The mammalian circadian timing system is composed of a central pacemaker in the suprachiasmatic nucleus of the brain that synchronizes countless subsidiary oscillators in peripheral tissues. The rhythm-generating mechanism is thought to rely on a feedback loop involving positively and negatively acting transcription factors. BMAL1 and CLOCK activate the expression of Period (Per) and Cryptochrome (Cry) genes, and once PER and CRY proteins accumulate to a critical level they form complexes with BMAL1-CLOCK heterodimers and thereby repress the transcription of their own genes. Here, we show that SIRT1, an NAD(+)-dependent protein deacetylase, is required for high-magnitude circadian transcription of several core clock genes, including Bmal1, Rorgamma, Per2, and Cry1. SIRT1 binds CLOCK-BMAL1 in a circadian manner and promotes the deacetylation and degradation of PER2. Given the NAD(+) dependence of SIRT1 deacetylase activity, it is likely that SIRT1 connects cellular metabolism to the circadian core clockwork circuitry.

The circadian clock, a highly specialized, hierarchical network of biological pacemakers, directs and maintains proper rhythms in endocrine and metabolic pathways required for organism homeostasis. The clock adapts to environmental changes, specifically daily light-dark cycles, as well as rhythmic food intake. Nutritional challenges reprogram the clock, while time-specific food intake has been shown to have profound consequences on physiology. Importantly, a critical role in the clock-nutrition interplay appears to be played by the microbiota. The circadian clock appears to operate as a critical interface between nutrition and homeostasis, calling for more attention on the beneficial effects of chrono-nutrition.

In mammals, most metabolic processes are influenced by biological clocks and feeding rhythms. The mechanisms that couple metabolism to circadian oscillators are just emerging. NAD-dependent enzymes (e.g., Sirtuins and poly[ADP-ribose] polymerases), redox- and/or temperature-dependent transcription factors (e.g., CLOCK, NPAS2, and HSF1), nutrient-sensing transcriptional regulatory proteins (e.g., CREB-CBP-CRCT2, FOXO-p300, nuclear receptors, PGC-1, and SP1 family members) and protein kinases (e.g., AMPK), are plausible candidates for conveying a cell's metabolic state to the core clock circuitry. The intertwining between these acute regulators and circadian clock components is so tight that the discrimination between metabolic and circadian oscillations may be somewhat arbitrary.

Protein degradation is an essential and highly regulated process. The proteasomal degradation of the tumor suppressors p53 and p73 is regulated by both polyubiquitination and by an ubiquitin-independent process. Here, we show that this ubiquitin-independent process is mediated by the 20S proteasomes and is regulated by NQO1. NQO1 physically interacts with p53 and p73 in an NADH-dependent manner and protects them from 20S proteasomal degradation. Remarkably, the vast majority of NQO1 in cells is found in physical association with the 20S proteasomes, suggesting that NQO1 functions as a gatekeeper of the 20S proteasomes. We further show that this pathway plays a role in p53 accumulation in response to ionizing radiation. Our findings provide the first evidence for in vivo degradation of p53 and p73 by the 20S proteasomes and its regulation by NQO1 and NADH level. Protein degradation determines the outcome of many cellular physiological processes (Coux et al. 1996). Degradation of proteins by the proteasomes occurs via various pathways (Verma and Deshaies 2000;Pickart and Cohen 2004). The most intensely studied one is the ubiquitin-26S proteasome pathway (Hershko 1996;Hershko and Ciechanover 1998;Goldberg 2003). The tumor suppressor p53 is a very labile protein that undergoes Mdm2 and ubiquitin-dependent 26S proteasomal degradation (Haupt et al. 1997;Kubbutat et al. 1997). Recently, we reported that degradation of p53 also occurs in an Mdm2 and ubiquitin-independent manner (Asher et al. 2002b). This pathway of p53 degradation is regulated by NAD(P)H quinone oxidoreductase 1 (NQO1) (Asher et al. 2001(Asher et al. , 2002a(Asher et al. ,b, 2003(Asher et al. , 2004), yet the underlying molecular mechanisms that control p53 degradation remained elusive. Results and DiscussionTo investigate the role of NQO1 in proteasomal degradation, we followed NQO1 distribution in fractionated mouse liver extracts. Ammonium sulfate precipitation and gel-filtration chromatography of liver extracts revealed that the majority of NQO1 cofractionated with the 20S proteasomes (Fig. 1A). These fractions are devoid of the 26S proteasomes that were excluded by the differential ammonium sulfate precipitation (Fig. 1A, IB: 26S, anti TBP1 a subunit of the 19S). These results suggest that the vast majority of cellular NQO1 is found in a large protein complex that possibly includes the 20S proteasomes.To further study this possibility, the 20S-containing fractions were pooled and fractionated by anion exchange chromatography according to a standard 20S purification protocol (Friguet et al. 2002). Remarkably, NQO1 was detected in the 0.3 M NaCl fraction containing the 20S proteasomes (Fig. 1B). Electrophoresis of the 0.3 M NaCl fraction on a nondenaturing PAGE, followed by peptidase activity assay, showed that the purified 20S is functional (Fig. 1C, Activity panel). Immunoblot analysis with anti NQO1 antibody revealed that NQO1 comigrated with the 20S proteasomes, but not with the 26S proteasomes (Fig. 1C). Finally, a coimmunopercipitation experim...

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