Misfolded Proteins Recognition Strategies of E3 UbiquitinLigases and Neurodegenerative Diseases

Deepak Chhangani & Nihar Ranjan Jana & Amit Mishra

Received: 24 July 2012 /Accepted: 12 September 2012 /Published online: 22 September 2012# Springer Science+Business Media New York 2012

Abstract Impairment in the clearance ofmisfolded proteins byfunctional proteins leads to various late-onset neurodegenera-tive diseases. Cell applies a strict quality control mechanismagainst malfunctioned proteins which may generate cellularproteoxicity. Under proteotoxic insults, cells immediately adopttwo major approaches to either refold the misfolded proteina-ceous species or degrade the unmanageable candidates. How-ever, the main cellular proteostasis quality control mechanism isnot clear. It is therefore important to understand the events andcellular pathways, which are implicated in the clearance ofrecalcitrant proteins. Ubiquitin proteasome system managesintracellular protein degradation. In this process, E3 ubiquitinligase enzyme provides specificity for recognition of clientproteins. In this review, we summarize various molecularapproaches governed by E3 ubiquitin ligases in the degradationof aberrant proteins. A clear understanding of E3 ubiquitinligases can offer a well tractable therapeutic approach againstneurodegenerative diseases.

Keywords Misfolded proteins . Protein aggregation .

Neurodegenerative diseases . E3 ubiquitin ligases

Introduction

Numerous evidences suggest that various neurodegenerativediseases have a common cause of accumulation of aberrantor misfolding proteins [1]. In postmitotic cells, such as

neurons, protein misfolding induces high neurotoxic threatbecause they cannot reduce the deposition of toxic speciesthrough cell division [2, 3]. Consequently, sequestration ofnormal proteins with misfolded proteinaceous species trig-gers aggregation cascade and initiates disturbance in normalcellular functions [4, 5]. To cope under such situation, cellspossibly try to make two major decisions: (1) degradation ofmisfolded proteins and (2) replacement of aberrant or dam-aged proteins with newly synthesized proteins. Both pro-cesses are highly selective in nature and tightly controlledby various cellular factors [6]. Cellular failures in theclearance of misfolded and damaged proteins lead toformation of intra- or extracellular insoluble depositssuch as aggresome- and amyloid-like structures [7].Misfolded protein accumulation has been observed inmost of the neurodegenerative diseases such as Parkin-son’s diseases, Alzheimer’s disease, amyotrophic lateralsclerosis, and Huntington’s disease [8]. How cellularmachinery challenges protein misfolding and degrada-tion of aberrant proteins to maintain protein homeostasisis a big question of debate?

Eukaryotic cells have evolved quality control system fordegradation of damaged proteins and simultaneously pre-vent cross talk between normal and misfolded proteins. Toachieve protein homeostasis, quality control system requiresinclusive regular activities of molecular chaperones andubiquitin proteasome system (UPS). Chaperones are thehighly conserved proteins that actively mediate protein fold-ing and prevent protein aggregation in cells [9]. Duringstress conditions, cells enormously increase the synthesisof a particular class of heat shock proteins (HSPs) [10].HSP family members act as chaperones and generate cyto-protective response against cellular toxicity induced by mis-folded protein aggregates [11]. Induction of chaperonesprevents accumulation of toxic species and provides neuro-protective response in various neurodegenerative diseases[12–14]. It is unclear how misfolded proteins are selectivelytargeted and eliminated from the dense cellular pool, and

D. Chhangani :A. Mishra (*)Cellular and Molecular Neurobiology Laboratory, Indian Instituteof Technology Rajasthan,Jodhpur 342011, Indiae-mail: amit@iitj.ac.in

N. R. JanaCellular and Molecular Neuroscience Laboratory, National BrainResearch Centre,Manesar,Gurgaon 122050, India

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cells retain an efficient quality control mechanism [15].Addition of a small (8⋅5 kDa) ubiquitin molecule to lysineresidue of target protein is a multistep process. Three differ-ent enzymatic components are required for protein ubiquiti-nylation process. These are ubiquitin-activating enzyme(E1), ubiquitin-conjugating enzyme (E2s), and ubiquitin-ligating enzymes (E3s). Degradation of aberrant proteinsvia UPS progresses through various steps: (1) covalenttagging of several ubiquitin molecules on the protein marksfor degradation. In UPS, E3 ubiquitin ligases exist in abroad range and play a key role in this complex process,i.e., specific substrate recognition and transferring ubiquitinmolecule to it [6, 16], and (2) polyubiquitin chain linkagerecognition by multicatalytic 26S proteasome complex [17].

