A glimpse into TriC's magic chamber of secrets.

Chaperones are important for protein folding, but visualizing this process has proven to be exceptionally difficult. In this issue of Cell, Frydman and colleagues have succeeded in watching tubulin being folded by its chaperonin TRiC at near-atomic resolution.

CCT2 is an aggrephagy receptor for clearance of solid protein aggregates.

Protein aggregation is a hallmark of multiple human pathologies. Autophagy selectively degrades protein aggregates via aggrephagy. How selectivity is achieved has been elusive. Here, we identify the chaperonin subunit CCT2 as an autophagy receptor regulating the clearance of aggregation-prone proteins in the cell and the mouse brain. CCT2 associates with aggregation-prone proteins independent of cargo ubiquitination and interacts with autophagosome marker ATG8s through a non-classical VLIR motif. In addition, CCT2 regulates aggrephagy independently of the ubiquitin-binding receptors (P62, NBR1, and TAX1BP1) or chaperone-mediated autophagy. Unlike P62, NBR1, and TAX1BP1, which facilitate the clearance of protein condensates with liquidity, CCT2 specifically promotes the autophagic degradation of protein aggregates with little liquidity (solid aggregates). Furthermore, aggregation-prone protein accumulation induces the functional switch of CCT2 from a chaperone subunit to an autophagy receptor by promoting CCT2 monomer formation, which exposes the VLIR to ATG8s interaction and, therefore, enables the autophagic function.

The Chaperonin TRiC/CCT Associates with Prefoldin through a Conserved Electrostatic Interface Essential for Cellular Proteostasis.

Maintaining proteostasis in eukaryotic protein folding involves cooperation of distinct chaperone systems. To understand how the essential ring-shaped chaperonin TRiC/CCT cooperates with the chaperone prefoldin/GIMc (PFD), we integrate cryoelectron microscopy (cryo-EM), crosslinking-mass-spectrometry and biochemical and cellular approaches to elucidate the structural and functional interplay between TRiC/CCT and PFD. We find these hetero-oligomeric chaperones associate in a defined architecture, through a conserved interface of electrostatic contacts that serves as a pivot point for a TRiC-PFD conformational cycle. PFD alternates between an open "latched" conformation and a closed "engaged" conformation that aligns the PFD-TRiC substrate binding chambers. PFD can act after TRiC bound its substrates to enhance the rate and yield of the folding reaction, suppressing non-productive reaction cycles. Disrupting the TRiC-PFD interaction in vivo is strongly deleterious, leading to accumulation of amyloid aggregates. The supra-chaperone assembly formed by PFD and TRiC is essential to prevent toxic conformations and ensure effective cellular proteostasis.

A hierarchical assembly pathway directs the unique subunit arrangement of TRiC/CCT.

How the essential eukaryotic chaperonin TRiC/CCT assembles from eight distinct subunits into a unique double-ring architecture remains undefined. We show TRiC assembly involves a hierarchical pathway that segregates subunits with distinct functional properties until holocomplex (HC) completion. A stable, likely early intermediate arises from small oligomers containing CCT2, CCT4, CCT5, and CCT7, contiguous subunits that constitute the negatively charged hemisphere of the TRiC chamber, which has weak affinity for unfolded actin. The remaining subunits CCT8, CCT1, CCT3, and CCT6, which comprise the positively charged chamber hemisphere that binds unfolded actin more strongly, join the ring individually. Unincorporated late-assembling subunits are highly labile in cells, which prevents their accumulation and premature substrate binding. Recapitulation of assembly in a recombinant system demonstrates that the subunits in each hemisphere readily form stable, noncanonical TRiC-like HCs with aberrant functional properties. Thus, regulation of TRiC assembly along a biochemical axis disfavors the formation of stable alternative chaperonin complexes.

How TriC folds tricky proteins.

How chaperonins orchestrate the successful folding of even the most elaborate of proteins is a question of central importance. In two recent studies in Cell by Joachimiak et al. and Freund et al., a new class of TRiC substrate is identified, and how the chaperonin exploits its different subunits to extend its substrate repertoire and direct productive folding is revealed.

