TCP1γ Subunit Is Indispensable for Growth and Infectivity of Leishmania donovani.

T-complex protein-1 (TCP1) is a ubiquitous group II chaperonin and is known to fold various proteins, such as actin and tubulin. In Leishmania donovani, the γ subunit of TCP1 (LdTCP1γ) has been cloned and characterized. It forms a high-molecular-weight homo-oligomeric complex that performs ATP-dependent protein folding. In the present study, we evaluated the essentiality of the LdTCP1γ gene. Gene replacement studies indicated that LdTCP1γ is essential for parasite survival. The LdTCP1γ single-allele-replacement mutants exhibited slowed growth and decreased infectivity in mouse macrophages compared to the growth and infectivity of the wild-type parasites. Modulation of LdTCP1γ expression in promastigotes also modulated cell cycle progression. Suramin, an antitrypanosomal drug, not only inhibited the luciferase refolding activity of the recombinant LdTCP1γ (rLdTCP1γ) homo-oligomeric complex but also exhibited potential antileishmanial efficacy both in vitro and in vivo The interaction of suramin and LdTCP1γ was further validated by isothermal titration calorimetry. The study suggests LdTCP1γ as a potential drug target and also provides a framework for the development of a new class of drugs.

An ensemble of cryo-EM structures of TRiC reveal its conformational landscape and subunit specificity.

TRiC/CCT assists the folding of ∼10% of cytosolic proteins through an ATP-driven conformational cycle and is essential in maintaining protein homeostasis. Here, we determined an ensemble of cryo-electron microscopy (cryo-EM) structures of yeast TRiC at various nucleotide concentrations, with 4 open-state maps resolved at near-atomic resolutions, and a closed-state map at atomic resolution, revealing an extra layer of an unforeseen N-terminal allosteric network. We found that, during TRiC ring closure, the CCT7 subunit moves first, responding to nucleotide binding; CCT4 is the last to bind ATP, serving as an ATP sensor; and CCT8 remains ADP-bound and is hardly involved in the ATPase-cycle in our experimental conditions; overall, yeast TRiC consumes nucleotide in a 2-ring positively coordinated manner. Our results depict a thorough picture of the TRiC conformational landscape and its allosteric transitions from the open to closed states in more structural detail and offer insights into TRiC subunit specificity in ATP consumption and ring closure, and potentially in substrate processing.

Structural and functional analysis of the role of the chaperonin CCT in mTOR complex assembly.

The mechanistic target of rapamycin (mTOR) kinase forms two multi-protein signaling complexes, mTORC1 and mTORC2, which are master regulators of cell growth, metabolism, survival and autophagy. Two of the subunits of these complexes are mLST8 and Raptor, ß-propeller proteins that stabilize the mTOR kinase and recruit substrates, respectively. Here we report that the eukaryotic chaperonin CCT plays a key role in mTORC assembly and signaling by folding both mLST8 and Raptor. A high resolution (4.0 Å) cryo-EM structure of the human mLST8-CCT intermediate isolated directly from cells shows mLST8 in a near-native state bound to CCT deep within the folding chamber between the two CCT rings, and interacting mainly with the disordered N- and C-termini of specific CCT subunits of both rings. These findings describe a unique function of CCT in mTORC assembly and a distinct binding site in CCT for mLST8, far from those found for similar ß-propeller proteins.

TRiC/CCT chaperonins are essential for organ growth by interacting with insulin/TOR signaling in Drosophila.

Organ size is regulated by intercellular signaling for cell growth and proliferation. The TOR pathway mediates a key signaling mechanism for controlling cell size and number in organ growth. Chaperonin containing TCP-1 (CCT) is a complex that assists protein folding and function, but its role in animal development is largely unknown. Here we show that the CCT complex is required for organ growth by interacting with the TOR pathway in Drosophila. Reduction of CCT4 results in growth defects by affecting both cell size and proliferation. Loss of CCT4 causes preferential cell death anterior to the morphogenetic furrow in the eye disc and within wing pouch in the wing disc. Depletion of any CCT subunit in the eye disc results in headless phenotype. Overgrowth by active TOR signaling is suppressed by CCT RNAi. The CCT complex physically interacts with TOR signaling components including TOR, Rheb, and S6K. Loss of CCT leads to decreased phosphorylation of S6K and S6 while increasing phosphorylation of Akt. Insulin/TOR signaling is also necessary and sufficient for promoting CCT complex transcription. Our data provide evidence that the CCT complex regulates organ growth by directly interacting with the TOR signaling pathway.

