Modulation of Nocturnin Phosphatase Activity through the Disordered Amino Terminus

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August 2021

Authors

Wickramaratne, Anushka Christobel

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Abstract

The endogenous circadian clock controls the rhythmicity of behavioral and physiological processes and this is entrained by the daily fluctuations in light and dark. Nocturnin (Noct) is a rhythmically expressed gene regulated by the circadian clock that belongs to the CCR4 family of endonuclease-exonuclease-phosphatase (EEP) enzymes. Its expression is induced by acute stimuli and loss of Nocturnin (Noct-/-) in mice results in resistance to diet-induced obesity on a high fat diet and confers a protective effect to oxidative stress in HEK cells. Modeling of full-length Nocturnin reveals a partially structured amino terminus that is disparate from its CCR4 family members. I show that Nocturnin functions as a phosphatase, catalyzing the removal of the 2′-phosphate from NADP(H). High sequence conservation of the leucine zipper (LZ)-like motif, the only structural element in the amino terminus, highlights the potential importance of this domain in modulating phosphatase activity. I use in vitro biochemical and biophysical techniques to demonstrate that the amino terminus and the LZ-like domain are necessary for preserving the active site cleft in an optimal conformation to promote efficient turnover of the substrate. This modulation occurs in cis and is additionally pivotal in maintaining the stability and conformational integrity of the enzyme. These new findings suggest an additional layer of modulating the activity of Nocturnin in addition to its rhythmicity in order to provide fine-tuned control over cellular levels of NADPH. This lays the essential groundwork necessary to further understand the role of the partially structured amino terminus in metabolism and the oxidative stress response through regulation of NADP(H) and NAD(H) levels.

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