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Journal of Electrostatics 72 (2014) 198e202

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Journal of Electrostatics

journal homepage: www.elsevier .com/locate/elstat

Review

Unifying effectors of circadian rhythm: Protein N-acetylation,phosphorylation, sulfation and other electrical effects

Peter Kovacic a,*, Ratnasamy Somanathan a,b

aDepartment of Chemistry and Biochemistry, San Diego State University, San Diego, CA 92182-1030, USAbCentro de Graduados e Investigación del Instituto Tecnológico de Tijuana, Apdo Postal 1166, Tijuana, BC, Mexico

a r t i c l e i n f o

Article history:Received 18 January 2013Accepted 7 March 2014Available online 21 March 2014

Keywords:Circadian rhythmPhosphorylationProtein N-acetylationSulfationElectro chemistrySignaling and magnetic field

Abbreviations: ET, electron transfer; ROS, reactivenitrogen species; OS, oxidative stress; NADPH, nicotiphosphate-oxidase; SCN, suprachiasmatic nucleus.* Corresponding author. Tel.: þ1 619 594 5595; fax

E-mail addresses: pkovacic@mail.sdsu.edu,(P. Kovacic).

http://dx.doi.org/10.1016/j.elstat.2014.03.0010304-3886/� 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

The vast literature concerning circadian rhythm is devoted mostly to forces that influence operation andharmful effects resulting from disturbances to the clock. The present review presents a novel, unifyingtheme for influences from protein N-acetylation, phosphorylation and sulfation based on electrochem-istry. The unifying theme entails formation of electrostatic fields in the various processes, namely fromformation of amide from protein amine in acetylation, presence of phosphate anions from phosphory-lation and sulfate anions from sulfation. The electrostatic fields may operate as bridges in communicationor in energetics derived from phosphorylation. Other electrochemical and magnetic effects arepresented.

� 2014 Elsevier B.V. All rights reserved.

1. Introduction

Circadian rhythm, driven by a clock, came into effect billions ofyears ago at the beginning of life in response to various external,adverse forces. Circadian rhythm involves an oscillation of about24 h in animals, plants, fungi and cyanobacteria. Much research hasbeen performed with cyanobacteria which possess a simple clock.The rhythm may function within a single cell, and cells maycommunicate with each other in a signaling manner. Based oncircadian rhythms, living matter can adjust to external, potentiallyharmful changes.

The circadian process participates in brain function, tempera-ture regulation, hormonal chemistry, photoperiodism, variouscellular activities, and time measurement. In mammals, the hypo-thalamus contains the main circadian clock. The hormone mela-tonin, secreted by the pineal gland, plays an important role. Manyreports document association of disturbances in the rhythmwith ahost of health problems, including various diseases, cancer devel-opment, sleep disorder, bipolar disorder, jet lag, renal failure,azotemia, uremia, and seasonal affective disorders.

oxygen species; RNS, reactivenamide adenine dinucleotide

: þ1 619 594 4634.pkovacic@sundown.sdsu.edu

In relation to the multiple influences operating in circadianrhythm, the present review presents a novel, unifying theme basedon electrochemical forces. Involved are protein N-acetylation,phosphorylation, sulfation, electrochemistry and magnetic fields.

2. Protein N-acetylation

This process plays an important role in various aspects of bio-logical action. The first part of this section deals with involvementof N-acetylation in the circadian process, which is followed by atreatment of the fundamental, molecular aspect of the process.

The protein clock, a central component of the circadian pace-maker, has histone acetyltransferase (HAT) activity [1] which isessential to rescue circadian rhythmicity and activation of clockgenes in mutant cells. The results reveal that chromatin remodelingis crucial for the core clock mechanism and identifies unforeseenlinks between histone acetylation and cellular physiology.

