Previously, I wrote about the circadian rhythm and its regulation in D. melanogaster, a fruit fly. It was largely regulated by the level of certain cytoplasmic proteins and by a protein that’s activated in response to light. These are examples of a transcriptional-translational feedback loop (TTFL) – a reoccurring theme among circadian mechanisms. Those TTFLs and the ones I’ll discuss in this post describe how the production of a protein exhibits a negative feedback loop on the transcription of its gene – that is, the protein inhibits its own production. This mechanism is present in nearly every cell of your body.
While the mammalian circadian rhythm is, understandably, much more complex than D. melanogaster’s, the basic TTFL is quite similar. The complexity, rather, stems from coordinating all the mammalian cells’ activities together. Unlike with what we saw in D. melanogaster, not all of your cells are capable of synchronizing to the light/dark cycle. This responsibility is passed off to the suprachiasmatic nucleus – a small region in your hypothalamus, just above your optic nerves. You can probably deduce why it’s situated there. Some cells, like red blood cells, don’t have a nucleus and thus cannot have transcriptional-translational feedback, yet they abide by a circadian rhythm. Several oxidation and phosphorylation rhythms and have been discovered in red blood cells and appear to be interconnected with the TTFL we know of in DNA-containing cells. There are likely many more mechanisms yet to be discovered that regulate this entire process. Here, I’ll be discussing the most well known of those.
If you haven’t read the introductory post on circadian clocks, I recommend that you do – I’ll be discussing mechanisms that mirror what was previously discussed. We have proteins that are analogous to period (PER) and timeless (TIM) in D. melanogaster. But, because we are much more complex than a fruit fly, there are several differences. First, we have three different PER proteins: PER 1,2, and 3. While there are some slight structural differences between these three (and thus, slight differences in their functions), for all intents and purposes of this post, they serve the same function.
Second, we don’t have TIM proteins. Rather, we have two cryptochrome (CRY) proteins: CRY 1 and 2. To complicate things further, these CRY proteins are not the same as what we saw in D. melanogaster. Their role isn’t to activate in response to light, but instead are analogous to TIM proteins in D. melanogaster – that is, they form heterodimers with the PER proteins. Together, these proteins rise and fall over a 24-hour course, similar to what we saw with PER and TIM, but exhibiting some key differences.
And, just as with CLK and CYC in D. melanogaster, we have proteins that heterodimerize to affect the transcription of PER and CRY. We still have CLK, but we also carry another protein with a similar function called NPAS2. We don’t have CYC, but rather BMAL1. BMAL1 will form a heterodimer with CLK or NPAS2. Here’s an interactive resource to see how the two dimerize on a biochemical level: http://biology.kenyon.edu/BMB/jmol2014/SpencerKyle/index.html?#DIMER
We saw the CLK/CYC heterodimer in D. melanogaster bind to specific gene control regions of DNA called E-boxes. Once bound, their role was to increase transcription of downstream genes. The BMAL1-CLK/NPAS2 heterodimers do the same, increasing the transcription of the per and cry genes, among many, many others. This is rhythmic binding is one reason why many of a cell’s behavior is dictated by a circadian pattern.
Just like before, PER and CRY proteins inhibit the BMAL1-CLK/NPAS2 heterodimer from forming, thereby inhibiting their own production. When levels are high enough, PER and CRY will heterodimerize and enter the nucleus, sequestering BMAL1 and CLK/NPAS2. This process takes time – there are a number of post-translational modifications that must take place in order for this to happen.
Forming the PER/CRY Heterodimer
Post-Translationally Modifying CRY
AMP kinase, a metabolic “sensor”, is an enzyme highly involved in communicating the cell’s energy status. It will, for example, play a critical role in increasing glucose and fatty acid uptake (and their breakdown) as a response to low energy levels. AMPK can phosphorylate CRY, thereby utilizing circadian clocks to transduce nutrient status to the cell. This is one “non-light” way circadian clocks are influenced.
