Cyclization of Polyubiquitin by the E2-25K Ubiquitin Conjugating Enzyme

For most substrates of ubiquitin (Ub)-dependent degradation, recognition by the proteasome is mediated by a covalently attached signal assembled from multiple ubiquitins linked to each other via the C terminus of one Ub and the ε-amine of Lys48 of another Ub. Among Ub-conjugating enzymes, E2-25K is unique in its ability to synthesizein vitro unanchored Lys48-linked poly-Ub chains from mono- or poly-Ub, E1, and ATP; thus, E2-25K has distinct binding sites for donor and acceptor (poly)Ub. During studies of chain assembly by E2-25K, we observed that Lys48-linked tri-Ub was efficiently converted to a new species that upon SDS-polyacrylamide gel electrophoresis migrated between linear di-Ub and tri-Ub. Analysis of this product by mass spectrometry and tryptic digestion showed that it was a cyclic form of tri-Ub. Cyclization of tri-Ub requires E1, E2-25K, ATP, and that the linear substrate has a free Gly76 C terminus on the proximal end Ub and a Lys48 side chain available on the distal end Ub. E2-25K similarly can catalyze the cyclization of longer poly-Ub chains, including tetra- and penta-Ub. Although cyclic tri-Ub resists hydrolysis by the PA700 or isopeptidase T deubiquitinating enzymes, it can be disassembled to Ub monomers by isopeptidase(s) in a red blood cell extract. Thus, if cyclic poly-Ub forms in vivo, it will not accumulate as a dead-end product. For most substrates of ubiquitin (Ub)-dependent degradation, recognition by the proteasome is mediated by a covalently attached signal assembled from multiple ubiquitins linked to each other via the C terminus of one Ub and the ε-amine of Lys48 of another Ub. Among Ub-conjugating enzymes, E2-25K is unique in its ability to synthesizein vitro unanchored Lys48-linked poly-Ub chains from mono- or poly-Ub, E1, and ATP; thus, E2-25K has distinct binding sites for donor and acceptor (poly)Ub. During studies of chain assembly by E2-25K, we observed that Lys48-linked tri-Ub was efficiently converted to a new species that upon SDS-polyacrylamide gel electrophoresis migrated between linear di-Ub and tri-Ub. Analysis of this product by mass spectrometry and tryptic digestion showed that it was a cyclic form of tri-Ub. Cyclization of tri-Ub requires E1, E2-25K, ATP, and that the linear substrate has a free Gly76 C terminus on the proximal end Ub and a Lys48 side chain available on the distal end Ub. E2-25K similarly can catalyze the cyclization of longer poly-Ub chains, including tetra- and penta-Ub. Although cyclic tri-Ub resists hydrolysis by the PA700 or isopeptidase T deubiquitinating enzymes, it can be disassembled to Ub monomers by isopeptidase(s) in a red blood cell extract. Thus, if cyclic poly-Ub forms in vivo, it will not accumulate as a dead-end product. ubiquitin ubiquitin aldehyde high pressure liquid chromatography polyacrylamide gel electrophoresis N-methylated andS-aminoethylated glutathione S-transferase ubiquitin C-terminalhydrolase ubiquitin-specificprotease bovine red blood cell fraction II Ub activating enzyme Ub conjugating enzyme Ub-protein ligase In eukaryotes, covalent attachment of the 76-residue protein ubiquitin (Ub)1 identifies a substrate for intracellular proteolysis by the 26 S proteasome (reviewed in Refs. 1Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6959) Google Scholar and 2Kirschner M. Trends Cell Biol. 1999; 9: M42-M45Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Usually, the C-terminal carboxyl group of Ub is linked to an internal lysine side chain of the substrate, and subsequently, multiple ubiquitins are added to form a poly-Ub chain extension. Its 8 primary amino groups (i.e. an unblocked N terminus plus 7 lysine residues) give Ub the potential to