Members of eukaryotic chaperonin family are essential for cell survival. Dysregulation of Chaperonin containing TCP-1 subunit 3 (CCT3) has been implicated in the development of several types of cancers. However, the role of CCT3 in the development of gastric cancer has yet to be determined.

Chaperonin-containing TCP-1 (CCT or TRiC) is a multi-subunit complex that folds many of the proteins essential for cancer development. CCT is expressed in diverse cancers and could be an ideal therapeutic target if not for the fact that the complex is encoded by eight distinct genes, complicating the development of inhibitors. Few definitive studies addressed the role of specific subunits in promoting the chaperonin's function in cancer. To this end, we investigated the activity of CCT2 (CCTβ) by overexpressing or depleting the subunit in breast epithelial and breast cancer cells. We found that increasing total CCT2 in cells by 1.3-1.8-fold using a lentiviral system, also caused CCT3, CCT4, and CCT5 levels to increase. Likewise, silencing cct2 gene expression by ~50% caused other CCT subunits to decrease. Cells expressing CCT2 were more invasive and had a higher proliferative index. CCT2 depletion in a syngeneic murine model of triple negative breast cancer (TNBC) prevented tumor growth. These results indicate that the CCT2 subunit is integral to the activity of the chaperonin and is needed for tumorigenesis. Hence CCT2 could be a viable target for therapeutic development in breast and other cancers.

In metazoans, heritable states of cell type-specific gene expression patterns linked with specialization of various cell types constitute transcriptional cellular memory. Evolutionarily conserved Polycomb group (PcG) and trithorax group (trxG) proteins contribute to the transcriptional cellular memory by maintaining heritable patterns of repressed and active expression states, respectively. Although chromatin structure and modifications appear to play a fundamental role in maintenance of repression by PcG, the precise targeting mechanism and the specificity factors that bind PcG complexes to defined regions in chromosomes remain elusive. Here, we report a serendipitous discovery that uncovers an interplay between Polycomb (Pc) and chaperonin containing T-complex protein 1 (TCP-1) subunit 7 (CCT7) of TCP-1 ring complex (TRiC) chaperonin in Drosophila. CCT7 interacts with Pc at chromatin to maintain repressed states of homeotic and non-homeotic targets of PcG, which supports a strong genetic interaction observed between Pc and CCT7 mutants. Depletion of CCT7 results in dissociation of Pc from chromatin and redistribution of an abundant amount of Pc in cytoplasm. We propose that CCT7 is an important modulator of Pc, which helps Pc recruitment at chromatin, and compromising CCT7 can directly influence an evolutionary conserved epigenetic network that supervises the appropriate cellular identities during development and homeostasis of an organism.

Chaperonin containing TCP-1 (T-complex protein 1) (CCT) is a large molecular weight complex that contains nine subunits (TCP1, CCT2, CCT3, CCT4, CCT5, CCT6A, CCT6B, CCT7, CCT8). This study aimed to reveal key genes which encode CCT subunits for prognosis and establish prognostic gene signatures based on CCT subunit genes. The data was downloaded from The Cancer Genome Atlas, International Cancer Genome Consortium and Gene Expression Omnibus. CCT subunit gene expression levels between tumor and normal tissues were compared. Corresponding Kaplan-Meier analysis displayed a distinct separation in the overall survival of CCT subunit genes. Correlation analysis, protein-protein interaction network, Gene Ontology analysis, immune cells infiltration analysis, and transcription factor network were performed. A nomogram was constructed for the prediction of prognosis. Based on multivariate Cox regression analysis and shrinkage and selection method for linear regression model, a three-gene signature comprising CCT4, CCT6A, and CCT6B was constructed in the training set and significantly associated with prognosis as an independent prognostic factor. The prognostic value of the signature was then validated in the validation and testing set. Nomogram including the signature showed some clinical benefit for overall survival prediction. In all, we built a novel three-gene signature and nomogram from CCT subunit genes to predict the prognosis of hepatocellular carcinoma, which may support the medical decision for HCC therapy.

