{"gene":"SPAG1","run_date":"2026-06-10T07:46:38","timeline":{"discoveries":[{"year":2013,"finding":"SPAG1 is present in human airway epithelial cell lysates but absent from isolated axonemes; immunofluorescence showed absence of ODA and IDA proteins in cilia from affected individuals with SPAG1 mutations, indicating SPAG1 plays a role in cytoplasmic assembly and/or trafficking of axonemal dynein arms rather than being a structural cilia component. Zebrafish morpholino knockdown of spag1 produced cilia-related phenotypes consistent with cytoplasmic assembly factor function.","method":"Subcellular fractionation, immunofluorescence, zebrafish morpholino knockdown, exome sequencing with loss-of-function mutation analysis","journal":"American journal of human genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (fractionation, IF, in vivo morpholino), replicated across multiple affected families","pmids":["24055112"],"is_preprint":false},{"year":2022,"finding":"SPAG1 interacts (by immunoprecipitation) with multiple DNAAFs (dynein axonemal assembly factors), dynein heavy chains (DHCs), dynein intermediate chains (DICs), and canonical components of the R2TP complex. In SPAG1 mutants, protein levels of DHCs were reduced and interactions between DHCs and DICs were diminished, demonstrating that SPAG1 scaffolds R2TP-like complexes to facilitate folding/binding of DHCs to the DIC complex during dynein arm assembly.","method":"Immunoprecipitation, protein interaction studies, Western blot in control vs. PCD human airway epithelia","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP with multiple interactors, functional consequence shown by reduced DHC levels and disrupted DHC-DIC interaction in mutant human tissue","pmids":["35178554"],"is_preprint":false},{"year":2022,"finding":"A previously uncharacterized 60 kDa SPAG1 isoform can partially compensate for the absence of full-length SPAG1 to assemble a reduced number of outer dynein arms, as identified in PCD subjects with atypical ultrastructural defects.","method":"Protein isoform identification in patient-derived airway epithelia, ultrastructural analysis","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — single lab, novel isoform identification with functional inference from patient ultrastructure","pmids":["35178554"],"is_preprint":false},{"year":2019,"finding":"The TPR domains of SPAG1 recruit HSP70 and HSP90 chaperones: only two of the three TPR domains are capable of recruiting these chaperones. NMR-driven docking and MD simulations defined the binding interface between SPAG1-TPR1 and the C-terminal tails of HSP70 and HSP90. Additionally, a SPAG1 sub-fragment containing a putative P-loop motif cannot efficiently bind or hydrolyze GTP in vitro, challenging prior claims of intrinsic GTPase activity.","method":"Biochemical assays, isothermal titration calorimetry (ITC), NMR spectroscopy, molecular dynamics simulations, in vitro GTPase assay","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structure, ITC, and in vitro enzymatic assay combined in single study; negative GTPase result explicitly tested","pmids":["31118266"],"is_preprint":false},{"year":2021,"finding":"Structural and biophysical analysis of the first TPR domain of human SPAG1 (using an optimized variant) revealed with atomistic precision how the C-terminal tails of HSP70 and HSP90 bind the SPAG1-TPR1 domain, identifying specific motifs in the TPR sequence that drive HSP peptide positioning.","method":"Protein sequence optimization, NMR structure determination, biophysical binding assays","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structure with functional binding validation, multiple biophysical methods in one study","pmids":["33739091"],"is_preprint":false},{"year":2001,"finding":"The HSD-3.8 protein (SPAG1) contains tetratricopeptide repeat (TPR) motifs, a P-loop sequence, and phosphorylation sites. GTP-binding was demonstrated by blot overlay assay with [α-32P]GTP; the protein possesses GTPase activity and is phosphorylated by PKC in vitro. The protein localizes to the postacrosomal zone surface of human spermatozoa and to germ cells in seminiferous epithelium.","method":"GTP-binding blot overlay assay, in vitro GTPase assay, in vitro PKC phosphorylation assay, immunostaining","journal":"Molecular human reproduction","confidence":"Medium","confidence_rationale":"Tier 1–3 / Weak — in vitro biochemical assays from a single lab; GTPase claim later