The most challenging question is to know how theseE3 ubiquitin ligases identify abnormal or misfolded sub-strates and distinguish them for degradation. Interestingly,there are few E3s known which actively participate incellular quality control mechanism. In this review, weconcise our current understanding of various cellulardefense approaches, planned by E3 ubiquitin ligase formisfolded protein degradation and their implication inneurodegeneration and protein conformation disorders.Together with the recent findings, in our current review,we elucidate critical substrates recognition process gov-erned by E3s, specifically employed for clearance ofmisfolded protein aggregation. As shown in Fig. 1, wepropose a model of cellular protein quality control mech-anism, and probably, failure of this system leads tovarious neurodegenerative diseases.

Is Hunting of Misfolded Protein by E3 Ubiquitin Ligasesa Reward or Cost?

Cells always keep doing their regular and efficient efforts tomaintain a proper proteostasis balance via quality controlmechanism [18]. Nascent polypeptides generation and re-placement of old proteins are common events in living cells.In an entire life-span from nascent to mature state, polypep-tide chains always stand with a persistent cytotoxic threat ofmisfolding and aggregation [15]. Under stress conditions,protein misfolding vulnerability and aggregation propensityexponentially rise in cells [19]. To avoid such cytotoxicpotential, cells continuously try to fold nonnative proteinsand degrade unsolved misfolded proteins through UPS [20].Aggregated proteinaceous structures represent a defectiveor exhaustive cellular quality control system in the cells.Inefficient degradation leads to overburden of misfoldedproteins and finally crosses the refolding or chaperonecapacity of a cell. This failure of protein quality controlmechanism leads to frequent probability of sequestrationof noncomplex polypeptides that are more prone towards

aggregation [21]. We do not know whether recognition ofan aberrant protein by E3 ubiquitin ligases is a realbeneficiary challenge or, probably, a mysterious risk. Itmay be possible that, after recognition, recruitment of E3ubiquitin ligases towards the site of aggregation from thesite of action unknowingly progresses to another side, i.e.ultrasensitive sequestration.

Apart from chaperones and UPS components, severalother essential proteins such as transcription factors, e.g.,heat shock transcription factor 1, CREB-binding protein(CBP), NF-Y transcriptional factor, tumor suppressor pro-tein p53, TATA-binding protein, specificity protein (Sp1),and transcription initiation factor TFIID subunit 4(TAFII130), sequester with misfolded proteinaceous speciesand consequently affect entire cellular proteostasis [22–24].Sequestration of proteins with aberrant complex aggregatesis a widespread mechanism and most probably influencesproteostasis network. Recently, in a quantitative proteomicanalysis, it was observed that numerous metastable proteins,including preexisting and newly synthesized proteins, se-quester with amyloidogenic aggregates. The same studysuggests that numerous proteins responsible for variouscellular functions interact with amyloid-like aggregatesand thereby generate toxicity and successive failure of crit-ical cellular functions [25]. Under stress conditions, mis-folded protein generation load dramatically increases, anddue to insufficient chaperone capacity, cells became unableto cope against these disastrous effects. Consequently, mis-folded and damaged proteins get actively sequestered in apericentriolar structure known as aggresomes [26, 27].EPM2B gene encodes an end product malin protein whichserves as really interesting new gene (RING) finger domainE3 ubiquitin ligase. Missense mutations in malin werereported with an autosomal recessive neurodegenerativeepilepsy disorder. Malin loss of function impairs the degra-dation of laforin [28]. Recently, it was investigated thatubiquitinated Lafora bodies are co-localized with Hsc/Hsp70 chaperones, 20S proteasome, and mutant malin. As-sociation of mutant malin, chaperones, and proteasomecomponents with Lafora bodies indicates failure of the qual-ity control system and one of the possible reasons of diseaseprogression [29]. Mutated form of laforin protein and E3ubiquitin ligase malin makes perinuclear-like structures andis nicely co-localized with ribosomes [30].