be assembled into a large variety of poly-Ub structures. Ub residues Lys6, Lys11, Lys29, Lys48, and Lys63 all can act as acceptors for ubiquitination, and poly-Ub with linkages through Lys29, Lys48, and Lys63 have been implicated in distinct physiological functions (reviewed in Refs. 3Pickart C.M. FASEB J. 1997; 11: 1055-1066Crossref PubMed Scopus (308) Google Scholar and 4Dubiel W. Gordon C. Curr. Biol. 1999; 9: R554-R557Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Nonetheless, chains with Ub−Ub isopeptide bonds made exclusively through Ub Lys48 appear to be the principal 26 S proteasome recognition signal (5Chau V. Tobias J.W. Bachmair A. Marriott D. Ecker D.J. Gonda D.K. Varshavsky A. Science. 1989; 243: 1576-1583Crossref PubMed Scopus (1122) Google Scholar, 6Finley D. Sadis S. Monia B.P. Boucher P. Ecker D.J. Crooke S.T. Chau V. Mol. Cell. Biol. 1994; 14: 5501-5509Crossref PubMed Scopus (303) Google Scholar). Ubiquitination generally entails multiple rounds of Ub transfer catalyzed by the sequential action of the Ub activating enzyme (E1), a Ub conjugating enzyme (E2), and a Ub-protein ligase (E3). Among the large number of E2 enzymes (7Haas A.L. Siepmann T.J. FASEB J. 1997; 11: 1257-1268Crossref PubMed Scopus (277) Google Scholar), mammalian E2-25K and its 20-kDa wheat homolog are unique in their ability to assemble Lys48-linked poly-Ub in the absence of an E3 in vitro (8Chen Z. Pickart C.M. J. Biol. Chem. 1990; 265: 21835-21842Abstract Full Text PDF PubMed Google Scholar, 9Mastrandrea L.D. Kasperek E.M. Niles E.G. Pickart C.M. Biochemistry. 1998; 37: 9784-9792Crossref PubMed Scopus (13) Google Scholar, 10Haldeman M.T. Xia G. Kasperek E.M. Pickart C.M. Biochemistry. 1997; 36: 10526-10537Crossref PubMed Scopus (123) Google Scholar, 11van Nocker S. Vierstra R.D. J. Biol. Chem. 1993; 268: 24766-24773Abstract Full Text PDF PubMed Google Scholar). E2-25K can utilize either mono-Ub or poly-Ub as the acceptor for ubiquitination, and in all cases the transfer is exclusively to Lys48 of the distal Ub unit. E2-25K is similarly flexible with respect to the donor substrate. Both Ub and poly-Ub can be activated by E1 to form a (poly)Ub−E1 thiol ester, and such adducts are competent as (poly)Ub donors for E2-25K-catalyzed chain elongation. However, whether E2-25K functions to assemble unanchored chains of Lys48-linked poly-Ub in vivo is unclear, and its kinetic properties make such a possibility unlikely (Ref. 8Chen Z. Pickart C.M. J. Biol. Chem. 1990; 265: 21835-21842Abstract Full Text PDF PubMed Google Scholar, and see “Discussion”). Nonetheless, the unique ability of E2-25K in vitro to assemble chainsde novo from Ub, E1, and ATP makes possible the synthesis of large amounts of unanchored Lys48-linked poly-Ub (11van Nocker S. Vierstra R.D. J. Biol. Chem. 1993; 268: 24766-24773Abstract Full Text PDF PubMed Google Scholar, 12Chen Z.J. Niles E.G. Pickart C.M. J. Biol. Chem. 1991; 266: 15698-15704Abstract Full Text PDF PubMed Google Scholar, 13Piotrowski J. Beal R. Hoffman L. Wilkinson K.D. Cohen R.E. Pickart C.M. J. Biol. Chem. 1997; 272: 23712-23721Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). The products of such ubiquitination reactions have been extremely useful in studies of poly-Ub structure (14Cook W.J. Jeffrey L.C. Carson M. Chen Z. Pickart C.M. J. Biol. Chem. 1992; 267: 16467-16471Abstract Full Text PDF PubMed Google Scholar, 15Cook W.J. Jeffrey L.C. Kasperek E. Pickart C.M. J. Mol. Biol. 1994; 236: 601-609Crossref PubMed Scopus (130) Google Scholar), poly-Ub binding proteins (16Deveraux V. Pickart C. M. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, R.E. D. P. M. Pickart C.M. Biochemistry. 