Nucleotide sequence of a cDNA encoding a Trypanosoma cruzi acidic ribosomal P1 type protein.
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Amino Acid Sequence↗
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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↗
Cucumber T-complex protein. Molecular cloning, bacterial expression and characterization within a 22-S cytosolic complex in cotyledons and hypocotyls.
T-complex protein (TCP) found in mammalian cells and yeast has been proposed as cytosolic folding machinery. We report here the cloning and initial characterization of a plant TCP cDNA. CSTCP-1 cDNA prepared from mRNA of cotyledons of germinating cucumber seeds encodes a polypeptide composed of 535 amino acid residues. The 59157-Da protein exhibits only 28% identity to both TCP-1p from yeast or and its homolog in Arabidopsis thaliana. Antibodies raised against the bacterially expressed plant protein were used to analyze the intracellular localization of TCP in two different plant tissues: fat-degrading non-dividing cotyledons and meristematic hypocotyls during seed germination. Cell fractionations included differential centrifugation and sedimentation of large complexes at 23000O x g for 4h. The latter fraction was further fractionated by sedimentation velocity centrifugation. This enrichment was required to detect by Western blotting cytosolic 59-kDa species as constituents of 22-S particles. From hypocotyls, a preparation of T-complex was obtained which consisted almost exclusively of proteins in the molecular range of 57-62 kDa. Likewise, the radioactive Cucumis sativus TCP-1 synthesized from CSTCP-1 mRNA in vitro using reticulocyte lysate was shown to migrate as a 61-kDa species.
Amino Acid Sequence↗
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↗
Principles of chaperone-assisted protein folding: differences between in vitro and in vivo mechanisms.
Molecular chaperones in the eukaryotic cytosol were shown to interact differently with chemically denatured proteins and their newly translated counterparts. During refolding from denaturant, actin partitioned freely between 70-kilodalton heat shock protein, the bulk cytosol, and the chaperonin TCP1-ring complex. In contrast, during cell-free translation, the chaperones were recruited to the elongating polypeptide and protected it from exposure to the bulk cytosol during folding. Posttranslational cycling between chaperone-bound and free states was observed with subunits of oligomeric proteins and with aberrant polypeptides; this cycling allowed the subunits to assemble and the aberrant polypeptides to be degraded. Thus, folding, oligomerization, and degradation are linked hierarchically to ensure the correct fate of newly synthesized polypeptides.
Actins↗
The two human organic cation transporter genes SLC22A1 and SLC22A2 are located on chromosome 6q26.
Polyspecific transporters for organic cations (OCT) belong to a new protein family which also include organic anion transporters. The first human transporters from this family (OCT1, OCT2) have been recently cloned. They translocate small cations like tetraethylammonium, choline and monoamine neurotransmitters and are involved in hepatic and renal cation excretion, respectively. We have localized the OCT1 and OCT2 genes (SLC22A1, SLC22A2) on chromosome 6q26.
Carrier Proteins↗
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↗
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↗
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↗
gamma-Tubulin in mammalian cells: the centrosomal and the cytosolic forms.
The centrosome is one of the cellular organelles for which the mechanism by which it operates still remains to be unlavelled. The finding of the association with the centrosome of gamma-tubulin, a protein which belongs to the tubulin superfamily, has provided a long sought after biochemical tool with which to address centrosome function. We have generated a specific anti-gamma-tubulin polyclonal antibody to study the biochemical properties and the cellular distribution of the human lymphoblastic gamma-tubulin. Using cell fractionation and mass isolation of centrosomes, we observed that in contrast to the figures suggested by immunofluorescence, a minimum figure of 80% of total gamma-tubulin exists as a cytosolic form. The centrosomal form, for which at least half is not strongly associated with the centrosome, behaves in two-dimensional gel electrophoresis identically to the soluble form (as at least two spots of a pI of around 6). Post-embedding immunolocalization reveals that gamma-tubulin is distributed in the pericentriolar matrix but is also closely associated with centrioles. Using a combination of gel filtration, ion exchange chromatography, equilibrium sucrose gradient centrifugation and immunoprecipitation, we show that the major part of cytosolic gamma-tubulin might be involved in complexes heavier than the Tcp1 particle. We further demonstrate, by co-immunoprecipitation of gamma-tubulin and Tcp1 with either anti-Tcp1 or anti-gamma-tubulin antibodies, that a small part of gamma-tubulin participates in Tcp1-gamma-tubulin particles. Interestingly, the soluble form of gamma-tubulin co-purifies with taxol-stabilized microtubules and its association with microtubules resisted salt, ATP and GTP treatments. The existence of a centrosomal form and a large pool of cytosolic gamma-tubulin-containing complexes in somatic cells suggests that the overall gamma-tubulin cellular distribution does not seem to be as straightforward as it was drawn earlier.
Amino Acid Sequence↗
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↗
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↗
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↗
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↗
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↗
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↗
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↗
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↗