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PubMed2001

Complete cDNA and deduced amino acid sequence of the chaperonin containing T-complex polypeptide 1 (CCT) delta subunit from Aedes triseriatus mosquitoes.

The chaperonin containing t-complex polypeptide 1 (CCT) assists in the ATP-dependent folding and assembly of newly translated actin and tubulin in the eukaryotic cytosol. CCT is composed of eight different subunits, each encoded by an independent gene. In this report, we used RT-PCR amplification and 5'- and 3'-rapid amplification of cDNA ends (RACE) to determine the complete cDNA sequence of the CCT delta subunit from Aedes triseriatus mosquitoes. The CCT delta cDNA is 1936 nucleotides in length and encodes a putative 533 amino acid protein with a calculated molecular mass of 57,179 daltons and pI of 7.15. Hydrophobic residues comprise 39.8% of the amino acid sequence and putative motifs for ATP-binding and ATPase-activity are present. The amino acid sequence displays strong sequence similarity to Drosophila melanogaster (92%), human (85%), puffer fish (84%) and mouse (84%) counterparts. CCT delta mRNA was detected in both biosynthetically active (embryonating) and dormant (diapausing) Ae. triseriatus embryos by RT-PCR analysis.

Aedes↗
PubMed1994

Tcp20, a subunit of the eukaryotic TRiC chaperonin from humans and yeast.

Members of the Hsp60 chaperonin family, such as Escherichia coli GroEL/S and the eukaryotic cytosolic chaperonin complex, TRiC (TCP ring complex), are double toroid complexes capable of assisting the folding of proteins in vitro in an ATP-dependent fashion. TRiC differs from the GroEL chaperonin in that it has a hetero rather than homo-oligomeric subunit composition and lacks a GroES-like regulatory cofactor. We have established greater than 57% identity between a protein encoded by the TCP20 gene from a human cDNA library and the newly identified protein encoded by the TCP20 gene located on the right arm of chromosome IV of the yeast Saccharomyces cerevisiae. These Tcp20 proteins showed approximately 30% identity to Tcp1, a known subunit of TRiC. Gel filtration, followed by Western analysis of purified bovine testis TRiC with a Tcp20-specific antibody, indicated that Tcp20 is a subunit of the hetero-oligomeric TRiC. Gene disruption experiments showed that TCP20, like TCP1, is an essential gene in yeast, consistent with the view that TRiC is required for folding of key proteins. The amino acid sequence similarities and the derived evolutionary relationships established that the human and yeast Tcp20 proteins represent members of a new family of subunits of TRiC chaperonins.

Amino Acid Sequence↗
PubMed1995

Molecular chaperones: physical and mechanistic properties.

Molecular chaperones can be broadly defined as proteins which interact with non-native states of other protein molecules. This activity is important in the folding of newly synthesized polypeptides and the assembly of multisubunit structures; the maintenance of proteins in unfolded states suitable for translocation across membranes; and the stabilization of inactive forms of proteins which are turned on by cellular signals; and the stabilization of proteins unfolded during cellular stress. The major chaperone classes are hsp60 (including TCP1), hsp70 and hsp90. All these proteins prevent the aggregation of unfolded proteins and the strength of interaction with their protein substrates is modified by the binding and hydrolysis of ATP. Hsp70 is a dimeric and ubiquitous protein which binds its substrates in an extended conformation through hydrophobic interactions. It binds to newly synthesized proteins and is required for protein transport. In its ATP-bound state it has a low protein affinity but when the nucleotide is hydrolysed to give the ADP state the affinity is increased. Hsp70 in E. coli (DnaK) is regulated by two co-proteins: DnaJ (of which there are homologues in eukaryotes) stimulates hydrolysis of ATP and GrpE promotes the dissociation of ADP to allow rebinding of ATP. Thus DnaJ promotes the association of substrate proteins and GrpE promotes dissociation. Hsp60 is a large, tetradecameric protein with a central cavity in which non-native protein structures are proposed to bind. It is essential for the folding of a huge spectrum of unrelated proteins and is present in all biological compartments except the ER. As in hsp70, the binding of ATP stimulates release of the substrate and its hydrolysis restores high binding affinity. It functions in conjunction with a co-protein, cpn10, which enhances its ability to eject proteins during the ATPase cycle. The enhancement of folding yields arises either from the prevention of irreversible aggregation or the ability to unfold misfolded structures and allow further attempts to arrive at the native state. Proteins of the hsp90 class are found associated with inactive or unstable substrate proteins within the cell, thus preventing their aggregation and/or permitting rapid activation.

