Rong Wang

Gamma-cleavage of beta-amyloid precursor protein (APP) in the middle of the cell membrane generates amyloid beta protein (Abeta), and epsilon-cleavage, approximately 10 residues downstream of the gamma-cleavage site, releases the APP intracellular domain (AICD). A significant link between generation of Abeta and AICD and failure to detect AICD41-99 led us to hypothesize that epsilon-cleavage generates longer Abetas, which are then processed to Abeta40/42. Using newly developed gel systems and an N-end-specific monoclonal antibody, we have identified the longer Abetas (Abeta1-43, Abeta1-45, Abeta1-46, and Abeta1-48) within the cells and in brain tissues. The production of these longer Abetas as well as Abeta40/42 is presenilin dependent and is suppressed by {1S-benzyl-4R-[1S-carbamoyl-2-phenylethylcarbamoyl-1S-3-methylbutylcarbamoyl]-2R-hydroxy-5-phenylpentyl}carbamic acid tert-butyl ester, a transition state analog inhibitor for aspartyl protease. In contrast, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, a potent dipeptide gamma-secretase inhibitor, builds up Abeta1-43 and Abeta1-46 intracellularly, which was also confirmed by mass spectrometry. Notably, suppression of Abeta40 appeared to lead to an increase in Abeta43, which in turn brings an increase in Abeta46, in a dose-dependent manner. We therefore propose an alpha-helical model in which longer Abeta species generated by epsilon-cleavage is cleaved at every three residues in its carboxyl portion.

Gamma-secretase is a member of an unusual class of proteases with intramembrane catalytic sites. This enzyme cleaves many type I membrane proteins, including the amyloid beta-protein (Abeta) precursor (APP) and the Notch receptor. Biochemical and genetic studies have identified four membrane proteins as components of gamma-secretase: heterodimeric presenilin (PS) composed of its N- and C-terminal fragments (PS-NTF/CTF), a mature glycosylated form of nicastrin (NCT), Aph-1, and Pen-2. Recent data from studies in Drosophila, mammalian, and yeast cells suggest that PS, NCT, Aph-1, and Pen-2 are necessary and sufficient to reconstitute gamma-secretase activity. However, many unresolved issues, in particular the possibility of other structural or regulatory components, would be resolved by actually purifying the enzyme. Here, we report a detailed, multistep purification procedure for active gamma-secretase and an initial characterization of the purified protease. Extensive mass spectrometry of the purified proteins strongly suggests that PS-NTF/CTF, mNCT, Aph-1, and Pen-2 are the components of active gamma-secretase. Using the purified gamma-secretase, we describe factors that modulate the production of specific Abeta species: (1) phosphatidylcholine and sphingomyelin dramatically improve activity without changing cleavage specificity within an APP substrate; (2) increasing CHAPSO concentrations from 0.1 to 0.25% yields a approximately 100% increase in Abeta42 production; (3) exposure of an APP-based recombinant substrate to 0.5% SDS modulates cleavage specificity from a disease-mimicking pattern (high Abeta42/43) to a physiological pattern (high Abeta40); and (4) sulindac sulfide directly and preferentially decreases Abeta42 cleavage within the purified complex. Taken together, our results define a procedure for purifying active gamma-secretase and suggest that the lipid-mediated conformation of both enzyme and substrate regulate the production of the potentially neurotoxic Abeta42 and Abeta43 peptides.

ChPrP is the chicken homologue of PrPC, the cellular isoform of the mammalian prion protein. We have used sequence-specific antibodies to immunoprecipitate and immunoblot chPrP derived from stably transfected cultures of neuroblastoma cells, as well as from chicken brain and cerebrospinal fluid. We have also used mass spectrometry to characterize fragments of the protein purified from conditioned medium. The majority of chPrP protein present in neuroblastoma cells and on isolated brain membranes can be released by incubation with phosphatidylinositol-specific phospholipase C, indicating that these molecules are attached to the cell surface by a glycosylphosphatidylinositol anchor. Surprisingly, most of the surface-anchored molecules are truncated at their N-terminus distal to the proline/glycine-rich repeats. The corresponding N-terminal fragments are found in medium conditioned by neuroblastoma cells, as well as in cerebrospinal fluid and a postmicrosomal supernatant of brain. One of these fragments extends from Lys25 to Phe116. 35-45-kDa forms of chPrP that can be metabolically labeled with [3H]ethanolamine can also be found in extracellular media. We propose that the chPrP molecule undergoes at least two cleavages as part of its normal metabolism: one within the glycosylphosphatidylinositol anchor and one within or just N-terminal to the central hydrophobic domain. The second cleavage lies within a region of 24 amino acids that is identical in chPrP and mammalian PrP, and represents a major processing event that may have physiological as well as pathological significance.

