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Functional assessment of the V390F mutation in the CCTδ subunit of chaperonin containing tailless complex polypeptide 1.

ABSTRACT: The chaperonin containing tailless complex polypeptide 1 (CCT) is a multi-subunit molecular chaperone. It is found in the cytoplasm of all eukaryotic cells, where the oligomeric form plays an essential role in the folding of predominantly the cytoskeletal proteins actin and tubulin. Both the CCT oligomer and monomeric subunits also display functions that extend beyond folding, which are often associated with microtubules and actin filaments. Here, we assess the functional significance of the CCTδ V390F mutation, reported in several cancer cell lines. Upon transfection into B16F1 mouse melanoma cells, GFP-CCTδ^V390F incorporates into the CCT oligomer more readily than GFP-CCTδ. Furthermore, unlike GFP-CCTδ, GFP-CCTδ^V390F does not interact with the dynactin complex component, p150^Glued. As CCTδ has previously been implicated in altered migration in wound healing assays, we assessed the behaviour of GFP-CCTδ^V390F and other mutants of CCTδ, previously used to assess functional interactions with p150^Glued, in chemotaxis assays. We developed the assay system to incorporate a layer of the inert hydrogel GrowDex® to provide a 3D matrix for chemotaxis assessment and found subtle differences in the migration of B16F1 cells, depending on the presence of the hydrogel.

SUBMITTER: Vallin J

PROVIDER: S-EPMC8578507 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Chaperonin-containing tailless complex polypeptide 1 subunit 6A correlates with increased World Health Organization grade, less isocitrate dehydrogenase mutation, and deteriorative survival of astrocytoma patients.

Project description:ObjectiveChaperonin-containing tailless complex polypeptide 1 subunit 6A (CCT6A) is reported to be an efficient prognostic biomarker in various cancers, but it is rarely reported in astrocytoma. Thus, this study aimed to evaluate the expression of CCT6A and its correlation with disease features and prognosis in astrocytoma patients.MethodsTotally, 198 astrocytoma patients who received surgery treatment were enrolled. CCT6A protein expression was determined in the tumor tissues fixed in formalin and embedded in paraffin (FFEP) by immunohistochemistry (IHC) assay. In addition, 133 out of 198 astrocytoma patients had fresh tumor tissues frozen in the liquid nitrogen for the determination of CCT6A mRNA expression by reverse transcription-quantitative polymerase chain reaction.ResultsSixty-nine (34.8%), 70 (35.4%), 46 (23.2%), and 13 (6.6%) astrocytoma patients had the CCT6A immunohistochemistry (IHC) score of 0-3, 4-6, 7-9, and 10-12, respectively. CCT6A protein expression was correlated with increased World Health Organization (WHO) grade (P < 0.001) and less isocitrate dehydrogenase (IDH) mutation (P = 0.002); meanwhile, CCT6A mRNA expression was only related to elevated WHO grade (P = 0.001). However, CCT6A protein and mRNA expression were not correlated with other clinical features and subsequent treatment modalities (all P > 0.05). Moreover, CCT6A protein high and CCT6A mRNA high were related to shorter accumulating overall survival (OS; both P < 0.05). CCT6A protein high was an independent factor for predicting the worse OS (hazard ratio: 1.821, P = 0.012).ConclusionChaperonin-containing tailless complex polypeptide 1 subunit 6A correlates with elevated WHO grade and less IDH mutation; besides, CCT6A high expression is independently associated with unfavorable accumulating OS of astrocytoma patients.

| S-EPMC8418478 | biostudies-literature

Chaperonin containing T-complex polypeptide (CCT) subunit expression in oral mucosal wounds and fibroblasts.

Project description:Mucosal wound healing in adults has been reported to feature diminished scar formation compared to healing skin wounds. We sought to determine if the expression pattern of chaperonin containing T-complex polypeptide (CCT) subunits in mucosal wounds and fibroblasts is different from that observed in skin wounds and fibroblasts. We found that CCT-beta is the only subunit message to be reduced in wounded mucosa versus unwounded control, and this reduction was confirmed at the protein level. In contrast, mRNA levels of CCT-zeta, -delta, -eta, and -epsilon were significantly increased in mucosal wounds. The increase in CCT-eta was also confirmed at the protein level. Expression levels of CCT-alpha, -beta, -delta; -epsilon, and -theta mRNAs were significantly increased in adult mucosal fibroblasts in culture compared to skin-derived fibroblasts. Western blot analyses confirmed a modest increase in CCT-beta in adult mucosal fibroblasts relative to skin fibroblasts, but CCT-eta protein was unaffected. These differences may contribute to the reported difference in healing outcomes between these two tissue types.

| S-EPMC3220385 | biostudies-literature

Programmed cell death protein 5 interacts with the cytosolic chaperonin containing tailless complex polypeptide 1 (CCT) to regulate β-tubulin folding.

