{"gene":"SPAG1","run_date":"2026-04-28T20:42:08","timeline":{"discoveries":[{"year":2013,"finding":"SPAG1 is present in human airway epithelial cell lysates but absent from isolated axonemes; immunofluorescence shows absence of both outer and inner dynein arm (ODA and IDA) proteins in cilia from SPAG1-mutant individuals, indicating SPAG1 functions in the cytoplasmic pre-assembly and/or trafficking of axonemal dynein arms rather than as a structural axonemal component. Zebrafish spag1 morpholino knockdown produced cilia-related phenotypes consistent with cytoplasmic assembly factors.","method":"Immunofluorescence, axoneme fractionation/cell lysate comparison, zebrafish morpholino knockdown","journal":"American journal of human genetics","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (fractionation, IF, in vivo morpholino), replicated across 14 affected individuals and zebrafish model","pmids":["24055112"],"is_preprint":false},{"year":2022,"finding":"SPAG1 interacts with multiple dynein axonemal assembly factors (DNAAFs), dynein heavy chains (DHCs), dynein intermediate chains (DICs), and canonical R2TP complex components (RUVBL1, RUVBL2, PIH1D2) by immunoprecipitation. SPAG1 loss reduces DHC protein levels and impairs DHC–DIC interactions, showing SPAG1 scaffolds R2TP-like complexes to facilitate folding/binding of DHCs to DIC complexes. A 60 kDa SPAG1 isoform can partially compensate for full-length SPAG1 loss to assemble a reduced number of outer dynein arms.","method":"Immunoprecipitation, quantitative proteomics (mass spectrometry), analysis of PCD patient airway epithelia","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 — reciprocal co-IPs with multiple partners and quantitative proteomic readouts, disease-relevant human cell system","pmids":["35178554"],"is_preprint":false},{"year":2019,"finding":"SPAG1 contains three TPR domains, but only two of them recruit the chaperones HSP70 and HSP90 via binding to their C-terminal tails. NMR structure of one TPR domain was solved, and NMR-driven docking plus molecular dynamics simulations defined the binding interface with HSP70 and HSP90 C-terminal peptides. A SPAG1 sub-fragment containing the putative P-loop motif cannot efficiently bind or hydrolyze GTP in vitro, challenging prior reports of SPAG1 GTPase activity and suggesting SPAG1 instead regulates nucleotide hydrolysis of HSP and RUVBL1/2 partners.","method":"NMR spectroscopy (3D structure), ITC, biochemical GTPase assays, molecular dynamics simulations","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 — NMR structure with in vitro binding and enzymatic assays, multiple orthogonal biophysical methods","pmids":["31118266"],"is_preprint":false},{"year":2021,"finding":"Structural and biophysical characterization of the first TPR domain of human SPAG1 using an optimized variant showed with atomistic precision how the C-terminal tails of HSP70 and HSP90 bind; specific motifs within the TPR sequence drive positioning of HSP peptides.","method":"Protein sequence optimization, NMR spectroscopy, biophysical binding assays","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 — structural determination combined with biophysical interaction analysis","pmids":["33739091"],"is_preprint":false},{"year":2001,"finding":"HSD-3.8 (SPAG1) encodes a GTP-binding protein with GTPase activity and is phosphorylated by PKC in vitro. The protein localizes to the postacrosomal zone of human spermatozoa and to pachytene primary spermatocytes. It contains TPR motifs and a P-loop sequence. Immunization of female rats with recombinant HSD-3.8 protein caused infertility.","method":"[α-32P]GTP blot overlay assay, in vitro GTPase assay, in vitro PKC phosphorylation assay, immunofluorescence/immunostaining","journal":"Molecular human reproduction","confidence":"Medium","confidence_rationale":"Tier 1–2 — in vitro enzymatic assays with direct biochemical demonstration, but later work challenges GTPase interpretation","pmids":["11517287"],"is_preprint":false},{"year":2006,"finding":"HSD-3.8 (SPAG1) interacts with the C-terminal 144 amino acids of G-protein β1 subunit (Gβ1), forming a complex in the cytoplasm in the presence of GDP (co-immunoprecipitation in HEK293 cells, yeast two-hybrid). Overexpression of HSD-0.7 (SPAG1 fragment) activates ERK1/2 via a PKC-dependent (not Ras-dependent) pathway; deletion of either the TPR domain or the P-loop abolished ERK1/2 activation.","method":"Yeast two-hybrid, co-transfection/co-immunoprecipitation in HEK293 cells, ERK1/2 activation assay, domain deletion analysis","journal":"Frontiers in bioscience","confidence":"Medium","confidence_rationale":"Tier 2–3 — yeast two-hybrid and co-IP with functional domain deletion, single lab","pmids":["16368546"],"is_preprint":false},{"year":2016,"finding":"In mouse oocytes, SPAG1 associates with meiotic spindles. RNAi depletion of SPAG1 impairs germinal vesicle breakdown (GVBD) via