Five Questions (with their Answers) on ER-Associated Degradation

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Five Questions (with their Answers) onER-Associated DegradationGiorgia Brambilla Pisoni1,2,3,∗ and Maurizio Molinari1,2,4

1Institute for Research in Biomedicine, CH-6500 Bellinzona, Switzerland2Università della Svizzera italiana, CH-6900 Lugano, Switzerland3ETH Zurich, D-BIOL, 8093 Zurich, Switzerland4School of Life Sciences, École Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland∗Corresponding author: Giorgia Brambilla Pisoni, [email protected]

Abstract

Production of a functional proteome is a major burden for our cells.

Native proteins operate inside and outside the cells to eventually war-

rant life and adaptation to metabolic and environmental changes, there

is no doubt that production and inappropriate handling of misfolded

proteins may cause severe disease states. This review focuses on protein

destruction, which is, paradoxically, a crucial event for cell and organ-

ism survival. It regulates the physiological turnover of proteins and the

clearance of faulty biosynthetic products. It mainly relies on the inter-

vention of two catabolic machineries, the ubiquitin proteasome system

and the (auto)lysosomal system. Here, we have selected five questions

dealing with how, why and when proteins produced in the mammalian

endoplasmic reticulum are eventually selected for destruction.

Keywords autophagy, endoplasmic reticulum-associated degradation,

ERAD tuning, protein folding, protein misfolding (conformational) dis-

eases, protein quality control, ubiquitin proteasome system, unfolded

protein response

Received 13 October 2015, revised and accepted for publication 6

January 2016, uncorrected manuscript published online 11 January

2016, published online 18 February 2016

Question 1: How Efficient is Protein Folding?

Many (most?) proteins may adopt multiple ‘native’ con-formations (1,2). Moreover, the very same gene productmay enter different supramolecular complexes, as in thecase of P97 (3,4), Gp78 (5) or protein disulfide isomerase(PDI) (6), which may have distinct functions and intra-cellular localizations. The percentage of nascent chainsthat eventually attain the native and functional structureor structures, and that will be delivered to the appropriatesite of activity, varies dramatically for each gene prod-uct and strictly depends on the cell and environmentalconditions. Essentially, for most polypeptides, if not all,there is no trustable prediction on the efficiency to attainthe functional shape. However, it is assumed that cellularchaperone machineries ensure that folding is efficient formost proteins (7,8), even though divergent opinions exist

(9,10) and classical examples of inefficient folders such asthe cystic fibrosis transmembrane conductance regulator(CFTR) are described in the literature (11). Despite thechallenges encountered by polypeptides emerging fromthe ribosome in the living cell, molecular crowding beinga major one (12), folding in vivo is in general much moreefficient than protein (re)folding in the more controlledand controllable environment of a test tube (13).

The fact that protein folding might already start during thesynthesis of the polypeptide chain and proceeds vectoriallyunder the assistance of general, as well as specific foldingfactors, substantially contributes to folding efficiency inthe living cell (14). Regardless of the overall efficiencyof protein biogenesis, biosynthetic compartments suchas the endoplasmic reticulum (ER), the cytosol and themitochondria must deal with the presence of non-native

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Brambilla Pisoni and Molinari

Figure 1: Protein folding effi-ciency in the ER. Newly synthe-sized polypeptides entering the ERas unfolded chains reach the foldedstate (in green) and are transportedto their site of activity (Export). Mis-folded proteins (in red) are selectedfor destruction (Degradation). Fold-ing efficiency is protein-specific andis represented as a scale that mea-sures the rate of transition betweenfolded versus misfolded states orexport versus degradation for eachprotein.

polypeptides that engage molecular chaperones and thatmay eventually undergo terminal misfolding with possibledeleterious effect on compartmental and cellular pro-teostasis (15,16). Protein misfolding does not only pertainto defective products arising from inherited errors in thegenetic material (15) or from the imperfect activity ofthe nano-machineries translating the genetic informationinto the polypeptide sequences (17–19). As discussedbelow, it may arise from the non-stoichiometric expressionof subunits of multimeric protein complexes. It is alsoa consequence of the crowded environment where pro-tein folding occurs and where native proteins eventuallyoperate (20), and of the relatively small free energy of sta-bilization of folded versus unfolded structures. This latterfactor leads to a dynamic equilibrium between folded andunfolded states (Figure 1) with the propensity of unfoldedconformers to enter aggregates (21) or other irreversible‘off’ pathways of folding programs (22). In this context, thecytosolic proteome is probably at higher risk of misfold-ing than secretory proteins because the latter tend to beglycosylated and stabilized by inter- and intra-moleculardisulfide bonds (23).

Question 2: How are Polypeptide ChainsSelected for Destruction?

