Review Article The E3 Ligase CHIP: Insights into Its Structure and … · 2019. 7. 31. · Review...

Review ArticleThe E3 Ligase CHIP Insights into Its Structure and Regulation

Indranil Paul and Mrinal K Ghosh

Cancer Biology and Inflammatory Disorder Division Council of Scientific and Industrial Research-Indian Institute ofChemical Biology (CSIR-IICB) 4 Raja SC Mullick Road Kolkata 700032 India

Correspondence should be addressed to Mrinal K Ghosh mrinalresgmailcom

Received 7 February 2014 Accepted 7 April 2014 Published 24 April 2014

Academic Editor Hiroyuki Tomiyama

Copyright copy 2014 I Paul and M K Ghosh This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The carboxy-terminus of Hsc70 interacting protein (CHIP) is a cochaperone E3 ligase containing three tandem repeats oftetratricopeptide (TPR) motifs and a C-terminal U-box domain separated by a charged coiled-coil region CHIP is known tofunction as a central quality control E3 ligase and regulates several proteins involved in a myriad of physiological and pathologicalprocesses Recent studies have highlighted varied regulatory mechanisms operating on the activity of CHIP which is crucial forcellular homeostasis In this review article we give a concise account of our current knowledge on the biochemistry and regulationof CHIP

1 Introduction

Maintaining the integrity of the proteome is essential fornormal cellular functions Although the native state of aprotein is energetically favoured the attainment of it is a ther-modynamic challenge The matured form of a given proteinremains in a dubious equilibrium oftenmisfolding to variousdegrees during its lifespan as a result of stochastic fluctua-tions presence of destabilizing mutations stress conditionsor unique metabolic challenges such as cancer or aging Inaddition a myriad of posttranslational modifications (suchas phosphorylation and acetylation) canmark native proteinsfor regulated degradation borne out of signaling necessities[1]

The carboxyl-terminus of Hsc70 interacting protein orCHIP (referred to hereafter as CHIP) is a U-box typechaperone associated E3 ligase CHIP was identified in aninteraction-cloning screen as a TPR-containing and Hscp70interacting protein It was found to be highly expressed intissues (such as skeletal muscle) with high metabolic activityand protein turnover [2]This dual-function cochaperoneE3ligase protein activates HSF1 under various stress conditionsand confers protection against apoptosis The presence ofCHIP is essential for eliciting a normal heat stress response[3 4] In contrast to cochaperones Hip Hsp40 and BAG1which promote substrate binding and release CHIP was

found to attenuate ATP hydrolysis and inhibit the forwardHsc70Hsp70 cycle at least in vitro [2 5] Predictive of its U-box CHIP was identified as the first chaperone associated E3ligase that targets Hsp90 (partially folded) and Hsp70 (mis-folded) clients for degradation through the 26S proteasome[6ndash9] and is suggested to be also involved in substrate deliveryto the proteasome [6 10]

Over the last decade CHIP has been demonstrated toregulate a number of biological processes Aberrations inprotein expression and activity of CHIP are observed inmanypathological conditions stressing the need to understand itsregulation In this review we attempt to comprehensivelysummarize the recent findings on CHIP focusing mostly onits structure and various regulatory mechanisms operatingon it Finally we evoke some important questions of CHIPbiology

2 Initial Discovery and Characterization

Tetratricopeptide repeats (TPR) are protein-protein interact-ing motifs that consist of loosely conserved 34-amino acidstretch usually present in tandem in proteins with diversecellular functions [11] particularly those that interact withchaperones including Hip Hop and the cyclophilins [12ndash14]

In an attempt to identify novel TPR-containing proteinsin the human heart a fragment corresponding to nucleotides

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 918183 12 pageshttpdxdoiorg1011552014918183

2 BioMed Research International

U-boxTPR H sapiens

U-boxTPR M musculus

U-boxTPR B taurus

U-boxTPR R norvegicus

U-boxTPR X tropicalis

U-box C familiaris

U-box

TPR

D rerio

26 127 230 293

29 126 228 300

32 125 227 299

29 126 228 300

34 131 234 306

124 189 291 363

12 110 208 280

TPR

Figure 1 Schematic diagram showing alignment of CHIP from different species The figure clearly depicts very high conservation in thecentral and C-terminal regions The N-terminal protein-protein interaction motif is apparently amenable to alterations probably for speciesspecific specialization of interaction

721 to 1150 of the human CyP-40 (cytochrome P-40) cDNAwas radiolabeled with [120572-32P] dCTP and used to screen aphage library of human heart cDNA at low stringency CHIPwas identified through this screen [2] CHIP cDNA encodesa 345-kDa protein Evolutionary CHIP is a well-conservedprotein with an amino acid sequence similarity of sim98 withmouse and sim60 with the fruit fly [11] The C-terminus 94residues (the U-box region) seem to be least altered withsim87 similarity among these speciesrsquo (Figure 1) Intracellu-larly CHIP was found to primarily localize to the cytoplasmunder quiescent conditions [2] although a fraction of CHIPwas later found to be present in the nucleus as well [10]

21 CHIP A Protein Quality Control E3 Ligase The classicalrole of chaperones was regarded as those of folding andsalvaging proteins However it was apparently clear that eachand every newly synthesized polypeptide that engages withchaperones for its folding could not reach the native stateand thus there must be a link between the chaperones andthe degradation pathways CHIP provided that link CHIPwas identified as a TPR-containing protein that inhibitedforward cycle of chaperones However the curtain on theactual role of CHIP was raised when it was shown to haveintrinsic E3 ligase activity owing to the C-terminus U-boxa domain related to the well-characterized RING-fingerExperimental studies with increased cellular levels of CHIPfound a marked shift towards degradation of the Hsp70Hsp90 clients glucocorticoid receptor (GR) [6 15] andCFTR [10 16] Definitive evidence for a quality control roleof CHIP was provided when it was shown to selectivelyubiquitinate thermally denatured luciferase (and not the

native form) when captured by Hsc70 and Hsp90 [6 8 17] Itcan be noted here that in bacteria which lack ubiquitinationATP-dependent proteolysis also depends on chaperones forsubstrate recognition by Lon proteases and ClpP as do yeastswhich are known to have CHIP In other words chaperonesare an excellent way to identify misfolded proteins