Project description:Programmed cell death protein 5 (PDCD5) has been proposed to act as a pro-apoptotic factor and tumor suppressor. However, the mechanisms underlying its apoptotic function are largely unknown. A proteomics search for binding partners of phosducin-like protein, a co-chaperone for the cytosolic chaperonin containing tailless complex polypeptide 1 (CCT), revealed a robust interaction between PDCD5 and CCT. PDCD5 formed a complex with CCT and β-tubulin, a key CCT-folding substrate, and specifically inhibited β-tubulin folding. Cryo-electron microscopy studies of the PDCD5·CCT complex suggested a possible mechanism of inhibition of β-tubulin folding. PDCD5 bound the apical domain of the CCTβ subunit, projecting above the folding cavity without entering it. Like PDCD5, β-tubulin also interacts with the CCTβ apical domain, but a second site is found at the sensor loop deep within the folding cavity. These orientations of PDCD5 and β-tubulin suggest that PDCD5 sterically interferes with β-tubulin binding to the CCTβ apical domain and inhibits β-tubulin folding. Given the importance of tubulins in cell division and proliferation, PDCD5 might exert its apoptotic function at least in part through inhibition of β-tubulin folding.

| S-EPMC3924310 | biostudies-literature

Functional characterization of chaperonin containing T-complex polypeptide-1 and its conserved and novel substrates in Arabidopsis.

Project description:Chaperonin containing T-complex polypeptide-1 (CCT) is an evolutionarily conserved chaperonin multi-subunit complex that mediates protein folding in eukaryotes. It is essential for cell growth and survival in yeast and mammals, with diverse substrate proteins. However, only a few studies on plant CCT have been reported to date, due to the essentiality of CCT subunit genes and the large size of the complex. Here, we have investigated the structure and function of the Arabidopsis CCT complex in detail. The plant CCT consisted of eight subunits that assemble to form a high-molecular-mass protein complex, shown by diverse methods. CCT-deficient cells exhibited depletion of cortical microtubules, accompanied by a reduction in cellular α- and β-tubulin levels due to protein degradation. Cycloheximide-chase assays suggested that CCT is involved in the folding of tubulins in plants. Furthermore, CCT interacted with PPX1, the catalytic subunit of protein phosphatase 4, and may participate in the folding of PPX1 as its substrate. CCT also interacted with Tap46, a regulatory subunit of PP2A family phosphatases, but Tap46 appeared to function in PPX1 stabilization, rather than as a CCT substrate. Collectively, our findings reveal the essential functions of CCT chaperonin in plants and its conserved and novel substrates.

| S-EPMC6506772 | biostudies-literature

Chaperonin containing T-complex polypeptide subunit eta is a potential marker of joint contracture: an experimental study in the rat.

Project description:Joint contracture is a fibroproliferative disorder that restricts joint mobility, resulting in tissue degeneration and deformity. However, the etiology of joint contracture is still unknown. Chaperonin containing T-complex polypeptide subunit eta (CCT-eta) is reported to increase in fibrotic diseases. The purpose of this study was to investigate whether CCT-eta is implicated in joint contracture and to determine the role of CCT-eta in the progression of joint contracture by analyzing a rat model. We immobilized the left knee joint of rat by internal fixation for 8 weeks. The non-immobilized right leg served as a control. The range of motion (ROM) of the knee was investigated. Fibroblasts were obtained from the posterior joint capsule of the joints. The outcome was followed by quantitative real-time polymerase chain reaction (qRT-PCR), Western blot, fibroblast migration assay, and collagen assay. The effect of CCT-eta on the functions of fibroblasts was observed by utilizing a short inhibitory RNA (siRNA) targeting CCT-eta. The ROM of the immobilized joints was significantly limited compared to the contralateral joints (p < 0.05). Fibroblasts derived from the contractive joints showed higher mRNA and protein expressions of CCT-eta in parallel with alpha-smooth muscle actin (α-SMA) compared to the cells from the contralateral knees (p < 0.05). siRNA-mediated downregulation of CCT-eta inhibited the expressions of both CCT-eta and α-SMA. Moreover, the reduction of CCT-eta also significantly decreased fibroblast functions such as cell mobility and collagen synthesis (all p < 0.05). Our findings indicate that CCT-eta appears to be a potential marker of joint contracture disease.

| S-EPMC4595434 | biostudies-literature

Investigating Chaperonin-Containing TCP-1 subunit 2 as an essential component of the chaperonin complex for tumorigenesis.

Project description: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 (CCT?) 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.

| S-EPMC6972895 | biostudies-literature

Overexpression of chaperonin containing T-complex polypeptide subunit zeta 2 (CCT6b) suppresses the functions of active fibroblasts in a rat model of joint contracture.

