Maturation and Assembly of mTOR Complexes by the HSP90-R2TP-TTT Chaperone System: Molecular Insights and Mechanisms.

The mechanistic target of rapamycin (mTOR) is a master regulator of cell growth and metabolism, integrating environmental signals to regulate anabolic and catabolic processes, regulating lipid synthesis, growth factor-induced cell proliferation, cell survival, and migration. These activities are performed as part of two distinct complexes, mTORC1 and mTORC2, each with specific roles. mTORC1 and mTORC2 are elaborated dimeric structures formed by the interaction of mTOR with specific partners. mTOR functions only as part of these large complexes, but their assembly and activation require a dedicated and sophisticated chaperone system. mTOR folding and assembly are temporarily separated with the TELO2-TTI1-TTI2 (TTT) complex assisting the cotranslational folding of mTOR into a native conformation. Matured mTOR is then transferred to the R2TP complex for assembly of active mTORC1 and mTORC2 complexes. R2TP works in concert with the HSP90 chaperone to promote the incorporation of additional subunits to mTOR and dimerization. This review summarizes our current knowledge on how the HSP90-R2TP-TTT chaperone system facilitates the maturation and assembly of active mTORC1 and mTORC2 complexes, discussing interactions, structures, and mechanisms.

The role of SPAG1 in the assembly of axonemal dyneins in human airway epithelia.

Mutations in SPAG1, a dynein axonemal assembly factor (DNAAF) that facilitates the assembly of dynein arms in the cytoplasm before their transport into the cilium, result in primary ciliary dyskinesia (PCD), a genetically heterogenous disorder characterized by chronic oto-sino-pulmonary disease, infertility and laterality defects. To further elucidate the role of SPAG1 in dynein assembly, we examined its expression, interactions and ciliary defects in control and PCD human airway epithelia. Immunoprecipitations showed that SPAG1 interacts with multiple DNAAFs, dynein chains and canonical components of the R2TP complex. Protein levels of dynein heavy chains (DHCs) and interactions between DHCs and dynein intermediate chains (DICs) were reduced in SPAG1 mutants. We also identified a previously uncharacterized 60 kDa SPAG1 isoform, through examination of PCD subjects with an atypical ultrastructural defect for SPAG1 variants, that can partially compensate for the absence of full-length SPAG1 to assemble a reduced number of outer dynein arms. In summary, our data show that SPAG1 is necessary for axonemal dynein arm assembly by scaffolding R2TP-like complexes composed of several DNAAFs that facilitate the folding and/or binding of the DHCs to the DIC complex.

TTT (Tel2-Tti1-Tti2) Complex, the Co-Chaperone of PIKKs and a Potential Target for Cancer Chemotherapy.

The heterotrimeric Tel2-Tti1-Tti2 or TTT complex is essential for cell viability and highly observed in eukaryotes. As the co-chaperone of ATR, ATM, DNA-PKcs, mTOR, SMG1, and TRRAP, the phosphatidylinositol 3-kinase-related kinases (PIKKs) and a group of large proteins of 300-500 kDa, the TTT plays crucial roles in genome stability, cell proliferation, telomere maintenance, and aging. Most of the protein kinases in the kinome are targeted by co-chaperone Cdc37 for proper folding and stability. Like Cdc37, accumulating evidence has established the mechanism by which the TTT interacts with chaperone Hsp90 via R2TP (Rvb1-Rvb2-Tah1-Pih1) complex or other proteins for co-translational maturation of the PIKKs. Recent structural studies have revealed the α-solenoid structure of the TTT and its interactions with the R2TP complex, which shed new light on the co-chaperone mechanism and provide new research opportunities. A series of mutations of the TTT have been identified that cause disease syndrome with neurodevelopmental defects, and misregulation of the TTT has been shown to contribute to myeloma, colorectal, and non-small-cell lung cancers. Surprisingly, Tel2 in the TTT complex has recently been found to be a target of ivermectin, an antiparasitic drug that has been used by millions of patients. This discovery provides mechanistic insight into the anti-cancer effect of ivermectin and thus promotes the repurposing of this Nobel-prize-winning medicine for cancer chemotherapy. Here, we briefly review the discovery of the TTT complex, discuss the recent studies, and describe the perspectives for future investigation.

