Sangdun ChoiEditor

Encyclopedia ofSignaling Molecules

Second Edition

With 1893 Figures and 247 Tables

EditorSangdun ChoiDepartment of Molecular Science and TechnologyAjou UniversitySuwon, Korea

ISBN 978-3-319-67198-7 ISBN 978-3-319-67199-4 (eBook)ISBN 978-3-319-67200-7 (print and electronic bundle)https://doi.org/10.1007/978-3-319-67199-4

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2512 IkBz

IkBz

Balachandran Manavalan1,2, Shaherin Basith1,3

and Sangdun Choi11Department of Molecular Science andTechnology, Ajou University, Suwon, Korea2Center for In Silico Protein Science, School ofComputational Sciences, Korea Institute forAdvanced Study, Seoul, Korea3National Leading Research Laboratory (NLRL)of Molecular Modeling and Drug Design, Collegeof Pharmacy and Graduate School ofPharmaceutical Sciences, Ewha WomansUniversity, Seoul, Korea

Synonyms

IkappaBzeta; IkB-zeta; IL-1 inducible nuclearankyrin-repeat protein (INAP); Inap; Mail; Mole-cule possessing ankyrin repeats induced by lipo-polysaccharide (MAIL); Molecule possessingankyrin-repeats induced by lipopolysaccharide;NF-kappa-B inhibitor zeta; NFKBIZ; Nuclearfactor of kappa light polypeptide gene enhancerin B-cells inhibitor, zeta

Historical Background

IkBζ is also known as molecule possessingankyrin repeats induced by lipopolysaccharide(MAIL) or interleukin (IL)-1 inducible nuclearankyrin-repeat protein (INAP) and it was firstdiscovered independently in two laboratories dur-ing 2000 as a protein containing ankyrin repeats.The gene encodes a protein with an amino-terminal region of 450 amino acids that containsa nuclear localization signal (NLS) and a trans-activation domain (TAD) followed by the sevenrecognizable ankyrin repeats at its carboxyl ter-minal. Treatment of cells with lipopolysaccha-rides (LPS) or the cytokine interleukin (IL)-1(Kitamura et al. 2000; Haruta et al. 2001;Yamazaki et al. 2001) induced the expression ofIkBζ, whereas tumor necrosis factor (TNF)-a(Totzke et al. 2006) treated cells did not show

any IkBζ expression. The induced IkBζ is local-ized in the nucleus, where it interacts with thenuclear factor (NF)-kB subunit and other nuclearproteins via their ankyrin repeat domain (ARD),leading to a positive or negative regulation of itstranscriptional activity depending on genes (Muta2006). Thus, the innate immune system utilizesNF-kB as a major transcription factor and modu-lates its activity in a gene-specific manner via theregulatory factor IkBζ, which is specificallyinduced upon stimulation of the innate immunesystem. This multistep regulation of the transcrip-tion would be fundamental in the selective expres-sion of genes upon cell activation. In this chapter,we summarize recent findings in nuclear IkBζwith an emphasis on its immunological aspects.

Induction of IkBζ and Its Functions

Many cellular responses are mediated by orches-trated gene expression. When cells are exposed todiverse inflammatory stimuli, such as microbialcomponents, a large number of genes are inducedto elicit inflammatory responses. These genesinclude cytokine/chemokine, antimicrobial pep-tides, and cell adhesion molecules; most of thegenes are known to be induced through activationof transcription factor NF-kB (Akira and Takeda2004; Hoffmann and Baltimore 2006; Haydenand Ghosh 2008). In resting cells, typical cyto-plasmic IkB proteins (IkB-a, -b, and -e) mask theNLS of NF-kB, thereby preventing its transloca-tion into the nucleus. The activation of cells withappropriate stimuli, particularly toll-like receptor(TLR) ligands or various host immune mediatorssuch as proinflammatory cytokines and IL-1superfamily proteins, induces activation of IkBkinase complex, which leads to the degradationof the cytoplasmic IkBs by the ubiquitin-proteosomal pathway. The NF-kB liberated fromthe IkBs is then translocated to the nucleus, whereit binds to the promoter/enhancer region of thetarget genes, resulting in the regulation of tran-scription via recruitment of several coactivatorsand corepressors. This transcriptional activationleads to the expression of primary/early responsegene A, depicted in Fig. 1, which includes three

