Human TRiC complex purified from HeLa cells contains all eight CCT subunits and is active in vitro

Knee, Kelly M.; Sergeeva, Oksana A.; King, Jonathan

doi:10.1007/s12192-012-0357-z

Cited by 27 publications

(31 citation statements)

References 57 publications

(82 reference statements)

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“…3). This elution was consistent with a 1-MDa complex and corresponding to the elution volume of both human TRiC and Mm-Cpn (39,41). The symmetry of the distribution and absence of a trailing edge indicate that the complexes were not dissociating into subunits under the conditions of the fractionation.…”

Section: Expression and Purification Of Cct Subunits-supporting

confidence: 67%

“…CCT4 and CCT5 homo-oligomers were assayed for refolding of luciferase (51), which we previously used to test the substrate refolding activity of human TRiC (39). In the experiment, unfolded luciferase was diluted into buffer with chaperonin (51).…”

Section: Cct4 Was Viewed In Cryo-emmentioning

confidence: 99%

“…Eukaryotic TRiC has been purified from yeast (22,23,36) and from bovine (18,31,37) and mouse (20,32,38) testes, and more recently from HeLa cells (39). The potential of TRiC as a target of therapeutic agent will benefit from access to human TRiC (39).…”

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confidence: 99%

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confidence: 99%

“…The potential of TRiC as a target of therapeutic agent will benefit from access to human TRiC (39). However, purification of human TRiC from HeLa cells is expensive (39), and recombinant co-expression of all eight CCT subunits has resulted in very low yields (40). To understand how individual human CCT subunits function (both in terms of ATP binding and hydrolysis, and substrate recognition and folding), we have successfully expressed single subunits in Escherichia coli.…”

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confidence: 99%

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Human CCT4 and CCT5 Chaperonin Subunits Expressed in Escherichia coli Form Biologically Active Homo-oligomers

Sergeeva

Chen

Haase-Pettingell

et al. 2013

Journal of Biological Chemistry

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Background:The subunit-specific roles of the CCT subunits in the chaperonin, TRiC, have not been elucidated. Results: When expressed in E. coli, CCT4 and CCT5 form TRiC-like homo-oligomeric rings. Conclusion: TRiC does not require all eight CCT subunits to form functional rings. Significance: The unexpected formation of homo-oligomeric CCT rings provides clues into the assembly of TRiC as a complex.

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Section: Expression and Purification Of Cct Subunits-supporting

confidence: 67%

Section: Cct4 Was Viewed In Cryo-emmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Human CCT4 and CCT5 Chaperonin Subunits Expressed in Escherichia coli Form Biologically Active Homo-oligomers

Sergeeva

Chen

Haase-Pettingell

et al. 2013

Journal of Biological Chemistry

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Mechanism and Function of the Eukaryotic Chaperonin CCT

Bueno-Carrasco

Cuéllar

2018

Encyclopedia of Life Sciences

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Chaperonins are a family of molecular chaperones that utilise the cavity formed by its two rings for the folding of substrates. The eukaryotic cytosolic chaperonin CCT or TRiC is thought to be involved in the folding of over 10% of newly synthesised proteins, although its major substrates are the cytoskeletal proteins such as actin and tubulin. CCT is the most complex of all chaperonins, with eight different subunits able to recognise specific sequences within substrate proteins, and it interacts with a variety of chaperones, cochaperones and cofactors to perform its activity. Substrate folding is mediated by an ATP‐driven cycle that induces conformational changes resulting in the opening or closing of the cavity. The different subunits are spatially arranged into an ATP‐binding and a substrate‐binding cluster, showing an asymmetric behaviour of the chaperonin. The increasing number of CCT substrates involved in cell signalling, cancer and neurodegenerative diseases suggest a central role of the chaperonin in the cellular proteostasis. Key Concepts CCT is a chaperonin composed of two rings of eight subunits each that form a cavity for substrate folding. Each CCT subunit is divided into apical, intermediate and equatorial domains with specialised functions. Actin and tubulin are the two main substrates of CCT and have driven the evolution of the chaperonin. ATP hydrolysis induces the closing of the cavity required for substrate folding. WD40 proteins have been described to be an important family of CCT substrates. The folding process often requires collaboration with other chaperones, cochaperones or cofactors that are the key for CCT specificity. CCT subunits display different affinities for ATP and substrates, leading to a spatial and functional clustering.

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Interaction of a Novel Chaperone PhLP2A With the Heat Shock Protein Hsp90

et al. 2016

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PhLP2 is a small cytosolic protein that belongs to the highly conserved phosducin-like family of proteins. In amniote genomes there are two PhLP2 homologs, PhLP2A and PhLP2B. It has been shown that mammalian PhLP2A modulates the CCT/TRiC chaperonin activity during folding of cytoskeletal proteins. In order to better understand the function of PhLP2A in cellular protein quality control system, in the present study we have searched for its protein targets. Applying immunoprecipitation followed by mass spectrometry analysis we have identified Hsp90 as a partner of PhLP2A. With pull down experiments, we have confirmed this interaction in protein lysate and using purified proteins we have shown that PhLP2A interacts directly with Hsp90. Furthermore, the proximity ligation assay (PLA) performed on mIMCD-3 cells has shown that PhLP2A forms complexes with Hsp90 which are mainly localized in the cytoplasm of these cells. Further analysis has indicated that the level of PhLP2A increases after heat shock or radicicol treatment, similarly as the level of Hsp90, and that expression of PhLP2A after heat shock is regulated at the transcriptional level. Moreover, using recombinant luciferase we have shown that PhLP2A stabilizes this enzyme in a folding competent state and prevents its denaturation and aggregation. In addition, overexpression of PhLP2A in HEK-293 cells leads to increased heat stress resistance. Altogether, our results have shown that PhLP2A interacts with Hsp90 and exhibits molecular chaperone activity toward denatured proteins. J. Cell. Biochem. 118: 420-429, 2017. © 2016 Wiley Periodicals, Inc.

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Human TRiC complex purified from HeLa cells contains all eight CCT subunits and is active in vitro

Cited by 27 publications

References 57 publications

Human CCT4 and CCT5 Chaperonin Subunits Expressed in Escherichia coli Form Biologically Active Homo-oligomers

Human CCT4 and CCT5 Chaperonin Subunits Expressed in Escherichia coli Form Biologically Active Homo-oligomers

Mechanism and Function of the Eukaryotic Chaperonin CCT

Interaction of a Novel Chaperone PhLP2A With the Heat Shock Protein Hsp90

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