That CHIP may have participation in protein turnoverwas hinted by initial observations depicting a relatively higherexpression of CHIP mRNA in tissues with a large proportionof terminally differentiated nonproliferating cells and highlevels of metabolic activity such as skeletal muscle heartand brain [2] The physiological importance of CHIP cameinto light with the observations that sim20 of CHIP null(CHIP minus minus) mice die at embryonic stages and 100 fail tosurvive thermal stress [18]

In mammals CHIP is located within chromosome 16 Noisoforms or transcript variants have been reported yet forCHIP

3 Structural Organization of CHIP

31 Primary Structure of CHIP The primary organizationof CHIPrsquos structure comprises two structured motifs anN-terminus TPR domain and a C-terminus U-box domainseparated by a central coiled-coil region [2 19] The entireTPR domain (residues 26ndash131) consists of three pairs of TPRrepeats where each TPR consists of two antiparallel 120572-helicesseparated by a turn such that apposed bulky and small sidechains form a ldquoknob and holerdquo structure The hydrophobicsurface of this structure imparts the ability of protein-proteininteraction to the TPR motifs [20 21] The N-terminus of

BioMed Research International 3

an elongated seventh helix (helix 7) packs against the thirdTPR repeat The structure of this helix 7 has significantconsequences for CHIP (see below) U-boxes are structurallyrelated to RING-finger domains except that U-boxes arestabilized by hydrogen bonds instead of zinc binding [22]Mechanistically there are two categories of E3 enzymes [23]HECT domain E3 ligases form transient thioester linkageswith ubiquitin to a C-terminus active-site cysteine residueand transfer the ubiquitin moiety to the substrate bound totheN-terminusOn the other hand RING-finger orU-boxE3ligases simply act as scaffolds or adaptors which position thesubstrate in precise proximity to the E2-ubiquitin thioester(E2-Ub) In CHIP the U-box domain (residues 232ndash298)consists of a pair of 120573-hairpins running into a short 120572-helixfollowed by a third hairpin leading to a C-terminal 120572-helixThe central region (residues 128ndash229) forms helical hairpinsand has been shown to be essential for dimerization of CHIPand consequently its function [19]

32 Structural Aspects of CHIP Activity Another interestingstructural feature of CHIP is that it forms an asymmetrichomodimer meaning that each protomer inherently adoptssignificantly different conformations in the dimer The struc-tural explanation is as follows First CHIP dimerizationinvolves two interacting interfaces between each monomertheU-box domain and the region linking the TPR andU-boxThe U-box forms a parallel dimer with a sim2-fold symmetrySimilarly the core of second interface the helical hairpinsalso displayssim2-fold symmetryHowever the local 2-fold axisrelating the respective symmetry-axes of U-box and helicalhairpins does not coincide but is tilted by 30∘ Second helix 7has a different structure in the two protomers of the CHIPdimer In one it is a straight 120572-helix while in the otherthe polypeptide is broken into two separate and mutuallyperpendicular120572-helicesThe functional consequence of thesearrangements is that only one of the U-boxes in the CHIPdimer is active thus effectively displaying a ldquohalf-of-sitesrdquoactivity which allows it to form monotonic polyubiquitinchains [24]

33 Interaction with E2s CHIP was previously shown tocooperate with the UbcH5 family of E2s to catalyze Lys-48-linked polyubiquitination UbcH5 is a stress associatedE2 UbcH5simUb conjugates have been shown to adopt bothinfinite spiral and linear staggered arrangement (backsideinteraction) Interestingly CHIP can directly interact withfour (or more) UbcH5simUb conjugates allowing wide confor-mational flexibility during polyubiquitination of substrates[25] One consequence of this is the possibility of formationof forked ubiquitin chains (see Section 4310)

Later Xu and coworkers [26] reported CHIP to interactwith the dimeric ubiquitin E2 complex Ubc13-Uev1A whichcatalyzes the synthesis of Lys-63-linked polyubiquitinationThey analyzed crystal structures of mouse CHIP U-box incomplex with Ubc13-Uev1a and found a common ldquoSer-Pro-Alardquo motif present in UbcH4 UbcH5 and Ubc13 that medi-ates and is necessary for their interaction with the CHIP U-box Although the catalysis of K63-linked polyubiquitinationis an inherent structural feature of Ubc13-Uev1A it is not

clear at present how the binding of CHIP to Ubc13-Uev1Afacilitates the process Interestingly CHIP only stimulatesthe formation of free K63-polyubiquitin by Ubc13-Uev1a andthus may have to interact sequentially with other E2 enzymesto attach K63-linked polyubiquitin chains on substratesCHIP binds 3- to 5-fold more strongly to uncharged Ubc13than UbcH5a It remains to be seen whether CHIP displayssimilar relative binding affinities towards ubiquitin chargedE2s [26]

The physiological ramifications of the fact that CHIPcan function with different E2s to catalyze distinct forms ofpolyubiquitination are yet to be studied in detail

4 Regulation of CHIP

41 CHIP Is Regulated at the Transcriptional and Posttran-scriptional Levels

411 Transcriptional Regulation A limited number of studieshave been devoted to understand the regulation of CHIPmRNA under different physiological and pathological con-texts Under normal conditions the basal levels of Hsc70Hsp90 and its cochaperones suffice to maintain proteinhomeostasis However it is not difficult to imagine that amassive build-up of misfolded polypeptides resulting fromcellular stresses would need rapid readjustments in the cellu-lar levels of these proteins Indeed themRNA levels of Hsp70andor CHIP are upregulated under various stress conditionssuch as heat-shock overexpression of the pathogenic form ofpolyQ [27] and oxidative damage [28] and have been shownin vivo or in vitro to provide protection (Figures 2(a) and2(b)) [29 30] From a clinical point of view such stressorsare an integral feature of various human pathologies includ-ing neurodegenerative disorders muscular dystrophies andheart ailments