increased intracellular cAMP and decreased ATP, activating AMPK. SPAG1 depletion also reduces MAPK phosphorylation and causes irregular distribution of phospho-MAPK, impairing γ-tubulin function and spindle morphogenesis. Additionally, SPAG1 RNAi reduces actin expression and disrupts cortical granule-free domains, actin caps, and the contractile ring.","method":"RNAi in mouse oocytes, live imaging of spindle morphology, cAMP/ATP measurement, AMPK activation assay, immunofluorescence of γ-tubulin and phospho-MAPK","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 — RNAi with multiple defined biochemical and cellular readouts, single lab","pmids":["27053660"],"is_preprint":false},{"year":2017,"finding":"miR-638 directly targets SPAG1 in porcine immature Sertoli cells; SPAG1 knockdown (siRNA) phenocopies miR-638 overexpression by downregulating p-PI3K, p-AKT, c-MYC, CCND1, CCNE1, and CDK4, inhibiting proliferation and promoting apoptosis. SPAG1 siRNA also suppresses SOX2 and POU5F1 mRNA levels.","method":"miRNA target validation (luciferase reporter), siRNA knockdown, Western blot for PI3K/AKT pathway components, cell cycle/proliferation assays","journal":"Cell cycle (Georgetown, Tex.)","confidence":"Medium","confidence_rationale":"Tier 2–3 — direct target validation with functional epistasis via siRNA, single lab, porcine (non-human) Sertoli cell model","pmids":["29119857"],"is_preprint":false},{"year":2022,"finding":"SPAG1 knockdown in AML cells reduces proliferation and survival, and regulates expression of SMC3 while activating the ERK/MAPK signaling pathway.","method":"RNA interference knockdown, proliferation/survival assays, Western blot for ERK/MAPK pathway","journal":"Neoplasma","confidence":"Low","confidence_rationale":"Tier 3 — single lab, knockdown with pathway readout but no direct molecular mechanism established","pmids":["35951456"],"is_preprint":false},{"year":2025,"finding":"The human R2SP complex (RUVBL1, RUVBL2, SPAG1, PIH1D2) has a 3D organization similar to the canonical R2TP complex, determined by cryo-EM, NMR, and structural mass spectrometry. SPAG1 and PIH1D2 act as adaptors recruiting specific clients, while the RUVBL1/2 ATPase core functions as the catalytic powerhouse; differences in RUVBL1/2 ATPase activity regulation and adaptor binding mode distinguish R2SP from canonical R2TP.","method":"Cryo-EM, NMR, structural mass spectrometry, ATPase activity assays","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 — multi-method structural study; preprint, not yet peer-reviewed","pmids":["bio_10.1101_2025.01.27.635100"],"is_preprint":true}],"current_model":"SPAG1 is a multidomain dynein axonemal assembly factor (DNAAF) that functions in the cytoplasm to scaffold the R2SP complex (with RUVBL1, RUVBL2, and PIH1D2) and recruit HSP70/HSP90 chaperones via its TPR domains, facilitating the pre-assembly of dynein heavy chains with intermediate chains before their transport into the cilium; loss of SPAG1 causes primary ciliary dyskinesia with combined outer and inner dynein arm defects, and SPAG1 also modulates ERK/MAPK and PI3K/AKT signaling in spermatogenic cells."},"narrative":{"teleology":[{"year":2001,"claim":"Initial biochemical characterization identified SPAG1 as a testis-expressed, TPR- and P-loop-containing protein with apparent GTP-binding and GTPase activity, establishing its domain architecture and spermatozoon localization but leaving its cellular function unknown.","evidence":"GTP overlay assay, in vitro GTPase/PKC assays, immunostaining of human spermatozoa","pmids":["11517287"],"confidence":"Medium","gaps":["GTPase activity was later challenged by NMR/biochemical studies","no physiological substrate or pathway identified","no loss-of-function data"]},{"year":2006,"claim":"Interaction with Gβ1 and demonstration that SPAG1 fragments activate ERK1/2 via PKC in a TPR- and P-loop-dependent manner suggested SPAG1 could modulate MAPK signaling, expanding its functional scope beyond sperm antigen to a signaling scaffold.","evidence":"Yeast two-hybrid, co-IP in HEK293 cells, ERK1/2 activation with domain deletion analysis","pmids":["16368546"],"confidence":"Medium","gaps":["single lab, not replicated independently","overexpression system; physiological relevance in vivo unclear","Gβ1 interaction not validated in motile cilia biology"]},{"year":2013,"claim":"Identification of SPAG1 mutations in primary ciliary dyskinesia families established that SPAG1 is a dynein axonemal assembly factor required for both outer and inner dynein arm assembly; fractionation showed it acts in the cytoplasm rather than as an axonemal structural component.","evidence":"Immunofluorescence, axoneme fractionation, analysis of 14 affected individuals, zebrafish morpholino knockdown","pmids":["24055112"],"confidence":"High","gaps":["precise molecular mechanism of dynein pre-assembly unknown","identity of direct dynein client proteins not