The PAQosome, an R2TP-Based Chaperone for Quaternary Structure Formation.

The Rvb1-Rvb2-Tah1-Pih1/prefoldin-like (R2TP/PFDL) complex is a unique chaperone that provides a platform for the assembly and maturation of many key multiprotein complexes in mammalian cells. Here, we propose to rename R2TP/PFDL as PAQosome (particle for arrangement of quaternary structure) to more accurately represent its unique function.

The Expression of the RUVBL1 Component of the R2TP Complex Correlates with Poor Prognosis in DLBCL.

INTRODUCTION: Diffuse large B-cell lymphoma (DLBCL) is the most prevalent subtype of non-Hodgkin's lymphoma (NHL) accounting for 30% of adult NHL worldwide and 50% in developing countries like India. DNA damage and Myc-induced transformation are well-known contributing factors towards development of DLBCL. A recently identified HSP90 co-chaperone complex R2TP has been shown to contribute towards DNA damage and Myc-induced transformation. This study aimed to analyse the immunohistochemical (IHC) expression of R2TP complex components RUVBL1, PIH1D1, and RPAP3 in DLBCL patients and correlate with prognosis. METHODS: DLBCL (n = 54) histological slides were retrieved from archives, and detailed histomorphological and clinical features were noted. IHC staining of R2TP complex components RUVBL1, PIH1D1, and RPAP3 was performed on 54 cases (FFPE) of DLBCL. Expression data were correlated with survival and clinical features. RESULTS: Out of the 54 DLBCL cases, 59.26% (n = 32) stained positive for RUVBL1. The RUVBL1 expression was associated with poor prognosis in both progression-free survival (PFS) (p = 0.0146) and overall survival (OS) (p = 0.0328). The expression was positively correlated with bone marrow involvement (p = 0.0525). The expression of PIH1D1 was observed in 68.51% (n = 32) of DLBCL cases, and positive correlation was observed with international prognostic index score (p = 0.0246); however, no correlation was observed with PFS or OS. Finally, RPAP3 was found immunopositive in only 1 case of DLBCL. CONCLUSIONS: Immunopositivity for RUVBL1 is associated with poor prognosis along with a higher relapse rate amongst the DLBCL patients. PIH1D1 immunopositivity correlated with a higher IPI score.

Identification and characterization of R2TP in the development of oral squamous cell carcinoma.

R2TP is a well-conserved molecular chaperone complex, composed of Pontin, Reptin, RPAP3, and PIH1D, in eukaryotes. Recent studies have suggested an involvement of R2TP in cancer development. However, it remains unclear if it is related to the development of oral squamous cell carcinoma (OSCC), which is the most common type of oral cancer. Here, we identify and investigate the function of R2TP in OSCC development. Immunohistochemical analysis reveals that all of the R2TP components are strongly expressed in normal oral epithelia and OSCC tissues, where actively proliferating cells are abundant. Co-immunoprecipitation assay identifies that R2TP components form a protein complex in OSCC-derived HSC4-cells. Knockdown experiments show that all R2TP components, except for RPAP3, are required for the cell proliferation and migration of HSC-4 cells. Furthermore, we reveal that Pontin contributes to a gain-of-function (GOF) activity of mutp53-R248Q in HSC-4 cells by regulating phosphorylation levels of mutp53 at Ser15 and Ser46. To our knowledge, this study is the first to report the functional involvement of R2TP and its components in the malignant characteristics of OSCC cells.

Upstream ORF-Encoded ASDURF Is a Novel Prefoldin-like Subunit of the PAQosome.

