The enzyme Nocturnin, which governs daily tasks such as fat metabolism and energy usage, works in an entirely different way than previously thought, reported a team of researchers at Princeton University.
The newly discovered mechanism reveals the molecular link between the enzyme’s daily fluctuations and its energy-regulating role in the body, according to a study published this week in Nature Communications.
“The realization that Nocturnin works in this manner will guide our thinking about sleep, oxidative stress and metabolism, and eventually may serve as a step toward finding better treatments for metabolic diseases,” said Alexei Korennykh, an associate professor of molecular biology at Princeton, who led the work.
Nocturnin is part of the circadian clock that alters the metabolism and behavior of living organisms to match the body’s needs at different times of the day.
For example, Nocturnin levels fluctuate throughout the day, dramatically peaking when the body first awakens. Nocturnin is also a critical regulator of metabolism; compared to regular mice, mice lacking the enzyme make less insulin, are protected from fatty liver disease and are less susceptible to weight gain.
The precise function of Nocturnin inside cells has remained unclear, however
For many years, the enzyme was thought to turn on and off cellular metabolism by degrading certain cellular messages made of ribonucleic acid, or mRNAs.
Last year, however, three groups of researchers – a group from the University of Michigan, a group from the University of Minnesota, and Korennykh’s team – discovered that Nocturnin is incapable of degrading RNAs.
To find out how Nocturnin can have such large effects on the body’s metabolism, Korennykh teamed up with Princeton’s Joshua Rabinowitz, a professor of chemistry and the Lewis-Sigler Institute for Integrative Genomics, and Paul Schedl, a professor of molecular biology.
The study was led by postdoctoral research associate Michael Estrella and graduate student Jin Du in the Alexei lab, and postdoctoral research associate Li Chen in the Rabinowitz lab.
Using methods pioneered by Rabinowitz to screen tissues for the presence of metabolites, the researchers discovered that Nocturnin plays a far more direct role in metabolism than previously appreciated.
Rather than degrading mRNAs, the enzyme regulates specific metabolites that help energy production and protect cells from damage.
The study determined that Nocturnin is located in the cell’s energy-producing structures, the mitochondria, suggesting that this is where the enzyme performs its function.
The team found that Nocturnin removes a phosphate group from two molecules important in metabolism, called NADP+ and NADPH.
These molecules allow the cell to modulate the levels of reactive oxygen species, which function both as harmful agents causing damage and as signaling molecules controlling metabolism and fat storage.
The researchers conclude that Nocturnin is the first known enzyme to perform this reaction on NADP+ and NADPH inside mitochondria.
Removing phosphate groups from NADP+ and NADPH produces two different but equally important molecules, NAD+ and NADH, which are essential for the function of metabolic enzymes – the molecular machines that produce energy by breaking down energy-rich biomolecules such as glucose.
Nocturnin upregulation when an animal first awakens might therefore kick the body’s energy production into high gear by providing more NAD+ and NADH.
“It is tempting to propose that one physiologic function of Nocturnin could be to maximize available NAD+ and NADH for energy generation in a search for food, using the elevated blood sugar that animals have at the time of awakening,” Korennykh said.
Korennykh and colleagues also deciphered the crystal structure of human Nocturnin bound to NADPH, showing at the atomic level how the reaction mediated by Nocturnin occurs. NADPH fits perfectly into Nocturnin’s active site so that the enzyme can easily remove the molecule’s phosphate group.
Finally, the researchers determined that the fruit fly version of Nocturnin, known as Curled, is also unable to cleave RNA. Instead, Curled uses the same mechanism as human Nocturnin and targets NADP+ and NADPH. The Curled gene was first described over 100 years ago by Thomas Hunt Morgan, the pioneering geneticist who won a Nobel Prize for demonstrating that genes are carried on chromosomes.
Though Curled has been studied by fruit fly researchers ever since, its biochemical mechanism was a mystery until now.
“Our work shows that even in the age of genomics and personalized medicine, basic biology still remains to be understood,” Korennykh said.
“In the example of Nocturnin and Curled, a pathway regulating some of the most important molecules in metabolism was hidden in plain sight for the past 100 years.”
The study, “The Metabolites NADP+ and NADPH are the Targets of the Circadian Protein Nocturnin (Curled),” by Michael A Estrella, Jin Du, Li Chen, Sneha Rath, Eliza Prangley, Alisha Chitrakar, Tsutomu Aoki, Paul Schedl, Joshua Rabinowitz and Alexei Korennykh, was published online in Nature Communications on May 30, 2019.
The circadian clock exerts central and peripheral effects that are vital for proper metabolic homeostasis
Obesity and the metabolic syndrome
A variety of conditions collectively grouped and diagnosed as the metabolic syndrome are on the rise world-wide and causing serious health concerns.
Nearly 40% of men and women in the United States qualified for such diagnosis as of 2005 [1]. Among the conditions associated with the metabolic syndrome are obesity and dysregulation of glucose levels [1].
Indeed, excess weight is the leading cause of poor health and is associated with development of cardiovascular disease and diabetes [2].
Interestingly, alterations in biological timing occurring through shift work and jet lag, result in major disruptions in physiology that manifest as symptoms of the metabolic syndrome [reviewed in 3]. Examining the regulation of rhythmic metabolic processes is therefore vital to our understanding and treatment of this disorder.
Research investigating the intimate relationship between daily “circadian” cycles and metabolic rhythms has benefitted from examining metabolic phenotypes in mutant mice with targeted disruptions to genes involved in both rhythm generation and rhythmic output.