challenged by Chagot et al. 2019","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), as identified by yeast two-hybrid and confirmed by co-immunoprecipitation in HEK293 cells (where both proteins co-localized in the cytoplasm). Overexpression of the HSD-0.7 fragment activated ERK1/2 in a PKC-dependent (not Ras-dependent) manner; deletion of either the TPR domain or P-loop abolished ERK1/2 activation.","method":"Yeast two-hybrid, co-immunoprecipitation, co-transfection in HEK293 cells, ERK1/2 activation assay, domain deletion analysis","journal":"Frontiers in bioscience","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — yeast two-hybrid confirmed by co-IP and functional ERK assay with domain deletions, single lab","pmids":["16368546"],"is_preprint":false},{"year":2002,"finding":"Yeast two-hybrid screening with the 0.7 kb fragment of HSD-3.8 (SPAG1) identified the C-terminal 144 amino acids of human G-protein β1 subunit as an interacting partner; truncated bait plasmids lacking parts of the HSD-0.7 sequence did not interact, indicating the interaction requires the intact bait domain.","method":"Yeast two-hybrid screen of human ovary cDNA library","journal":"Acta Academiae Medicinae Sinicae","confidence":"Low","confidence_rationale":"Tier 3 / Weak — yeast two-hybrid only, single lab, no in-cell confirmation in this paper","pmids":["12905684"],"is_preprint":false},{"year":2016,"finding":"In mouse oocytes, SPAG1 associates with meiotic spindles. RNAi-mediated depletion of SPAG1 impaired germinal vesicle breakdown (GVBD) by increasing intracellular cAMP and decreasing ATP production, activating AMPK. SPAG1 depletion also disrupted spindle morphogenesis by reducing γ-tubulin function and MAPK phosphorylation at spindle poles, and decreased actin expression with disruption of actin caps, cortical granule-free domains, and the contractile ring.","method":"RNAi knockdown in mouse oocytes, live imaging, immunofluorescence, cAMP/ATP measurement, AMPK activation assay, MAPK phosphorylation analysis","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RNAi with multiple orthogonal readouts (cAMP, ATP, AMPK, MAPK, actin), single lab","pmids":["27053660"],"is_preprint":false},{"year":2017,"finding":"SPAG1 is a direct target of miR-638 in porcine immature Sertoli cells; SPAG1 RNAi reduced phospho-PI3K and phospho-AKT levels, and downregulated cell cycle factors (c-MYC, CCND1, CCNE1, CDK4), demonstrating that SPAG1 sustains PI3K/AKT pathway activation to promote Sertoli cell proliferation.","method":"miRNA target validation (luciferase or equivalent), SPAG1 siRNA knockdown, Western blot for PI3K/AKT pathway components","journal":"Cell cycle","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — direct target validation plus functional pathway readout, single lab with multiple downstream markers","pmids":["29119857"],"is_preprint":false},{"year":2022,"finding":"SPAG1 knockdown in AML cells reduced proliferation and survival, downregulated SMC3 expression, and suppressed ERK/MAPK signaling pathway activation. Inhibiting SPAG1 also altered AML cell susceptibility to venetoclax.","method":"RNA interference knockdown in AML cell lines, proliferation/survival assays, Western blot for ERK/MAPK pathway","journal":"Neoplasma","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, RNAi with downstream pathway readout but no mechanistic detail on how SPAG1 engages SMC3 or ERK","pmids":["35951456"],"is_preprint":false},{"year":2025,"finding":"Cryo-EM and structural mass spectrometry revealed the 3D organization of the human R2SP complex (RUVBL1, RUVBL2, SPAG1, PIH1D2), showing a structure similar to the canonical R2TP complex but with differences in RUVBL1/2 ATPase mode of action and in how adaptors SPAG1 and PIH1D2 bind. SPAG1 and PIH1D2 function as adaptors that interact with specific clients to promote quaternary assembly.","method":"Cryo-EM, NMR, structural mass spectrometry, biochemical reconstitution, ATPase assay","journal":"bioRxiv","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cryo-EM structure with multiple orthogonal structural and biochemical methods in single study; preprint not yet peer-reviewed","pmids":[],"is_preprint":true}],"current_model":"SPAG1 is a cytoplasmic dynein axonemal assembly factor (DNAAF) that scaffolds an R2SP quaternary chaperone complex (with RUVBL1, RUVBL2, and PIH1D2) via its TPR domains—which recruit HSP70 and HSP90—to facilitate folding and assembly of dynein heavy chains onto intermediate chains before their transport into cilia; loss of SPAG1 causes primary ciliary dyskinesia with combined outer and inner dynein arm defects, and in other cellular contexts SPAG1 also participates in AMPK/MAPK signaling during meiosis and PI3K/AKT signaling in Sertoli cells through interactions with Gβ1 and PKC-dependent ERK activation."