Earlier studies suggested that few E3 ubiquitin ligases’aberrant function would generate serious imbalance incellular quality control events [31, 32]. On beneficiaryside, E3 ubiquitin ligases try to solve the problem ofaggregation and play a major role in protein qualitycontrol mechanism [33], but in the same pool, absenceat the site of origin due to major recruitment with anearlier aggregate possibly leads to accumulation of unat-tended client proteins. We are not sure, but most

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probably, those unattended accumulated substrates aremore prone to sequestration with preformed aggregateswhich are already targeted by respective E3 ubiquitinligase. This situation is likely to be aggravated because of theloss of function of E3 ubiquitin ligase due to sequestration withpreformed aggregates as we depict in a proposed model inFig. 2. In support of our proposed model, there is anotherinteresting study which states that, like expanded polyglutamineaggregates, chimera GFP170* protein also forms cytoplasmicand nuclear aggregates. GFP170* protein recruits promyelo-cytic leukemia protein (bodies), chaperones, proteasomal com-ponents, SUMO-1, and transcription factors, e.g., CBP and p53[34]. In our previous studies, we observed that E3 ubiquitinligase E6–AP regulates the turnover of expanded polyglut-amine, p53 and p27 proteins act as a quality control E3 ubiquitinligase [35–37]. However, earlier reports suggest that variousessential cellular proteins get attracted towardsmisfolded proteininclusions such as vimentin [38], tubulin [39], elongation factor1 alpha [40], and ubiquitin protein [41]. In support of cellulardefense line mechanism, recently, it was shown that a stress

response lipid mediator neuroprotectin D1 (NPD1) attenuatesataxin-1 poly(Q)-induced proteotoxic stress, and aspirin-triggered NPD1 treatment suppresses cerebral ischemic injury[42, 43]. It may be possible that stress response-mediatedchaperones and other proteins also participate in sequestrationprocess. Still, it is not clear that what typical molecular featuresor signatures are responsible for discrimination between mis-folded protein and native protein. However, the most prominentassumption is that these E3 ubiquitin ligases may be recruitedfor the clearance of aberrant proteins.

Misfolded Proteins Recognition Tactics of E3 UbiquitinLigases

Possibly, without much disturbing normal cellular homeo-stasis, cells apply different strategies for identification anddegradation of a damaged protein in crowded milieu ofcells. In this section, we focus on the emerging roles of E3ubiquitin ligases associated with specific selection of their

Fig. 1 Cellular and molecular steps of protein quality control mecha-nism primarily implicated in various neurodegenerative diseases. Inliving cells, DNA transcribed into messenger RNA; this information istranslated into polypeptide chains. Ribosome is a large macromolecularcellular machine responsible for the synthesis of nascent polypeptidechains as per information reserved in mRNA transcripts. A newlytranslated emerging nascent polypeptide chains from the ribosome facea constant risk of misfolding and aggregation. To overcome this prob-lem, molecular chaperones govern immediate protein folding into theirnative structure for proper cellular functions. This is a challenging task,and lack of chaperone capacity and various cellular insults generate acumulative imbalance in protein homeostasis, which leads to misfoldedprotein aggregation in cells. Misfolded protein aggregation is a pivotal

hallmark in several neurodegenerative diseases. In living cells, ubiq-uitin proteasome pathway is responsible for the intracellular proteindegradation. In this pathway, E3 ubiquitin ligases provide variouscellular strategies to select specific substrates or misfolded proteins.Neurons are postmitotic cells that are more prone towards the aggre-gation of misfolded proteinaceous structures. Still, the molecular path-omechanism of various neurodegenerative diseases linked withmalfunctioned/damaged proteins is not known. Even though it istempting to speculate that the E3 ubiquitin ligases provide variouscellular approaches for specific substrate selection to regulate proteinaggregation, still, it is not clear how these E3 ubiquitin ligases sensemisfolded protein aggregation phenomenon

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critical substrates and misfolded proteins. Here, we discussand summarize various basic E3 ubiquitin ligases based oncellular strategies for the clearance of misfolded proteins toavoid interference with normal cellular functions. In thissection, we summarize most possible tactics govern by E3ubiquitin ligases in the clearance of misfolded proteins orcritical substrates as shown in Fig. 3.

