Figure - available from: Nature Communications
This content is subject to copyright. Terms and conditions apply.
Identification of R2TP-like complexes. a LUMIER interaction assays between SPAG1 and RUVBL1/2. Legend as in Fig. 3c excepted that an untagged PIH1D2 was co-expressed (lanes+), or SMD1 as control (lanes−). b SILAC proteomic analysis of the partners of GFP-PIH1D2, GFP-DNAAF2, GFP-CCDC103, and GFP-PIH1D3, performed in HeLa cells. Legend as in Fig. 2a. The color code is indicated between the graphs. c Models of possible R2TP-related complexes
Source publication
R2TP is an HSP90 co-chaperone that assembles important macro-molecular machineries. It is composed of an RPAP3-PIH1D1 heterodimer, which binds the two essential AAA+ATPases RUVBL1/RUVBL2. Here, we resolve the structure of the conserved C-terminal domain of RPAP3, and we show that it directly binds RUVBL1/RUVBL2 hexamers. The human genome encodes tw...
Citations
... Bound proteins were eluted by adding 1% SDS to the beads and boiling them for 10 min. Proteomic analysis was performed as previously described (Maurizy et al, 2018). MS data were analyzed on MaxQuant software v 2.1.0.0 (Cox & Mann, 2008) with standard parameters and the UniProt database of human canonical protein isoforms (www.uniprot.org). ...
The signal recognition particle is essential for targeting transmembrane and secreted proteins to the endoplasmic reticulum. Remarkably, because they work together in the cytoplasm, the SRP and ribosomes are assembled in the same biomolecular condensate: the nucleolus. How important is the nucleolus for SRP assembly is not known. Using quantitative proteomics, we have investigated the interactomes of SRP components. We reveal that SRP proteins are associated with scores of nucleolar proteins important for ribosome biogenesis and nucleolar structure. Having monitored the subcellular distribution of SRP proteins upon controlled nucleolar disruption, we conclude that an intact organelle is required for their proper localization. Lastly, we have detected two SRP proteins in Cajal bodies, which indicates that previously undocumented steps of SRP assembly may occur in these bodies. This work highlights the importance of a structurally and functionally intact nucleolus for efficient SRP production and suggests that the biogenesis of SRP and ribosomes may be coordinated in the nucleolus by common assembly factors.
... To examine utility of dMP in a real-life scenario, we applied dMP to optimize XL conditions before MS analysis of R2SP, a multicomponent protein complex that includes two hexamers of 3 AAA+ ATPases RuvB-Like1 (R1) and 3 RuvB-Like2 (R2) 31 each, 1 SPAG1 (S) molecule, and 1 PIH1D2 (P) molecule 32,33 . The first nMP measurement of the R2SP preparation allowed us to identify R1R2 hexamers at 513 ± 1.1 kDa (42 ± 10% of total counts, over triplicates, Supplementary Fig. 6) coexisting with R2SP complexes at 549 ± 4 kDa (28 ± 10% of total counts, over triplicatesexpected mass ∼540 kDa, Fig. 5). ...
Chemical cross-linking reactions (XL) are an important strategy for studying protein-protein interactions (PPIs), including low abundant sub-complexes, in structural biology. However, choosing XL reagents and conditions is laborious and mostly limited to analysis of protein assemblies that can be resolved using SDS-PAGE. To overcome these limitations, we develop here a denaturing mass photometry (dMP) method for fast, reliable and user-friendly optimization and monitoring of chemical XL reactions. The dMP is a robust 2-step protocol that ensures 95% of irreversible denaturation within only 5 min. We show that dMP provides accurate mass identification across a broad mass range (30 kDa–5 MDa) along with direct label-free relative quantification of all coexisting XL species (sub-complexes and aggregates). We compare dMP with SDS-PAGE and observe that, unlike the benchmark, dMP is time-efficient (3 min/triplicate), requires significantly less material (20–100×) and affords single molecule sensitivity. To illustrate its utility for routine structural biology applications, we show that dMP affords screening of 20 XL conditions in 1 h, accurately identifying and quantifying all coexisting species. Taken together, we anticipate that dMP will have an impact on ability to structurally characterize more PPIs and macromolecular assemblies, expected final complexes but also sub-complexes that form en route.