Recently CHIP has been intimately linked to variousinflammatory responses such as regulation of IL-4 andTLR signaling T cell activation and DALIS formation [31ndash36] Treatment of RAW2647 cells by peptidoglycan acti-vates TLR2 receptors and through JNK pathway upregulatesexpression of CHIP Interestingly although CHIP is crucialfor proper TLR2479 signaling its expression seems to becontrolled by only TLR2 signaling as LPS (TLR4 ligand) orCpG ODN (TLR9 ligand) does not stimulate endogenousCHIP overexpression [37 38] (Figure 2(c))

The expression of CHIP is also altered in human malig-nancies Both mRNA and protein levels of CHIP have beenfound to be lower than corresponding normal tissue in breast[38 39] colorectal [40 41] and gastric cancer [42] andcorrelate highly with tumour prognosis

412 Posttranscriptional Regulation An instance of post-transcriptional regulation of CHIP mRNA has been reportedin the context of bone morphogenesis CHIP is downreg-ulated in calvarial and osteoblast progenitor cells duringosteoblast differentiation In MC3T3-E1 cells the microRNAmiR-764-5p has been shown to inhibit the translation ofCHIP mRNA by binding at its 31015840-UTR Perturbations ofmiR-764-5p could be rescued by a concomitant and opposite


(a)

miR-764-5p

Osteoblastdifferentiation

HS

Peptidoglycan

Heat-shockresponse

TLR2PolyQ

PolyQ SepsisCardiovascular diseases

(b) (d)(c)

Figure 2 Schematic representation of known transcriptional and posttranscriptional regulation of CHIP under various conditions See textfor details

Ub

Substrate

Substrate UbUbUb

Ub

Ataxin-3

Ube2w

Ataxin-3

(a)

IGF-1

Erk5

ICER

Myocardialinfarction

(b)

Figure 3 Schematic representation of known posttranslational modes of regulation of CHIP (a) Monoubiquitination (b) phosphorylationSee text for details

extraneous expression of CHIP Translational repression ofCHIP mRNA by miR-764-5p was found to be essential forproper osteoblast differentiation (Figure 2(d)) [43] Clearlyregulation of CHIP at the mRNA level is an importantstrategy for regulating its biological activity

42 Posttranslational Modifications of CHIP Are a PotentialMode of Regulation The activity of CHIP is also regulatedthrough posttranslational modifications [44] Such modi-fications of CHIP likely dictate recruitment of cofactorswhich in turn may participate in restricting the type andlength of ubiquitin chains produced [45 46] CHIP is knownto undergo regulatory ubiquitination in cells and in vitro[8 47 48] which facilitates targeting of its substrates forproteasomal degradation [5] For example the proteasomalsubunit S5a is a ubiquitin-interactingmotif (UIM) containingprotein which stimulates turnover of CHIP substrates bypreventing the formation of forked ubiquitin chains [49]Interestingly ubiquitination of CHIP increases under con-ditions in which CHIP levels and activity are increased

[50] The mechanistic details have been recently workedout [51] Ataxin-3 a specialized ubiquitin-interacting motif(UIM) containing deubiquitinase (DUB) associates withCHIP monoubiquitinated by the initiator E2 Ube2w (andperhaps other E2s) at Lys 2 of CHIP [51] This modificationoccurs before or just after polyubiquitin chain initiationConsequently ataxin-3 provides a chain editing activityeffectively determining the dynamics of substrate ubiquitina-tion by CHIP Upon completion of substrate ubiquitinationas determined by chain length ataxin-3 presumably bindsubiquitinated substrate through itsUIMs and deubiquitinatesCHIP thus terminating the reaction (Figure 3(a)) [51]

Although direct evidence showing similar posttransla-tional modifications other than ubiquitination in regulatingthe activity of CHIP is still lacking the possibility of suchevents cannot be ruled out Indeed the N- and C-terminalregions of CHIP have been proposed to contain functionalphosphorylation sites (Figure 4) [52] In an interesting studyan association of the phosphatase laforin and the E3 ligasemalin with CHIP has been shown to be essential to achieve


U-boxTPR

26

1 303

127 230 293

S25S23

S19 T276S273

T271

Figure 4 Schematic representation of various conserved putativephosphorylation sites on CHIP (source httpwwwhprdorg) Seetext for details

cellular heat-shock response mediated through HSF1 Eitherlaforin or malin does not affect the nuclear localizationhyperphosphorylation or the trimerization property of HSF1Although the nuclear translocation of CHIP seems to bepromoted by the complex [53] what effects does the complexhave on the phosphorylation status of CHIP has not beenreported yet but presents an interesting possibility

Associations between CHIP and kinases such as ERK5and Lim kinase 1 (LIMK1) have also been reported [54 55]The ERK5-CHIP interaction was shown to be an essentialmodule operating downstream of IGF-1 induction havinga cardioprotective effect mediated through inducible cAMP(cyclic adenosine monophosphate) early repressor (ICER)destabilization ERK5 activation leads to increased CHIPubiquitin ligase activity possibly through a conformationalchange in CHIP [55] (Figure 3(b)) LIMK1 has been shownto associate with CHIP in a complex of which Parkin is also amember [54] Parkin ubiquitinated LIMK1 in dopaminergicneuronal BE(2)-M17 cells but not in kidney-derived HEK293cells and attenuated LIMK1-induced cofilin phosphorylationand subsequent accumulation of actin filaments ConverselyLIMK1 attenuated Parkin autoubiquitination and its activityin a phosphorylation independent manner [54] In both ofthese cases the possibility of a phosphorylation dependentregulation of the activity of CHIP seems to be an interestingpossibility and needs further investigation

421 Half-Life Other than the posttranslational modifica-tions that directly affect the function of CHIP the half-life orprotein stability of CHIP may also be a possible mode of itsregulation For instance a study specifically devoted to thisaspect of CHIP found that the TPR domain of CHIP whenisolated was monomeric and stable while its U-box domainformed dimers and had very low stability [56] Thus morebiochemical studies are clearly needed to shed light on thisimportant aspect of CHIP biology

43 Activity of CHIP Is Regulated by Interactions with OtherProteins A number of proteins have been shown to interactwith CHIP and regulate its activity