IkBz, Fig. 1 Roles of IkBζ in inflammatory response.Activation of the TIR-containing receptors by TLR ligandselicits phosphorylation and ubiquitination-induced degra-dation of the cytosolic IkB proteins, which allows nucleartranslocation of NF-kB. In the nucleus, NF-kB activatestranscription of a subset of genes A, which includes IkBζ.

The expression of IkBζ also requires a specific mRNAstabilization signal that comes from the TIR-containingreceptor as well as activation of NF-kB. The expressedIkBζ associates with NF-kB, and the complex engagestranscription of another subset of genes B. Simultaneously,IkBζ inhibits transcription of the subset of genes A

IkBz 2513

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atypical members, IkBζ, Bcl-3, and IkBNS. Theinduced IkBζ associates with NF-kB and thenactivates another subset of inflammatory geneB (Fig. 1). Simultaneously, IkBζ inhibits the tran-scriptional regulation of gene A.

The predominantly expressed cytoplasmic IkBproteins, IkB-a, -b, and -e, act exclusively asNF-kB inhibitors, whereas the nuclear IkB

proteins, Bcl-3, IkBNS, and IkBζ, can both actas either a positive or negative regulator of NF-kBtarget genes. Bcl-3 can act as a positive regulatorof NF-kB either by removing transcriptionallyinactive p50 and p52 dimers from the IkB sites,thus allowing transcriptionally active hetero-dimers to take their places, or by forming a ternarycomplex with DNA-binding p50 and p52

2514 IkBz

homodimers, thereby facilitating gene expression(Fujita et al. 1993; Dechend et al. 1999; Yamazakiet al. 2001; Yamamoto et al. 2004; Totzkeet al. 2006). IkBNS enters the nucleus where itpreferentially interacts with p50 and functions asthe repressor of NF-kB. Unlike other classical IkBproteins, IkBζ is strongly expressed in response totreatment with different proinflammatory stimuli.This list includes LPS, IL-1, peptidoglycan, bac-terial and mycoplasmal lipopeptides, flagellin,R-848 (an imidazoquinoline derivative) as wellas CpG oligonucleotides, and ligands for TLR2,-5, -7, and -9. The physiological role of IkBζ hasbeen shown either as inhibiting the transcriptionalactivity of NF-kB by associating with the p50/p65heterodimer or activating the expression of genessuch as IL-6, CCL2, IL10, Lcn2, and IL-12p40 asbinding with the p50 homodimer.

IkBζ is encoded by a primary responsive gene,Nfkbiz, and its induction depends on NF-kB acti-vation, suggesting that IkBζ-regulated genes areinduced via two-step machinery (Motoyamaet al. 2005; Yamazaki and Takeshige 2008). Verylittle IkBζ is detected in unstimulated cells, and itis induced by various microbial substances thatstimulate TLRs and IL-1b receptors but not byTNF-a. Actually, TNF-a induces transcription ofNfkbiz gene, but IkBζ mRNA did not stabilizeduring posttranscriptional process, indicatingthat the stimulus-specific expression of IkBζ isdetermined posttranscriptionally. All TLRs pos-sess ectodomains that recognize ligands frommicrobial substances and endodomains that facil-itate the downstream signaling, which has beenshown to be associated with the ectodomainthrough a transmembrane segment. With theexception of TLR3, which mediates downstreamsignaling through TRIF/TRAM (which is Myd88independent), the other TLRs share common sig-naling pathways initiated by an adaptor protein.The signal either through the Myd88 (Myd88-dependent pathway) or TRAF6 or both leads tothe induction of IkBζ. In macrophage cells, LPSstimulation leads to three variants (L, S, and D)that are generated by alternative splicing of IkBζ.The longer form, IkBζ (L, 1–728), is predomi-nantly expressed upon LPS stimulation, whilethe shorter form IkBζ (S) has been observed at