Conformational Plasticity of Disorder

Eukaryotic cells achieve intracellular degradation throughubiquitin proteasome system [6]. E3s enzymes provide spec-ificity for protein degradation in ubiquitination-based qualitycontrol (QC) system. In crowded cellular environment, nearly

30 % of the nascent polypeptide chains are misfolded due toposttranslational slips related with folding process [44, 45].Because disordered proteins tend to form cytotoxic aggre-gates, cellular quality control system immediately recruitsE3 ubiquitin ligases for clearances of misfolded protein viaUPS [46], but how these E3 ubiquitin ligases discriminatebetween normal and misfolded proteins is not clear. Recently,it was shown that Sir Antagonist 1 (San1) E3 ubiquitin ligasecontributes in both nuclear and cytoplasmic protein qualitycontrol mechanisms [47, 48]. Mostly, functional proteins arenatively organized into three-dimensional structure with well-ordered structural motifs, thus easily accessible for recogni-tion by other interacting proteins, but misfolded proteins losetheir native structures and adopt irregular shapes, so how is it

Fig. 2 A schematic diagram proposed to represent sequestration ofnumerous cellular components and aggravate the aggregation processwith a preformed misfolded protein structure. Nascent polypeptidechains fold through different intermediates to achieve their three-dimensional structure for their proper cellular functions. Partiallyfolded or misfolded polypeptide chain may provide an attractive tem-plate for deposition of other essential cellular proteins and probablydevelop a metastable inclusion structure enriched with several needfulcellular components. Transational errors or proteotoxic insults maycause protein misfolding and leads to huge aggregates in variouscellular compartments. The detailed structure and nature of theseaggregated inclusions is not well understood. Previous reports suggestthat, during failure of cellular quality control mechanism, more intrigu-ing is the possibility that misfolded intermediates can attract various

transcription factors, chaperone family members, UPS components,and microtubules to increased amounts of aberrant proteins in cells asearlier discussed in the same review. To overcome uncontrolled anddisastrous aggregation process, how E3 ubiquitin ligases govern mis-folded protein degradation and promote protein disaggregation is notcompletely known. Why these essential cellular proteins are recruitedtowards metastable amorphous aggregates is an open question fordebate. Maybe interaction of E3 ubiquitin ligases with preformedaggregates generates lack of function at the previous site of actionand consequently promotes global disturbance linked with their clientproteins and leads to impairment in normal cellular functions. Actionof chaperons and UPS components tries to stimulate disaggregationand degradation process to make various cellular defense lines againstproteotoxic species

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possible to recognize and separate them from crowded envi-ronment? Interestingly, a recent report suggests that E3 ubiq-uitin ligase San1 applies a novel conformational plasticitystrategy in the recognition of toxic abnormal proteins. San1targets misfolded substrates through highly disordered N- andC-terminal regions that retain substrates identification mod-ules [49, 50]. It is reasonable to believe that, probably, otherE3 ubiquitin ligases exhibit such capability and may contrib-ute in protein quality control mechanism.

Ribosomal Association of Quality Control Mechanism

Protein synthesis cellular machinery, i.e., ribosomes, trans-forms messenger RNA (mRNA) transcript into nascentpolypeptide chains. Ribosomes hold about 30 % of entirecell bulk, and approximately 105 and 106 ribosomes exist in

bacterial and mammalian cells, respectively [51]. Polysomesarrange in such a fashion that polypeptide exit faces outwardand thereby probably inhibits unwanted interactions amongthem [52]. In addition to this arrangement to minimizeunfavorable early misfolding and aggregation, cells haveevolved ribosomal-associated chaperones, which promoteprotein folding [53]. In bacteria, trigger factor, the archaealand eukaryotic nascent polypeptide-associated complex,and particular heat shock proteins serve as ribosomal-associated chaperones [9, 54, 55]. Because of high localconcentration of nonnative proteins, most probably, aggre-gation propensity of nascent polypeptides chains sharplyincreased at polyribosomal site.