},"narrative":{"mechanistic_narrative":"SPAG1 is a cytoplasmic dynein axonemal assembly factor (DNAAF) required for the preassembly of ciliary dynein arms; in airway epithelia it is present in the cytoplasm but absent from assembled axonemes, and loss-of-function mutations cause primary ciliary dyskinesia with combined loss of outer and inner dynein arm proteins from cilia [PMID:24055112]. Mechanistically, SPAG1 acts as a scaffold for an R2TP-like chaperone assembly, interacting with multiple DNAAFs, dynein heavy chains (DHCs), dynein intermediate chains (DICs), and canonical R2TP components; in SPAG1-mutant tissue DHC levels fall and DHC–DIC association is disrupted, establishing that SPAG1 promotes the folding and loading of dynein heavy chains onto the intermediate-chain complex during dynein arm assembly [PMID:35178554]. SPAG1 carries tetratricopeptide-repeat (TPR) domains, two of whose three TPR units recruit the C-terminal tails of HSP70 and HSP90, an interaction defined at atomic resolution and providing the chaperone-recruitment basis for client folding [PMID:31118266, PMID:33739091]; structural analysis of the SPAG1-containing R2SP complex (RUVBL1, RUVBL2, SPAG1, PIH1D2) shows SPAG1 and PIH1D2 serving as client-binding adaptors that drive quaternary assembly. Beyond ciliary assembly, RNAi studies implicate SPAG1 in meiotic spindle morphogenesis and germinal vesicle breakdown in oocytes through AMPK and MAPK signaling [PMID:27053660] and in PI3K/AKT-driven Sertoli cell proliferation [PMID:29119857].","teleology":[{"year":2001,"claim":"Initial characterization established SPAG1 as a TPR- and P-loop-containing, PKC-phosphorylated protein expressed in male germ cells, framing it as a candidate signaling/scaffold molecule.","evidence":"GTP-binding blot overlay, in vitro GTPase and PKC phosphorylation assays, and immunostaining of human spermatozoa and seminiferous epithelium","pmids":["11517287"],"confidence":"Medium","gaps":["GTPase activity inferred from blot overlay was not validated by quantitative kinetics","No cellular function assigned at this stage","Reproductive localization not connected to a defined pathway"]},{"year":2002,"claim":"A yeast two-hybrid screen began to define SPAG1 binding partners, identifying the G-protein β1 subunit C-terminus as an interactor and mapping the interaction to an intact bait domain.","evidence":"Yeast two-hybrid screen of a human ovary cDNA library with the HSD-0.7 fragment","pmids":["12905684"],"confidence":"Low","gaps":["Yeast two-hybrid only, with no in-cell confirmation in this study","Functional consequence of the interaction not tested","Single bait fragment used"]},{"year":2006,"claim":"The SPAG1–Gβ1 interaction was validated in cells and linked to a functional signaling output, showing SPAG1 fragments activate ERK1/2 through a PKC-dependent route requiring both TPR and P-loop motifs.","evidence":"Co-immunoprecipitation and co-localization in HEK293 cells with ERK1/2 activation and domain-deletion assays","pmids":["16368546"],"confidence":"Medium","gaps":["Used overexpressed fragments rather than full-length endogenous protein","ERK activation not connected to ciliary or reproductive phenotype","Single lab"]},{"year":2013,"claim":"Discovery of loss-of-function SPAG1 mutations in primary ciliary dyskinesia, combined with its cytoplasmic (non-axonemal) localization, repositioned SPAG1 as a cytoplasmic dynein arm assembly/trafficking factor rather than a structural cilia component.","evidence":"Exome sequencing, subcellular fractionation, immunofluorescence of patient cilia, and zebrafish morpholino knockdown","pmids":["24055112"],"confidence":"High","gaps":["Did not define molecular partners or the biochemical assembly step","Mechanism linking SPAG1 loss to absent ODA and IDA proteins unresolved"]},{"year":2016,"claim":"RNAi in oocytes extended SPAG1 function beyond cilia, implicating it in meiotic spindle morphogenesis and germinal vesicle breakdown via cAMP/ATP balance, AMPK activation, and MAPK signaling.","evidence":"RNAi knockdown in mouse oocytes with live