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Journal ArticleDOI

Peroxiredoxins are conserved markers of circadian rhythms

24 May 2012-Nature (Nature Publishing Group)-Vol. 485, Iss: 7399, pp 459-464

TL;DR: It is shown that oxidation–reduction cycles of peroxiredoxin proteins constitute a universal marker for circadian rhythms in all domains of life, by characterizing their oscillations in a variety of model organisms and exploring the interconnectivity between these metabolic cycles and transcription–translation feedback loops of the clockwork in each system.

AbstractCellular life emerged ∼3.7 billion years ago. With scant exception, terrestrial organisms have evolved under predictable daily cycles owing to the Earth’s rotation. The advantage conferred on organisms that anticipate such environmental cycles has driven the evolution of endogenous circadian rhythms that tune internal physiology to external conditions. The molecular phylogeny of mechanisms driving these rhythms has been difficult to dissect because identified clock genes and proteins are not conserved across the domains of life: Bacteria, Archaea and Eukaryota. Here we show that oxidation–reduction cycles of peroxiredoxin proteins constitute a universal marker for circadian rhythms in all domains of life, by characterizing their oscillations in a variety of model organisms. Furthermore, we explore the interconnectivity between these metabolic cycles and transcription–translation feedback loops of the clockwork in each system. Our results suggest an intimate co-evolution of cellular timekeeping with redox homeostatic mechanisms after the Great Oxidation Event ∼2.5 billion years ago. Daily oxidation–reduction cycles of peroxiredoxin proteins are shown to be conserved in all domains of life, including Bacteria, Archaea and Eukaryota. Most living organisms possess an endogenous circadian clock that ties their metabolism to a 24-hour day–night cycle. 'Clock genes' have been studied in many organisms and their variety has encouraged the view that each clock evolved independently. But there is a unifying factor: a non-transcriptionally based form of circadian oscillation, involving the oxidation–reduction cycles of peroxiredoxin proteins, has been identified in human red blood cells and algae. This study demonstrates that these redox cycles are conserved in all domains of life, including Bacteria, Archaea and Eukaryota, pointing to the possibility that this type of cellular timekeeping has co-evolved with redox homeostatic mechanisms across organisms for billions of years. The link may go back 2.5 billion years, to the Great Oxidation Event that consigned anaerobic metabolism to the margins of evolutionary history.

Topics: Bacterial circadian rhythms (60%), Circadian clock (58%), CLOCK (56%)

Summary (3 min read)

Peroxiredoxins are conserved markers of

  • The molecular phylogeny of mechanisms driving these rhythms has been difficult to dissect because identified clock genes and proteins are not conserved across the domains of life: Bacteria, Archaea and Eukaryota.
  • Therefore, across phylogenetic kingdoms, there are apparently no common ‘clock’ components, suggesting that daily timekeeping evolved independently within different lineages.
  • The authors previously characterized circadian cycles of peroxiredoxin oxidation using antiserum directed against the oxidized active site, which recognizes both over- (PRX-SO2) and hyper- (PRX-SO3) oxidized forms9,10,14.
  • 7MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.

Peroxiredoxin rhythms in eukaryotes

  • Using the PRX-SO2/3 antiserum, the authors first examined circadian time courses from a range of eukaryotes under constant conditions (that is, in the absence of external timing cues).
  • To extend these findings beyond vertebrates, the authors examined peroxiredoxin rhythms in the fruitfly Drosophila melanogaster.
  • The authors pooled whole heads from insects maintained in constant darkness over two circadian cycles after they had been stably entrained to 12 h light, 12 h dark cycles.
  • Again, circadian oscillations in PRX-SO2/3 immunoreactivity were observed, as well as in the clock protein Timeless (TIM) (Fig. 2b).
  • Similarly, seedlings from the plant Arabidopsis thaliana exhibited robust PRX-SO2/3 oscillations in free-running conditions of constant light, which were also seen in the filamentous fungus Neurospora crassa—another well-characterized clock model system (Fig. 2c and Supplementary Fig. 2).

Peroxiredoxin rhythms in prokaryotes

  • Having observed ,24 h peroxiredoxin oxidation rhythms in organisms with nucleated cells, the authors next sought to examine representative prokaryotes from each major domain—Bacteria and Archaea.
  • Because all three Kai proteins are expressed together in only a small number of bacterial species, and in no known archaea18, this system cannot represent a general prokaryotic clock mechanism.
  • The authors postulated that, regardless of the timekeeping mechanism, the peroxiredoxin oxidation cycles they observe reflect an absolutely conserved rhythmic cellular output.
  • This indicates the presence of an underlying capacity for circadian timing in both mutant strains, which was clearly perturbed by the absence of functional transcriptional feedback circuitry (Fig. 4a).
  • To establish the wider relevance of these findings, the authors also examined similar mutants in the fungus Neurospora crassa.

Implications for clock evolution

  • The authors have observed ,24 h cycles of peroxiredoxin oxidation–reduction in all domains of life and consequently, the possibility that cellular rhythms share a common molecular origin seems increasingly plausible.
  • Approximately 2.5 billion years ago, photosynthetic bacteria acquired the capacity for photo-dissociation of water, leading to the geologically rapid accumulation of molecular oxygen during the Great Oxidation Event (GOE), when anaerobic life underwent a catastrophic decline35.
  • If there was considerable pressure for the co-evolution of metabolic/ROS pathways with cellular timekeeping systems, then evidence for this should exist in the phylogenetic trees of their component mechanisms.
  • See Supplementary Table 9 for cycle period estimates (by harmonic regression) and detailed statistics (by ANOVA), as well as Supplementary Fig. 10 for TIM and FRQ immunoblots for fruitfly and fungus, respectively.
  • This suggests that similarities in the evolutionary profiles of these cellular mechanisms go beyond those that would be expected simply based on the time since a common ancestry, because even highly conserved proteins had considerably inferior correlations to peroxiredoxin (Supplementary Fig. 6 and Supplementary Table 8)42.