One such rhythmic output gene is Nocturnin (Noc), which codes for a circadian deadenylase and is the focus of this review.
Deadenylases participate in post-transcriptional mRNA regulation through destabilization of target transcripts, and circadian control of deadenylase activity is one mechanism whereby the clock regulates rhythmic gene expression.
Noc has been implicated in many aspects of lipid metabolism presumably through the post-transcriptional circadian regulation of genes involved in metabolizing fat [4]. More recently, its roles in metabolism have expanded to include lipogenesis, adipogenesis and osteogenesis [4–7].
Here we highlight the recent advances that place Nocturnin at a unique intersection between both circadian clocks and metabolism, and we emphasize how these two important processes interact to maintain proper balance in the face of metabolic challenges.
The circadian clock and metabolism
The circadian clock drives rhythmic processes in both physiology and behavior and synchronizes them to the environment. In mammals, clocks are found within cells throughout the body and are coordinated by a “master” pacemaker located within the hypothalamic suprachiasmatic nucleus(SCN) [reviewed in 8].
The circadian clock mechanism consists of auto-regulatory transcription/translation feedback loops resulting in rhythmic production and degradation of core “clock” genes/proteins (Figure 1A).
In one loop, the proteins CLOCK and BMAL1 form a heterodimer that binds to E-box enhancer elements of target genes, including other core clock genes such as Period and Cryptochrome.
The protein products PERIOD (PER) and CRYPTOCHROME (CRY) accumulate in the cytoplasm and form a complex with Casein Kinase 1 (CK1), followed by translocation back into the nucleus where they repress the CLOCK/BMAL1 activity and thus turn off their own transcription. In an interlocking loop of the clock, the nuclear receptors RORα and REV-ERBα direct alternating activation and repression, respectively, of Bmal1 expression (Figure 1A).
Together, these loops drive a 24 h oscillation that coordinates the timing of processes, e.g. metabolic reactions, centrally in the brain and peripherally throughout the body. In turn, the core clock genes receive inputs from the periphery to ensure proper between-function synchronization.
For example, NAD+, a major contributor of cell metabolism, shows 24-hour oscillations [9] and has been shown to modulate the circadian activity of different key metabolites such as SIRT1 [10], AMPK [11] or PGC1-α [12].
Overall, there is growing evidence that circadian rhythms and metabolic processes maintain intricate interactions in order to ensure energy homeostasis [13–16].
The molecular circadian clock is responsive to environmental and metabolic cues while maintaining tight control over gene expression(A) Cells from the suprachiasmatic nucleus (SCN) within the brain receive external environmental (e.g. light) and internal (e.g. nutrients and hormones) cues that influence gene expression of core “clock” genes. The CLOCK/BMAL1 heterodimer binds to E-box enhancer elements in the promoter of core clock genes, such as Period (Per) and Cryptochrome (Cry), and other clock-controlled genes (Ccg) responsible for clock output. PER and CRY proteins accumulate in the cytoplasm where they complex with Casein Kinase 1 (CK1) and translocate back into the nucleus inhibiting their own transcription. Clock output is responsible for synchronizing rhythms in peripheral clocks and influencing processes such as rhythmic nutrient metabolism and uptake, and the sleep/wake and body temperature cycles. Nutrient signals (e.g. NAD+, AMPK) participate in crosstalk with the core clock by feeding back and influencing nuclear receptors such as Rev-erbαand the retinoic acid-related orphan receptor α (RORα), thus contributing to molecular rhythm generation. Positive and negative regulatory mechanisms are represented with green and red broken lines, respectively. (B) The circadian clock generates rhythms in gene expression, but post-transcriptional mechanisms such as deadenylation can also alter rhythmic mRNA processing. mRNA stability is maintained in part through polyadenylation, a process involving the addition of 3’ adenosine residues creating a polyA tail on messenger transcripts. Conversely, polyA tail removal through deadenylation leads to transcript degradation or silencing. The clock and metabolic cues influence rhythmic expression of the gene Nocturnin (Noc), encoding a circadian deadenylase. This could be one mechanism whereby the clock exerts tight control over expression of genes involved in nutrient metabolism through regulating post-transcriptional modifications.
Peripheral tissues can respond directly to clock output, or local oscillators within the tissues can be entrained by systemic cues and affect tissue-specific gene expression [14]. Likewise, metabolic perturbations such as high fat diet (HFD) feeding produce nutrient signals that can feedback and influence clock gene expression [17].
This intimate relationship between clocks and metabolism was notably exhibited in mice harboring a mutation in the Clock gene sequence (ClockΔ19). These mice display many physiological disruptions associated with the metabolic syndrome including hyperlipidemia, hyperglycemia and hypoinsulinemia [18].
Disruptions in other core clock genes such as Bmal1 or Per2 lead to disruptions in metabolism as well [reviewed in 3, 19]. While the autoregulatory feedback loops comprising the core oscillator affect gene transcription, there is increasing evidence for circadian control over post-transcriptional modifications [20, 21].
Examples of these modifications include mRNA splicing, silencing and deadenylation [21] which allow for the precise temporal control of gene expression at times that are most beneficial to the energetics, and thus survival, of an organism. Study of the circadian deadenylase Nocturnin is therefore vital to understanding this link between the circadian clock and metabolism.
More information: “The Metabolites NADP+ and NADPH are the Targets of the Circadian Protein Nocturnin (Curled),” Nature Communications(2019). DOI: 10.1038/s41467-019-10125-z
Journal information: Nature Communications
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