{"gene":"PIH1D1","run_date":"2026-06-10T06:43:35","timeline":{"discoveries":[{"year":2008,"finding":"Pih1/Nop17 (yeast PIH1D1 ortholog) is an unstable protein that is stabilized by Hsp90 acting together with the Tah1 cofactor; together Tah1 and Pih1 bind the essential helicases Rvb1 and Rvb2 to form the R2TP complex, which is required for correct accumulation of box C/D snoRNPs (and also box H/ACA snoRNAs).","method":"Genetic interaction screens, co-immunoprecipitation, in vivo stability assays, snoRNA accumulation assays in yeast","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, in vivo functional assays, replicated across multiple labs subsequently","pmids":["18268103"],"is_preprint":false},{"year":2010,"finding":"Pih1 and Tah1 form a stable heterodimeric complex; alone Pih1 is unstable and degraded from its N terminus. The Pih1-Tah1 heterodimer binds Hsp90 with similar affinity and stoichiometry as Tah1 alone, but antagonizes Tah1's stimulatory effect and inhibits Hsp90 ATPase activity. The region within Pih1 responsible for Tah1 interaction and Hsp90 ATPase inhibition was identified.","method":"Analytical ultracentrifugation, isothermal titration calorimetry (ITC), noncovalent mass spectrometry, ATPase activity assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — multiple orthogonal biophysical methods (AUC, ITC, MS) plus enzymatic assay in one study","pmids":["20663878"],"is_preprint":false},{"year":2011,"finding":"NMR structure of Tah1 reveals two TPR motifs followed by a C-helix and unstructured region; Tah1 binds Hsp90 via a two-carboxylate clamp engaging the EEVD C-terminal residues of Hsp90, and binds the C terminus of Pih1 through its C-helix and unstructured region. Tah1 binding to Pih1 C terminus stabilizes Pih1 by forming a stable complex.","method":"NMR structure determination, binding assays, mutagenesis, in vitro/in vivo stability assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structure plus mutagenesis and stability assays in one study","pmids":["22179618"],"is_preprint":false},{"year":2012,"finding":"The C terminus of Pih1 contains multiple destabilization/degron elements including two intrinsically disordered regions and five hydrophobic clusters; IDR2 is required for Tah1 binding. The Pih1 N-terminal domain (residues 1–230) is sufficient to bind the Rvb1/Rvb2 heterocomplex and complement Pih1 physiological function at 37°C, while the sequence between the two disordered regions significantly enhances Rvb1/Rvb2 binding.","method":"Site-directed mutagenesis, in vitro binding assays, in vivo complementation assays, secondary structure analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — mutagenesis combined with in vitro binding and in vivo complementation assays","pmids":["23139418"],"is_preprint":false},{"year":2012,"finding":"Human PIH1D1 interacts directly with histone H4 and recruits the Brg1-SWI/SNF complex (via SNF5) to rRNA gene promoters, mediates DNase I-hypersensitive chromatin remodeling at the core promoter, enhances Pol I complex occupancy and transcription initiation of rRNA genes, and displaces TIP5 (NoRC component) from the core region to derepress rRNA gene silencing.","method":"Co-immunoprecipitation, ChIP assays, RNAi knockdown with rRNA transcription readout, DNase I hypersensitivity assay","journal":"Journal of molecular cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus ChIP plus functional KD readouts, single lab","pmids":["22368283"],"is_preprint":false},{"year":2012,"finding":"RPAP3 isoform 1 (but not isoform 2) interacts with PIH1D1; knockdown of RPAP3 isoform 1 reduces PIH1D1 protein level without affecting PIH1D1 mRNA, indicating post-transcriptional stabilization of PIH1D1 by RPAP3 isoform 1.","method":"Co-immunoprecipitation, siRNA knockdown, western blot","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP and mRNA vs protein-level distinction, single lab, two methods","pmids":["23159623"],"is_preprint":false},{"year":2013,"finding":"PIH1D1 specifically associates with mTORC1 (co-IP of Raptor but not Rictor); PIH1D1 knockdown decreases mTORC1 assembly, reduces S6 kinase phosphorylation (mTORC1 activity indicator), and decreases rRNA transcription without affecting mTORC2.","method":"Co-immunoprecipitation, siRNA knockdown, S6K phosphorylation assay, rRNA transcription assay","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus functional KD with specific signaling readouts, single lab","pmids":["24036451"],"is_preprint":false},{"year":2014,"finding":"Crystal structure of the PIH1D1 N-terminal domain (PIH-N) bound to a CK2-phosphorylated TEL2 peptide reveals a phosphopeptide-binding domain that specifically recognizes a DpSDD motif; Lys57 and Lys64 in PIH1D1 are essential for binding. Proteomic analysis identified additional R2TP substrates recruited by PIH-N in a sequence-specific, phosphorylation-dependent manner.","method":"Crystal structure (co-crystal with phosphopeptide), mutagenesis of Lys57/Lys64, phosphoproteomics, binding assays","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus mutagenesis plus proteomics; replicated by independent structural study (PMID:24794838)","pmids":["24656813"],"is_preprint":false},{"year":2014,"finding":"Structural and biochemical analysis of Pih1D1-Tel2 complex confirms that Pih1D1 contains a domain specific for binding CK2 phosphorylation sites; structural characterization of Hsp90-Tah1-Pih1, Hsp90-Spagh (RPAP3), and Pih1D1-Tel2 complexes defines the structural basis by which the R2TP complex connects Hsp90 to the TTT complex.","method":"X-ray crystallography, biochemical binding assays","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure independently confirming phosphopeptide-binding domain, consistent with PMID:24656813","pmids":["24794838"],"is_preprint":false},{"year":2015,"finding":"NMR solution structure of the Tah1p:Pih1p complex shows that the C-terminal tail (S93-S111) of Tah1p binds the CS domain (Pih1p264-344) forming two intermolecular β-sheets and one covering loop. Pih1p has two direct binding partners beyond Tah1p: the assembly factor Rsa1p and the snoRNP core protein Nop58p; these two interactions are mutually exclusive. Additionally, the Pih1p257-344 CS domain contacts the Hsp82 middle-C-terminal domain in the presence of Tah1p.","method":"NMR structure, ITC, co-expression/pulldown in E. coli, in vitro and in vivo binding assays","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structure plus ITC plus co-expression pulldown in one study with multiple binding partners characterized","pmids":["26210662"],"is_preprint":false},{"year":2015,"finding":"Nop17/Pih1 (yeast PIH1D1 ortholog) interacts with Nop58 (snoRNP core protein) and ATP modulates the interaction between Nop17 and the ATPases Rvb1/Rvb2; R2TP complex reduces affinity of Nop58 for snoRNA to facilitate binding of other snoRNP subunits.","method":"Co-immunoprecipitation, in vitro binding assays, domain mapping","journal":"BMC molecular biology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP and in vitro binding with domain mapping, single lab, multiple interactions tested","pmids":["25888478"],"is_preprint":false},{"year":2016,"finding":"Pih1 is degraded via a ubiquitin-independent proteasome pathway: the proteasome subunit Rpn8 directly interacts with the Pih1 C terminus (specifically the last 30 amino acids of Rpn8 bind Pih1 C terminus); Pih1(282-344) acts as a degron sufficient to induce ubiquitin-independent degradation. Truncation of the Rpn8 C-terminal disordered region does not affect proteasome assembly but specifically inhibits degradation of GFP-Pih1(282-344) in vivo and Pih1 in vitro.","method":"Co-immunoprecipitation, truncation mutagenesis, in vitro and in vivo degradation assays, ubiquitin-independent proteasome assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro degradation assay plus mutagenesis plus in vivo complementation, single lab, multiple orthogonal approaches","pmids":["27053109"],"is_preprint":false},{"year":2009,"finding":"PIH1D1 stabilizes the SWI/SNF core subunit SNF5 by attenuating its proteasome-mediated degradation; overexpression of PIH1D1 increases SNF5 protein level.","method":"Cycloheximide chase assay, proteasome inhibitor (MG132) treatment, PIH1D1 overexpression with western blot readout","journal":"Acta Academiae Medicinae Sinicae","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, single method, limited controls reported in abstract","pmids":["20078948"],"is_preprint":false},{"year":2010,"finding":"PIH1D1 interacts with both RPAP3 and Monad (Reptin/RUVBL2) in human cells; siRNA knockdown of PIH1D1 enhances doxorubicin-induced apoptosis and caspase-3 activation, indicating PIH1D1 functions as a modulator of the apoptosis pathway.","method":"Co-immunoprecipitation in HEK293/U2OS cells, siRNA knockdown, caspase-3 activity assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP plus functional siRNA KD with specific apoptosis readout, single lab","pmids":["21078300"],"is_preprint":false},{"year":2025,"finding":"In Trypanosoma brucei, PIH1D1 concentrates at co-translational assembly sites where outer dynein arm (ODA) heavy chain (HC) translation occurs; without PIH1D1, HC protein levels are reduced and the IC-LC complex is stranded in the cytoplasm, indicating PIH1D1 generates specialized compartments to assist co-translational folding of HCs and enable their assembly with other ODA subunits.","method":"Live fluorescence imaging, genetic knockdown with western blot and localization readouts in Trypanosoma brucei","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — preprint, single organism model (Trypanosoma), single lab, imaging plus KD without mechanistic reconstitution","pmids":["bio_10.1101_2025.07.26.666928"],"is_preprint":true}],"current_model":"PIH1D1 is the defining subunit of the R2TP co-chaperone complex that bridges the Hsp90 chaperone system to diverse macromolecular assemblies: its N-terminal PIH-N domain recognizes CK2-phosphorylated substrates (including TEL2 of the TTT complex) via a structurally defined phosphopeptide-binding mechanism requiring Lys57/Lys64, while its C-terminal CS domain interacts with Tah1/RPAP3 (which connects to Hsp90 via TPR-EEVD contacts) and directly binds Rvb1/Rvb2 ATPases and snoRNP core proteins such as Nop58; as a complex with Tah1, PIH1D1 inhibits Hsp90 ATPase activity and facilitates assembly of box C/D snoRNPs, mTORC1, RNA Pol II, and other clients, while in the absence of Hsp90/Tah1 support its intrinsically unstable C terminus is degraded via a ubiquitin-independent proteasome pathway mediated by direct interaction with proteasome subunit Rpn8."