Chaperonin containing TCP1 (CCT/TRiC) is a multi-subunit protein folding complex that enables the cancer phenotype to emerge from the mutational landscape that drives oncogenesis. We and others linked increased expression of CCT subunits to advanced tumor stage and invasiveness that inversely correlates with cancer patient outcomes. In this study, we examined the expression of the second CCT subunit, CCT2, using genomic databases of adult and pediatric tumors and normal tissues, and found that it was highly expressed in pediatric cancers, showing a significant difference compared to normal tissues. Histologic staining confirmed that CCT subunits are highly expressed in tumor tissues, which was exemplified in neuroblastoma. Using two neuroblastoma cells, MYCN-amplified, IMR-32 cells, and non-amplified, SK-N-AS cells, we assessed baseline levels for CCT subunits and found expressions comparable to the highly invasive triple-negative breast cancer (TNBC) cell line, MDA-MB-231. Exogenous expression of CCT2 in both SK-N-AS and IMR-32 cells resulted in morphological changes, such as larger cell size and increased adherence, with significant increases in the CCT substrates, actin, and tubulin, as well as increased migration. Depletion of CCT2 reversed these effects and reduced cell viability. We evaluated CCT as a therapeutic target in IMR-32 cells by testing a novel peptide CCT inhibitor, CT20p. Treatment with CT20p induced cell death in these neuroblastoma cells. The use of CCT2 as a biological indicator for detection of neuroblastoma cells shed in blood was examined by spiking IMR-32 cells into human blood and using an anti-CCT2 antibody for the identification of spiked cancer cells with the CellSearch system. Results showed that using CCT2 for the detection of neuroblastoma cells in blood was more effective than the conventional approach of using epithelial markers like cytokeratins. CCT2 plays an essential role in promoting the invasive capacity of neuroblastoma cells and thus offers the potential to act as a molecular target in the development of novel therapeutics and diagnostics for pediatric cancers.

Chaperonin-containing TCP-1 (CCT) is a chaperonin composed of eight subunits that participates in intracellular protein folding. Here, we showed that increased levels of subunits of CCT, particularly CCT-β, were significantly correlated with lower survival rates for cancer patients. Endogenously high expression of CCT-β was found in cancer cell lines, such as the triple-negative breast cancer cell line MDA-MB-231 and the highly metastatic non-small-cell lung cancer cell line CL1-5. Knocking down CCT-β in these cancer cells led to decreased levels of anti-apoptotic proteins, such as XIAP, as well as inhibited phosphorylation of Ser473-AKT and GSK3, resulting in decrease of the nucleus-entering form of β-catenin; these changes reduced the chemoresistance and migration/invasion of the cells. Conversely, overexpression of CCT-β recovered the chemoresistance and cell migration/invasion by promoting the AKT-GSK3β-β-catenin and XIAP-Survivin pathways. Coimmunoprecipitation data revealed that the CCT complex might directly bind and stabilize XIAP and β-catenin. This study not only elucidates the roles of CCT in chemoresistance and metastasis, which are two major obstacles for current cancer therapy, but also provides a possible therapeutic strategy against cancers with overexpressed CCT-β.

Herein we report the use of Chaperonin-Containing TCP-1 (CCT or TRiC) as a marker to detect circulating tumor cells (CTCs) that are shed from tumors during oncogenesis. Most detection methods used in liquid biopsy approaches for enumeration of CTCs from blood, employ epithelial markers like cytokeratin (CK). However, such markers provide little information on the potential of these shed tumor cells, which are normally short-lived, to seed metastatic sites. To identify a marker that could go beyond enumeration and provide actionable data on CTCs, we evaluated CCT. CCT is a protein-folding complex composed of eight subunits. Previously, we found that expression of the second subunit (CCT2 or CCTβ) inversely correlated with cancer patient survival and was essential for tumorigenesis in mice, driving tumor-promoting processes like proliferation and anchorage-independent growth. In this study, we examined CCT2 expression in cancer compared to normal tissues and found statistically significant increases in tumors. Because not all blood samples from cancer patients contain detectable CTCs, we used the approach of spiking a known number of cancer cells into blood from healthy donors to test a liquid biopsy approach using CCT2 to distinguish rare cancer cells from the large number of non-cancer cells in blood. Using a clinically validated method for capturing CTCs, we evaluated detection of intracellular CCT2 staining for visualization of breast cancer and small cell lung (SCLC) cancer cells. We demonstrated that CCT2 staining could be incorporated into a CTC capture and staining protocol, providing biologically relevant information to improve detection of cancer cells shed in blood. These results were confirmed with a pilot study of blood from SCLC patients. Our studies demonstrate that detection of CCT2 could identify rare cancer cells in blood and has application in liquid biopsy approaches to enhance the use of minimally invasive methods for cancer diagnosis.