AMPK phosphorylates a specific serine residue of CRY – a modification that will, through a series of events, decrease its stability. When CRY is in this phosphorylated state, its ability to bind to PER is lessened. Instead, the nuclear protein F-box/LRR-repeat protein 3 (FBXL3), gains a higher affinity for CRY, occupying PER’s spot (Lamia, 2010). FBXL3 binds to a flavin adenine dinucleotide (FAD) pocket within CRY. FAD is a redox intermediate involved in metabolism, and the discovery of this site within CRY would suggest that FBXL3 binding could be regulated through multiple mechanisms.
FBXL3 is part of a larger complex of enzymes that catalyze the ubiquitination of proteins, called the SCF complex. FBXL3 recognizes the phosphorylated CRY protein, binds to it, and brings it into proximity with the other enzymes of the complex. These enzymes function together to ubiquitinate CRY, tagging it for degradation.
Another F-box protein that’s highly similar to FBXL3, competes for this same binding site, but has an opposite function. The (primarily) cytosolic protein, FBXL21, stabilizes the CRY proteins.
FBXL21 also forms an SCF complex and ubiquitinates CRY, but the pattern is different. Ubiquitination isn’t solely a tag for degradation – it can be involved in signal transduction, protein stability, and subcellular localization. It would appear then, that the different ubiquitin chain conferred by FBXL21 stabilizes CRY or interferes with its ability to be degraded. And so, the two proteins work to maintain circadian stability by acting as a counterbalance to one another.
Other Post-Translational Modifications
CRY stability is clearly a crucial variably for circadian period length. But, the PER proteins are the rate-limiting factor for the formation of the PER/CRY heterodimer. As such, their regulation is even more dynamic. As we saw with D. melanogaster, casein kinase 2 alpha was a key regulator of clock gene translocation into the nucleus. We, as mammals, also have casein kinases that affect circadian cycle length. Casein kinases 1 delta (CK1δ) and epsilon (CK1ɛ) phosphorylate clock proteins and, although playing a similar role, have distinct functions. CK1δ phosphorylates the PER proteins while CK1ɛ phosphorylates the PER, CRY, and BMAL1 proteins. While the mechanism of action isn’t fully elucidated yet, we do know that mutations in CK1ɛ affect circadian period length and that loss of CK1δ reduces the rate of PER turnover, increasing circadian length (Etchegaray, 2009).
When PER and CRY Come Together
As we’ve seen, there are some regulatory roadblocks PER and CRY must overcome to form a dimer. It is worthwhile then, to know a little bit about how they accomplish this. Their increasing concentration throughout the daytime increases their likelihood of interacting. When they do, a part of the PER protein attaches to CRY, near a pocket that binds to CLK and BMAL1 (Nangle, 2014). This would suggest that PER has some say over CRY’s ability to bind to CLK and BMAL1. PER and CRY “hug” each other, preventing other enzymes from binding and affecting their stability. A critical zinc ion coordinates this first, stabilizing step, until stronger disulfide bonds form between the two (Schmalen, 2014).
Binding of PER to CRY lends to further regulation of the protein, affecting entry into the nucleus and dictating the length of time it can downregulate the activity of CLK and BMAL1. And, as previously mentioned, PER’s binding prevents degradation of CRY via FBXL3. Thus, PER proteins are often described as the “master timekeepers” in circadian literature.
In the Nucleus
When PER/CRY heterodimers enter into the nucleus (around midday), they bind BMAL1-CLK/NPAS2 heterodimers, inhibiting their ability to bind E-boxes. This halts protein production on all of the genes BMAL1-CLK/NPAS2 was bound to. And so, the PER and CRY protein production rate will drop below that of their degradation, resulting in an overall decrease in levels leading into the night. This is a timely process – while PER and CRY proteins are being degraded in the cytoplasm, there are still PER/CRY heterodimers within the nucleus. It isn’t until around midnight (normally) that these protein’s levels hit their minimum, allowing BMAL1-CLK/NPAS2 to upregulate gene expression and starting the cycle over.
This mechanism is nearly identical to what I described in my introductory post. However, there’s more to the story – much more. In the next post, I’ll introduce another layer of regulation on top of this mechanism, further adding to its complexity.
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