Chaperonin Containing TCP-1↗
PubMed1998

Interaction of p59fyn kinase with the dynein light chain, Tctex-1, and colocalization during cytokinesis.

The protein tyrosine kinase p59fyn (Fyn) plays important roles in both lymphocyte Ag receptor signaling and cytokinesis of proB cells. We utilized yeast two-hybrid cloning to identify the product of the tctex-1 gene as a protein that specifically interacts with Fyn, but not with other Src family kinases. Tctex-1 was recently identified as a component of the dynein cytoskeletal motor complex. The capacity of a Tctex-1-glutathione S-transferase fusion protein to effectively bind Fyn from cell lysates confirmed the authenticity of this interaction. Tctex-1 binding required the first 19 amino acids of Fyn and integrity of two lysine residues within this sequence that were previously shown to be important for Fyn interactions with the immunoreceptor tyrosine-based activation motifs (ITAMs) of lymphocyte Ag receptors. Expression of tctex-1 mRNA and protein was observed in all lymphoma lines analyzed, and immunofluorescence confocal microscopy localized the protein to the perinuclear region. Analysis of a T cell hybridoma revealed prominent colocalization of Tctex-1 and Fyn at the cleavage furrow and mitotic spindles in cells undergoing cytokinesis. Our results provide a unique insight into a mechanism by which Tctex-1 might mediate specific recruitment of Fyn to the dynein complex in lymphocytes, which may be a critical event in mediating the previously defined role of Fyn in cytokinesis.

Animals↗
PubMed1995

The chaperonin containing t-complex polypeptide 1 (TCP-1). Multisubunit machinery assisting in protein folding and assembly in the eukaryotic cytosol.

Many proteins in the cell require assistance from molecular chaperones at stages in their life cycles in order to attain correctly folded states and functional conformations during protein synthesis or during recovery from denatured states. A recently discovered molecular chaperone, which is abundant in the eukaryotic cytosol and is called the chaperonin containing TCP-1 (CCT), has been shown to assist the folding of some proteins in cytosol. This chaperone is a member of the chaperonin family which includes GroEL, 60-kDa heat shock protein (Hsp60), Rubisco subunit binding protein (RBP) and thermophilic factor 55 (TF55), but is distinct from the other members in several respects. Presently the most intriguing feature is the hetero-oligomeric nature of the CCT; at least eight subunit species which are encoded by independent and highly diverged genes are known. These genes are calculated to have diverged around the starting point of the eukaryotic lineage and they are maintained in all eukaryotes investigated, suggesting a specific function for each subunit species. The amino acid sequences of these subunits share approximately 30% identity and have some highly conserved motifs probably responsible for ATPase function, suggesting this function is common to all subunits. Thus, each subunit is thought to have both specific and common functions. These observations, in conjunction with biochemical and genetic analysis, suggest that CCT functions as a very complex machinery for protein folding in the eukaryotic cell and that its chaperone activity may be essential for the folding and assembly of various newly synthesized polypeptides. This complex behaviour of CCT may have evolved to cope with the folding and assembly of certain highly evolved proteins in eukaryotic cells.

Amino Acid Sequence↗
PubMed1993

Eukaryotic cytosolic chaperonin contains t-complex polypeptide 1 and seven related subunits.

We have characterized the cytosolic chaperonin from both rabbit reticulocyte lysate and bovine testis. The heteromeric complex contains eight subunits. Partial amino acid sequence data reveal that one of these is t-complex polypeptide 1 (TCP-1), while the other seven are TCP-1-related polypeptides, implicating the existence of a multigene family of TCP-1 homologues. We provide evidence that TCP-1 ring complex from bovine testis can facilitate the folding of both actin and tubulin, although, as in the case of chaperonin from reticulocyte lysate, two cofactors are required for the generation of properly folded tubulin. An additional molecule of TCP-1 may associate with the chaperonin depending on the purification procedure used. We propose that a highly conserved region in these polypeptides and in other chaperonins of the cpn60 chaperone family participates in ATP binding.