Identifying new druggable targets is desired to meet the needs for effective cancer treatments. To this end, we previously reported the efficacy of a therapeutic peptide called CT20p that displays selective cytotoxicity through inhibition of a multi-subunit, protein-folding complex called Chaperonin-Containing TCP-1 (CCT). To investigate the role of CCT in cancer progression, we examined protein levels of CCT subunits in liver, prostate, and lung cancer using human tissue microarrays. We found that these cancers expressed higher levels of CCT2 as compared to normal tissues. Small cell lung cancer (SCLC) stood out as having statistically significant difference in CCT2. Higher levels of CCT2 in tumors from lung cancer patients were also associated with decreased survival. Using SCLC cell lines, we observed detectable amounts of CCT subunits and cells were susceptible to killing by CT20p. Treatment with CT20p, delivered to cells using polymeric nanoparticles, was cytotoxic to all SCLC cell lines, decreasing the levels of CCT client proteins like STAT3. In contrast, treatment with a STAT3 inhibitor was effective in one of the SCLC cell lines. While we found that CCT levels could vary in cell lines, normal tissues had low levels of CCT and minimal toxicity to liver or kidney function was observed in mice treated with CT20p. These results indicate that in SCLC, changes in CCT levels could be used as a biomarker for diagnosis and that targeting CCT for inhibition with CT20p is a promising treatment approach for those cancers such as SCLC that currently lack targeted therapeutics.

The eukaryotic cytosolic chaperonin containing TCP-1 (CCT) has an important function in maintaining cellular homoeostasis by assisting the folding of many proteins, including the cytoskeletal components actin and tubulin. Yet the nature of the proteins and cellular pathways dependent on CCT function has not been established globally. Here, we use proteomic and genomic approaches to define CCT interaction networks involving 136 proteins/genes that include links to the nuclear pore complex, chromatin remodelling, and protein degradation. Our study also identifies a third eukaryotic cytoskeletal system connected with CCT: the septin ring complex, which is essential for cytokinesis. CCT interactions with septins are ATP dependent, and disrupting the function of the chaperonin in yeast leads to loss of CCT-septin interaction and aberrant septin ring assembly. Our results therefore provide a rich framework for understanding the function of CCT in several essential cellular processes, including epigenetics and cell division.

Viruses are molecular machines sustained through a life cycle that requires replication within host cells. Throughout the infectious cycle, viral and cellular components interact to advance the multistep process required to produce progeny virions. Despite progress made in understanding the virus-host protein interactome, much remains to be discovered about the cellular factors that function during infection, especially those operating at terminal steps in replication. In an RNA interference screen, we identified the eukaryotic chaperonin T-complex protein-1 (TCP-1) ring complex (TRiC; also called CCT for chaperonin containing TCP-1) as a cellular factor required for late events in the replication of mammalian reovirus. We discovered that TRiC functions in reovirus replication through a mechanism that involves folding the viral σ3 major outer-capsid protein into a form capable of assembling onto virus particles. TRiC also complexes with homologous capsid proteins of closely related viruses. Our data define a critical function for TRiC in the viral assembly process and raise the possibility that this mechanism is conserved in related non-enveloped viruses. These results also provide insight into TRiC protein substrates and establish a rationale for the development of small-molecule inhibitors of TRiC as potential antiviral therapeutics.

