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Circadian Rhythms

Circadian Rhythms and the Biological Clock

Many human behaviors are cyclic with a repeat period of 24 hours (such as sleep/wake cycles). These circadian rhythms, which can be phase-shifted by alternations in environmental cues (such as light intensity, sleep deprivation) must have some biochemical basis. Understanding the mechanisms underlying circadian rhythms and its regulations would prove helpful in developing new ways to help people minimize the influence of jet lag or shift work. In humans, light intensity information is transmitted from the retina to the suprachiasmatic nucleus in the hypothalmus (in the brain), which then signals the pineal gland (behind the hypothalmus) to secrete the hormone melatonin. Its levels rise at night and fall in the day, but its biochemical mechanism of action is still being determined. Other hormones are also involved. What are the biochemical targets of these hormones? What is the basis of circadian changes in individual cells?

One such protein is the membrane enzyme (Hydroquinone) NADH Oxidase (NOX). NADH is a small cellular reducing agent that we will discuss in the future. This enzyme is found in the external plasma membrane of all human cells, including tumor cells. NOX proteins are very usual in that they possess two activities: a NADH oxidase activity (measured by the disappearance of NADH) and a protein disulfide isomerase (thiol interchange) activity (measured by the renaturation of RNase A using cCMP as a substrate or the cleavage of dithiodipyridine). These activities alternate in a temporal sense with a 24 minute period! The normal cellular form of the protein is constitutively expressed and responsive to hormones. A form found specifically on tumor cells, tNOX, is inhibited by certain chemotherapeutic drugs and by capsaicin (the active ingredient in hot peppers), and is not responsive to hormones (suggesting unregulated activity). tNOX has a 22 minute period. In cancer patients, a truncated form of tNOX (ttNOX), formed by limited proteolysis from tNOX, is found in the serum. NOX proteins are also resistant to proteases and can self aggregate to form "amyloid" type fibrils similar to those found in prion diseases. They can also interact with other proteins and render them protease resistant. The temporal periods of both proteins are independent of temperature and can be "entrained" through the appropriate stimuli. What structural features of the protein can account for unique properties? Early evidence (using FTIR and CD) suggests that changes in secondary structure (similar to prion proteins) occurs involving changes from alpha helices to beta sheets. Perhaps the protein can exist in two distinct, yet similar conformations, each with a different activity.

The tNOX gene has been cloned and expressed. It encodes a protein of 610 amino acids, and its activities, when expressed in bacteria, cycle in 22 minutes. Site-specific mutations produce periods of 36 minutes (for Cys575Ala) and 42 minutes (for Cys558Ala) mutants. These proteins show the same activities and periods when produced in transformed eukaryotic COS-1 cells. This cell surface proteins affects the circadian rhythms of the whole cell. A normal protein in the cell, glyceraldehyde -3-phosphate dehydrogenase, (GAPDH) exhibits a 24 hour activity circadian rhythm in normal COS cells (which have a constitutively expressed NOX gene product). When transformed with the mutants, the demonstrate not only a 24 hour GAPDH activity, but in addition, a 22, 36, or 42 hour activity when transformed with the gene for tNOX, the tNOX Cys575Ala mutant, and the tNOX Cys558Ala mutant respectively.