Actins↗
PubMed1994

A novel cochaperonin that modulates the ATPase activity of cytoplasmic chaperonin.

The folding of alpha- and beta-tubulin requires three proteins: the heteromeric TCP-1-containing cytoplasmic chaperonin and two additional protein cofactors (A and B). We show that these cofactors participate in the folding process and do not merely trigger release, since in the presence of Mg-ATP alone, alpha- and beta-tubulin target proteins are discharged from cytoplasmic chaperonin in a nonnative form. Like the prokaryotic cochaperonin GroES, which interacts with the prototypical Escherichia coli chaperonin GroEL and regulates its ATPase activity, cofactor A modulates the ATPase activity of its cognate chaperonin. However, the sequence of cofactor A derived from a cloned cDNA defines a 13-kD polypeptide with no significant homology to other known proteins. Moreover, while GroES functions as a heptameric ring, cofactor A behaves as a dimer. Thus, cofactor A is a novel cochaperonin that is structurally unrelated to GroES.

Adenosine Triphosphatases↗
PubMed2025

CCT8 drives colorectal cancer progression via the RPL4-MDM2-p53 axis and immune modulation.

PURPOSE: Colorectal cancer (CRC) ranks high in global mortality, emphasizing the need for effective interventions. The aim of the research is to elucidate the oncogenic role of CCT8 in CRC and its interaction with RPL4 in the RPL4-MDM2-p53 axis. METHODS: TIMER 2.0, TCGA, and GTEx databases were used to analyze CCT8 expression patterns in CRC. Immunohistochemistry was performed to examine CCT8 distribution in CRC tissues and adjacent non-tumor tissues. Functional assays, including CCK-8, transwell, wound-healing, and flow cytometry, were conducted using DLD-1 and HCT116 cell lines to assess the effects of CCT8 on cell proliferation, migration, invasion, and apoptosis. Gene set enrichment analysis, protein-protein interaction network analysis, and co-immunoprecipitation were performed to explore the interaction between CCT8 and RPL4 and their role in the RPL4-MDM2-p53 pathway. Additionally, gene set variation analysis was applied to investigate the relationship between CCT8/RPL4 expression and immune infiltration patterns in CRC. RESULTS: CCT8 was significantly upregulated in CRC and associated with tumor progression. Mechanistically, CCT8 potentially synergizes with RPL4 concluded from their positive correlation and similar immune infiltration patterns, influencing the RPL4-MDM2-p53 axis and contributing to p53 ubiquitination and degradation. CONCLUSION: These findings underscore the oncogenic significance of CCT8 in CRC and shed light on its molecular mechanisms, paving the way for potential therapeutic applications.

Humans↗
PubMed2026

CCT2 defines a highly cisplatin-resistant and poor-prognosis subtype of lung adenocarcinoma.

Cisplatin-based chemotherapy is a standard treatment for lung adenocarcinoma (LUAD), yet acquired cisplatin resistance remains a marked cause of treatment failure. The molecular mechanisms driving cisplatin resistance in LUAD have not been fully elucidated. The present study integrated bulk transcriptomic data, genomic mutation profiles and single-cell RNA sequencing data to systematically investigate cisplatin resistance in LUAD. Resistance-associated genes were identified through differential expression, survival analysis and database integration. Unsupervised clustering was used to define cisplatin resistance-associated subtypes. Functional characteristics were explored using pathway enrichment, immune infiltration, tumor mutation burden and weighted gene co-expression network analysis. A machine learning framework incorporating 101 algorithms was applied to identify key genes and construct a prognostic model. Single-cell analyses and in vitro experiments were performed to validate the biological role of the core gene. Molecular docking and molecular dynamics simulations were conducted to identify potential therapeutic compounds. A total of two molecular subtypes with distinct cisplatin resistance levels and prognostic outcomes were identified. The high-resistance subtype exhibited enhanced cell cycle activity, DNA repair signaling and immune heterogeneity. Machine learning analysis revealed a five-gene signature, with chaperonin-containing TCP1 subunit 2 (CCT2) emerging as a key regulator of cisplatin resistance. Single-cell analyses showed that CCT2 was predominantly enriched in resistant epithelial cell subpopulations. Functional experiments demonstrated that CCT2 knockdown significantly inhibited cell proliferation and enhanced cisplatin sensitivity in LUAD cell lines. A number of candidate compounds targeting CCT2 exhibited stable binding in silico. The present findings identified CCT2 as a key mediator of cisplatin resistance in LUAD and provided potential therapeutic strategies to overcome chemotherapy resistance.

