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Healthy sleep is vital for humans to achieve optimal health and longevity. Poor sleep and sleep disorders are strongly associated with increased morbidity and mortality. However, the importance of good sleep continues to be underrecognized. Mechanisms regulating sleep and its functions in humans remain mostly unclear even after decades of dedicated research. Advancements in gene sequencing techniques and computational methodologies have paved the way for various genetic analysis approaches, which have provided some insights into human sleep genetics. This review summarizes our current knowledge of the genetic basis underlying human sleep traits and sleep disorders. We also highlight the use of animal models to validate genetic findings from human sleep studies and discuss potential molecular mechanisms and signaling pathways involved in the regulation of human sleep.

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Neuropeptide S (NPS) was postulated to be a wake-promoting neuropeptide with unknown mechanism, and a mutation in its receptor (NPSR1) causes the short sleep duration trait in humans. We investigated the role of different NPS+ nuclei in sleep/wake regulation. Loss-of-function and chemogenetic studies revealed that NPS+ neurons in the parabrachial nucleus (PB) are wake-promoting, whereas peri-locus coeruleus (peri-LC) NPS+ neurons are not important for sleep/wake modulation. Further, we found that a NPS+ nucleus in the central gray of the pons (CGPn) strongly promotes sleep. Fiber photometry recordings showed that NPS+ neurons are wake-active in the CGPn and wake/REM-sleep active in the PB and peri-LC. Blocking NPS-NPSR1 signaling or knockdown of Nps supported the function of the NPS-NPSR1 pathway in sleep/wake regulation. Together, these results reveal that NPS and NPS+ neurons play dichotomous roles in sleep/wake regulation at both the molecular and circuit levels.

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Circadian rhythms form a self-sustaining, endogenous, time-keeping system that allows organisms to anticipate daily environmental changes. The core of the clock network consists of interlocking transcriptional-translational feedback loops that ensures that metabolic, behavioral, and physiological processes run on a 24 h timescale. The hierarchical nature of the clock manifests itself in multiple points of control on the daily cell division cycle, which relies on synthesis, degradation, and post-translational modification for progression. This relationship is particularly important for understanding the role of clock components in sensing stress conditions and triggering checkpoint signals that stop cell cycle progression. A case in point is the interplay among the circadian factor PERIOD2 (PER2), the tumor suppressor p53, and the oncogenic mouse double minute-2 homolog protein (MDM2), which is the p53's negative regulator. Under unstressed conditions, PER2 and p53 form a stable complex in the cytosol and, along with MDM2, a trimeric complex in the nucleus. Association of PER2 to the C-terminus end of p53 prevents MDM2-mediated ubiquitylation and degradation of p53 as well as p53's transcriptional activation. Remarkably, when not bound to p53, PER2 acts as substrate for the E3-ligase activity of MDM2; thus, PER2 is degraded in a phosphorylation-independent fashion. Unexpectedly, the phase relationship between PER2 and p53 are opposite; however, a systematic modeling approach, inferred from the oscillatory time course data of PER2 and p53, aided in identifying additional regulatory scenarios that explained, a priori, seemingly conflicting experimental data. Therefore, we advocate for a combined experimental/mathematical approach to elucidating multilevel regulatory cellular processes.

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The circadian clock relies on posttranslational modifications to set the timing for degradation of core regulatory components, which drives clock progression. Ubiquitin-modifying enzymes that target clock components for degradation mainly recognize phosphorylated substrates. Degradation of the circadian clock component PERIOD 2 (PER2) is mediated by its phospho-specific recognition by ß-transducin repeat-containing proteins (ß-TrCPs), which are F-box-containing proteins that function as substrate recognition subunits of the SCFß-TRCP ubiquitin ligase complex. However, this mode of regulating PER2 stability falls short of explaining the persistent oscillatory phenotypes reported in biological systems lacking functional elements of the phospho-dependent PER2 degradation machinery. We identified PER2 as a previously uncharacterized substrate for the ubiquitin ligase mouse double minute 2 homolog (MDM2) and found that MDM2 targeted PER2 for degradation in a manner independent of PER2 phosphorylation. Deregulation of MDM2 plays a major role in oncogenesis by contributing to the accumulation of genomic and epigenomic alterations that favor tumor development. MDM2-mediated PER2 turnover was important for defining the circadian period length in mammalian cells, a finding that emphasizes the connection between the circadian clock and cancer. Our results not only broaden the range of specific substrates of MDM2 beyond the cell cycle to include circadian components but also identify a previously unknown regulator of the clock as a druggable node that is often found to be deregulated during tumorigenesis.

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We report the preparation of S-aroylthiooxime (SATO) functionalized amphiphilic block copolymer micelles that release hydrogen sulfide (H2S), a gaseous signaling molecule of relevance to various physiological and pathological conditions. The micelles release H2S in response to cysteine with a half-life of 3.3 h, which is substantially slower than a related small molecule SATO. Exogenous administration of H2S impacts growth and proliferation of cancer cells; however, the limited control over H2S generation from inorganic sulfide sources results in conflicting reports. Therefore, we compare the cellular cytotoxicity of SATO-functionalized micelles, which release H2S in a sustained manner, to Na2S, which releases H2S in a single dose. Our results show that H2S-releasing micelles significantly reduce the survival of HCT116 colon cancer cells relative to Na2S, GYY4137, and a small molecule SATO, indicating that release kinetics may play an important role in determining toxicity of H2S toward cancer cells. Furthermore, H2S-releasing micelles are well tolerated by immortalized fibroblasts (NIH/3T3 cells), suggesting a selective toxicity of H2S toward cancer cells.

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