A major function of the ER is to synthesize proteinsthat will then function either in the compartment itself,

in other cellular organelles, in membranes or in theextracellular space. As such, the ER contains native res-ident proteins as well as a variety of non-native newlysynthesized polypeptides and unpaired subunits ofmultimeric complexes that do not (yet) fulfill qual-ity control requirements for transport at the site ofactivity.

Clearance of native proteinsNative, ER-resident proteins are characterized by a physio-logic life span that can be short [<3 h for the ER-AssociatedDegradation (ERAD) factors homocysteine-responsiveendoplasmic reticulum-resident ubiquitin-like domainmember 1 protein (Herp) (24) and Gp78 (25)] or verylong [for the vast majority of chaperones such as BiP,calnexin and many others (26,27)]. It has recently beenproposed that modulation of ER-resident factors turnover,especially of those factors such as Herp that have shorthalf-lives, might be part of the first line of response tovariations in ER homeostasis. Posttranslational eventssuch as ERAD tuning (i.e., stabilization of short livingfactors resulting in immediate level increase, regulation ofsupramolecular complexes’ assembly/disassembly (28)),reversible ADP-ribosylation to activate/inactivate chap-erone functions (29), reversible palmitoylation to changechaperone suborganellar distribution (30,31) allow rapidadaptations of ERAD and folding activities to changes inenvironmental conditions.

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Endoplasmic Reticulum-Associated Degradation

Figure 2: Selection for degrada-tion of native ER proteins fol-lowing pathogen attack. Infectionby human cytomegalovirus (HCMV,Pathogen) represents one exampleof pathogen-induced degradation ofnative polypeptides (here illustratedas a red tornado). Cells infected byHCMV generate viral products that tar-get for degradation newly synthesizedclass I and class II MHC molecules (ingreen).

The regulated clearance of native proteins plays funda-mental roles in ER and cell physiology by modulating,for example, lipid biosynthesis (32). In this context,the intraluminal level of a rate limiting enzyme in thesterol synthesis (3-hydroxy-3-methylglutaryl-coenzymeA reductase (HMGCR)) inversely correlates with the cel-lular sterol levels in a posttranslational feedback regulationthat relies on HMGCR clearance upon engagement of theE3 ubiquitin-protein ligase synoviolin (SYVN1) and theubiquitin proteasome system, UPS (33). Clearance of cel-lular proteins and presentation at the cell surface of classI major histocompatibility complex (MHC)-associatedproteasomal products allows sampling of individual cell’sproteome to identify the non-self and thereby the elimi-nation of cells that express mutant or viral peptides (34).Selection for degradation of native proteins from the ERcan also be a consequence of pathogen’s attack (Figure 2).Clearance of class I or class II MHC and CD4 moleculesare common strategies adopted by many viruses such ascytomegalovirus, human immunodeficiency virus andhepatitis B virus to evade immunosurveillance (35).

Clearance of non-native proteinsA major focus of the research in the field is the character-ization of mechanisms regulating disposal of polypeptidechains that do not attain the native structure or that donot find their partners in the biosynthetic organelle. Gen-erally, these by-products of protein biogenesis are activelyretained in the ER lumen to complete the maturationprogram. Certain unfolded chains are selected for

degradation during or immediately after their synthe-sis, as in the case of ApoB (36). Others can spend severalhours as immature proteins in the ER without beingselected for degradation, as for the gp160 of HIV (37). Thetime allocated for folding is characteristic for each geneproduct. A delay in the folding program may lead to inap-propriate destruction of proteins that, if given additionaltime, would eventually have achieved their functionalconformation (22). Such delays may result from inheritedor sporadic mutations of the polypeptide sequence (22,38),from non-stoichiometric synthesis of multimeric com-plex’s subunits, for example, due to the gene duplicationsfound in some cancer types (39) or from perturbationsin the folding environment. It is crucial to understandthe fate of folding-defective polypeptides, i.e. if they areselected for degradation, and in this case, which degrada-tion pathway they engage and why, or if they will undergoaggregation leading to intracellular or extracellular accu-mulation. In fact, loss-of-function and gain-of-toxicfunction diseases are direct consequences of disposal oraggregation of mutant polypeptides. Understanding themolecular mechanisms governing these processes allowsdesign of chemical and pharmacologic chaperones thatmay substantially alleviate disease phenotypes (15,40).

