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Recognition and Cleavage of Related to Ubiquitin 1 (Rub1) and Rub1-Ubiquitin Chains by Components of the Ubiquitin-Proteasome System

Recognition and Cleavage of Related to Ubiquitin 1 (Rub1) and Rub1-Ubiquitin Chains by Components of the Ubiquitin-Proteasome System
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  Recognition and Cleavage of Relatedto Ubiquitin 1 (Rub1) and Rub1-UbiquitinChains by Components of the Ubiquitin-Proteasome System* □ S Rajesh K. Singh‡, Sylvia Zerath§, Oded Kleifeld¶, Martin Scheffner  ,Michael H. Glickman§**, and David Fushman‡** Of all ubiquitin-like proteins, Rub1 (Nedd8 in mammals) isthe closest kin of ubiquitin. We show via NMR that struc-turally, Rub1 and ubiquitin are fundamentally similar aswell. Despite these profound similarities, the prevalenceof Rub1/Nedd8 and of ubiquitin as modifiers of the pro-teome is starkly different, and their attachments to spe-cific substrates perform different functions. Recently,some proteins, including p53, p73, EGFR, caspase-7, andParkin, have been shown to be modified by both Rub1/ Nedd8 and ubiquitin within cells. To understand whetherand how it might be possible to distinguish among thesame target protein modified by Rub1 or ubiquitin or both,we examined whether ubiquitin receptors can differenti-ate between Rub1 and ubiquitin. Surprisingly, Rub1 inter-acts with proteasome ubiquitin-shuttle proteins compa-rably to ubiquitin but binds more weakly to a proteasomalubiquitin receptor Rpn10. We identified Rub1-ubiquitinheteromers in yeast and Nedd8-Ub heteromers in humancells. We validate that in human cells and  in vitro , humanRub1 (Nedd8) forms chains with ubiquitin where it acts asa chain terminator. Interestingly, enzymatically assem-bled K48-linked Rub1-ubiquitin heterodimers are recog-nized by various proteasomal ubiquitin shuttles and re-ceptors comparably to K48-linked ubiquitin homodimers.Furthermore, these heterologous chains are cleaved by COP9 signalosome or 26S proteasome. A derubylationfunction of the proteasome expands the repertoire of itsenzymatic activities. In contrast, Rub1 conjugates may be somewhat resilient to the actions of other canonicaldeubiquitinating enzymes. Taken together, these find-ings suggest that once Rub1/Nedd8 is channeled intoubiquitin pathways, it is recognized essentially likeubiquitin.  Molecular & Cellular Proteomics 11: 10.1074/ mcp.M112.022467, 1595–1611, 2012. The selective degradation of many proteins in eukaryoticcells is a highly specific and irreversible process required toperform vital cellular functions such as cell cycle progression(1, 2), differentiation and development (3, 4), and transcrip-tional control (5). One of the major pathways involved in thisselective degradation is the ubiquitin-proteasome pathway. Inthis pathway, ubiquitin (Ub), a 76-amino-acid protein, is at-tached to the substrate, and this is followed by the recognitionand degradation of the substrate by a protein complex col-lectively known as proteasome (6–8).The attachment of Ub (ubiquitination) to a substrate proteinis achieved via a cascade of enzymatic reactions (involvingE1, E2, and E3 enzymes) that results in the formation of anisopeptide bond between the C-terminal glycine of Ub and alysine residue of the substrate (9–11). Sequential repetition ofthis cascade can result in the attachment of a chain of Ubmolecules (polyubiquitin) to the substrate protein. The lengthand topology of the polyubiquitin (polyUb) tag decide the fateof the substrate protein. For example, we and others haveshown that a K48-linked tetraubiquitin (tetraUb) chain thatacts as a proteasomal degradation signal forms a “closed”structure (12, 13), whereas a K63-linked Ub chain forms an“extended ” structure (14) and has been implicated in non-proteolytic functions including ribosomal functioning (15, 16)and post-replicational DNA repair (17, 18).Yeast protein Rub1 and its