Substrate specificity of the ubiquitin and Ubl proteases.pdf

REVIEWnpgCell Research(2016)26:441- specificity of the ubiquitin and Ubl proteasesJudith A Ronau1,John F Beckmann1,Mark Hochstrasser1,21Department of Molecular Biophysics and Biochemistry,2Department of Molecular,Cellular and Developmental Biology,Yale University,266 Whitney Avenue,New Haven,CT 06520,USAConjugation and deconjugation of ubiquitin and ubiquitin-like proteins(Ubls)to cellular proteins are highly regu-lated processes integral to cellular homeostasis.Most often,the C-termini of these small polypeptides are attached to lysine side chains of target proteins by an amide(isopeptide)linkage.Deubiquitinating enzymes(DUBs)and Ubl-spe-cific proteases(ULPs)comprise a diverse group of proteases that recognize and remove ubiquitin and Ubls from their substrates.How DUBs and ULPs distinguish among different modifiers,or different polymeric forms of these mod-ifiers,remains poorly understood.The specificity of ubiquitin/Ubl-deconjugating enzymes for particular substrates depends on multiple factors,ranging from the topography of specific substrate features,as in different polyubiquitin chain types,to structural elements unique to each enzyme.Here we summarize recent structural and biochemical studies that provide insights into mechanisms of substrate specificity among various DUBs and ULPs.We also discuss the unexpected specificities of non-eukaryotic proteases in these families.Keywords:ubiquitin;DUBs;Ubl proteases;SUMO;Nedd8;ULPsCell Research(2016)26:441-456.doi:10.1038/cr.2016.38;published online 25 March 2016Correspondence:Mark HochstrasserE-mail:mark.hochstrasseryale.eduIntroduction Covalent attachment of ubiquitin to proteins governs a wide array of cellular processes,including cell divi-sion,DNA repair,endocytosis,cellular signaling,and protein quality control 1-4.The sequential action of three enzymes E1 ubiquitin-activating enzyme,E2 ubiquitin-conjugating enzyme,and E3 ubiquitin ligase results in attachment of ubiquitin to a substrate pro-tein,usually via an amide(isopeptide)bond that links the C-terminal carboxyl group of ubiquitin with a lysine side chain(s)of the protein substrate(Figure 1)5,6.Ubiquitin itself possesses seven lysine residues(Lys6,11,27,29,33,48,and 63),enabling it to form ubiquitin polymers;chains with different linkages signal different functional outcomes for the tagged proteins 3,7-9.All seven lysines are used for chain formation in vivo,as is the N-terminal-amino group,the latter leading to linear ubiquitin chains.Chains can be homopolymeric or,less commonly,of mixed linkage.Branched ubiquitin chains,which use different lysines of a single ubiquitin for chain extension,can also form 10-13.Following the discovery of ubiquitin,related proteins called ubiquitin-like proteins(Ubls)were also identified;these proteins share a common core architecture called the-grasp fold 14.As with ubiquitin conjugation,a similar cascade of E1,E2 and E3 enzymes,specific to each Ubl,is utilized to covalently link Ubl and substrate 15.Besides ubiquitin,the most frequently employed Figure 1 The ubiquitin(Ub)modification cycle.442Substrate specificity of the ubiquitin and Ubl proteasesnpgCell Research|Vol 26 No 4|April 2016Ubl is SUMO(small ubiquitin-related modifier).In humans,SUMO is present as four isoforms,each en-coded by a distinct gene 16.Interestingly,hybrid SU-MO-ubiquitin chains have also been described 17.The variety of covalently ligated ubiquitin/Ubls and their polymeric forms creates significant challenges for the deconjugating enzymes in identifying and cleaving the appropriate substrates.Deubiquitinating enzymes(DUBs)and Ubl-specific proteases(ULPs)catalyze the cleavage of ubiquitin or Ubls from substrate proteins and also process C-termi-nally extended precursor forms of these modifiers(Figure 1)18.DUBs and ULPs can be classified into one of two mechanistic classes:(1)thiol proteases,which are mechanistically and structurally related to the well-stud-ied cysteine protease papain and rely on a nucleophilic cysteine in the active site for catalysis,and(2)metal-loproteases,which coordinate a Zn2+ion in the active site and use a nucleophilic water ligated to the metal to hydrolyze the isopeptide linkage 19.The JAB1/MPN/MOV34 metalloproteases(JAMMs)include multiple DUBs and at least one ULP(a deneddylase,i.e.,an en-zyme that cleaves the Nedd8 Ubl from substrates).The thiol protease class includes the great majority of DUBs and ULPs.They are categorized into distinct families according to the structure of their catalytic domains.As with the JAMMs,the same family may have members that cleave ubiquitin,Ubls,or both.It remains difficult to predict these specificities based on enzyme primary sequence alone.The four known eukaryotic thiol protease DUB fam-ilies are the ubiquitin C-terminal hydrolases(UCHs),ubiquitin-specific proteases(USPs),ovarian tumor(OTU)proteases,and Machado-Josephin-domain proteases 20,21.As will be discussed later,bacterial and