Question 3: How are Futile Folding AttemptsInterrupted?The ER produces polypeptides that are co-translationallymodified with pre-assembled 14-sugar unit high mannose

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Figure 3: The hourglass model for timing glycoprotein fate. Removal of α1,2-linked mannose residues from protein-boundoligosaccharides by the sequential intervention of ER-resident glycosidases and their recognition by specific folding or ERAD lectinsdetermines the fate of newly synthesized glycopolypeptides.

oligosaccharides on side chains of asparagine (N) residues.It also generates proteins that do not display these N-linkedoligosaccharides (41). All newly synthesized polypeptidesare allocated a certain time that they can spend in thelumen of the ER to reach the native conformation (22).To avoid clogging of the folding compartment withpolypeptides that cannot attain a transport-competentstructure, folding-defective structures are eventuallyselected for dislocation across the ER membrane anddegradation by cytosolic 26S proteasomes or for deliv-ery to the autolysosomal degradation pathway (42). Fornon-glycoproteins, the rules that determine the lengthof the folding-attempt phase preceding selection for dis-posal are not known. For N-glycosylated proteins, theserely on the engagement of ER-resident glycosidases thatsequentially remove the three terminal glucose residuesand two to four α1,2-bonded mannose residues from theprotein-bound oligosaccharides (Figure 3). Removal ofmannoses is the true timer that prevents longer reten-tions of folding-defective proteins in a folding chaperonesystem named the calnexin/calreticulin/ERp57 foldingcycle (41,43,44). Removal of the terminal mannose residuefrom the central branch B of the polypeptide-boundoligosaccharide possibly facilitates removal of mannoseresidues from the other two oligosaccharide branchesA and C (Figure 4). Removal of the terminal mannose

residue(s) from the branch A impedes the action of theUDP-glucose:glycoprotein glucosyltransferase (UGT1),which is required for retention of folding proteins in thecalnexin cycle (Figure 4) (45). Removal of the terminalmannose from branch C exposes a terminal α1,6-bondedmannose residue that recruits OS-9 and XTP3-B, twoER-resident lectins (Figure 4). OS-9 and XTP3-B deliverterminally misfolded proteins to dislocation sites in theER membrane that are engaged to transport the unfoldedcargo into the cytosol for polyubiquitylation and proteaso-mal degradation (46–49). Identification of the ER-residentmannosidases regulating terminal α1,2-bonded mannoseremoval revealed the enzymatic intervention of one ormore members of the glycoside hydrolase 47 family,namely the ER α1,2-mannosidase I (50), EDEM1 (51),EDEM2 (52) and/or EDEM3 (53,54) (Figure 4).

Question 4: Who is Degrading Misfoldedor Orphan Proteins Produced in the ER?

As far as is known, the ER lumen does not contain pro-teolytic machineries that are able to clear the biosyntheticorganelle of misfolded proteins. For instance, proteases enroute for the lysosomal system are made in a pro-formto ensure that they are inactive in the ER (55). The ERcontains, however, putative proteases such as Lonp2 and

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Figure 4: The sweetest N-glycan. The 14-unit high mannose oligosaccharide is composed of three glucoses (light blue candies),nine mannoses (light and dark green candies) and two N-acetylglucosamines (blue cupcakes). The oligosaccharide branches are definedas A, B and C. Processing enzymes and glycosidic bond types are indicated on the right.

CPVL that associate with the ERAD lectins OS-9 andXTP3-B to ensure clearance of soluble ERAD substrates(56). Nevertheless, in current models, soluble polypep-tides must be dislocated across the ER membrane andmembrane-anchored polypeptides must be extracted fromthe membrane to be delivered into the cytosol, which con-tains several proteolytic systems (Figure 5) (see below). Theprocesses of misfolded protein dislocation across the ERmembrane or of misfolded protein extraction from the ERmembrane are largely unknown and several more or lessconvincing theories have been proposed from transportthrough large proteinaceous retrotranslocation channels tothe intervention of clipping intramembrane proteases, lipiddroplets, extracting factors and others, each one of whichmay apply to only a subset of misfolded proteins (57–61).

During or after transport into the cytosol, ERAD sub-strates might undergo polyubiquitylation by a set of atleast 24 ER membrane-embedded E3 ubiquitin ligases(62) to be eventually degraded by cytosolic 26S pro-teasomes (Figure 5). Alternatively, if they cannot enterthe proteasomal tunnel, or if they undergo aggregation,aberrant polypeptides are engulfed in autophagosomesthat deliver them for autolysosomal destruction (Figure 5)(63). As proteasome inhibitors as well as lysosomotropic

drugs only partially and temporarily block the disposal ofmisfolded polypeptides from the ER, it is likely that manyproteolytic systems are engaged contemporarily or can beactivated upon blockade of one or the other pathway. Infact the cytosol, or the cytosolic face of organellar mem-branes contains several proteolytic systems such as theUPS (64,65), the autophagic machinery (66,67), tricornprotease-like hydrolases (68), calpains (69), Zmpste24 (70)and many others (Figure 5). Consistently, at least the UPSand the autophagic pathways are linked by compensatorymechanisms that increase activity of one, when the otheris impaired or defective (71–73).

Question 5: What is ERAD for?