mRNA and protein levels in minor species.Although IkBζ (D) mRNA has been detected inmacrophages, its corresponding protein level hasnot been found (Yamazaki et al. 2005). Overall,IkBζ (L and S) are functionally active whenexpressed in the cells.

IkBζ (1–728 amino acids) can be divided intoN-terminal (1–450) and C-terminal (451–728)portions. The N-terminal region composed ofNLS and TAD, whereas the C-terminal regioncontains the ANK repeat domain, which plays animportant role in interaction with NF-kB subunits,thereby regulating its functions (Fig. 2a). Trun-cated mutation studies of IkBζ (especially itsN-terminal region) have shown -K 163-R 164-X

12-K 177-R 178- to be indispensable for NLS, andthe mutation of this portion has revealed that IkBζlocalized in the cytosol and effectively inhibitedNF-kB, whose function is similar to the cytosolicIkB proteins. IkBζ was initially characterized as anegative regulator of NF-kB, but subsequent stud-ies demonstrated that it could also act as a positiveregulator of NF-kB. Analysis using GAL4-fusionprotein of IkBζ revealed that its N-terminalregion (329–403) exhibits transcriptional activityafter association with NF-kB (p50/p50) subunit(Yamazaki et al. 2005). To date, numerous func-tions of IkBζ have been reported, when bound toother nuclear proteins: (1) Overexpression ofIkBζ augmented IL-6 production in response toLPS by interacting with p50 homodimer, whereasTNF-a production is inhibited through interactionwith p50/p65 heterodimer, indicating specific tar-get gene activity (Motoyama et al. 2005). (2) IkBζphysically and functionally interacts with STAT3,which is a member of the NF-kB signaling path-way, thereby inhibiting the transcriptional activa-tion of STAT3 (Wu et al. 2009). (3) Human IkBζexpressed in response to TNF-a binds to theDNA-binding region of p50/p65 heterodimer,consequently leading to the inhibition of theTNF-a response (Totzke et al. 2006). (4) IkBζmediates preinitiation complex assembly and his-tone H3K4 methylation, leading to the activationof secondary response genes, thereby suggesting arole of IkBζ in the nucleosome remodeling(Kayama et al. 2008). (5) IkBζ is expressed inIL-17-producing helper T (T H17) cells that play

ANK7 ANK1

N

C

ANK2ANK3

ANK4

ANKΙκΒαa

b

ΙκΒβ

ΙκΒε

ΙκΒζ

ΙκBNS

Bcl-3

NLS

NLS

NLS

NLS

NLS TAD

PEST

PEST

NLS

ANK5

ANK6

IkBz, Fig. 2 IkB familymembers. (a) All IkBproteins harbor ankyrinrepeats at their carboxyterminal region. TheN-terminal regulatoryregion of IkB-a, -b, and -econtains specific sequencesthat are phosphorylated andubiquitinated. TheN-terminal region of IkBζcontains the nuclearlocalization andtransactivation domains thatare necessary for thetranscriptional process.(b) The predicted 3D modelof the IkBζ ARD domainare shown in bright pink.The 28 amino acid residuesoccurring within the ANK4are shown in red

IkBz 2515

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an important role in resistance to experimentalautoimmune encephalomyelitis (EAE) (Okamotoet al. 2010). (6) IkBζ controls the proliferation anddifferentiation of epidermal keratinocytes throughNF-kB-independent mechanisms (Ishiguro-Oonuma et al. 2015).

