockdown), multiple orthogonal methods, single lab\",\n      \"pmids\": [\"24424245\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"SPAG5 physically interacts with centrosomal protein CEP55, and this interaction triggers phosphorylation of AKT at Ser473, activating PI3K/AKT signaling to promote hepatocellular carcinoma cell growth and metastasis; inhibition of PI3K/AKT markedly attenuates SPAG5-mediated cell growth.\",\n      \"method\": \"Co-immunoprecipitation; in vitro and in vivo functional assays; PI3K/AKT inhibitor treatment; miR-363-3p luciferase reporter assay\",\n      \"journal\": \"Molecular cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — co-IP plus pathway inhibitor rescue, single lab\",\n      \"pmids\": [\"30089483\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"SPAG5 promotes HCC progression by downregulating SCARA5 through modulating β-catenin degradation, engaging the β-catenin/TCF4 signaling pathway; SCARA5 expression is inversely correlated with SPAG5 in HCC tissues.\",\n      \"method\": \"shRNA knockdown; overexpression; cell proliferation/apoptosis assays; Western blot for β-catenin pathway components; in vivo xenograft\",\n      \"journal\": \"Journal of experimental & clinical cancer research : CR\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Weak — loss-of-function with defined signaling outcome, single lab, mechanism inferred from pathway markers without biochemical reconstitution\",\n      \"pmids\": [\"30249289\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"SPAG5 interacts with c-MYC binding protein (MYCBP), increasing MYCBP protein levels and enhancing c-MYC transcriptional activity, which in turn promotes expression of CDC20, CDC25C, BRCA1, BRCA2, and RAD51 in triple-negative breast cancer; knockdown of MYCBP or c-MYC abolishes SPAG5-induced cell-cycle progression and proliferation.\",\n      \"method\": \"Co-immunoprecipitation; Western blot; siRNA knockdown epistasis; in vitro and in vivo functional assays\",\n      \"journal\": \"Journal of hematology & oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — co-IP plus epistasis knockdown, single lab\",\n      \"pmids\": [\"30736840\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"p53-p21 axis transcriptionally suppresses SPAG5 expression: MDM2 inhibitor Nutlin-3a restores p53/p21 and suppresses SPAG5 in wild-type p53 cells but not p53-null cells; p53 or p21 knockdown attenuates Nutlin-3a-induced repression of SPAG5, establishing p53-p21 as required for SPAG5 repression.\",\n      \"method\": \"MDM2 inhibitor treatment; siRNA knockdown of p53/p21; Western blot; qRT-PCR\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — pharmacological and genetic perturbation with consistent results, single lab\",\n      \"pmids\": [\"30955859\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SPAG5 is a direct transcriptional target of the YAP/TAZ/TEAD axis (Hippo pathway); depletion of YAP, TAZ, or TEAD strongly reduces SPAG5 expression; SPAG5 is also directly targeted post-transcriptionally by miR-10b-3p; SPAG5 depletion impairs cancer cell cycle progression, proliferation, and migration.\",\n      \"method\": \"ChIP/luciferase reporter assays for TEAD binding to SPAG5 promoter; YAP/TAZ/TEAD siRNA knockdown; miR-10b-3p target validation; cell cycle and proliferation assays\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — transcriptional target validated by reporter assay and genetic knockdown, single lab\",\n      \"pmids\": [\"33230261\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SPAG5-AS1 (a lncRNA neighbor of SPAG5) interacts with USP14, leading to de-ubiquitination and stabilization of SPAG5 protein; YY1 transcriptionally upregulates both SPAG5-AS1 and SPAG5; SPAG5 promotes AKT/mTOR signaling, inhibiting autophagy and aggravating apoptosis in podocytes under high glucose.\",\n      \"method\": \"Pulldown; RIP; co-IP; ChIP; luciferase reporter assay; Western blot; flow cytometry; immunofluorescence\",\n      \"journal\": \"Cell proliferation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — multiple biochemical assays (co-IP, pulldown, RIP, ChIP), single lab, involves lncRNA regulation of the SPAG5 protein\",\n      \"pmids\": [\"31957155\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SPAG5 promotes osteosarcoma metastasis by stabilizing FOXM1 protein (reducing its degradation), which in turn upregulates MMP2 expression, promoting invasion and migration; SPAG5 silencing inhibits the SPAG5-FOXM1-MMP2 axis and suppresses lung metastasis in vivo.\",\n      \"method\": \"siRNA/shRNA knockdown; Western blot for FOXM1 protein stability; in vitro invasion/migration assays; in vivo metastasis model\",\n      \"journal\": \"The international journal of biochemistry & cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Weak — loss-of-function with defined molecular pathway, single lab, mechanism inferred from protein levels without direct ubiquitination assay\",\n      \"pmids\": [\"32668328\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Splicing factor SF3B4 promotes SPAG5 expression by ensuring effective splicing of SPAG5 pre-mRNA; SF3B4 knockdown causes intron retention in SPAG5 pre-mRNA, reducing mature SPAG5 mRNA and protein, and the pro-tumorigenic effects of SF3B4 overexpression in cervical cancer are impaired by SPAG5 deficiency.