biological sequence analysis 1 v12 circadian rhythms are coupled to metabolism review: the...

Biological Sequence Analysis1

V12 Circadian rhythms are coupled to metabolism

Review:

The suprachiasmatic nuclei (SCN) of the hypothalamus are the principal

circadian pacemaker in mammals,

They drive the sleepwake cycle and coordinate subordinate clocks in other

tissues.

Current understanding:

The molecular clockwork within the SCN is being modeled as a combination of

transcriptional and posttranslational negative feedback loops.

Protein products of Period and Cryptochrome genes periodically suppress their

own expression.

O‘Neill et al.Science, 320, 949 (2008)

SS 2009 lecture 12


Circadian rhythms are coupled to metabolism

Open question: It is unclear how long-term, high-amplitude oscillations with a

daily period are maintained.

In particular, transcriptional feedback loops are typically less precise than the

oscillation of the circadian clock and oscillate at a higher frequency than one

cycle per day.

Possible explanations given in V11:

- phosphorylation causes delay,

- secondary loops give stabilization.


SS 2009 lecture 12

Ouzonis, Karp, Genome Res. 10, 568 (2000)

Each distinct substrate occurs in an average of 2.1 reactions.

Intro: Metabolites in E. coli

Intro: Metabolism: Citrate Cycle (TCA cycle) in E.coli

Intro: Coupling of gene transcription and metabolites

Solid arrows indicate direct associations between genes and proteins (via transcription and translation), between proteins and proteins (via direct physical interactions), between proteins and metabolites (via direct physical interactions or with proteins acting as enzymatic catalysts), and the effect of metabolite binding to genes (via direct interactions).

Lines show direct effects, with arrows standing for activation, and bars for inhibition.

The dashed lines represent indirect associations between genes that result from the projection onto 'gene space'. For example, gene 1 deactivates gene 2 via protein 1 resulting in an indirect interaction between gene 1 and gene 2 (drawn after [Brazhnik00]).


Review (V11): circadian rhythms in mammals

Ko & Takahashi Hum Mol Genet 15, R271 (2006)

SS 2009 lecture 11

Evidence for coupling of circadian clocks with metabolism(1) Recombinant cyanobacterial proteins can sustain circadian cycles of

autophosphorylation in vitro, in the absence of transcription,

(2) intracellular signaling molecules cyclic adenosine diphosphate–ribose

(cADPR) and Ca2+ are essential regulators of circadian oscillation in

Arabidopsis and Drosophila.

This indicates that transcriptional mechanisms may not be the sole, or principal,

mediator of circadian pacemaking.


Example of a gene regulatory networkO’Neill and co-workers now show that the transcriptional feedback loops of

theSCN are sustained by cytoplasmic cAMP signaling.

cAMP signaling determines their canonical properties of amplitude, phase, and

period.

Roles of cAMP?

In molluscs, birds, and the mammalian SCN, cAMP is implicated in entrainment or

maintenance of clocks, or both, or mediation of clock output. It has not been

considered as part of the core oscillator.

This extends the concept of the mammalian pacemaker beyond transcriptional

feedback to incorporate its integration with rhythmic cAMP-mediated cytoplasmic

signaling.


What is cAMP

Cyclic adenosine monophosphate (cAMP) is a second

messenger that is important in many biological processes.

cAMP is derived from ATP and used for intracellular signal

transduction in many different organisms, conveying the

cAMP dependent pathway.

In humans, cyclic AMP works by activating cAMP-

dependent protein kinase.

Cyclic AMP binds to specific locations on the regulatory

units of the protein kinase, and causes dissociation

between the regulatory and catalytic subunits

Thus it activates the catalytic units and enables them to

phosphorylate substrate proteins. www.wikipedia.org

Side functions of cAMP

There are some minor PKA-independent functions of cAMP, e.g. activation of

calcium channels.

This provides a minor pathway by which growth hormone releasing hormone

causes release growth hormone

Picture: Epinephrine (adrenaline) binds its receptor, that associates with an

heterotrimeric G protein. The G protein associates with adenylyl cyclase that

converts ATP to cAMP, spreading the signal

www.wikipedia.org

Cyclic cAMP levels in mouse brain

We tracked the molecular oscillations

of the SCN as circadian emission of

bioluminescence by organo-typical

slices from transgenic mouse brain.

