Circadian Regulation of Protein Homeostasis

Abstract

Circadian rhythms orchestrate physiology across all kingdoms of life to anticipate the differing demands of day and night. Circadian timekeeping has a cell-autonomous basis, amenable to study in cultured mammalian cells, where daily rhythms have been shown to affect most biological processes. In various models, rhythms in protein abundance of 10-20% of the proteome have been reported. However, regulation of these rhythms is not well understood, as most of them are not accounted for at the transcript or even ribosomal occupancy level. It is also unclear how circadian rhythmicity in protein translation and abundance affects protein homeostasis, maintenance of which is crucial for cell viability.

In this thesis, I report the investigation of post-transcriptional temporal control of protein abundance, and the interplay between protein homeostasis and the circadian clock. I was particularly interested in proteasomal degradation, as it has been implicated in a variety of circadian model systems, and is likely to have a conserved role both feeding into and being regulated by cellular timekeeping. I provide data in support of this in the first results chapter, showing that proteasome activity is essential for temporal organisation of circadian transcriptional networks, and that cellular proteasome activity is circadian-regulated.

In the subsequent chapter, using various protein labelling strategies, I describe circadian variation in global protein degradation. A rhythm in global protein synthesis has been reported previously, but I found this occurs in-phase with the rhythm in degradation. It suggests that protein turnover and proteome renewal is temporally consolidated. Data from cells deficient in circadian transcriptional feedback, where proteostasis is perturbed and more proteins have rhythms in abundance, supported the idea that circadian rhythms could function to maintain protein homeostasis, rather than to generate protein abundance rhythms.

To explore this new model proteome-wide, I then optimised and performed a complex dynamic proteomics study, using a cutting-edge pulsed SILAC-TMT methodology to measure protein synthesis, degradation and abundance across circadian time. This provided a detailed picture of temporal protein regulation, and, among other things, showed that protein synthesis varies more over time than does steady-state abundance, in terms of number of rhythmic proteins and relative amplitude.

Finally, I also looked into the consequences of rhythms in protein turnover. I predicted that at times of higher turnover, cells would be more sensitive to proteotoxic stress. This appeared true at the level of stress response markers and protein aggregation, as well as cell viability after a proteostasis challenge. Time-of-day variation in turnover was also observed in animal tissues, as was evidence for time-of-day variation in the effect of bortezomib, a clinically relevant proteasomal inhibitor.

Altogether, this work provides new insights into temporal control of protein homeostasis. It points to a novel model where circadian timekeeping acts to promote proteostasis and to co-ordinate proteome renewal, which would be advantageous in terms of bioenergetic efficiency and osmotic homeostasis.