The Circadian Clock: Regulating the Rhythm of Life

Transcription

1 The Circadian Clock: Regulating the Rhythm of Life Christopher G. Gomez 1 1BIOC 462aH, University of Arizona

2 Abstract The human body follows a daily cycle of sleep, of hunger which is independent of the environment. Many people obey the cycle of the sun, remaining active during the light hours and sleeping during the dark hours. The circadian clock system is based on the function of an internal oscillator which dictates the biological time. The most basic and well-understood circadian system is that of the cyanobacteria Synechococcus elongatus and is composed of three clock proteins: KaiA, KaiB, and KaiC. KaiC dictates the circadian rhythm by phosphorylation and dephosphorylation of Ser431 and Thr432. KaiA forms a complex with KaiC and stimulates phosphorylation, while KaiB inhibits the activity of KaiA and allows for dephosphorylation to occur. The direct structure-function relationship between the phosphorylation of the KaiC protein remains unknown, as are the complexities of the oscillator systems of multicellular organisms. However, the dysfunction of the circadian clock system has now been linked to diseases and disorders, such as low blood pressure and some sleep disorders. Further understanding of the function of this system may lead to the successful treatment of these health issues.

3 Introduction The circadian rhythm follows an approximately 24 hour period for many organisms and dictates rhythmic biological processes such as hunger and sleep. This rhythm is controlled by one or more molecular oscillators which can be affected by certain environmental cues while keeping consistent function through fluctuations of others. Temperature, for example, will not drastically affect the consistency of these circadian clocks. On the other hand, light cues and changes in activity or feeding schedule can reset the circadian rhythm to match the local time referred to as entrainment (Harmer et al., 2001). For example, this phenomenon is apparent with long distance travel in the form of jet lag. Much work has been attempted to solve the complex mechanisms that dictate the rhythm of the internal clock. The circadian clock mechanism of the cyanobacteria Synechococcus elongatus has been found to be relatively simple compared to many multicellular organisms and makes for an excellent model organism. S. elongatus employs a three protein system which makes up its core oscillating system (Dong et al., 2010). The circadian system of this cyanobacteria is composed of KaiA, KaiB, and KaiC clock proteins. KaiC dictates the circadian rhythm by phosphorylation and dephosphorylation of Ser431 and Thr432. KaiA forms a complex with KaiC and stimulates phosphorylation, while KaiB inhibits the activity of KaiA and allows for dephosphorylation to occur. The intricacies of other more complex organisms circadian clocks have yet to be fully understood. Many involve systems of several oscillators whose mechanisms affect each other, further delaying our understanding.

4 Even so, correlations are being made between abnormalities in the circadian clock system and some diseases and disorders (Jones et al., 2013). Dysfunctions in the circadian system have been related to abnormalities such as low blood pressure and some sleep disorders. Further understanding of the circadian clock system may lead to the development of treatments for some of these disorders. Understanding the Basic Circadian Clock: S. elongatus The cyanobacterial circadian clock of Synechococcus elongatus is composed of three proteins. The largest is KaiC (PDB ID: 2GBL, crystal structure by Pattanayek et. al, 2004), a hexameric enzyme shown in Figure 1. Composed largely of alpha helices, KaiC is able to autophosphorylate and autodephosphrylate at specific amino acid side chains, Ser431 and Thr432 in response to free protein concentration levels of the other two primary clock proteins, KaiA and KaiB (Rust et al., 2007). KaiA (PDB ID: 1R8J, crystal structure by Ye et. al, 2004), shown in Figure 2, is a homodimer which stimulates KaiC to phosphorylate while KaiB (PDB ID: 1R5P, crystal structure by Garces et al., 2004), shown in Figure 3, inhibits the activity of KaiA. As seen in Figure 3, KaiB appears to be homodimeric (Mackey and Golden, 2007). The phosphorylation and dephosphorylation of the KaiC protein occurs rhythmically over an approximately 24 hour period in response to stimulation and inhibition by the KaiA and KaiB proteins; it controls genetic expression patterns for the bacterium (Paddock et al., 2013). KaiA binds to the C-terminus of the KaiC protein, stimulating the phosphorylation of Thr432 (Pattanayek et al., 2006), shown in Figure 5. Pattanayek et al. explored the KaiA-KaiC interaction by deleting 25 amino acids from