The answer appears quite simple: the physiologic role ofERAD is to contribute to the maintenance of ER and cellhomeostasis by removing faulty and potentially toxic geneproducts or subunits of multimeric complexes expressedin non-stoichiometric quantity (35,60,74–76). The lattermight result, for example, from aneuploidy, i.e. abnor-mal chromosome number with missing or supernumerarygenes that may correlate with proteotoxic stress leading tomental retardation or several types of cancer (39,77,78).It is believed that the occurrence of late onset protein

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Figure 5: Proteolytic systems and ER cleaning. Classical ERAD substrates undergo polyubiquitylation and are degradedby cytosolic 26S proteasomes. Misfolded proteins that get into aggregates might enter autophagosomes for eventual lysosomaldestruction. Other proteolytic systems comprise putative intraluminal and intramembrane proteases, tricorn protease-like hydrolasesand calpains.

misfolding diseases could be related to the age-dependentdecline of quality control capacity (79–82).

Moreover, ERAD regulates turnover and intracellularcontent of various functional gene products therebydetermining their function at the posttranslational level(35,61,64,74,76,83,84) in processes that can collectively bedefined as ERAD tuning (28).

However, we still do not know most of the endogenoussubstrates and of the pathways that might be controlledby regulated ERAD or ERAD tuning (28). Certainly, afew endogenous ERAD substrates have been reported,e.g. the HMGCR (85), Herp (86) and E3 ubiquitin ligases(25,87), CD147 (88) and ATF6 (89), the degradation ofwhich might play important regulatory roles in processessuch as sterol synthesis, ERAD, assembly of various mul-timeric complexes or unfolded protein response (UPR),respectively. The proteolytic activities by cytosolic andnuclear proteasomes or by (auto)lysosomal enzymes,ERAD as well, may eventually generate amino acids to be

used for protein biogenesis (90,91). Moreover, a fractionof the proteolytic fragments derived from proteasomalactivity is imported back into the ER lumen, where, upontrimming by amino peptidases to the appropriate length,they are loaded onto class I MHC and presented at thecell surface (self-reactivity of the immune system). In thismanner, CD8+ T cells can recognize and eliminate cellsthat present viral- or bacterial-derived peptides, as symp-toms of pathogenic infection, as well as cells displayingtumor-specific antigens (10).

The importance of the regulatory role of ERAD or ofprotein degradation in the control of cells or organismshomeostasis appears to clash with the observation thatmost proteins in Escherichia coli (92), Saccharomyces cere-visiae (76,93–96) and Metazoa cells (27,97) are extremelystable. Their abundance must therefore be controlledat the translational level (97,98) and, in the case ofsupramolecular complexes, cells must know how manymolecules of individual complex subunits are needed

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Figure 6: Regulatory role ofERAD. The posttranslational regula-tion of the oligosaccharyltransferasecomplex (OST) complex assemblyhas recently been reported (76).

to eventually assemble the functional protein (76,98)(Figure 6). This is a nice way to spare energy by preventingthe production of excessive amounts of subunits that mustthen be destroyed. However, it should not be forgottenthat the modulation of protein turnover could represent,together with other posttranslational modifications (29),the first line of response to variations in cellular home-ostasis requiring regulation of the intracellular level andactivity of proteins in the absence of induction of tran-scriptional programs such as the UPR (35). According tothis hypothesis, the identification of rapidly turned-overER factors, amongst the bulk of stable proteins (76), couldidentify candidate proteins whose expression level canbe rapidly enhanced posttranslationally upon regulatedprolongation of the half-life.

Which Questions do Remain to be Answered?

There are obviously many questions to be tackled in thefuture years. Some derive from the imprecise and contro-versial answers available to the five questions addressedabove. Others have to do with the hijacking of ERAD path-ways by viral and bacterial pathogens aiming to promotetheir replication and escape immunosurveillance (99,100).Discovering the identity and the mechanisms of hijackedpathways and the molecular components targeted by bac-terial toxins or viral gene products will pave the way forthe discovery of novel antibacterial or antiviral therapies

and may indicate how cancer cells can exploit modulationof degradation activities to acquire resistance to antitumortherapy or higher malignancy. Another question is whatsignals and which modulators activate the clearance ofentire ER portions with their content, a type of ER-specificautophagy defined as ER-phagy (101), that possibly occursin many different, still insufficiently appreciated flavors(e.g. to maintain the ER volume, to help it return to thephysiologic size at the end of a phase of expansion resultingfrom stresses, to eliminate subdomains containing inex-tricable aggregates of misfolded proteins and to provideamino acids in starved cells).

Acknowledgments

This manuscript is dedicated to Ari Helenius, whose sitting around inthe post-doc offices remains unforgettable. M. M. is supported by SignoraAlessandra, the Foundation for Research on Neurodegenerative Diseases,the Swiss National Science Foundation (SNF), the Sinergia program of theSNF and the Comel, Gabriele and Gelu Foundations.

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Five Questions (with their Answers) on ER-Associated Degradation