1998; 37: PubMed Scopus Google Scholar, Nocker S. M. Vierstra R.D. S. A. PubMed Scopus Google Scholar, Nocker S. Sadis S. M. D. Vierstra R.D. Mol. Cell. Biol. PubMed Scopus Google Scholar), recognition and by the 26 S proteasome J. Beal R. Hoffman L. Wilkinson K.D. Cohen R.E. Pickart C.M. J. Biol. Chem. 1997; 272: 23712-23721Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, Hoffman L. M. Pickart C.M. J. PubMed Scopus Google Scholar), and poly-Ub by deubiquitinating enzymes W. Cohen R.E. 1997; PubMed Scopus Google Scholar, Pickart C.M. Cohen R.E. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). During the of Lys48-linked poly-Ub, of tri-Ub and longer poly-Ub chains to the of the E2-25K-catalyzed observed as the of mono-Ub and di-Ub a new protein that by to have a between di-Ub and tri-Ub. of this protein showed that it was a cyclic by an isopeptide that the proximal and Ub of a linear tri-Ub cyclization products for and penta-Ub. gel with of poly-Ub chains (11van Nocker S. Vierstra R.D. J. Biol. Chem. 1993; 268: 24766-24773Abstract Full Text PDF PubMed Google Scholar, V. Pickart C. M. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, R.E. D. P. M. Pickart C.M. Biochemistry. 1998; 37: PubMed Scopus Google and the potential for from cyclic in to the of cyclic poly-Ub are as are to or cyclization in E2-25K-catalyzed the cyclic chains most deubiquitinating enzymes, an in a red blood cell was that can cyclic Lys48-linked tri-Ub and it to form Ub Ub, and from E2-25K and M.T. Xia G. Kasperek E.M. Pickart C.M. Biochemistry. 1997; 36: 10526-10537Crossref PubMed Scopus (123) Google Scholar), as the Ub and M.T. Xia G. Kasperek E.M. Pickart C.M. Biochemistry. 1997; 36: 10526-10537Crossref PubMed Scopus (123) Google Scholar, 13Piotrowski J. Beal R. Hoffman L. Wilkinson K.D. Cohen R.E. Pickart C.M. J. Biol. Chem. 1997; 272: 23712-23721Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). the form of E2-25K M.T. Xia G. Kasperek E.M. Pickart C.M. Biochemistry. 1997; 36: 10526-10537Crossref PubMed Scopus (123) Google Scholar), and enzyme from C. Pickart Ub on A.L. Wilkinson K.D. Biochem. PubMed Scopus Google was by Ub with in for and the product was by with a linear of in The bovine PA700 was by G. of and the Ub isopeptidase of the was in by W. W. of a by into the D. The of Scholar). bovine isopeptidase T was as Z.J. Niles E.G. Pickart C.M. J. Biol. Chem. 1991; 266: 15698-15704Abstract Full Text PDF PubMed Google Scholar). of E. from either and or was by M. of Lys48-linked poly-Ub chains to Chen and Pickart (8Chen Z. Pickart C.M. J. Biol. Chem. 1990; 265: 21835-21842Abstract Full Text PDF PubMed Google Scholar). chain synthesis E1, E2-25K or the or ATP, and of or For cyclization reactions that chains, the that poly-Ub chains added of of poly-Ub with deubiquitinating enzymes in and The products from a chain synthesis by with a linear of in chains of and by gel with as the of linear from cyclic poly-Ub was by on a with a linear of in and and by the of cell was from S. in with in by and by the cell for with of and The fraction was as the II from a bovine red blood cell was by chromatography as for II A. S. Ciechanover A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The by P. of of was through a of with The most of the and Ub. with the the fraction was with The was and was to R. R.E. in Scholar). polyacrylamide and proteins with species by through a polyacrylamide gel and to a was to with as in A.L. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and the of mass spectrometry a in linear a and with an of was for the and and as internal for mass The was from the of Ub and Lys48-linked di-Ub and The cyclic was by of the isopeptide in the di-Ub structure and of the Ub by the of new isopeptide The structure was by all bonds and Lys48 side chain The of a chain synthesis catalyzed by E2-25K and with Ub as the substrate was by with high mass poly-Ub chains and the of Ub the free Ub was a new species that migrated between di-Ub and tri-Ub was the of this new to as the linear tri-Ub we that was a or of tri-Ub. for we sequential to it from the poly-Ub product chains of by chromatography by gel The tri-Ub was to Although was not from linear tri-Ub by either or gel chromatography not was with a The and as linear tri-Ub and by observed that the of the new species the chain synthesis was to a of a this was with tri-Ub we that tri-Ub was converted to the new and that the on E1, E2-25K, and on the of E1 and E2-25K (8Chen Z. Pickart C.M. J. Biol. Chem. 1990; 265: 21835-21842Abstract Full Text PDF PubMed Google Scholar, A.L. A. J. Biol. Chem. Full Text PDF PubMed Google Scholar), the was that E2-25K catalyzed of an isopeptide in tri-Ub that the C terminus of the proximal Ub to the of the distal Ub. be a cyclic form of tri-Ub isopeptide was by of mass For linear tri-Ub and mass from we the and the of their was to have a mass that of linear tri-Ub mass was with the of one that from chain Ub, E1, E2-25K, and an ATP was and 8 added to and by polyacrylamide the a a new product a between di-Ub and tri-Ub. The of Ub and poly-Ub of are on the that tri-Ub and E2-25K tri-Ub is a of the product. tri-Ub was with of E1, E2-25K, and ATP, as and by as and of tri-Ub into was to E1, E2-25K, and ATP of linear and mass for a cyclic form of linear and by of mass The and of the are on the observed in mass for a cyclic form of tri-Ub. in a new linear and by of mass The and of the are on the observed in mass a mono-Ub is by K.D. J. Biol. Chem. Full Text PDF PubMed Google Scholar). was a cyclic form of a by the The product from linear tri-Ub in that and from the Ub di-Ub the proximal and distal of the we that a cyclic tri-Ub in this linear tri-Ub with respect to its upon that a of with a product that migrated the and with linear tri-Ub. the of linear it was by as by the of a new species that migrated the of mono-Ub and In that a of that linear tri-Ub is the cyclic form to by not with this we that the was a in the digestion reactions and for of the if the new in was an isopeptide between the proximal Ub and Lys48 of the distal Ub, we forms of and The tri-Ub is from by E1 by the of a C-terminal Ub J. Beal R. Hoffman L. Wilkinson K.D. Cohen R.E. Pickart C.M. J. Biol. Chem. 1997; 272: 23712-23721Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar), the chain is from as an acceptor for by E2-25K by the distal end M.T. Xia G. Kasperek E.M. Pickart C.M. Biochemistry. 1997; 36: 10526-10537Crossref PubMed Scopus (123) Google Scholar). of tri-Ub chains be converted by E2-25K into the species is with the that poly-Ub chains by E2-25K are linked exclusively through isopeptide bonds (8Chen Z. Pickart C.M. J. Biol. Chem. 1990; 265: 21835-21842Abstract Full Text PDF PubMed Google Scholar). For to the new in we of a form of Ub, in this Ub the primary is on the side chain of a Ub in a chain synthesis a species to was observed the products with the that E2-25K can a isopeptide into tri-Ub by Gly76 the proximal end to Lys48 the distal Thus, is the product of an Ub by of is a acceptor in E2-25K-catalyzed chain synthesis reactions C.M. M.T. Kasperek E.M. Chen Z. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). Although is activated by E1 and can be to form a thiol the of E2-25K, of of (poly)Ub the acceptor of an thiol Thus, E2-25K will to the distal not the proximal of a poly-Ub that if is not a competent acceptor in linear chain it will not be an acceptor in and Lys48-linked di-Ub with E2-25K in a chain synthesis In this poly-Ub product with an number of ubiquitins an its distal The that was into linear tri-Ub that was that chain cyclization by E2-25K the substrate acceptor as is in chain elongation. Cyclization of tri-Ub that tri-Ub has a structure that (i.e. the distal Ub and the thiol proximal to into the of However, it is possible that longer poly-Ub chains are flexible to binding for In to we linear Lys48-linked and as potential substrates for chains with E1 and E2-25K in the of In the of chains to the a to was observed by not However, a was by the new product that to this and linear it was longer to by isopeptidase T a deubiquitinating enzyme that requires a free proximal Ub for and see The is that the was by E2-25K, the with the the cyclic was not by cyclization by E2-25K of an longer substrate, linear to a protein as by that cyclization by E2-25K is not to that longer chains are to this In poly-Ub synthesis by E2-25K is For transfer of donor Ub to acceptor the enzyme of and a high for the acceptor substrate M.T. Xia G. Kasperek E.M. Pickart C.M. Biochemistry. 1997; 36: 10526-10537Crossref PubMed Scopus (123) Google Scholar). to this ubiquitination the cyclization of poly-Ub is in E1 was we that linear tri-Ub is converted to cyclic tri-Ub the of with linear a that is for Ub the to cyclic tri-Ub of the for di-Ub M.T. Xia G. Kasperek E.M. Pickart C.M. Biochemistry. 1997; 36: 10526-10537Crossref PubMed Scopus (123) Google Scholar). for the high of the E2-25K-catalyzed cyclization is that binding of the poly-Ub proximal end to the E2 donor the of the acceptor the distal Ub of the we that by substrates the E2-25K donor poly-Ub that the be to the acceptor The in this was as a donor substrate, cyclization of linear tri-Ub was in an acceptor for ubiquitination by E2-25K M.T. Xia G. Kasperek E.M. Pickart C.M. Biochemistry. 1997; 36: 10526-10537Crossref PubMed Scopus (123) Google Scholar), tri-Ub cyclization was to a the linear amounts of (i.e. and cyclic tri-Ub not to the cyclization with the of linear tri-Ub was is such a that tri-Ub has a that the cyclization However, we have observed that and are as as tri-Ub in the of “Discussion”). whether cyclic poly-Ub, if in vivo, be a dead-end product. Among the deubiquitinating enzymes isopeptidase T and the isopeptidase in the PA700 of the 26 S proteasome are to Lys48-linked poly-Ub, W. Cohen R.E. 1997; PubMed Scopus Google Scholar, Pickart C.M. Cohen R.E. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, K.D. Kasperek E. Pickart C.M. Biochemistry. PubMed Scopus Google Scholar). isopeptidase T the proximal Ub from an unanchored the PA700 isopeptidase Ub from the distal that enzymes Ub from a chain we not to that one cyclic tri-Ub 7 from the PA700 the isopeptidase of PA700 has a substrate in it can adducts from the proximal end of (poly)Ub. and R. in linear cyclic tri-Ub was by 7 are with the deubiquitinating enzymes, and free enzymes adducts from the proximal end of Ub or poly-Ub Wilkinson K.D. Biochemistry. PubMed Scopus Google Cohen R.E. J. 1999; PubMed Scopus Google Scholar). cyclic tri-Ub it be by E2-25K to form longer as an cyclic tri-Ub not as a donor in E2-25K-catalyzed chain synthesis 7 cyclic tri-Ub was not an acceptor for the transfer of are in with that all Lys48 and Gly76 residues in the cyclic are in isopeptide Thus, cyclic tri-Ub be as a donor for chain it has free C terminus for by E1 or of a thiol with an on the (8Chen Z. Pickart C.M. J. Biol. Chem. 1990; 265: 21835-21842Abstract Full Text PDF PubMed Google of E2-25K for transfer to Lys48 in cyclic tri-Ub was not to be an acceptor substrate with this by the possibility of cyclic poly-Ub a dead-end we for a cyclic poly-Ub that of the Ub by chain of the deubiquitinating enzymes from S. K.D. FASEB J. 1997; 11: PubMed Scopus Google of mammalian isopeptidase T A. S. Wilkinson K.D. M. J. 1997; PubMed Scopus Google in E. with as a substrate, all of the enzymes showed the and the in vitro the substrate L.C. Biochemistry. 