Structural and functional insights into TRiC chaperonin from a psychrophilic yeast, Glaciozyma antarctica.

Studies on TCP1-1 ring complex (TRiC) chaperonin have shown its indispensable role in folding cytosolic proteins in eukaryotes. In a psychrophilic organism, extreme cold temperature creates a low-energy environment that potentially causes protein denaturation with loss of activity. We hypothesized that TRiC may undergo evolution in terms of its structural molecular adaptation in order to facilitate protein folding in low-energy environment. To test this hypothesis, we isolated G. antarctica TRiC (GaTRiC) and found that the expression of GaTRiC mRNA in G. antarctica was consistently expressed at all temperatures indicating their importance in cell regulation. Moreover, we showed GaTRiC has the ability of a chaperonin whereby denatured luciferase can be folded to the functional stage in its presence. Structurally, three categories of residue substitutions were found in α, ß, and δ subunits: (i) bulky/polar side chains to alanine or valine, (ii) charged residues to alanine, and (iii) isoleucine to valine that would be expected to increase intramolecular flexibility within the GaTRiC. The residue substitutions observed in the built structures possibly affect the hydrophobic, hydrogen bonds, and ionic and aromatic interactions which lead to an increase in structural flexibility. Our structural and functional analysis explains some possible structural features which may contribute to cold adaptation of the psychrophilic TRiC folding chamber.

Chaperonin CCT checkpoint function in basal transcription factor TFIID assembly.

TFIID is a cornerstone of eukaryotic gene regulation. Distinct TFIID complexes with unique subunit compositions exist and several TFIID subunits are shared with other complexes, thereby conveying precise cellular control of subunit allocation and functional assembly of this essential transcription factor. However, the molecular mechanisms that underlie the regulation of TFIID remain poorly understood. Here we use quantitative proteomics to examine TFIID submodules and assembly mechanisms in human cells. Structural and mutational analysis of the cytoplasmic TAF5-TAF6-TAF9 submodule identified novel interactions that are crucial for TFIID integrity and for allocation of TAF9 to TFIID or the Spt-Ada-Gcn5 acetyltransferase (SAGA) co-activator complex. We discover a key checkpoint function for the chaperonin CCT, which specifically associates with nascent TAF5 for subsequent handover to TAF6-TAF9 and ultimate holo-TFIID formation. Our findings illustrate at the molecular level how multisubunit complexes are generated within the cell via mechanisms that involve checkpoint decisions facilitated by a chaperone.

Sequential allosteric mechanism of ATP hydrolysis by the CCT/TRiC chaperone is revealed through Arrhenius analysis.

Knowing the mechanism of allosteric switching is important for understanding how molecular machines work. The CCT/TRiC chaperonin nanomachine undergoes ATP-driven conformational changes that are crucial for its folding function. Here, we demonstrate that insight into its allosteric mechanism of ATP hydrolysis can be achieved by Arrhenius analysis. Our results show that ATP hydrolysis triggers sequential ?conformational waves." They also suggest that these waves start from subunits CCT6 and CCT8 (or CCT3 and CCT6) and proceed clockwise and counterclockwise, respectively.

Role of CCT chaperonin in the disassembly of mitotic checkpoint complexes.