5 the C-terminal end of the KaiC protein. They found that this deletion did not inhibit the KaiC protein from folding and forming the hexameric structure, but it did prevent KaiA from forming the activating complex with KaiC. The initial phosphorylation reaction then stimulates a second phosphorylation of the adjacent serine amino acid, Ser431 (Dong et al., 2010). Phosphorylated Ser431 and Thr432 are shown in Figure 4. In order for dephosphorylation to completely occur, the activity of KaiA must be inhibited therefore preventing the rephosphorylation of KaiC, and KaiB serves as this necessary inhibitor. The fully phosphorylated KaiC (where both Ser431 and Thr432 are phosphorylated) will spontaneously dephosphorylate. KaiB binds to the KaiA:KaiC complex and inactivates KaiA, allowing for the dephosphorylation of the KaiC residues Ser431 and Thr432 (Mackey and Golden, 2007). These complexes form throughout the circadian cycle, and the proportions of the different complexes fluctuate with the circadian rhythm. The level of KaiC phosphorylation reaches the same point twice each cycle once when phosphorylation levels are increasing, and the second time when levels are decreasing so the concentration of phosphorylated KaiC cannot be solely responsible for the clock s function (Rust et al., 2007). To test this, Rust et al. isolated different phosphoroforms of the KaiC protein to prepare solutions with different levels of KaiC phosphorylated at Ser431 (S-KaiC) and KaiC phosphorylated at Thr432 (T-KaiC). KaiB was incubated in each of the above KaiC solutions followed by the addition of KaiA to investigate the conditions which stimulate further phosphorylation. They found that the solution with high T-KaiC concentration underwent phosphorylation upon the addition of KaiA and KaiB, and that the solution with high S-KaiC concentration began the dephosphorylation

6 process. Terauchi et al. (2007) conducted a similar study to understand the timing of the S. elongatus circadian rhythm. They found that ATP concentration in their reaction mixtures fluctuated by approximately 60 μm in a 24 hour period, with peaks at 0, 24, and 48 hours. They also found that kinase activity fluctuated as a result of changing ATP concentrations and ATPase activity. The phosphorylation process seems to affect the KaiC protein much in the way that oxygenation affects hemoglobin: when one of the KaiC monomers is phosphorylated, it affects the phosphorylation of the other subunits, much like cooperative binding. Eguchi et al. (2008) suggest that KaiC monomers not only affect the phosphorylation of one another, but also shuffle between hexamers in order to keep the KaiC proteins synchronized. They postulate that this also keeps the circadian rhythm synchronized, as asynchrous KaiC proteins would throw off the rhythm. Circadian Clocks of Multicellular Organisms Unlike the cyanobacteria S. elongatus, the circadian clock mechanisms of multicellular organisms seem to function based on multiple oscillators instead of a single oscillator system. In mammals, the central oscillator mechanism seems to be located in the brain, in a portion called the suprachiasmatic nucleus, or the SCN (Harmer et al., 2001). With specialized tissues and organ systems, certain rhythmic biological functions are also controlled by peripheral oscillators; of these systems, though, only the SCN oscillator is sensitive to entrainment by light (Bell-Pedersen et al., 2005). The mammalian circadian clock system, like that of the cyanobacteria, is a system of positive and negative feedback loops.

7 Defective Circadian Clock Systems While the workings of the mammalian circadian clock system are not as deeply understood as that of the cyanobacteria S. elongatus, research is being conducted concerning the role of the circadian rhythm in disease. In mice, a study by Sahar and Sassone-Corsi (2012) has linked conditions such as low blood pressure, altered renal function, and diabetes mellitus to defects in the proteins associated with the circadian clock system. Irregularities in the human circadian rhythm have also been linked to some sleep disorders. One example is the advanced sleep phase disorder in which a person s circadian cycle is shifted a few hours ahead of the solar time in which he or she lives and causes him or her to fall asleep earlier in the evening and wake up much earlier in the day than if their circadian rhythm was synchronized with the solar cycle (Jones et al., 2013). Disorders like this might become treatable, even correctable, if more can be learned about the complexities of the human circadian rhythm. One method which has been explored is the use of melatonin in resynchronizing abnormal circadian cycles. Melatonin is a hormone which functions naturally in regulating the sleep cycle of many organisms, including humans. Arendt and Skene (2005) have found that melatonin may be used to entrain the circadian clock to follow a desired cycle based on the time intervals in which the hormone is administered. Their studies suggest that such treatment may be effective in treating disorders related to the internal circadian rhythm. However, further understanding of the human circadian clock system will allow more targeted approaches for regulating the self-sustaining oscillating system which governs the rhythm of our lives.