Concluding remarks

  • It has long been recognized that oxygen-sensing PAS-domain proteins are involved in the clockwork of many eukaryotes, but the rationale behind this has remained elusive23,43–45.
  • In light of their current findings, the authors speculate that sensing and responding to oxidative cycles in cellular environments could have driven the evolution of circadian rhythms, and maintained the intrinsic link between clocks and metabolism (Fig. 6).
  • A direct prediction therefore, is that organisms that lack ROS detoxification systems will not have circadian rhythms.
  • At least one such class of organism exists on Earth, an example being the hyperthermophilic archaea Methanopyri (NCBI taxonomy accession 183988).
  • Given its distinct anoxic environmental niche and methanogenic metabolism46, there may never have been a selective evolutionary pressure to develop circadian timekeeping as the authors know it.

METHODS SUMMARY

  • A. thaliana, D. melanogaster, H. salinarum sp. NRC-1, M. musculus, N. crassa, O. tauri and S. elongatus were bred, grown or cultured in appropriate conditions, synchronized by specific methods normally used in each organisms, and then sampled under constant conditions of either darkness or light, depending on the organism.
  • Immunoblotting was performed after protein transfer to nitrocellulose membranes.
  • Interspecies plots were generated, which contain a simplified representation of the correlation between the interprotein distances in phylogenetic trees for each protein being compared.
  • Statistical comparisons between Mirrortree correlation coefficients were performed as detailed above.
  • C, Co-evolution of cyanobacteria oscillator components and peroxiredoxin proteins.

Published online 16 May 2012.

  • The adaptive value of circadian clocks; an experimental assessment in cyanobacteria.
  • The oligomeric conformation of peroxiredoxins links redox state to function.

METHODS

  • Equal protein loading was confirmed by gel electrophoresis and Coomassie staining of gels loaded with equal volumes of lysate from each time point in each replicate set.
  • Circadian time (CT) 0 was operationally defined as the nadir in bioluminescence signal, and CT12 was taken to be at the peak.
  • Correlation coefficients obtained for analyses of KaiC distance versus other conserved bacterial proteins (including KaiB, KaiC and PRX2) were compared by converting them to a normally distributed metric using Fisher’s r-to-z transformation: r0~ 1 2 ln 1zr 1{r in which r is the Mirrortree correlation coefficient; and r9 is the Fisher-transformed correlation coefficient.

CORRECTIONS & AMENDMENTS

  • In the author list of this Article, the names of Gerben van Ooijen and Maria Olmedo should also have been asterisked, indicating their equal contributions.
  • This error has been corrected in the HTML and PDF versions of the original paper.
  • *These authors contributed equally to this work.