431Ca2+S100 Proteins S100 proteins are a family of at least25 low molecular weight multifunctional members foundin vertebrates with regulatory roles in a variety of cellularprocesses [57 58] Recently S100A2 and S100P were foundto associate in vitro and in vivo (Huh-7 and Hep3B cells)with TPR-containing cochaperones CHIP (and also Hop)

in a Ca2+ dependent manner This interaction seems tocompete with substrate binding and consequently suppressthe E3 ligase activity of CHIP towards Hsp70 Hsp90 p53HSF1 and Smad1 Interestingly S100 has no effect on CHIP-UbcH5a interaction Further Lys-30 and Pro-269 were foundto contribute to S100-CHIP interactionThis report identifiesa novel mechanism of protein quality control in response tointracellular Ca2+ signaling (Figure 5(a)) [59]

432 Xap2 The hepatitis B virus X-associated protein(Xap2) is a TPR-containing protein with poorly definedfunctions except that it plays a role in dioxin receptor (DR)metabolism Along with Hsp90 and p23 Xap2 retains the DRin a latent state until it is activated upon binding of dioxinsor structurally related forms of xenobiotics and translocatesto the nucleus for activating transcription of target genesinvolved in xenobiotic metabolism [60ndash62] CHIP has beenproposed to degrade DR efficiently in the nucleus an activitythat can be overcome by overexpression of XAP2 ApparentlyXAP2 which also contains TPR domain competes withCHIP for binding with Hsp90 thus shifting the balance ofthe triage towards folding (Figure 5(b)) [48]

433 OLA-1 Obg-like ATPase 1 or OLA-1 is a highly con-served sim45 kDa cytosolic ATPase and belongs to theTRAFAC class Obg family and YchF subfamily of P-loopNTPases YchF subfamily of NTPases binds and hydrolyzesATP more efficiently than GTP [63] Recently OLA-1 wasshown to positively regulate the heat-shock response byprotecting Hsp70 from CHIP mediated degradation Thehalf-life of Hsp70 is shortened in OLA-1minusminus MEFs OLA-1 competes with CHIP for binding at the C-terminal ofHsp70 and thus precludes CHIP activity towards Hsp70(Figure 5(b)) [64]

434 HspBP1 It is a nucleotide release factor of Hsc70 thathas similar expression patterns as of CHIP and competeswith BAG1 and BAG2 for the ATPase domain of Hsc70[65] In HeLa cells HspBP1 apparently inhibits the ubiquitinligase activity of CHIP bound to Hsc70 but not Hsp90in a noncompetitive manner HspBP1 presumably inducesconformational changes of the chaperone complex that keepubiquitin acceptor sites of the substrate from the reach ofCHIP HspBP1 itself is not recognized as a substrate proteinby CHIP Interestingly although overexpression of HspBP1influenced CFTR biogenesis it did not have any effect onglucocorticoid hormone receptor turnover pointing towardsa yet poorly understood mechanism of client specificity(Figure 5(c)) [16]

435 BAG1 Human Bcl-2-associated athanogen (BAG) is afamily of six known members (BAG1 to BAG6) with diversecellular functions [66ndash68] They are characterized by thepresence of a BAG domain implicated in direct interactionwith the ATPase domain of Hscp70 and can regulate chap-erone activity in both positive and negative manners [69]During the sorting of chaperone clients to the proteasomeCHIP can cooperate with the Hsc70 cochaperone BAG1


Xap2OLA-1

HspBP1 BAG2 HSJ1aS5a

BAG1BAG3

S100A2S100P

Hsp7090

CHIP

E2

Substrate

(a) (b) (c) (d) (e) (f)

Figure 5 Schematic representation of regulatory protein interactions between CHIP and various reported proteins (a) Competition withsubstrate binding (b) competition with chaperone binding (c) conformational modification of CHIP (d) disruption of CHIP and E2interaction (e) facilitation of chaperone binding and (f) facilitation of interaction with E2 See text for details

The two cochaperones interact simultaneously with Hsc70as BAG1 binds to the amino terminal ATPase domain ofthe chaperone and leaves the carboxy-terminus of Hsc70available for an association with CHIP [7 70] BAG1 isintimately involved in the regulation of the ATP-dependentpeptide binding and release cycle of Hsc70 by stimulatingnucleotide exchange [71 72] Furthermore BAG1 associateswith the proteasome through an integrated ubiquitin-likedomain and thereby recruits Hsc70 chaperone complexesto the proteasome [73] As a consequence BAG1 stimulatesCHIP-induced degradation of the glucocorticoid hormonereceptor (Figure 5(e)) [7]

436 BAG2 It is a protein of sim34 kDa and exists as dimersunder physiological conditions Immunoprecipitates of over-expressed BAG2 fromHeLa cells contained CHIP which wasreleased upon ATP treatment a reflection of ATP-regulatedchaperonecochaperone interactions BAG2 inhibits theubiquitin ligase activity of CHIP bound to Hsc70 by disrupt-ing the interaction between CHIP and its E2 and UbcH5bBAG2 itself is not ubiquitinated by CHIP (Figure 5(d)) [7475]

437 BAG3 There is a functional reciprocality betweenBAG1 and BAG3 during conditions of acute stress or agingoften referred to as the ldquoBAG1-BAG3 switchrdquo BAG3 hasrecently gained much popularity as a mediator of a novelmacroautophagy pathway termed chaperone-assisted selec-tive autophagy or CASA that exploits Hsp70 and cochaper-ones including CHIP Both BAG3 and CHIP have been foundto induce the association of the phagophore-interacting ubiq-uitin adaptor p62 withHsp70 P62 acts as an adaptor betweenubiquitinated substrates and phagophore membranes BAG3also indirectly stimulates the binding of CHIP to Hsp70

complex Reciprocally CHIP ubiquitinates BAG3 furtherfacilitating its interaction with p62 eventually resulting inengulfment by phagophore (Figure 5(e)) [76 77]

438 BAG5 BAG5 protein was shown to interact with CHIPthrough Hsp70 and inhibit the E3 ubiquitin ligase activity ofCHIP towards 120572-synuclein The mechanism by which BAG5inhibits CHIP remains to be elucidated [75 78]