Proteostatic control of telomerase function through TRiC-mediated folding of TCAB1.

Telomere maintenance by telomerase is impaired in the stem cell disease dyskeratosis congenita and during human aging. Telomerase depends upon a complex pathway for enzyme assembly, localization in Cajal bodies, and association with telomeres. Here, we identify the chaperonin CCT/TRiC as a critical regulator of telomerase trafficking using a high-content genome-wide siRNA screen in human cells for factors required for Cajal body localization. We find that TRiC is required for folding the telomerase cofactor TCAB1, which controls trafficking of telomerase and small Cajal body RNAs (scaRNAs). Depletion of TRiC causes loss of TCAB1 protein, mislocalization of telomerase and scaRNAs to nucleoli, and failure of telomere elongation. DC patient-derived mutations in TCAB1 impair folding by TRiC, disrupting telomerase function and leading to severe disease. Our findings establish a critical role for TRiC-mediated protein folding in the telomerase pathway and link proteostasis, telomere maintenance, and human disease.

The structural basis of substrate recognition by the eukaryotic chaperonin TRiC/CCT.

The eukaryotic chaperonin TRiC (also called CCT) is the obligate chaperone for many essential proteins. TRiC is hetero-oligomeric, comprising two stacked rings of eight different subunits each. Subunit diversification from simpler archaeal chaperonins appears linked to proteome expansion. Here, we integrate structural, biophysical, and modeling approaches to identify the hitherto unknown substrate-binding site in TRiC and uncover the basis of substrate recognition. NMR and modeling provided a structural model of a chaperonin-substrate complex. Mutagenesis and crosslinking-mass spectrometry validated the identified substrate-binding interface and demonstrate that TRiC contacts full-length substrates combinatorially in a subunit-specific manner. The binding site of each subunit has a distinct, evolutionarily conserved pattern of polar and hydrophobic residues specifying recognition of discrete substrate motifs. The combinatorial recognition of polypeptides broadens the specificity of TRiC and may direct the topology of bound polypeptides along a productive folding trajectory, contributing to TRiC's unique ability to fold obligate substrates.

Recognition of the CCT5 di-Glu degron by CRL4DCAF12 is dependent on TRiC assembly.

Assembly Quality Control (AQC) E3 ubiquitin ligases target incomplete or incorrectly assembled protein complexes for degradation. The CUL4-RBX1-DDB1-DCAF12 (CRL4DCAF12 ) E3 ligase preferentially ubiquitinates proteins that carry a C-terminal double glutamate (di-Glu) motif. Reported CRL4DCAF12 di-Glu-containing substrates include CCT5, a subunit of the TRiC chaperonin. How DCAF12 engages its substrates and the functional relationship between CRL4DCAF12 and CCT5/TRiC is currently unknown. Here, we present the cryo-EM structure of the DDB1-DCAF12-CCT5 complex at 2.8 Å resolution. DCAF12 serves as a canonical WD40 DCAF substrate receptor and uses a positively charged pocket at the center of the ß-propeller to bind the C-terminus of CCT5. DCAF12 specifically reads out the CCT5 di-Glu side chains, and contacts other visible degron amino acids through Van der Waals interactions. The CCT5 C-terminus is inaccessible in an assembled TRiC complex, and functional assays demonstrate that DCAF12 binds and ubiquitinates monomeric CCT5, but not CCT5 assembled into TRiC. Our biochemical and structural results suggest a previously unknown role for the CRL4DCAF12 E3 ligase in overseeing the assembly of a key cellular complex.

Molecular, structural, and functional characterization of delta subunit of T-complex protein-1 from Leishmania donovani.