Still, it is an unsolved puzzle that, under such macromo-lecular crowded environment, how it is possible to maintainproper quality control mechanism? Recently, it was

Fig. 3 Proposed diagrammatic representation of comprehend cellulartactics adopted by various E3 ubiquitin ligases implicated in theclearance of misfolded and other client proteins. Newly synthesizedprotein folds into their correct three-dimensional structure and trans-ported into various cellular compartments or extracellular environmentfor normal cellular functions. Improperly or poorly folded proteinsneed immediate assistance from chaperones to further fold into theirnative form. Absence of sufficient chaperone capacity leads to proteinmisfolding and aggregation. In neuronal cells, it is a well-establishedfact that, generally, protein misfolding leads to neurotoxicity, and theaggravation of such situation can cause neurodegenerative disorderlinked with protein aggregation. Here, we have proposed seven

comprehensive tactics of different E3 ubiquitin ligases from variousfamily members that induce the clearance or degradation of misfoldedproteins as serially discussed in the above review section. Most likely,E3 ubiquitin ligases can assist various protein quality control-disapproved candidates/client proteins in different manners accordingto their specificity and subcellular localization. Caption of various E3ubiquitin ligases tactics as shown in the figure include the following: 1conformational plasticity of disorder, 2 ribosomal association of qual-ity control mechanism, 3 harmonious interaction of E3 ubiquitinligases with chaperones, 4 disposal of endoplasmic reticulum-anchored misfolded proteins, 5 hand to hand coordination, 6modulatedrecruitment with misfolded proteins, 7 sugar chains recognition

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demonstrated that ribosomal-associated Ltn1, a really inter-esting new gene domain containing E3 ubiquitin ligase, pro-vides quality control mechanism against newly synthesizednonstop proteins [56]. Recently, it was shown that mutationsin LISTERIN, which is a homolog of Ltn1, cause neurode-generation in mice and critically involved in embryonic de-velopment [57]. It was reported that proteasomal subunit 19Scoimmunoprecipitates with ubiquitin ligases [58]. Saccharo-myces cerevisiae Not4 RING finger domain E3 ubiquitinligase, involved in transcriptional regulation and transcription-al elongation, has been detectable in polysome fractions[59, 60]. These studies provide a clue that protein qualitycontrol mechanism and degradation of nascent polypeptideschains are possible during protein synthesis. However, we arefar from understanding the exact molecular mechanism ofribosomal-associated E3 ubiquitin ligases that are implicatedin early quality control events.

Harmonious Interaction of E3 Ubiquitin Ligaseswith Chaperones

Capacity of refolding of misfolded proteins via chaperonesor degradation through E3 ubiquitin ligases determines theoverall efficiency of cellular quality control system in cells[61]. During stress conditions, cells need extensive protein-folding capacity through chaperones to overcome the expo-nential load of misfolded proteins [62]. Because all foldingattempts are not successful in one go and to avoid unsolic-ited aggregation of this failure, UPS immediately governsQC E3 ubiquitin ligases for intracellular degradation ofmisfolded proteins. Under such improper protein-foldingcapacity, it is not well known that how chaperones help E3ubiquitin ligases in aberrant protein recognition process andmake their job easier. Earlier studies and models suggestthat the best instantaneously available help is possiblethrough chaperones in this triage process [63–66]. CHIP(C-terminus of Hsc70-interacting protein) is a tetratricopep-tide repeat containing co-chaperone protein, which controlschaperone activities of Hsc70 [67]. This U-box domaincontaining E3 ubiquitin ligase CHIP cooperates with HSPfamily chaperones and facilitates the ubiquitinylation ofunfolded proteins [68].