Project description:BackgroundJoint contracture is a fibrous disease characterized as joint capsule fibrosis that results in joint dysfunction and disability. The purpose of this study was to analyze the biological activities of chaperonin containing T-complex polypeptide (CCT) subunits and to determine the role of CCT chaperone in joint contracture in a rat model.MethodsIn this study, the rat model of joint contracture was established by immobilizing the rat knee for 8 weeks. Then, fibroblasts were isolated from the posterior joint capsule and were cultured for functional analysis such as qRT-PCR, Western blot, transwell assay, and collagen assay. The effect of CCT subunit was determined by employing a lentivirus containing target gene and transfecting it into fibroblasts.ResultsResults of qRT-PCR and Western blot showed that among all CCT subunits, CCT6b significantly decreased in the fibroblasts from contractive joints compared to cells from normal joints (p < 0.05). Overexpression of CCT6b by transfection of lentivirus containing CCT6b gene to active fibroblasts significantly inhibited fibrous marker (α-SMA, COL-1) expressions, fibroblast migration, and collagen synthesis (all p < 0.05). Moreover, fibrosis-related chaperone CCT7 expression was decreased with CCT6b overexpression (p < 0.05).ConclusionThe biological activities of CCT subunits in fibroblasts from the joint contracture rat model were analyzed in this study. CCT6b significantly decreased in the active fibroblasts, and overexpression of CCT6b significantly inhibited fibroblast functions. These findings indicate that CCT6b appears to be a potential molecular biomarker and therapeutic target for the novel therapies of joint contracture.

| S-EPMC6507144 | biostudies-literature

Chaperonin structure: the large multi-subunit protein complex.

Project description:The multi sub-unit protein structure representing the chaperonins group is analyzed with respect to its hydrophobicity distribution. The proteins of this group assist protein folding supported by ATP. The specific axial symmetry GroEL structure (two rings of seven units stacked back to back - 524 aa each) and the GroES (single ring of seven units - 97 aa each) polypeptide chains are analyzed using the hydrophobicity distribution expressed as excess/deficiency all over the molecule to search for structure-to-function relationships. The empirically observed distribution of hydrophobic residues is confronted with the theoretical one representing the idealized hydrophobic core with hydrophilic residues exposure on the surface. The observed discrepancy between these two distributions seems to be aim-oriented, determining the structure-to-function relation. The hydrophobic force field structure generated by the chaperonin capsule is presented. Its possible influence on substrate folding is suggested.

| S-EPMC2672005 | biostudies-literature

Mutation in the epsilon subunit of the cytosolic chaperonin-containing t-complex peptide-1 (Cct5) gene causes autosomal recessive mutilating sensory neuropathy with spastic paraplegia.

Project description:BACKGROUND: Mutilating sensory neuropathy with spastic paraplegia is a very rare disease with both autosomal dominant and recessive modes of inheritance. We previously mapped the locus of the autosomal recessive form to a 25 cM interval between markers D5S2048 and D5S648 on chromosome 5p. In this candidate interval, the Cct5 gene encoding the epsilon subunit of the cytosolic chaperonin-containing t-complex peptide-1 (CCT) was the most obvious candidate gene since mutation in the Cct4 gene encoding the CCT delta subunit has been reported to be associated with autosomal recessive mutilating sensory neuropathy in mutilated foot (mf) rat mutant. METHODS: A consanguineous Moroccan family with four patients displaying mutilating sensory neuropathy associated with spastic paraplegia was investigated. To identify the disease causing gene, the 11 coding exons of the Cct5 gene were screened for mutations by direct sequencing in all family members including the four patients, parents, and six at risk relatives. RESULTS: Sequence analysis of the Cct5 gene revealed a missense A492G mutation in exon 4 that results in the substitution of a highly conserved histidine for arginine amino acid 147. Interestingly, R147 was absent in 384 control matched chromosomes tested. CONCLUSION: This is the first disease causing mutation that has been identified in the human CCT subunit genes; the mf rat mutant could serve as an animal model for studying these chaperonopathies.

| S-EPMC2564519 | biostudies-literature

Eukaryotic chaperonin containing T-complex polypeptide 1 interacts with filamentous actin and reduces the initial rate of actin polymerization in vitro.

Project description:We have previously observed that subunits of the chaperonin required for actin production (type-II chaperonin containing T-complex polypeptide 1 [CCT]) localize at sites of microfilament assembly. In this article we extend this observation by showing that substantially substoichiometric CCT reduces the initial rate of pyrene-labeled actin polymerization in vitro where eubacterial chaperonin GroEL had no such effect. CCT subunits bound selectively to F-actin in cosedimentation assays, and CCT reduced elongation rates from both purified actin filament "seeds" and the short and stabilized, minus-end blocked filaments in erythrocyte membrane cytoskeletons. These observations suggest CCT might remain involved in biogenesis of the actin cytoskeleton, by acting at filament (+) ends, beyond its already well-established role in producing new actin monomers.

| S-EPMC514823 | biostudies-literature

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