Cytosolic Chaperonin Containing TCP-1 Research Articles

Maize tubulin folding cofactor B is required for cell division and cell growth through modulating microtubule homeostasis.

Tubulin folding cofactors (TFCs) are required for tubulin folding, α/β tubulin heterodimer formation, and microtubule (MT) dynamics in yeast and mammals. However, the functions of their plant counterparts remain to be characterized. We identified a natural maize crumpled kernel mutant, crk2, which exhibits reductions in endosperm cell number and size, as well as embryo/seedling lethality. Map-based cloning and functional complementation confirmed that ZmTFCB is causal for the mutation. ZmTFCB is targeted mainly to the cytosol. It facilitates α-tubulin folding and heterodimer formation through sequential interactions with the cytosolic chaperonin-containing TCP-1 ε subunit ZmCCT5 and ZmTFCE, thus affecting the organization of both the spindle and phragmoplast MT array and the cortical MT polymerization and array formation, which consequently mediated cell division and cell growth. We detected a physical association between ZmTFCB and the maize MT plus-end binding protein END-BINDING1 (ZmEB1), indicating that ZmTFCB1 may modulate MT dynamics by sequestering ZmEB1. Our data demonstrate that ZmTFCB is required for cell division and cell growth through modulating MT homeostasis, an evolutionarily conserved machinery with some species-specific divergence.

The prefoldin complex stabilizes the von Hippel-Lindau protein against aggregation and degradation.

Loss of von Hippel-Lindau protein pVHL function promotes VHL diseases, including sporadic and inherited clear cell Renal Cell Carcinoma (ccRCC). Mechanisms controlling pVHL function and regulation, including folding and stability, remain elusive. Here, we have identified the conserved cochaperone prefoldin complex in a screen for pVHL interactors. The prefoldin complex delivers non-native proteins to the chaperonin T-complex-protein-1-ring (TRiC) or Cytosolic Chaperonin containing TCP-1 (CCT) to assist folding of newly synthesized polypeptides. The pVHL-prefoldin interaction was confirmed in human cells and prefoldin knock-down reduced pVHL expression levels. Furthermore, when pVHL was expressed in Schizosaccharomyces pombe, all prefoldin mutants promoted its aggregation. We mapped the interaction of prefoldin with pVHL at the exon2-exon3 junction encoded region. Low levels of the PFDN3 prefoldin subunit were associated with poor survival in ccRCC patients harboring VHL mutations. Our results link the prefoldin complex with pVHL folding and this may impact VHL diseases progression.

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

Intestinal epithelial cells have unique apical membrane structures, known as microvilli, that contain bundles of actin microfilaments. In this study, we report that Caenorhabditis elegans cytosolic chaperonin containing TCP-1 (CCT) is essential for proper formation of microvilli in intestinal cells. In intestinal cells of cct-5(RNAi) animals, a substantial amount of actin is lost from the apical area, forming large aggregates in the cytoplasm, and the apical membrane is deformed into abnormal, bubble-like structures. The length of the intestinal microvilli is decreased in these animals. However, the overall actin protein levels remain relatively unchanged when CCT is depleted. We also found that CCT depletion causes a reduction in the tubulin levels and disorganization of the microtubule network. In contrast, the stability and localization of intermediate filament protein IFB-2, which forms a dense filamentous network underneath the apical surface, appears to be superficially normal in CCT-deficient cells, suggesting substrate specificity of CCT in the folding of filamentous cytoskeletons in vivo. Our findings demonstrate physiological functions of CCT in epithelial cell morphogenesis using whole animals.

New insights into the functions and localization of nuclear CCT protein complex in K562 leukemia cells

The eukaryotic cytosolic chaperonin containing TCP-1 (CCT) plays an important role in maintaining cellular homeostasis by assisting the folding of many proteins and is also well known for the critical roles in disease. However, the functions of CCT complex have not been established globally, especially when translocating into nuclear. The purpose of this study is to explore the function of CCT in nuclear and present a strategy in clinical proteomics studies. Blue native polyacrylamide gel electrophoresis (BN-PAGE) combined with mass spectrometry was applied to separate and identify CCT protein complexes. We isolated the CCT complex in K562 nucleus and identified a novel CCT complex containing 40 protein components involved in protein folding, RNA processing, apoptosis, and cell metabolism. The interactions between four candidate proteins and CCT were confirmed by immunoblotting. Computational biological analyses and independent biochemical assays validated the overall quality of interactions. Our results support clues that CCT might play an unexpected role in various biological processes including RNA processing. And we envision future applications for this system searching for new clues of CCT in disease and readily be applied to the clinic.