},"narrative":{"mechanistic_narrative":"PIH1D1 is the defining substrate-recognition subunit of the R2TP co-chaperone complex, which couples the Hsp90 chaperone machinery to the assembly of diverse macromolecular complexes [PMID:18268103, PMID:24656813]. Its N-terminal PIH domain is a phosphopeptide-binding module that specifically recognizes CK2-phosphorylated substrates such as TEL2 of the TTT complex through a DpSDD motif, an interaction that requires Lys57 and Lys64 and recruits additional clients in a phosphorylation-dependent manner [PMID:24656813, PMID:24794838]. Through its C-terminal CS domain, PIH1D1 binds the TPR cofactor Tah1/RPAP3 — which in turn engages the Hsp90 C-terminal EEVD via a two-carboxylate clamp — and together the Pih1-Tah1 heterodimer inhibits Hsp90 ATPase activity [PMID:20663878, PMID:22179618, PMID:26210662]. PIH1D1 also binds the Rvb1/Rvb2 (RUVBL1/RUVBL2) AAA+ ATPases via its N-terminal region to form the R2TP complex, which is required for accumulation of box C/D snoRNPs, acting in part by lowering Nop58 affinity for snoRNA to permit ordered subunit assembly [PMID:18268103, PMID:23139418, PMID:26210662, PMID:25888478]. Beyond snoRNP biogenesis, PIH1D1 promotes mTORC1 assembly and activity through association with Raptor [PMID:24036451] and regulates RNA polymerase I transcription of rRNA genes by recruiting the Brg1-SWI/SNF complex to rRNA promoters [PMID:22368283]. The intrinsically unstable C terminus of PIH1D1, which carries multiple disordered degron elements, is stabilized by Tah1/RPAP3 binding and, when unsupported, is destroyed by a ubiquitin-independent proteasome pathway mediated by direct interaction with the proteasome subunit Rpn8 [PMID:22179618, PMID:23139418, PMID:23159623, PMID:27053109].","teleology":[{"year":2008,"claim":"Established PIH1D1 (yeast Pih1/Nop17) as an unstable protein that, with Tah1, binds the Rvb1/Rvb2 helicases to form the R2TP complex required for snoRNP accumulation, defining its core function in ribonucleoprotein assembly.","evidence":"Genetic interaction screens, co-IP, in vivo stability and snoRNA accumulation assays in yeast","pmids":["18268103"],"confidence":"High","gaps":["Did not define the structural basis of substrate or partner recognition","Mechanism by which R2TP promotes snoRNP assembly not resolved"]},{"year":2010,"claim":"Showed the Pih1-Tah1 heterodimer binds Hsp90 and inhibits its ATPase activity, revealing R2TP as a negative modulator of the chaperone cycle rather than a passive adaptor.","evidence":"AUC, ITC, noncovalent MS, ATPase assays on reconstituted complexes","pmids":["20663878"],"confidence":"High","gaps":["Did not provide atomic structure of the inhibitory interaction","Physiological consequence of ATPase inhibition for client assembly not tested"]},{"year":2010,"claim":"Connected human PIH1D1 to RPAP3 and RUVBL2 and to cell survival, indicating an apoptosis-modulating role.","evidence":"Co-IP in HEK293/U2OS cells, siRNA knockdown, caspase-3 activity assay","pmids":["21078300"],"confidence":"Medium","gaps":["Mechanistic link between R2TP function and apoptosis not defined","Direct versus indirect effect on caspase activation unresolved"]},{"year":2012,"claim":"Mapped the modular architecture of PIH1D1: an N-terminal Rvb1/Rvb2-binding region sufficient for function and a C-terminal disordered degron region required for Tah1 binding, separating partner recognition from instability.","evidence":"Mutagenesis, in vitro binding and in vivo complementation in yeast","pmids":["23139418"],"confidence":"High","gaps":["Did not identify the substrate-recognition mechanism of the N domain","Degradation machinery acting on the degron not yet known"]},{"year":2012,"claim":"Linked human PIH1D1 to rRNA gene activation by recruiting Brg1-SWI/SNF to rRNA promoters and displacing the silencing factor TIP5, extending its role to chromatin remodeling at the rDNA.","evidence":"Co-IP, ChIP, RNAi with rRNA transcription readout, DNase I hypersensitivity","pmids":["22368283"],"confidence":"Medium","gaps":["Direct histone H4 versus SNF5 binding contributions not separated","Whether this depends on R2TP/Hsp90 unclear"]},{"year":2012,"claim":"Demonstrated that RPAP3 isoform 1 post-transcriptionally stabilizes PIH1D1 protein, linking the human cofactor to PIH1D1 abundance control.","evidence":"Co-IP, siRNA knockdown, western blot vs mRNA comparison","pmids":["23159623"],"confidence":"Medium","gaps":["Did not identify the degradation pathway counteracted by RPAP3","Isoform specificity mechanism not defined"]},{"year":2013,"claim":"Identified PIH1D1 as a factor promoting mTORC1 assembly and activity, broadening R2TP clients to a major signaling complex.","evidence":"Co-IP of Raptor, siRNA knockdown, S6K phosphorylation and rRNA transcription assays","pmids":["24036451"],"confidence":"Medium","gaps":["Direct binding site within mTORC1 not mapped","Whether assembly requires Hsp90/Tah1 not tested"]},{"year":2014,"claim":"Solved the structural basis of substrate recognition: the PIH-N domain binds CK2-phosphorylated DpSDD motifs (e.g., TEL2) via Lys57/Lys64, defining R2TP as a phosphorylation-dependent client-selection machine.","evidence":"Co-crystal structure with phosphopeptide, Lys57/Lys64 mutagenesis, phosphoproteomics","pmids":["24656813","24794838"],"confidence":"High","gaps":["Full repertoire of phosphorylated clients incompletely defined","How recognition triggers downstream chaperone-assisted assembly unresolved"]},{"year":2015,"claim":"Defined the CS-domain interactions of PIH1D1, showing Tah1 binds via β-sheet contacts and that binding of the snoRNP protein Nop58 and the assembly factor Rsa1 is mutually exclusive, indicating switchable client engagement.","evidence":"NMR structure, ITC, co-expression pulldown in E. coli, in vitro/in vivo binding","pmids":["26210662","25888478"],"confidence":"High","gaps":["Functional consequence of the Nop58/Rsa1 switch in vivo not established","How ATP-modulated Rvb1/Rvb2 binding integrates with CS-domain switching unclear"]},{"year":2016,"claim":"Revealed that the PIH1D1 C-terminal degron is destroyed by a ubiquitin-independent proteasome route through direct binding to the proteasome subunit Rpn8, explaining its intrinsic instability when cofactor-unsupported.","evidence":"Co-IP, truncation mutagenesis, in vitro and in vivo ubiquitin-independent degradation assays in