-ATPase (V-ATPase) by its specific inhibitor can abrogate tumor metastasis, prevent autophagy, and reduce cellular signaling responses. Bafilomycin A1, a member of macrolide antibiotics and an autophagy inhibitor, serves as a specific and potent V-ATPases inhibitor. Although there are many V-ATPase structures reported, the molecular basis of specific inhibitors on V-ATPase remains unknown. Here, we report the cryo-EM structure of bafilomycin A1 bound intact bovine V-ATPase at an overall resolution of 3.6-Å. The structure reveals six bafilomycin A1 molecules bound to the c-ring. One bafilomycin A1 molecule engages with two c subunits and disrupts the interactions between the c-ring and subunit a, thereby preventing proton translocation. Structural and sequence analyses demonstrate that the bafilomycin A1-binding residues are conserved in yeast and mammalian species and the 7'-hydroxyl group of bafilomycin A1 acts as a unique feature recognized by subunit c.

Nedd8 activates ubiquitination by increasing the efficiency of polyubiquitin chain assembly through its covalent conjugation to cullin molecules. Here we report the isolation, cloning, and characterization of a novel human Nedd8-specific protease called DEN1. Human DEN1 is encoded by AAH31411.1, a previously uncharacterized protein of 212 amino acids that shares homology with the Ulp1 cysteinyl SUMO deconjugating enzyme family. Recombinant human DEN1, purified from bacteria, selectively binds to Nedd8 and hydrolyzes C-terminal derivatives of Nedd8. Interestingly, DEN1 deconjugates cullin 1 (CUL1)-Nedd8 in a concentration-dependent manner. At a low concentration, DEN1 processes hyper-neddylated CUL1 to yield a mononeddylated form, which presumably contains the Lys-720CUL1-Nedd8 linkage. At elevated concentrations, DEN1 is able to complete the removal of Nedd8 from CUL1. These activities distinguish DEN1 from the COP9 signalosome, which is capable of efficiently cleaving the Lys-720CUL1-Nedd8 conjugate, but lacks Nedd8 C-terminal hydrolytic activity and poorly processes hyperneddylated CUL1. These results suggest a DEN1 in the of by Nedd8. Nedd8 activates ubiquitination by increasing the efficiency of polyubiquitin chain assembly through its covalent conjugation to cullin molecules. Here we report the isolation, cloning, and characterization of a novel human Nedd8-specific protease called DEN1. Human DEN1 is encoded by AAH31411.1, a previously uncharacterized protein of 212 amino acids that shares homology with the Ulp1 cysteinyl SUMO deconjugating enzyme family. Recombinant human DEN1, purified from bacteria, selectively binds to Nedd8 and hydrolyzes C-terminal derivatives of Nedd8. Interestingly, DEN1 deconjugates cullin 1 (CUL1)-Nedd8 in a concentration-dependent manner. At a low concentration, DEN1 processes hyper-neddylated CUL1 to yield a mononeddylated form, which presumably contains the Lys-720CUL1-Nedd8 linkage. At elevated concentrations, DEN1 is able to complete the removal of Nedd8 from CUL1. These activities distinguish DEN1 from the COP9 signalosome, which is capable of efficiently cleaving the Lys-720CUL1-Nedd8 conjugate, but lacks Nedd8 C-terminal hydrolytic activity and poorly processes hyperneddylated CUL1. These results suggest a DEN1 in the of by Nedd8. Nedd8 is a cullin COP9 cullin COP9 protein that a in and Nedd8 is Nedd8 is of the Nedd8 in results in of Nedd8 is by its activity a protein to of the cullin is of the ubiquitination by the of the of a the of a cullin to the of Nedd8 enzyme of the a Nedd8-specific enzyme of the enzyme and the protein in that Nedd8 activates the ubiquitination of through its conjugation to cullin 1 These by in which CUL1 a that of by Nedd8 Nedd8 to that the protein in These suggest a Nedd8 in the assembly of that conjugation of Nedd8 to CUL1 the of a the to polyubiquitin in a by the enzyme we that the of Nedd8 in polyubiquitin chain assembly is its that Nedd8 to the of with that of cullin in polyubiquitin and Nedd8 conjugation