Cullin5 RING E3 ubiquitin ligase interacts with Hsp90chaperone complex and promotes the degradation of itsclient proteins, i.e., ErbB2 and HIF1-α [69]. Similarly,another homologous to E6-AP C-terminus (HECT) domainE3 ubiquitin ligase E6-AP also promotes the ubiquitinyla-tion of misfolded proteins captured by Hsp70 molecularchaperone [70]. Undoubtedly, these observations clearlyindicate that E3 ubiquitin ligases take help from variouschaperones. Most possibly, interaction of various E3 ubiq-uitin ligases with chaperones facilitate their misfolded pro-teins recognition mechanism, and these cumulative efforts

design an accurate scheme for the clearance of misfoldedproteins.

Disposal of Endoplasmic Reticulum-Anchored MisfoldedProteins

Endoplasmic reticulum (ER) is an important cell organellefor the posttranslational modifications of nascent polypep-tide chains synthesized by ribosomes. ER membrane-anchored gp78 RING finger domain E3 ubiquitin ligase isencoded by tumor autocrine motility factor receptor gene.Gp78 targets ataxin-3 and superoxide dismutase-1 for pro-teasomal degradation involved in Machado–Joseph disease/spinocerebellar ataxia type 3 and familial amyotrophic lat-eral sclerosis neurodegenerative disease, respectively [71].ER-associated gp78 E3 ubiquitin ligase also recognizesCFTRΔF508 and promotes their polyubiquitylation [72].Apart from correct folding of nascent polypeptide chains,to ensure proper function of a protein, ER cellular networkhelps in the placement of polypeptide chains into theircorrect subcellular localization. Gene SMAD ubiquitinationregulatory factor 1 (Smurf1) encodes Smurf1 E3 ubiquitinligase [73]. Smurf1 targets Wolfram syndrome (WFS1) pro-tein for proteasomal degradation at ER, and its endogenouslevels are elevated against ER stress condition [74].

Misfolded protein aggregation interrupts the normalstructure and function of ER and generates ER stress, whichconsequently leads to cell death governed by proteasome[75, 76]. BAX-induced apoptosis inhibitor, bifunctional ap-optosis regulator, is an ER-linked RING finger type E3ubiquitin ligase, which is mainly expressed in neurons andprotects cell death against various cell death stimuli, e.g.,ER stress [77]. In the same context, ER-linked E3 ubiquitinligase Der3/Hrd1p contains six transmembrane domains,and membrane topology of Hrd1p is implicated in endoplas-mic reticulum-associated degradation (ERAD) pathway[78]. Under ER stress conditions, to avoid aggravation ofproteoxicity, cells actively governs ER-associated E3s forthe clearance of damaged proteins through endoplasmicreticulum-associated degradation pathway. ERAD-E3 ubiq-uitin ligase ret finger protein 2 is a family member of RBCC(RING finger, B-box, and coiled-coil) proteins that associatewith valosin-containing protein (VCP), T cell receptor sub-unit CD3-δ, and Ubc6 [79]. Several E3 ubiquitin ligaseshave been found to be involved in mammalian endoplasmicreticulum-associated degradation pathway. RING fingerprotein 103 Kf-1 is an ER-localized E3 ubiquitin ligase,and its expression level has been found increased in Alz-heimer’s disease patient; Kf-1 interacts with Derlin–VCPcomplex and acts as component of ERAD pathway [80,81]. Human ER membrane E3 ubiquitin ligase TEB4(MARCH-VI) is an ortholog of Doa10 and induces thedegradation of type 2 iodothyronine deiodinase [82, 83].