yeast","pmids":["27053109"],"confidence":"High","gaps":["Whether the human PIH1D1-Rpn8 axis operates identically not shown","How Tah1 binding kinetically shields the degron not quantified"]},{"year":2025,"claim":"Extended PIH1D1 function to co-translational assembly, showing in Trypanosoma it concentrates at sites of dynein heavy-chain translation to support folding and assembly of axonemal motor subunits.","evidence":"Live fluorescence imaging and genetic knockdown in Trypanosoma brucei (preprint)","pmids":["bio_10.1101_2025.07.26.666928"],"confidence":"Low","gaps":["Preprint, single organism, no in vitro reconstitution","Whether mammalian PIH1D1 acts at co-translational assembly sites untested","Molecular mechanism of compartment formation undefined"]},{"year":null,"claim":"How PIH1D1-mediated substrate recognition is mechanistically coupled to Hsp90/RUVBL ATP cycling to drive client maturation remains unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No reconstituted assembly cycle linking phosphosubstrate capture to client folding","Full client spectrum and selection rules in human cells incompletely mapped"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,7,9]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[1]},{"term_id":"GO:0042393","term_label":"histone binding","supporting_discovery_ids":[4]}],"localization":[{"term_id":"GO:0005730","term_label":"nucleolus","supporting_discovery_ids":[4]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[4]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,10]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[1,2,11]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[4]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[6]}],"complexes":["R2TP complex","Pih1-Tah1 (RPAP3) heterodimer"],"partners":["TAH1","RPAP3","RUVBL1","RUVBL2","TELO2","NOP58","RPTOR","SMARCB1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9NWS0","full_name":"PIH1 domain-containing protein 1","aliases":["Nucleolar protein 17 homolog"],"length_aa":290,"mass_kda":32.4,"function":"Involved in the assembly of C/D box small nucleolar ribonucleoprotein (snoRNP) particles (PubMed:17636026). Recruits the SWI/SNF complex to the core promoter of rRNA genes and enhances pre-rRNA transcription (PubMed:22368283, PubMed:24036451). Mediates interaction of TELO2 with the R2TP complex which is necessary for the stability of MTOR and SMG1 (PubMed:20864032). Positively regulates the assembly and activity of the mTORC1 complex (PubMed:24036451)","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9NWS0/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PIH1D1","classification":"Not Classified","n_dependent_lines":29,"n_total_lines":1208,"dependency_fraction":0.024006622516556293},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"POLR2A","stoichiometry":10.0},{"gene":"POLR2H","stoichiometry":10.0},{"gene":"POLR2B","stoichiometry":4.0},{"gene":"POLR2E","stoichiometry":4.0},{"gene":"FKBP5","stoichiometry":0.2},{"gene":"NOP58","stoichiometry":0.2},{"gene":"POLR1C","stoichiometry":0.2},{"gene":"POLR2J","stoichiometry":0.2},{"gene":"PTGES3","stoichiometry":0.2},{"gene":"RPAP2","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/PIH1D1","total_profiled":1310},"omim":[{"mim_id":"611480","title":"PIH1 DOMAIN-CONTAINING PROTEIN 1; PIH1D1","url":"https://www.omim.org/entry/611480"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Cytosol","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/PIH1D1"},"hgnc":{"alias_symbol":["FLJ20643","Pih1","MOT48","DNAAF14"],"prev_symbol":["NOP17"]},"alphafold":{"accession":"Q9NWS0","domains":[{"cath_id":"-","chopping":"47-180","consensus_level":"high","plddt":88.928,"start":47,"end":180},{"cath_id":"2.60.40.790","chopping":"210-285","consensus_level":"high","plddt":86.3489,"start":210,"end":285}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NWS0","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NWS0-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NWS0-F1-predicted_aligned_error_v6.png","plddt_mean":78.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PIH1D1","jax_strain_url":"https://www.jax.org/strain/search?query=PIH1D1"},"sequence":{"accession":"Q9NWS0","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9NWS0.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9NWS0/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NWS0"}},"corpus_meta":[{"pmid":"18268103","id":"PMC_18268103","title":"Molecular chaperone Hsp90 stabilizes Pih1/Nop17 to maintain R2TP complex activity that regulates snoRNA accumulation.","date":"2008","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/18268103","citation_count":151,"is_preprint":false},{"pmid":"24794838","id":"PMC_24794838","title":"Structural basis for phosphorylation-dependent recruitment of Tel2 to Hsp90 by Pih1.","date":"2014","source":"Structure (London, England : 1993)","url":"https://pubmed.ncbi.nlm.nih.gov/24794838","citation_count":77,"is_preprint":false},{"pmid":"24656813","id":"PMC_24656813","title":"Phosphorylation-dependent PIH1D1 interactions define substrate specificity of the R2TP cochaperone complex.","date":"2014","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/24656813","citation_count":75,"is_preprint":false},{"pmid":"20663878","id":"PMC_20663878","title":"The Pih1-Tah1 cochaperone complex inhibits Hsp90 molecular chaperone ATPase activity.","date":"2010","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/20663878","citation_count":61,"is_preprint":false},{"pmid":"22179618","id":"PMC_22179618","title":"Structure of minimal tetratricopeptide repeat domain protein Tah1 reveals mechanism of its interaction with Pih1 and