Glucocorticoids influence organ functions through their recep-tor, a protein acylated and decetylated by several histone acetyl-transferases and deacetylases [2]. The circadian rhythm-relatedtranscription factor “clock,” a key component of the biological clockwith inherent histone acetyltransferase activity, and acetyl gluco-corticoid receptor lysines within its hinge region, represses itstranscriptional activity. This clock-induced repression of theglucocorticoid receptor activity is inversely phased to the diura-nally circulating glucocorticoids and may act as a local counterregulatory mechanism to the actions of these hormones. Thus,

Fig. 1. Amide resonance hybrid structure.

P. Kovacic, R. Somanathan / Journal of Electrostatics 72 (2014) 198e202 199

acetylation-mediated epigenetic regulation of the glucocorticoidreceptor may be essential for the maintenance of proper time-integrated glucocorticoid action, significantly influencing humanwell-being and longevity.

Glucocorticoids influence functions of virtually all organs andtissues through their receptor (GR) [3]. Circulating levels of gluco-corticoids fluctuate naturally in a circadian fashion and regulate thetranscriptional activity of GR in target tissues. The basic helix-loop-helix protein clock, a histone acetyltransferase (HAT), and its het-erodimer partner BMAL1 are self-oscillating transcription factorsthat generate circadian rhythms in both the central nervous systemand periphery. Clock suppressed binding of GR to its DNA recog-nition sequences by acetylating multiple lysine residues located inits hinge region. These findings indicate that clock/BMAL1 func-tions as a reverse-phase negative regulator of glucocorticoid actionin target tissues.

Regulation of circadian physiology relies on the interplay ofinterconnected transcriptional-translational feedback loops. Theclock-MBAL1 complex activates clock-controlled genes [4]. Clockpossesses intrinsic histone acetyltransferase activity, and thisenzymatic function contributes to chromatin-remodeling eventsimplicated in circadian control of gene expression. Clock alsoacetylates a non-histone substrate; its own partner, BMAL1, spe-cifically acetylates on a unique, hugely conserved Lys 537 residue.BMAL1 undergoes rhythmic acetylation in mouse liver, with atiming that parallels the downregulation of circadian transcriptionof clock-controlled genes. BMAL1 acetylation facilitates recruit-ment of crytochrome 1 to clock-BMAL1, thereby promoting tran-scriptional repression.

In the mouse circadian clock, a transcriptional feedback loop isat the center of the clockworkmechanism [5]. Clock and BMAL1 areessential transcription factors that drive the expression of threeperiod genes (Perl-3) and two cryptochrome genes (Cry1 and Cry2). The Cry proteins feedback to inhibit clock/BMAL1-mediatedtranscription by a mechanism that does not alter clock/BMAL1binding to DNA. Transcriptional regulation of the core clockmechanism in mouse liver is accompanied by rhythms in H3 his-tone acetylation, and H3 acetylation is a potential target of theinhibitory action of Cry. The promoter regions of the Per1, Per2 andCryl genes exhibit circadian rhythms in H3 acetylation that aresynchronous with the corresponding steady-state messenger RNArhythms.

A report deals with functional implication of a transcriptionfactor in the circadian control of histone acetylation [6].

Circadian clock genes are regulated through a transcriptional-translational feedback loop [7]. Alterations of the chromatinstructure by histone acetyltransferases and histone deacetylasesare commonly implicated in the regulation of gene transcription.The state of acetylated histones fluctuated in parallel with therhythm of mouse Per1. These data indicate that the rhythmictranscription and light induction of clock genes are regulated byhistone acetylation and deacetylation.

Lysine acetylation plays a key role in regulating gene expression[8]. Mass spectrometry identified that 3600 lysine acetylationpreferentially targets large macromolecular complexes involved indiverse cellular processes. The results demonstrate that the regu-latory scope of lysine acetylation is broad and comparable with thatof other major posttranslational modifications.

Protein lysine acetylation has emerged as a key posttranslationalmodification in cellular regulation, in particular through tran-scription regulators [9]. Lysine acetylation is a prevalent modifica-tion in a host of enzymes that catalyze intermediate metabolism,which is believed to play a major role in metabolic regulation.