1998; 37: PubMed Scopus Google Scholar). Among the and showed with However, each was with cyclic hydrolysis was was hydrolysis for with of a cell not Although it a E2 structure with the and enzymes, E2-25K a unique that is for its and has in S. Xia G. R.D. Pickart C.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). it possible that an isopeptidase to cyclic poly-Ub be in we II from bovine red blood as a potential of such an with cyclic tri-Ub was disassembled to Ub monomers was by a of Ub C.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar, A. S. A. PubMed Scopus Google Scholar, Cohen R.E. 1997; PubMed Google Scholar), that the observed hydrolysis was not to the of the the hydrolysis product was to be Ub as the of with cyclic was added to the hydrolysis and the was with E1, E2-25K, and an that the hydrolysis product be to the acceptor and di-Ub if the of cyclic tri-Ub Ub with an C 7 C that all of the hydrolysis product that migrated as mono-Ub in be converted by the enzymes to the of di-Ub Thus, have the ability to cyclic forms of have that E2-25K not can catalyze chain by Ub−Ub isopeptide that it can chains of or Ub by an isopeptide with the chain (8Chen Z. Pickart C.M. J. Biol. Chem. 1990; 265: 21835-21842Abstract Full Text PDF PubMed Google Scholar), cyclization is to the of Gly76 from a donor Ub to Lys48 of an acceptor Ub. by E2-25K been observed to as or upon (11van Nocker S. Vierstra R.D. J. Biol. Chem. 1993; 268: 24766-24773Abstract Full Text PDF PubMed Google Scholar, V. Pickart C. M. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, R.E. D. P. M. Pickart C.M. Biochemistry. 1998; 37: PubMed Scopus Google Scholar), the of this was that the of cyclic chains in poly-Ub product for the by In vitro synthesis by mammalian E2-25K or its homolog (8Chen Z. Pickart C.M. J. Biol. Chem. 1990; 265: 21835-21842Abstract Full Text PDF PubMed Google Scholar, 11van Nocker S. Vierstra R.D. J. Biol. Chem. 1993; 268: 24766-24773Abstract Full Text PDF PubMed Google Scholar, 13Piotrowski J. Beal R. Hoffman L. Wilkinson K.D. Cohen R.E. Pickart C.M. J. Biol. Chem. 1997; 272: 23712-23721Abstract Full Text Full Text PDF PubMed Scopus (191) Google is the available for the of unanchored Lys48-linked that chains made by E2-25K be with upon the of synthesis cyclic poly-Ub a of the it is to that is not a for the of cyclic chains longer Ub of the linear and cyclic forms is and is by in gel and electrophoresis for and for longer chains that have an number of the cyclic and linear forms not this is observed in the gel and mass chains and have each into 11van Nocker S. Vierstra R.D. J. Biol. Chem. 1993; 268: 24766-24773Abstract Full Text PDF PubMed Google Scholar). each to the linear and cyclic forms of a poly-Ub the forms is an number is is that the most by poly-Ub on whether or chains, the linear and cyclic forms be with respect to binding or that upon this is for the the is to a of the linear or cyclic of chains to be be useful to have an that can cyclic from linear poly-Ub a of For chains or can the cyclic and linear forms and However, the can be to with chain the cyclic and linear forms of chains longer are unlikely to be In a to cyclic from linear poly-Ub is by with cyclic chains are unanchored linear poly-Ub can be disassembled from the proximal or distal end by isopeptidase T or the PA700 see 7 that the to of a poly-Ub with either of can the of cyclic In we have it to of that poly-Ub chains not the for with chains (i.e. or that have a of chain can be to or cyclic products from in vitro poly-Ub synthesis by The on the that chains with or Ub the donor on E2-25K in for cyclization to mono-Ub efficiently for binding to this cyclization will be this was in 7 by the of tri-Ub