Cellular accumulation of misfolded proteins is a hallmark of ageing. In young cells, the proteostasis network limits toxicity by activating one or more systems for misfolded protein clearance. We focus on how these clearance systems are integrated within the network to maintain proteome health during youth, and how their dis-integration contributes to cellular senescence—another ageing hallmark with strong links to chronic inflammation and organismal frailty.
We currently use two evolutionarily distant cell types—budding yeast and primary human fibroblasts—to identify common, conserved lines of communication between different clearance systems of the proteostasis network, and how these get re-wired during replicative ageing (yeast) and senescence (mammals). Our lab employs multi-disciplinary approaches such as super-resolution imaging, flow cytometry, and mass-spectrometry-based proteomics to measure proteostasis capacity and senescence phenotypes as quantitatively and robustly as possible. As proteostasis modulators hold therapeutic promise in ageing-associated pathologies—with renewed interest in ‘senolytics’ specifically targeting senescent cells—we hope to drive fundamental discoveries that have a direct impact on promoting lifelong health.
We use a multi-disciplinary approach (left panel), using high-resolution imaging, flow cytometry, and mass spectrometry-based proteomics, to probe the relationship between loss of proteostasis and onset of senescence—two of the hallmarks of ageing (middle—adapted from Lopéz-Otín et al., 2013). Our current focus is on the interplay between different protein clearance systems in young vs. senescent cells (right).
Check out Rahul’s talk from Methuselah Health UK’s Conference on Why We Age (2020), entitled “Why we turnover our proteins (and how it gets done)"
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.
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.
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.