Rahul Samant

Native mass spectrometry analyses of chaperonin complex TRiC/CCT reveal subunit N-terminal processing and re-association patterns.
Collier MP, Moreira KB, Li KH, Chen YC, Itzhak D, Samant R, Leitner A, Burlingame A, Frydman J

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.

Scientific reports, 11, 1, 22 Jun 2021

Alternative systems for misfolded protein clearance: life beyond the proteasome.
Johnston HE, Samant RS

Protein misfolding is a major driver of ageing-associated frailty and disease pathology. Although all cells possess multiple, well-characterised protein quality control systems to mitigate the toxicity of misfolded proteins, how they are integrated to maintain protein homeostasis ('proteostasis') in health-and how their dis-integration contributes to disease-is still an exciting and fast-paced area of research. Under physiological conditions, the predominant route for misfolded protein clearance involves ubiquitylation and proteasome-mediated degradation. When the capacity of this route is overwhelmed-as happens during conditions of acute environmental stress, or chronic ageing-related decline-alternative routes for protein quality control are activated. In this review, we summarise our current understanding of how proteasome-targeted misfolded proteins are re-trafficked to alternative protein quality control routes such as juxta-nuclear sequestration and selective autophagy when the ubiquitin-proteasome system is compromised. We also discuss the molecular determinants of these alternative protein quality control systems, attempt to clarify distinctions between various cytoplasmic spatial quality control inclusion bodies (e.g., Q-bodies, p62-bodies, JUNQ, aggresomes, and aggresome-like induced structures 'ALIS'), and speculate on emerging concepts in the field that we hope will spur future research-with the potential to benefit the rational development of healthy ageing strategies.

The FEBS journal, 1, 1, 01 Nov 2020

Dosage compensation plans: protein aggregation provides additional insurance against aneuploidy.
Samant RS, Masto VB, Frydman J

Gene dosage alterations caused by aneuploidy are a common feature of most cancers yet pose severe proteotoxic challenges. Therefore, cells have evolved various dosage compensation mechanisms to limit the damage caused by the ensuing protein level imbalances. For instance, for heteromeric protein complexes, excess nonstoichiometric subunits are rapidly recognized and degraded. In this issue of , Brennan et al. (pp. 1031-1047) reveal that sequestration of nonstoichiometric subunits into aggregates is an alternative mechanism for dosage compensation in aneuploid budding yeast and human cell lines. Using a combination of proteomic and genetic techniques, they found that excess proteins undergo either degradation or aggregation but not both. Which route is preferred depends on the half-life of the protein in question. Given the multitude of diseases linked to either aneuploidy or protein aggregation, this study could serve as a springboard for future studies with broad-spanning implications.

Genes & development, 33, 1549-5477, 2019