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University of Groningen
Peroxiredoxins are conserved markers of circadian rhythms
Edgar, Rachel S.; Green, Edward W.; Zhao, Yuwei; van Ooijen, Gerben; Olmedo, Maria; Qin,
Ximing; Xu, Yao; Pan, Min; Valekunja, Utham K.; Feeney, Kevin A.
Published in:
Nature
DOI:
10.1038/nature11088
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Citation for published version (APA):
Edgar, R. S., Green, E. W., Zhao, Y., van Ooijen, G., Olmedo, M., Qin, X., Xu, Y., Pan, M., Valekunja, U.
K., Feeney, K. A., Maywood, E. S., Hastings, M. H., Baliga, N. S., Merrow, M., Millar, A. J., Johnson, C. H.,
Kyriacou, C. P., O'Neill, J. S., Reddy, A. B., & O’Neill, J. S. (2012). Peroxiredoxins are conserved markers
of circadian rhythms.
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ARTICLE
doi:10.1038/nature11088
Peroxiredoxins are conserved markers of
circadian rhythms
Rachel S. Edgar
1
*, Edward W. Green
2
*, Yuwei Zhao
3
*, Gerben van Ooijen
4
*, Maria Olmedo
5
*, Ximing Qin
3
,YaoXu
3
, Min Pan
6
,
Utham K. Valekunja
1
, Kevin A. Feeney
1
, Elizabeth S. Maywood
7
, Michael H. Hastings
7
, Nitin S. Baliga
6
, Martha Merrow
5
,
Andrew J. Millar
4,8
, Carl H. Johnson
3
, Charalambos P. Kyriacou
2
, John S. O’Neill
1
& Akhilesh B. Reddy
1
Cellular life emerged 3.7 billion years ago. With scant exception, terrestrial organisms have evolved under predictable
daily cycles owing to the Earth’s rotation. The advantage conferred on organisms that anticipate such environmental
cycles has driven the evolution of endogenous circadian rhythms that tune internal physiology to external conditions.
The molecular phylogeny of mechanisms driving these rhythms has been difficult to dissect because identified clock
genes and proteins are not conserved across the domains of life: Bacteria, Archaea and Eukaryota. Here we show that
oxidation–reduction cycles of peroxiredoxin proteins constitute a universal marker for circadian rhythms in all domains
of life, by characterizing their oscillations in a variety of model organisms. Furthermore, we explore the
interconnectivity between these metabolic cycles and transcription–translation feedback loops of the clockwork in
each system. Our results suggest an intimate co-evolution of cellular timekeeping with redox homeostatic
mechanisms after the Great Oxidation Event
2.5 billion years ago.
Circadian rhythms are considered to be a feature of almost all living
cells. When isolated from external stimuli, organisms exhibit self-
sustained cycles in behaviour, physiology and metabolism, with a
period of approximately 24 h
1
. Circadian clocks afford competitive
selective advantages that have been observed experimentally
2,3
, and
disturbance of circadian timing in humans, as seen in rotational shift
work and jet lag, carries long-term health costs
4
. For all organisms in
which the molecular timing mechanism has been investigated, a com-
mon model has arisen, namely a transcription–translation feedback
loop (TTFL). TTFL components are not, however, shared between
organisms. For example, the cyanobacterial clock is modelled around
three proteins: KaiA, B and C. In the fungus Neurospora crassa, a loop
involving the protein FREQUENCY (FRQ) and the WHITE
COLLAR (WC) complex is thought to drive cellular rhythms, whereas
the plant TTFL involves elements including TOC1 and CCA1 (refs 1,
5). Furthermore, although some multicellular organisms such as
Drosophila and humans possess homologous components (for
example, the Period proteins), their functions seem to differ between
organisms
6–8
. Therefore, across phylogenetic kingdoms, there are
apparently no common ‘clock’ components, suggesting that daily
timekeeping evolved independently within different lineages. The
converse, however, could equally be true, and the primary premise
of this study was therefore to test the hypothesis that circadian clocks
may instead have a common ancestry.
Conservation of peroxiredoxin in circadian systems
Recent studies show that the oxidation state of highly conserved per-
oxiredoxin (PRX) proteins exhibit circadian oscillations in cells from
humans, mice and marine algae
9,10
, probably reflecting an endogenous
rhythm in the generation of reactive oxygen species (ROS)
10
. Because
virtually all living organisms possess peroxiredoxins
11
, we proposed
that this marker for circadian rhythms in metabolism may be
functionally conserved across all three phylogenetic domains:
Archaea, Bacteria and Eukaryota. Peroxiredoxins are peroxidases,
the activity of which is dependent on the oxidation of a key
‘peroxidatic’ cysteine residue (C
p
) in the active site, that is absolutely
conserved, as are neighbouring proline and threonine/serine residues:
conforming to a PXXX(T/S)XXC
p
consensus
12
. Crucially, the catalytic
cysteine can become over- or hyperoxidized (PRX-SO
2/3
), rendering
the peroxiredoxin catalytically inactive, but able to participate in ROS
signalling and chaperone activity
13
. Once overoxidized, peroxiredoxin
can be recycled by sulphiredoxin.
We previously characterized circadian cycles of peroxiredoxin oxida-
tion using antiserum directed against the oxidized active site, which
recognizes both over- (PRX-SO
2
) and hyper- (PRX-SO
3
)oxidized
forms
9,10,14
. To determine whether this antiserum (raised against an
oxidized DFTFVCPTEI peptide) could be used to assay over-/hyper-
oxidation in diverse species, we performed several sequence alignments
to compare peroxiredoxin protein sequences across a variety of cir-
cadian model organisms. This revealed a remarkable degree of conser-
vation across all phylogenetic domains, especially within the active site
(Fig. 1a, Supplementary Fig. 1 and Supplementary Tables 1–7). Even
when we examined the structure of HyrA, the most distantly related
peroxiredoxin orthologue in the archaeon Halobacterium salinarum sp.
NRC-1, we found that amino acid substitutions would not perturb the
geometry of the active site (Fig. 1b). Together, these findings suggested
that the same antiserum could be used to probe oxidation rhythms in
potentially any organism expressing a peroxiredoxin protein. This was
confirmed by gene knockout and peroxide treatment in several repres-
entative organisms (Supplementary Figs 3 and 7–9).
*These authors contributed equally to this work.
1
Department of Clinical Neurosciences, University of Cambridge Metabolic Research Laboratories, NIHR Biomedical Research Centre, Institute of Metabolic Science, University of Cambridge,
Addenbrooke’s Hospital, Cambridge CB2 0QQ, UK.
2
Department of Genetics, University of Leicester, Leicester LE1 7RH, UK.
3
Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee
37235-1634, USA.
4
Synthetic and Systems Biology (SynthSys), Mayfield Road, Edinburgh EH9 3JD, UK.
5
Department of Molecular Chronobiology, Center for Life Sciences, University of Groningen, 9700
CC Groningen, The Netherlands.
6
Institute for Systems Biology, 401 Terry Avenue North, Seattle, Washington 98109, USA.
7
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.
8
School of Biological Sciences, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JR, UK.
24 MAY 2012 | VOL 485 | NATURE | 459
Macmillan Publishers Limited. All rights reserved
©2012

