In a study published today in Molecular Cell, a team led by Harvard Medical School researcher Charles Weitz shows that a set of core clock proteins organize themselves into a handful of molecular machines that control the precise workings of circadian rhythms. The results begin to explain how circadian clocks run and shed light on the variety of conditions that can develop when something in the clock machinery goes awry.
In the late 1990s, Weitz, the Robert Henry Pfeiffer Professor of Neurobiology at Harvard, and researchers from other labs discovered several key proteins involved in the clock system. These include three different period proteins (PER), two different cryptochrome proteins (CRY), and casein kinase-1 (CK1). When these proteins accumulate inside cells and enter the cell nucleus, they bind to a protein called CLOCK-BMAL1 that is attached to DNA responsible for making more PER and CRY. The influx and accumulation of these proteins inside the nucleus effectively shut down the production of PER and CRY. However, when the levels of PER and CRY drop, the CLOCK-BMAL1 can once again resume work unhindered so that the DNA responsible for making PER and CRY can do its job.
To figure out precisely how these proteins might run the clock, Weitz and colleagues selectively pulled out proteins from the nuclei of mouse cells at the peak of PER and CRY negative feedback. Their findings turned up a single large protein complex that incorporated each of the six important clock proteins: the three PERs, two CRYs, and CK1, along with about thirty other accessory proteins. Additionally, the protein complex was associated with CLOCK-BMAL1, the experiments showed.
Although their initial experiments were done in mouse livers, experiments in other tissues, including kidney and brain, detected the presence of the same large protein complex. The results suggest that this complex, which the researchers named the PER complex, is universal in tissues throughout the body. They also suggest that the six key clock proteins probably don't operate individually; instead, they seem to organize themselves to work in concert to run the circadian clock's negative feedback loop.
To determine when this organization happens, the researchers looked for the presence of the six main clock proteins in the cytoplasm. There, they found four other complexes composed of different groups of the six proteins, one with all six, named the upper complex, and three others missing one or more of these key proteins. The researchers hypothesized that these complexes were in various states of assembly, but that the six key proteins entered the nucleus as a group.
The upper complex also had a seventh protein called GAPVD1, known from other studies to help shepherd chemicals to different locations inside cells. Although the role of GAPVD1 in the circadian clock remains somewhat unclear, Weitz said, experiments in which this protein was trimmed out of the upper complex caused disruption in circadian cycle, an observation that suggests GAPVD1 plays a key role in the clock.
Weitz cautions that the precise orchestration performed by this constellation of proteins in running the body's clock remains yet to be teased out. However, he said, learning more about how these proteins interact has given researchers a clearer clue into inner workings of the system overall.