439 HSJ1a Human HSJ1 isoforms HSJ1a and HSJ1b arecochaperones which facilitate forward cycle of Hsc70 protectpolyubiquitin chains against trimming and promote pro-teasomal sorting of ubiquitinated proteins [79 80] Coex-pression of CHIP and HSJ1a in HeLa cells led to a sig-nificant increase in the amount of HSJ1a bound ubiquiti-nated polypeptidesHSJ1a directly stimulatedCHIPmediatedubiquitination Further CHIP mediated ubiquitination ofHSJ1a promoted client sorting to the proteasome machinery(Figure 5(f)) [81]

4310 S5a Under in vitro conditions many ring-finger andU-box E3 enzymes including CHIP in conjunction withUbcH5 have a tendency to form forked ubiquitin chainsin which two Ubs are linked to adjacent lysines on theproximal Ub instead of standard isopeptide linkages in whichthe C-terminal carboxyl group of a Ub is coupled throughan isopeptide bond to the 120576-amino group on one of the 7possible lysines on the proximal Ub These forked linkagesresist proteasomal isopeptidases and bind only weakly to the26S proteasome compared to standard linkages [82] In astudy to identify cellular Ub-binding proteins that preventformation of these nondegradable conjugates S5a (Rpn10)was identified S5a was found to enhance proteasomal degra-dation of firefly luciferase ubiquitinated by CHIPUbcH5


by preventing forked chain formation S5a occurs free inlarge amounts in the cytosol Cells lacking S5a are viablebut show defects in proteasomal degradation of a specificsubset of cellular proteins (specifically where UbcH5 is theE2) resulting in increased levels of Ub conjugates as oftenfound in many neurodegenerative diseases Interestingly S5agets ubiquitinated by CHIPUbcH5 and seems to be essentialfor eliciting a proper heat-shock or oxidative stress response(Figure 5(f)) [49 83]

4311 Ataxin-3 Thedeubiquitinase (DUB) ataxin-3 containsan amino-terminal protease domain followed by three UIMsthat bind chains of four or more ubiquitins [84 85] Unlikeother characterized DUBs that function to deubiquitinatesubstrates and rescue them from proteasomal delivery [86]ataxin-3 promotes the flux of substrates through degradationpathways [87 88] Recently ataxin-3 has been shown as anexample of a CHIP-associated DUB (Figure 3(a) and abovefor details) In pathological conditions such as spinocerebel-lar ataxia type 3 the polyQ-expanded ataxin-3 has higheraffinity forCHIP and correlateswith decreased levels of CHIPultimately causing proteotoxicity [51]

44 Collaboration with Other E3rsquosmdashAn E4 Activity of CHIPApart from a direct ubiquitin ligase activity CHIP is alsoknown to facilitate ubiquitination mediated through otherE3rsquos in effect functioning as an E4 ligase The defining exam-ple is Parkin Parkin is a RING-finger type E3 ligase initiallyidentified as the culprit (due to an inactivating mutation) inan autosomal recessive form of juvenile Parkinsonrsquos disease(AR-JP) [89] Later the Pael-R membrane receptor wasfound to be a substrate of Parkin and its accumulation inendoplasmic reticulum (ER) caused ER-stress induced neu-ronal death and hence the disease [90 91] In an interestingstudy CHIP was found to potentiate Parkin mediated Pael-R ubiquitination both in vivo and in vitro SurprisinglyHsp70 bound Pael-R is a poor substrate of Parkin and it isonly when CHIP displaces Hsp70 (presumably by blockingits ATPase activity) from ParkinPael-R complex can Parkinfully exercise its E3 activity towards Pael-R Although CHIPcan bind to both Parkin and Pael-R it showed no ubiquitinligase activity for Pael-R and thus functions purely as an E4ligase in this scenario [47 92 93]

Another E3 ligase with which CHIP has been foundto interact is the E3 ligase complex SCFSkp2 (Skp1-Cullin-F boxS-phase kinase associated protein 2) Among many others p27c-Myc E47 and Tal1Scl (T cell acute leukemia 1stem cellleukemia) are well known substrates of SCFSkp2 [94ndash98] Inthe context of Tal1Scl and E47 which forms heterodimers toactivate transcription the role of CHIP has been found to becrucial Although both Tal1Scl and E47 associate with Skp2independently and are apparently ubiquitinated by SCFSkp2overexpression of chaperone-noninteracting and E3 ligasedeficient forms of CHIP or knockdown of endogenous CHIPdiminishes Tal1Scl and E47 ubiquitination Interestinglydespite Skp2 interacting with Hsc70 and CHIP with cullin1the association of CHIP with Hsc70 seems to be critical forefficient degradation Thus it has been suggested that CHIP

could promote activity of some factors required for SCFSkp2mediated ubiquitination of at least a few of its substrates ina manner dependent on chaperone binding and ldquoE4rdquo activityof CHIP [97]

45 Subcellular Localization of CHIP Is Regulated Despitebeing initially characterized as a cytoplasmic protein thenuclear existence of CHIP is now well established Usingtechniques such as immunofluorescence microscopy (IFM)immunohistochemistry (IHC) and immunoblotting (IB) ofcytoplasmic and nuclear fractions CHIP has been shown tolocalize to the nucleus under different physiological contexts[50] Consistently we found that CHIP (both endogenousand exogenous) is expressed in both the cytoplasm andthe nucleus of DBTRG-05MG [99] HEK293 C6 U87MGU118MG U138MG MCF7 and HepG2 cells (unpublished)

Examples exist to support the notion that subcellularlocalization of CHIP is regulated and in turn can regulateits functional activity Upon heat-shock of murine fibroblastsHSF1 and CHIP are known to rapidly translocate to thenucleus as a part of the response and reaccumulate inthe cytoplasm during recovery from heat-shock [18] Inthis context malin and laforin have recently been shownto promote the nuclear translocation of CHIP and to beessential for providing a complete protection against heat-shock [53] In rodent brain and primary cortical neuronsCHIP rapidly (within 5ndash10 minutes) and transiently (for upto 60 minutes) accumulated in the nucleus following heat-shock and oxygen-glucose deprivation which was linked tothe capacity and capability of these cells to recover andsurvive [100] Although the identities of nuclear substratesof CHIP are obscure a few have nevertheless been reportedFor instance endogenous CHIP and mutant AR were foundto colocalize in the nuclei of spinal anterior horn neurons ofthe AR-97Q mice and SBMA (spinal and bulbar muscularatrophy) patients [101] Similarly CHIP has been foundto colocalize with ataxin-1 nuclear inclusions [102 103]Coimmunoprecipitation experiments using cytoplasmic andnuclear fractions of mammalian cells showed that CHIPinteracted with p65 [104] RUNX1 [105] and RUNX2 [106]proteins only in the nuclear extracts but not from thecytoplasmic fractions Furthermore under heat-stress CHIPnot only assisted p53 to regain its native and transcriptionallyactive form but also comigrated with it into the nucleus andwas found tethered to p21 and p53 promoters along with p53as revealed by chromatin immunoprecipitation [107]