Store-operated Ca2+ entry (SOCE) encodes a range of cellular responses downstream of Ca2+ influx through the SOCE channel Orai1. Orai1 recycles at the plasma membrane (PM), with ~40% of the total Orai1 pool residing at the PM at steady state. The mechanisms regulating Orai1 recycling remain poorly understood. We map the domains in Orai1 that are required for its trafficking to and recycling at the PM. We further identify, using biochemical and proteomic approaches, the CCT [chaperonin-containing TCP-1 (T-complex protein 1)] chaperonin complex as a novel regulator of Orai1 recycling by primarily regulating Orai1 endocytosis. We show that Orai1 interacts with CCT through its intracellular loop and that inhibition of CCT-Orai1 interaction increases Orai1 PM residence. This increased residence is functionally significant as it results in prolonged Ca2+ signaling, early formation of STIM1-Orai1 puncta, and more rapid activation of NFAT (nuclear factor of activated T cells) downstream of SOCE. Therefore, the CCT chaperonin is a novel regulator of Orai1 trafficking and, as such, a modulator of Ca2+ signaling and effector activation kinetics.

The deep ocean is the largest biome on Earth and yet it is among the least studied environments of our planet. Life at great depths requires several specific adaptations; however, their molecular mechanisms remain understudied. We examined patterns of positive selection in 416 genes from four brittle star (Ophiuroidea) families displaying replicated events of deep-sea colonization (288 individuals from 216 species). We found consistent signatures of molecular convergence in functions related to protein biogenesis, including protein folding and translation. Five genes were recurrently positively selected, including chaperonin-containing TCP-1 subunit α (CCTα), which is essential for protein folding. Molecular convergence was detected at the functional and gene levels but not at the amino-acid level. Pressure-adapted proteins are expected to display higher stability to counteract the effects of denaturation. We thus examined in silico local protein stability of CCTα across the ophiuroid tree of life (967 individuals from 725 species) in a phylogenetically corrected context and found that deep-sea-adapted proteins display higher stability within and next to the substrate-binding region, which was confirmed by in silico global protein stability analyses. This suggests that CCTα displays not only structural but also functional adaptations to deep-water conditions. The CCT complex is involved in the folding of ∼10% of newly synthesized proteins and has previously been categorized as a "cold-shock" protein in numerous eukaryotes. We thus propose that adaptation mechanisms to cold and deep-sea environments may be linked and highlight that efficient protein biogenesis, including protein folding and translation, is a key metabolic deep-sea adaptation.

The eukaryotic chaperonin CCT (chaperonin containing TCP-1) uses cavities built into its double-ring structure to encapsulate and to assist folding of a large subset of proteins. CCT can inhibit amyloid fibre assembly and toxicity of the polyQ extended mutant of huntingtin, the protein responsible for Huntington's disease. This raises the possibility that CCT modulates other amyloidopathies, a still-unaddressed question. We show here that CCT inhibits amyloid fibre assembly of α-synuclein A53T, one of the mutants responsible for Parkinson's disease. We evaluated fibrillation blockade in α-synuclein A53T deletion mutants and CCT interactions of full-length A53T in distinct oligomeric states to define an inhibition mechanism specific for α-synuclein. CCT interferes with fibre assembly by interaction of its CCTζ and CCTγ subunits with the A53T central hydrophobic region (NAC). This interaction is specific to NAC conformation, as it is produced once soluble α-synuclein A53T oligomers form and blocks the reaction before fibres begin to grow. Finally, we show that this association inhibits α-synuclein A53T oligomer toxicity in neuroblastoma cells. In summary, our results and those for huntingtin suggest that CCT is a general modulator of amyloidogenesis via a specific mechanism.

Canine parvovirus (CPV) is one of the most common lethal viruses in canines. The virus disease is prevalent throughout the year, with high morbidity and mortality rate, causing serious harm to dogs and the dog industry. Previously, yeast two hybrid method was used to screen the protein chaperonin containing TCP-1 (CCT7) that interacts with VP2. However, the mechanism of interactions between CCT7 and VP2 on CPV replication remains unclear. In this study, we first verified the interaction between CCT7 and viral VP2 proteins using yeast one-to-one experiment and co-immunoprecipitation (CoIP) experiment. Laser confocal microscopy observation showed that CCT7 and VP2 were able to co-localize and were mostly localized in the cytoplasm. In addition, the study of VP2 truncated mutant found that the interaction region of VP2 with CCT7 was located between amino acids 231 and 320. Cycloheximide (CHX) chase experiments showed that CCT7 can improve the stability of VP2 protein. After further regulation of CCT7 expression in F81 cells, it was found that the expression level of VP2 protein was significantly reduced after knocking down CCT7 expression by RNA interference (RNAi) or HSF1A inhibitor, and increased after overexpressing host CCT7. The study reveals the role of VP2 interacting protein CCT7 in the replication process of CPV, which could provide a potential target for the prevention and control of CPV.

