CRYPTOCHROME suppresses the circadian proteome and promotes protein homeostasis

Abstract

13Circadian timekeeping in mammalian cells involves daily cycles of CRYPTOCHROME-dependent 14 transcriptional feedback repression. Ablation of CRY in mice leads to reduced growth and numerous 15 other phenotypes for reasons that are not well understood. Here, we find that cells adapt to CRY 16 deficiency by extensive remodelling of the proteome, phosphoproteome and ionome, with twice the 17 number of circadian-regulated proteins and phosphopeptides as well as increased rhythmic ion 18 transport compared to wild-type cells. CRY-deficient cells also have increased protein synthesis and 19 reduced proteasomal degradation, as well as an altered energetic state. These adaptations render cells 20 more sensitive to stress, and may provide an explanation for the wide-ranging phenotypes of CRY-21 deficient mice. We suggest that daily rhythms in cellular protein abundance are damped by CRY-22 mediated repression to facilitate daily cycles of proteome renewal whilst maintaining protein 23 homeostasis. 24 25 complex containing the activating transcription factors (BMAL1 and CLOCK or NPAS2). The 52 stability, interactions and nucleocytoplasmic shuttling of the encoded PER and CRY proteins is 53 regulated post-translationally until, many hours later, they repress the activity of BMAL1-containing 54 complexes. This transcriptional-translational feedback loop (TTFL) is proposed as the basis of 55 circadian timekeeping in mammalian cells [15]. Genes whose transcription are regulated by core 56 TTFL factors are thought to drive circadian rhythms in the encoded proteins to control myriad cellular 57 functions [13,14]. 58 59 Within the TTFL model of circadian rhythm generation, CRY proteins are the essential repressors of 60 CLOCK/BMAL1 activity [16,17], and have long been considered indispensable for circadian 61 rhythms in vivo and cells ex vivo [18][19][20][21][22]. In contrast, PER proteins play critical signalling and 62 scaffolding roles, required for the nuclear import and targeting of CRY to BMAL1-containing 63 complexes [17]. Recently however, we found that CRY-deficient cells, tissues and mice retain the 64 capacity for circadian timing, in the absence of canonical TTFL function [23]. Similarly, circadian 65 oscillations were retained in cells and tissue slices lacking BMAL1 [24]. Whilst we cannot exclude 66 the presence of some unknown transcriptional feedback-driven oscillation, many previous 67 observations argue against this [12,17,19,21,22,[25][26][27][28]. Recent evidence supports the hypothesis that 68 a conserved post-translational cytosolic oscillator (PTO, or "cytoscillator") may be responsible for 69 generating the oscillation from which circadian transcriptional cycles derive [12,29,30]. Thus, while 70 the TTFL is crucial for rhythmic robustness and co-ordinating outputs, it is not required for generation 71 of rhythms. 72 73The complex phenotype of mice lacking CRY proteins has been interpreted to mean that circadian 74 timekeeping is crucial for organismal physiology. The emerging observations that circadia...

Differential effects of environmental and endogenous 24h rhythms within a deep-coverage spatiotemporal proteome

“…However, rhythmic proteins were significantly more abundant than arrhythmic proteins under entrained conditions (Fig. 4A, left), consistent with observations in mouse primary fibroblasts 36 . Interestingly, the opposite is true under LL conditions: rhythmic proteins were significantly less abundant than arrhythmic proteins (Fig.…”

“…It seems more likely that transcript rhythmicity presents a means of achieving proteostasis, to counteract a rhythmic requirement driven by rhythmic function and associated rhythmic protein turnover. This interpretation would be supported by the recent observation in mammalian cells that more rhythmic proteins are observed without a transcriptional 'core clock' than there are observed with one 36 . Therefore, we suggest that perhaps the canonical transcriptional/translational clock model might be back to front: it is more consistent with available data that rhythmic function generates rhythmic gene expression as a consequence of rhythmic post-translational regulation and protein degradation.…”

“…Samples were randomised before allocating to TMT runs, and the operator was blinded to the sample IDs. Samples were trypsin-digested as reported previously 36 . Lyophilized peptides were resuspended in 20 µl of 175 mM triethylammonium bicarbonate and labelled with a distinct TMT tag, 12µl, from a stock prepared as per manufacturer's instructions (Thermo Scientific), for 60 minutes at room temperature.…”

Compensatory ion transport buffers daily protein rhythms to regulate osmotic balance and cellular physiology

“…4a). Moreover, several recent phospho-proteomics studies indicate daily variation in the phosphorylation of WNK isoforms in several tissues, including cultured fibroblasts 44 , mouse liver 12 and forebrain 27 ( Supplementary Fig. 4b-d), as well as rhythms in the phosphorylation of NKCC and KCC isoforms 27,31 , suggesting that similar mechanisms operate in vivo.…”

“…3c). In a parallel investigation, on a different clock mutant cell line, we found that increased amplitude of soluble protein levels resulted in a decreased relative amplitude of K + oscillations as compared to WT cells 44 . These data suggest that cell-autonomous circadian clock mechanisms coordinate antiphasic oscillations of cytosolic ion and protein content.…”