Hsp90.","date":"2011","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/22179618","citation_count":43,"is_preprint":false},{"pmid":"26210662","id":"PMC_26210662","title":"Structure/Function Analysis of Protein-Protein Interactions Developed by the Yeast Pih1 Platform Protein and Its Partners in Box C/D snoRNP Assembly.","date":"2015","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/26210662","citation_count":26,"is_preprint":false},{"pmid":"24036451","id":"PMC_24036451","title":"PIH1D1 interacts with mTOR complex 1 and enhances ribosome RNA transcription.","date":"2013","source":"FEBS 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biology","url":"https://pubmed.ncbi.nlm.nih.gov/22368283","citation_count":13,"is_preprint":false},{"pmid":"27053109","id":"PMC_27053109","title":"The Proteasome Subunit Rpn8 Interacts with the Small Nucleolar RNA Protein (snoRNP) Assembly Protein Pih1 and Mediates Its Ubiquitin-independent Degradation in Saccharomyces cerevisiae.","date":"2016","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/27053109","citation_count":6,"is_preprint":false},{"pmid":"41424038","id":"PMC_41424038","title":"PIH1D1 and RPAP3, Components of the PAQosome: Emerging Roles in Cellular Physiology.","date":"2025","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/41424038","citation_count":2,"is_preprint":false},{"pmid":"20078948","id":"PMC_20078948","title":"[Effect of PIH1D1 on the degradation of its binding protein SNF5].","date":"2009","source":"Zhongguo yi xue ke xue yuan xue bao. Acta Academiae Medicinae Sinicae","url":"https://pubmed.ncbi.nlm.nih.gov/20078948","citation_count":1,"is_preprint":false},{"pmid":"41819055","id":"PMC_41819055","title":"Methylated PIH1D1 as a Heart-Specific Biomarker for Anthracycline-Induced Cardiac Remodeling in Breast Cancer Patients.","date":"2026","source":"JACC. Basic to translational science","url":"https://pubmed.ncbi.nlm.nih.gov/41819055","citation_count":1,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.09.11.675549","title":"CCDC103-mediated assembly of the R2C complex links RUVBL1-RUVBL2 to Primary Ciliary Dyskinesia","date":"2025-09-13","source":"bioRxiv","url":"https://doi.org/10.1101/2025.09.11.675549","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.07.26.666928","title":"Spatiotemporal regulation of ciliary outer dynein arm biogenesis and the role of PIH homologs","date":"2025-07-30","source":"bioRxiv","url":"https://doi.org/10.1101/2025.07.26.666928","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":10729,"output_tokens":4017,"usd":0.046221,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":11589,"output_tokens":3928,"usd":0.078073,"stage2_stop_reason":"end_turn"},"total_usd":0.124294,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2008,\n      \"finding\": \"Pih1/Nop17 (yeast PIH1D1 ortholog) is an unstable protein that is stabilized by Hsp90 acting together with the Tah1 cofactor; together Tah1 and Pih1 bind the essential helicases Rvb1 and Rvb2 to form the R2TP complex, which is required for correct accumulation of box C/D snoRNPs (and also box H/ACA snoRNAs).\",\n      \"method\": \"Genetic interaction screens, co-immunoprecipitation, in vivo stability assays, snoRNA accumulation assays in yeast\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, in vivo functional assays, replicated across multiple labs subsequently\",\n      \"pmids\": [\"18268103\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Pih1 and Tah1 form a stable heterodimeric complex; alone Pih1 is unstable and degraded from its N terminus. The Pih1-Tah1 heterodimer binds Hsp90 with similar affinity and stoichiometry as Tah1 alone, but antagonizes Tah1's stimulatory effect and inhibits Hsp90 ATPase activity. The region within Pih1 responsible for Tah1 interaction and Hsp90 ATPase inhibition was identified.\",\n      \"method\": \"Analytical ultracentrifugation, isothermal titration calorimetry (ITC), noncovalent mass spectrometry, ATPase activity assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — multiple orthogonal biophysical methods (AUC, ITC, MS) plus enzymatic assay in one study\",\n      \"pmids\": [\"20663878\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"NMR structure of Tah1 reveals two TPR motifs followed by a C-helix and unstructured region; Tah1 binds Hsp90 via a two-carboxylate clamp engaging the EEVD C-terminal residues of Hsp90, and binds the C terminus of Pih1 through its C-helix and unstructured region. Tah1 binding to Pih1 C terminus stabilizes Pih1 by forming a stable complex.\",\n      \"method\": \"NMR structure determination, binding assays, mutagenesis, in vitro/in vivo stability assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure plus mutagenesis and stability assays in one study\",\n      \"pmids\": [\"22179618\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The C terminus of Pih1 contains multiple destabilization/degron elements including two intrinsically disordered regions and five hydrophobic clusters; IDR2 is required for Tah1 binding. The Pih1 N-terminal domain (residues 1–230) is sufficient to bind the Rvb1/Rvb2 heterocomplex and complement Pih1 physiological function at 37°C, while the sequence between the two disordered regions significantly enhances Rvb1/Rvb2 binding.\",\n      \"method\": \"Site-directed mutagenesis, in vitro binding assays, in vivo complementation assays, secondary structure analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis combined with in vitro binding and in vivo complementation assays\",\n      \"pmids\": [\"23139418\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Human PIH1D1 interacts directly with histone H4 and recruits the Brg1-SWI/SNF complex (via SNF5) to rRNA gene promoters, mediates DNase I-hypersensitive chromatin remodeling at the core promoter, enhances Pol I complex occupancy and transcription initiation of rRNA genes, and displaces TIP5 (NoRC component) from the core region to derepress rRNA gene silencing.