viral thiol proteases outside of these families have been found to have DUB activity;it is noteworthy that these bacterial and viral ubiquitin-cleaving enzymes all function within eukaryotic cells.Similar to the DUBs,there are multi-ple SUMO-specific thiol protease families:the SUMO/sentrin-specific proteases(SENPs;which are related to yeast Ulp1),desumoylating isopeptidases(DESIs),and ubiquitin-specific protease-like 1(USPL1)16.SUMO proteases of the SENP/ULP class are the most phyloge-netically widespread.Nearly 100 DUBs have been identified in humans.Since the capacity for ubiquitin deconjugation in cells is extremely high,these activities must be kept under tight control.Accordingly,DUB(and ULP)activity is regulat-ed by a host of factors.In many cases,DUBs and ULPs have domains extending from either end of their catalytic domains that help regulate their activity.Such regulation may occur by facilitating enzyme-cofactor interactions,targeting enzymes to specific cellular compartments,maintaining enzymes in an auto-inhibited state,and/or altering their affinity for substrate 20.DUBs and ULPs display high specificity toward their substrates.They can differentiate between ubiquitin and Ubls,show preferences for particular polymeric forms of ubiquitin or Ubls,and distinguish among distinct conju-gated substrates.The molecular basis of this specificity is the subject of the present review.We focus on revelations about specificity derived from recent structural studies.We also discuss non-eukaryotic DUBs and ULPs and their sometimes surprising specificities.Ubiquitin and Ubl recognition by DUBs and ULPsModifier propertiesThe modifier proteins sport surface features that aid in their recognition by DUBs and ULPs.Although ubiquitin is a small,compact protein with a rigid core,it contains several important motifs for interactions with other proteins.The two motifs most commonly observed con-tacting DUBs are the so-called Ile44 patch(comprising Ile44,Leu8,Val70 and His68)and the Ile36 patch(Ile36,Leu71 and Leu73)22,23.Other protein-binding el-ements utilized by ubiquitin are the Phe4 patch(Gln2,Phe4 and Thr14),the TEK box(Lys6,Lys11,Thr12,Thr14 and Glu34),and the Asp58 patch(Arg54,Thr55,Ser57 and Asp58)3.Sequence alignment of ubiquitin and the Ubls SUMO,Nedd8,ISG15 and Fat10 reveals that,aside from Nedd8,the other Ubls exhibit very little sequence conservation with ubiquitin in these motifs(Figure 2).Both the Ile36 and Ile44 patches are conserved in Nedd8,and the Nedd8 Ile44 patch binds directly to the deneddylase Den1/SENP8 24,25.However,the Ile44 patch is not always a key DUB/ULP contact spot.SdeA,a bacterial DUB,does not engage in any interactions with this patch when bound to a ubiquitin suicide substrate 26.Likewise,new crystal structures of the USP CYLD bound to either Met1-or Lys63-linked diubiquitin revealed that the Ile44 patch of the distal ubiquitin(the one with its C-terminal carboxyl in amide linkage)has no direct interactions with the enzyme 27.A similar observation was made previously for USP7 bound to ubiquitin aldehyde 28.Perhaps the most significant feature of ubiquitin and Ubls for cognate protease recognition is their flexible C-terminal tail 29.The ubiquitin/Ubl tail is stabilized by several interactions in the protease active site cleft.DUB and ULP deconjugation of their cognate modifier proteins depends heavily on these C-terminal residues(labeled P6-P1 in Figure 2).For example,a single ami-Judith A Ronau et al.443npgwww.cell-|Cell Researchno-acid exchange of the Ala at position P5 in Nedd8 to an Arg,which is the ubiquitin P5 residue,markedly de-creased the affinity of Den1 for the mutated Ubl,likely due to steric interference with the ULP 25.Conversely,for USP21,which exhibits dual specificity for ubiquitin and ISG15,Arg72 at P5 of ubiquitin is stabilized through formation of a salt bridge with an invariant Glu in USP21 30.This Arg residue is present in the sequence of ISG15 but not Nedd8.Were Arg72 not engaged in DUB binding,one might predict that discrimination against Nedd8 would not be seen;this is borne out for the pro-karyotic DUB SdeA 26.Other ubiquitin/Ubl features also contribute to binding of their cognate deconjugating enzymes.In the-grasp fold,a central-helix is cradled by a curved-sheet(Fig-ure 3)31.Most co-crystal structures of ubiquitin-DUB complexes reveal that the two-residue loop(Leu8-Thr9)that connects the 1 and 2 strands nestles into a binding pocket in DUBs well away from the active site 28,32,33.Binding by this loop in Nedd8 also generates several key van der Waals contacts with the ULP Den1 24,25.By contrast,inspection of co-crystal structures of SUMO with SUMO proteases indicates that the SUMO 1-2 loop has little direct involvement in binding to the prote-Figure 2 Sequence alignment of ubiquitin and Ubls(A)and ubiquitin surface elements important for protein binding(B).The various patches that ubiquitin(PDB code:1UBQ 31)uses to bind other proteins are highlighted as follows:the C-terminal