In addition to the nucleus studies have pointed towardsa role for CHIP in the endoplasmic reticulum (ER) CHIPhas been shown to colocalize with CFTR and Hsp70 at theER membrane indicating a role in CFTR biogenesis Laterinactivation of CHIP was shown to permit a subpopulationof CFTR-ΔF508 to fold escape the ER and accumulate asa maturely glycosylated species [10 108] Growth hormonereceptor (GHR) is efficiently folded in the ER and underconditions of CHIP depletion both precursor and matureGHR accumulate inside the cells [109] Recently a studylinking ER stress and tauopathy reported that upon elicitationof ER stress (using glucose deprivation) the interaction


between tau a known target of CHIP and CHIP significantlydecreases leading to an accumulation of tau and consequenttauopathy [110]

However the details of the molecular mechanisms thatmodulate the intercompartmental distribution of CHIP needto be further explored which might provide new insights intothe significant role of CHIP inside the various organelles inregulating biological phenomena

46 Substrate Recognition by CHIP Is Specific The intimatefunctional association of CHIP with the chaperones hasraised concerns regarding the specificity of CHIP towardsits substrates The suspicion that CHIP may promiscuouslyubiquitinate any protein fulfilling the criteria of being struc-turally disorganized to be eligible for being recognized bythe chaperones may not be correct On the contrary severalreports have documented varying degrees of dependency ofauxiliary factors that potentially determine the response ofCHIP towards its substrates However it should be men-tioned that the extent of involvement of the chaperones inevery such case was not determined conclusively

461 Posttranslational Modification (PTM) of SubstratesPhosphorylation is one of the most prolific PTMs in thecell PKA (protein kinase A) mediated phosphorylation ofHDAC8 (histone deacetylase 8) under normal and tumorconditions recruits EST1B (ever shorter telomeres 1B) andHsp70Hsp90 in a complex in a manner which precludesCHIP from ubiquitinating EST1B which is otherwise asubstrate of CHIP [111]

In prostate cancers hormone-ablation therapy in mostcases leads to more aggressive androgen-refractory diseasewhich is characterized by overactivated androgen receptor(AR) mediated signaling and accumulation of cells withneuroendocrine characteristics such as elevation of PTHrP(parathyroid hormone-related protein) levels PTHrP acti-vates EGFR and Src kinase (through an unknown mecha-nism)which in turn phosphorylates AR onTyrosine 534Thisphosphorylation event reduces the interaction between ARand CHIP leading to AR stabilization and enhanced growthof prostate cancer cells even at very low levels of androgen[112]

Similarly under oxidative stress the protein kinase c-Abl phosphorylates another kinaseMST1 (mammalian Ste20-like kinase 1) at Tyrosine 433 which in turn accomplishestwo roles first the event inhibits the degradation of MST1through CHIP and second triggers association betweenMST1 and FOXO3 (Forkhead box O3) thereby activating theMST1-FOXO signaling leading to cell death in both primaryculture neurons and rat hippocampal neurons [113]

Another case of regulated engagement of CHIP forsubstrate ubiquitination is provided by CYP2E1 a liver ERcytochrome P450 responsible for xenobiotic metabolismCYP2E1 displays a biphasic turnover with respect to pres-ence or absence of its substrates (low molecular weightxenobiotics carcinogens and endogenous ketones) BothUBC7gp78 and UbcH5aCHIP were found to ubiquitinateCYP2E1 in vitro a process blocked by intracellular inhibition

of PKA or PKC Several phosphorylationubiquitinationclusters in CYP2E1 were also identified [114]

Apart from regulatory phosphorylation on substratesthe association of ERK5 and CHIP has been shown to beessential for CHIP mediated cardioprotection (see above)Specifically diabetic mice subjected to myocardial infarction(MI) overexpress ICER protein which reduces antiapoptoticBcl-2 levels thus leading to apoptosis Under these con-ditions IGF-1 (insulin-like growth factor 1) induction orMEK5120572 overexpression results in a specific and obligatoryassociation between ERK5 and CHIP this increases theE3 ligase activity of CHIP through a hitherto unknownmechanism subsequently degrading ICER and inhibitingapoptosis (Figure 3(b)) [55] The importance of ERK5CHIPassociation in CHIP mediated cardioprotection is furthersupported by showing that p90RSK mediated inhibitionof ERK5CHIP axis accelerates cardiac apoptosis after MIa phenomenon fully reversible by activating ERK5 Twodistinct mechanisms have been identified for the actionof p90RSK on ERK5CHIP axis first it directly inhibitsERK5CHIP interaction by competing for binding site onERK5 and second it phosphorylates ERK5 at S496 phospho-S496-ERK5 facilitates angiotensin-II mediated inhibition ofCHIP [115]

462 Availability of Ubiquitination Motifs Further supportfor a functional regulation of CHIP mediated ubiquitinationwas provided by Landre and coworkers [116] In the contextof IRF-1 (interferon regulatory factor-1) protein stability theyrecently reported that both CHIP and MDM2 docked at theMf2 domain of IRF-1 and subsequent ubiquitination occurredonly in the specific lysine residues found predominantly inloop structures that extend from the DNA-binding domainThis E3 docking site is not available when IRF-1 is in itsDNA-bound conformation thus linking IRF-1 function andturnover [116]

5 Future Directions

Recent studies provide a plethora of evidences to emphasizethe critical role of CHIP in regulating varied biologicalphenomena Hence an obvious question to be addressed iswhether and how CHIP itself is regulated Evidence gatheredtill date projects CHIP as a highly regulated protein whosefunction in the cell is critically monitored