Peroxiredoxin rhythms in eukaryotes
Using the PRX-SO
2/3
antiserum, we first examined circadian time
courses from a range of eukaryotes under constant conditions (that
is, in the absence of external timing cues). In mice, PRX-SO
2/3
and
total PRX1 exhibited a daily cycle in liver tissue and also in the central
pacemaker, the suprachiasmatic nuclei (SCN) of the hypothalamus
(Fig. 2a). Interestingly, between the two tissues, peroxiredoxin oxida-
tion rhythms were in distinct, and different, phase relationships with
respect to total PRX1 and BMAL1 protein, suggesting a difference
between brain and peripheral tissue (Fig. 2a) , as observed previously
for other clock components
15
.
To extend these findings beyond vertebrates, we examined perox-
iredoxin rhythms in the fruitfly Drosophila melanogaster. We pooled
whole heads from insects maintained in constant darkness over two
circadian cycles after they had been stably entrained to 12 h light, 12 h
dark cycles. Again, circadian oscillations in PRX-SO
2/3
immuno-
reactivity were observed, as well as in the clock protein Timeless
(TIM) (Fig. 2b).Similarly,seedlingsfrom the plant Arabidopsis thaliana
exhibited robust PRX-SO
2/3
oscillations in free-running conditions of
constant light, which were also seen in the filamentous fungus
Neurospora crassa—another well-characterized clock model system
(Fig. 2c and Supplementary Fig. 2). Therefore, just as in tissue from
‘complex’ vertebrates, this range of ‘simpler’ eukaryotic systems had
robust peroxiredoxin oxidation cycles, with peaks tending to occur
around anticipated dawn.
Peroxiredoxin rhythms in prokaryotes
Having observed ,24 h peroxiredoxin oxidation rhythms in
organisms with nucleated cells, we next sought to examine represent-
ative prokaryotes from each major domain—Bac teria and Archaea.
Time (h)
0 9 15 3 6 12 18 21
PRX1
PRX-SO
2/3
BMAL1
β-actin
Mouse
a
FRQ
Time (h)
0 12 20 4 8 16 24 28 32 40 36 44 48
Coomassie blue
PRX6-SO
2/3
Fungus
c
Fly
b
Time (h)
0 12 20 4 8 16 24 28 32 40 36 44
β-actin
PRX1
PRX-SO
2/3
BMAL1
β-actin
Time (h)
0 12 20 4 8 16
PRX-SO
2/3
TIM
Liver
SCN
48
0 4 8 12162024283236404448
0
20
40
60
80
100
Time (h)
Normalized
Abundance (%)
TIM
PRX-SO
2/3
0 3 6 9 12 15 18 21 24
0
20
40
60
80
100
Time (h)
Normalized
abundance (%)
PRX-SO
2/3
BMAL1
PRX1
Time (h)
Time (h)
Normalized
abundance (%)
0 4 8 12162024283236404448
0
20
40
60
80
100
PRX6-SO
2/3
FRQ
0 4 8 12162024
0
20
40
60
80
100
Normalized
abundance (%)
PRX-SO
2/3
BMAL
1
PRX1
Figure 2
|
Peroxiredoxin oxidation cycles are conserved in eukaryotic
models of the circadian clock. ac, Representative immunoblots probed for
oxidized/hyperoxidized 2-Cys peroxiredoxin (PRX-SO
2/3
or PRX6-SO
2/3
) are
shown for mouse (Mus musculus; a), fruitfly (D. melanogaster; b), and fungus
(N. crassa; c). For each model system, the organism was sampled under free-
running conditions. Loading controls show either b-actin immunoblots or
Coomassie blue-stained gels loaded with identical samples used for
immunoblotting. Immunoblot quantification by densitometry is shown below
each panel (mean 6 s.e.m.) for n 5 3 biological replicates. See Supplementary
Fig. 2 for plant rhythms, and Supplementary Table 9 for cycle period estimates
(by harmonic regression) and detailed statistics (by ANOVA).
a
Thr
Pro
Cys
Thr
Pro
Cys
Ser
Pro
Cys
Generic
(PRX-V)
PGAFTPGCSKTH PLDFTFVCPTEI PFDFSPVCATEL
b
Human
(PRDX2)
Archaea
(HyrA)
Ot
Se
At
Hs
Mm
Ce
Se
Dm
Nc
Has
Figure 1
|
The peroxiredoxin active site is highly conserved in all domains of
life. a, Multiple sequence alignment showing peroxiredoxin amino acid
sequences. The highly conserved active site is underlined. Representatives
shown from Eukaryota (At, A. thaliana; Ce, Caenorhabditis elegans; Dm, D.
melanogaster; Hs, Homo sapiens; Mm, M. musculus; Nc, N. crassa; Ot, O. tauri;
Sc, S. cerevisiae), Bacteria (Se, S. elongatus sp. PCC7942) and Archaea (Has, H.
salinarum sp. NRC-1). b, Critical residues in the active site of 2-Cys
peroxiredoxins (in bold) are conserved in all organisms. Structures were
derived from human PRX-V (Protein Data Bank (PDB) accession 1HD2)
47
and
human PRDX2 (PDB accession 1QMV)
48
, and modified with PyMOL to show
the predicted structure for archaeal peroxiredoxin (HyrA, GenBank accession
NP_280562.1).
RESEARCH ARTICLE
460 | NATURE | VOL 485 | 24 MAY 2012
Macmillan Publishers Limited. All rights reserved
©2012