... Co-immunoprecipitation with GST and His-tagged proteins in human cell lysates show that DNAAF2 (PF13), a PIHdomain-containing protein also interacts with DNAAF4 (PF23) [43]. In addition, PF13 forms part of a non-canonical R2TP complex that is involved in many cellular processes [44,45]. In addition to interacting with several DNAAFs, the WDR (tryptophan-aspartic-repeat) protein WDR92 (Monad) is a part of multiple co-chaperone complexes including R2TP and prefoldins [45,46]. ...
... In addition, PF13 forms part of a non-canonical R2TP complex that is involved in many cellular processes [44,45]. In addition to interacting with several DNAAFs, the WDR (tryptophan-aspartic-repeat) protein WDR92 (Monad) is a part of multiple co-chaperone complexes including R2TP and prefoldins [45,46]. A different group of proteins involved in dynein assembly are called 'maturation' factors. ...
Motile cilia assembly utilizes over 800 structural and cytoplasmic proteins. Variants in approximately 58 genes cause primary ciliary dyskinesia (PCD) in humans, including the dynein arm (pre)assembly factor (DNAAF) gene DNAAF4 . In humans, outer dynein arms (ODAs) and inner dynein arms (IDAs) fail to assemble motile cilia when DNAAF4 function is disrupted. In Chlamydomonas reinhardtii , a ciliated unicellular alga, the DNAAF4 ortholog is called PF23 . The pf23-1 mutant assembles short cilia and lacks IDAs, but partially retains ODAs. The cilia of a new null allele ( pf23-4 ) completely lack ODAs and IDAs and are even shorter than cilia from pf23-1 . In addition, PF23 plays a role in the cytoplasmic modification of IC138, a protein of the two-headed IDA (I1/f). As most PCD variants in humans are recessive, we sought to test if heterozygosity at two genes affects ciliary function using a second-site non-complementation (SSNC) screening approach. We asked if phenotypes were observed in diploids with pairwise heterozygous combinations of 21 well-characterized ciliary mutant Chlamydomonas strains. Vegetative cultures of single and double heterozygous diploid cells did not show SSNC for motility phenotypes. When protein synthesis is inhibited, wild-type Chlamydomonas cells utilize the pool of cytoplasmic proteins to assemble half-length cilia. In this sensitized assay, 8 double heterozygous diploids with pf23 and other DNAAF mutations show SSNC; they assemble shorter cilia than wild-type. In contrast, double heterozygosity of the other 203 strains showed no effect on ciliary assembly. Immunoblots of diploids heterozygous for pf23 and wdr92 or oda8 show that PF23 is reduced by half in these strains, and that PF23 dosage affects phenotype severity. Reductions in PF23 and another DNAAF in diploids affect the ability to assemble ODAs and IDAs and impedes ciliary assembly. Thus, dosage of multiple DNAAFs is an important factor in cilia assembly and regeneration.
... The R2TP/Prefoldin-like (R2TP/PFDL) complex acts as a co-chaperone complex and plays a crucial role in assembling important protein complexes, including snoRNPs and nuclear RNA polymerases. [18] During snoRNP assembly, the RUVBL1/RUVBL2 complex, along with SHQ1, stabilizes NAP57, a snoRNP component. SHQ1 binds to NAP57, interacting with its CS domain, while its specific domain binds to the opposite surface. ...
A BSTRACT
Background
H/ACA small nucleolar ribonucleoproteins (snoRNP) form a complex with multiple proteins to accomplish the pseudouridylation of rRNA. The assembly of H/ACA small nucleolar ribonucleoproteins (snoRNP) is initiated by H/ACA ribonucleoprotein Assembly factor, that is, SHQ1. Mutations in SHQ1 have been reported to cause two disorders namely, dystonia-35 childhood onset (OMIM*619921) and neurodevelopmental disorder with seizures and dystonia (OMIM*619922), both of which are inherited in an autosomal recessive manner. Considering the high genetic and clinical diversity of SHQ1-related clinical features and the importance of SHQ1 in the assembly of the H/ACA snoRNP complex, it is important to take a systematic approach to delineate the genetic diagnosis and impact of mutations on protein structure and stability.