Exploring the Role of Topological Frustration in Actin Refolding with Molecular Simulations

Actin plays crucial roles in the life of the cell while being notorious for its inability to reach a functional conformation without the help of assistant proteins. In eukaryotes, for example, the cytosolic chaperonin containing TCP-1 (CCT) and prefoldin (PFD) are required for actin folding assistance and prevention of protein aggregation in the crowded cellular environment. The folding of non-native actin is known to occur in a number of steps, but the reasons underlying its folding difficulty are unknown. Because a full, atomistic-level, investigation of the kinetics and thermodynamics of folding of such a large molecule is beyond computational reach, we focused our investigation on the role of topological frustration on the folding of actin. Namely, we studied the (re)folding of actin using simulations of a variant self-organized polymer model (SOP-DH) starting from a stretched state, leading to results that correlate well with experimentally driven conclusions and allowing us to make a number of testable predictions. Primarily, our simulations reveal that the successful refolding of the C-terminus end of actin occurs through a zipping process in which the α-helices wind up turn by turn upon formation of their native tertiary contacts. In turn, an early formation of the helical structure in this region of the chain has deleterious effects for actin's refolding fitness. Moreover, the C-terminus refolding is a very rare event in our simulations, in agreement with the large activation barrier predicted on the basis of experimental studies of actin unfolding in EDTA. We also discovered that subdomain 4 has a low refolding probability, which can help explain why many of the non-native actin target binding sites for CCT and PFD are located within this subdomain.

Association of the Influenza Virus RNA Polymerase Subunit PB2 with the Host Chaperonin CCT

The RNA polymerase of influenza A virus is a host range determinant and virulence factor. In particular, the PB2 subunit of the RNA polymerase has been implicated as a crucial factor that affects cell tropism as well as virulence in animal models. These findings suggest that host factors associating with the PB2 protein may play an important role during viral replication. In order to identify host factors that associate with the PB2 protein, we purified recombinant PB2 from transiently transfected mammalian cells and identified copurifying host proteins by mass spectrometry. We found that the PB2 protein associates with the cytosolic chaperonin containing TCP-1 (CCT), stress-induced phosphoprotein 1 (STIP1), FK506 binding protein 5 (FKBP5), alpha- and beta-tubulin, Hsp60, and mitochondrial protein p32. Some of these binding partners associate with each other, suggesting that PB2 might interact with these proteins in multimeric complexes. More detailed analysis of the interaction of the PB2 protein with CCT revealed that PB2 associates with CCT as a monomer and that the CCT binding site is located in a central region of the PB2 protein. PB2 proteins from various influenza virus subtypes and origins can associate with CCT. Silencing of CCT resulted in reduced viral replication and reduced PB2 protein and viral RNA accumulation in a ribonucleoprotein reconstitution assay, suggesting an important function for CCT during the influenza virus life cycle. We propose that CCT might be acting as a chaperone for PB2 to aid its folding and possibly its incorporation into the trimeric RNA polymerase complex.

The interaction network of the chaperonin CCT

The eukaryotic cytosolic chaperonin containing TCP-1 (CCT) has an important function in maintaining cellular homoeostasis by assisting the folding of many proteins, including the cytoskeletal components actin and tubulin. Yet the nature of the proteins and cellular pathways dependent on CCT function has not been established globally. Here, we use proteomic and genomic approaches to define CCT interaction networks involving 136 proteins/genes that include links to the nuclear pore complex, chromatin remodelling, and protein degradation. Our study also identifies a third eukaryotic cytoskeletal system connected with CCT: the septin ring complex, which is essential for cytokinesis. CCT interactions with septins are ATP dependent, and disrupting the function of the chaperonin in yeast leads to loss of CCT-septin interaction and aberrant septin ring assembly. Our results therefore provide a rich framework for understanding the function of CCT in several essential cellular processes, including epigenetics and cell division.

Modeling of possible subunit arrangements in the eukaryotic chaperonin TRiC

The eukaryotic cytosolic chaperonin TRiC (TCP-1 Ring Complex), also known as CCT (Cytosolic Chaperonin containing TCP-1), is a hetero-oligomeric complex consisting of two back-to-back rings of eight different subunits each. The general architecture of the complex has been determined, but the arrangement of the subunits within the complex remains an open question. By assuming that the subunits have a defined arrangement within each ring, we constructed a simple model of TRiC that analyzes the possible arrangements of individual subunits in the complex. By applying the model to existing data, we find that there are only four subunit arrangements consistent with previous observations. Our analysis provides a framework for the interpretation and design of experiments to elucidate the quaternary structure of TRiC/CCT. This in turn will aid in the understanding of substrate binding and allosteric properties of this chaperonin.

Type II chaperonns, prefoldin, and the tubulin-specific chaperones

Publisher Summary This chapter describes several experiments that give a qualitative feel for how Type II chaperonins might facilitate protein folding. Imaging of CCT/actin complexes and experiments on actin and tubulin folding in vitro suggest that target proteins bind to chaperonin in a surprisingly native-like state, probably consisting of only one or a few kinetically trapped folding intermediates. Chaperonins are a specialized but ubiquitous group of molecular chaperones that are characterized by a multisubunit ring structure. Chaperonins exist in all kingdoms of life, in all cases using cycles of ATP binding and hydrolysis to generate allosteric changes that promote the correct folding of bound target protein. Type I chaperonins are present in prokaryotes (exemplified by GroEL/GroES), in mitochondria (exemplified by Hsp60/hspl0), and in chloroplasts (exemplified by Rubisco subunit binding protein), while Type II chaperonins include the chaperonin present in archaea (exemplified by the thermosome) and the chaperonin present in the cytosol of eukaryotes (referred to here as CCT, for cytosolic chaperonin-containing TCP-1, but which is also termed c-cpn or TriC). Indeed, chaperones in general appear to function by releasing and rebinding target proteins, allowing a kinetic partition of folding intermediates among the chaperone proteins present in the cell, and leading to their folding, subcellular sorting, or degradation.