mammalian orthologue Nedd8(for simplicity, we refer to the molecule as Rub1 regardless ofits source unless specifically highlighting a unique property ofthe mammalian orthologue, in which case we prefer the nameNedd8), also a 76-amino-acid-long polypeptide, are the clos-est kin of Ub in the family of Ub-like proteins. Rub1 is  53%identical and  77% similar to Ub at the amino acid level. Thestructures are incredibly similar as well, as the three-dimen-sional folds of Rub1 orthologues from mammals or plants arelargely superimposable with Ub (19–21). Key residues are con-served as well (Fig. 1  A  ), suggesting that the overall surface and From the ‡Department of Chemistry and Biochemistry, Center forBiomolecularStructureandOrganization,UniversityofMaryland,Col-lege Park, MD 20742; §Department of Biology, Technion-Israel Insti-tute of Technology, 32000 Haifa, Israel; ¶Department of Biochemistry& Molecular Biology, Monash University, Clayton, VIC 3800, Australia;  Department of Biology, University of Konstanz, D-78457 Konstanz,GermanyReceived July 20, 2012, and in revised form, October 8, 2012Published, MCP Papers in Press, October 26, 2012, DOI10.1074/mcp.M112.022467  Research © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. This paper is available on line at  Molecular & Cellular Proteomics 11.12  1595  functional residues are maintained. Despite these striking sim-ilarities, Rub1/Nedd8 and Ub employ their own cognate E1 andE2 to post-translationally modify their specific substrates (22,23). The recognition of the cognate E2s by their respective E1paralogues is critical in avoiding cross-talk between these twoparallel regulatory pathways (24, 25). The Ub pathway involvesthe attachment of monomeric Ub or polyUb chains to sub-strates and has been implicated in degradative and regulatoryor protein sorting/trafficking functions. In contrast, the Rub1pathway exclusively attaches monomeric Rub1 to substratesand performs mainly regulatory functions (26). The attachmentof Rub1 (rubylation) to cullin proteins and the subsequent reg-ulation of a multi-subunit E3 ligase, the Skp, cullin, and F-boxcontaining complex, is probably the most prevalent, and clearlythe best studied, biological function reported so far (22, 23, 27,28).However,someotherproteinshaverecentlybeenshowntobe modified by Nedd8, including p53 and its E3 ligase Mdm2(29–31), p73 (a homolog of p53) (32), ribosomal protein L11 (33,34), epidermal growth factor receptor (EGFR) (35), caspase-7(36, 37), and parkin (38, 39), though the effects are still vague,particularly as the ratio of modified to unmodified forms of eachprotein is extremely low. Complicating matters, many of theseproteins are also reported to be targets for ubiquitination, and inmany cases the same E3 enzymes are required for both ubiq-uitination and rubylation. Moreover, under certain stress condi-tions, such as those that occur when the level of free Ub islimitingincells,theextentofneddylationincreasesdramatically,in some cases resulting in Rub1/Nedd8 and Ub on the sametarget (40, 41). This raises the important yet unaddressed ques-tion of how the proteasomal and the non-proteasomal Ub re-ceptors differentiate among the same target proteins modifiedby either Ub or Rub1 or both. To address this question, weexaminedtheinteractionsofRub1withtheUb-bindingdomainsof these receptors. Surprisingly, Rub1 showed binding to all thenon-proteasomal receptors/shuttles tested. However, it inter-acts weakly with a proteasomal Ub-receptor, Rpn10. As thesereceptors are well known for their preference for Ub chains (42),we tested the ability of human Rub1 (Nedd8) to form chains inH1299 cells. Interestingly, Nedd8 forms a heterologous chainwith Ub. Our analysis revealed that in the substrate-freeNedd8-Ub heterodimers, Nedd8 acts as a chain terminator.Moreover, we were able to synthesize these heterodimers  invitro  using the E1 and E2 enzymes from the Ub pathway. Herewe also show that a K48-linked