Physiological Roles of IkBζ

Studies of IkBζ-deficient mice have demonstratedthat IkBζ plays a role as a positive and nega-tive regulator of NF-kB-mediated transcription(Yamamoto et al. 2004). Microarray studies haveshown that IkBζ is an indispensable componentof the LPS-induced transcription of genesrepresented by IL-6 and of the genes listed inTable 1. It should also be noted that IL-6 playsan important role in many inflammatory diseasesincluding sepsis, heart attacks, and stroke as wellas in many human cancers including

hepatocarcinoma, multiple carcinoma, and ovar-ian cancer. However, the transcription complex onIL-6 appears to differ depending on the stimuli.The role of IkBζ as a negative regulator was notevident in the isolated cells, probably because ofredundant negative regulators of NF-kB. Theinhibitory roles of IkBζ in NF-kB-mediated tran-scription are critical in fine-tuning to balanceinflammatory reactions to maintain homeostasisin vivo. IkBζ knockout animals have atopic-likedermatitis and eye inflammation that supports arole of IkBζ in innate host defense. However, theinflammation caused by IkBζ deficiency is not soclear. Accordingly, it will be important to testwhether the skin and eye inflammation in IkBζknockout mice is due to the lack of host defensemolecules such as lipocalin or lack of essentialcytokines such as IL-6. However, the other func-tion of IkBζ, suppression of NF-kB activity, can-not be excluded. Atopic dermatitis and ocularinflammation may also occur due to the

IkBz, Table 1 Genes that require IkBζ for LPS-mediated induction

Category Subset of genes

Cytokines IL-6, IL-12 p40 subunit, IL-18, IL10, granulocyte macrophage stimulating factor (GM-CSF),granulocyte colony stimulating factor (G-CSF), growth-differentiation factor (GDF)15, Epstein-Barr-virus-induced gene (EBI) 3

Chemokines CXC chemokine ligand (CXCL)5, CXCL13, chemokine ligand (CCL)7, CCL17, CCL2

Enzymes Histidine decarboxylase, caspase11, inositol polyphosphate-5-phosphatase B, deltex 2B,glutathione reductase, guanylate nucleotide-binding protein (GDP) 1

Receptors Formyl peptide receptor 1, macrophage receptor with collagenous structure (MARCO)

Biological activepeptides

Endothelin 1, ghrelin

Transcriptionfactors

Basic leucine zipper transcription factor (BATF), CCAAT/enhancer-binding protein (C/EBP)-d

Antimicrobialsubstances

Lipocalin 2/neutrophil gelatinase-associated lipocalin (NGAL), Pentraxin 3

Others Tax-1 binding protein, extracellular proteinase inhibitor, solute carrier family 11 member2 (S1c11a2), Src-like adaptor protein (SLAP), immunoglobulin heavy chain, immunoglobulinlight chain, membrane spanning 4-domains (MS4A1), thrombospondin 1, immediate earlyresponse 3 (IER3/IEX1), disabled-2

2516 IkBz

overexpression of cytokines that IkBζ mightinhibit via the C-terminal ankyrin repeat.

The factors that distinguish genes that are acti-vated or inhibited by IkBζ appear to be present inthe promoter of each gene. Biochemical studieshave indicated that in addition to NF-kB bindingsites, IkBζ is also required for transcriptional acti-vation. Since the transcriptional activation activityof IkBζ on the GAL4-reporter system is muchweaker than that of the NF-kB p65 subunit,other transcription factors are necessary for effi-cient IkBζ-mediated transcription (Yamazakiet al. 2008). IkBζ acts as a negative regulator ofthe promoter harboring canonical NF-kB-bindingsequences alone. Moreover, all the nuclear IkBproteins are homologous to each other; therefore,these nuclear proteins may act as competitors forIkBζ or vice versa. In fact, IkBNS has beenreported to inhibit LPS-mediated IL-6, IL-12 p40,and IL-18 production (Kuwata et al. 2006).