For bacteria, we used the best characterized prokaryotic clock model
system, Synechococcus elongatus sp. PCC7942 (ref. 16). The major
proteins involved in the cyanobacterial clockwork are encoded by
the KaiABC cluster from which, remarkably, circadian oscillations
of phosphorylation can be reconstituted in vitro
17
. However, because
all three Kai proteins are expressed together in only a small number of
bacterial species, and in no known archaea
18
, this system cannot
represent a general prokaryotic clock mechanism. We postulated that,
regardless of the timekeeping mechanism, the peroxiredoxin oxida-
tion cycles we observe reflect an absolutely conserved rhythmic
cellular output. We tested this by assaying PRX-SO
2/3
under free-
running conditions (constant light) for 48 h, and observed cycles of
cyanobacterial peroxiredoxin oxidation, peaking later than phos-
phorylated KaiC (Fig. 3a).
Furthermore, we extended our studies to the third phylogenetic
domain, Archaea, assaying rhythms of peroxiredoxin oxidation in
H. salinarum sp. NRC-1. Although no clock mechanisms have been
identified for any archaeon, diurnal transcriptional rhythms were
recently observed in H. salinarum
19
, and it thus represents an ideal
platform to test whether peroxiredoxin oxidation constitutes a
universal marker for cellular rhythms. After entraining the archaea
for three cycles in 12 h light, 12 h dark cycles, we placed them into
constant light at constant temperature. We observed robust, high
amplitude circadian oscillations of PRX-SO
2/3
for three cycles
(Fig. 3b). Together, our findings in evolutionarily diverse prokaryotes
provide compelling evidence that rhythmic peroxiredoxin oxidation
is a conserved circadian marker across phylogenetic domains.
Relations between peroxiredoxin cycles and TTFLs
In all circadian model systems, the proposed clock mechanism
revolves around a TTFL
5,20,21
. How the metabolic rhythms observable
through peroxiredoxin oxidation relate to, and interact with, the
known transcriptional clockwork in different organisms is unclear.
Again, we used several model organisms, with available ‘clock’
mutants, to address this question.
The Drosophila transcriptional clockwork is structurally similar to
the mammalian TTFL, although there are some important differ-
ences
22
. In flies, Clock and Cycle (orthologous to mammalian
BMAL1, also known as ARNTL) comprise the positive limb, driving
oscillatory expression of Period (PER) and Timeless (TIM). PER and
TIM negatively regulate their own expression, closing the loop
5,6
. This
circuit can be disrupted by many mutations
23
. Two mutants, per
01
and
Clk
Jrk
, are behaviourally arrhythmic, and show non-cycling expres-
sion of circadian components, including PER and TIM
24,25
.To
examine peroxiredoxin oxidation patterns in these mutants, they were
entrained as described above for wild-type (Canton-S) flies. We
observed two circadian cycles of PRX-SO
2/3
oscillation, with an
altered circadian phase relative to wild type. This indicates the pres-
ence of an underlying capacity for circadian timing in both mutant
strains, which was clearly perturbed by the absence of functional
transcriptional feedback circuitry (Fig. 4a).
To establish the wider relevance of these findings, we also examined
similar mutants in the fungus Neurospora crassa. The frequency (frq)
locus encodes a critical element in the TTFL of Neurospora,in
addition to the Per–Arnt–Sim (PAS)-containing WC transcription
factors
26
. In the long-period frq
7
mutant
27
, peroxiredoxin oxidation
rhythms showed a similarly lengthened period, with an altered phase
relative to rhythms in FRQ protein abundance (Fig. 4b). Deletion of
the frq locus characterizes the frq
10
strain, and measurable markers of
clock output, such as its spore-forming (‘conidiation’) rhythm, are
profoundly perturbed in these fungi, although apparently stochastic
oscillations can re-emerge under various growth conditions
27–29
.
Circadian rhythms of peroxiredoxin oxidation were, however, clearly
seen in frq
10
mutants sampled in constant darkness (Fig. 4b), with a
delayed phase relative to wild-type (bd) fungi. This illustrates that
peroxiredoxin rhythms represent an alternative readout for an
oscillator that persists in the absence of a FRQ-dependent clock.
We next examined the phenotypes of mutant circadian transcrip-
tional regulators in photosynthetic eukaryotes and prokaryotes. The
transcriptional clockwork of the plant Arabidopsis thaliana and the
alga Ostreococcus tauri are very similar and rely on circadian oscil-
lation of TOC1. Accordingly, overexpressing TOC1 in either species
disrupts transcriptional rhythms
30,31
. In such strains, under constant
light, we observed persistent oscillations of peroxiredoxin oxidation,
albeit with altered amplitude and phase relative to controls (Sup-
plementary Figs 2 and 3). Furthermore, in cyanobacteria we assayed
peroxiredoxin oxidation in the arrhythmic KaiA deletion strain,
AMC702 (ref. 32). Notably, an approximately 24-h rhythm of
peroxiredoxin oxidation persisted despite a functional Kai-based
oscillator being absent, again in an altered phase relative to wild type
(Fig. 5a). Taken together, these observations indicate that metabolic
rhythms remain closely aligned to transcriptional feedback mechan-
isms when those mechanisms are present. Crucially, however,
metabolic rhythms persist even when cycling clock gene transcription
is abolished (summarized in Supplementary Table 9).
Having determined the TTFL influence on peroxiredoxin oxida-
tion rhythms, we reciprocally tested whether rhythmic peroxiredoxin
oxidation is required for timekeeping, using TTFL components as
markers of the clockwork. We assayed reporter bioluminescence
and delayed fluorescence in mutant S. elongatus and A. thaliana lines,
respectively, that were deficient in 2-CysPRX (Synechococcus D2-
CysPRX, GenBank accession AAP49028; Arabidopsis double mutant:
D2-CysPRXA D2-CysPRXB, GenBank accessions NM_111995 and
NM_120712)
33
. In these mutants, circadian rhythms persisted with
wild-type period, albeit significantly perturbed in either phase or
amplitude, relative to controls (Fig. 5b and Supplementary Fig. 9).
This suggests that peroxiredoxins are not required for oscillator
Time (h)
Archaea
Bacteria
a
b
Coomassie blue
PRX-SO
2/3
Time (h)
KaiC
PRX-SO
2/3
Coomassie blue
0 12 20 4 8 16 24 28 32 40 36 44
48
P
NP
0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68
0
20
40
60
80
100
Time (h)
Normalized
abundance (%)
PRX-SO
2/3
0 4 8 12162024283236404448
0
20
40
60
80
100
Time (h)
Normalized
Abundance (%)
PRX-SO
2/3
Phospho-KaiC
0 12 20 4 8 16 24 28 32 40 36 44 48 52 56 60 64 68
Figure 3
|
Peroxiredoxin oxidation cycles are conserved in prokaryotic
models of the circadian clock. a, b, Representative immunoblots probed for
oxidized/hyperoxidized 2-Cys peroxiredoxin (PRX-SO
2/3
) are shown for
bacteria (S. elongatus sp. PCC7942; a), and archaea (H. salinarum sp. NRC-1;
b). Before sampling under free-running conditions (constant light),
cyanobacteria were synchronized with a 12 h dark pulse, whereas archaea were
stably entrained to 12 h light, 12 h dark cycles. Loading controls show
Coomassie blue-stained gels loaded with identical samples used for
immunoblotting. Immunoblot quantification by densitometry is shown below
each panel (mean 6 s.e.m.) for n 5 3 biological replicates. See Supplementary
Table 9 for cycle period estimates and detailed statistics. P, phosphorylated
KaiC; NP, non-phosphorylated KaiC.
ARTICLE RESEARCH
24 MAY 2012 | VOL 485 | NATURE | 461
Macmillan Publishers Limited. All rights reserved
©2012