\",\n      \"method\": \"RNA-seq with alternative splicing analysis; SF3B4 siRNA knockdown; SPAG5 rescue experiments; in vitro and in vivo functional assays\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — RNA-seq splicing analysis plus genetic epistasis rescue, single lab\",\n      \"pmids\": [\"35853859\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"SPAG5 promotes ferroptosis in colorectal cancer cells by upregulating intracellular reactive oxygen species (ROS); SPAG5 depletion reduces ROS levels and attenuates ferroptosis, diminishing chemotherapy efficacy; ROS inhibition reverses the chemotherapy-sensitizing effect of SPAG5.\",\n      \"method\": \"SPAG5 overexpression and knockdown; ROS measurement; ferroptosis assays; chemotherapy sensitivity assays in vitro and patient cohort analysis\",\n      \"journal\": \"Apoptosis : an international journal on programmed cell death\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Weak — loss- and gain-of-function with mechanistic ROS/ferroptosis readouts, single lab\",\n      \"pmids\": [\"41774258\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In triple-negative breast cancer, high SPAG5 expression is regulated by coordinated activity of YAP, mutant p53, and MYC; depletion of YAP or mutant p53 reduces SPAG5 expression and reduces MYC recruitment to the SPAG5 promoter; MYC targeting reduces SPAG5 expression and TNBC tumorigenicity in a SPAG5-expression-dependent manner.\",\n      \"method\": \"siRNA knockdown of YAP, mutant p53, MYC; ChIP for MYC at SPAG5 promoter; functional cell and in vivo assays; chemotherapy combination experiments\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — ChIP plus multiple genetic knockdowns, single lab\",\n      \"pmids\": [\"39164278\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"SPAG5 knockdown activates autophagy and inhibits PI3K/Akt/mTOR signaling in endothelial cells treated with ox-LDL, reducing atherosclerotic plaque formation in ApoE-/- mice; autophagy inhibitor 3-MA reverses the protective effects of SPAG5 silencing, placing SPAG5 upstream of autophagy via PI3K/Akt/mTOR.\",\n      \"method\": \"siRNA/shRNA knockdown; Western blot for PI3K/Akt/mTOR phosphorylation and autophagy markers; GFP-LC3 puncta assay; 3-MA pharmacological rescue; in vivo ApoE-/- mouse model\",\n      \"journal\": \"BMC cardiovascular disorders\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — genetic and pharmacological epistasis in vitro and in vivo, single lab\",\n      \"pmids\": [\"38807081\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SPAG5 (also known as hMAP126/Astrin/Deepest) is a microtubule-associated mitotic spindle protein phosphorylated by CDK1 (p34cdc2) that localizes to the spindle in dividing cells and to the endoplasmic reticulum/microtubules in interphase somatic cells, but to outer dense fibers in spermatozoa; its C-terminal spindle-targeting domain is essential for proper Sertoli cell mitosis in vivo, and in cancer contexts it drives proliferation and survival by interacting with CEP55 to activate PI3K/AKT signaling, stabilizing FOXM1 to upregulate MMP2, engaging MYCBP to amplify c-MYC transcriptional output, and modulating β-catenin degradation, while being transcriptionally regulated downstream of YAP/TAZ/TEAD and suppressed by the p53-p21 axis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SPAG5 (hMAP126/Astrin) is a microtubule-associated mitotic spindle protein that is phosphorylated by p34(cdc2)/CDK1 and localizes to the mitotic spindle in dividing cells while distributing to the endoplasmic reticulum and microtubules in interphase somatic cells [#0, #3]. Its localization and binding partners are cell-type specific: in spermatids and sperm it associates with outer dense fibers through a C-terminal leucine-zipper interaction with ODF1 rather than with axonemal microtubules [#1, #3], and in interphase cells it can be recruited onto ER membranes by ORP8, downstream of which SPAG5 contributes to oxysterol-mediated G2/M cell cycle accumulation [#6]. A C-terminal spindle-targeting domain is essential for SPAG5's function in cell division: a natural truncating mutation in rats abolishes spindle formation and causes abnormal Sertoli cell mitosis, apoptosis, and testicular dysplasia, although complete Spag5 knockout in mice leaves spermatogenesis and fertility intact, indicating functional compensation in the germline [#2, #4]. In cancer, SPAG5 acts as a proliferation- and survival-promoting hub: it interacts with CEP55 to activate PI3K/AKT signaling [#7], engages MYCBP to amplify c-MYC transcriptional output driving cell-cycle genes [#9], stabilizes FOXM1 to upregulate MMP2 and promote metastasis [#13], and modulates beta-catenin degradation to repress SCARA5 [#8]. SPAG5 expression is itself a convergence point of oncogenic transcriptional control, being a direct target of the YAP/TAZ/TEAD (Hippo) axis and, in triple-negative breast cancer, of coordinated YAP, mutant p53, and MYC activity, while the wild-type p53-p21 axis suppresses it [#10, #11, #16]. Across multiple disease contexts SPAG5 also regulates PI3K/AKT/mTOR-dependent autophagy and ROS/ferroptosis balance [#15, #17].