Rhythmic luciferase activity controlled

by the Per1 promoter (Per1::luciferase)

revealed circadian transcription, and a

fusion protein of mPER2 and

LUCIFERASE (mPER2::LUC) reported

circadian protein synthesis rhythms.

O‘Neill et al.Science, 320, 949 (2008) Interpretation: Under these conditions, the

cAMP content of the SCN was circadian.

Circadian oscillation of cAMP concentration (blue) and PER2::LUC bioluminescence (red), as well as cAMP concentration in SCN slices treated with MDL-12,330A (MDL) or with forskolin plus IBMX.

Effect of MDL

Idea: can one show that cAMP is the

reason for the oscillations?

Realization: need to suppress

cAMP-production in the cell.

Experiment: treat SCN slices with

MDL, a potent, irreversible inhibitor

of adenylyl cyclase (that synthesizes

cAMP) to reduce concentrations of

cAMP to basal levels.


Interpretation: MDL rapidly suppressed

circadian CRE::luciferase activity,

presumably through loss of cAMP-

dependent activation of CRE sequences.

This caused a dose-dependent decrease in

the amplitude of cycles of circadian

transcription and protein synthesis

observed with mPer1::luciferase and

mPER2::LUC.

MDL also affects the synchronization of the clock

Prolonged exposure to mild

levels of MDL (1.0 mM)

suppressed and desynchro-

nized the transcriptional cycles

of SCN cells.


Can one block cAMP action?


Idea: If cAMP sustains the clock,

interference with cAMP effectors should

compromise pacemaking.

PlanA: treat brain slices with inhibitors

of cAMP-dependent protein kinase. This

had no effect, however, on circadian

gene expression in the SCN.

PlanB: But cAMP also acts through

hyperpolarizing cyclic nucleotide–gated

ion (HCN) channels and through the

guanine nucleotide–exchange factors

Epac1 and Epac2 (Epac, exchange

protein directly activated by cAMP).

The irreversible HCN channel blocker

ZD7288, which would be expected to

hyperpolarize the neuronal membrane,

dose-dependently damped circadian

gene expression in the SCN.

This is consistent with disruption of

transcriptional feedback rhythms by

other manipulations that hyperpolarize

clock neurons.

Time of application of ZD7288

Can cAMP stimulation be recoved?

Idea: Direct activation of the

effectors might compensate,

therefore, for inactivation of

Adenylate Cyclase by MDL.

Observation: A hydrolysis-resistant

Epac agonist transiently activated

oscillations in transcriptional activity

in SCN treated with MDL.


slowing cAMP synthesis

Idea: if cAMP signaling is an integral

component of the SCN pacemaker,

altering the rate of cAMP synthesis

should affect circadian period.

Experiment: 9-(Tetrahydro-2-furyl)-

adenine (THFA) is a noncompetitive

inhibitor of adenylate cyclase that slows

the rate of Gs-stimulated cAMP

synthesis, which attenuates peak

concentrations.


Interpretation: THFA dose-

dependently increased the period of

circadian pacemaking in the SCN, from

24 to 31 hours, with rapid reversal upon

washout

Conclusions on cAMP-coupling


Circadian pacemaking in mammals is sustained.

Its canonical properties of amplitude, phase, and period are determined by

a reciprocal interplay in which transcriptional and posttranslational feedback

loops drive rhythms of cAMP signaling.

Dynamic changes in cAMP signaling, in turn, regulate transcriptional cycles.

Thus, output from the current cycle constitutes an input into subsequent cycles.

The interdependence between nuclear and cytoplasmic oscillator elements we

describe for cAMP also occurs in the case of Ca2+ and cADPR.

This highlights an important newly recognized common logic to circadian

pacemaking in widely divergent taxa.

Implications?

Harrising & NitabachScience, 320, 879 (2008)

These studies raise the question of which mechanisms couple oscillations of

intracellular signaling molecules to the transcriptional feedback loops of

circadian clocks.

Of the cAMP effectors studied by O’Neill et al., only inhibition of - the hyperpolarization-activated cyclic nucleotide–gated ion channel or - the guanine nucleotide–exchange factors Epac 1 and Epac 2

suppressed circadian gene expression.

Implications?

Harrising & NitabachScience, 320, 879 (2008)

Application of an Epac agonist resulted in the phosphorylation and increased

activity of cAMP response element–binding (CREB) protein, a transcription

factor.

This suggests that changes in cAMP signaling could feed into the circadian

transcriptional oscillator by regulating the expression of genes that contain

binding sites for CREB.