Methods
Whole exome sequencing followed by Sanger validation was performed in an individual with the clinical features of neurodevelopmental disorder with seizures and dystonia (OMIM*619922). Protein modeling studies of all the reported SHQ1 variants to date were performed using freely available web servers Interactive Tree of Life, String, BioGrid, ShinyGO, DAVID, and Pathvix. Protein structures were visualized using Pymol.
Results and Discussion
We identified compound heterozygous variants, one known frameshift deletion c. 828_831del, p.(Asp277Serfs*27) and the other novel missense variant c. 1157A>C, p.(Tyr386Ser) found in an individual with neurodevelopmental disorder, seizures, movement disorder, and hypomyelination leukodystrophy on neuroimaging. Protein-interactome studies identified potential genetic interactors that include GAR1, NAF1, TRUB1, UTP15, DKC1, NOP10, NPHOSPH 10, KRR1, NOP58, NOP56, FBL, RRP9, NHP2, RUVBL1 , and RUVBL2 . Ribosome biogenesis in eukaryotes, RNA polymerase, RNA transport, spliceosome, ribosome, cytosolic DNA-sensing pathway, DNA replication, mismatch repair, base excision repair, nucleotide excision repair, and basal transcription factors process were identified as the linked pathways with the prioritized genes.
Conclusion
In conclusion, a sophisticated genotype and phenotype correlation followed by linking the genes to the key biological pathways opens new avenues to understand disease pathology and plan for therapeutic interventions.
... For example, some are PIH proteins that recruit heat-shock proteins and interact with specific scaffolding factors to generate variant forms of the R2TP complex that contain the RuvBL1 and RuvBL2 AAA ATPases [55,56]. Different dynein types require distinct PIH proteins [57,58], and there is also evidence that distinct assembly proteins, including variant R2TP scaffolds [59] and chaperone recruiters, act sequentially during HC assembly, e.g., [60]. Other assembly factors such as WDR92 (DNAAF10), HEATR2 (DNAAF5), and LRRC6 (DNAAF11) provide protein-protein interaction domains, e.g., [58,61,62]. ...
Axonemal dyneins are highly complex microtubule motors that power ciliary motility. These multi-subunit enzymes are assembled at dedicated sites within the cytoplasm. At least nineteen cytosolic factors are specifically needed to generate dynein holoenzymes and/or for their trafficking to the growing cilium. Many proteins are subject to N-terminal processing and acetylation, which can generate degrons subject to the AcN-end rule, alter N-terminal electrostatics, generate new binding interfaces, and affect subunit stoichiometry through targeted degradation. Here, we have used mass spectrometry of cilia samples and electrophoretically purified dynein heavy chains from Chlamydomonas to define their N-terminal processing; we also detail the N-terminal acetylase complexes present in this organism. We identify four classes of dynein heavy chain based on their processing pathways by two distinct acetylases, one of which is dependent on methionine aminopeptidase activity. In addition, we find that one component of both the outer dynein arm intermediate/light chain subcomplex and the docking complex is processed to yield an unmodified Pro residue, which may provide a setpoint to direct the cytosolic stoichiometry of other dynein complex subunits that contain N-terminal degrons. Thus, we identify and describe an additional level of processing and complexity in the pathways leading to axonemal dynein formation in cytoplasm.
... SILAC experiments were performed as previously described (65). Six 15-cm diameter plates per condition of HEK293 Flp-In T-REx cells inducibly expressing the GFP-tagged proteins were grown for 15 days in each isotopically labeled media (CIL/Eurisotop), to ensure complete incorporation of isotopically L-lysine-2 HCl/L-arginine-HCl light label (K0R0 or L condition, corresponding to the control), L-lysine-2 HCl ( 2 H4, 96-98%)/L-arginine-HCl ( 13 C6, 99%) semi-heavy label (K4R6 or M condition), or L-lysine-2 HCl ( 13 C6, 99%; 15 N2, 99%)/L-arginine-HCl ( 13 C6, 99%; 15 N4, 99%) heavy label (K8R10 or H condition) (percentages represent the isotopic purity of the labeled amino acids). ...