chaperonin-containing TCP-1 subunit 2↗
PubMed2001

The 'sequential allosteric ring' mechanism in the eukaryotic chaperonin-assisted folding of actin and tubulin.

Folding to completion of actin and tubulin in the eukaryotic cytosol requires their interaction with cytosolic chaperonin CCT [chaperonin containing tailless complex polypeptide 1 (TCP-1)]. Three-dimensional reconstructions of nucleotide-free CCT complexed to either actin or tubulin show that CCT stabilizes both cytoskeletal proteins in open and quasi-folded conformations mediated through interactions that are both subunit specific and geometry dependent. Here we find that upon ATP binding, mimicked by the non-hydrolysable analog AMP-PNP (5'-adenylyl-imido-diphosphate), to both CCT-alpha-actin and CCT- beta-tubulin complexes, the chaperonin component undergoes concerted movements of the apical domains, resulting in the cavity being closed off by the helical protrusions of the eight apical domains. However, in contrast to the GroE system, generation of this closed state does not induce the release of the substrate into the chaperonin cavity, and both cytoskeletal proteins remain bound to the chaperonin apical domains. Docking of the AMP-PNP-CCT-bound conformations of alpha-actin and beta-tubulin to their respective native atomic structures suggests that both proteins have progressed towards their native states.

3T3 Cells↗
PubMed1995

Cloning and sequencing of the chaperonin-encoding Cctd gene from Fugu rubripes rubripes.

CCT, a chaperonin containing t-complex polypeptide 1 (TCP-1), is a cytosolic molecular chaperone involved in the folding of proteins. We have isolated the Cctd gene from a Fugu rubripes rubripes (Frr) genomic library using a rat Ccta cDNA as a probe, and cloned its cDNA by reverse transcription-polymerase chain reaction (RT-PCR) using a pair of oligodeoxyribonucleotides corresponding to the 5' and 3' non-coding regions of Frr Cctd. Cctd spans a region of 4.7 kb and consists of at least 13 exons with small introns of about 144 bp on average. The Cctd cDNA sequence revealed a deduced polypeptide of 536 amino acids sharing a high degree of homology with that of the mouse Cctd cDNA (88%). Cctd is present as a single-copy gene, as shown by genomic Southern blot analysis, and can be used for evolutionary and classification analyses of Fugu species.

Amino Acid Sequence↗
PubMed1994

Reversible interaction of beta-actin along the channel of the TCP-1 cytoplasmic chaperonin.

The cytoplasm of eukaryotes contains a heteromeric toroidal chaperonin assembled from the t-complex TCP-1 and several other related polypeptides. The structure of the TCP-1 cytoplasmic chaperonin and that of the binary complex formed between this chaperonin and unfolded beta-actin have been studied using electron microscopy and image processing techniques. Two-dimensional averaging of front views reveals a circular stain-excluding mass surrounding a central stain-penetrating region in which the stain is excluded upon actin binding. Sections of a three-dimensional reconstruction of the chaperonin show that the inner core is an empty channel that becomes filled upon binary complex formation with unfolded beta-actin. Upon incubation with Mg-ATP, the beta-actin:chaperonin complex discharges the actin such that the chaperonin central cavity reappears. Side views from different forms of TCP-1 reveals that upon Mg-ATP binding, the cytoplasmic chaperonin undergoes a structural rearrangement that is confirmed using a new classification method.

Actins↗
PubMed1992

Function in protein folding of TRiC, a cytosolic ring complex containing TCP-1 and structurally related subunits.