function in systems that possess an alternative timing mechanism,
namely a TTFL. On the other hand, our findings in TTFL mutants
reveal that cellular components that are required for rhythmic outputs
are not essential to rhythms in redox metabolism. Given the informa-
tion that we have about the above model organisms, used commonly
to study clock biology, we suggest that both peroxiredoxin and TTFL
components of the circadian system are important, but potentially
individually dispensable for circadian rhythms at the cellular level.
Moreover, the phenotypes of these mutants suggest that the cellular
ROS balance is important for robust clock function, as was described
recently in Neurospora
34
.
Implications for clock evolution
We have observed ,24 h cycles of peroxiredoxin oxidation–reduction
in all domains of life and consequently, the possibility that cellular
rhythms share a common molecular origin seems increasinglyplausible.
Because the cellular role of peroxiredoxins principally involves the
removal of toxic metabolic by-products (that is, ROS), we proposed that
the ability to survive cycles of oxidative stress may have contributed a
selective advantage from the beginnings of aerobic life.
Approximately 2.5 billion years ago, photosynthetic bacteria
acquired the capacity for photo-dissociation of water, leading to the
geologically rapid accumulation of molecular oxygen during the Great
Oxidation Event (GOE), when anaerobic life underwent a cata-
strophic decline
35
. Evidently, organisms that survived the transition
to an aerobic environment were those that respired and/or evolved
oxygen. As electron transport chains involving oxygen inevitably pro-
duce toxic superoxide anions as by-products
36
, during the GOE, suc-
cessful organisms had to acquire ROS removal systems or were
relegated to anaerobic niches
35
. Superoxide dismutase, which converts
superoxide to hydrogen peroxide, is ubiquitous and, like peroxire-
doxin (the major cellular H
2
O
2
‘sink’), is estimated to have arisen
around the time of the GOE
37
, the same era during which the most
ancient known clock mechanism (the Kai oscillator) evolved.
Importantly, we note that (1) during the GOE, rhythms of O
2
pro-
duction/consumption and ROS generation would have been driven by
the solar cycle, as they are today
38–40
; (2) metabolic/oxidation rhythms
seem to be present in every organism with a circadian clock, all of
which are aerobes, and these rhythms persist in the absence of tran-
scriptional cycles; and (3) circadian timekeeping confers a selective
advantage when it facilitates anticipation of environmental change
(noxious or otherwise).
We believe that the most reasonable interpretation is that cellular
metabolism in the most competitive early aerobes adapted to confer
anticipation of, and resonate with, environmental cycles in energy
supply and oxidative stress. We presume that the echoes of this
ancient evolutionary adaptation are revealed by the conserved
peroxiredoxin oxidation cycles we now observe in disparate
organisms, indicating that during the past 2.5 billion years, ROS
and metabolic pathways must have co-evolved with the cellular clock-
work, and are probably interlinked. On top of this, further cellular
mechanisms seem to have been incorporated, when advantageous, as
they arose over time. For example, in eukaryotes, the timekeeping
contribution made by certain post-translational mechanisms (such
as casein kinase) is conserved across several disparate organisms,
whereas clock gene transcription factors are widely divergent, having
evolved and been introduced more recently (see Fig. 6).
If there was considerable pressure for the co-evolution of meta-
bolic/ROS pathways with cellular timekeeping systems, then evidence
for this should exist in the phylogenetic trees of their component
mechanisms. To substantiate this we used the Mirrortree algorithm
41
to assess the degree of co-evolution between the 2-Cys peroxiredoxin
family, representing metabolism/ROS pathways, with the most
ancient characterized clock mechanism: the three cyanobacterial
Kai proteins. Because the Kai proteins are found exclusively in pro-
karyotes, we focused on these for our analysis
18
. All three components
of the cyanobacterial oscillator seem to have co-evolved with 2-Cys
peroxiredoxins, as shown by the strong correlation between the dis-
tances of respective proteins within each phylogenetic tree (KaiA,
r 5 0.784; KaiB, r 5 0.883; KaiC, r 5 0.865; P , 1 3 10
26
for all)
(Fig. 5c and Supplementary Fig. 4). Notably, when evolution of
KaiC (the most ancient member) was compared with other absolutely
conserved protein families, the three highest correlations observed
were for the other two clock components (KaiA and KaiB) and for
Time (h)
0 12 20 4 8 16 24 28 32 40 36 44 48
Coomassie blue
PRX6-SO
2/3
Fungus
b
Coomassie blue
PRX6-SO
2/3
frq
7
frq
10
0 4 8 12162024283236404448
0
20
40
60
80
100
Normalized
abundance (%)
Time (h)
WT
frq
10
frq
7
Fly
a
PRX-SO
2/3
β-actin
PRX-SO
2/3
β-actin
Clk
Jrk
per
01
0 4 8 12 16 20 24 28 32 36 40 44 48
0
20
40
60
80
100
Normalized
abundance (%)
Time (h)
Canton-S (WT)
Clk
Jrk
per
01
Time (h)
0 12 20 4 8 16 24 28 32 40 36 44 48
Figure 4
|
Peroxiredoxin oxidation cycles in circadian clock mutants.
a, b, Representative immunoblots probed for oxidized/hyperoxidized
peroxiredoxin (PRX-SO
2/3
or PRX6-SO
2/3
) are shown for fruitfly (D.
melanogaster; a) and fungus (N. crassa; b). For each model system, organisms
were sampled under free-running conditions (constant darkness). Loading
controls show either b-actin immunoblots or Coomassie blue-stained gels
loaded with identical samples used for immunoblotting. Immunoblot
quantification by densitometry is shown below each panel (mean 6 s.e.m.) for
n 5 3 biological replicates. See Supplementary Table 9 for cycle period
estimates (by harmonic regression) and detailed statistics (by ANOVA), as well
as Supplementary Fig. 10 for TIM and FRQ immunoblots for fruitfly and
fungus, respectively. WT, wild type.
RESEARCH ARTICLE
462|NATURE|VOL485|24MAY2012
Macmillan Publishers Limited. All rights reserved
©2012