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Established SPAG5's identity as a cell-cycle-regulated spindle protein by showing it associates with the mitotic spindle and is a CDK1 substrate, linking it to mitotic regulation.\",\n      \"evidence\": \"Polyclonal antibody immunolocalization and in vitro p34(cdc2) kinase assay\",\n      \"pmids\": [\"11549262\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vitro kinase phosphorylation site not mapped\", \"Functional consequence of phosphorylation on spindle binding undefined\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Identified a tissue-specific binding partner, showing SPAG5 binds the outer dense fiber protein ODF1 via a C-terminal leucine zipper, implicating it in sperm tail structure.\",\n      \"evidence\": \"Yeast two-hybrid screen with leucine-zipper domain mapping in rat\",\n      \"pmids\": [\"11468777\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Interaction not biochemically reconstituted\", \"Physiological role of ODF1 binding untested\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Tested whether SPAG5 is required for spermatogenesis; null mice were fertile, revealing functional redundancy in the germline despite the ODF1 interaction.\",\n      \"evidence\": \"Homologous-recombination knockout and phenotypic analysis of null mice\",\n      \"pmids\": [\"11884588\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Compensating proteins not identified\", \"Somatic/mitotic phenotypes not examined in this knockout\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Resolved SPAG5's cell-type-specific localization, distinguishing ER/microtubule association in somatic cells from outer-dense-fiber association in sperm, defining context-dependent function.\",\n      \"evidence\": \"Immunofluorescence, in vitro microtubule-binding, immuno-EM, and fractionation in rat\",\n      \"pmids\": [\"16211599\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Microtubule-binding determinant not mapped at residue level\", \"Isoform-specific functions unresolved\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Established the C-terminal spindle-targeting domain as essential for cell division, since a natural truncation caused defective Sertoli cell mitosis and testicular dysplasia.\",\n      \"evidence\": \"Positional cloning of hgn/hgn rat mutant with marker immunostaining and phenotyping\",\n      \"pmids\": [\"16816335\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular partners of the spindle-targeting domain not defined\", \"Reconciliation with fertile mouse knockout phenotype unresolved\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Connected SPAG5 to ciliary/basal body biology by placing it in the Usher protein network at photoreceptor basal bodies.\",\n      \"evidence\": \"Yeast two-hybrid and co-localization in retinal photoreceptors\",\n      \"pmids\": [\"23351521\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Yeast two-hybrid interaction not biochemically confirmed\", \"Functional role at basal bodies untested\", \"Relevance to disease unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Showed SPAG5 acts downstream of ORP8 in oxysterol-driven cell cycle control, integrating it into lipid-sensing G2/M regulation at the ER.\",\n      \"evidence\": \"Yeast two-hybrid, pull-down, reciprocal co-IP, siRNA knockdown, and flow cytometry\",\n      \"pmids\": [\"24424245\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism linking ER recruitment to G2/M arrest unresolved\", \"Direct binding interface not mapped\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defined an oncogenic mechanism whereby SPAG5-CEP55 binding activates PI3K/AKT to drive HCC growth and metastasis.\",\n      \"evidence\": \"Co-IP, PI3K/AKT inhibitor rescue, and in vitro/in vivo HCC assays\",\n      \"pmids\": [\"30089483\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How CEP55 binding triggers AKT Ser473 phosphorylation not mechanistically resolved\", \"Single lab without reciprocal structural validation\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Linked SPAG5 to Wnt/beta-catenin signaling, showing it represses SCARA5 by modulating beta-catenin degradation to promote HCC.\",\n      \"evidence\": \"shRNA/overexpression with Western blot of beta-catenin pathway and xenografts\",\n      \"pmids\": [\"30249289\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct effect of SPAG5 on beta-catenin turnover not biochemically demonstrated\", \"Mechanism inferred from pathway markers only\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Established a SPAG5-MYCBP-c-MYC axis amplifying c-MYC transcriptional output and cell-cycle gene expression in TNBC.