Structure-Based Activationand Inhibition Mechanism of NuclearIkBζ

The primary sequence of human and mouse IkBζshare about 70% homology with the N-terminalregion (1–450) and 97% sequence identity with

the C-terminal ARD. Although there is sequencevariation at the N-terminal region, the NLS andTAD are conserved. There has been speculationabout the sequence variations in the N-terminalregion between human and mouse. IkBζmay playa significant role to bind with specific NF-kBsubunits. Mouse IkBζ binds only to p50/p50homodimer, whereas human IkBζ binds to boththe p65 and p50 subunits. Sequence analysis ofthe N-terminal region did not identify anysequences known to correlate with specific struc-ture or function. Moreover, secondary structureprediction showed that the N-terminal has noANK repeat followed by the C-terminal ARD.This type of architecture has also been reportedin proteins such as the yeast ribosomal bindingprotein yar-1 (Lycan et al. 1996). No crystal struc-ture is yet available for IkBζ ARD, but recentmodeling studies have shown the IkBζ three-dimensional structure, which was built basedupon the Bcl-3 crystal structure (Michel et al.2001) (Fig. 2b). Each ANK repeat of the IkBζmodels depicted two antiparallel a-helices,followed by a loop of variable length at a rightangle. Each repeat began and ended with shortb-hairpin turns that protruded away from thea-helix. This nonglobular fold was stabilizedthrough intra- and interrepeat hydrophobic inter-actions. The represented structural motifs stack

IkBz, Fig. 3 Docking studies predicted that the IkBζARDbinds at the side of the p50/p65 heterodimer interface.(a) The p50/p65 heterodimers, represented as a ribbondiagram, are shown in magenta and cyan, respectively.Docked IkBζ is green in the ribbon diagram.(b) p65-IkBζ binding interface. Side chains of the amino

acid contributing to the hydrogen bonding formation(marked as black) are represented by a stick model withthe residue name and numbers shown next to them. (c) Thep50-IkBζ binding interface is shown in a similar fashion asin (b)

IkBz 2517

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upon one another in a linear fashion to form acurved architecture. These motifs are known tofacilitate protein–protein interaction but have noknown enzymatic activity (Mosavi et al. 2004).The presence of ankyrin repeats and their role ininflammatory signaling immediately suggestedstructural and functional homology with theARD-containing classical IkB proteins. Despitethe similarities, IkBζ differs structurally from theclassical IkB inhibitors in its unique amino-terminal region, the number of ankyrin repeats,the presence of a unique 28 amino acids insertionwithin the helices of the a1 and a2 of ANK4(similar to one that has been observed in IkBNS),and the complete absence of a PEST-like regionwithin the IkBζ carboxy-terminus (Fig. 2b).

Recent studies have indicated that humanTNF-a-induced IkBζ is associated with both thep50/p65 subunits of NF-kB in the nucleus andinhibits the transcriptional activity of antiapoptoticprotein (Totzke et al. 2006). The structural basis ofthis inhibitory and activation mechanism wasexplained by docking studies (Manavalanet al. 2011), which have shown that IkBζ ARDassociates with the DNA-binding domain ofp50/p65 subunits of NF-kB and contains importantresidues that interact with the bases as well as sugarphosphate backbone present in the p50 and p65subunit (Fig. 3). Hence, there will be no morep50/p65 subunits available to the promoter region,

which ultimately results in inhibition of the tran-scription mediated by p50/p65 subunit. Generally,p65 subunits contain the TAD at the C-terminalend, which is important for its transcriptional activ-ity (O’Shea and Perkins 2008). IkBζ inhibits p65transactivation activity through its binding with theN-terminal DNA-binding domain.