established","relationship to R2TP/chaperone machinery not yet defined"]},{"year":2016,"claim":"RNAi depletion in mouse oocytes revealed a role for SPAG1 in meiotic spindle morphogenesis via regulation of MAPK phosphorylation, γ-tubulin distribution, and AMPK-dependent metabolic signaling, extending SPAG1 function beyond motile cilia.","evidence":"RNAi in mouse oocytes, live imaging, cAMP/ATP measurement, immunofluorescence","pmids":["27053660"],"confidence":"Medium","gaps":["single lab; mechanism linking SPAG1 to AMPK/cAMP unclear","whether this reflects TPR/chaperone scaffolding or an independent function is unknown","no genetic knockout confirmation"]},{"year":2017,"claim":"Validation of SPAG1 as a miR-638 target in Sertoli cells showed that SPAG1 knockdown suppresses PI3K/AKT signaling and proliferation, indicating SPAG1 supports spermatogenic cell survival through this pathway.","evidence":"Luciferase reporter assay, siRNA knockdown, Western blot for PI3K/AKT in porcine Sertoli cells","pmids":["29119857"],"confidence":"Medium","gaps":["porcine model; relevance to human spermatogenesis not confirmed","mechanism by which SPAG1 activates PI3K/AKT not defined","single lab"]},{"year":2019,"claim":"NMR structure of a SPAG1 TPR domain and biophysical analysis resolved how two of three TPR domains recruit HSP70/HSP90 via their C-terminal tails, while demonstrating that SPAG1 itself lacks efficient GTPase activity — redefining SPAG1 as a chaperone-recruiting scaffold rather than an enzyme.","evidence":"NMR spectroscopy, ITC, biochemical GTPase assays, molecular dynamics simulations","pmids":["31118266"],"confidence":"High","gaps":["role of the third non-chaperone-binding TPR domain unknown","how chaperone recruitment couples to dynein folding not shown","full-length SPAG1 structure not available"]},{"year":2021,"claim":"Atomistic structural detail of the first TPR domain binding HSP70/HSP90 C-terminal peptides provided a high-resolution map of the chaperone recruitment interface, refining the structural basis for SPAG1's adaptor function.","evidence":"Optimized TPR variant, NMR spectroscopy, biophysical binding assays","pmids":["33739091"],"confidence":"High","gaps":["binding measured with isolated domains; full-length context and client-loaded complexes not characterized","no mutagenesis-based functional validation in cells"]},{"year":2022,"claim":"Proteomic and co-IP studies in PCD patient airway epithelia demonstrated that SPAG1 directly scaffolds the R2SP complex (RUVBL1, RUVBL2, PIH1D2) and is required for dynein heavy chain stability and heavy chain–intermediate chain interactions, providing the molecular mechanism for its dynein assembly role.","evidence":"Reciprocal co-IPs, quantitative mass spectrometry in human airway epithelia","pmids":["35178554"],"confidence":"High","gaps":["how the 60 kDa isoform partially compensates is unclear","order of assembly steps (client loading, chaperone release) not resolved","whether SPAG1 participates in dynein transport to cilia or only cytoplasmic pre-assembly is unknown"]},{"year":null,"claim":"The full 3D architecture of the client-loaded R2SP complex, the precise order of dynein arm assembly steps mediated by SPAG1, and the mechanistic basis for SPAG1's roles in meiotic spindle morphogenesis and MAPK/PI3K signaling remain to be determined.","evidence":"","pmids":[],"confidence":"Low","gaps":["no full-length SPAG1 structure in complex with dynein clients","no reconstituted in vitro dynein assembly assay with R2SP","non-ciliary signaling functions lack mechanistic connection to known TPR/R2SP scaffolding"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,2,3,9]},{"term_id":"GO:0044183","term_label":"protein folding chaperone","supporting_discovery_ids":[1,2]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,1]},{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[0]}],"pathway":[{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[0]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[0,1]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[5,6,7]}],"complexes":["R2SP (RUVBL1/RUVBL2/SPAG1/PIH1D2)"],"partners":["RUVBL1","RUVBL2","PIH1D2","HSPA1A","HSP90AA1","GNB1"],"other_free_text":[]},"mechanistic_narrative":"SPAG1 is a cytoplasmic dynein axonemal assembly factor that scaffolds the R2SP complex (with RUVBL1, RUVBL2, and PIH1D2) to facilitate the pre-assembly and folding of axonemal dynein heavy chains and their association with intermediate chains before transport into cilia [PMID:24055112, PMID:35178554]. SPAG1 contains three TPR domains, two of which recruit HSP70 and HSP90 chaperones by binding their C-terminal tails, as defined by NMR structures and biophysical assays; SPAG1 itself lacks efficient GTPase activity and instead coordinates the ATPase activities of its RUVBL1/2 and chaperone partners [PMID:31118266, PMID:33739091]. Loss-of-function mutations in SPAG1 cause primary ciliary dyskinesia with combined outer and inner dynein arm defects [PMID:24055112]. In reproductive cells, SPAG1 knockdown impairs meiotic spindle morphogenesis in oocytes and modulates PI3K/AKT and ERK/MAPK signaling in Sertoli cells, indicating additional roles in germ cell proliferation and meiotic progression [PMID:27053660, PMID:29119857]."