Native mass spectrometry analyses of chaperonin complex TRiC/CCT reveal subunit N-terminal processing and re-association patterns

Abstract

The eukaryotic chaperonin TRiC/CCT is a large ATP-dependent complex essential for cellular protein folding. Its subunit arrangement into two stacked eight-membered hetero-oligomeric rings is conserved from yeast to man. A recent breakthrough enables production of functional human TRiC (hTRiC) from insect cells. Here, we apply a suite of mass spectrometry techniques to characterize recombinant hTRiC. We find all subunits CCT1-8 are N-terminally processed by combinations of methionine excision and acetylation observed in native human TRiC. Dissociation by organic solvents yields primarily monomeric subunits with a small population of CCT dimers. Notably, some dimers feature non-canonical inter-subunit contacts absent in the initial hTRiC. This indicates individual CCT monomers can promiscuously re-assemble into dimers, and lack the information to assume the specific interface pairings in the holocomplex. CCT5 is consistently the most stable subunit and engages in the greatest number of non-canonical dimer pairings. These findings confirm physiologically relevant post-translational processing and function of recombinant hTRiC and offer quantitative insight into the relative stabilities of TRiC subunits and interfaces, a key step toward reconstructing its assembly mechanism. Our results also highlight the importance of assigning contacts identified by native mass spectrometry after solution dissociation as canonical or non-canonical when investigating multimeric assemblies.

Revisiting the chaperonin T‐complex protein‐1 ring complex in human health and disease: A proteostasis modulator and beyond

“…22 CCT5 can form dimers with all eight subunits except for CCT8, indirectly supporting this viewpoint. 24 The processes of TRiC/CCT disassembly and degradation are less well understood than those of its assembly. Early studies demonstrated that two octameric rings disassemble one ring at the same time to generate microcom-plexes and monomers, not double rings.…”

Targeting HSF1 for cancer treatment: mechanisms and inhibitor development

“…TRiC/CCT consists of two stacked anti-parallel rings of eight hetero-oligomers and forms a hexadecamer. Each subunit possesses its own function and CCT5 is thought to play a master role in assembly and disassembly of the complex 48 .…”

The TRiCky Business of Protein Folding in Health and Disease

“…This idea is supported by mass spectrometry analysis of human CCT purified from insect cells, in which CCT5 was the most stable subunit observed under chemical destabilization conditions, hence, the most likely to self-assemble or co-assemble with other subunits. Importantly, CCT5 formed dimers with all subunits except for CCT8 ( Collier et al, 2021 ). However, since CCT subunits are independently transcribed in the crowded cellular environment, bringing together all the subunits for assembly could be challenging.…”

“…N-terminal modifications may also affect the formation of the CCT complex since the N-termini of subunit apical domains mediate inter-ring cooperativity. Truncation of N-termini through methionine loss or acetylation could lead to degradation of CCT subunits ( Collier et al, 2021 ). Evident from these experiments is that assembly/disassembly of the CCT complex may be linked to the monomer/oligomer pool in a dynamic process that ultimately controls protein folding activity and critical cell functions.…”