The PAQosome is an 11-subunit chaperone involved in the biogenesis of several human protein complexes. We show that ASDURF, a recently discovered upstream open reading frame (uORF) in the 5' UTR of ASNSD1 mRNA, encodes the 12th subunit of the PAQosome. ASDURF displays significant structural homology to ß-prefoldins and assembles with the five known subunits of the prefoldin-like module of the PAQosome to form a heterohexameric prefoldin-like complex. A model of the PAQosome prefoldin-like module is presented. The data presented here provide an example of a eukaryotic uORF-encoded polypeptide whose function is not limited to cis-acting translational regulation of downstream coding sequence and highlights the importance of including alternative ORF products in proteomic studies.

Axonemal dynein assembly requires the R2TP complex component Pontin.

Pontin (Ruvbl1) and Reptin (Ruvbl2) are closely related AAA ATPases. They are components of the Ruvbl1-Ruvbl2-Tah1-Pih1 (R2TP) complexes that function as co-chaperones for the assembly of multiple macromolecular protein complexes. Here, we show that Pontin is essential for cilia motility in both zebrafish and mouse and that Pontin and Reptin function cooperatively in this process. Zebrafish pontin mutants display phenotypes tightly associated with cilia defects, and cilia motility is lost in a number of ciliated tissues along with a reduction in the number of outer and inner dynein arms. Pontin protein is enriched in cytosolic puncta in ciliated cells in zebrafish embryos. In mouse testis, Pontin is essential for the stabilization of axonemal dynein intermediate chain 1 (DNAI1) and DNAI2, the first appreciated step in axonemal dynein arm assembly. Strikingly, multiple dynein arm assembly factors show structural similarities to either Tah1 or Pih1, the other two components of the R2TP complex. Based on these results, we propose that Pontin and Reptin function to facilitate dynein arm assembly in cytosolic foci enriched with R2TP-like complexes.

Role of the Novel Hsp90 Co-Chaperones in Dynein Arms' Preassembly.

The outer and inner dynein arms (ODAs and IDAs) are composed of multiple subunits including dynein heavy chains possessing a motor domain. These complex structures are preassembled in the cytoplasm before being transported to the cilia. The molecular mechanism(s) controlling dynein arms' preassembly is poorly understood. Recent evidence suggests that canonical R2TP complex, an Hsp-90 co-chaperone, in cooperation with dynein axonemal assembly factors (DNAAFs), plays a crucial role in the preassembly of ODAs and IDAs. Here, we have summarized recent data concerning the identification of novel chaperone complexes and their role in dynein arms' preassembly and their association with primary cilia dyskinesia (PCD), a human genetic disorder.

Role of the PAQosome in Regulating Arrangement of Protein Quaternary Structure in Health and Disease.

The PAQosome, formerly known as the R2TP/PFDL complex, is an eleven-subunit cochaperone complex that assists HSP90 in the assembly of numerous large multisubunit protein complexes involved in essential cellular functions such as protein synthesis, ribosome biogenesis, transcription, splicing, and others. In this review, we discuss possible mechanisms of action and role of phosphorylation in the assembly of client complexes by the PAQosome as well as its potential role in cancer, ciliogenesis and ciliopathies.

The Multiple Functions of the PAQosome: An R2TP- and URI1 Prefoldin-Based Chaperone Complex.

The PAQosome (Particle for Arrangement of Quaternary structure) is a large multisubunit chaperone complex that is essential for the assembly and stabilization of other macromolecular complexes. It also interacts with several chaperones including Hsp90, Hsp70, and CCT. The PAQosome is comprised of the R2TP complex, the URI1 prefoldin complex (also known as the non-canonical prefoldin-like complex), the RNA polymerase subunit RPB5, and the WD40 repeat protein WDR92. The R2TP complex is conserved among eukaryotes and has been comprehensively studied over the last 13 years. The R2TP complex is known for its involvement in the assembly and stabilization of L7Ae ribonucleoproteins, U5 small nuclear ribonucleoprotein, RNA polymerase II, phosphatidylinositol-3-kinase-related proteins (PIKKs), and the tuberous sclerosis complex (TSC1-TSC2). By contrast, the URI1 prefoldin complex has evolved exclusively in higher metazoans. Although the URI1 prefoldin complex was initially reported more than 15 years ago, little is known about its function and its role within the PAQosome. Given that URI1 is overexpressed in many types of cancer, it is surprising that the URI1 prefoldin complex has been overlooked. This chapter provides an update on the recent progress uncovering the physiological roles of each PAQosome subunit and provides an overview of the potential functions of the URI1 prefoldin complex.