},"prefetch_data":{"uniprot":{"accession":"Q07617","full_name":"Sperm-associated antigen 1","aliases":["HSD-3.8","Infertility-related sperm protein Spag-1"],"length_aa":926,"mass_kda":103.6,"function":"May play a role in the cytoplasmic assembly of the ciliary dynein arms (By similarity). May play a role in fertilization. Binds GTP and has GTPase activity","subcellular_location":"Cytoplasm; Dynein axonemal particle","url":"https://www.uniprot.org/uniprotkb/Q07617/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SPAG1","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":[{"gene":"HSP90AA1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/SPAG1","total_profiled":1310},"omim":[{"mim_id":"617014","title":"NEUTROPENIA, SEVERE CONGENITAL, 7, AUTOSOMAL RECESSIVE; SCN7","url":"https://www.omim.org/entry/617014"},{"mim_id":"615505","title":"CILIARY DYSKINESIA, PRIMARY, 28; CILD28","url":"https://www.omim.org/entry/615505"},{"mim_id":"603395","title":"SPERM-ASSOCIATED ANTIGEN 1; SPAG1","url":"https://www.omim.org/entry/603395"},{"mim_id":"244400","title":"CILIARY DYSKINESIA, PRIMARY, 1; CILD1","url":"https://www.omim.org/entry/244400"},{"mim_id":"138971","title":"COLONY-STIMULATING FACTOR 3 RECEPTOR, GRANULOCYTE; CSF3R","url":"https://www.omim.org/entry/138971"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Primary cilium","reliability":"Supported"},{"location":"Cytosol","reliability":"Supported"},{"location":"Principal piece","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/SPAG1"},"hgnc":{"alias_symbol":["SP75","FLJ32920","HSD-3.8","TPIS","CT140","CILD28","DNAAF13"],"prev_symbol":[]},"alphafold":{"accession":"Q07617","domains":[{"cath_id":"-","chopping":"23-74","consensus_level":"high","plddt":88.686,"start":23,"end":74},{"cath_id":"1.25.40.10","chopping":"203-328","consensus_level":"medium","plddt":92.8911,"start":203,"end":328},{"cath_id":"1.25.40.10","chopping":"446-579","consensus_level":"high","plddt":90.9902,"start":446,"end":579},{"cath_id":"1.25.40.10","chopping":"653-744","consensus_level":"high","plddt":91.1659,"start":653,"end":744},{"cath_id":"1.25.40","chopping":"807-925","consensus_level":"high","plddt":85.1948,"start":807,"end":925}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q07617","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q07617-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q07617-F1-predicted_aligned_error_v6.png","plddt_mean":73.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SPAG1","jax_strain_url":"https://www.jax.org/strain/search?query=SPAG1"},"sequence":{"accession":"Q07617","fasta_url":"https://rest.uniprot.org/uniprotkb/Q07617.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q07617/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q07617"}},"corpus_meta":[{"pmid":"24055112","id":"PMC_24055112","title":"Mutations in SPAG1 cause primary ciliary dyskinesia associated with defective outer and inner dynein arms.","date":"2013","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/24055112","citation_count":113,"is_preprint":false},{"pmid":"7838169","id":"PMC_7838169","title":"Polymorphism of SPAG-1, a candidate antigen for inclusion in a sub-unit vaccine against Theileria annulata.","date":"1994","source":"Molecular and biochemical parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/7838169","citation_count":44,"is_preprint":false},{"pmid":"8388029","id":"PMC_8388029","title":"Immunoglobulin M reactivity towards the immunologically active region sp75 of the core protein of hepatitis C virus (HCV) in chronic HCV infection.","date":"1993","source":"Journal of medical virology","url":"https://pubmed.ncbi.nlm.nih.gov/8388029","citation_count":42,"is_preprint":false},{"pmid":"29119857","id":"PMC_29119857","title":"miR-638 Inhibits immature Sertoli cell growth by indirectly inactivating PI3K/AKT pathway via SPAG1 gene.","date":"2017","source":"Cell cycle (Georgetown, Tex.)","url":"https://pubmed.ncbi.nlm.nih.gov/29119857","citation_count":36,"is_preprint":false},{"pmid":"7517029","id":"PMC_7517029","title":"Theileria annulata sporozoite surface antigen (SPAG-1) contains neutralizing determinants in the C terminus.","date":"1994","source":"Parasite immunology","url":"https://pubmed.ncbi.nlm.nih.gov/7517029","citation_count":26,"is_preprint":false},{"pmid":"16870344","id":"PMC_16870344","title":"Vaccination of calves with an attenuated cell line of Theileria annulata and the sporozoite antigen SPAG-1 produces a synergistic effect.","date":"2006","source":"Veterinary parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/16870344","citation_count":24,"is_preprint":false},{"pmid":"27053660","id":"PMC_27053660","title":"The GTPase SPAG-1 orchestrates meiotic program by dictating meiotic resumption and cytoskeleton architecture in mouse oocytes.","date":"2016","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/27053660","citation_count":20,"is_preprint":false},{"pmid":"11517287","id":"PMC_11517287","title":"Expression and function of the HSD-3.8 gene encoding a testis-specific protein.","date":"2001","source":"Molecular human reproduction","url":"https://pubmed.ncbi.nlm.nih.gov/11517287","citation_count":15,"is_preprint":false},{"pmid":"8760935","id":"PMC_8760935","title":"SP75 is encoded by the DP87 gene and belongs to a family of modular Dictyostelium discoideum outer layer spore coat proteins.","date":"1996","source":"Microbiology (Reading, England)","url":"https://pubmed.ncbi.nlm.nih.gov/8760935","citation_count":13,"is_preprint":false},{"pmid":"10527845","id":"PMC_10527845","title":"A tetratricopeptide repeat-containing protein gene, tpis, whose expression is induced with differentiation of spermatogenic cells.","date":"1999","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/10527845","citation_count":13,"is_preprint":false},{"pmid":"16368546","id":"PMC_16368546","title":"A sperm component, HSD-3.8 (SPAG1), interacts with G-protein beta 1 subunit and activates extracellular signal-regulated kinases (ERK).","date":"2006","source":"Frontiers in bioscience : a journal and virtual library","url":"https://pubmed.ncbi.nlm.nih.gov/16368546","citation_count":13,"is_preprint":false},{"pmid":"31118266","id":"PMC_31118266","title":"Binding properties of the quaternary assembly protein SPAG1.","date":"2019","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/31118266","citation_count":12,"is_preprint":false},{"pmid":"35178554","id":"PMC_35178554","title":"The role of SPAG1 in the assembly of axonemal dyneins in human airway epithelia.","date":"2022","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/35178554","citation_count":12,"is_preprint":false},{"pmid":"33739091","id":"PMC_33739091","title":"Optimizing the First TPR Domain of the Human SPAG1 Protein Provides Insight into the HSP70 and HSP90 Binding Properties.","date":"2021","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/33739091","citation_count":7,"is_preprint":false},{"pmid":"35951456","id":"PMC_35951456","title":"SPAG1 promotes the development of AML by activating the ERK/MAPK signaling pathway and affects the chemotherapy sensitivity of venetoclax.","date":"2022","source":"Neoplasma","url":"https://pubmed.ncbi.nlm.nih.gov/35951456","citation_count":3,"is_preprint":false},{"pmid":"12905684","id":"PMC_12905684","title":"[Study on the function of HSD-3.8 gene encoding a testis-specific protein with yeast two-hybrid system].","date":"2002","source":"Zhongguo yi xue ke xue yuan xue bao. Acta Academiae Medicinae Sinicae","url":"https://pubmed.ncbi.nlm.nih.gov/12905684","citation_count":1,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.07.01.25330014","title":"Immune Cell-Based Transcriptomic Mendelian Randomization and Colocalization Study on Type 1 Diabetes","date":"2025-07-02","source":"bioRxiv","url":"https://doi.org/10.1101/2025.07.01.25330014","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.01.27.635100","title":"R2TP-like Quaternary Chaperones: a comprehensive overview to understand the dynamic R2SP complex","date":"2025-01-28","source":"bioRxiv","url":"https://doi.org/10.1101/2025.01.27.635100","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9683,"output_tokens":2767,"usd":0.035277},"stage2":{"model":"claude-opus-4-6","input_tokens":6097,"output_tokens":2388,"usd":0.135277},"total_usd":0.170554,"stage1_batch_id":"msgbatch_011rkcQdVV1RaFq9XhcG6df6","stage2_batch_id":"msgbatch_01RzgrJafdF4EWHhj5mNi6dh","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2013,\n      \"finding\": \"SPAG1 is present in human airway epithelial cell lysates but absent from isolated axonemes; immunofluorescence shows absence of both outer and inner dynein arm (ODA and IDA) proteins in cilia from SPAG1-mutant individuals, indicating SPAG1 functions in the cytoplasmic pre-assembly and/or trafficking of axonemal dynein arms rather than as a structural axonemal component. Zebrafish spag1 morpholino knockdown produced cilia-related phenotypes consistent with cytoplasmic assembly factors.\",\n      \"method\": \"Immunofluorescence, axoneme fractionation/cell lysate comparison, zebrafish morpholino knockdown\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (fractionation, IF, in vivo morpholino), replicated across 14 affected individuals and zebrafish model\",\n      \"pmids\": [\"24055112\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SPAG1 interacts with multiple dynein axonemal assembly factors (DNAAFs), dynein heavy chains (DHCs), dynein intermediate chains (DICs), and canonical R2TP complex components (RUVBL1, RUVBL2, PIH1D2) by immunoprecipitation. SPAG1 loss reduces DHC protein levels and impairs DHC–DIC interactions, showing SPAG1 scaffolds R2TP-like complexes to facilitate folding/binding of DHCs to DIC complexes. A 60 kDa SPAG1 isoform can partially compensate for full-length SPAG1 loss to assemble a reduced number of outer dynein arms.