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TL;DR: The sources of ROS within cells and what is known regarding how intracellular oxidant levels are regulated are discussed, with the recent observations that reduction–oxidation (redox)-dependent regulation has a crucial role in an ever-widening range of biological activities.
Abstract: Reactive oxygen species (ROS), which were originally characterized in terms of their harmful effects on cells and invading microorganisms, are increasingly implicated in various cell fate decisions and signal transduction pathways. The mechanism involved in ROS-dependent signalling involves the reversible oxidation and reduction of specific amino acids, with crucial reactive Cys residues being the most frequent target. In this Review, we discuss the sources of ROS within cells and what is known regarding how intracellular oxidant levels are regulated. We further discuss the recent observations that reduction-oxidation (redox)-dependent regulation has a crucial role in an ever-widening range of biological activities - from immune function to stem cell self-renewal, and from tumorigenesis to ageing.

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Abstract: Hydrogen peroxide emerged as major redox metabolite operative in redox sensing, signaling and redox regulation. Generation, transport and capture of H2O2 in biological settings as well as their biological consequences can now be addressed. The present overview focuses on recent progress on metabolic sources and sinks of H2O2 and on the role of H2O2 in redox signaling under physiological conditions (1–10 nM), denoted as oxidative eustress. Higher concentrations lead to adaptive stress responses via master switches such as Nrf2/Keap1 or NF-κB. Supraphysiological concentrations of H2O2 (>100 nM) lead to damage of biomolecules, denoted as oxidative distress. Three questions are addressed: How can H2O2 be assayed in the biological setting? What are the metabolic sources and sinks of H2O2? What is the role of H2O2 in redox signaling and oxidative stress?

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Book ChapterDOI
TL;DR: This work has shown that there are other molecular circadian oscillators which can act independently of the transcription-based clock in all species which have been tested.
Abstract: Mammals synchronize their circadian activity primarily to the cycles of light and darkness in the environment This is achieved by ocular photoreception relaying signals to the suprachiasmatic nucleus (SCN) in the hypothalamus Signals from the SCN cause the synchronization of independent circadian clocks throughout the body to appropriate phases Signals that can entrain these peripheral clocks include humoral signals, metabolic factors, and body temperature At the level of individual tissues, thousands of genes are brought to unique phases through the actions of a local transcription/translation-based feedback oscillator and systemic cues In this molecular clock, the proteins CLOCK and BMAL1 cause the transcription of genes which ultimately feedback and inhibit CLOCK and BMAL1 transcriptional activity Finally, there are also other molecular circadian oscillators which can act independently of the transcription-based clock in all species which have been tested

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Cites background from "Peroxiredoxins are conserved marker..."

  • ...The peroxiredoxin oscillators are remarkably conserved among all phyla that have been examined (Edgar et al. 2012)....

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Journal ArticleDOI
23 Oct 2014-Cell
TL;DR: Evidence of coordinated metaorganism diurnal rhythmicity is provided and jet-lag-induced dysbiosis in both mice and humans promotes glucose intolerance and obesity that are transferrable to germ-free mice upon fecal transplantation.
Abstract: All domains of life feature diverse molecular clock machineries that synchronize physiological processes to diurnal environmental fluctuations. However, no mechanisms are known to cross-regulate prokaryotic and eukaryotic circadian rhythms in multikingdom ecosystems. Here, we show that the intestinal microbiota, in both mice and humans, exhibits diurnal oscillations that are influenced by feeding rhythms, leading to time-specific compositional and functional profiles over the course of a day. Ablation of host molecular clock components or induction of jet lag leads to aberrant microbiota diurnal fluctuations and dysbiosis, driven by impaired feeding rhythmicity. Consequently, jet-lag-induced dysbiosis in both mice and humans promotes glucose intolerance and obesity that are transferrable to germ-free mice upon fecal transplantation. Together, these findings provide evidence of coordinated metaorganism diurnal rhythmicity and offer a microbiome-dependent mechanism for common metabolic disturbances in humans with aberrant circadian rhythms, such as those documented in shift workers and frequent flyers.

683 citations


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  • ...All three domains of life —archaea, bacteria, and eukarya—have evolved different methods of developing molecular machineries to coordinate this task (Edgar et al., 2012)....

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TL;DR: Sleep appears to have not only a short‐term, use‐dependent function; it also serves to enforce rest and fasting, thereby supporting the optimization of metabolic processes at the appropriate phase of the 24‐h cycle.
Abstract: In the last three decades the two-process model of sleep regulation has served as a major conceptual framework in sleep research. It has been applied widely in studies on fatigue and performance and to dissect individual differences in sleep regulation. The model posits that a homeostatic process (Process S) interacts with a process controlled by the circadian pacemaker (Process C), with time-courses derived from physiological and behavioural variables. The model simulates successfully the timing and intensity of sleep in diverse experimental protocols. Electrophysiological recordings from the suprachiasmatic nuclei (SCN) suggest that S and C interact continuously. Oscillators outside the SCN that are linked to energy metabolism are evident in SCN-lesioned arrhythmic animals subjected to restricted feeding or methamphetamine administration, as well as in human subjects during internal desynchronization. In intact animals these peripheral oscillators may dissociate from the central pacemaker rhythm. A sleep/fast and wake/feed phase segregate antagonistic anabolic and catabolic metabolic processes in peripheral tissues. A deficiency of Process S was proposed to account for both depressive sleep disturbances and the antidepressant effect of sleep deprivation. The model supported the development of novel non-pharmacological treatment paradigms in psychiatry, based on manipulating circadian phase, sleep and light exposure. In conclusion, the model remains conceptually useful for promoting the integration of sleep and circadian rhythm research. Sleep appears to have not only a short-term, use-dependent function; it also serves to enforce rest and fasting, thereby supporting the optimization of metabolic processes at the appropriate phase of the 24-h cycle.

