Epsilon subunit of T-complex protein-1 from Leishmania donovani: A tetrameric chaperonin.

The cytosolic T-complex protein-1 ring complex (TRiC), also referred as chaperonin containing TCP-1(CCT), comprising eight different subunits stacked in double toroidal rings, binds to around 10 % of newly synthesized polypeptides and facilitates their folding in ATP dependent manner. In Leishmania, among five subunits of TCP1 complex, identified either by transcriptome or by proteome analysis, only LdTCP1γ has been well characterized. It forms biologically active homo-oligomeric complex and plays role in protein folding and parasite survival. Lack of information regarding rest of the TCP1 subunits and its structural configuration laid down the necessity to study individual subunits and their role in parasite pathogenicity. The present study involves the cloning, expression and biochemical characterization of TCP1ε subunit (LdTCP1ε) of Leishmania donovani, the causative agent of visceral leishmaniasis. LdTCP1ε exhibited significant difference in primary structure as compared to LdTCP1γ and was evolutionary close to LdTCP1 zeta subunit. Recombinant protein (rLdTCP1ε) exhibited two major bands of 132 kDa and 240 kDa on native-PAGE that corresponds to the dimeric and tetrameric assembly of the epsilon subunit, which showed the chaperonin activity (ATPase and luciferase refolding activity). LdTCP1ε also displayed an increased expression upto 2.7- and 1.8-fold in the late log phase and stationary phase promastigotes and exhibited majorly vesicular localization. The study, thus for the first time, provides an insight for the presence of highly diverge but functionally active dimeric/tetrameric TCP1 epsilon subunit in Leishmania parasite.

Compound heterozygous mutations in a mouse model of Leber congenital amaurosis reveal the role of CCT2 in photoreceptor maintenance.

TRiC/CCT is a chaperonin complex required for the folding of cytoplasmic proteins. Although mutations in each subunit of TRiC/CCT are associated with various human neurodegenerative diseases, their impact in mammalian models has not yet been examined. A compound heterozygous mutation in CCT2 (p.[Thr400Pro]; p.[Arg516His]) is causal for Leber congenital amaurosis. Here, we generate mice carrying each mutation and show that Arg516His (R516H) homozygosity causes photoreceptor degeneration accompanied by a significant depletion of TRiC/CCT substrate proteins in the retina. In contrast, Thr400Pro (T400P) homozygosity results in embryonic lethality, and the compound heterozygous mutant (T400P/R516H) mouse showed aberrant cone cell lamination and died 2 weeks after birth. Finally, CCDC181 is identified as a interacting protein for CCTß protein, and its localization to photoreceptor connecting cilia is compromised in the mutant mouse. Our results demonstrate the distinct impact of each mutation in vivo and suggest a requirement for CCTß in ciliary maintenance.

Protocol to study CCT-mediated folding of Gß5 by single-particle cryo-EM.

The chaperonin CCT mediates folding of many cytosolic proteins, including G protein ß subunits (Gßs). Here, we present a protocol for isolating Gß5 bound to CCT and its co-chaperone PhLP1 and determining the CCT-mediated folding trajectory of Gß5 using single-particle cryoelectron microscopy (cryo-EM) techniques. We describe steps for purifying CCT-Gß5-PhLP1 from human cells, stabilizing the closed CCT conformation, preparing and imaging cryo-EM specimens, and processing data to recover multiple Gß5 folding intermediates. This protocol permits visualization of protein folding by CCT. For complete details on the use and execution of this protocol, please refer to Sass et al.1.

CCT and Cullin1 Regulate the TORC1 Pathway to Promote Dendritic Arborization in Health and Disease.

The development of cell-type-specific dendritic arbors is integral to the proper functioning of neurons within their circuit networks. In this study, we examine the regulatory relationship between the cytosolic chaperonin CCT, key insulin pathway genes, and an E3 ubiquitin ligase (Cullin1) in dendritic development. CCT loss of function (LOF) results in dendritic hypotrophy in Drosophila Class IV (CIV) multi-dendritic larval sensory neurons, and CCT has recently been shown to fold components of the TOR (Target of Rapamycin) complex 1 (TORC1) in vitro. Through targeted genetic manipulations, we confirm that an LOF of CCT and the TORC1 pathway reduces dendritic complexity, while overexpression of key TORC1 pathway genes increases the dendritic complexity in CIV neurons. Furthermore, both CCT and TORC1 LOF significantly reduce microtubule (MT) stability. CCT has been previously implicated in regulating proteinopathic aggregation, thus, we examine CIV dendritic development in disease conditions as well. The expression of mutant Huntingtin leads to dendritic hypotrophy in a repeat-length-dependent manner, which can be rescued by Cullin1 LOF. Together, our data suggest that Cullin1 and CCT influence dendritic arborization through the regulation of TORC1 in both health and disease.