T-complex polypeptide 1 (TCP-1) was analyzed as a potential chaperonin (GroEL/Hsp60) equivalent of the eukaryotic cytosol. We found TCP-1 to be part of a hetero-oligomeric 970 kDa complex containing several structurally related subunits of 52-65 kDa. These members of a new protein family are assembled into a TCP-1 ring complex (TRiC) which resembles the GroEL double ring. The main function of TRiC appears to be in chaperoning monomeric protein folding: TRiC binds unfolded polypeptides, thereby preventing their aggregation, and mediates the ATP-dependent renaturation of unfolded firefly luciferase and tubulin. At least in vitro, TRiC appears to function independently of a small co-chaperonin protein such as GroES. Folding of luciferase is mediated by TRiC but not by GroEL/ES. This suggests that the range of substrate proteins interacting productively with TRiC may differ from that of GroEL. We propose that TRiC mediates the folding of cytosolic proteins by a mechanism distinct from that of the chaperonins in specific aspects.

Adenosine Triphosphatases↗
PubMed2000

RNA differential display of scarless wound healing in fetal rabbit indicates downregulation of a CCT chaperonin subunit and upregulation of a glycophorin-like gene transcript.

BACKGROUND/PURPOSE: Scars form as wounds heal in adult organisms. In addition to disrupting cosmetic appearance, scar tissue can cause significant morbidity, and even death if it blocks vital organ function. Previous work has established that fetal wounds, especially in early to midgestation, can heal without scarring. Because such inherent physiological mechanisms ultimately are under genetic control, a study was initiated to elucidate the differences in gene expression that produce scarless wound healing in the mammalian fetus but scarring in postnatal wounds. Reverse transcription polymerase chain reaction (RT-PCR) differential display (DD) was used to detect differentially expressed mRNA transcripts in a rabbit model of wound healing. METHODS: Adult and 21-day fetal full-thickness rabbit skin specimens from wounded and unwounded sites were harvested 12 hours postwounding. RNA extracted from the tissue was used as a template in DD reactions using anchoring and random primers to generate tissue-specific gene expression fingerprints. The over 2,000 resulting amplimers (gene transcripts) were screened for differential expression among the 4 types of specimens: fetal control (unwounded), fetal wound, adult control, and adult wound. Selected bands distinctly upregulated or downregulated in fetal wound lanes on the DD gels were excised, and the cDNA was extracted, reamplified, cloned into vectors, and sequenced. DD results were confirmed by limiting-dilution RT-PCR using sequence-specific primers. RESULTS: Differential display (DD) showed 22 amplimers that were significantly upregulated in all fetal wound samples as compared with little or no expression in fetal control, adult control, or adult wound tissues. Conversely, 5 transcripts were downregulated in the fetal wound specimens but highly expressed in the 3 comparison tissues. Reamplification of selected transcripts by PCR, followed by cloning and DNA sequencing, yielded 7 distinct sequences, each representing a gene expressed differently in fetal wound than in the other 3 tissues. A transcript that was downregulated in fetal wound showed very high sequence homology to part of the human gene for the eta subunit of the hetero-oligomeric particle CCT (the chaperonin containing T-complex polypeptide 1 or TCP-1). An upregulated amplimer showed significant DNA sequence homology to glycophorins A and B. One sequence was identified as 28S rRNA. The remaining 4 candidate sequences showed no significant homology to known genes, but 1 had high homology to expressed sequence tags of unknown function. CONCLUSIONS: With careful experimental design and proper controls and verifications, differential display of RNA expression is a potentially powerful method of finding genes that specifically regulate a particular physiological process such as fetal wound healing. No a priori knowledge of what genes might be involved, or why, is necessary. This study indicates that downregulation of a gene that codes for a chaperonin subunit and upregulation of several other genes may be involved in the striking scarless character of wound healing in the mammalian fetus. Results suggest the hypothesis that downregulation of the CCT chaperonin in fetal wound may inhibit the formation of myofibroblasts, a cell type that correlates highly with scarring in postnatal wound healing, by preventing the folding of sufficient alpha-smooth muscle actin to form the stress fibers characteristic of these cells.