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Hand to Hand Coordination

Deregulation of ubiquitin proteasome system can lead tointracellular accumulation of damaged or abnormal proteinsand consequently leads to neurodegeneration [84]. To chal-lenge this condition, cells possess highly evolved proteinquality control system which is well organized with variousquality control E3 ubiquitin ligases. Interestingly, few stud-ies suggest that cooperation of various E3 ligases amongthemselves may play a critical role in substrate recognitionand, most probably, promotes an efficient degradation viaubiquitin system. Ubr1 gene encodes UBR box-containingUbr1 (ubiquitin-protein ligase E3 component n-recognin 1),a 225-kDa RING finger domain protein [85]. Lack of func-tion of UBR1 ubiquitin ligase causes mental retardationJohanson–Blizzard syndrome [86]. Ufd4 is a HECT domain168-kDa E3 ubiquitin ligase, joins ubiquitin carrier 4 (Ubc4)or ubiquitin carrier 5 (Ubc5) E2 enzymes for functionalactivity in ubiquitin-fusion degradation pathway [87, 88].Dual proteolytic pathways co-target Mgt1 O6meG-DNAalkyltransferase protein for polyubiquitylation, mediatedby both Ubr1 and Ufd4 E3 ubiquitin ligases, and this coop-eration enhances the yield of polyubiquitylated Mgt1 [89].Physical and functional interaction complexes of HECT-type Ufd4 and RING-type Ubr1 are more effective to pro-duce longer polyubiquitin chain linked with substrates withtheir E2 ubiquitin carrier enzymes Ubc4/Ubc5 and Rad6,respectively [90].

To reduce proteoxicity, misfolded protein degradationemerges as a cellular adaptability for clearance of unwantedaggregates in cells. Ubr1 and Ubr2 ubiquitin ligases promotethe ubiquitinylation of unfolded polypeptides and stimulatethe degradation of damaged proteins. Ubr1 specifically pro-motes the ubiquitinylation of damaged/aberrant proteins [91,92]. To ensure correct cellular functioning, three-dimensionalnatively folded protein structures are essential in crowdedmilieu. E3 ubiquitin ligase San1 degrades misfolded nuclearproteins, and Ubr1 is involved in “N-end rule” pathway[93–96]. Recently, it was shown that both Ubr1 and San1regulate proteotoxic stress via different approaches for cyto-plasmic QC process. San1 needs chaperones function fornuclear delivery of substrates, instead of this Ubr1 that gov-erns chaperones for direct substrates ubiquitination [47].

Modulated Recruitment with Misfolded Proteins

Protein misfolding, amyloid fibrils accumulation, and ag-gregation are well-known conformation changes in protein-associated neurodegenerative diseases [97]. Recent evi-dence suggests that aggravation of these aberrant proteina-ceous species cause neuronal apoptosis [98]. But howprotein misfolding initiates neurodegeneration and whatother factors are recruited with those preformed aggregates

are not completely known? Earlier studies indicate that themost suspicious interacting proteins are chaperones, com-ponent of UPS, and transcriptional factors [99]. As we knowthat few QC E3 ubiquitin ligases are dedicated for misfoldedprotein clearance process, but the reason of recruitment withmisfolded protein is not known yet. Still, it is a big questionof debate whether QC E3 ubiquitin ligase recruitment withdamaged proteins facilitates their degradation or may un-knowingly generate a mysterious problem.

UBE3A gene encodes a conserved HECT domain familyE3 ubiquitin ligase, i.e., E6-AP, mutated in Angelman men-tal retardation syndrome [100]. Recently, we have observedthat E6-AP retains QC properties and actively recruits withcystic fibrosis transmembrane conductance regulator aggre-somes. E6-AP recruitment/co-localization facilitates theubiquitination of misfolded proteins anchored by Hsp70chaperone [70]. In another study, we have demonstrated thatE6-AP recruits to neuronal intranuclear inclusions in Hun-tington’s disease transgenic mice model. E6-AP alleviatesproteoxicity mediated by expanded polyglutamine proteinsvia degradation through ubiquitination [35]. Lafora diseaseis a progressive neurodegenerative disease caused by muta-tion in NHLRC1 gene, which encodes RING finger malinE3 ubiquitin ligase [28, 101]. Proteasomal inhibition treat-ment leads to generation of aggresomes which are positivewith malin and other UPS components [102]. In the samecontext, several other studies report that parkin is another E3ubiquitin ligase, which co-localizes with aggresome afterproteasomal inhibition [103–105].