... Bound proteins were eluted by adding 1% SDS to the beads and boiling them for 10 min. Proteomic analysis was performed as previously described (65). Enrichment is calculated as a SILAC ratio M/L or H/L. ...
... Our observation raises the possibility that the entire PAQosome can play a role in the assembly/disassembly of some of the H/ACA intermediate complexes during H/ACA biogenesis. DPCD, which was also well associated with GFP-SHQ1, might also intervene since this protein has been proposed to be a co-factor of the R2TP (65,80,81). ...
The conserved H/ACA RNPs consist of one H/ACA RNA and 4 core proteins: dyskerin, NHP2, NOP10, and GAR1. Its assembly requires several assembly factors. A pre-particle containing the nascent RNAs, dyskerin, NOP10, NHP2 and NAF1 is assembled co-transcriptionally. NAF1 is later replaced by GAR1 to form mature RNPs. In this study, we explore the mechanism leading to the assembly of H/ACA RNPs. We performed the analysis of GAR1, NHP2, SHQ1 and NAF1 proteomes by quantitative SILAC proteomic, and analyzed purified complexes containing these proteins by sedimentation on glycerol gradient. We propose the formation of several distinct intermediate complexes during H/ACA RNP assembly, notably the formation of early protein-only complexes containing at least the core proteins dyskerin, NOP10, and NHP2, and the assembly factors SHQ1 and NAF1. We also identified new proteins associated with GAR1, NHP2, SHQ1 and NAF1, which can be important for box H/ACA assembly or function. Moreover, even though GAR1 is regulated by methylations, the nature, localization, and functions of these methylations are not well known. Our MS analysis of purified GAR1 revealed new sites of arginine methylations. Additionally, we showed that unmethylated GAR1 is correctly incorporated in H/ACA RNPs, even though with less efficiency than methylated ones.
... Liprin-α2, encoded by PPFIA2, is located in the mature hippocampal presynapses. Liprin-α2 plays an important role in regulating neuronal activity and is highly expressed in the urinary sediments of patients with prostate cancer [9,[32][33][34]. PPFIA3 can be methylated in gastric cancer but is rarely methylated in normal gastric tissues. ...
Purpose: The PPFIA gene family (PPFIA1, PPFIA2, PPFIA3, and PPFIA4) is associated with multiple human diseases, particularly malignant tumors. However, the expression and prognostic value of the PPFIA family in human colorectal cancers (CRCs) have not been reported.
Materials and methods: In this study, several databases, including Oncomine, UALCAN, and the cancer cell line encyclopedia, were used to compare differences in PPFIA1, PPFIA2, PPFIA3, and PPFIA4 expression between normal colon samples and CRCs. The expression levels of these four proteins were used to evaluate the survival of patients with CRC, as determined by the Cancer Genome Atlas Program (TCGA) portal and gene expression profiling interactive analysis (GEPIA) databases. Western blotting and reverse transcription-polymerase chain reaction were performed to detect protein and mRNA levels of PPFIA1, PPFIA3, and PPFIA4, respectively. Immunohistochemical (IHC) staining was used to detect the correlation between PPFIA4 expression and the degree of CRC malignancy. Furthermore, potential miRNAs targeting PPFIA4 in CRCs were studied and confirmed.