Amino Acid Sequence↗
PubMed1994

Cloning and expression of a new mammalian chaperonin gene from a multipotent hematopoietic progenitor clone.

Molecular chaperones assist in the folding and assembly of proteins in cells. Although chaperonins have been shown in prokaryotes, mitochondria, and chloroplasts long-ago, a cytoplasmic heteromeric chaperonin complex was isolated only recently and found to contain at least five to six polypeptides, one of which was identified as the product of the T complex polypeptide-1 (TCP-1) gene. We have isolated and cloned a novel gene called A45 from a cDNA library constructed from poly (A)+ RNA of a multipotent hematopoietic progenitor clone. The A45 cDNA encodes a predicted polypeptide of M(r) 58,118 that exhibits 32% overall amino acid sequence identity to TCP-1 and contains the putative adenosine triphosphate-binding domain and two characteristic consensus regions that are conserved in all chaperonins. The A45 gene is expressed in hematopoietic precursors cells at a much higher level than in nonhematopoietic cells and tissues. We conclude that A45 represents a new member of the mammalian chaperonins that is involved in the folding and assembly of polypeptides.

Amino Acid Sequence↗
PubMed1995

Evolution of the chaperonin families (Hsp60, Hsp10 and Tcp-1) of proteins and the origin of eukaryotic cells.

The members of the 10 kDa and 60 kDa heat-shock chaperonin proteins (Hsp10 and Hsp60 or Cpn10 and Cpn60), which form an operon in bacteria, are present in all eubacteria and eukaryotic cell organelles such as mitochondria and chloroplasts. In archaebacteria and eukaryotic cell cytosol, no close homologues of Hsp10 or Hsp60 have been identified. However, these species (or cell compartments) contain the Tcp-1 family of proteins (distant homologues of Hsp60). Phylogenetic analysis based on global alignments of Hsp60 and Hsp10 sequences presented here provide some evidence regarding the evolution of mitochondria from a member of the alpha-subdivision of Gram-negative bacteria and chloroplasts from cyanobacterial species, respectively. This interference is strengthened by the presence of sequence signatures that are uniquely shared between Hsp60 homologues from alpha-purple bacteria and mitochondria on one hand, and the chloroplasts and cyanobacterial hsp60s on the other. Within the alpha-purple subdivision, species such as Rickettsia and Ehrlichia, which live intracellularly within eukaryotic cells, are indicated to be the closest relatives of mitochondrial homologues. In the Hsp60 evolutionary tree, rooted using the Tcp-1 homologue, the order of branching of the major groups was as follows: Gram-positive bacteria--cyanobacteria and chloroplasts--chlamydiae and spirochaetes--beta- and gamma-Gram-negative purple bacteria--alpha-purple bacteria--mitochondria. A similar branching order was observed independently in the Hsp10 tree. Multiple Hsp60 homologues, when present in a group of species, were found to be clustered together in the trees, indicating that they evolved by independent gene-duplication events. This review also considers in detail the evolutionary relationship between Hsp60 and Tcp-1 families of proteins based on two different models (viz. archaebacterial and chimeric) for the origin of eukaryotic cell nucleus. Some predictions of the chimeric model are also discussed.

Amino Acid Sequence↗
PubMed1994

A yeast TCP-1-like protein is required for actin function in vivo.

We previously identified the ANC2 gene in a screen for mutations that enhance the defects caused by yeast actin mutations. Here we report that ANC2 is an essential gene that encodes a member of the TCP-1 family. TCP-1-related proteins are subunits of cytosolic heteromeric protein complexes referred to as chaperonins. These complexes can bind to newly synthesized actin and tubulin in vitro and can convert these proteins into an assembly-competent state. We show that anc2-1 mutants contain abnormal and disorganized actin structures, are defective in cellular morphogenesis, and are hypersensitive to the microtubule inhibitor benomyl. Furthermore, overexpression of wild-type Anc2p ameliorates defects in actin organization and cell growth caused by actin overproduction. Mutations in BIN2 and BIN3, two other genes that encode TCP-1-like proteins, also enhance the phenotypes of actin mutants. Taken together, these findings demonstrate that TCP-1-like proteins are required for actin and tubulin function in vivo.

Actins↗