Circadian (daily) rhythms are a crucial component of human health that regulates sleep, alertness, hormones, metabolism, and many other biological processes. The ultimate explanation for the mechanism of circadian oscillators will require characterizing the structures, functions, and interactions of the molecular components of these clocks. The current project is to elucidate the basic principles of circadian clocks at a biophysical/molecular level in a model system, the prokaryotic cyanobacteria, where genetic/biochemical studies have identified three key clock proteins, KaiA, KaiB, and KaiC. These three proteins can reconstitute a circadian oscillator in vitro; this remarkable demonstration has led to a re-evaluation of our understanding of circadian clocks in all organisms, including mammals. Moreover, the crystal structures of the KaiA, KaiB, and KaiC proteins have been reported-these are the first clock proteins to have their 3-D structures determined. The advent of atomic resolution structures of the molecular components of this circadian pacemaker marks a dramatic watershed in circadian research by ushering in truly molecular analyses of circadian mechanisms. The current project will determine the molecular basis of the core clockwork by biochemical/biophysical, genetic, and structural approaches. Three critically important unanswered questions in chronobiology are to explain how the biochemical mechanism (i) can be temperature compensated, (ii) operates rhythmic outputs under some conditions but not others, and (iii) is able to keep time accurately in the face of changes in metabolism. This project will face these issues head-on. Temperature compensation of this biological clock will be investigated by screening for temperature dependent mutants of KaiC, KaiB, and KaiA in vivo. These mutations will be mapped onto the 3-D structures of the proteins to generate specific hypotheses that will be tested by novel in vitro biochemical analyses and targeted mutations. The rate constants and other biochemical data that result from the analyses of these mutants will be integrated with our previous data to generate models that account for the temperature-compensated, 24 h time constant of the in vitro oscillator. Differential expression of circadian rhythms under some conditions but not others (conditionality) is based on novel mechanisms of codon usage in cyanobacteria, and the mechanism and adaptiveness of this fascinating phenomenon will be analyzed as well as recruited to maximize cost-effective production of biopharmaceuticals. Finally, a novel hypothesis with far-reaching implications will be analyzed, namely that accurate circadian timekeeping requires compensation for metabolic perturbations, of which temperature change is only one among many such perturbations. The answers to these questions will lead to wide-ranging insights into the mechanisms and applications of biological timekeeping.

Public Health Relevance

This project will clarify circadian mechanisms at molecular levels that were heretofore unreachable. Biological clocks have been found to be crucial for physical and mental health; cancer, metabolic disorders, cardiovascular disease, and depression are associated with the disruption of these timing systems by genetic and/or environmental disturbance (i.e., by shiftwork or 'jet-lag'). Knowledge of circadian mechanisms along with the development of therapies to properly phase sleep will allow us to enhance health, performance, and well-being in addition to improving the quality of life for depressed subjects.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM067152-14
Application #
8990985
Study Section
Cellular Signaling and Regulatory Systems Study Section (CSRS)
Program Officer
Sesma, Michael A
Project Start
2003-01-01
Project End
2019-12-31
Budget Start
2016-01-01
Budget End
2016-12-31
Support Year
14
Fiscal Year
2016
Total Cost
$334,157
Indirect Cost
$121,253
Name
Vanderbilt University Medical Center
Department
Physiology
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Mori, Tetsuya; Sugiyama, Shogo; Byrne, Mark et al. (2018) Revealing circadian mechanisms of integration and resilience by visualizing clock proteins working in real time. Nat Commun 9:3245
Johnson, Carl Hirschie; Zhao, Chi; Xu, Yao et al. (2017) Timing the day: what makes bacterial clocks tick? Nat Rev Microbiol 15:232-242
Tackenberg, Michael C; Johnson, Carl H; Page, Terry L et al. (2017) Revealing Oft-cited but Unpublished Papers of Colin Pittendrigh and Coworkers. J Biol Rhythms 32:291-294
Jazmin, Lara J; Xu, Yao; Cheah, Yi Ern et al. (2017) Isotopically nonstationary 13C flux analysis of cyanobacterial isobutyraldehyde production. Metab Eng 42:9-18
Zhang, Yunfei; Robertson, J Brian; Xie, Qiguang et al. (2016) Monitoring Intracellular pH Change with a Genetically Encoded and Ratiometric Luminescence Sensor in Yeast and Mammalian Cells. Methods Mol Biol 1461:117-30
Ma, Peijun; Mori, Tetsuya; Zhao, Chi et al. (2016) Evolution of KaiC-Dependent Timekeepers: A Proto-circadian Timing Mechanism Confers Adaptive Fitness in the Purple Bacterium Rhodopseudomonas palustris. PLoS Genet 12:e1005922
Shi, S-q; White, M J; Borsetti, H M et al. (2016) Molecular analyses of circadian gene variants reveal sex-dependent links between depression and clocks. Transl Psychiatry 6:e748
Qin, Ximing; Mori, Tetsuya; Zhang, Yunfei et al. (2015) PER2 Differentially Regulates Clock Phosphorylation versus Transcription by Reciprocal Switching of CK1? Activity. J Biol Rhythms 30:206-16
Veatch, Olivia J; Pendergast, Julie S; Allen, Melissa J et al. (2015) Genetic variation in melatonin pathway enzymes in children with autism spectrum disorder and comorbid sleep onset delay. J Autism Dev Disord 45:100-10
Egli, Martin; Johnson, Carl H (2015) Biochemistry that times the day. Biochemistry 54:104-9

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