Results: Bioinformatic analysis showed that the mRNA levels of PPFIA1, PPFIA3, and PPFIA4 were higher in CRC tissue samples than in normal colon tissue. Both mRNA and protein expression of PPFIA1, PPFIA3, and PPFIA4 were increased in the CRC cell lines LoVo and Hct116 compared with the normal colon epithelial cell line. Only PPFIA4 was associated with the prognosis of patients with CRC, which was confirmed by TCGA portal and GEPIA. IHC staining confirmed that the expression of PPFIA4 was higher in CRC tissues than in normal colon tissues and also increased in poorly differentiated CRC tissues and lymph node metastatic foci in comparison with well-differentiated CRC tissues and moderately differentiated CRC tissues. Functional annotation enrichment analysis indicated that the top 100 genes co-expressed with PPFIA4 were enriched in the G-protein coupled peptide receptor activity, leukotrience B4 receptor activity, and peroxisome proliferator-activated receptors and hypoxia-inducible factor-1 signaling pathways. In addition, miR-485-5p negatively regulates the expression of PPFIA4.
Conclusion: PPFIA4 expression is associated with the development of CRCs and may be a novel potential prognostic marker for human CRCs.
... Chaperones are important for many cellular functions including the assembly of large multi-subunit complexes like axonemal dynein motors. For several DNAAFs, such a function is strongly indicated by DNAAF sequence relationships with a known HSP90 co-chaperone, the R2TP complex (Maurizy et al., 2018). This co-chaperone was discovered in S. cerevisiae as facilitating RNA polymerase II assembly (Zhao et al., 2005). ...
... Much is known of the structural features of R2TP: for RPAP3, the TPR domains directly recruit HSP70 and HSP90 while the RPAP3_C domain binds to RUVBL2 (Martino et al., 2018). For PIH1D1, the PIH domain recruits client proteins, while the CS domain binds to a region of RPAP3 C-terminal to the TPR domain (Kakihara and Houry, 2012;Martino et al., 2018;Maurizy et al., 2018). ...
... Similarly, the CS and PIH domains of PIH1D1 are also present in several other PIH proteins: PIH1D2, DNAAF2 (KTU), and DNAAF6 (PIH1D3) (Dong et al., 2014). There is biochemical evidence that SPAG1 complexes with PIH1D2 and DNAAF2 (Maurizy et al., 2018;Smith et al., 2022). Different isoforms of DNAAF4 complex with DNAAF2 and DNAAF6 Maurizy et al., 2018). ...
... SPAG1 is expressed in both types in cancer cells and undifferentiated respiratory epithelial cells (Neesse et al., 2007;Smith, et al., 2022), and only 50 kDa isoform in sperm (Kanazawa et al., 2003), and is considered to be a cancer-testis antigen (CTA) (Siliņa et al., 2011). SPAG1-2 provides a platform for quaternary protein folding of proteins via the TPR domain (Takaishi et al., 1999;Allan et al., 2011) that is involved in protein-protein interactions as a member of the R2SP complex (SPAG1, PIH1D2, RUVB1 / 2), which is a co-chaperone complex, and is involved in the assembly of protein complexes such as dynein arms (Smith, et al., 2022;Maurizy et al., 2018) (its mutation causes primary ciliary dyskinesia due to dysplasia of the axoneme dynein arm). The R2SP complex is identified as a ubiquitous R2TP (RPAP3, PIH1D1, RUVB1 / 2) -like chaperone, where RPAP3 is located at SPAG1-2 of R2SP, and is particularly strongly expressed in the testis, and its assembly function is strongest at the proper temperature of the testis, 32 °C, and is optimized for the testis environment (Maurizy et al., 2018). ...
... SPAG1-2 provides a platform for quaternary protein folding of proteins via the TPR domain (Takaishi et al., 1999;Allan et al., 2011) that is involved in protein-protein interactions as a member of the R2SP complex (SPAG1, PIH1D2, RUVB1 / 2), which is a co-chaperone complex, and is involved in the assembly of protein complexes such as dynein arms (Smith, et al., 2022;Maurizy et al., 2018) (its mutation causes primary ciliary dyskinesia due to dysplasia of the axoneme dynein arm). The R2SP complex is identified as a ubiquitous R2TP (RPAP3, PIH1D1, RUVB1 / 2) -like chaperone, where RPAP3 is located at SPAG1-2 of R2SP, and is particularly strongly expressed in the testis, and its assembly function is strongest at the proper temperature of the testis, 32 °C, and is optimized for the testis environment (Maurizy et al., 2018). This means that R2TP malfunctions in high temperature environments. ...