\",\n      \"method\": \"Co-immunoprecipitation, ChIP assays, RNAi knockdown with rRNA transcription readout, DNase I hypersensitivity assay\",\n      \"journal\": \"Journal of molecular cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus ChIP plus functional KD readouts, single lab\",\n      \"pmids\": [\"22368283\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"RPAP3 isoform 1 (but not isoform 2) interacts with PIH1D1; knockdown of RPAP3 isoform 1 reduces PIH1D1 protein level without affecting PIH1D1 mRNA, indicating post-transcriptional stabilization of PIH1D1 by RPAP3 isoform 1.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, western blot\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP and mRNA vs protein-level distinction, single lab, two methods\",\n      \"pmids\": [\"23159623\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"PIH1D1 specifically associates with mTORC1 (co-IP of Raptor but not Rictor); PIH1D1 knockdown decreases mTORC1 assembly, reduces S6 kinase phosphorylation (mTORC1 activity indicator), and decreases rRNA transcription without affecting mTORC2.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, S6K phosphorylation assay, rRNA transcription assay\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus functional KD with specific signaling readouts, single lab\",\n      \"pmids\": [\"24036451\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Crystal structure of the PIH1D1 N-terminal domain (PIH-N) bound to a CK2-phosphorylated TEL2 peptide reveals a phosphopeptide-binding domain that specifically recognizes a DpSDD motif; Lys57 and Lys64 in PIH1D1 are essential for binding. Proteomic analysis identified additional R2TP substrates recruited by PIH-N in a sequence-specific, phosphorylation-dependent manner.\",\n      \"method\": \"Crystal structure (co-crystal with phosphopeptide), mutagenesis of Lys57/Lys64, phosphoproteomics, binding assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus mutagenesis plus proteomics; replicated by independent structural study (PMID:24794838)\",\n      \"pmids\": [\"24656813\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Structural and biochemical analysis of Pih1D1-Tel2 complex confirms that Pih1D1 contains a domain specific for binding CK2 phosphorylation sites; structural characterization of Hsp90-Tah1-Pih1, Hsp90-Spagh (RPAP3), and Pih1D1-Tel2 complexes defines the structural basis by which the R2TP complex connects Hsp90 to the TTT complex.\",\n      \"method\": \"X-ray crystallography, biochemical binding assays\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure independently confirming phosphopeptide-binding domain, consistent with PMID:24656813\",\n      \"pmids\": [\"24794838\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"NMR solution structure of the Tah1p:Pih1p complex shows that the C-terminal tail (S93-S111) of Tah1p binds the CS domain (Pih1p264-344) forming two intermolecular β-sheets and one covering loop. Pih1p has two direct binding partners beyond Tah1p: the assembly factor Rsa1p and the snoRNP core protein Nop58p; these two interactions are mutually exclusive. Additionally, the Pih1p257-344 CS domain contacts the Hsp82 middle-C-terminal domain in the presence of Tah1p.\",\n      \"method\": \"NMR structure, ITC, co-expression/pulldown in E. coli, in vitro and in vivo binding assays\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure plus ITC plus co-expression pulldown in one study with multiple binding partners characterized\",\n      \"pmids\": [\"26210662\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Nop17/Pih1 (yeast PIH1D1 ortholog) interacts with Nop58 (snoRNP core protein) and ATP modulates the interaction between Nop17 and the ATPases Rvb1/Rvb2; R2TP complex reduces affinity of Nop58 for snoRNA to facilitate binding of other snoRNP subunits.\",\n      \"method\": \"Co-immunoprecipitation, in vitro binding assays, domain mapping\",\n      \"journal\": \"BMC molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP and in vitro binding with domain mapping, single lab, multiple interactions tested\",\n      \"pmids\": [\"25888478\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Pih1 is degraded via a ubiquitin-independent proteasome pathway: the proteasome subunit Rpn8 directly interacts with the Pih1 C terminus (specifically the last 30 amino acids of Rpn8 bind Pih1 C terminus); Pih1(282-344) acts as a degron sufficient to induce ubiquitin-independent degradation. Truncation of the Rpn8 C-terminal disordered region does not affect proteasome assembly but specifically inhibits degradation of GFP-Pih1(282-344) in vivo and Pih1 in vitro.\",\n      \"method\": \"Co-immunoprecipitation, truncation mutagenesis, in vitro and in vivo degradation assays, ubiquitin-independent proteasome assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro degradation assay plus mutagenesis plus in vivo complementation, single lab, multiple orthogonal approaches\",\n      \"pmids\": [\"27053109\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"PIH1D1 stabilizes the SWI/SNF core subunit SNF5 by attenuating its proteasome-mediated degradation; overexpression of PIH1D1 increases SNF5 protein level.