imaging, immunofluorescence, cAMP/ATP measurement, and AMPK/MAPK readouts","pmids":["27053660"],"confidence":"Medium","gaps":["Direct molecular targets of SPAG1 at the spindle not identified","Relationship to the ciliary assembly role unclear","Single lab"]},{"year":2017,"claim":"SPAG1 was identified as a miR-638 target that sustains PI3K/AKT signaling to drive Sertoli cell proliferation, adding a proliferative signaling role.","evidence":"miRNA target validation and SPAG1 siRNA knockdown with Western blot of PI3K/AKT and cell cycle factors in porcine Sertoli cells","pmids":["29119857"],"confidence":"Medium","gaps":["How SPAG1 engages the PI3K/AKT pathway mechanistically not defined","Direct binding partners in this context unknown"]},{"year":2019,"claim":"Biochemical and NMR analysis established the chaperone-recruitment basis of SPAG1, showing two of its three TPR domains bind HSP70/HSP90 C-terminal tails, and directly challenged the earlier intrinsic GTPase claim.","evidence":"ITC, NMR spectroscopy, MD simulations, and an in vitro GTPase assay on the P-loop fragment","pmids":["31118266"],"confidence":"High","gaps":["Did not resolve full-length complex architecture","Client specificity of chaperone recruitment not addressed"]},{"year":2021,"claim":"Atomic-resolution structure of SPAG1-TPR1 detailed the specific motifs positioning HSP70/HSP90 peptides, refining the chaperone-binding mechanism.","evidence":"NMR structure determination of an optimized SPAG1-TPR1 variant with biophysical binding assays","pmids":["33739091"],"confidence":"High","gaps":["Single TPR domain in isolation","Does not capture client (dynein) engagement"]},{"year":2022,"claim":"Co-IP in human airway epithelia placed SPAG1 within an R2TP-like scaffold that promotes DHC folding and DHC–DIC assembly, and identified a 60 kDa SPAG1 isoform capable of partial ODA rescue.","evidence":"Reciprocal immunoprecipitation, Western blot in control vs PCD epithelia, and isoform/ultrastructural analysis","pmids":["35178554"],"confidence":"High","gaps":["Stoichiometry and assembly order not fully resolved","Functional role of the 60 kDa isoform inferred from patient ultrastructure only"]},{"year":2025,"claim":"Cryo-EM of the human R2SP complex defined how SPAG1 and PIH1D2 act as client-binding adaptors and how RUVBL1/2 ATPase behavior differs from canonical R2TP, providing the structural framework for SPAG1-mediated quaternary assembly.","evidence":"Cryo-EM, NMR, structural mass spectrometry, reconstitution, and ATPase assays (preprint)","pmids":[],"confidence":"High","gaps":["Preprint, not yet peer-reviewed","Dynein client engagement within the intact complex not directly visualized"]},{"year":null,"claim":"How SPAG1's chaperone-scaffolding role mechanistically integrates with its reported signaling functions (AMPK/MAPK, PI3K/AKT, ERK) across reproductive and proliferative contexts remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No unifying mechanism connecting dynein assembly scaffold to signaling outputs","Direct signaling substrates/effectors of SPAG1 undefined","Tissue-specific isoform contributions not mapped"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1,11]},{"term_id":"GO:0044183","term_label":"protein folding chaperone","supporting_discovery_ids":[3,4]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,11]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,6]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[8]}],"pathway":[{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[0,1]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[1,3]}],"complexes":["R2SP complex (RUVBL1, RUVBL2, SPAG1, PIH1D2)","R2TP-like dynein assembly scaffold"],"partners":["RUVBL1","RUVBL2","PIH1D2","HSP70","HSP90","GNB1"],"other_free_text":[]}},"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":37,"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 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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":10471,"output_tokens":3198,"usd":0.039691,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10646,"output_tokens":3653,"usd":0.072277,"stage2_stop_reason":"end_turn"},"total_usd":0.111968,"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\": 2013,\n      \"finding\": \"SPAG1 is present in human airway epithelial cell lysates but absent from isolated axonemes; immunofluorescence showed absence of ODA and IDA proteins in cilia from affected individuals with SPAG1 mutations, indicating SPAG1 plays a role in cytoplasmic assembly and/or trafficking of axonemal dynein arms rather than being a structural cilia component. Zebrafish morpholino knockdown of spag1 produced cilia-related phenotypes consistent with cytoplasmic assembly factor function.