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Title: Circadian orchestration of the cellular proteome
Author: Wong, David Chuen Soong
ISNI:       0000 0004 8500 3883
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2020
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Circadian rhythms are biological oscillations with a period length of roughly 24 hours. They confer an adaptive advantage as they enable the prediction of the regular environmental changes caused by the rotation of the Earth, and so they are highly conserved. Circadian coordination of biology is an important feature not only across kingdoms, but also at multiple levels of biological scale, from behaviour to physiology and cell biology. On the cellular level it is thought that mammalian circadian rhythms are driven by a delayed feedback loop, involving the transcription and translation of a core set of 'clock proteins'. This cellular timekeeping system is remarkably precise and robust to perturbations. In the first chapter of this thesis I explored the mechanism of action of picrotoxin, a drug that shortens circadian period through unknown means. I uncovered features that are not readily explained by the canonical transcriptional-translational-feedback loop (TTFL) model of circadian rhythm generation. In the second part of this thesis I used mass spectrometry-based proteomics to characterise the cellular circadian proteome, and I uncovered the reciprocal, rhythmic regulation of protein and ion concentrations in cultured cells. Strikingly, by using a genetic knock-out of the CRYPTOCHROME (CRY) proteins I found that the generation of these rhythms was not dependent upon the TTFL, strongly supporting an alternative model of circadian rhythm generation - the post-translational oscillator (PTO) model. In the next chapter I explored the circadian regulation of the PERIOD 2 (PER2) protein in CRY-deficient cells, suggesting that a key determinant of PER2 behaviour in the absence of the TTFL may be cellular protein and ion concentrations. In the final chapter I investigated the functional consequences of CRY knock-out. I found that CRY-deficient cells and animals have disrupted proteostasis and metabolism, and found that impaired proteostasis attenuates circadian rhythmicity at the cellular and organism levels. Altogether, the work presented in this thesis provides evidence for a PTO model of circadian rhythm generation. I have uncovered circadian regulation of the cellular proteome even in the absence of the canonical TTFL. I propose that rather than driving circadian rhythms, the TTFL confers robustness to cellular timekeeping and facilitates proteostasis.
Supervisor: O'Neill, John Stuart Sponsor: MRC ; Frank Edward Elmore Fund
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: circadian ; proteome ; proteomics ; cell ; proteostasis ; protein homeostasis