However we are just beginning to understand variousfacets of CHIP and several mechanistic questions remainwaiting to be answered For example we do not understandthe mechanisms behind the transcriptional regulation ofCHIP although we do have sufficient published data showingdirect relationships between cancers and expression levels ofCHIP Another important issue is substrate specificity Sep-arating the chaperone dependent and independent activitiesof CHIP may also be important to explain the occurrence ofsuch a diverse array of its substrates In this context is CHIPfunction regulated through interaction with adaptors andormodulators Further as CHIP is known to catalyse multipletypes of polyubiquitination reactions it would be necessary


to have enough knowledge of the various substrates of CHIPbeing targeted by these different modifications Also whatare the criteria for selection of E2s that CHIP selects under aparticular condition Finally a mechanistic understanding ofhow the intracellular distribution of CHIP is regulated undervarious physiological conditions will also be critical

Therefore understanding how CHIP is regulated interms of transcriptional posttranscriptional posttransla-tionalmodifications and interacting partners and subcellularlocalization is expected to provide new insights into theoverall biological role of CHIP

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The authorsrsquo lab is supported by Grants provided by Councilof Scientific and Industrial Research (CSIR) (EMPOWER(OLP-2) MEDCHEM (BSC-0108) miND (BSC-0115) andCSIR-MAYO Clinic (MLP-0017))

References

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[3] S-A Kim J-H Yoon D-K Kim S-G Kim and S-G AhnldquoCHIP interacts with heat shock factor 1 during heat stressrdquoFEBS Letters vol 579 no 29 pp 6559ndash6563 2005

[4] S-B Qian H McDonough F Boellmann D M Cyr and CPatterson ldquoCHIP-mediated stress recovery by sequential ubiq-uitination of substrates and Hsp70rdquo Nature vol 440 no 7083pp 551ndash555 2006

[5] H McDonough and C Patterson ldquoCHIP a link between thechaperone and proteasome systemsrdquo Cell Stress amp Chaperonesvol 8 no 4 pp 303ndash308 2003

[6] P Connell C A Ballinger J Jiang et al ldquoThe co-chaperoneCHIP regulates protein triage decisionsmediated by heat-shockproteinsrdquo Nature Cell Biology vol 3 no 1 pp 93ndash96 2001

[7] J Demand S Alberti C Patterson and J Hohfeld ldquoCoopera-tion of a ubiquitin domain protein and an E3 ubiquitin ligaseduring chaperoneproteasome couplingrdquo Current Biology vol11 no 20 pp 1569ndash1577 2001

[8] J Jiang C A Ballinger Y Wu et al ldquoCHIP is a U-box-dependent E3 ubiquitin ligase identification ofHsc70 as a targetfor ubiquitylationrdquoThe Journal of Biological Chemistry vol 276no 46 pp 42938ndash42944 2001

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[13] J Hohfeld Y Minami and F-U Hartl ldquoHip a novel cochap-erone involved in the eukaryotic Hsc70Hsp40 reaction cyclerdquoCell vol 83 no 4 pp 589ndash598 1995

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[15] X Wang and D B DeFranco ldquoAlternative effects of theubiquitin-proteasome pathway on glucocorticoid receptordown-regulation and transactivation are mediated by CHIP anE3 ligaserdquoMolecular Endocrinology vol 19 no 6 pp 1474ndash14822005

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[97] L Nie HWu and X-H Sun ldquoUbiquitination and degradationof Tal1SCL are induced by notch signaling and depend on Skp2and CHIPrdquo The Journal of Biological Chemistry vol 283 no 2pp 684ndash692 2008

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[101] H Adachi M Waza K Tokui et al ldquoCHIP overexpressionreducesmutant androgen receptor protein and ameliorates phe-notypes of the spinal and bulbar muscular atrophy transgenicmouse modelrdquo The Journal of Neuroscience vol 27 no 19 pp5115ndash5126 2007

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[104] S Wang X Wu J Zhang et al ldquoCHIP functions as a novelsuppressor of tumour angiogenesis with prognostic significancein human gastric cancerrdquo Gut vol 62 no 4 pp 496ndash508 2012

[105] Y Shang X Zhao X Xu et al ldquoCHIP functions an E3 ubiquitinligase of Runx1rdquo Biochemical and Biophysical Research Commu-nications vol 386 no 1 pp 242ndash246 2009

[106] X Li M Huang H Zheng et al ldquoCHIP promotes Runx2degradation and negatively regulates osteoblast differentiationrdquoJournal of Cell Biology vol 181 no 6 pp 959ndash972 2008

[107] V Tripathi A Ali R Bhat and U Pati ldquoCHIP chaperones wildtype p53 tumor suppressor proteinrdquo The Journal of BiologicalChemistry vol 282 no 39 pp 28441ndash28454 2007

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[110] Y Sakagami T Kudo H Tanimukai et al ldquoInvolvement ofendoplasmic reticulum stress in tauopathyrdquo Biochemical andBiophysical Research Communications vol 430 no 2 pp 500ndash504 2013

[111] H Lee N Sengupta A Villagra N Rezai-Zadeh and ESeto ldquoHistone deacetylase 8 safeguards the human ever-shortertelomeres 1B (hEST1B) protein from ubiquitin-mediated degra-dationrdquoMolecular and Cellular Biology vol 26 no 14 pp 5259ndash5269 2006

[112] J DaSilva D Gioeli M J Weber and S J Parsons ldquoTheneuroendocrine-derived peptide parathyroid hormone-relatedprotein promotes prostate cancer cell growth by stabilizing theandrogen receptorrdquo Cancer Research vol 69 no 18 pp 7402ndash7411 2009

[113] L Xiao D Chen P Hu et al ldquoThe c-Abl-MST1 signalingpathwaymediates oxidative stress-induced neuronal cell deathrdquoThe Journal of Neuroscience vol 31 no 26 pp 9611ndash9619 2011

[114] Y Wang S Guan P Acharya et al ldquoUbiquitin-dependentproteasomal degradation of human liver cytochrome P450 2E1identification of sites targeted for phosphorylation and ubiqui-tinationrdquoThe Journal of Biological Chemistry vol 286 no 11 pp9443ndash9456 2011

