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PubMed1995

Conformational cycle of the archaeosome, a TCP1-like chaperonin from Sulfolobus shibatae.

The major heat shock proteins in the archaeon Sulfolobus shibatae are similar to the cytosolic eukaryotic chaperonin and form an 18-subunit bitoroidal complex. Two sequence-related subunits constitute a functional complex, named the archaeosome. The archaeosome exists in two distinct conformational states that are part of chaperonin functional cycle. The closed archaeosome complex binds ATP and forms an open complex. Upon ATP hydrolysis, the open complex dissociates into subunits. Free subunits reassemble into a two-ring structure. The equilibrium between the complexes and free subunits is affected by ATP and temperature. Denatured proteins associate with both conformational states as well as with free subunits that form an intermediate complex. These unexpected observations suggest a new mechanism of archaeosome-mediated thermotolerance and protein folding.

Adenosine Triphosphate↗
PubMed1994

The essential yeast Tcp1 protein affects actin and microtubules.

Previously, we showed that the yeast Saccharomyces cerevisiae cold-sensitive mutation tcp1-1 confers growth arrest concomitant with cytoskeletal disorganization and disruption of microtubule-mediated processes. We have identified two new recessive mutations, tcp1-2 and tcp1-3, that confer heat- and cold-sensitive growth. Cells carrying tcp1 alleles were analyzed after exposure to the appropriate restrictive temperatures by cell viability tests, differential contrast microscopy, fluorescent, and immunofluorescent microscopy of DNA, tubulin, and actin and by determining the DNA content per cell. All three mutations conferred unique phenotypes indicative of cytoskeletal dysfunction. A causal relationship between loss of Tcp1p function and the development of cytoskeletal abnormalities was established by double mutant analyses. Novel phenotypes indicative of allele-specific genetic interactions were observed when tcp1-1 was combined in the same strain with tub1-1, tub2-402, act1-1, and act1-4, but not with other tubulin or actin mutations or with mutations in other genes affecting the cytoskeleton. Also, overproduction of wild-type Tcp1p partially suppressed growth defects conferred by act1-1 and act1-4. Furthermore, Tcp1p was localized to the cytoplasm and the cell cortex. Based on our results, we propose that Tcp1p is required for normal development and function of actin and microtubules either through direct or indirect interaction with the major cytoskeletal components.

Actins↗
PubMed1993

Protein folding in the cell: functions of two families of molecular chaperone, hsp 60 and TF55-TCP1.

Two families of molecular chaperone, the hsp 60-GroEL family and the TF55-TCP1 family, have been discovered in evolutionarily related cellular compartments. A member of one of these families, hsp 60, has been shown to play a global role in polypeptide chain folding in mitochondria. We review here studies of both hsp 60 and other family members, discussing their essential physiological roles and mechanism of action.

Animals↗
PubMed1993

Identification of chaperonin particles in mammalian brain cytosol and of T-complex polypeptide 1 as one of their components.

An approximately 950-kDa heteromeric particle was purified from guinea-pig and rat brain by sucrose gradient fractionation of post-mitochondrial supernatants. Further purification, by affinity chromatography on ATP-Sepharose and anion exchange FPLC on MonoQ, yielded a particle with typical chaperonin ultrastructure. One of the component polypeptides was recognized by a monoclonal antibody to murine T-complex polypeptide 1. Brain cytosolic chaperonin particles formed a binary complex with unfolded tubulin subunits. The polypeptide compositions of the cytosolic chaperonin particles appeared very similar between brain and testicular tissues of the same animal, but differed subtly between the guinea-pig and rat.

Animals↗
PubMed1994

TRiC-P5, a novel TCP1-related protein, is localized in the cytoplasm and in the nuclear matrix.

