Assembly and substrate recognition properties of human CCT subunits of the TRiC chaperonin

Abstract

Group II chaperonins are large multi-subunit complexes that fold cytosolic proteins to their native structures. They are composed of two back-to-back rings of 7-9 subunits. The eukaryotic cytosolic type II chaperonin Tailless Complex Polypeptide-1 (TCP-1) Ring Complex (TRiC) consists of eight different subunits identified as Chaperonin Containing TCP-1 (CCT) [alpha] (1) - [theta] (8). TRiC is necessary for folding about 10% of newly synthesized proteins and is essential for folding actin and tubulin. Most of the research on TRiC in the last 20 years has focused on yeast and bovine TRiC. However, recently, there has been inquiry into TRiC as a target for disease therapy for Huntington's disease, cataract, and some cancers. Consequently, to understand human TRiC, we purified endogenous TRiC from HeLa cells for characterization. These complexes contained all eight of the CCT subunits as determined by immunoblot. The structures were well organized as double-rings of eight subunits each, using negative stain electron microscopy (EM). Human TRiC was active in suppressing aggregation and refolding two different substrates: luciferase (a model substrate) and human [gamma]D-crystallin (H[gamma]D-Crys; a physiological substrate found in the eye lens). To further understand human TRiC, we expressed all of the human CCT subunits, one at a time in E. coli. This was done so that the subunit specificities of the CCT subunits could be studied and so we could have a system where these proteins could be genetically manipulated. Theoretically, all eight subunits in the mature TRiC-complex are needed to successfully recognize all substrates that need to be folded in the cell. We found that two CCT subunits, CCT4 and CCT5, but not the others, formed TRiC-like homo-oligomeric rings in the absence of the other CCT subunits. Purification of these complexes and subsequent structural assays by negative stain and cryo-EM showed that they formed double rings of eight subunits per ring. Biochemically, we found that CCT4 and CCT5 hydrolyzed ATP at the same rate as human TRiC, could refold luciferase to the same level as human TRiC, and suppressed aggregation of H[gamma]D-Crys. The homo-oligomeric complexes also assisted the refolding of H[gamma]D-Crys, a property not observed in the lens specific [alpha]-crystallin chaperone. On the substrates studied, CCT4 and CCT5 homo-oligomers worked as efficiently as hetero-oligomeric TRiC. More stringent substrates such as actin and tubulin need to be studied to further understand CCT specificity. Two mutations, one in CCT4 (C450Y) and one in CCT5 (H147R), have been implicated in hereditary sensory neuropathy. In order to study the defective mutant proteins, we introduced these mutations into the CCT4 and CCT5 constructs. We found that for CCT4, the newly translated mutant polypeptide chains aggregated much more than wild-type (WT) CCT4. While the mutant formed some rings in the E. coli lysate, as assayed by sucrose ultracentrifugation gradients and negative stain EM, they were not stable throughout the purification and the final purified sample contained few homo-oligomers. The mutant CCT5 polypeptide chains were properly folded and assembled in homo-oligomers. H147R CCT5 was able to hydrolyze ATP at a similar rate as WT CCT5. However, in the H[gamma]D-Crys aggregation suppression and refolding assay, mutant huntingtin aggregation suppression assay, and actin refolding assay, mutant CCT5 was not as efficient in suppression or refolding as WT CCT5. Therefore, the H147R mutation in CCT5 led to a chaperoning defect while the C450Y mutation in CCT4 led to a folding/stability defect. In order to understand features of partially folded intermediates that group II chaperonins recognize in a substrate, we investigated whether the archaeal group II chaperonin from Methanococcus maripaludis (Mm-Cpn) could recognize a variety of H[gamma]D-Crys mutants. These mutations were in regions of the protein that could act as recognition signals of substrate - unpaired aromatics, domain interface, and buried core residues. We found that Mm-Cpn was able to recognize all of these mutants, better than it recognized WT H[gamma]D-Crys. In addition, Mm-Cpn could refold most of the mutants to levels higher than WT H[gamma]D-Crys. Therefore, we concluded that Mm-Cpn doesn't recognize any of the proposed recognition signals but recognizes some [beta]-sheet interface exposed in these mutants. These studies further our knowledge of group II chaperonins and specifically human TRiC, and open up some new avenues for the investigation of the folding, assembly and function of this eukaryotic protein essential for the reproduction of all cells.