\",\n      \"method\": \"Subcellular fractionation, immunofluorescence, zebrafish morpholino knockdown, exome sequencing with loss-of-function mutation analysis\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (fractionation, IF, in vivo morpholino), replicated across multiple affected families\",\n      \"pmids\": [\"24055112\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SPAG1 interacts (by immunoprecipitation) with multiple DNAAFs (dynein axonemal assembly factors), dynein heavy chains (DHCs), dynein intermediate chains (DICs), and canonical components of the R2TP complex. In SPAG1 mutants, protein levels of DHCs were reduced and interactions between DHCs and DICs were diminished, demonstrating that SPAG1 scaffolds R2TP-like complexes to facilitate folding/binding of DHCs to the DIC complex during dynein arm assembly.\",\n      \"method\": \"Immunoprecipitation, protein interaction studies, Western blot in control vs. PCD human airway epithelia\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP with multiple interactors, functional consequence shown by reduced DHC levels and disrupted DHC-DIC interaction in mutant human tissue\",\n      \"pmids\": [\"35178554\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"A previously uncharacterized 60 kDa SPAG1 isoform can partially compensate for the absence of full-length SPAG1 to assemble a reduced number of outer dynein arms, as identified in PCD subjects with atypical ultrastructural defects.\",\n      \"method\": \"Protein isoform identification in patient-derived airway epithelia, ultrastructural analysis\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — single lab, novel isoform identification with functional inference from patient ultrastructure\",\n      \"pmids\": [\"35178554\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The TPR domains of SPAG1 recruit HSP70 and HSP90 chaperones: only two of the three TPR domains are capable of recruiting these chaperones. NMR-driven docking and MD simulations defined the binding interface between SPAG1-TPR1 and the C-terminal tails of HSP70 and HSP90. Additionally, a SPAG1 sub-fragment containing a putative P-loop motif cannot efficiently bind or hydrolyze GTP in vitro, challenging prior claims of intrinsic GTPase activity.\",\n      \"method\": \"Biochemical assays, isothermal titration calorimetry (ITC), NMR spectroscopy, molecular dynamics simulations, in vitro GTPase assay\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure, ITC, and in vitro enzymatic assay combined in single study; negative GTPase result explicitly tested\",\n      \"pmids\": [\"31118266\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Structural and biophysical analysis of the first TPR domain of human SPAG1 (using an optimized variant) revealed with atomistic precision how the C-terminal tails of HSP70 and HSP90 bind the SPAG1-TPR1 domain, identifying specific motifs in the TPR sequence that drive HSP peptide positioning.\",\n      \"method\": \"Protein sequence optimization, NMR structure determination, biophysical binding assays\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure with functional binding validation, multiple biophysical methods in one study\",\n      \"pmids\": [\"33739091\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"The HSD-3.8 protein (SPAG1) contains tetratricopeptide repeat (TPR) motifs, a P-loop sequence, and phosphorylation sites. GTP-binding was demonstrated by blot overlay assay with [α-32P]GTP; the protein possesses GTPase activity and is phosphorylated by PKC in vitro. The protein localizes to the postacrosomal zone surface of human spermatozoa and to germ cells in seminiferous epithelium.\",\n      \"method\": \"GTP-binding blot overlay assay, in vitro GTPase assay, in vitro PKC phosphorylation assay, immunostaining\",\n      \"journal\": \"Molecular human reproduction\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–3 / Weak — in vitro biochemical assays from a single lab; GTPase claim later challenged by Chagot et al. 2019\",\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), as identified by yeast two-hybrid and confirmed by co-immunoprecipitation in HEK293 cells (where both proteins co-localized in the cytoplasm). Overexpression of the HSD-0.7 fragment activated ERK1/2 in a PKC-dependent (not Ras-dependent) manner; deletion of either the TPR domain or P-loop abolished ERK1/2 activation.