We have recently reported the cloning of a novel protein, TRiC-P5, with significant homology with protein 1 of the t-complex (TCP1). In the present study, the cellular localization of TRiC-P5 in Raji cells has been determined using an antiserum raised against a 18.5 kDa fusion protein. Results from cell fractionation and immunoblot studies indicate that TRiC-P5 is mainly localized in the cytoplasm. In addition, a significant part of TRiC-P5 is also found in the nucleus where it is attached to the nuclear matrix, a complex filament network involved in essential cellular functions such as DNA replication, and RNA transcription and maturation. Immunofluorescence experiments using the anti-TRiC-P5 antibodies confirm these results. We also provide evidence that, in the cytoplasm, TRiC-P5 is part of a large protein complex, most probably the TCP1-ring complex (TRiC), a hetero-oligomeric ring complex that plays a role of molecular chaperone in the folding of actin and tubulin.

Chaperonin Containing TCP-1↗
PubMed1995

Identification of intrinsic dimer and overexpressed monomeric forms of gamma-tubulin in Sf9 cells infected with baculovirus containing the Chlamydomonas gamma-tubulin sequence.

A new member of the tubulin superfamily, gamma-tubulin, is localized at microtubule-organizing centers (MTOCs) in a variety of organisms. Chlamydomonas cDNA coding for the full-length sequence of gamma-tubulin was expressed in insect ovarian Sf9 cells using the baculovirus expression system. Approximately half of the induced 52 kDa gamma-tubulin was recovered in the supernatant after centrifugation of Sf9 cell lysates at 18,000 g for 15 minutes. When the cell supernatant was analyzed by FPLC on a Superdex 200 sizing column, Chlamydomonas gamma-tubulin separated into two major peaks. The lagging peak contained a monomeric form of gamma-tubulin with a sedimentation coefficient of 2.5 S, which interacted with the Superdex column in a salt-dependent manner. The leading peak, with an apparent molecular mass of 900 kDa, corresponded to a molecular chaperonin complex, and TCP1 chaperonin released folded gamma-tubulin polypeptide from the complex in the presence of MgATP. The released gamma-tubulin monomers were capable of binding to microtubules in vitro and biochemical quantities of active monomers were further purified using a combination of size-exclusion and ion-exchange column chromatography. The endogenous Sf9 cell gamma-tubulin migrated faster than Chlamydomonas gamma-tubulin with an apparent molecular mass of 49 kDa on gels. Analyses on gel filtration and sucrose density gradient centrifugation showed that, while overexpressed Chlamydomonas gamma-tubulin was present in a monomeric form, endogenous gamma-tubulin from Sf9 and HeLa cells exists as a dimer. These results may suggest the possibility that gamma-tubulin could form a heterodimer with hitherto unknown molecule(s).

Amino Acid Sequence↗
PubMed1995

Cytoplasmic chaperonin complexes enter neurites developing in vitro and differ in subunit composition within single cells.

Chaperonins containing t-complex polypeptide-1 (CCT) are cytosolic molecular chaperone particles implicated especially in the biogenesis of cytoskeletal proteins by promoting the correct folding of the major ubiquitous cytoskeletal components, tubulin and actin. We have purified cytosolic chaperonins from the ND7/23 cell line, determined their subunit composition and examined changes in the intracellular locations of their components during differentiation of ND7/23 cells to a neuronal phenotype by using immunocytochemistry and immunoblots. Chaperonins containing the CCT alpha (TCP1) subunit enter neuritic processes and are particularly noticeable at the leading edge of growth cone-like structures where they co-localise with actin. Chaperonins containing three other components (CCT beta, epsilon and gamma), however, remain predominantly restricted to perikaryal cytoplasm. These findings suggest a heterogeneous population of chaperonin particles within single differentiated ND7/23 cells and this may reflect specialisation of chaperonin function in different cytoplasmic compartments of a neurone. Further, since ribosomes do not enter neurites while CCT alpha-containing chaperonins do, the latter may play roles, subsequent to translation, which influence cytoskeletal elaboration during neuritogenesis.

Actins↗
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

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↗
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↗
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↗