Intestinal epithelial cells have unique apical membrane structures, known as microvilli, that contain bundles of actin microfilaments. In this study, we report that Caenorhabditis elegans cytosolic chaperonin containing TCP-1 (CCT) is essential for proper formation of microvilli in intestinal cells. In intestinal cells of cct-5(RNAi) animals, a substantial amount of actin is lost from the apical area, forming large aggregates in the cytoplasm, and the apical membrane is deformed into abnormal, bubble-like structures. The length of the intestinal microvilli is decreased in these animals. However, the overall actin protein levels remain relatively unchanged when CCT is depleted. We also found that CCT depletion causes a reduction in the tubulin levels and disorganization of the microtubule network. In contrast, the stability and localization of intermediate filament protein IFB-2, which forms a dense filamentous network underneath the apical surface, appears to be superficially normal in CCT-deficient cells, suggesting substrate specificity of CCT in the folding of filamentous cytoskeletons in vivo. Our findings demonstrate physiological functions of CCT in epithelial cell morphogenesis using whole animals.

Chaperonin-containing TCP-1 (TRiC or CCT) was demonstrated to be involved in oncogenesis of cancers carcinogenesis and development of various malignancies. Increasing experimental evidence indicated that dysregulation of TRiC was implicated in the tumor progression of breast cancer (BCa). However, few definitive studies have addressed the diverse expression patterns and prognostic values of eight TRiC subunits. Thus, we aimed to investigate the clinical significance of TRiC subunit expression and prognostic values for their possible implications in diagnosis and treatment of BCa.

The eukaryotic cytoplasmic chaperonin-containing TCP-1 (CCT) is a complex formed by two back-to-back stacked hetero-octameric rings that assists the folding of actins, tubulins, and other proteins in an ATP-dependent manner. Here, we tested the significance of the hetero-oligomeric nature of CCT in its function by introducing, in each of the eight subunits in turn, an identical mutation at a position that is conserved in all the subunits and is involved in ATP hydrolysis, in order to establish the extent of 'individuality' of the various subunits. Our results show that these identical mutations lead to dramatically different phenotypes. For example, Saccharomyces cerevisiae yeast cells with the mutation in subunit CCT2 display heat sensitivity and cold sensitivity for growth, have an excess of actin patches, and are the only strain here generated that is pseudo-diploid. By contrast, cells with the mutation in subunit CCT7 are the only ones to accumulate juxtanuclear protein aggregates that may reflect an impaired stress response in this strain. System-level analysis of the strains using RNA microarrays reveals connections between CCT and several cellular networks, including ribosome biogenesis and TOR2, that help to explain the phenotypic variability observed.

Dendritic morphology is inextricably linked to neuronal function. Systematic large-scale screens combined with genetic mapping have uncovered several mechanisms underlying dendrite morphogenesis. However, a comprehensive overview of participating molecular mechanisms is still lacking. Here, we conducted an efficient clonal screen using a collection of mapped P-element insertions that were previously shown to cause lethality and eye defects in Drosophila melanogaster. Of 280 mutants, 52 exhibited dendritic defects. Further database analyses, complementation tests, and RNA interference validations verified 40 P-element insertion genes as being responsible for the dendritic defects. Twenty-eight mutants presented severe arbor reduction, and the remainder displayed other abnormalities. The intrinsic regulators encoded by the identified genes participate in multiple conserved mechanisms and pathways, including the protein folding machinery and the chaperonin-containing TCP-1 (CCT) complex that facilitates tubulin folding. Mutant neurons in which expression of CCT4 or CCT5 was depleted exhibited severely retarded dendrite growth. We show that CCT localizes in dendrites and is required for dendritic microtubule organization and tubulin stability, suggesting that CCT-mediated tubulin folding occurs locally within dendrites. Our study also reveals novel mechanisms underlying dendrite morphogenesis. For example, we show that Drosophila Nogo signaling is required for dendrite development and that Mummy and Wech also regulate dendrite morphogenesis, potentially via Dpp- and integrin-independent pathways. Our methodology represents an efficient strategy for identifying intrinsic dendrite regulators, and provides insights into the plethora of molecular mechanisms underlying dendrite morphogenesis.