Lysine regulates many eukaryotic processes, but its function inprokaryotes is largely unknown [10]. The relative activities of key

enzymes were regulated by acetylation. The metabolic regulatorymechanism involving acetylation is conserved from bacteria tomammals.

N-Acetylation of histone has attracted appreciable attention(e.g., Ref. [9]). The literature on histone is quite extensive. An articledemonstrates that histone acetylation on lysine controls chromatinstructure and proteininteractions [11]. Facilitation of acetylation atlysine of histone by trichostatin A, a histone deacetylase inhibitor,may play a critical regulatory role in chromatin remodeling andgene expression [12].

In other aspects, N-acetylation in the Chinese population wasstudied in relation to carcinogenesis [13]. Slow acetylators aresignificantly associated with bladder cancer. N-Acetylation appearsto be an important regulatory process for modulating the behav-ioral activity of peptides [14].

In a recent article, a novel mechanism is presented for the role ofprotein N-acetylation [15]. Investigation of the process began morethan a century ago [16]. Comparison with phosphorylation hasbeen made over the years. The present review provides anotherexample involving an integrated, interdisciplinary approach.

A 2009 article points out the importance of acetylation in biologyby designation as “remarkably ubiquitous” [17]. Earlier studiesfocused on histone modification and gene transcription. Morerecent reports involve non-histone proteins and enzymes. Identifi-cation of the acetylome revealed acetylation events at the whole-proteome level. The complexity rivals that of the phosphopro-teome. These data underline the regulatory power of acetylation.

An electrostatic mechanism was proposed as playing animportant role in protein ET [15]. On examination of the electro-chemical literature on proteins, one finds reports on charged re-gions in the peptide matrix, many of which arise from ions derivedfrom acid and base substituents, in addition to dipoles from theomnipresent peptide bond and other substituents [18]. Hence,N-acetylation provides another comparatively strong electrostaticfield (EF) capable of interacting with the other fields and forproviding a bridge for species, such as radicals and electronsinvolved in cell signaling [19,20] Amide formation arising fromN-acetylation of lysine results in replacement of the relatively weakdipole mement (DM) of the lysine primary amine (1.22 D for themodel ethylamine) by the considerably stronger dipole of 3.76 D forthe amine moiety. Also, the amide dipole is appreciably greaterthan those for the related ketone acetone (2.88 D), the ester ethylacetate (2.08 D), and acetic acid (1.70 D) [21].

It is important to recognize the nature of the amide bond inrelation to EFs. The functionality is a resonance hybrid as shown inFig. 1. In addition, hydrogen bonding may play a role involvinghydroxyl and amino groups from protein substituents or prevalentwater associated with protein [18]. Hydrogen bonding with theamide carbonyl enhances the cationic character of the nitrogen,which should increase the electrostatic force field. Protonationyields an iminium type ion (Fig. 2) which has been discussed inrelation to protein ET [18]. Hence, by analogy, the amide fromN-acetylation might play a part im ET.

Suggestions have been made concerning the role of ETs,including energetics and function as a conduit [20]. It is significantthat the term energy-rich has been applied to acetylation [16].

Fig. 2. Iminium from protonated amide.

P. Kovacic, R. Somanathan / Journal of Electrostatics 72 (2014) 198e202200

Since cell signaling is widely involved in biological processes, itis not surprising to find considerable relevant literature associatedwith N-acetylation. Representative examples are provided [15]. Themechanism of cell signaling has been addressed in a recent reviewwith emphasis on ROS [19].