The mitotic checkpoint system prevents premature separation of sister chromatids in mitosis and thus ensures the fidelity of chromosome segregation. When this checkpoint is active, a mitotic checkpoint complex (MCC), composed of the checkpoint proteins Mad2, BubR1, Bub3, and Cdc20, is assembled. MCC inhibits the ubiquitin ligase anaphase promoting complex/cyclosome (APC/C), whose action is necessary for anaphase initiation. When the checkpoint signal is turned off, MCC is disassembled, a process required for exit from checkpoint-arrested state. Different moieties of MCC are disassembled by different ATP-requiring processes. Previous work showed that Mad2 is released from MCC by the joint action of the TRIP13 AAA-ATPase and the Mad2-binding protein p31comet Now we have isolated from extracts of HeLa cells an ATP-dependent factor that releases Cdc20 from MCC and identified it as chaperonin containing TCP1 or TCP1-Ring complex (CCT/TRiC chaperonin), a complex known to function in protein folding. Bacterially expressed CCT5 chaperonin subunits, which form biologically active homooligomers [Sergeeva, et al. (2013) J Biol Chem 288(24):17734-17744], also promote the disassembly of MCC. CCT chaperonin further binds and disassembles subcomplexes of MCC that lack Mad2. Thus, the combined action of CCT chaperonin with that of TRIP13 ATPase promotes the complete disassembly of MCC, necessary for the inactivation of the mitotic checkpoint.

Regulation of GPCR expression through an interaction with CCT7, a subunit of the CCT/TRiC complex.

Mechanisms that prevent aggregation and promote folding of nascent G protein-coupled receptors (GPCRs) remain poorly understood. We identified chaperonin containing TCP-1 subunit eta (CCT7) as an interacting partner of the ß-isoform of thromboxane A2 receptor (TPß) by yeast two-hybrid screening. CCT7 coimmunoprecipitated with overexpressed TPß and ß2-adrenergic receptor (ß2AR) in HEK 293 cells, but also with endogenous ß2AR. CCT7 depletion by small interfering RNA reduced total and cell-surface expression of both receptors and caused redistribution of the receptors to juxtanuclear aggresomes, significantly more so for TPß than ß2AR. Interestingly, Hsp90 coimmunoprecipitated with ß2AR but virtually not with TPß, indicating that nascent GPCRs can adopt alternative folding pathways. In vitro pull-down assays showed that both receptors can interact directly with CCT7 through their third intracellular loops and C-termini. We demonstrate that Trp334 in the TPß C-terminus is critical for the CCT7 interaction and plays an important role in TPß maturation and cell-surface expression. Of note, introducing a tryptophan in the corresponding position of the TPα isoform confers the CCT7-binding and maturation properties of TPß. We show that an interaction with a subunit of the CCT/TCP-1 ring complex (TRiC) chaperonin complex is involved in regulating aggregation of nascent GPCRs and in promoting their proper maturation and expression.

The role of the Chaperonin containing t-complex polypeptide 1, subunit 8 (CCT8) in B-cell non-Hodgkin's lymphoma.

The chaperonin containing t-complex polypeptide 1 (CCT) is known to mediate folding of proteins. CCT, subunit 8 (CCT8), is the θ subunit of CCT complex chaperonin. CCT8 has been reported to be dysregulated in several tumor tissues. In this study, we investigated the role of CCT8 in B-cell non-Hodgkin's lymphoma (NHL). Clinically, the expression levels of CCT8 in reactive lymphoid hyperplasia (RLH) and B-cell NHL specimens were investigated using immunohistochemical analysis. We found that CCT8 was highly expressed in proliferating germinal center cells compared with the quiescent cells of the follicular mantle zone. Furthermore, CCT8 was highly expressed in progressive lymphomas than in indolent lymphomas. Kaplan-Meier curve showed that high expression of CCT8 was significantly associated with shorter overall survival in patients with diffuse large B-cell lymphoma. Moreover, we demonstrated that CCT8 could promote the proliferation of B-cell NHL cells. In addition, we found that CCT8 could accelerate the G1/S transition in B-cell NHL. Finally, we demonstrated that overexpression of CCT8 could reverse cell adhesion-mediated drug resistance (CAM-DR) phenotype. Our study may shed new insights into the important role of CCT8 in cancer development.

Two members of the TRiC chaperonin complex, CCT2 and TCP1 are essential for survival of breast cancer cells and are linked to driving oncogenes.