\",\n      \"evidence\": \"Co-IP, Western blot, and siRNA epistasis with functional assays\",\n      \"pmids\": [\"30736840\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which SPAG5 raises MYCBP levels unknown\", \"Direct vs indirect target gene effects not distinguished\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identified the p53-p21 axis as a required suppressor of SPAG5 transcription, defining tumor-suppressor control of SPAG5.\",\n      \"evidence\": \"Nutlin-3a treatment with p53/p21 knockdown, Western blot, and qRT-PCR\",\n      \"pmids\": [\"30955859\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs indirect transcriptional mechanism not resolved\", \"p53/p21 binding to SPAG5 locus not demonstrated\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Placed SPAG5 as a direct YAP/TAZ/TEAD Hippo-pathway target additionally repressed by miR-10b-3p, defining its upstream transcriptional/post-transcriptional regulation.\",\n      \"evidence\": \"ChIP/luciferase reporter for TEAD, YAP/TAZ/TEAD knockdown, and miRNA target validation\",\n      \"pmids\": [\"33230261\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relative contribution of transcriptional vs miRNA control unquantified\", \"Single lab\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Defined post-translational stabilization of SPAG5 via the SPAG5-AS1/USP14 axis and downstream AKT/mTOR-driven autophagy suppression in podocytes.\",\n      \"evidence\": \"Pulldown, RIP, co-IP, ChIP, luciferase, and functional assays under high glucose\",\n      \"pmids\": [\"31957155\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct USP14 deubiquitination of SPAG5 not enzymatically reconstituted\", \"Relevance beyond podocytes untested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Established a SPAG5-FOXM1-MMP2 axis driving osteosarcoma metastasis through FOXM1 protein stabilization.\",\n      \"evidence\": \"siRNA/shRNA knockdown, Western blot for FOXM1 stability, and in vivo metastasis model\",\n      \"pmids\": [\"32668328\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct ubiquitination assay for FOXM1 stabilization\", \"Mechanism of SPAG5-FOXM1 interaction undefined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Showed SPAG5 expression depends on SF3B4-mediated splicing, identifying a splicing-level control point for SPAG5 in cervical cancer.\",\n      \"evidence\": \"RNA-seq splicing analysis, SF3B4 knockdown, and SPAG5 rescue assays\",\n      \"pmids\": [\"35853859\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct SF3B4 binding to SPAG5 pre-mRNA not shown\", \"Specificity of splicing effect not established\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Linked SPAG5 to redox-dependent cell death, showing it promotes ROS-driven ferroptosis and chemosensitivity in colorectal cancer.\",\n      \"evidence\": \"Gain/loss-of-function, ROS measurement, ferroptosis and chemosensitivity assays with patient cohort\",\n      \"pmids\": [\"41774258\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which SPAG5 elevates ROS unknown\", \"Connection to mitotic function unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Extended SPAG5's PI3K/Akt/mTOR-autophagy role to vascular disease, where SPAG5 silencing activates autophagy and reduces atherosclerosis.\",\n      \"evidence\": \"Knockdown with PI3K/Akt/mTOR and autophagy readouts, 3-MA rescue, and ApoE-/- mouse model\",\n      \"pmids\": [\"38807081\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct molecular link between SPAG5 and PI3K/Akt activation undefined\", \"Cell-autonomous vs systemic effects not separated\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Refined SPAG5 transcriptional control in TNBC, showing coordinated YAP, mutant p53, and MYC activity with MYC recruitment to the SPAG5 promoter.\",\n      \"evidence\": \"siRNA knockdown of YAP/mutant p53/MYC, ChIP at SPAG5 promoter, and in vivo assays\",\n      \"pmids\": [\"39164278\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct interplay among YAP, mutant p53, and MYC at the locus not fully resolved\", \"Single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How SPAG5's core mitotic spindle function mechanistically connects to its diverse oncogenic signaling roles (PI3K/AKT, c-MYC, beta-catenin, FOXM1, ROS/ferroptosis) remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model linking spindle role to signaling roles\", \"Structural basis of partner interactions undefined\", \"Direct enzymatic activities, if any, unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [7, 9, 13]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005819\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [3, 6]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [0, 4, 6]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [7, 11, 17]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [7, 8, 13]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"ODF1\", \"ORP8\", \"CEP55\", \"MYCBP\", \"USP14\", \"NINL\", \"USH2A\", \"FOXM1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"tie","faith_supported":6,"faith_total":6,"faith_pct":100.0}}