... XY-type organisms ( Fig. 2 ) It is considered that SPAG1-2 and SPAG1-1 interact with the TPR domain, and some of the multiple domains are the same due to the splicing variant (Hayashida et al., 2008;Maurizy et al., 2018). Therefore, it is predicted that within the same species, it will not become unresponsive even if the domain is mutated. ...
Charles Darwin proposed the theory of evolution that natural selection leads to the evolution of organisms in "On the Origin of Species”, but did not show the mechanism by which new species differentiate and fix. Speciation requires a system in which genes are not mixed by interspecific hybridization, and reproductive isolation, especially postmating reproductive isolation, is considered to be the most reliable as guarantee. Haldane proposed that heterogametic sex was absent, rare or sterile in interspecific hybrid F1. Dobzhansky and Muller predicted that postmating reproductive isolation occurs when mutations occurring at two or more interacting loci exhibit incompatibility in the hybrid. Genes that satisfy these observation and prediction are considered speciation genes. Here, I would like to review the findings on reproductive isolation and speciation to consider the candidate conditions for the speciation genes, and present the genes that fit these conditions.
... Several DNAAFs have been found to associate with HSP90 by immunoprecipitation [8], suggesting that DNAAFs function in recruiting dynein clients to HSP90. DNAAFs that directly interact with HSP90 from experimental evidence or prediction include TPR domain containing proteins DNAAF4/DYX1C1, DNAAF13/SPAG1 and its paralog RPAP3 [16]. DYX1C1 interacts with DNAAF2/PF13 or DNAAF6/TWI1 [16,17]. ...
... DNAAFs that directly interact with HSP90 from experimental evidence or prediction include TPR domain containing proteins DNAAF4/DYX1C1, DNAAF13/SPAG1 and its paralog RPAP3 [16]. DYX1C1 interacts with DNAAF2/PF13 or DNAAF6/TWI1 [16,17]. SPAG1/RPAP3 are homologues of yeast Tah1, a component of a R2TP complex (Rvb1, Rvb2, Tah1 and Pih1 in yeast and RuvBL1, RuvBL2, RPAP3 and PIH1D1 in mammal), which is a HSP90 co-chaperone [16,18,19]. ...
... DYX1C1 interacts with DNAAF2/PF13 or DNAAF6/TWI1 [16,17]. SPAG1/RPAP3 are homologues of yeast Tah1, a component of a R2TP complex (Rvb1, Rvb2, Tah1 and Pih1 in yeast and RuvBL1, RuvBL2, RPAP3 and PIH1D1 in mammal), which is a HSP90 co-chaperone [16,18,19]. Mutations in the components of R2TP or its like complex in different organisms lead to instability of axonemal dyneins [16,[20][21][22][23][24][25][26][27]. ...
Assembly of dynein arms requires cytoplasmic processes which are mediated by dynein preassembly f actors (DNAAFs). CFAP298, which is conserved in organisms with motile cilia, is required for assembly of dynein arms but with obscure mechanisms. Here, we show that FBB18, a Chlamydomonas homologue of CFAP298, localizes to the cytoplasm and functions in folding/stabilization of almost all axonemal dyneins at the early steps of dynein preassembly. Mutation of FBB18 causes no or short cilia accompanied with partial loss of both outer and inner dynein arms. Comparative proteomics using ¹⁵ N labeling suggests partial degradation of almost all axonemal dynein heavy chains (DHCs). A mutant mimicking a patient variant induces particular loss of DHCα. FBB18 associates with 9 DNAAFs and 14 out of 15 dynein HCs but not with IC1/IC2. FBB18 interacts with RuvBL1/2, components of the HSP90 co-chaperone R2TP complex but not the holo-R2TP complex. Further analysis suggests simultaneous formation of multiple DNAAF complexes involves dynein folding/stability and thus provides new insights into axonemal dynein preassembly.