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, co-transfection in HEK293 cells, ERK1/2 activation assay, domain deletion analysis\",\n      \"journal\": \"Frontiers in bioscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — yeast two-hybrid confirmed by co-IP and functional ERK assay with domain deletions, single lab\",\n      \"pmids\": [\"16368546\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Yeast two-hybrid screening with the 0.7 kb fragment of HSD-3.8 (SPAG1) identified the C-terminal 144 amino acids of human G-protein β1 subunit as an interacting partner; truncated bait plasmids lacking parts of the HSD-0.7 sequence did not interact, indicating the interaction requires the intact bait domain.\",\n      \"method\": \"Yeast two-hybrid screen of human ovary cDNA library\",\n      \"journal\": \"Acta Academiae Medicinae Sinicae\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — yeast two-hybrid only, single lab, no in-cell confirmation in this paper\",\n      \"pmids\": [\"12905684\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In mouse oocytes, SPAG1 associates with meiotic spindles. RNAi-mediated depletion of SPAG1 impaired germinal vesicle breakdown (GVBD) by increasing intracellular cAMP and decreasing ATP production, activating AMPK. SPAG1 depletion also disrupted spindle morphogenesis by reducing γ-tubulin function and MAPK phosphorylation at spindle poles, and decreased actin expression with disruption of actin caps, cortical granule-free domains, and the contractile ring.\",\n      \"method\": \"RNAi knockdown in mouse oocytes, live imaging, immunofluorescence, cAMP/ATP measurement, AMPK activation assay, MAPK phosphorylation analysis\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNAi with multiple orthogonal readouts (cAMP, ATP, AMPK, MAPK, actin), single lab\",\n      \"pmids\": [\"27053660\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"SPAG1 is a direct target of miR-638 in porcine immature Sertoli cells; SPAG1 RNAi reduced phospho-PI3K and phospho-AKT levels, and downregulated cell cycle factors (c-MYC, CCND1, CCNE1, CDK4), demonstrating that SPAG1 sustains PI3K/AKT pathway activation to promote Sertoli cell proliferation.\",\n      \"method\": \"miRNA target validation (luciferase or equivalent), SPAG1 siRNA knockdown, Western blot for PI3K/AKT pathway components\",\n      \"journal\": \"Cell cycle\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — direct target validation plus functional pathway readout, single lab with multiple downstream markers\",\n      \"pmids\": [\"29119857\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SPAG1 knockdown in AML cells reduced proliferation and survival, downregulated SMC3 expression, and suppressed ERK/MAPK signaling pathway activation. Inhibiting SPAG1 also altered AML cell susceptibility to venetoclax.\",\n      \"method\": \"RNA interference knockdown in AML cell lines, proliferation/survival assays, Western blot for ERK/MAPK pathway\",\n      \"journal\": \"Neoplasma\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, RNAi with downstream pathway readout but no mechanistic detail on how SPAG1 engages SMC3 or ERK\",\n      \"pmids\": [\"35951456\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Cryo-EM and structural mass spectrometry revealed the 3D organization of the human R2SP complex (RUVBL1, RUVBL2, SPAG1, PIH1D2), showing a structure similar to the canonical R2TP complex but with differences in RUVBL1/2 ATPase mode of action and in how adaptors SPAG1 and PIH1D2 bind. SPAG1 and PIH1D2 function as adaptors that interact with specific clients to promote quaternary assembly.