Advances on the Structure of the R2TP/Prefoldin-like Complex.

Cellular stability, assembly and activation of a growing list of macromolecular complexes require the action of HSP90 working in concert with the R2TP/Prefoldin-like (R2TP/PFDL) co-chaperone. RNA polymerase II, snoRNPs and complexes of PI3-kinase-like kinases, a family that includes the ATM, ATR, DNA-PKcs, TRAPP, SMG1 and mTOR proteins, are among the clients of the HSP90-R2TP system. Evidence links the R2TP/PFDL pathway with cancer, most likely because of the essential role in pathways commonly deregulated in cancer. R2TP forms the core of the co-cochaperone and orchestrates the recruitment of HSP90 and clients, whereas prefoldin and additional prefoldin-like proteins, including URI, associate with R2TP, but their function is still unclear. The mechanism by which R2TP/PFLD facilitates assembly and activation of such a variety of macromolecular complexes is poorly understood. Recent efforts in the structural characterization of R2TP have started to provide some mechanistic insights. We summarize recent structural findings, particularly how cryo-electron microscopy (cryo-EM) is contributing to our understanding of the architecture of the R2TP core complex. Structural differences discovered between yeast and human R2TP reveal unanticipated complexities of the metazoan R2TP complex, and opens new and interesting questions about how R2TP/PFLD works.

Roles and Functions of the Unconventional Prefoldin URI.

Almost 15 years ago, the URI prefoldin-like complex was discovered by Krek and colleagues in immunoprecipitation experiments conducted in mammalian cells with the aim of identifying new binding partners of the E3 ubiquitin-protein ligase S-phase kinase-associated protein 2 (SKP2) (Gstaiger et al. Science 302(5648):1208-1212, 2003). The URI prefoldin-like complex is a heterohexameric chaperone complex comprising two α and four ß subunits (α2ß4). The α subunits are URI and STAP1, while the ß subunits are PFDN2, PFDN6, and PFDN4r, one of which is probably present in duplicate. Elucidating the roles and functions of these components in vitro and in vivo will help to clarify the mechanistic behavior of what appears to be a remarkably important cellular machine.

The Proteasome Subunit Rpn8 Interacts with the Small Nucleolar RNA Protein (snoRNP) Assembly Protein Pih1 and Mediates Its Ubiquitin-independent Degradation in Saccharomyces cerevisiae.

Pih1 is a scaffold protein of the Rvb1-Rvb2-Tah1-Pih1 (R2TP) protein complex, which is conserved in fungi and animals. The chaperone-like activity of the R2TP complex has been implicated in the assembly of multiple protein complexes, such as the small nucleolar RNA protein complex. However, the mechanism of the R2TP complex activity in vivo and the assembly of the complex itself are still largely unknown. Pih1 is an unstable protein and tends to aggregate when expressed alone. The C-terminal fragment of Pih1 contains multiple destabilization factors and acts as a degron when fused to other proteins. In this study, we investigated Pih1 interactors and identified a specific interaction between Pih1 and the proteasome subunit Rpn8 in yeast Saccharomyces cerevisiae when HSP90 co-chaperone Tah1 is depleted. By analyzing truncation mutants, we identified that the C-terminal 30 amino acids of Rpn8 are sufficient for the binding to Pih1 C terminus. With in vitro and in vivo degradation assays, we showed that the Pih1 C-terminal fragment Pih1(282-344) is able to induce a ubiquitin-independent degradation of GFP. Additionally, we demonstrated that truncation of the Rpn8 C-terminal disordered region does not affect proteasome assembly but specifically inhibits the degradation of the GFP-Pih1(282-344) fusion protein in vivo and Pih1 in vitro We propose that Pih1 is a ubiquitin-independent proteasome substrate, and the direct interaction with Rpn8 C terminus mediates its proteasomal degradation.

