Cells respond to stress by up-regulating chaperone proteins that correct protein misfolding to maintain function. However, protein homeostasis is lost in ageing, leading to aggregates characteristic of protein-folding diseases. While much is known about how these diseases progress, discovering what causes protein-folding to deteriorate could be key to their prevention. Studying ageing in humans introduces a key temporal obstacle, however results in model organisms suggest ageing has cellular origins. In this work, primary human mesenchymal stem cells (hMSCs) have been cultured to a point of replicative senescence and subjected to proteotoxic stress, as an in vitro model to investigate changes to the stress response during cellular ageing. Through -omic analyses it has been shown that the maintenance of protein homeostasis deteriorated in senescent cells through the attenuation of processes downstream of chaperone transcription. This was coincident with lowered levels of a functional module of chaperone proteins associated with stress-inducible heat shock protein 70 kDa (HSPA1A). Further analysis of the temporal dynamics of the transcriptomic and proteomic stress responses revealed a lack of translational capacity to be a limiting factor in the ability of senescent cells to mitigate proteotoxic stress, leading to a loss of speed, magnitude, and efficacy of the cellular stress response. Furthermore, here it has been shown that senescent cells experience partial loss of the E3 ubiquitin ligase CHIP. CHIP is known to regulate HSPA1A stability during stress and cooperates with the chaperone to mark misfolded proteins for turnover. By building a mathematical model of the proteotoxic stress response, in silico simulations have predicted that a decrease in cellular CHIP leads to a greater accumulation of misfolded proteins during proteotoxic stress. Using a cysteine-reactive label which binds to residues typically buried in correctly folded proteins, this prediction has been validated by showing significant conformational compromise to the proteomes of senescent cells in response to stress, whereas no perturbation was seen in proliferating cell populations. Prime among the conformationally compromised proteins in senescent cells were several cytoskeletal proteins. Here it has been shown that this is likely due to the senescence-associated down-regulation of another functional module of chaperone proteins responsible for maintaining cytoskeletal protein homeostasis, the chaperonin-containing tailless (CCT) complex. These results demonstrate multiple mechanisms which drive the attenuation of the proteotoxic stress response in cellular ageing. As such, these represent a step forward in our understanding of the underlying link between ageing and a loss of protein homeostasis. Given the therapeutic potential of hMSCs, this work also holds relevance to the emerging field of regenerative medicine. In this context, this study gives a comprehensive evaluation of consequential, but often neglected, changes to the hMSC stress response following extended culture in vitro.
|Date of Award||31 Dec 2021|
- The University of Manchester
|Supervisor||Simon Hubbard (Supervisor) & Joe Swift (Supervisor)|
- Human mesenchymal stem cells
- Stress response