\",\n      \"method\": \"Cryo-EM, NMR, structural mass spectrometry, biochemical reconstitution, ATPase assay\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM structure with multiple orthogonal structural and biochemical methods in single study; preprint not yet peer-reviewed\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"SPAG1 is a cytoplasmic dynein axonemal assembly factor (DNAAF) that scaffolds an R2SP quaternary chaperone complex (with RUVBL1, RUVBL2, and PIH1D2) via its TPR domains—which recruit HSP70 and HSP90—to facilitate folding and assembly of dynein heavy chains onto intermediate chains before their transport into cilia; loss of SPAG1 causes primary ciliary dyskinesia with combined outer and inner dynein arm defects, and in other cellular contexts SPAG1 also participates in AMPK/MAPK signaling during meiosis and PI3K/AKT signaling in Sertoli cells through interactions with Gβ1 and PKC-dependent ERK activation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SPAG1 is a cytoplasmic dynein axonemal assembly factor (DNAAF) required for the preassembly of ciliary dynein arms; in airway epithelia it is present in the cytoplasm but absent from assembled axonemes, and loss-of-function mutations cause primary ciliary dyskinesia with combined loss of outer and inner dynein arm proteins from cilia [#0]. Mechanistically, SPAG1 acts as a scaffold for an R2TP-like chaperone assembly, interacting with multiple DNAAFs, dynein heavy chains (DHCs), dynein intermediate chains (DICs), and canonical R2TP components; in SPAG1-mutant tissue DHC levels fall and DHC–DIC association is disrupted, establishing that SPAG1 promotes the folding and loading of dynein heavy chains onto the intermediate-chain complex during dynein arm assembly [#1]. SPAG1 carries tetratricopeptide-repeat (TPR) domains, two of whose three TPR units recruit the C-terminal tails of HSP70 and HSP90, an interaction defined at atomic resolution and providing the chaperone-recruitment basis for client folding [#3, #4]; structural analysis of the SPAG1-containing R2SP complex (RUVBL1, RUVBL2, SPAG1, PIH1D2) shows SPAG1 and PIH1D2 serving as client-binding adaptors that drive quaternary assembly [#11]. Beyond ciliary assembly, RNAi studies implicate SPAG1 in meiotic spindle morphogenesis and germinal vesicle breakdown in oocytes through AMPK and MAPK signaling [#8] and in PI3K/AKT-driven Sertoli cell proliferation [#9].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Initial characterization established SPAG1 as a TPR- and P-loop-containing, PKC-phosphorylated protein expressed in male germ cells, framing it as a candidate signaling/scaffold molecule.\",\n      \"evidence\": \"GTP-binding blot overlay, in vitro GTPase and PKC phosphorylation assays, and immunostaining of human spermatozoa and seminiferous epithelium\",\n      \"pmids\": [\"11517287\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"GTPase activity inferred from blot overlay was not validated by quantitative kinetics\", \"No cellular function assigned at this stage\", \"Reproductive localization not connected to a defined pathway\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"A yeast two-hybrid screen began to define SPAG1 binding partners, identifying the G-protein \\u03b21 subunit C-terminus as an interactor and mapping the interaction to an intact bait domain.\",\n      \"evidence\": \"Yeast two-hybrid screen of a human ovary cDNA library with the HSD-0.7 fragment\",\n      \"pmids\": [\"12905684\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Yeast two-hybrid only, with no in-cell confirmation in this study\", \"Functional consequence of the interaction not tested\", \"Single bait fragment used\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"The SPAG1\\u2013G\\u03b21 interaction was validated in cells and linked to a functional signaling output, showing SPAG1 fragments activate ERK1/2 through a PKC-dependent route requiring both TPR and P-loop motifs.\",\n      \"evidence\": \"Co-immunoprecipitation and co-localization in HEK293 cells with ERK1/2 activation and domain-deletion assays\",\n      \"pmids\": [\"16368546\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Used overexpressed fragments rather than full-length endogenous protein\", \"ERK activation not connected to ciliary or reproductive phenotype\", \"Single lab\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Discovery of loss-of-function SPAG1 mutations in primary ciliary dyskinesia, combined with its cytoplasmic (non-axonemal) localization, repositioned SPAG1 as a cytoplasmic dynein arm assembly/trafficking factor rather than a structural cilia component.\",\n      \"evidence\": \"Exome sequencing, subcellular fractionation, immunofluorescence of patient cilia, and zebrafish morpholino knockdown\",\n      \"pmids\": [\"24055112\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define molecular partners or the biochemical assembly step\", \"Mechanism linking SPAG1 loss to absent ODA and IDA proteins unresolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"RNAi in oocytes extended SPAG1 function beyond cilia, implicating it in meiotic spindle morphogenesis and germinal vesicle breakdown via cAMP/ATP balance, AMPK activation, and MAPK signaling.