\",\n      \"method\": \"Cycloheximide chase assay, proteasome inhibitor (MG132) treatment, PIH1D1 overexpression with western blot readout\",\n      \"journal\": \"Acta Academiae Medicinae Sinicae\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, single method, limited controls reported in abstract\",\n      \"pmids\": [\"20078948\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"PIH1D1 interacts with both RPAP3 and Monad (Reptin/RUVBL2) in human cells; siRNA knockdown of PIH1D1 enhances doxorubicin-induced apoptosis and caspase-3 activation, indicating PIH1D1 functions as a modulator of the apoptosis pathway.\",\n      \"method\": \"Co-immunoprecipitation in HEK293/U2OS cells, siRNA knockdown, caspase-3 activity assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP plus functional siRNA KD with specific apoptosis readout, single lab\",\n      \"pmids\": [\"21078300\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In Trypanosoma brucei, PIH1D1 concentrates at co-translational assembly sites where outer dynein arm (ODA) heavy chain (HC) translation occurs; without PIH1D1, HC protein levels are reduced and the IC-LC complex is stranded in the cytoplasm, indicating PIH1D1 generates specialized compartments to assist co-translational folding of HCs and enable their assembly with other ODA subunits.\",\n      \"method\": \"Live fluorescence imaging, genetic knockdown with western blot and localization readouts in Trypanosoma brucei\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — preprint, single organism model (Trypanosoma), single lab, imaging plus KD without mechanistic reconstitution\",\n      \"pmids\": [\"bio_10.1101_2025.07.26.666928\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"PIH1D1 is the defining subunit of the R2TP co-chaperone complex that bridges the Hsp90 chaperone system to diverse macromolecular assemblies: its N-terminal PIH-N domain recognizes CK2-phosphorylated substrates (including TEL2 of the TTT complex) via a structurally defined phosphopeptide-binding mechanism requiring Lys57/Lys64, while its C-terminal CS domain interacts with Tah1/RPAP3 (which connects to Hsp90 via TPR-EEVD contacts) and directly binds Rvb1/Rvb2 ATPases and snoRNP core proteins such as Nop58; as a complex with Tah1, PIH1D1 inhibits Hsp90 ATPase activity and facilitates assembly of box C/D snoRNPs, mTORC1, RNA Pol II, and other clients, while in the absence of Hsp90/Tah1 support its intrinsically unstable C terminus is degraded via a ubiquitin-independent proteasome pathway mediated by direct interaction with proteasome subunit Rpn8.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PIH1D1 is the defining substrate-recognition subunit of the R2TP co-chaperone complex, which couples the Hsp90 chaperone machinery to the assembly of diverse macromolecular complexes [#0, #7]. Its N-terminal PIH domain is a phosphopeptide-binding module that specifically recognizes CK2-phosphorylated substrates such as TEL2 of the TTT complex through a DpSDD motif, an interaction that requires Lys57 and Lys64 and recruits additional clients in a phosphorylation-dependent manner [#7, #8]. Through its C-terminal CS domain, PIH1D1 binds the TPR cofactor Tah1/RPAP3 — which in turn engages the Hsp90 C-terminal EEVD via a two-carboxylate clamp — and together the Pih1-Tah1 heterodimer inhibits Hsp90 ATPase activity [#1, #2, #9]. PIH1D1 also binds the Rvb1/Rvb2 (RUVBL1/RUVBL2) AAA+ ATPases via its N-terminal region to form the R2TP complex, which is required for accumulation of box C/D snoRNPs, acting in part by lowering Nop58 affinity for snoRNA to permit ordered subunit assembly [#0, #3, #9, #10]. Beyond snoRNP biogenesis, PIH1D1 promotes mTORC1 assembly and activity through association with Raptor [#6] and regulates RNA polymerase I transcription of rRNA genes by recruiting the Brg1-SWI/SNF complex to rRNA promoters [#4]. The intrinsically unstable C terminus of PIH1D1, which carries multiple disordered degron elements, is stabilized by Tah1/RPAP3 binding and, when unsupported, is destroyed by a ubiquitin-independent proteasome pathway mediated by direct interaction with the proteasome subunit Rpn8 [#2, #3, #5, #11].\",\n  \"teleology\": [\n    {\n      \"year\": 2008,\n      \"claim\": \"Established PIH1D1 (yeast Pih1/Nop17) as an unstable protein that, with Tah1, binds the Rvb1/Rvb2 helicases to form the R2TP complex required for snoRNP accumulation, defining its core function in ribonucleoprotein assembly.\",\n      \"evidence\": \"Genetic interaction screens, co-IP, in vivo stability and snoRNA accumulation assays in yeast\",\n      \"pmids\": [\"18268103\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the structural basis of substrate or partner recognition\", \"Mechanism by which R2TP promotes snoRNP assembly not resolved\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Showed the Pih1-Tah1 heterodimer binds Hsp90 and inhibits its ATPase activity, revealing R2TP as a negative modulator of the chaperone cycle rather than a passive adaptor.\",\n      \"evidence\": \"AUC, ITC, noncovalent MS, ATPase assays on reconstituted complexes\",\n      \"pmids\": [\"20663878\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not provide atomic structure of the inhibitory interaction\", \"Physiological consequence of ATPase inhibition for client assembly not tested\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Connected human PIH1D1 to RPAP3 and RUVBL2 and to cell survival, indicating an apoptosis-modulating role.\",\n      \"evidence\": \"Co-IP in HEK293/U2OS cells, siRNA knockdown, caspase-3 activity assay\",\n      \"pmids\": [\"21078300\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanistic link between R2TP function and apoptosis not defined\", \"Direct versus indirect effect on caspase activation unresolved\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Mapped the modular architecture of PIH1D1: an N-terminal Rvb1/Rvb2-binding region sufficient for function and a C-terminal disordered degron region required for Tah1 binding, separating partner recognition from instability.