3. Phosphorylation

Phosphorylation appears to be an important regulatory step inmaintaining the robustness of the circadian clock [22,23]. proteinphosphorylation and modulation of circadian behavior are thesubject of a recent report [24]. A morning-induced phosphoryla-tion-gated repressor times evening gene expression [25]. A smallmolecule modulates circadian rhythms through phosphorylation ofthe period proteins [26]. A report deals with a time-delay phos-phorylation circuit that acts on circadian clock speed [27].Phosphorylation influences half-life, subcellular localization, tran-scriptional activity and conformation of circadian clock compo-nents [28]. Phosphorylation of different sites on the clock proteinplays a prominent, although complex role [29]. The dual role andthe effect on period and phase of the clock are discussed. Clockproteins are regulated by the phosphorylation-dependent modu-lations of rapid shuttling cycles that alter subcellular localization ina time-of-day specific manner [30]. A phosphorylation cascadereveals novel roles for proline-directed kinases [31]. A study shedlight on regulation of circadian phosphorylation rhythm of cyano-bacterial proteins [32]. Another report addresses regulation ofprotein stability and circadian function by protein-mediatedphosphorylation [22]. Reversible phosphorylation subserves circa-dian rhythms by creating a switch in inactivating the positiveelement [33]. The regulatory roles of phosphorylation sites en-hances our understanding of the molecular mechanism underlyingcircadian rhythm generation [34]. Phosphate-activated protein ki-nase phosphorylates and destabilizes a clock component [35].Sequential and compartment-specific phosphorylation controls thelife cycle of the circadian CLOCK protein [36]. Post-translationalregulation of the circadian clock was studies through selectiveproteolysis and phosphorylation of pseudo-response regulatorproteins [37]. Reversible protein phosphorylation was found toregulate circadian rhythms [38]. In a related report, reversiblephosphorylation of certain proteins within the negative feedbackloop seems to exert a key role for the correct timing of nuclearrepression [39]. Regulation of circadian clock components byphosphorylation plays essential roles in clock functions, beingconserved from fungi to mammals [40]. Various kinases areinvolved in mediating sequential phosphorylation events in thecircadian negative feedback loop. An investigation deals withautonomous synchronization of circadian phosphorylation rhythm[41]. Circadian control of clock retina photoreceptors appears toentail elevated daytime tyrosine phosphorylation of protein [42].Findings indicate prominent roles for reversible phosphorylation ofclock proteins in the core oscillatory mechanism [43]. The pro-cesses affect key properties of the clock, namely period, amplitudeand phase. Cyanobacteria are the simplest organisms known toexhibit circadian rhythms [44]. The clock gene products regulatephosphorylation. Phosphorylation plays a vital role in the precisetiming of circadian clock [45]. Daily rhythms of phosphorylation in

the Drosophila clock were first described about two decades ago.Precise site phosphorylation is reported. Negative feedback in theclock of Neurospora appears to be mediated by clock proteinswhich rhythmically promote phosphorylation [46]. The enigma ofthe circadian clock has been studied in vitro by examination of theinteractions between three crucial proteins [47]. Phisphorylation ofspecific protein sites negatively regulates function in the circadianfeedback and is important for function of the Neurospora clock [48].Key phosphorylation sites have been identified in clock protein byseveral analytical methods [49]. Phosphorylation states are regu-lated by the clock, which is important for maintenance of a stable,oscillatory clock [50]. Phosphorylation of clock protein regulatescircadian degradation in human fibroblasts [51]. Protein phos-phorylation by casein kinase is necessary for the function of theNeurospora clock [52]. Phosphorylation regulates potential stabilityand circadian function [53].

Ordered phosphorylation and dephosphorylation of two Kai Camino acids are key to cyanobacterial clock oscillation [54]. Auto-phosphorylation involves threonine and serine residues. Light-induced resetting of the clock is associated with changes incellular amounts of adenosine triphosphate (ATP) and adenosinediphosphate (ADP). Kai C phosphorylation is influenced by the ATP/ADP ratio [55]. Stimulation of Kai C phosphorylation is blocked byquinones. Manipulation of the ATP/ADP ratio can reset timing ofphosphorylation peaks in the oscillator.

A recent review presents a novel mechanism for phosphoryla-tion action based on electrostatics [20]. Phosphate anions providestrong electrostatic fields that are believed to be of major impor-tance. It is probably not coincidental that the phosphates involvedare mono- or di- esters containing at least one hydroxyl in anionform. The field may serve as a link that connects other electrostaticfields. Energetics may play a role.