\",\n      \"method\": \"Immunoprecipitation, quantitative proteomics (mass spectrometry), analysis of PCD patient airway epithelia\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IPs with multiple partners and quantitative proteomic readouts, disease-relevant human cell system\",\n      \"pmids\": [\"35178554\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"SPAG1 contains three TPR domains, but only two of them recruit the chaperones HSP70 and HSP90 via binding to their C-terminal tails. NMR structure of one TPR domain was solved, and NMR-driven docking plus molecular dynamics simulations defined the binding interface with HSP70 and HSP90 C-terminal peptides. A SPAG1 sub-fragment containing the putative P-loop motif cannot efficiently bind or hydrolyze GTP in vitro, challenging prior reports of SPAG1 GTPase activity and suggesting SPAG1 instead regulates nucleotide hydrolysis of HSP and RUVBL1/2 partners.\",\n      \"method\": \"NMR spectroscopy (3D structure), ITC, biochemical GTPase assays, molecular dynamics simulations\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — NMR structure with in vitro binding and enzymatic assays, multiple orthogonal biophysical methods\",\n      \"pmids\": [\"31118266\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Structural and biophysical characterization of the first TPR domain of human SPAG1 using an optimized variant showed with atomistic precision how the C-terminal tails of HSP70 and HSP90 bind; specific motifs within the TPR sequence drive positioning of HSP peptides.\",\n      \"method\": \"Protein sequence optimization, NMR spectroscopy, biophysical binding assays\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — structural determination combined with biophysical interaction analysis\",\n      \"pmids\": [\"33739091\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"HSD-3.8 (SPAG1) encodes a GTP-binding protein with GTPase activity and is phosphorylated by PKC in vitro. The protein localizes to the postacrosomal zone of human spermatozoa and to pachytene primary spermatocytes. It contains TPR motifs and a P-loop sequence. Immunization of female rats with recombinant HSD-3.8 protein caused infertility.\",\n      \"method\": \"[α-32P]GTP blot overlay assay, in vitro GTPase assay, in vitro PKC phosphorylation assay, immunofluorescence/immunostaining\",\n      \"journal\": \"Molecular human reproduction\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro enzymatic assays with direct biochemical demonstration, but later work challenges GTPase interpretation\",\n      \"pmids\": [\"11517287\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"HSD-3.8 (SPAG1) interacts with the C-terminal 144 amino acids of G-protein β1 subunit (Gβ1), forming a complex in the cytoplasm in the presence of GDP (co-immunoprecipitation in HEK293 cells, yeast two-hybrid). Overexpression of HSD-0.7 (SPAG1 fragment) activates ERK1/2 via a PKC-dependent (not Ras-dependent) pathway; deletion of either the TPR domain or the P-loop abolished ERK1/2 activation.\",\n      \"method\": \"Yeast two-hybrid, co-transfection/co-immunoprecipitation in HEK293 cells, ERK1/2 activation assay, domain deletion analysis\",\n      \"journal\": \"Frontiers in bioscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — yeast two-hybrid and co-IP with functional domain deletion, single lab\",\n      \"pmids\": [\"16368546\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In mouse oocytes, SPAG1 associates with meiotic spindles. RNAi depletion of SPAG1 impairs germinal vesicle breakdown (GVBD) via increased intracellular cAMP and decreased ATP, activating AMPK. SPAG1 depletion also reduces MAPK phosphorylation and causes irregular distribution of phospho-MAPK, impairing γ-tubulin function and spindle morphogenesis. Additionally, SPAG1 RNAi reduces actin expression and disrupts cortical granule-free domains, actin caps, and the contractile ring.\",\n      \"method\": \"RNAi in mouse oocytes, live imaging of spindle morphology, cAMP/ATP measurement, AMPK activation assay, immunofluorescence of γ-tubulin and phospho-MAPK\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — RNAi with multiple defined biochemical and cellular readouts, single lab\",\n      \"pmids\": [\"27053660\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"miR-638 directly targets SPAG1 in porcine immature Sertoli cells; SPAG1 knockdown (siRNA) phenocopies miR-638 overexpression by downregulating p-PI3K, p-AKT, c-MYC, CCND1, CCNE1, and CDK4, inhibiting proliferation and promoting apoptosis. SPAG1 siRNA also suppresses SOX2 and POU5F1 mRNA levels.