Such genes include the circadian clock genes Per1 and Per2.

Effect of cADPR in plantsDodd et al. determined that cADPR concentration peaks during the early hours of the day.

This fluctuation was abolished in plants with defective clock function, indicating that the circadian clock regulates cADPR concentration.

cADPR is synthesized from nicotinamide adenine dinucleotide by the enzyme ADP ribosyl cyclase. Nicotinamide, at 10 to 50 mM concentrations, inhibited ADP ribosyl cyclase and weakened circadian [Ca2+]i oscillation in plant cells.

Dodd et al. also found a correlation between the expression of circadian- and cADPR-regulated genes. Moreover, decreasing the cellular concentration of cADPR lengthened the period of circadian gene expression.

The authors suggest that circadian- regulated cADPR-derived Ca2+ signaling may configure part of the feedback loop that controls the clock (see the figure).

Imaizumi et al.Science, 318, 1730 (2007)

Example of a gene regulatory networkThe results of Dodd et al. raise interesting questions.

The phytohormone abscisic acid, thought to lengthen the clock period, induces

cADPR production, and cADPR gene expression overlaps with that of genes

controlled by abscisic acid.

Does abscisic acid affect the clock partly through cADPR derived signals?

Also, assuming that both IP3-and cADPR-dependent pathways are involved in

generating circadian [Ca2+]i oscillation, do they interact with each other?


Example of a gene regulatory network

Dodd et al. found that a pharmacological inhibitor (U73122 at 1 μM) of

IP3 production did not affect daily [Ca2+]i oscillation.

Because IP3 concentrations were not analyzed, more research is needed to

understand the relative roles of both cADPR and IP3.

In particular, identification of the plant genes that encode the enzymes that

produce cADPR and the proteins that control Ca2+ release by cADPR and IP3 are

required to analyze the functions of these signaling molecules in plants.


Current evidence

Eckel-Mahan & Sassone-Corsi,Nat Struct Mol Biol. 16, 462 (2009)

Recent finding: activity of sirtuin-1 (Sirt1),a longevity-associated protein belongingto a family of NAD+-activated histonedeacetylases oscillates in a circadian fashion.

Outlook1


Whether there are mammalian metabolite oscillations analogous to those of the

yeast metabolic cycle is still unclear, but it remains a tantalizing possibility.

The fact that cellular demands are met temporally as a function of the cell’s

metabolic cycle is likely to be true for all cells, regardless of the organism.

In the context of mammalian Sirt1 circadian activity, it seems likely that

metabolite oscillations in the coenzyme NAD+ must also occur in a cyclical,

circadian manner.

If metabolite fluctuations are organized temporally in a circadian manner, what

might this mean physiologically?

Outlook 2


The central functions of NAD+ in DNA repair, gene silencing, the cell cycle and

circadian control indicate that the consequences of its aberrant regulation could

be numerous and physiologically severe.

It is conceivable that food restriction impinges on circadian rhythms because it

disrupts NAD+-NADH cycling, essentially allowing the redox state of individual

cells and tissues to alter rhythmicity.

Outlook 3


The absence of Clock–Bmal1 dimerization in the presence of increased levels

of oxidized NAD is one piece of evidence supporting this idea.

As such, it is easy to imagine sophisticated schemes coordinating SCN-driven

rhythms with those of a phase-shifted periphery for drug administration and

efficacy.

Already there are numerous drugs, perhaps most commonly known within

cancer chemotherapeutic strategies, administered following a circadian

protocol so that the maximal benefit might be achieved from their use.

Additional slides

Cross-talk


By activating Sirt1, NAD+ conjoins two feedback loops necessary for cross-talk between the circadian clock and metabolite production. The NAD+-salvage pathway is important for regulating intracellular NAD+ levels. After the conversion of nicotinamide (NAM) into nicotinamide mononucleotide (NMN) by NAM phosphoribosyl transferase (NAMPT), NMN is further modified into NAD+ by the nicotinamide mononucleotide adenylyl transferases (Nmnat1, –2 and –3).

Whereas NAM inhibits Sirt1 activity, NAD+-activated Sirt1 feeds back into the NAD+-salvage pathway by directly regulating Nampt gene expression in a Clock–Bmal1-dependent manner. By this mechanism, NAD+ conjoins the two feedback loops, contributing to the fine tuning necessary for achieving energy balance.

biological sequence analysis 1 v12 circadian rhythms are coupled to metabolism review: the...

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