Gene amplification is a common mechanism of oncogene activation in cancer. Several large-scale efforts aimed at identifying the comprehensive set of genomic regions that are recurrently amplified in cancer have been completed. In breast cancer, these studies have identified recurrently amplified regions containing known drivers such as HER2 and CCND1 as well as regions where the driver oncogene is unknown. In this study, we integrated RNAi-based functional genetic data with copy number and expression data to identify genes that are recurrently amplified, overexpressed and also necessary for the growth/survival of breast cancer cells. Further analysis using clinical data from The Cancer Genome Atlas specifically identified candidate genes that play a role in determining patient outcomes. Using this approach, we identified two genes, TCP1 and CCT2, as being recurrently altered in breast cancer, necessary for growth/survival of breast cancer cells in vitro, and determinants of overall survival in breast cancer patients. We also show that expression of TCP1 is regulated by driver oncogene activation of PI3K signaling in breast cancer. Interestingly, the TCP1 and CCT2 genes both encode for components of a multi-protein chaperone complex in the cell known as the TCP1 Containing Ring Complex (TRiC). Our results demonstrate a role for the TRiC subunits TCP1 and CCT2, and potentially the entire TRiC complex, in breast cancer and provide rationale for TRiC as a novel therapeutic target in breast cancer.

Caenorhabditis elegans chaperonin CCT/TRiC is required for actin and tubulin biogenesis and microvillus formation in intestinal epithelial cells.

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.

The chaperonin CCT interacts with and mediates the correct folding and activity of three subunits of translation initiation factor eIF3: b, i and h.

eIF3 (eukaryotic initiation factor 3) is the largest and most complex eukaryotic mRNA translation factor in terms of the number of protein components or subunits. In mammals, eIF3 is composed of 13 different polypeptide subunits, of which five, i.e. a, b, c, g and i, are conserved and essential in vivo from yeasts to mammals. In the present study, we show that the eukaryotic cytosolic chaperonin CCT [chaperonin containing TCP-1 (tailless complex polypeptide 1)] binds to newly synthesized eIF3b and promotes the correct folding of eIF3h and eIF3i. Interestingly, overexpression of these last two subunits is associated with enhanced translation of specific mRNAs over and above the general enhancement of global translation. In agreement with this, our data show that, as CCT is required for the correct folding of eIF3h and eIF3i subunits, it indirectly influences gene expression with eIF3i overexpression enhancing both cap- and IRES (internal ribosome entry segment)-dependent translation initiation, whereas eIF3h overexpression selectively increases IRES-dependent translation initiation. Importantly, these studies demonstrate the requirement of the chaperonin machinery for the correct folding of essential components of the translational machinery and provide further evidence of the close interplay between the cell environment, cell signalling, cell proliferation, the chaperone machinery and translational apparatus.

Involvement of multimeric protein complexes in mediating the capacitation-dependent binding of human spermatozoa to homologous zonae pellucidae.

The recognition and binding of a free-swimming spermatozoon to an ovulated oocyte is one of the most important cellular interactions in biology. While traditionally viewed as a simple lock and key mechanism, emerging evidence suggests that this event may require the concerted action of several sperm proteins. In this study we examine the hypothesis that the activity of such proteins may be coordinated by their assembly into multimeric recognition complexes on the sperm surface. Through the novel application of blue native polyacrylamide gel electrophoresis (BN-PAGE), we tender the first direct evidence that human spermatozoa do indeed express a number of high molecular weight protein complexes on their surface. Furthermore, we demonstrate that a subset of these complexes displays affinity for homologous zonae pellucidae. Proteomic analysis of two such complexes using electrospray ionization mass spectrometry identified several of the components of the multimeric 20S proteasome and chaperonin-containing TCP-1 (CCT) complexes. The latter complex was also shown to harbor at least one putative zona pellucida binding protein, ZPBP2. Consistent with a role in the mediation of sperm-zona pellucida interaction we demonstrated that antibodies directed against individual subunits of these complexes were able to inhibit sperm binding to zona-intact oocytes. Similarly, these results were able to be recapitulated using native sperm lysates, the zona affinity of which was dramatically reduced by antibody labeling of the complex receptors, or in the case of the 20S proteasome the ubiquitinated zonae ligands. Overall, the strategies employed in this study have provided novel, causal insights into the molecular mechanisms that govern sperm-egg interaction.

Association of CRISP2, CCT8, PEBP1 mRNA abundance in sperm and sire conception rate in Holstein bulls.