A recent review proposes a novel hypothesis based on electro-static forces for part of the action involving phosphorylation andsulfation [20]. In sulfation, monoesterification preserves an anionresidue with associated electrostatic field. This aspect is treated inmore detail in the phosphorylation section.

4. Sulfation

This process is related to phosphorylation, but plays a lessimportant role, including circadian rhythms. A study investigatedthe diurnal variations in the activity of the hepatic enzymes cata-lyzing conjugation and sulfation of bile acids [56]. A circadianrhythm was noted in cholic acid, CoA ligase and glycolithocholatesulfotransferase activity. There was no diurnal variations in lith-ocolate and sulfotransferase activity, raising the possibility ofmultiple bile acid sulfotransferases in liver.

Proteoglycans are dominant glycoconjugates located on the cellsurface and in extracellular spaces and consist of a core proteinwith one or more glycosaminoglycan side chains linked covalently.Heparan sulfate (HS) belongs to the family of glycosaminoglycans[57]. HS has been assigned a variety of physiological and patho-logical functions. Light induced changes in pineal HS fine structureand occurrence of the rare 3-O sulfation catalyzed by HS 3-O-sul-fotransferase are predominantly restricted to daytime pinealglands.

A study investigated the diurnal variations in the activity of thehepatic enzymes catalyzing conjugation and sulfation of bile acids[56]. A circadian rhythm was noted in cholic acid, CoA ligase andglycolithocholate sulfotransferase activity. There was no diurnalvariations in lithocholate and sulfotransferase activity, raising thepossibility of multiple bile acid sulfotransferases in liver.

A recent review proposes a novel hypothesis based on electro-static forces for part of the action involving phosphorylation and

P. Kovacic, R. Somanathan / Journal of Electrostatics 72 (2014) 198e202 201

sulfation [20]. In sulfation, monoesterification preserves an anionresidue with associated electrostatic field. This aspect is treated inmore detail in the phosphrylation section.

5. Electrochemistry and signaling

Physical forces, such as electrostatic and electromagnetic fields,and gravity, display a daily cyclic behavior and can function assecondary time-cues [58]. Clock responsiveness to external timecues is central to the cellular clock mechanism. Lysine acetylationapparently enhances binding in the circadian clock by affectingelectrostatic interactions [59]. The core clock gene ELF4 forms analpha-helical homodimer with a likely electrostatic interface thatcould be structurally modeled [60]. Effects were studied of a lightpulse given after lights off [61]. Cells were depolarized and spikefiring rates increased. Neurons were hyperpolarized. Membranehyperpolarization may result from suppression of a leakage cur-rent. Results show that the circadian clock has electrochemicaloscillators which can activate ion channels in electrochemical cir-cuits [62e64]. Studies with the biological clock were performedinvolving the effect of electrical stimulation [65,66]. Circadiantimekeeping in mammals is driven by feedback loops that areactive within the superchiasmetic nuclei (SCN) [67]. A polypeptideis an intrinsic SCN factor implicated in activation and electricalsynchronization of SCN neurons and coordination of behavioralrhythms. Neuropeptidergic interneuronal signaling confers a can-oncal property upon the SCN: spontaneous synchronization of theintracellular molecular clockwork of individual neurons. In mam-mals, an internal timingmechanism located in the SCN orchestratesa diverse array of neuroendocrine and physiological parameters toanticipate the cyclical environmental fluctuations that occur everyday [68]. Electrophysiological recording techniques have provedinvaluable for our understanding of how this clock becomes syn-chronized and coordinates the timing of rhythm. Studies indicatethat electrical activity within individual clock cells is a vital part ofSCN clockwork. These results are in accord with the importance ofelectrochemistry in diverse body functions [69e73].

6. Magnetic fields

During the course of evolution humans have been exposed tothe magnetic field of the earth, which has exerted various in-fluences. Herein, involvement with the circadian clock is addressed[74e86].

Acknowledgment

Editorial assistance by Thelma Chavez is acknowledged.

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