Rub1-Ub heterodimer is recog-nized by the proteasomal and non-proteasomal Ub-receptorscomparablytoK48-linkedUbhomodimerandiscleaved  invitro by either purified 26S proteasome or the COP9 signalosome. EXPERIMENTAL PROCEDURES Expression Constructs— The DNA encoding 1–76 amino acids ofRub1wasclonedintopTXB1vector(NEB)usingstandardtechniques,and the construct was verified by DNA sequencing. Details of thecloning procedure and primer sequences are provided in the supple-mental information. All constructs used in this study are listed insupplemental Table S1. Protein Expression and Purification—  All proteins used in this studywere expressed in the BL21 (DE3) strain of  E. coli   except for Ub con-structs, which were expressed in the pJY2 strain. Proteins were purifiedusing standard chromatographic techniques (see supplemental TableS2 ). Synthesis and Purification of Rub1-Ub Dimer— Purified Rub1 andUb monomers (100  M  each) were incubated with 500 n M  of E1 of Ub,20   M  of E2–25K (or 20   M  each of Ubc13 and Mms2), 5 m M  ATP, 5m M  MgCl 2 , phosphocreatin, and phosphocreatine kinase in 50 m M Tris-HCl buffer (pH 8) for 16 h. Rub1-Ub dimer fractions were purifiedvia gel filtration chromatography. The  15 N-labeled Rub1-Ub het-erodimer for NMR studies was assembled from  15 N-enriched Rub1and  15 N-enriched Ub monomers using the same procedure. Afterpurification, the protein was exchanged into 20 m M  phosphate buffer(pH 6.8) containing 0.02% (v/v) NaN 3  and 7% D 2 O for NMR studies. NMR— NMR samples for Rub1 monomer were prepared in 20 m M phosphate buffer (pH 6.0), which was used for structural character-ization. All NMR binding studies were done in 20 m M  phosphate buffer(pH 6.8). NMR data were acquired at 22.5 °C on a Bruker Avance III600 MHz spectrometer equipped with a cryoprobe. Details of theexperiments performed and data analysis can be found in the sup-plemental information. Mammalian Cell Culture, Transfection, and Ni-affinity Purification— H1299 cells were cultured in Dulbecco’s modified Eagle’s mediumwith 10% (v/v) fetal bovine serum. Cells at 90% confluency in 6 cmplates were transfected using Lipofectamine 2000 as described else-where (43). Twenty to twenty-four hours after transfection, the cellswere lysed in 500   l Gd-HCl buffer (6  M  guanidinium HCl, 100 m M phosphate buffer pH 8.0, 10 m M  imidazol, 10 m M   -mercaptoethanol,1 m M  pefabloc, 1   g/ml apeptin/leupeptin mixture), and 20   l ofprotein A Sepharose beads (equilibrated in Gd-HCl buffer) wereadded. The samples were incubated at 4 °C with rotation for 1 h andthen centrifuged, and 50   l of Ni-agarose beads (equilibrated inGd-HCl buffer) were added to the supernatant; this was followed byincubation at 4 °C for 3 to 4 h or overnight. The beads were thenwashed two times with Gd-HCl buffer, and this was followed by twomore washes in a buffer containing one part Gd-HCl buffer and fourparts 50 m M  Tris-Cl (pH 6.8) containing 20 m M  imidazol. Finally, thesamples were washed two times with 50 m M  Tris-Cl (pH 6.8) buffercontaining 20 m M  imidazol. The samples were boiled at 95 °C for 5min in 100   l of Laemmli buffer containing 200 m M  imidazole andloaded onto SDS-PAGE gels. Cleavage of Rub1-Ub Dimer— 0.1 m M  of K48-linked Rub1-Ub 74dimer was incubated with either 26S proteasome or COP9 signalo-some or with both in the presence of 100 m M  Tris-Cl buffer (pH 7.4),20% glycerol, 20 m M  MgCl 2 , 0.5  M  phosphocreatine, 0.2 mg/ml phos-phocreatinine kinase, 0.5  M  ATP, and 1  M  DTT. The reaction wasincubated for 12 to 16 h at 30 °C. Stable Isotope Labeling of Amino Acids in Cell Culture and Data Analysis— RGS-His 6 -Rub1-expressing yeast cells were grown in min-imal synthetic defined (SD) media containing yeast nitrogen base andglucose supplemented with 4 mg/l  13 C 615 N 2  lysine and 2 mg/l 13 C 615 N 4  arginine (Cambridge Isotope Laboratories Inc, Andover, MA,USA.), and RGS-His 6 -empty vector expressing cells in SD