\",\n      \"evidence\": \"Mutagenesis, in vitro binding and in vivo complementation in yeast\",\n      \"pmids\": [\"23139418\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the substrate-recognition mechanism of the N domain\", \"Degradation machinery acting on the degron not yet known\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Linked human PIH1D1 to rRNA gene activation by recruiting Brg1-SWI/SNF to rRNA promoters and displacing the silencing factor TIP5, extending its role to chromatin remodeling at the rDNA.\",\n      \"evidence\": \"Co-IP, ChIP, RNAi with rRNA transcription readout, DNase I hypersensitivity\",\n      \"pmids\": [\"22368283\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct histone H4 versus SNF5 binding contributions not separated\", \"Whether this depends on R2TP/Hsp90 unclear\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Demonstrated that RPAP3 isoform 1 post-transcriptionally stabilizes PIH1D1 protein, linking the human cofactor to PIH1D1 abundance control.\",\n      \"evidence\": \"Co-IP, siRNA knockdown, western blot vs mRNA comparison\",\n      \"pmids\": [\"23159623\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not identify the degradation pathway counteracted by RPAP3\", \"Isoform specificity mechanism not defined\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identified PIH1D1 as a factor promoting mTORC1 assembly and activity, broadening R2TP clients to a major signaling complex.\",\n      \"evidence\": \"Co-IP of Raptor, siRNA knockdown, S6K phosphorylation and rRNA transcription assays\",\n      \"pmids\": [\"24036451\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct binding site within mTORC1 not mapped\", \"Whether assembly requires Hsp90/Tah1 not tested\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Solved the structural basis of substrate recognition: the PIH-N domain binds CK2-phosphorylated DpSDD motifs (e.g., TEL2) via Lys57/Lys64, defining R2TP as a phosphorylation-dependent client-selection machine.\",\n      \"evidence\": \"Co-crystal structure with phosphopeptide, Lys57/Lys64 mutagenesis, phosphoproteomics\",\n      \"pmids\": [\"24656813\", \"24794838\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full repertoire of phosphorylated clients incompletely defined\", \"How recognition triggers downstream chaperone-assisted assembly unresolved\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defined the CS-domain interactions of PIH1D1, showing Tah1 binds via β-sheet contacts and that binding of the snoRNP protein Nop58 and the assembly factor Rsa1 is mutually exclusive, indicating switchable client engagement.\",\n      \"evidence\": \"NMR structure, ITC, co-expression pulldown in E. coli, in vitro/in vivo binding\",\n      \"pmids\": [\"26210662\", \"25888478\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of the Nop58/Rsa1 switch in vivo not established\", \"How ATP-modulated Rvb1/Rvb2 binding integrates with CS-domain switching unclear\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Revealed that the PIH1D1 C-terminal degron is destroyed by a ubiquitin-independent proteasome route through direct binding to the proteasome subunit Rpn8, explaining its intrinsic instability when cofactor-unsupported.\",\n      \"evidence\": \"Co-IP, truncation mutagenesis, in vitro and in vivo ubiquitin-independent degradation assays in yeast\",\n      \"pmids\": [\"27053109\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the human PIH1D1-Rpn8 axis operates identically not shown\", \"How Tah1 binding kinetically shields the degron not quantified\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Extended PIH1D1 function to co-translational assembly, showing in Trypanosoma it concentrates at sites of dynein heavy-chain translation to support folding and assembly of axonemal motor subunits.\",\n      \"evidence\": \"Live fluorescence imaging and genetic knockdown in Trypanosoma brucei (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.07.26.666928\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Preprint, single organism, no in vitro reconstitution\", \"Whether mammalian PIH1D1 acts at co-translational assembly sites untested\", \"Molecular mechanism of compartment formation undefined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How PIH1D1-mediated substrate recognition is mechanistically coupled to Hsp90/RUVBL ATP cycling to drive client maturation remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No reconstituted assembly cycle linking phosphosubstrate capture to client folding\", \"Full client spectrum and selection rules in human cells incompletely mapped\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 7, 9]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"GO:0042393\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005730\", \"supporting_discovery_ids\": [4]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 10]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 2, 11]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [4]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"complexes\": [\n      \"R2TP complex\",\n      \"Pih1-Tah1 (RPAP3) heterodimer\"\n    ],\n    \"partners\": [\n      \"TAH1\",\n      \"RPAP3\",\n      \"RUVBL1\",\n      \"RUVBL2\",\n      \"TELO2\",\n      \"NOP58\",\n      \"RPTOR\",\n      \"SMARCB1\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}