Chaperonins/Heat shock protein 60 are ubiquitous multimeric protein complexes that assist in the folding of partially and/or misfolded proteins using metabolic energy into their native stage. The eukaryotic group II chaperonin, also referred as T-complex protein-1 ring complex (TRiC)/T-complex protein-1 (TCP1)/chaperonin containing T-complex protein (CCT), contains 8-9 paralogous subunits, arranged in each of the two rings of hetero-oligomeric complex. In Leishmania, till date, only one subunit, LdTCP1γ, has been well studied. Here, we report the molecular, structural, and functional characterization of TCP1δ subunit of Leishmania donovani (LdTCP1δ), the causative agent of Indian kala-azar. LdTCP1δ gene exhibited only 27.9% identity with LdTCP1γ and clustered in a separate branch in the phylogenic tree of LdTCP1 subunits. The purified recombinant protein formed a high molecular weight complex (0.75 MDa), arranged into 16-mer assembly, and performed in vitro chaperonin activity as assayed by ATP-dependent luciferase folding. LdTCP1δ exhibits 1.8-fold upregulated expression in metabolically active, rapidly dividing log phase promastigotes. Over-expression of LdTCP1δ in promastigotes results in increased infectivity and rate of multiplication of intracellular amastigotes. The study thus establishes the existence of an individual functionally active homo-oligomeric complex of LdTCP1δ chaperonin with its role in parasite infectivity and multiplication.

Chaperonin TRiC/CCT Participates in Mammarenavirus Multiplication in Human Cells via Interaction with the Viral Nucleoprotein.

The eukaryotic chaperonin containing tailless complex polypeptide 1 ring complex (CCT, also known as TCP-1 Ring Complex, TRiC/CCT) participates in the folding of 5% to 10% of the cellular proteome and has been involved in the life cycle of several viruses, including dengue, Zika, and influenza viruses, but the mechanisms by which the TRiC/CCT complex contributes to virus multiplication remain poorly understood. Here, we document that the nucleoprotein (NP) of the mammarenavirus lymphocytic choriomeningitis virus (LCMV) is a substrate of the human TRiC/CCT complex, and that pharmacological inhibition of TRiC/CCT complex function, or RNAi-mediated knockdown of TRiC/CCT complex subunits, inhibited LCMV multiplication in human cells. We obtained evidence that the TRiC/CCT complex is required for the production of NP-containing virus-like particles (VLPs), and the activity of the virus ribonucleoprotein (vRNP) responsible for directing replication and transcription of the viral genome. Pharmacological inhibition of the TRIC/CCT complex also restricted multiplication of the live-attenuated vaccine candidates Candid#1 and ML29 of the hemorrhagic fever causing Junin (JUNV) and Lassa (LASV) mammarenaviruses, respectively. Our findings indicate that the TRiC/CCT complex is required for mammarenavirus multiplication and is an attractive candidate for the development of host directed antivirals against human-pathogenic mammarenaviruses. IMPORTANCE Host-directed antivirals have gained great interest as an antiviral strategy to counteract the rapid emergence of drug-resistant viruses. The chaperonin TRiC/CCT complex has been involved in the life cycle of several viruses, including dengue, Zika, and influenza viruses. Here, we have provided evidence that the chaperonin TRiC/CCT complex participates in mammarenavirus infection via its interaction with the viral NP. Importantly, pharmacological inhibition of TRiC/CCT function significantly inhibited multiplication of LCMV and the distantly related mammarenavirus JUNV in human cells. Our findings support that the TRiC/CCT complex is required for multiplication of mammarenaviruses and that the TRiC/CCT complex is an attractive host target for the development of antivirals against human-pathogenic mammarenaviruses.

CCT6A facilitates lung adenocarcinoma progression and glycolysis via STAT1/HK2 axis.

BACKGROUND: Chaperonin Containing TCP1 Subunit 6 A (CCT6A) is a prominent protein involved in the folding and stabilization of newly synthesized proteins. However, its roles and underlying mechanisms in lung adenocarcinoma (LUAD), one of the most aggressive cancers, remain elusive. METHODS: Our study utilized in vitro cell phenotype experiments to assess CCT6A's impact on the proliferation and invasion capabilities of LUAD cell lines. To delve into CCT6A's intrinsic mechanisms affecting glycolysis and proliferation in lung adenocarcinoma, we employed transcriptomic sequencing and liquid chromatography-mass spectrometry analysis. Co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (CHIP) assays were also conducted to substantiate the mechanism. RESULTS: CCT6A was found to be significantly overexpressed in LUAD and associated with a poorer prognosis. The silencing of CCT6A inhibited the proliferation and migration of LUAD cells and elevated apoptosis rates. Mechanistically, CCT6A interacted with STAT1 protein, forming a complex that enhances the stability of STAT1 by protecting it from ubiquitin-mediated degradation. This, in turn, facilitated the transcription of hexokinase 2 (HK2), a critical enzyme in aerobic glycolysis, thereby stimulating LUAD's aerobic glycolysis and progression. CONCLUSION: Our findings reveal that the CCT6A/STAT1/HK2 axis orchestrated a reprogramming of glucose metabolism and thus promoted LUAD progression. These insights position CCT6A as a promising candidate for therapeutic intervention in LUAD treatment.