LRLRGG motif is colored green,the Ile44 patch is red,the Ile36 patch is blue,the Phe4 patch is cyan,the TEK box is orange,the Asp58 patch is purple,and Ser65,which is phosphorylated by the kinase PINK1,is black.444Substrate specificity of the ubiquitin and Ubl proteasesnpgCell Research|Vol 26 No 4|April 2016ases 34-36.Interestingly,ubiquitin co-crystal structures with UCH and USP family DUBs suggest that this ubiq-uitin loop adopts UCH-specific and USP-specific confor-mations 37.A newly identified feature of ubiquitin,that it can be phosphorylated at Ser65 by PINK1 38,39,provides an added element to consider in substrate specificity.Al-though yeast lacks an ortholog for PINK1,phosphoryla-tion of ubiquitin Ser65 has also been shown in yeast and may serve a conserved regulatory function 40.Phos-phorylated ubiquitin can adopt two states in solution;one may limit accessibility of its tail 41.Most DUBs tested so far have only weak activity toward ubiquitin chains composed of phosphorylated ubiquitin 41,42.Ser65 is conserved in both ISG15 and Nedd8,suggesting that they may also be susceptible to phosphorylation.Recognition of polyubiquitin chainsFor depolymerization of polyubiquitinated substrates,an important structural feature is the isopeptide linkage between ubiquitin monomers.The isopeptide bond link-ing the proximal(lysine-donating)and distal(lysine-ac-cepting)ubiquitin must be stabilized in the active site of the DUB.Comparison of Lys63-and Lys48-linked polyubiquitin chains shows that Lys63 linkages adopt an extended beads-on-a-string conformation in which the only interaction between the ubiquitin moieties is through the isopeptide linkage 43,44.This confor-mation is shared by Met1-linked diubiquitin 45.By contrast,the ubiquitin moieties in Lys48-linked poly-ubiquitin pack closely in a closed conformation through interactions of their Ile44 patches 46,47.Similarly,Lys11-and Lys6-linked diubiquitins also adopt more compact conformations,although the Ile44 patch is ex-posed on both ubiquitins in Lys11-linked diubiquitin and one of the ubiquitins in Lys6-linked diubiquitin 48-50.Recent structural studies revealed that K33-linked ubiquitin chains adopt open and closed conformations in triubiquitin and diubiquitin,respectively 51.Neverthe-less,polyubiquitin chains are dynamic in solution,and a chain can adopt both closed and open conformations 52,53.Compact chains likely cannot be recognized by DUBs unless they undergo significant conformational changes to expose the isopeptide bond.In the case of Lys48-link-ages,this remodeling probably also involves exposure of the Ile44 patch so that it is free to interact with the DUB.To our knowledge,no DUB bound to a Lys48-linked diubiquitin has been crystallized.Specificity of DUBs toward different ubiquitin linkages varies among family members.Many JAMM proteases,such as AMSH,are only active against Lys63-linked chains 54,55,primar-ily due to interactions with the proximal ubiquitin moiety 33,56.On the other hand,most DUBs belonging to the USP family will hydrolyze many chain types,albeit with different preferences 20,57.The OTU DUBs show a striking range of chain linkage preferences.Structural analyses reveal that proper positioning of the proximal ubiquitin on the OTU DUB surface is paramount for this selectivity 58 and that the length of the ubiquitin poly-mer also contributes to specificity 58,59.Ubiquitin/Ubl-induced active-site rearrangement Thiol protease DUBs and ULPs utilize variations of the classic papain-like Cys-His-Asp/Asn catalytic triad to catalyze hydrolysis of peptide or isopeptide bonds 18.To facilitate activation of the nucleophilic Cys by His(serving as the general base),these residues must be precisely oriented in the active site with a His-Cys hydrogen-bond distance within 3.8.However,a com-mon theme emerging from structural studies of these proteases is that the catalytic residues are often in unpro-ductive orientations in the absence of substrate.This mis-alignment involves displacement of the Cys,the His,or both 28,60-64.Substrate binding causes the active site residues to rearrange into a catalytically competent ori-entation 32,63,65-67.The earliest example of such a substrate-induced realignment of active site residues was the DUB USP7,which has been discussed extensively 20,28,68.Figure 3 Structural comparison depicting the conserved-grasp fold of ubiquitin and Ubl proteins(PDB codes:1UBQ 31,1NDD 146,1WM3 147,1Z2M 148,and 2KWC 149).ISG15 has tandem ubiquitin folds.The C-terminal glycine of ubiquitin and Ubls is colored red,except for SUMO2 because it was not or-dered in the crystal structure.Judith A Ronau et al.445npgwww.cell-|Cell Research A striking example of how remote substrate binding induces realignment of the active-site His to a catalyti-cally productive orientation can be seen with the free and ubiquitin-bound forms of UCHL1.Defects in UCHL1,the smallest member of the UCH family of DUBs,have been linked to a variety of diseases including Parkinsons d