\",\n      \"method\": \"miRNA target validation (luciferase reporter), siRNA knockdown, Western blot for PI3K/AKT pathway components, cell cycle/proliferation assays\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — direct target validation with functional epistasis via siRNA, single lab, porcine (non-human) Sertoli cell model\",\n      \"pmids\": [\"29119857\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SPAG1 knockdown in AML cells reduces proliferation and survival, and regulates expression of SMC3 while activating the ERK/MAPK signaling pathway.\",\n      \"method\": \"RNA interference knockdown, proliferation/survival assays, Western blot for ERK/MAPK pathway\",\n      \"journal\": \"Neoplasma\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single lab, knockdown with pathway readout but no direct molecular mechanism established\",\n      \"pmids\": [\"35951456\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The human R2SP complex (RUVBL1, RUVBL2, SPAG1, PIH1D2) has a 3D organization similar to the canonical R2TP complex, determined by cryo-EM, NMR, and structural mass spectrometry. SPAG1 and PIH1D2 act as adaptors recruiting specific clients, while the RUVBL1/2 ATPase core functions as the catalytic powerhouse; differences in RUVBL1/2 ATPase activity regulation and adaptor binding mode distinguish R2SP from canonical R2TP.\",\n      \"method\": \"Cryo-EM, NMR, structural mass spectrometry, ATPase activity assays\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — multi-method structural study; preprint, not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.01.27.635100\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"SPAG1 is a multidomain dynein axonemal assembly factor (DNAAF) that functions in the cytoplasm to scaffold the R2SP complex (with RUVBL1, RUVBL2, and PIH1D2) and recruit HSP70/HSP90 chaperones via its TPR domains, facilitating the pre-assembly of dynein heavy chains with intermediate chains before their transport into the cilium; loss of SPAG1 causes primary ciliary dyskinesia with combined outer and inner dynein arm defects, and SPAG1 also modulates ERK/MAPK and PI3K/AKT signaling in spermatogenic cells.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"SPAG1 is a cytoplasmic dynein axonemal assembly factor that scaffolds the R2SP complex (with RUVBL1, RUVBL2, and PIH1D2) to facilitate the pre-assembly and folding of axonemal dynein heavy chains and their association with intermediate chains before transport into cilia [PMID:24055112, PMID:35178554]. SPAG1 contains three TPR domains, two of which recruit HSP70 and HSP90 chaperones by binding their C-terminal tails, as defined by NMR structures and biophysical assays; SPAG1 itself lacks efficient GTPase activity and instead coordinates the ATPase activities of its RUVBL1/2 and chaperone partners [PMID:31118266, PMID:33739091]. Loss-of-function mutations in SPAG1 cause primary ciliary dyskinesia with combined outer and inner dynein arm defects [PMID:24055112]. In reproductive cells, SPAG1 knockdown impairs meiotic spindle morphogenesis in oocytes and modulates PI3K/AKT and ERK/MAPK signaling in Sertoli cells, indicating additional roles in germ cell proliferation and meiotic progression [PMID:27053660, PMID:29119857].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Initial biochemical characterization identified SPAG1 as a testis-expressed, TPR- and P-loop-containing protein with apparent GTP-binding and GTPase activity, establishing its domain architecture and spermatozoon localization but leaving its cellular function unknown.\",\n      \"evidence\": \"GTP overlay assay, in vitro GTPase/PKC assays, immunostaining of human spermatozoa\",\n      \"pmids\": [\"11517287\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"GTPase activity was later challenged by NMR/biochemical studies\", \"no physiological substrate or pathway identified\", \"no loss-of-function data\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Interaction with Gβ1 and demonstration that SPAG1 fragments activate ERK1/2 via PKC in a TPR- and P-loop-dependent manner suggested SPAG1 could modulate MAPK signaling, expanding its functional scope beyond sperm antigen to a signaling scaffold.\",\n      \"evidence\": \"Yeast two-hybrid, co-IP in HEK293 cells, ERK1/2 activation with domain deletion analysis\",\n      \"pmids\": [\"16368546\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"single lab, not replicated independently\", \"overexpression system; physiological relevance in vivo unclear\", \"Gβ1 interaction not validated in motile cilia biology\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identification of SPAG1 mutations in primary ciliary dyskinesia families established that SPAG1 is a dynein axonemal assembly factor required for both outer and inner dynein arm assembly; fractionation showed it acts in the cytoplasm rather than as an axonemal structural component.