The cytosolic class II chaperonin CCT recognizes delineated hydrophobic sequences in its target proteins.

The nonhomologous proteins actin and alpha- and beta-tubulin need the assistance of the cytosolic chaperonin containing TCP-1 (CCT) to reach their correct native state, and their folding requires a transient binary complex formation with CCT. We show that separate or combined deletion of three delineated hydrophobic sequences in actin disturbs the interaction with CCT. These sites are situated between residues 125-179, 244-285, and 340-375. Also, alpha- and beta-tubulin contain at least one recognition region, and intriguingly, it has a similar distribution of hydrophobic residues as region 244-285 in actin. Internal deletion of the sites in actin favor a model for cooperative binding of target proteins to CCT. Peptide mimetics, representing the binding regions, inhibit target polypeptide binding to CCT, suggesting that actin and tubulin contact similar CCT subunits. In addition, we show that actin recognition by class II chaperonins is different from that by class I.

The Chaperone Cofactor Hop/p60 Interacts with the Cytosolic Chaperonin-containing TCP-1 and Affects Its Nucleotide Exchange and Protein Folding Activities

The folding of protein structures often requires the presence of molecular chaperones and/or chaperonin complexes. We here investigated the inhibitory effects of the chaperone cofactors Hop/p60 and Hap46. By coimmunoprecipitation, we observed a direct interaction of the eukaryotic chaperonin-containing TCP-1 (CCT) purified from rabbit reticulocyte lysate with Hop/p60. By contrast, Hap46 was not coprecipitated. Binding of Hop/p60 to CCT is dependent on the presence of ATP or ADP and occurs through carboxyl-terminal sequences of Hop/p60. Hop/p60 significantly stimulates nucleotide exchange on CCT but not its ATPase activity, while Hap46 has no effects. We used denatured firefly luciferase as a model protein and found decreased binding to CCT in the presence of Hop/p60 and ATP. This coincides with the inhibitory effect of Hop/p60 on luciferase reactivation in an assay using purified CCT in combination with hsc70 and hsp40. We also observed that an antibody directed against one of the subunits of CCT efficiently inhibits refolding in a system which depends on crude reticulocyte lysate.

Developmental and light-dependent changes of the cytosolic chaperonin containing TCP-1 (CCT) subunits in maize seedlings, and the localization in coleoptiles.

The cytosolic chaperonin containing TCP-1 (CCT) is known to keep fold cytoskeletal proteins and is involved in the proper organization of the cytoskeleton. These studies are based on the assumption that growth responses linked to structural rearrangement of the plant cytoskeleton include the action of CCT and the need for newly synthesized tubulin. The presence of the alpha- and epsilon-subunits of CCT was investigated in soluble fractions of protein extracts from maize mesocotyls and coleoptiles at distinct growth stages. The CCT-subunits, tubulins and actin decreased in the coleoptile in response to far-red light. In addition, independent from light treatment, the amount of CCT epsilon abundance declined with age in coleoptiles and mesocotyls between 2 and 4.5 days after sowing. In contrast to CCT epsilon, no significant light regulation of CCT alpha was found in the mesocotyl. In two day old, light-grown rapidly elongating coleoptiles part of the CCT alpha subunit and the bulk of actin and tubulin was found shifted into fractions of high molecular weight complexes when compared to slowly elongating, dark grown coleoptiles. In 4.5 day old, etiolated and elongating coleoptiles, part of both CCT-subunits and cytoskeleton proteins were found in fractions of high molecular weight. A complete disappearance of these polypeptides was observed in old far-red irradiated growth-arrested coleoptiles. CCT epsilon was found to be colocalized to microtubular structures and to the nucleus. We conclude from our data that abundance of CCT-subunits in soluble extracts is dependent on age and light treatment, but independent from the growth stage of mesocotyl and coleoptile.

Tissue-specific subunit of the mouse cytosolic chaperonin-containing TCP-1

We have cloned a novel Tcp-1-related mouse testis cDNA encoding a polypeptide of 531 amino acids which shares 81.2% identity with the ζ subunit of the mouse cytosolic chaperonin-containing TCP-1 (CCT). Immunoblot analysis of mouse testis CCT subunits separated by 2-dimensional gel electrophoresis indicates that this novel gene, Cctz-2, encodes a CCT subunit of M r 57 000 and pI 7.1. Cctz-2 mRNA is detected only in testis whereas the other Cctz gene, Cctz-1, is expressed in all tissues investigated. The CCTζ-2 subunit may have specific functions in the folding of testicular proteins and for interactions with testicular molecular chaperones.