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

Massachusetts Institute of Technology, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 04/2013; 288(24). DOI: 10.1074/jbc.M112.443929
Source: PubMed


Chaperonins are a family of chaperones that encapsulate their substrates and assist their folding in an ATP-dependent manner. The ubiquitous eukaryotic chaperonin, TCP-1 Ring Complex (TRiC), is a hetero-oligomeric complex composed of two rings each formed from eight different CCT (Chaperonin Containing TCP-1) subunits. Each CCT subunit may have distinct substrate recognition and ATP-hydrolysis properties. We have expressed each human CCT subunit individually in E. coli to investigate whether they form chaperonin-like double ring complexes. CCT4 and CCT5, but not the other six CCT subunits, formed high molecular weight complexes within the E.coli cells that sedimented about 20S in sucrose gradients. When CCT4 and CCT5 were purified, they were both organized as two back-to-back rings of eight subunits each, as seen by negative stain and cryo-electron microscopy. This morphology is consistent with that of the hetero-oligomeric double-ring TRiC purified from bovine testes and HeLa cells. Both CCT4 and CCT5 homo-oligomers hydrolyzed ATP at a rate similar to human TRiC, and were active as assayed by luciferase refolding and human γD-crystallin aggregation suppression and refolding. Thus both CCT4 and CCT5 homo-oligomers have the property of forming eight-fold double rings absent the other subunits, and these complexes carry out chaperonin reactions without other partner subunits.

Download full-text


Available from: Jonathan King, Nov 16, 2015
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The βγ-crystallins are among the most stable and long-lived proteins in the human body. With increasing age, however, they transform to high molecular weight light-scattering aggregates, resulting in cataracts. This occurs despite the presence in the lens of high concentrations of the a-crystallin chaperones. Aggregation of crystallins can be induced in vitro by a variety of stresses, including acidic pH, ultraviolet light, oxidative damage, heating or freezing, and specific amino acid substitutions. Accumulating evidence points to the existence of specific biochemical pathways of protein: protein interaction and polymerization. We review the methods used for studying crystallin stability and aggregation and discuss the sometimes counterintuitive relationships between factors that favor native state stability and those that favor non-native aggregation. We discuss the behavior of βγ-crystallins in mixtures and their chaperone ability; the consequences of missense mutations and covalent damage to the side-chains; and the evolutionary strategies that have shaped these proteins. Efforts are ongoing to reveal the nature of cataractous crystallin aggregates and understand the mechanisms of aggregation in the context of key models of protein polymerization: amyloid, native-state, and domain-swapped. Such mechanistic understanding is likely to be of value for the development of therapeutic interventions and draw attention to unanswered questions about the relationship between a protein's native state stability and its transformation to an aggregated state.
    Progress in Biophysics and Molecular Biology 05/2014; 115(1). DOI:10.1016/j.pbiomolbio.2014.05.002 · 2.27 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The features in partially folded intermediates that allow the group II chaperonins to distinguish partially folded from native states remain unclear. The Archaeal group II chaperonin from Methanococcus Mauripaludis (Mm-Cpn) assists the in vitro refolding of the well-characterized β-sheet lens protein human γD-Crystallin (HγD-Crys). The domain interface and buried cores of this Greek key conformation include side chains, which might be exposed in partially folded intermediates. We sought to assess whether particular features buried in the native state, but absent from the native protein surface, might serve as recognition signals. The features tested were a) paired aromatic side chains; b) side chains in the interface between the duplicated domains of HγD-Crys, and c) side chains in the buried core which result in congenital cataract when substituted. We tested the Mm-Cpn suppression of aggregation of these HγD-Crys mutants upon dilution out of denaturant. Mm-Cpn was capable of suppressing the off-pathway aggregation of the three classes of mutants indicating that the buried residues were not recognition signals. In fact, Mm-Cpn recognized the HγD-Crys mutants better than WT and refolded most mutant HγD-Crys to levels twice that of WT HγD-Crys. This presumably represents the increased population or longer lifetimes of the partially folded intermediates of the mutant proteins. The results suggest that Mm-Cpn does not recognize the features of HγD-Crys tested – paired aromatics, exposed domain interface, or destabilized core – but rather recognizes other features of the partially folded β-sheet conformation that are absent or inaccessible in the native state of HγD-Crys.
    Protein Science 06/2014; 23(6). DOI:10.1002/pro.2452 · 2.85 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Hereditary sensory neuropathies are a class of disorders marked by degeneration of the nerve fibers in the sensory periphery neurons. Recently, two mutations were identified in the subunits of the eukaryotic cytosolic chaperonin, TRiC, a protein machine responsible for folding actin and tubulin the cell. C450Y CCT4 was identified in a stock of Sprague-Dawley rats, while H147R CCT5 was found in a human Moroccan family. As with many genetically identified mutations associated with neuropathies, the underlying molecular basis of the mutants was not defined. We investigated the biochemical properties of these mutants using an expression system in E. coli that produces homo-oligomeric rings of CCT4 and CCT5. Full-length versions of both mutant protein chains were expressed in E. coli at levels approaching that of the wild-type (WT) chains. Sucrose gradient centrifugation revealed chaperonin-sized complexes of both WT and mutant chaperonins, but with reduced recovery of C450Y CCT4 soluble subunits. Electron microscopy of negatively stained samples of C450Y CCT4 revealed few ring-shaped species, while WT CCT4, H147R CCT5, and WT CCT5 revealed similar ring structures. CCT5 complexes were assayed for their ability to suppress aggregation of and refold the model substrate γD-crystallin, suppress aggregation of mutant huntingtin, and refold the physiological substrate β-actin in vitro. H147R CCT5 was not as efficient in chaperoning these substrates as WT CCT5. The subtle effects of these mutations is consistent with the homozygous disease phenotype, in which most functions are carried out during development and adulthood, but some selective function is lost or reduced.
    Journal of Biological Chemistry 08/2014; 289(40). DOI:10.1074/jbc.M114.576033 · 4.57 Impact Factor
Show more