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Cites background from "Peroxiredoxins are conserved marker..."

  • ...The oxidation–reduction cycles of peroxiredoxin proteins has been proposed to constitute a universal marker for circadian rhythms in all domains of life (Edgar et al., 2012)....

    [...]


References
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TL;DR: It used to be that research in chronobiology moved biochemical functions [transcriptional activators], the along at a gentlemanly pace, but by mid 1997 the word in determining what the authors perceive as time was PASWCCLK.
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"Peroxiredoxins are conserved marker..." refers background in this paper

  • ...Importantly, we note that (1) during the GOE, rhythms of O2 production/consumption and ROS generation would have been driven by the solar cycle, as they are today; (2) metabolic/oxidation rhythms seem to be present in every organism with a circadian clock, all of which are aerobes, and these rhythms persist in the absence of transcriptional cycles; and (3) circadian timekeeping confers a selective advantage when it facilitates anticipation of environmental change (noxious or otherwise)....

    [...]


Journal ArticleDOI
TL;DR: It is demonstrated that peripheral tissues express self-sustained, rather than damped, circadian oscillations and the existence of organ-specific synchronizers of circadian rhythms at the cell and tissue level is suggested.
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1,788 citations


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TL;DR: Three unique and highly compartmentalized mammalian superoxide dismutases have been biochemically and molecularly characterized to date and a molecular understanding of each of these genes has proven useful toward the deciphering of their biological roles.
Abstract: Superoxide dismutases are an ubiquitous family of enzymes that function to efficiently catalyze the dismutation of superoxide anions. Three unique and highly compartmentalized mammalian superoxide dismutases have been biochemically and molecularly characterized to date. SOD1, or CuZn-SOD (EC 1.15.1.1), was the first enzyme to be characterized and is a copper and zinc-containing homodimer that is found almost exclusively in intracellular cytoplasmic spaces. SOD2, or Mn-SOD (EC 1.15.1.1), exists as a tetramer and is initially synthesized containing a leader peptide, which targets this manganese-containing enzyme exclusively to the mitochondrial spaces. SOD3, or EC-SOD (EC 1.15.1.1), is the most recently characterized SOD, exists as a copper and zinc-containing tetramer, and is synthesized containing a signal peptide that directs this enzyme exclusively to extracellular spaces. What role(s) these SODs play in both normal and disease states is only slowly beginning to be understood. A molecular understanding of each of these genes has proven useful toward the deciphering of their biological roles. For example, a variety of single amino acid mutations in SOD1 have been linked to familial amyotrophic lateral sclerosis. Knocking out the SOD2 gene in mice results in a lethal cardiomyopathy. A single amino acid mutation in human SOD3 is associated with 10 to 30-fold increases in serum SOD3 levels. As more information is obtained, further insights will be gained.

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25 Apr 2003-Science
TL;DR: It is suggested that this adaptation allows 2-Cys Prxs to act as floodgates, keeping resting levels of hydrogen peroxide low, while permitting higher levels during signal transduction, and is proposed to be the structural origins of sensitivity.
Abstract: Eukaryotic 2-Cys peroxiredoxins (2-Cys Prxs) not only act as antioxidants, but also appear to regulate hydrogen peroxide-mediated signal transduction. We show that bacterial 2-Cys Prxs are much less sensitive to oxidative inactivation than are eukaryotic 2-Cys Prxs. By identifying two sequence motifs unique to the sensitive 2-Cys Prxs and comparing the crystal structure of a bacterial 2-Cys Prx at 2.2 angstrom resolution with other Prx structures, we define the structural origins of sensitivity. We suggest this adaptation allows 2-Cys Prxs to act as floodgates, keeping resting levels of hydrogen peroxide low, while permitting higher levels during signal transduction.

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22 Jul 2005-Science
TL;DR: It is shown that a substantial photosynthetic advantage is conferred by correct matching of the circadian clock period with that of the external light-dark cycle, which explains why plants gain advantage from circadian control.
Abstract: Circadian clocks are believed to confer an advantage to plants, but the nature of that advantage has been unknown. We show that a substantial photosynthetic advantage is conferred by correct matching of the circadian clock period with that of the external light-dark cycle. In wild type and in long- and short-circadian period mutants of Arabidopsis thaliana, plants with a clock period matched to the environment contain more chlorophyll, fix more carbon, grow faster, and survive better than plants with circadian periods differing from their environment. This explains why plants gain advantage from circadian control.

1,141 citations


"Peroxiredoxins are conserved marker..." refers background in this paper

  • ...Importantly, we note that (1) during the GOE, rhythms of O2 production/consumption and ROS generation would have been driven by the solar cycle, as they are today; (2) metabolic/oxidation rhythms seem to be present in every organism with a circadian clock, all of which are aerobes, and these rhythms persist in the absence of transcriptional cycles; and (3) circadian timekeeping confers a selective advantage when it facilitates anticipation of environmental change (noxious or otherwise)....

    [...]


Frequently Asked Questions (1)
Q1. What contributions have the authors mentioned in the paper "Peroxiredoxins are conserved markers of circadian rhythms" ?

Ed Edgar et al. this paper showed that peroxiredoxins are conserved markers of circadian rhythms.