T cell asymmetry and metabolic crosstalk can fine-tune immunological synapses.

T cell asymmetry upon specific cell-cell interactions during mammalian immunological synapse (IS) contacts requires mammalian target of rapamycin complex (mTORC) activation and chaperones, such as the eukaryotic chaperonin containing TCP1 (CCT) for protein synthesis and folding. This mechanism can control cytoskeleton dynamics, and regulate mitochondrial fate, respiration, and metabolic rates, ultimately underlying cell reprogramming events that are relevant for CD4+ T cell functional outcomes.

A genome-wide CRISPR screen identifies the CCT chaperonin as a critical regulator of vesicle trafficking.

Vesicle trafficking is a fundamental cellular process that controls the transport of various proteins and cargos between cellular compartments in eukaryotes. Using a combination of genome-wide CRISPR screening in mammalian cells and RNAi screening in Caenorhabditis elegans, we identify chaperonin containing TCP-1 subunit 4 (CCT4) as a critical regulator of protein secretion and vesicle trafficking. In C. elegans, deficiency of cct-4 as well as other CCT subunits impairs the trafficking of endocytic markers in intestinal cells, and this defect resembles that of dyn-1 RNAi worms. Consistent with these findings, the silencing of CCT4 in human cells leads to defective endosomal trafficking, and this defect can be rescued by the dynamin activator Ryngo 1-23. These results suggest that the cytosolic chaperonin CCT may regulate vesicle trafficking by promoting the folding of dynamin in addition to its known substrate tubulin. Our findings establish an essential role for the CCT chaperonin in regulating vesicle trafficking, and provide new insights into the regulation of vesicle trafficking and the cellular function of the cytosolic chaperonin.

CCT2 prevented ß-catenin proteasomal degradation to sustain cancer stem cell traits and promote tumor progression in epithelial ovarian cancer.

BACKGROUND: Epithelial ovarian cancer (EOC) is featured by rapid progression and dismal outcomes clinically. Chaperonin Containing TCP1 Subunit 2 (CCT2) was identified as a crucial regulator for tumor progression, however, its exact role in EOC remained largely unknown. METHODS: CCT2 expression and prognostic value in EOC samples were assessed according to TCGA dataset. Proliferation and mobility potentials were assessed by CCK8, colony-formation, wound healing, and Transwell assays. Cancer stem cell (CSC) traits were evaluated by RT-PCR, WB assays, sphere-forming assay and chemoresistance analysis. Bioinformatic analysis, co-IP assays and ubiquitin assays were performed to explore the mechanisms of CCT2 on EOC cells. RESULTS: CCT2 highly expressed in EOC tissues and predicted poor prognosis of EOC patients by TCGA analysis. Silencing CCT2 significantly restrained cell proliferation, migration, and invasion. Moreover, CCT2 could effectively trigger epithelial-mesenchymal transition to confer extensive invasion potentials to EOC cells, Importantly, CCT2 positively correlated with CSC markers in EOC, and CCT2 knockdown impaired CSC traits and sensitize EOC cells to conventional chemotherapy regimens. Contrarily, overexpressing CCT2 achieved opposite results. Mechanistically, CCT2 exerted its pro-oncogene function by triggering Wnt/ß-catenin signaling. Specifically, CCT2 could recruit HSP105-PP2A complex, a well-established dephosphorylation complex, to ß-catenin via direct physical interaction to prevent phosphorylation-induced proteasomal degradation of ß-catenin, resulting in intracellular accumulation of active ß-catenin and increased signaling activity. CONCLUSIONS: CCT2 was a novel promotor for EOC progression and a crucial sustainer for CSC traits mainly by preventing ß-catenin degradation. Targeting CCT2 may represent a promising therapeutic strategy for EOC.