[115] N-T Le Y Takei T Shishido et al ldquoP90RSK targets the ERK5-CHIP ubiquitin E3 ligase activity in diabetic hearts and pro-motes cardiac apoptosis and dysfunctionrdquoCirculation Researchvol 110 no 4 pp 536ndash550 2012

[116] V Landre E Pion V Narayan D P Xirodimas and K L BallldquoDNA-binding regulates site-specific ubiquitination of IRF-1rdquoBiochemical Journal vol 449 no 3 pp 707ndash717 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

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Zoology


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Microbiology


U-boxTPR H sapiens

U-boxTPR M musculus

U-boxTPR B taurus

U-boxTPR R norvegicus

U-boxTPR X tropicalis

U-box C familiaris

U-box

TPR

D rerio

26 127 230 293

29 126 228 300

32 125 227 299

29 126 228 300

34 131 234 306

124 189 291 363

12 110 208 280

TPR

Figure 1 Schematic diagram showing alignment of CHIP from different species The figure clearly depicts very high conservation in thecentral and C-terminal regions The N-terminal protein-protein interaction motif is apparently amenable to alterations probably for speciesspecific specialization of interaction

721 to 1150 of the human CyP-40 (cytochrome P-40) cDNAwas radiolabeled with [120572-32P] dCTP and used to screen aphage library of human heart cDNA at low stringency CHIPwas identified through this screen [2] CHIP cDNA encodesa 345-kDa protein Evolutionary CHIP is a well-conservedprotein with an amino acid sequence similarity of sim98 withmouse and sim60 with the fruit fly [11] The C-terminus 94residues (the U-box region) seem to be least altered withsim87 similarity among these speciesrsquo (Figure 1) Intracellu-larly CHIP was found to primarily localize to the cytoplasmunder quiescent conditions [2] although a fraction of CHIPwas later found to be present in the nucleus as well [10]

21 CHIP A Protein Quality Control E3 Ligase The classicalrole of chaperones was regarded as those of folding andsalvaging proteins However it was apparently clear that eachand every newly synthesized polypeptide that engages withchaperones for its folding could not reach the native stateand thus there must be a link between the chaperones andthe degradation pathways CHIP provided that link CHIPwas identified as a TPR-containing protein that inhibitedforward cycle of chaperones However the curtain on theactual role of CHIP was raised when it was shown to haveintrinsic E3 ligase activity owing to the C-terminus U-boxa domain related to the well-characterized RING-fingerExperimental studies with increased cellular levels of CHIPfound a marked shift towards degradation of the Hsp70Hsp90 clients glucocorticoid receptor (GR) [6 15] andCFTR [10 16] Definitive evidence for a quality control roleof CHIP was provided when it was shown to selectivelyubiquitinate thermally denatured luciferase (and not the

native form) when captured by Hsc70 and Hsp90 [6 8 17] Itcan be noted here that in bacteria which lack ubiquitinationATP-dependent proteolysis also depends on chaperones forsubstrate recognition by Lon proteases and ClpP as do yeastswhich are known to have CHIP In other words chaperonesare an excellent way to identify misfolded proteins

That CHIP may have participation in protein turnoverwas hinted by initial observations depicting a relatively higherexpression of CHIP mRNA in tissues with a large proportionof terminally differentiated nonproliferating cells and highlevels of metabolic activity such as skeletal muscle heartand brain [2] The physiological importance of CHIP cameinto light with the observations that sim20 of CHIP null(CHIP minus minus) mice die at embryonic stages and 100 fail tosurvive thermal stress [18]

In mammals CHIP is located within chromosome 16 Noisoforms or transcript variants have been reported yet forCHIP

3 Structural Organization of CHIP

31 Primary Structure of CHIP The primary organizationof CHIPrsquos structure comprises two structured motifs anN-terminus TPR domain and a C-terminus U-box domainseparated by a central coiled-coil region [2 19] The entireTPR domain (residues 26ndash131) consists of three pairs of TPRrepeats where each TPR consists of two antiparallel 120572-helicesseparated by a turn such that apposed bulky and small sidechains form a ldquoknob and holerdquo structure The hydrophobicsurface of this structure imparts the ability of protein-proteininteraction to the TPR motifs [20 21] The N-terminus of















(a)

miR-764-5p


HS

Peptidoglycan

Heat-shockresponse

TLR2PolyQ


(b) (d)(c)


Ub

Substrate

Substrate UbUbUb

Ub

Ataxin-3

Ube2w

Ataxin-3

(a)

IGF-1

Erk5

ICER


(b)







U-boxTPR

26

1 303

127 230 293

S25S23

S19 T276S273

T271













Xap2OLA-1


BAG1BAG3

S100A2S100P

Hsp7090

CHIP

E2

Substrate

(a) (b) (c) (d) (e) (f)





























5 Future Directions








Acknowledgment


References
































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















(a)

miR-764-5p


HS

Peptidoglycan

Heat-shockresponse

TLR2PolyQ


(b) (d)(c)


Ub

Substrate

Substrate UbUbUb

Ub

Ataxin-3

Ube2w

Ataxin-3

(a)

IGF-1

Erk5

ICER


(b)







U-boxTPR

26

1 303

127 230 293

S25S23

S19 T276S273

T271













Xap2OLA-1


BAG1BAG3

S100A2S100P

Hsp7090

CHIP

E2

Substrate

(a) (b) (c) (d) (e) (f)





























5 Future Directions








Acknowledgment


References
































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


U-boxTPR

26

1 303

127 230 293

S25S23

S19 T276S273

T271













Xap2OLA-1


BAG1BAG3

S100A2S100P

Hsp7090

CHIP

E2

Substrate

(a) (b) (c) (d) (e) (f)





























5 Future Directions








Acknowledgment


References
































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Xap2OLA-1


BAG1BAG3

S100A2S100P

Hsp7090

CHIP

E2

Substrate

(a) (b) (c) (d) (e) (f)





























5 Future Directions








Acknowledgment


References
































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





















5 Future Directions








Acknowledgment


References
































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






Acknowledgment


References
































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Review Article The E3 Ligase CHIP: Insights into Its Structure and … · 2019. 7. 31. · Review...

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Transcript of Review Article The E3 Ligase CHIP: Insights into Its Structure and … · 2019. 7. 31. · Review...