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.

Targeting the molecular chaperone CCT2 inhibits GBM progression by influencing KRAS stability.

Proper protein folding relies on the assistance of molecular chaperones post-translation. Dysfunctions in chaperones can cause diseases associated with protein misfolding, including cancer. While previous studies have identified CCT2 as a chaperone subunit and an autophagy receptor, its specific involvement in glioblastoma remains unknown. Here, we identified CCT2 promote glioblastoma progression. Using approaches of coimmunoprecipitation, mass spectrometry and surface plasmon resonance, we found CCT2 directly bound to KRAS leading to increased stability and upregulated downstream signaling of KRAS. Interestingly, we found that dihydroartemisinin, a derivative of artemisinin, exhibited therapeutic effects in a glioblastoma animal model. We further demonstrated direct binding between dihydroartemisinin and CCT2. Treatment with dihydroartemisinin resulted in decreased KRAS expression and downstream signaling. Highlighting the significance of CCT2, CCT2 overexpression rescued the inhibitory effect of dihydroartemisinin on glioblastoma. In conclusion, the study demonstrates that CCT2 promotes glioblastoma progression by directly binding to and enhancing the stability of the KRAS protein. Additionally, dihydroartemisinin inhibits glioblastoma by targeting the CCT2 and the following KRAS signaling. Our findings overcome the challenge posed by the undruggable nature of KRAS and offer potential therapeutic strategies for glioblastoma treatment.

Revisiting the chaperonin T-complex protein-1 ring complex in human health and disease: A proteostasis modulator and beyond.

BACKGROUND: Disrupted protein homeostasis (proteostasis) has been demonstrated to facilitate the progression of various diseases. The cytosolic T-complex protein-1 ring complex (TRiC/CCT) was discovered to be a critical player in orchestrating proteostasis by folding eukaryotic proteins, guiding intracellular localisation and suppressing protein aggregation. Intensive investigations of TRiC/CCT in different fields have improved the understanding of its role and molecular mechanism in multiple physiological and pathological processes. MAIN BODY: In this review, we embark on a journey through the dynamic protein folding cycle of TRiC/CCT, unraveling the intricate mechanisms of its substrate selection, recognition, and intriguing folding and assembly processes. In addition to discussing the critical role of TRiC/CCT in maintaining proteostasis, we detail its involvement in cell cycle regulation, apoptosis, autophagy, metabolic control, adaptive immunity and signal transduction processes. Furthermore, we meticulously catalogue a compendium of TRiC-associated diseases, such as neuropathies, cardiovascular diseases and various malignancies. Specifically, we report the roles and molecular mechanisms of TRiC/CCT in regulating cancer formation and progression. Finally, we discuss unresolved issues in TRiC/CCT research, highlighting the efforts required for translation to clinical applications, such as diagnosis and treatment. CONCLUSION: This review aims to provide a comprehensive view of TRiC/CCT for researchers to inspire further investigations and explorations of potential translational possibilities.

Dysfunction of CCT3-associated network signals for the critical state during progression of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors and is a serious threat to human health; thus, early diagnosis and adequate treatment are essential. However, there are still great challenges in identifying the tipping point and detecting early warning signals of early HCC. In this study, we aimed to identify the tipping point (critical state) of and key molecules involved in hepatocarcinogenesis based on time series transcriptome expression data of HCC patients. The phase from veHCC (very early HCC) to eHCC (early HCC) was identified as the critical state in HCC progression, with 143 genes identified as key candidate molecules by combining the DDRTree (dimensionality reduction via graph structure learning) and DNB (dynamic network biomarker) methods. Then, we ranked the candidate genes to verify their mRNA levels using the diethylnitrosamine (DEN)-induced HCC mouse model and identified five early warning signals, namely, CCT3, DSTYK, EIF3E, IARS2 and TXNRD1; these signals can be regarded as the potential early warning signals for the critical state of HCC. We identified CCT3 as an independent prognostic factor for HCC, and functions of CCT3 involving in the "MYCtargets_V1" and "E2F-Targets" are closely related to the progression of HCC. The predictive method combining the DDRTree and DNB methods can not only identify the key critical state before cancer but also determine candidate molecules of critical state, thus providing new insight into the early diagnosis and preemptive treatment of HCC.