\",\n      \"evidence\": \"RNAi knockdown in mouse oocytes with live imaging, immunofluorescence, cAMP/ATP measurement, and AMPK/MAPK readouts\",\n      \"pmids\": [\"27053660\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct molecular targets of SPAG1 at the spindle not identified\", \"Relationship to the ciliary assembly role unclear\", \"Single lab\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"SPAG1 was identified as a miR-638 target that sustains PI3K/AKT signaling to drive Sertoli cell proliferation, adding a proliferative signaling role.\",\n      \"evidence\": \"miRNA target validation and SPAG1 siRNA knockdown with Western blot of PI3K/AKT and cell cycle factors in porcine Sertoli cells\",\n      \"pmids\": [\"29119857\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How SPAG1 engages the PI3K/AKT pathway mechanistically not defined\", \"Direct binding partners in this context unknown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Biochemical and NMR analysis established the chaperone-recruitment basis of SPAG1, showing two of its three TPR domains bind HSP70/HSP90 C-terminal tails, and directly challenged the earlier intrinsic GTPase claim.\",\n      \"evidence\": \"ITC, NMR spectroscopy, MD simulations, and an in vitro GTPase assay on the P-loop fragment\",\n      \"pmids\": [\"31118266\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve full-length complex architecture\", \"Client specificity of chaperone recruitment not addressed\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Atomic-resolution structure of SPAG1-TPR1 detailed the specific motifs positioning HSP70/HSP90 peptides, refining the chaperone-binding mechanism.\",\n      \"evidence\": \"NMR structure determination of an optimized SPAG1-TPR1 variant with biophysical binding assays\",\n      \"pmids\": [\"33739091\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Single TPR domain in isolation\", \"Does not capture client (dynein) engagement\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Co-IP in human airway epithelia placed SPAG1 within an R2TP-like scaffold that promotes DHC folding and DHC\\u2013DIC assembly, and identified a 60 kDa SPAG1 isoform capable of partial ODA rescue.\",\n      \"evidence\": \"Reciprocal immunoprecipitation, Western blot in control vs PCD epithelia, and isoform/ultrastructural analysis\",\n      \"pmids\": [\"35178554\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and assembly order not fully resolved\", \"Functional role of the 60 kDa isoform inferred from patient ultrastructure only\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Cryo-EM of the human R2SP complex defined how SPAG1 and PIH1D2 act as client-binding adaptors and how RUVBL1/2 ATPase behavior differs from canonical R2TP, providing the structural framework for SPAG1-mediated quaternary assembly.\",\n      \"evidence\": \"Cryo-EM, NMR, structural mass spectrometry, reconstitution, and ATPase assays (preprint)\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Preprint, not yet peer-reviewed\", \"Dynein client engagement within the intact complex not directly visualized\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How SPAG1's chaperone-scaffolding role mechanistically integrates with its reported signaling functions (AMPK/MAPK, PI3K/AKT, ERK) across reproductive and proliferative contexts remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No unifying mechanism connecting dynein assembly scaffold to signaling outputs\", \"Direct signaling substrates/effectors of SPAG1 undefined\", \"Tissue-specific isoform contributions not mapped\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 11]},\n      {\"term_id\": \"GO:0044183\", \"supporting_discovery_ids\": [3, 4]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 11]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 6]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 3]}\n    ],\n    \"complexes\": [\"R2SP complex (RUVBL1, RUVBL2, SPAG1, PIH1D2)\", \"R2TP-like dynein assembly scaffold\"],\n    \"partners\": [\"RUVBL1\", \"RUVBL2\", \"PIH1D2\", \"HSP70\", \"HSP90\", \"GNB1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":4,"faith_total":4,"faith_pct":100.0}}