mediasupplemented with 4 mg/l  12 C 614 N 2  lysine and 2 mg/l  12 C 614 N 4  argi-nine (Sigma Aldrich). Cells were lysed and enriched for Rub1 via theuse of a mini nickel-nitrilotriacetic acid column (Qiagen, Valencia, CA,USA). Eluted proteins were separated via SDS-PAGE and cut into 12gel slices. The gel slices were incubated with modified trypsin (Pro-mega, Madison, WI, USA), and the resulting tryptic peptides wereidentified by means of mass spectrometry. Details of the in-gel trypticdigestion and mass spectrometric analysis are included in the sup-plemental information. Recognition and Cleavage of Rub1-Ubiquitin Chains 1596  Molecular & Cellular Proteomics 11.12  Pulldowns of Ub Receptors with Rub1— Purified recombinantHis 6 -Rub1 or His 6 -Ub, immobilized on CH Sepharose 4B beads,were incubated with whole cell extract prepared from natively lysed   rub1  cells. 2 ml of cell extract were incubated with 150   l slurry(Rub1 or Ub conjugated, or mock) overnight at 4 °C. The beadswere washed with 20 column volumes of lysis/wash buffer, followedby elutions with 1  M  NaCl or 8  M  urea. Hydrophilic and hydrophobiceluates were separated via SDS-PAGE and immunoblotted againstRpn10 and Dsk2. RESULTS Rub1 Is Structurally Similar to Ubiquitin—  Although Rub1 isan important protein in  Sachharomyces cerevisiae , its three-dimensional structure was not known. NMR spectra of Rub1indicate a well-folded protein and show striking similarity toanalogous spectra of Ub (Fig. 1 B  ). To determine the structureof Rub1, we obtained a nearly complete NMR resonance F IG . 1.  Rub1 is structurally similar to Ub.  A , amino acid sequence comparison of Ub and Rub1/Nedd8 from  Saccharomyces cerevisiae  (Sc)and human (Hs). Lysine residues that are conserved in both Ub and Rub1 are represented by empty boxes. The hydrophobic patch residues(L8, I44, and V70) are shaded red. Shown here are sequences of mature proteins; the sequences of the non-processed proteins contain anadditional C-terminal N for Rub1 and residues GGLGQ for Nedd8.  B ,  1 H- 15 N HSQC spectrum of Rub1 (blue) overlaid with the similar spectrumof Ub (red). Selected residues that have similar chemical shifts in both Rub1 and Ub are marked with numbers and indicated by arrows.  C , anensemble of the nine lowest energy structures of Rub1 (backbone trace) generated by CS-Rosetta.  D , surface representation of one of theCS-Rosetta-generated structures of Rub1 (Rub1_798). The conserved hydrophobic patch residues are painted red and are indicated.  E  – G , theagreement between experimentally measured residual dipolar couplings (RDCs) of Rub1 and the back-calculated RDCs using the structuresof Rub1_798 (  E   ), Ub (  F  ; PDB ID: 1D3Z), or Nedd8 (  G ; PDB ID: 2KO3). The diagonal represents absolute agreement. Pearson’s correlationcoefficient (Corr. Coeff.) and the quality factor (R) are indicated. Recognition and Cleavage of Rub1-Ubiquitin Chains  Molecular & Cellular Proteomics 11.12  1597  assignment of  1 H,  13 C (C  , C  , C   ), and amide  15 N nuclei ofthis protein. Using these resonances along with predictions ofprotein backbone dihedral angles made by TALOS  (44), wecomputed a model structure of Rub1 using the CS-Rosettaapproach (45, 46), which resulted in an ensemble of nineclosely related low-energy structures (Fig. 1 C  ). To verify thesepredicted structures, we measured residual dipolar couplings(RDCs) for backbone amide groups of Rub1 in a weaklyaligned liquid-crystalline medium. Comparison of these RDCdata with the predictions based on CS-Rosetta-generatedstructures showed good general agreement for all structures,with the Pearson’s correlation coefficient values ranging from0.743 to 0.921 ( supplemental Table S3 ). This suggests that (i) these structural models provide a good representation of theRub1 structure and (ii) our NMR signal assignments can beused to map Rub1’s interactions with other proteins. Thestructure that gave the best agreement (shown in Figs. 1 D  and1 E   ) is used throughout this paper as the representative struc-ture of Rub1. Furthermore, the RDC data for Rub1 are also ingood agreement with the published structures of Ub andNedd8 (Figs. 1 F   and 1 G  ), directly indicating that Rub1 isstructurally very similar to Ub and Nedd8. Indeed, the pre-dicted model structure of Rub1 can be superimposed on thepublished structures of Ub and Nedd8 with backbone (C   )root-mean-square deviations of 0.998 Å and 1.185 Å, respec-tively ( supplemental Fig. S1  A  ). The distribution of surfacecharges on the   -sheet side of Rub1 encompassing the hy-drophobic-patch residues L8, I44, and V70 (functionally im-portant in Ub) is very similar to that of Ub and Nedd8 ( sup-plemental Fig. S1 B  ), suggesting similar ligand bindingproperties. Rub1 Binds to Non-proteasomal Ub Receptors/Shuttles— There are three well-studied non-proteasomal Ub receptors/ shuttles: Rad23 (its human orthologue is known as hHR23a)(47, 48), Dsk2 (known as hPLIC1 or Ubiquilin 1 (UQ1) inhumans) (49), and Ddi1 1 (50, 51). All of them are unique in thatthey contain an N-terminal Ub-like (UBL) domain that binds tothe proteasome via Rpn1 (42, 52, 53) and a C-terminal Ub-associated (UBA) domain that binds to Ub chains (54, 55).These UBL-UBA proteins have been seen to cycle on and offthe proteasome while delivering the ubiquitinated cargo fromthe cytoplasm and the nucleus to the proteasome, which iswhy they are also known as shuttle proteins (56, 57). Althougha Ub chain is the preferred binding partner of these shuttleproteins, monoubiquitin (monoUb) has been shown to interactwith them as well, albeit with lower affinity (58–62). More-over, as Rub1 is documented to form monomeric modifica-tions on its targets (26, 28), comparison of its binding prefer-ences with monoUb might unlock a clue as to whatdistinguishes the two. To assess whether Rub1 interacts withthe UBA domains of these receptors,  15 N labeled Rub1 wastitrated with the UBA domains of hHR23a, human UQ1, andyeast Ddi1, and the interaction was monitored using NMRspectroscopy (see Fig. 2 and below for further details).NMR signals are highly sensitive to the local electronicenvironment in a molecule (63, 64) and therefore have beenwidely used to map the interacting surfaces (13, 14, 58, 65,66) and assess the strength of binding (14, 58, 59, 66–68). A perturbation in the local environment in a protein could resultin a shift of the NMR signal from its srcinal position (referredto as chemical shift perturbation (CSP)) or broadening (atten-uation) of the NMR signal, or both. As both CSPs and signalattenuations are direct consequences of a change in the localenvironment in a protein caused by complex formation, theycan be used as markers to map the binding interface. Rub1 Interaction with the UBA2 Domain of hHR23a— Rad23 contains two UBA domains: a central UBA1 domainand a C-terminal UBA2 domain. Both UBA domains interactwith monoUb and have a binding preference for K48-linkedchains (61, 69, 70). To test whether the UBA2 domain inter-acts with Rub1, unlabeled UBA2 domain of the human ortho-logue of Rad23 (hHR23a) was added to  15 N labeled Rub1. Analysis of the NMR spectra revealed site-specific CSPs formany Rub1 residues, indicating a Rub1–UBA2 interaction(Fig. 2  A  ). The observed CSPs increased with increasingamounts of UBA2 and almost saturated at UBA2:Rub1 molarratios in the range of 3:1 to 4:1 (Fig. 2 C  ). Additionally, manyamides showed signal attenuations indicative of intermediateor slow exchange on the NMR chemical shift time scale (Fig.2  A  ). Mapping the perturbations onto the surface of Rub1revealed that they were clustered on one side of the moleculeand included the hydrophobic patch residues L8, I44, and V70(Fig. 2 B  ), in striking similarity to the UBA2-interacting surfaceof monoUb (71).To assess the stoichiometry of the UBA2:Rub1 complex,we measured the longitudinal  15 N relaxation time (  T  