The eukaryotic chaperonin-containing TCP-1 (CCT) folds the cytoskeletal protein actin. The folding mechanism of this 16-subunit, 1-MDa machine is poorly characterised due to the absence of quantitative in vitro assays. We identified phosducin-like protein 2, Plp2p (=PLP2), as an ATP-elutable binding partner of yeast CCT while establishing the CCT interactome. In a novel in vitro CCT-ACT1 folding assay that is functional under physiological conditions, PLP2 is a stimulatory co-factor. In a single ATP-driven cycle, PLP2-CCT-ACT1 complexes yield 30-fold more native actin than CCT-ACT1 complexes. PLP2 interacts directly with ACT1 through the C-terminus of its thioredoxin fold and the CCT-binding subdomain 4 of actin. The in vitro CCT-ACT1-PLP2 folding cycle of the preassembled complex takes 90 s at 30 degrees C, several times slower than the canonical chaperonin GroEL. The specific interactions between PLP2, CCT and ACT1 in the yeast-component in vitro system and the pronounced stimulatory effect of PLP2 on actin folding are consistent with in vivo genetic approaches demonstrating an essential and positive role for PLP2 in cellular processes involving actin in Saccharomyces cerevisiae. In mammalian systems, however, several members of the PLP family, including human PDCL3, the orthologue of PLP2, have been shown to be inhibitory toward CCT-mediated folding of actin in vivo and in vitro. Here, using a rabbit-reticulocyte-derived in vitro translation system, we found that inhibition of beta-actin folding by PDCL3 can be relieved by exchanging its acidic C-terminal extension for that of PLP2. It seems that additional levels of regulatory control of CCT activity by this PLP have emerged in higher eukaryotes.

Dengue virus (DENV) NS1 is a multifunctional protein essential for viral replication. To gain insights into NS1 functions in mosquito cells, the protein interactome of DENV NS1 in C6/36 cells was investigated using a proximity biotinylation system and mass spectrometry. A total of 817 mosquito targets were identified as protein-protein interacting with DENV NS1. Approximately 14% of them coincide with interactomes previously obtained in vertebrate cells, including the oligosaccharide transferase complex, the chaperonin containing TCP-1, vesicle localization, and ribosomal proteins. Notably, other protein pathways not previously reported in vertebrate cells, such as epigenetic regulation and RNA silencing, were also found in the NS1 interactome in mosquito cells. Due to the novel and strong interactions observed for NS1 and the epigenetic regulator DIDO1 (Death-Inducer Obliterator 1), the role of DIDO1 in viral replication was further explored. Interactions between NS1 and DIDO1 were corroborated in infected mosquito cells, by colocalization and proximity ligation assays. Silencing DIDO1 expression results in a significant reduction in DENV and ZIKV replication and progeny production. Comparison of transcription analysis of mock or DENV infected cells silenced for DIDO1 revealed variations in multiple gene expression pathways, including pathways associated with DENV infection such as RNA surveillance, IMD, and Toll. These results suggest that DIDO1 is a host factor involved in the negative modulation of the antiviral response necessary for flavivirus replication in mosquito cells. Our findings uncover novel mechanisms of NS1 to promote DENV and ZIKV replication, and add to the understanding of NS1 as a multifunctional protein. IMPORTANCE Dengue is the most important mosquito-borne viral disease to humans. Dengue virus NS1 is a multifunctional protein essential for replication and modulation of innate immunity. To gain insights into NS1 functions, the protein interactome of dengue virus NS1 in Aedes albopictus cells was investigated using a proximity biotinylation system and mass spectrometry. Several protein pathways, not previously observed in vertebrate cells, such as transcription and epigenetic regulation, were found as part of the NS1 interactome in mosquito cells. Among those, DIDO1 was found to be a necessary host factor for dengue and Zika virus replication in mosquito cells. Transcription analysis of infected mosquito cells silenced for DIDO1 revealed alterations of the IMD and Toll pathways, part of the antiviral response in mosquitoes. The results suggest that DIDO1 is a host factor involved in modulation of the antiviral response and necessary for flavivirus replication.