A structural vista of phosducin-like PhLP2A-chaperonin TRiC cooperation during the ATP-driven folding cycle.

Proper cellular proteostasis, essential for viability, requires a network of chaperones and cochaperones. ATP-dependent chaperonin TRiC/CCT partners with cochaperones prefoldin (PFD) and phosducin-like proteins (PhLPs) to facilitate folding of essential eukaryotic proteins. Using cryoEM and biochemical analyses, we determine the ATP-driven cycle of TRiC-PFD-PhLP2A interaction. PhLP2A binds to open apo-TRiC through polyvalent domain-specific contacts with its chamber's equatorial and apical regions. PhLP2A N-terminal H3-domain binding to subunits CCT3/4 apical domains displace PFD from TRiC. ATP-induced TRiC closure rearranges the contacts of PhLP2A domains within the closed chamber. In the presence of substrate, actin and PhLP2A segregate into opposing chambers, each binding to positively charged inner surface residues from CCT1/3/6/8. Notably, actin induces a conformational change in PhLP2A, causing its N-terminal helices to extend across the inter-ring interface to directly contact a hydrophobic groove in actin. Our findings reveal an ATP-driven PhLP2A structural rearrangement cycle within the TRiC chamber to facilitate folding.

[The Effect of TCP1 Expression on the Proliferation and the Accumulation of Intracellular Drug of HL60/A and HL60 Cell and Its Mechanism].

OBJECTIVE: To investigate the effect of TCP1 expression on the proliferation and the accumulation of intracellular drug of HL60/A and HL60 cells and its possible molecular mechanism. METHODS: Lentiviral transfection technology was used to construct HL60/A and HL60 cells with knocked down or overexpressed TCP1 and their control cells. The efficiency of knockdown and overexpression was evaluated by Western blot. The cell proliferation was detected by CCK-8 assay. The intracellular drug accumulation was detected by laser confocal detection and flow cytometry. The expression levels of MRP1, P-gP and p-AKT were evaluated by flow cytometry and Western blot. RESULTS: After TCP1 was knocked down,the proliferation ability of HL60/A cells was significantly reduced, the accumulation of intracellular drug was significantly increased and the expression of MRP1 and P-gP protein were decreased. After TCP1 was overexpressed, the proliferation ability of HL60 was significantly increased, the accumulation of intracellular drug was significantly decreased and the expression of MRP1 and P-gP protein were increased. Intervention of LY294002 significantly antagonized the promotion on cell proliferation, the inhibition on intracellular drug accumulation and the expression of MRP1 and P-gP mediated by TCP1 overexpressing in HL60 cells. CONCLUSION: TCP1 can promote cell proliferation, improve the expression of MRP1 and P-gP by activating PI3K/AKT signal, and reduce intracellular drug accumulation.

The structural basis of eukaryotic chaperonin TRiC/CCT: Action and folding.

Accurate folding of proteins in living cells often requires the cooperative support of molecular chaperones. Eukaryotic group II chaperonin Tailless complex polypeptide 1-Ring Complex (TRiC) accomplishes this task by providing a folding chamber for the substrate that is regulated by an Adenosine triphosphate (ATP) hydrolysis-dependent cycle. Once delivered to and recognized by TRiC, the nascent substrate enters the folding chamber and undergoes folding and release in a stepwise manner. During the process, TRiC subunits and cochaperones such as prefoldin and phosducin-like proteins interact with the substrate to assist the overall folding process in a substrate-specific manner. Coevolution between the components is supposed to consult the binding specificity and ultimately expand the substrate repertoire assisted by the chaperone network. This review describes the TRiC chaperonin and the substrate folding process guided by the TRiC network in cooperation with cochaperones, specifically focusing on recent progress in structural analyses.