Structural and functional dissection of reovirus capsid folding and assembly by the prefoldin-TRiC/CCT chaperone network.

Intracellular protein homeostasis is maintained by a network of chaperones that function to fold proteins into their native conformation. The eukaryotic TRiC chaperonin (TCP1-ring complex, also called CCT for cytosolic chaperonin containing TCP1) facilitates folding of a subset of proteins with folding constraints such as complex topologies. To better understand the mechanism of TRiC folding, we investigated the biogenesis of an obligate TRiC substrate, the reovirus σ3 capsid protein. We discovered that the σ3 protein interacts with a network of chaperones, including TRiC and prefoldin. Using a combination of cryoelectron microscopy, cross-linking mass spectrometry, and biochemical approaches, we establish functions for TRiC and prefoldin in folding σ3 and promoting its assembly into higher-order oligomers. These studies illuminate the molecular dynamics of σ3 folding and establish a biological function for TRiC in virus assembly. In addition, our findings provide structural and functional insight into the mechanism by which TRiC and prefoldin participate in the assembly of protein complexes.

Chaperonins in cancer: Expression, function, and migration in extracellular vesicles.

The chaperonins CCT and Hsp60 are molecular chaperones, members of the chaperone system (CS). Chaperones are cytoprotective but if abnormal in quantity or quality they may cause diseases, the chaperonopathies. Here, recent advances in the understanding of CCT and Hsp60 in cancerology are briefly discussed, focusing on breast and brain cancers. CCT subunits, particularly CCT2, were increased in breast cancer cells and this correlated with tumor progression. Experimental induction of CCT2 increase was accompanied by an increase of CCT3, 4, and 5, providing another evidence for the interconnection between the members of the CS and the difficulties expected while manipulating one member with therapeutic purposes. Another in silico study demonstrated a direct correlation between the increase in the tumor tissue of the mRNA levels of all CCT subunits, except CCTB6, with bad prognosis. Studies with glioblastomas demonstrated an increase in the CCT subunits in the tumor tissue and in extracellular vesicles (EVs) derived from them. Expression levels of CCT1, 2, 6A, and 7 were the most increased and markers of bad prognosis, particularly CCT6A. A method for measuring Hsp60 and related miRNA in exosomes from blood of patients with glioblastomas or other brain tumors was discussed, and the results indicate that the triad Hsp60-related miRNAs-exosomes has potential regarding diagnosis and patient monitoring. All these data provide a strong foundation for future studies on the role played by chaperonins in carcinogenesis and for fully developing their theranostics applications along with exosomes.

CCT3 as a Diagnostic and Prognostic Biomarker in Cervical Cancer.

The chaperonin-containing TCP1 complex subunit 3 (CCT3) has been reported to be involved in the development and prognosis of many tumors, including cervical cancer (CC). This study aimed to analyze the expression and prognostic value of CCT3 in CC by bioinformatics and retrospective study. CCT3 gene expression profiles and clinical information in CC were downloaded from the cancer genome atlas (TCGA) and gene expression omnibus (GEO) databases. CCT3 expression was verified by quantitative real-time polymerase chain reaction (RT-qPCR), Western blot, and immunohistochemistry (IHC). Logistic regression and chi-square testing were used to analyze the relationship between CCT3 expression and the clinical characteristics of CC. Kaplan-Meier and Cox analyses were used to evaluate whether CCT3 affects the prognosis of CC. Nomogram and calibration curves were used to test the predictive value of CCT3. The expression of CCT3 in CC tissues was significantly upregulated compared with that in adjacent benign tissues, and was related to HPV16/18 infection, grade, and positive lymph nodes. High expression of CCT3 is associated with poor prognosis of CC and can be used as an independent risk factor for CC. The prognostic model based on CCT3 and CC clinical features has good predictive ability. CCT3 is overexpressed in CC, which is related to poor prognosis and expected to become a biomarker for CC.