Sugar Chains Recognition

In protein quality control process, newly synthesized pro-teins enter into ER through a channel termed as “translocon”for N-glycosylation [106–108]. Earlier, it has been reportedthat many E3s are implicated in the ER-associated degrada-tion pathway. E3 ubiquitin ligases also help in the selectiveelimination of glycoproteins. However, the molecular mech-anism underlying the ability of E3 ubiquitin ligases torecognize target glycoproteins remains to be understood.Recently, a novel approach adopted by few ubiquitin ligasesin the recognition and ubiquitylation of N-linked glycopro-teins that act as major players in ERAD pathway has beenhighlighted. Skp1-Cullin1-Fbx2-Roc1 (SCFFbx2) ubiquitinligase complex utilizes N-glycan signal for degradation. Inthis complex ubiquitin ligase, Fbs2 protein interacts withendogenous N-linked high-mannose oligosaccharides con-taining glycoproteins and promotes their proteasomal deg-radation [109]. Fbx2 expression levels are very high in theorgan of Corti [110]. Fbx2 lack of function leads to degen-eration in epithelial support cells of the organ of Corti, andhearing loss in Fbxo2-/- mice began, which may be due toaberrant quality control mechanism of glycoprotein [111].

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Another F-box protein Fbs2 acts as E3 ubiquitin ligase,which is bound with N-glycan of T cell receptor α subunit,and promotes its degradation through ERAD pathway [112].In SCFFbs1.2 ubiquitin ligase complex, Fbs1 and Fbs2proteins preferentially associate with denatured glycopro-teins as compared to properly folded proteins. This studysuggests that, due to exposed chitobiose structure, Fbs canrecognize and discriminate folded glycoproteins over un-folded glycoproteins [113]. Interaction of HRD1 E3 ubiq-uitin ligase and SCFFbs2 ubiquitin ligase complex withuncleaved precursor of asialoglycoprotein receptor H2a pro-motes its degradation [114].

Key Questions and Perspectives

Exposure to various cellular insults generates proteotoxicstress and leads to overburden on cellular protein qualitycontrol system; however, the molecular mechanism of proteinquality control is unclear. Earlier studies suggest that molec-ular chaperones and quality control E3 ubiquitin ligases play apivotal role in protein quality control mechanism. Individualquality control E3 ubiquitin ligase retains their own particularfunction, but the interaction with different quality controlmembers, such as Hsp/chaperones, transcriptional factors,and UPS members, seems to be a critical role in cellularproteome. Many questions remain unanswered, for example,how E3 ubiquitin ligases determine the specificity for a criticalsubstrate in a crowded cellular milieu? How quality control E3ubiquitin ligases specifically recognize and differentiate be-tween nonnative/damaged proteins as compared to nativeproteins? To understand why aberrant function of E3 ubiquitinligases is implicated in the progression of misfolded proteinconformation disorders, it is mandatory to identify the molec-ular pathways that involve E3 ubiquitin ligases. It is veryimportant to understand how E3 ubiquitin ligases sense stressconditions and recognize misfolded proteins for further clear-ance by various approaches?

It is noteworthy that several neurodegenerative diseasesare associated with the failure in proteolytic machinery,possibly due to failure in their recognition and selectivedegradation through E3 ubiquitin ligases. Future studiesshould be devoted to understand the problem of aberrantprotein interactions, their involvement in neuronal dysfunc-tion, and investigating how E3 ubiquitin ligases rescue theireffect? Principally, detailed study of aberrant protein inte-grators and analysis of their interaction with other cellularpathways would enable us to answer these questions. Weexpect that various experimental studies of the cross talkbetween E3 ubiquitin ligases and other proteotoxic responsemechanisms in cells will suggest a further understanding ofcytoprotective responses. A better understanding of involve-ment of E3 ubiquitin ligases in the misfolded protein

identification and selective degradation will help in thedevelopment of new proteotoxicity-associated therapies.

Acknowledgments This work was supported by the Department ofBiotechnology, Government of India. AM was supported by Ramalin-ganswami Fellowship from the Department of Biotechnology, Govern-ment of India. The authors would like to thank Mr. Bharat Pareek forhis support and management during manuscript preparation.

Conflict of interest None.

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