The objective was to determine the association of mRNA expression of cystine rich secretary protein 2 (CRISP2), chaperonin containing T-complex protein 1, subunit 8 (CCT8), and phosphatidylethanolamine-binding protein 1 (PEBP1), in sperm of Holstein bulls with Sire Conception Rate (SCR) scores between -4 and +4. These proteins were involved in sperm capacitation and sperm-egg fusion. Samples of sperm obtained on a single day from Holstein bulls (N = 34) in a commercial AI centre were used to evaluate relative mRNA expression of CRISP2, CCT8, and PEBP1. The mRNA abundance of CRISP2 was positively correlated (r = 0.88; P < 0.002), CCT8 was negatively correlated (r = -0.87; P < 0.002), and PEBP1 was positively correlated (r = 0.83; P < 0.006) with SCR-scores. The means of CRISP2 mRNA abundance was greater among positive SCR-score bulls (2.5 to 8 fold), the means of CCT8 mRNA abundance was greater among the negative SCR-score bulls (9.5 to 3.5 fold), and the means of PEBP1 mRNA abundance was greater for the positive SCR-score bulls (5.4 to 7.7 fold). In multivariate regression models predicting SCR-scores, mRNA abundance of CCT8 was significantly associated with SCR-score in all models. In the presence of CRISP2 mRNA abundance in the model, the SCR score's predictability of PEBP1 was insignificant. However, in the absence of CRISP2 mRNA abundance in the model, the SCR-score's predictability of PEBP1 was significant. In multivariate regression models, CRISP2 and CCT8 mRNA expression in sperm accounted for 95% of the variance in Holstein bull's SCR-scores. In conclusion, Holstein bulls with greater CRISP2 and lower CCT8 mRNA expression in sperm had higher probabilities of siring calves.

Equivalent mutations in the eight subunits of the chaperonin CCT produce dramatically different cellular and gene expression phenotypes.

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.

Chaperonin containing T-complex polypeptide subunit eta (CCT-eta) is a specific regulator of fibroblast motility and contractility.

Integumentary wounds in mammalian fetuses heal without scar; this scarless wound healing is intrinsic to fetal tissues and is notable for absence of the contraction seen in postnatal (adult) wounds. The precise molecular signals determining the scarless phenotype remain unclear. We have previously reported that the eta subunit of the chaperonin containing T-complex polypeptide (CCT-eta) is specifically reduced in healing fetal wounds in a rabbit model. In this study, we examine the role of CCT-eta in fibroblast motility and contractility, properties essential to wound healing and scar formation. We demonstrate that CCT-eta (but not CCT-beta) is underexpressed in fetal fibroblasts compared to adult fibroblasts. An in vitro wound healing assay demonstrated that adult fibroblasts showed increased cell migration in response to epidermal growth factor (EGF) and platelet derived growth factor (PDGF) stimulation, whereas fetal fibroblasts were unresponsive. Downregulation of CCT-eta in adult fibroblasts with short inhibitory RNA (siRNA) reduced cellular motility, both basal and growth factor-induced; in contrast, siRNA against CCT-beta had no such effect. Adult fibroblasts were more inherently contractile than fetal fibroblasts by cellular traction force microscopy; this contractility was increased by treatment with EGF and PDGF. CCT-eta siRNA inhibited the PDGF-induction of adult fibroblast contractility, whereas CCT-beta siRNA had no such effect. In each of these instances, the effect of downregulating CCT-eta was to modulate the behavior of adult fibroblasts so as to more closely approximate the characteristics of fetal fibroblasts. We next examined the effect of CCT-eta modulation on alpha-smooth muscle actin (alpha-SMA) expression, a gene product well known to play a critical role in adult wound healing. Fetal fibroblasts were found to constitutively express less alpha-SMA than adult cells. Reduction of CCT-eta with siRNA had minimal effect on cellular beta-actin but markedly decreased alpha-SMA; in contrast, reduction of CCT-beta had minimal effect on either actin isoform. Direct inhibition of alpha-SMA with siRNA reduced both basal and growth factor-induced fibroblast motility. These results indicate that CCT-eta is a specific regulator of fibroblast motility and contractility and may be a key determinant of the scarless wound healing phenotype by means of its specific regulation of alpha-SMA expression.