LINC01234 promoted malignant behaviors of breast cancer cells via hsa-miR-30c-2-3p/CCT4/mTOR signaling pathway.

OBJECTIVE: Despite continuous progress in treatment, recurrence and metastasis limit further improvement in the prognosis of breast cancer (BC) patients. Our aim was to search for a crucial prognostic biomarker of BC. MATERIALS AND METHODS: Patient data were selected from The Cancer Genome Atlas (TCGA) and GTEx databases. Several online public databases, including Gene Expression Profiling Interactive Analysis (GEPIA), miRWalk, miRDB, and LncBase Predicted v.2, were used to identify potential upstream miRNAs and lncRNAs. These findings were validated through in vitro experiments. RESULTS: A total of 1, 097 invasive BC samples and 572 normal breast tissues (including 113 samples from TCGA and 459 samples from GTEx) were collected for the study. CCT4 was not only significantly overexpressed in BC compared with normal breast tissues but also had important prognostic significance (P < 0.001). By intersecting miRWalk and miRDB and conducting correlation analysis, hsa-miR-30c-2-3p was identified as the most probable upstream miRNA of CCT4. Following an extensive assessment that included survival analysis, correlation analysis, and common binding-site prediction, LINC01234 was chosen as the most likely upstream lncRNA. In vitro experiments showed that LINC01234-siRNA inhibited the proliferation, invasion, and migration abilities of BC cells. Western blot analysis further confirmed that LINC01234 promoted malignant behaviors of BC cells via the CCT4/mTOR signaling pathway. CONCLUSION: The LINC01234/hsa-miR-30c-2-3p/CCT4/mTOR axis was identified as a potential ceRNA regulatory mechanism in BC. These findings established the foundation for systematically unveiling the pathological mechanisms of BC and provided new insights for targeted therapy of BC patients.

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.

The Chaperonin TRiC/CCT Inhibitor HSF1A Protects Cells from Intoxication with Pertussis Toxin.

Pertussis toxin (PT) is a bacterial AB5-toxin produced by Bordetella pertussis and a major molecular determinant of pertussis, also known as whooping cough, a highly contagious respiratory disease. In this study, we investigate the protective effects of the chaperonin TRiC/CCT inhibitor, HSF1A, against PT-induced cell intoxication. TRiC/CCT is a chaperonin complex that facilitates the correct folding of proteins, preventing misfolding and aggregation, and maintaining cellular protein homeostasis. Previous research has demonstrated the significance of TRiC/CCT in the functionality of the Clostridioides difficile TcdB AB-toxin. Our findings reveal that HSF1A effectively reduces the levels of ADP-ribosylated Gαi, the specific substrate of PT, in PT-treated cells, without interfering with enzyme activity in vitro or the cellular binding of PT. Additionally, our study uncovers a novel interaction between PTS1 and the chaperonin complex subunit CCT5, which correlates with reduced PTS1 signaling in cells upon HSF1A treatment. Importantly, HSF1A mitigates the adverse effects of PT on cAMP signaling in cellular systems. These results provide valuable insights into the mechanisms of PT uptake and suggest a promising starting point for the development of innovative therapeutic strategies to counteract pertussis toxin-mediated pathogenicity.

CCT6A dysregulation in surgical prostate cancer patients: association with disease features, treatment information, and prognosis.

OBJECTIVE: Chaperonin-containing tailless complex polypeptide 1 subunit 6A (CCT6A) involves several solid cancers' development and progression, while its clinical utility in prostate cancer management is rarely revealed. Consequently, the present study intended to investigate the linkage of CCT6A with disease features, treatment information, and prognosis of surgical prostate cancer patients. METHODS: CCT6A in 220 surgical prostate cancer patients was determined via immunohistochemistry. Additionally, survival analyses on data from the public databases were performed to validate the prognostic value of CCT6A further. RESULTS: CCT6A expression was upregulated in tumor tissue than in adjacent tissue (P < 0.001). Increased CCT6A was related to elevated Gleason score (P < 0.001) and pathological T stage (P = 0.029). CCT6A was increased in patients with positive surgical margin status (vs. negative) (P = 0.029) and patients with adjuvant external-beam radiation therapy (vs. no) (P = 0.001). Concerning the prognostic value, high tumor CCT6A was linked with shortened disease-free survival (DFS) (P = 0.009), which was also validated through further Cox's proportional hazard regression model analyses (hazard ratio: 2.695, 95% CI: 1.086-6.683, P = 0.032), whereas CCT6A was not correlated with overall survival (OS) (P > 0.050). Additionally, the Gene Expression Profiling Interactive Analysis database indicated that high tumor CCT6A was related to shortened DFS (P = 0.036), but it was not associated with OS (P > 0.050); meanwhile, the Human Protein Atlas database suggested that high tumor CCT6A was linked with reduced OS (P = 0.048). CONCLUSION: Tumor CCT6A high expression correlates with the elevated Gleason score, pathological T stage, and shortened DFS in surgical prostate cancer patients.