8 References ARENDT, J., & SKENE, D. (2005). Melatonin as a chronobiotic. Sleep Medicine Reviews.9, BELL-PEDERSEN D, CASSONE VM, EARNEST DJ, GOLDEN SS, HARDIN PE, THOMAS TL, & ZORAN MJ. (2005). Circadian rhythms from multiple oscillators: lessons from diverse organisms. Nature Reviews. Genetics. 6, DONG G, KIM YI, & GOLDEN SS. (2010). Simplicity and complexity in the cyanobacterial circadian clock mechanism. Current Opinion in Genetics & Development. 20, EGUCHI K, YODA M, TERADA TP, & SASAI M. (2008). Mechanism of robust circadian oscillation of KaiC phosphorylation in vitro. Biophysical Journal. 95, GARCES RG, WU N, GILLON W, & PAI EF. (2004). Anabaena circadian clock proteins KaiA and KaiB reveal a potential common binding site to their partner KaiC. The EMBO Journal. 23, HARMER SL, PANDA S, & KAY SA. (2001). Molecular bases of circadian rhythms.annual Review of Cell and Developmental Biology. 17, JONES CR, HUANG AL, PTA C EK LJ, & FU YH. (2013). Genetic basis of human circadian rhythm disorders. Experimental Neurology. 243, MACKEY SR, & GOLDEN SS. (2007). Winding up the cyanobacterial circadian clock.trends in Microbiology. 15, PADDOCK ML, BOYD JS, ADIN DM, & GOLDEN SS. (2013). Active output state of the Synechococcus Kai circadian oscillator. Proceedings of the National Academy of Sciences of the United States of America. 110,

9 PATTANAYEK R, WANG J, MORI T, XU Y, JOHNSON CH, & EGLI M. (2004). Visualizing a circadian clock protein: crystal structure of KaiC and functional insights.molecular Cell. 15, PATTANAYEK R, WILLIAMS DR, PATTANAYEK S, XU Y, MORI T, JOHNSON CH, STEWART PL, & EGLI M. (2006). Analysis of KaiA-KaiC protein interactions in the cyano-bacterial circadian clock using hybrid structural methods. The EMBO Journal. 25, RUST, M. J., MARKSON, J. S., LANE, W. S., FISHER, D. S., & O'SHEA, E. K. (2007). Ordered Phosphorylation Governs Oscillation of a Three-Protein Circadian Clock.Science. 318, TERAUCHI, K., KITAYAMA, Y., NISHIWAKI, T., MIWA, K., MURAYAMA, Y., OYAMA, T., & KONDO, T. (2007). ATPase activity of KaiC determines the basic timing for circadian clock of cyanobacteria. Proceedings of the National Academy of Sciences. 104, YE S, VAKONAKIS I, IOERGER TR, LIWANG AC, & SACCHETTINI JC. (2004). Crystal structure of circadian clock protein KaiA from Synechococcus elongatus. The Journal of Biological Chemistry. 279,

10 Figure Legends Figure 1: Ribbon diagram of S. elongatus KaiC protein as seen from the side. One of the six monomers is colored red to show the structure of one of the monomeric units. Figure 2: Ribbon diagram of S. elongatus KaiA protein. A homodimer, the two monomeric chains are colored differently to differentiate between the two identical subunits. Figure 3: Ribbon diagram of S. elongatus KaiB protein. Also a homodimer, the two identical chains are colored separately for differentiation. Figure 4: Ribbon diagram of the phosphorylation sites on one of the KaiC subunits. Colored green in the full-view of the protein, the close-up view shows the phosphorylated side chains Ser 431 and Thr 432. Figure 5: Ribbon diagram of KaiA-KaiC complex. KaiA is shown in green, and KaiC is shown in blue.

11 Figure 1

12 Figure 2

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14 Figure 4 TPO 432 SEP 431

15 Figure 5