Furthermore, IkBζ-p50/p50 complex revealedthat ANK3-7 interact with the dimerizationdomain of the p50 subunit (Fig. 4). The bindingorientation of IkBζwith this homodimer is similarto that of the classical IkBa-p50/p65 heterodimer.Although the binding orientation is the same,there might be some differences in the regulationof NF-kB-dependent gene expression by IkBaand IkBζ. Activation of p65-containing NF-kBheterodimer by LPS or IL-1 leads to the expres-sion of NF-kB-dependent genes, including IkBproteins, IkBa and IkBζ. Following translocation,IkBa enters the nucleus, where it targets NF-kBp50/p65 dimers and removes them from DNAthrough the acidic PEST motif of IkBa and thebasic DNA-containing surfaces of the NF-kB p65subunit that likely disrupt protein/DNA binding.In contrast, IkBζ enters into the nucleus and tar-gets the p50/p50 homodimer, which is alreadybound to the promoter region, thereby blockingthe transcription due to the unavailability ofthe TAD. Overexpression experiments havesuggested that IkBζ exhibited transactivation

IkBz, Fig. 4 IkBζ ARD-p50 homodimer interface.(a) The p50/p50 dimers are blue and red in the ribbondiagram. Docked IkBζ is green in the ribbon diagram.(b) The p50 (chain A)-IkBζ binding interface. Side chainsof the amino acid contributing to the hydrogen bonding

formation (marked as black) are represented by a stickmodel with the residue name and numbers shown next tothem. (c) The p50 (chain B)-IkBζ binding interface isshown in a similar fashion as in (b)

2518 IkBz

potential (Motoyama et al. 2005); hence IkBζmediates transcriptional activity by binding withDNA-bound p50/p50 homodimer, thereby pro-viding a transactivation domain to the NF-kBcomplex. Such IkBζ-mediated transcription isimportant for the production of IL-6, antimicro-bial peptides, lipocalin, hDB-2, and the geneslisted in Table 1. Finally, it should be noted that,when compared with other IkB proteins, IkBζpossesses numerous functions that occur via bind-ing with different nuclear proteins. Recent studiesof MD (molecular dynamics) simulation of IkBhave revealed that IkBζ possesses more thermo-dynamically flexible residues than other IkBmembers. These findings demonstrate that struc-tural flexibility is the major factor that enablesIkBζ to interact with different sets of nuclear pro-teins (Manavalan et al. 2010; Basith et al. 2013).

Summary

Studies of IkBζ have provided evidence for multi-step regulation of inflammatory responses in TLRsignaling. Upon cell activation by appropriatestimuli, primary responses are induced by rapidactivation of the major transcription factor(NF-kB), which is activated through posttransla-tional modifications such as phosphorylation

without de novo protein synthesis. During thisperiod, transcriptional regulators such as IkBζare induced via stimuli-specific mechanisms. Sec-ondary response genes are activated, and primaryresponses are gradually diminished via the com-binations of major transcription factors and induc-ible regulators. Since the genes that are activatedvia secondary responses also include other tran-scription factors, stimulus-specific transcriptionalactivation would proceed in a multistep fashionwith time after the stimulation. In vitro studieshave shown that these nuclear IkB proteins inter-act with the p50 or p52 subunits of NF-kB. Onlyp50/p52 double knockout mice, but not singleknockout mice, exhibit severely defectiveimmune disorders such as osteopetrosis. How-ever, some immunological phenotypes occur inmice lacking only one nuclear IkB protein. Thiscondition may be compensated by utilizingother IkB proteins. Further studies are requiredto clarify and discover new and detailed physio-logical aspects of the nuclear IkB proteins inthe future by using mice devoid of two or allthree nuclear IkB proteins. In conclusion, it canbe seen that nuclear IkBζ not only contributes toNF-kB mediated transcription but also plays animportant role in innate immune responses bymodulating the expression of proinflammatorycytokines.

IkB-Zeta 2519

Acknowledgments This work was supported by theNational Research Foundation of Korea (NRF-2015R1A2A2A09001059).

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IkB-Zeta

▶ IkBz