1  ), which isa sensitive indicator of the overall tumbling rate of the mole-cule/complex and therefore is directly related to its apparentsize. The average  T  1  value measured for the UBA2:Rub1complex at a 4:1 molar ratio was 634    31 ms (see Table I),which corresponds to a molecular weight of 14 to 15 kDa (58).This result is consistent with a 1:1 stoichiometry of the UBA2/ Rub1 complex (the expected molecular mass  14 kDa). Wethen assessed the affinity of the interaction by fitting ourtitration data to a one-site binding model, which yielded a  K  d  value of 72  17   M , averaged over six residues (Table II, and 1 The abbreviations used are: Ddi1, DNA damage inducible protein1; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme;E3, isopeptide ligase enzyme; HA, hemagglutinin; hHR23a, humanhomologue of Rad23 protein A; Nedd8, neuronal precursor cell ex-pressed developmentally down-regulated protein 8; NMR, nuclearmagnetic resonance; NUB1, NEDD8 ultimate buster 1; NUB1L,NEDD8 ultimate buster 1-long; PDB, Protein Data Bank; Rub1,related to ubiquitin protein 1; SILAC, stable isotope labeling ofamino acids in cell culture; Ub, ubiquitin; UBA, ubiquitin-associatedprotein; UBL, ubiquitin-like protein; UIM, ubiquitin-interacting motif;UQ1, ubiquilin 1. Recognition and Cleavage of Rub1-Ubiquitin Chains 1598  Molecular & Cellular Proteomics 11.12  also see Fig. 2 C  ). This affinity is  4- to 5-fold higher than thereported affinity of UBA2 for monoUb (400  100   M  (71, 72)). Rub1 Interaction with the UBA Domain of UQ1— UnlikehHR23a, human UQ1 (also known as hPLIC1) contains onlyoneUBAdomain,locatedattheCterminus(73),andhasbeenshown to be the strongest Ub binder among the family ofUBL-UBA proteins (61, 74). We detected a relatively stronginteraction between Rub1 and the UBA2 domain of hHR23a,and we were interested to know whether Rub1 binds also tothe UBA domain of UQ1 (UBA-UQ1). Indeed, our NMR dataconfirmed this interaction. Moreover, NMR signal perturba-tions (CSPs and signal attenuations) in Rub1 caused by UBA-UQ1 binding indicate that this interaction is highly specificand involves predominantly residues clustered around thehydrophobic patch on Rub1’s surface (Figs. 2 D  and 2 E   ). TheUBA-UQ1-interaction surface on Rub1 is similar to that on Ub(59), as well as to the Rub1 surface involved in hHR23a UBA2binding (Figs. 2  A  and 2 B  ). A   15 N  T  1  of 647    44 ms for theRub1/UBA-UQ1 complex suggests a 1:1 binding (Table I). Thequantitative analysis of the CSPs of Rub1 upon titration with F IG . 2.  Interaction of Rub1 with the UBA domains of the shuttle proteins.  Chemical shift perturbations (CSPs) (black bars) and significantsignal attenuations (   75%; gray bars) in Rub1, plotted as a function of the residue number, at the end point of titration with UBA2-hHR23a(   A  ), UBA-UQ1 (  D  ), or UBA-Ddi1 (  G  ). Mapping of the Rub1 residues (red) exhibiting CSPs higher than indicated and/or significant signalattenuations upon binding to UBA2-hHR23a (  B  ), UBA-UQ1 (  E   ), or UBA-Ddi1 (  H  ). Representative titration curves showing the normalized CSPs(in Rub1) as a function of ligand/protein molar ratio for UBA2-hHR23a (  C  ), UBA-UQ1 (  F   ), or UBA-Ddi1 (  I  ) interactions with Rub1. The linesrepresent the results of fitting.T  ABLE  I Longitudinal   15 N relaxation time (T  1  , in ms) for the unbound proteins or the protein/ligand complexes at the endpoint of the titration Protein Unbound   UBA-UQ1   UBA2-hHR23a   UBA-Ddi1   UIM-Rpn10MonoRub1 459  33 647  44 634  31 543  27 499  33Proximal-Ub, heterodimer 694  42 1000  62 1054  85 874  56 1103  192Distal-Rub1, heterodimer 680  45 974  67 1040  88 829  57 968  90The  T  1  data reported here represent the mean and the standard deviation calculated over several backbone amide signals. Recognition and Cleavage of Rub1-Ubiquitin Chains  Molecular & Cellular Proteomics 11.12  1599
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