Thus, for chain that Ub as the substrate, cyclic products can be to by the reactions the of its cyclization can be by the reactions to to of the Ub and di-Ub and to cyclization is by assembly of chains from poly-Ub to proximal or distal groups and E2-25K can catalyze the transfer from donor to acceptor to longer chains the substrates product can form the isopeptide for that the and of such poly-Ub chains, as as to the proximal or distal have been J. Beal R. Hoffman L. Wilkinson K.D. Cohen R.E. Pickart C.M. J. Biol. Chem. 1997; 272: 23712-23721Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). poly-Ub of and by cyclic species can be The ability to catalyze chain cyclization has for the and kinetic properties of E1 and poly-Ub synthesis is a that requires E2-25K to an activated (poly)Ub as the donor from E1, another (poly)Ub as the and catalyze isopeptide between the C-terminal carboxyl group of the donor and the Lys48 ε-amine of the of the thiol in the between the E2 and the was by the that a of E2-25K that the can form the thiol M.T. Xia G. Kasperek E.M. Pickart C.M. Biochemistry. 1997; 36: 10526-10537Crossref PubMed Scopus (123) Google Scholar). However, the ability to Ub or poly-Ub as an acceptor to form Lys48-linked chains is unique to E2-25K and requires the and the L.D. Kasperek E.M. Niles E.G. Pickart C.M. Biochemistry. 1998; 37: 9784-9792Crossref PubMed Scopus (13) Google M.T. Xia G. Kasperek E.M. Pickart C.M. Biochemistry. 1997; 36: 10526-10537Crossref PubMed Scopus (123) Google Scholar). the of E2-25K for acceptor Ub is M.T. Xia G. Kasperek E.M. Pickart C.M. Biochemistry. 1997; 36: 10526-10537Crossref PubMed Scopus (123) Google Scholar), a is the Ub in vivo A.L. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Thus, it was that in vivo E2-25K requires another to its physiological M.T. Xia G. Kasperek E.M. Pickart C.M. Biochemistry. 1997; 36: 10526-10537Crossref PubMed Scopus (123) Google Scholar). kinetic studies that the high for acceptor Ub the cyclization of poly-Ub of cyclization is another to that E2-25K in in with other is that a chain as as tri-Ub can a In to possible to tri-Ub we a of the cyclic the and with the of Ub and Lys48-linked di-Ub In the free was all and Lys48 side chain the of each Ub that with tri-Ub cyclization showed that cyclization with chains longer and it is that the of longer chains the In in we have observed that is by E2-25K a tri-Ub for the as with for chains we that the will as the distal end of the chain to the of the acceptor The of cyclic poly-Ub a for deubiquitinating large number of deubiquitinating enzymes have been in their is available for a of is their physiological substrates K.D. FASEB J. 1997; 11: PubMed Scopus Google Scholar). In deubiquitinating enzymes, including most enzymes Cohen R.E. J. 1999; PubMed Scopus Google Scholar, Wilkinson K.D. Biochemistry. 1998; 37: PubMed Scopus Google Scholar), adducts from the C terminus of such as S. C. J. 1998; PubMed Scopus Google and R. E. can chains from large each isopeptide Lys48-linked poly-Ub is on each side by the of a enzyme a large Ub, on of the the enzymes, the PA700 isopeptidase and isopeptidase from of a poly-Ub chain and as are not that isopeptidase has been that can act on poly-Ub in an to an isopeptide that is by or The of cyclic poly-Ub an as as a to for such an cyclic tri-Ub as a substrate, we have a in a bovine red blood cell extract. to be it is by a for deubiquitinating the hydrolysis product from cyclic tri-Ub is it is by E1 and E2-25K, and it is competent to as a donor Ub for on deubiquitinating enzymes, the poly-Ub be a by or a that can poly-Ub The and physiological of this new isopeptidase are for are to Pickart for and for for the of and for with the mass for bovine for bovine and for the E.