A chaperonin containing T-complex polypeptide-1 facilitates the formation of the PbWoxT1-PbPTB3 ribonucleoprotein complex for long-distance RNA trafficking in Pyrus betulaefolia.

Numerous plant endogenous mRNAs move via phloem and thus affect the growth and development of long-distant organs. mRNAs are transported with RNA-binding proteins forming a ribonucleoprotein complex. However, it remains elusive how such RNP complex assembles and facilitates mRNA trafficking. Protease digestion and RNA immunoprecipitation were used to investigate the RNP assembly function of the complete Chaperonin Containing T-complex Polypeptide-1. In situ hybridization, hairy root transformation, microprojectile bombardment, and grafting experiments demonstrate the role of CCT complex in the transport of a PbWoxT1-PbPTB3 RNP complex in Pyrus betulaefolia. PbCCT5 silenced caused defective movement of GFP-PbPTB3 and GFP-PbWoxT1 from hairy roots to new leaves via the phloem. PbCCT5 is shown to interact with PbPTB3. PbCCT complex enhanced PbPTB3 stabilization and permitted assembly of PbWoxT1 and PbPTB3 into an RNP complex. Furthermore, silencing of individual CCT subunits inhibited the intercellular movement of GFP-PbPTB3 and long-distance trafficking of PbWoxT1 and PbPTB3 in grafted plants. Taken together, the CCT complex assembles PbPTB3 and PbWoxT1 into an RNP complex in the phloem in order to facilitate the long-distance trafficking of PbWoxT1 in P. betulaefolia. This study therefore provides important insights into the mechanism of RNP complex formation and transport.

Circular RNA CCT3 is a unique molecular marker in bladder cancer.

This study surveyed circular RNA CCT3 in bladder cancer (BCa). We recruited 85 BCa patients and 40 normal controls (Normal) and collected clinical specimens for analysis. circRNA CCT3 expression was analyzed by RT-qPCR, diagnostic accuracy was calculated by ROC curves, and survival outcomes were evaluated by survival curves. CircRNA CCT3 was overexpressed or knocked down in cells, thereafter to observe the changes in cell malignant phenotypes. The downstream molecules of circRNA CCT3 were detected. Our data suggest that circRNA CCT3 was upregulated in human BCa and was associated with poor survival outcomes of BCa patients. In cell experiments, overexpressing circRNA CCT3 promoted BCa cell malignancy, whereas silencing circRNA CCT3 did the opposite. In addition, circRNA CCT3 modulated PP2A expression by miR-135a-5p. This study demonstrates that circRNA CCT3 is a diagnostic and prognostic biomarker in BCa patients and is a tumor promoter in BCa.

Plasmodium chaperonin TRiC/CCT identified as a target of the antihistamine clemastine using parallel chemoproteomic strategy.

The antihistamine clemastine inhibits multiple stages of the Plasmodium parasite that causes malaria, but the molecular targets responsible for its parasite inhibition were unknown. Here, we applied parallel chemoproteomic platforms to discover the mechanism of action of clemastine and identify that clemastine binds to the Plasmodium falciparum TCP-1 ring complex or chaperonin containing TCP-1 (TRiC/CCT), an essential heterooligomeric complex required for de novo cytoskeletal protein folding. Clemastine destabilized all eight P. falciparum TRiC subunits based on thermal proteome profiling (TPP). Further analysis using stability of proteins from rates of oxidation (SPROX) revealed a clemastine-induced thermodynamic stabilization of the Plasmodium TRiC delta subunit, suggesting an interaction with this protein subunit. We demonstrate that clemastine reduces levels of the major TRiC substrate tubulin in P. falciparum parasites. In addition, clemastine treatment leads to disorientation of Plasmodium mitotic spindles during the asexual reproduction and results in aberrant tubulin morphology suggesting protein aggregation. This clemastine-induced disruption of TRiC function is not observed in human host cells, demonstrating a species selectivity required for targeting an intracellular human pathogen. Our findings encourage larger efforts to apply chemoproteomic methods to assist in target identification of antimalarial drugs and highlight the potential to selectively target Plasmodium TRiC-mediated protein folding for malaria intervention.