Chaperonin containing TCP1 subunit 6A may activate Notch and Wnt pathways to facilitate the malignant behaviors and cancer stemness in oral squamous cell carcinoma.

Chaperonin containing TCP1 subunit 6A (CCT6A) was recently discovered to be involved in cancer pathogenesis and stemness; however, its role in oral squamous cell carcinoma (OSCC) has not been reported. The current study aimed to investigate the impact of CCT6A on OSCC cell malignant behaviors and stemness and to explore its potentially interreacted pathways. SCC-15 and HSC-3 cells were transfected with the plasmid loading control overexpression, CCT6A overexpression, control knockout, or CCT6A knockout. Wnt4 overexpression or Notch1 overexpression plasmids were transfected into CCT6A-knockout SCC-15 cells. Cell proliferation, apoptosis, invasion, stemness, Notch, and Wnt pathways were detected in both cell lines, whereas RNA sequencing was only performed in SCC-15 cells. CCT6A was upregulated in five OSCC cell lines, including SCC-15, HSC-3, SAT, SCC-9, and KON, compared to that in the control cell line. In SCC-15 and HSC-3 cells, CCT6A overexpression increased cell proliferation, invasion, sphere formation, CD133, and Sox2 expression, but decreased cell apoptosis; on the contrary, CCT6A knockout exhibited an opposite effect on the above indexes. RNA-sequencing data revealed that the Wnt and Notch pathways were involved in the CCT6A'effect on SCC-15 cell functions. CCT6A positively regulates the Wnt and Notch pathways in SCC-15 and HSC-3 cells. Importantly, it was shown that activation of the Wnt or Notch pathways attenuated the effect of CCT6A knockout on SCC-15 cell survival, invasion, and stemness. CCT6A may promote OSCC malignant behavior and stemness by activating the Wnt and Notch pathways.

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.

Molecular Dynamics Mappings of the CCT/TRiC Complex-Mediated Protein Folding Cycle Using Diffracted X-ray Tracking.

The CCT/TRiC complex is a type II chaperonin that undergoes ATP-driven conformational changes during its functional cycle. Structural studies have provided valuable insights into the mechanism of this process, but real-time dynamics analyses of mammalian type II chaperonins are still scarce. We used diffracted X-ray tracking (DXT) to investigate the intramolecular dynamics of the CCT complex. We focused on three surface-exposed loop regions of the CCT1 subunit: the loop regions of the equatorial domain (E domain), the E and intermediate domain (I domain) juncture near the ATP-binding region, and the apical domain (A domain). Our results showed that the CCT1 subunit predominantly displayed rotational motion, with larger mean square displacement (MSD) values for twist (χ) angles compared with tilt (θ) angles. Nucleotide binding had a significant impact on the dynamics. In the absence of nucleotides, the region between the E and I domain juncture could act as a pivotal axis, allowing for greater motion of the E domain and A domain. In the presence of nucleotides, the nucleotides could wedge into the ATP-binding region, weakening the role of the region between the E and I domain juncture as the rotational axis and causing the CCT complex to adopt a more compact structure. This led to less expanded MSD curves for the E domain and A domain compared with nucleotide-absent conditions. This change may help to stabilize the functional conformation during substrate binding. This study is the first to use DXT to probe the real-time molecular dynamics of mammalian type II chaperonins at the millisecond level. Our findings provide new insights into the complex dynamics of chaperonins and their role in the functional folding cycle.

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.