Assembly and trafficking of box C/D and H/ACA snoRNPs.

Box C/D and box H/ACA snoRNAs are abundant non-coding RNAs that localize in the nucleolus and mostly function as guides for nucleotide modifications. While a large pool of snoRNAs modifies rRNAs, an increasing number of snoRNAs could also potentially target mRNAs. ScaRNAs belong to a family of specific RNAs that localize in Cajal bodies and that are structurally similar to snoRNAs. Most scaRNAs are involved in snRNA modification, while telomerase RNA, which contains H/ACA motifs, functions in telomeric DNA synthesis. In this review, we describe how box C/D and H/ACA snoRNAs are processed and assembled with core proteins to form functional RNP particles. Their biogenesis involve several transport factors that first direct pre-snoRNPs to Cajal bodies, where some processing steps are believed to take place, and then to nucleoli. Assembly of core proteins involves the HSP90/R2TP chaperone-cochaperone system for both box C/D and H/ACA RNAs, but also several factors specific for each family. These assembly factors chaperone unassembled core proteins, regulate the formation and disassembly of pre-snoRNP intermediates, and control the activity of immature particles. The AAA+ ATPase RUVBL1 and RUVBL2 belong to the R2TP co-chaperones and play essential roles in snoRNP biogenesis, as well as in the formation of other macro-molecular complexes. Despite intensive research, their mechanisms of action are still incompletely understood.

Drosophila Spag is the homolog of RNA polymerase II-associated protein 3 (RPAP3) and recruits the heat shock proteins 70 and 90 (Hsp70 and Hsp90) during the assembly of cellular machineries.

The R2TP is a recently identified Hsp90 co-chaperone, composed of four proteins as follows: Pih1D1, RPAP3, and the AAA(+)-ATPases RUVBL1 and RUVBL2. In mammals, the R2TP is involved in the biogenesis of cellular machineries such as RNA polymerases, small nucleolar ribonucleoparticles and phosphatidylinositol 3-kinase-related kinases. Here, we characterize the spaghetti (spag) gene of Drosophila, the homolog of human RPAP3. This gene plays an essential function during Drosophila development. We show that Spag protein binds Drosophila orthologs of R2TP components and Hsp90, like its yeast counterpart. Unexpectedly, Spag also interacts and stimulates the chaperone activity of Hsp70. Using null mutants and flies with inducible RNAi, we show that spaghetti is necessary for the stabilization of snoRNP core proteins and target of rapamycin activity and likely the assembly of RNA polymerase II. This work highlights the strong conservation of both the HSP90/R2TP system and its clients and further shows that Spag, unlike Saccharomyces cerevisiae Tah1, performs essential functions in metazoans. Interaction of Spag with both Hsp70 and Hsp90 suggests a model whereby R2TP would accompany clients from Hsp70 to Hsp90 to facilitate their assembly into macromolecular complexes.

DPCD is a regulator of R2TP in ciliogenesis initiation through Akt signaling.

R2TP is a chaperone complex consisting of the AAA+ ATPases RUVBL1 and RUVBL2, as well as RPAP3 and PIH1D1 proteins. R2TP is responsible for the assembly of macromolecular complexes mainly acting through different adaptors. Using proximity-labeling mass spectrometry, we identified deleted in primary ciliary dyskinesia (DPCD) as an adaptor of R2TP. Here, we demonstrate that R2TP-DPCD influences ciliogenesis initiation through a unique mechanism by interaction with Akt kinase to regulate its phosphorylation levels rather than its stability. We further show that DPCD is a heart-shaped monomeric protein with two domains. A highly conserved region in the cysteine- and histidine-rich domains-containing proteins and SGT1 (CS) domain of DPCD interacts with the RUVBL2 DII domain with high affinity to form a stable R2TP-DPCD complex both in cellulo and in vitro. Considering that DPCD is one among several CS-domain-containing proteins found to associate with RUVBL1/2, we propose that RUVBL1/2 are CS-domain-binding proteins that regulate complex assembly and downstream signaling.