\",\n      \"evidence\": \"Immunofluorescence, axoneme fractionation, analysis of 14 affected individuals, zebrafish morpholino knockdown\",\n      \"pmids\": [\"24055112\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"precise molecular mechanism of dynein pre-assembly unknown\", \"identity of direct dynein client proteins not established\", \"relationship to R2TP/chaperone machinery not yet defined\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"RNAi depletion in mouse oocytes revealed a role for SPAG1 in meiotic spindle morphogenesis via regulation of MAPK phosphorylation, γ-tubulin distribution, and AMPK-dependent metabolic signaling, extending SPAG1 function beyond motile cilia.\",\n      \"evidence\": \"RNAi in mouse oocytes, live imaging, cAMP/ATP measurement, immunofluorescence\",\n      \"pmids\": [\"27053660\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"single lab; mechanism linking SPAG1 to AMPK/cAMP unclear\", \"whether this reflects TPR/chaperone scaffolding or an independent function is unknown\", \"no genetic knockout confirmation\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Validation of SPAG1 as a miR-638 target in Sertoli cells showed that SPAG1 knockdown suppresses PI3K/AKT signaling and proliferation, indicating SPAG1 supports spermatogenic cell survival through this pathway.\",\n      \"evidence\": \"Luciferase reporter assay, siRNA knockdown, Western blot for PI3K/AKT in porcine Sertoli cells\",\n      \"pmids\": [\"29119857\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"porcine model; relevance to human spermatogenesis not confirmed\", \"mechanism by which SPAG1 activates PI3K/AKT not defined\", \"single lab\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"NMR structure of a SPAG1 TPR domain and biophysical analysis resolved how two of three TPR domains recruit HSP70/HSP90 via their C-terminal tails, while demonstrating that SPAG1 itself lacks efficient GTPase activity — redefining SPAG1 as a chaperone-recruiting scaffold rather than an enzyme.\",\n      \"evidence\": \"NMR spectroscopy, ITC, biochemical GTPase assays, molecular dynamics simulations\",\n      \"pmids\": [\"31118266\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"role of the third non-chaperone-binding TPR domain unknown\", \"how chaperone recruitment couples to dynein folding not shown\", \"full-length SPAG1 structure not available\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Atomistic structural detail of the first TPR domain binding HSP70/HSP90 C-terminal peptides provided a high-resolution map of the chaperone recruitment interface, refining the structural basis for SPAG1's adaptor function.\",\n      \"evidence\": \"Optimized TPR variant, NMR spectroscopy, biophysical binding assays\",\n      \"pmids\": [\"33739091\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"binding measured with isolated domains; full-length context and client-loaded complexes not characterized\", \"no mutagenesis-based functional validation in cells\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Proteomic and co-IP studies in PCD patient airway epithelia demonstrated that SPAG1 directly scaffolds the R2SP complex (RUVBL1, RUVBL2, PIH1D2) and is required for dynein heavy chain stability and heavy chain–intermediate chain interactions, providing the molecular mechanism for its dynein assembly role.\",\n      \"evidence\": \"Reciprocal co-IPs, quantitative mass spectrometry in human airway epithelia\",\n      \"pmids\": [\"35178554\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"how the 60 kDa isoform partially compensates is unclear\", \"order of assembly steps (client loading, chaperone release) not resolved\", \"whether SPAG1 participates in dynein transport to cilia or only cytoplasmic pre-assembly is unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The full 3D architecture of the client-loaded R2SP complex, the precise order of dynein arm assembly steps mediated by SPAG1, and the mechanistic basis for SPAG1's roles in meiotic spindle morphogenesis and MAPK/PI3K signaling remain to be determined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"no full-length SPAG1 structure in complex with dynein clients\", \"no reconstituted in vitro dynein assembly assay with R2SP\", \"non-ciliary signaling functions lack mechanistic connection to known TPR/R2SP scaffolding\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 2, 3, 9]},\n      {\"term_id\": \"GO:0044183\", \"supporting_discovery_ids\": [1, 2]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [5, 6, 7]}\n    ],\n    \"complexes\": [\n      \"R2SP (RUVBL1/RUVBL2/SPAG1/PIH1D2)\"\n    ],\n    \"partners\": [\n      \"RUVBL1\",\n      \"RUVBL2\",\n      \"PIH1D2\",\n      \"HSPA1A\",\n      \"HSP90AA1\",\n      \"GNB1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}