Unveiling the Role of Sorghum RPAP3 in the Function of R2TP Complex: Insights into Protein Assembly in Plants.

The chaperone R2TP has multiple subunits that assist in the proper folding, assembly, and stabilization of various protein complexes in cells and its study can offer valuable insights into the regulation and maintenance of protein assemblies in plant systems. The 'T' component of R2TP is Tah1 in yeast, consisting of 111 residues, while its counterpart in humans is RPAP3, with 665 residues. RPAP3 acts as a co-chaperone of Hsp90 and facilitates interactions between RUVBL proteins and other complex components, enhancing the recruitment of client proteins by the R2TP complex. These facts further underscore the relevance of studying this complex in different organisms. The putative gene corresponding to the RPAP3 in Sorghum bicolor, a monocotyledon plant, was cloned, and the protein (396 residues) purified for biochemical characterization. SbRPAP3 exists as a folded monomer and has a RPAP3 domain, which is present in human RPAP3 but absent in yeast Tah1. SbRPAP3 retains its functional capabilities, including binding with RUVBLs, Hsp90, and Hsp70. By elucidating the role of RPAP3 in plant R2TP complex, we can further comprehend the molecular mechanisms underlying plant-specific protein assembly and contribute to advancements in plant biology and biotechnological applications.

The multi-faceted roles of R2TP complex span across regulation of gene expression, translation, and protein functional assembly.

Macromolecular complexes play essential roles in various cellular processes. The assembly of macromolecular assemblies within the cell must overcome barriers imposed by a crowded cellular environment which is characterized by an estimated concentration of biological macromolecules amounting to 100-450 g/L that take up approximately 5-40% of the cytoplasmic volume. The formation of the macromolecular assemblies is facilitated by molecular chaperones in cooperation with their co-chaperones. The R2TP protein complex has emerged as a co-chaperone of Hsp90 that plays an important role in macromolecular assembly. The R2TP complex is composed of a heterodimer of RPAP3:P1H1DI that is in turn complexed to members of the ATPase associated with diverse cellular activities (AAA +), RUVBL1 and RUVBL2 (R1 and R2) families. What makes the R2TP co-chaperone complex particularly important is that it is involved in a wide variety of cellular processes including gene expression, translation, co-translational complex assembly, and posttranslational protein complex formation. The functional versatility of the R2TP co-chaperone complex makes it central to cellular development; hence, it is implicated in various human diseases. In addition, their roles in the development of infectious disease agents has become of interest. In the current review, we discuss the roles of these proteins as co-chaperones regulating Hsp90 and its partnership with Hsp70. Furthermore, we highlight the structure-function features of the individual proteins within the R2TP complex and describe their roles in various cellular processes.

Assembly principles of the human R2TP chaperone complex reveal the presence of R2T and R2P complexes.

R2TP is a highly conserved chaperone complex formed by two AAA+ ATPases, RUVBL1 and RUVBL2, that associate with PIH1D1 and RPAP3 proteins. R2TP acts in promoting macromolecular complex formation. Here, we establish the principles of R2TP assembly. Three distinct RUVBL1/2-based complexes are identified: R2TP, RUVBL1/2-RPAP3 (R2T), and RUVBL1/2-PIH1D1 (R2P). Interestingly, we find that PIH1D1 does not bind to RUVBL1/RUVBL2 in R2TP and does not function as a nucleotide exchange factor; instead, RPAP3 is found to be the central subunit coordinating R2TP architecture and linking PIH1D1 and RUVBL1/2. We also report that RPAP3 contains an intrinsically disordered N-terminal domain mediating interactions with substrates whose sequences are primarily enriched for Armadillo repeat domains and other helical-type domains. Our work provides a clear and consistent model of R2TP complex structure and gives important insights into how a chaperone machine concerned with assembly of folded proteins into multisubunit complexes might work.