to the and the that the covalent of Nedd8 CUL1 Nedd8 of a is to These a that Nedd8 activates ubiquitination by increasing the of the assembly of polyubiquitin suggest a of in CUL1 and called from and a COP9 in the of Nedd8 from CUL1. a of in a that the Nedd8 activity is the the of that a activities from that Nedd8 from we purified a novel cysteinyl protease we called DEN1 DEN1 selectively binds efficiently processes the of and deconjugates hyper-neddylated CUL1. These suggest a DEN1 in the Nedd8 of DEN1 and protease from its to by and of previously and 1 and to through with the protein by in of the of with a and protein by the by through a and a protein with and of to 1 and the a of in and 1 1 and to through a with a of in of and a by protein a DEN1, the from the and by by and previously with the to purified from that and a previously of and the purified a the to the protein with in 1 and a of and by from a human from of the a protease and the the the and by DEN1 a protein in the and and previously that and from the previously with that protease DEN1, with of and by the through and DEN1 purified by protein the of DEN1 of DEN1 the with that with purified of protein of with by with by a the and the Nedd8 from the and the Nedd8 in the through to with a by from a human the and a the the by with and and a protein in and purified the protein by with by with CUL1 of and and previously purified through and Nedd8 in a that of and of to a to of and of of the the to and the protein by in 1 and activity of DEN1 with CUL1. DEN1 DEN1 in a and concentration-dependent manner. DEN1 in a concentration-dependent manner. and the and of with the of to efficiently protein and by and hyper-neddylated purified in a and Nedd8 the and the with in 1 and by and hyper-neddylated in deconjugates hyper-neddylated CUL1. Nedd8 1 of hyper-neddylated and DEN1 in by and by a of 1 of hyper-neddylated of by and and that with and by with and with a that the by the by and in and purified the 1 purified in and DEN1 by and by 1 and DEN1 in by and by of a Human Nedd8 protease activities capable of deconjugating Nedd8 from a protein we a a Nedd8 that to a CUL1 by the Nedd8 to the purified of CUL1 C-terminal results with CUL1 by the purified the and a of with that in a of with Nedd8 the conjugation of Nedd8 to is by the assembly of a chain to by the conjugation of Nedd8 to and CUL1 C-terminal of DEN1. of DEN1 by of by the and the with DEN1 and the by and by of by 1 contains of purified DEN1 in the we and purified a Nedd8 protease activity from of that we DEN1 of the with a purified DEN1 in of the of and of the Nedd8 1 and of by DEN1 of the low of enzyme in the of the DEN1 by a of Nedd8 activity to that DEN1 is a its the in the DEN1 activity by and by results that the protein of a of that to 1 and a previously uncharacterized and the a protein that shares a homology with the Ulp1 of the a of that with the DEN1 activity These with the of Nedd8 protease activities by the protein that DEN1 is encoded by by DEN1 contains a the a of cysteinyl protease and the of the of DEN1 in and the of which and amino with the human suggest that DEN1 is cysteinyl Nedd8 and the of Nedd8 DEN1 with Nedd8. DEN1 and purified a protein from with by of to the protein from the through and by results that but a Nedd8 we Nedd8 with to with by that of the Nedd8 of DEN1 binds Nedd8 with Nedd8. but binds Nedd8. with increasing of with a and and with by the with and of the by to 1 of the DEN1 with Nedd8 but with with of and and and the is in and previously by in a to that the of we the of DEN1 to the of Nedd8. Nedd8 is a which is the form, by a C-terminal hydrolytic the Nedd8 is to cullin which to the of the Nedd8 C-terminal hydrolytic the DEN1 with a Nedd8 protein that the by DEN1 the of a of the to the Nedd8 1 of 1 and of hydrolytic activity of results we that DEN1 hydrolyzes C-terminal derivatives of Nedd8 is a Nedd8 C-terminal DEN1 but the Nedd8 in a and concentration-dependent manner. contains of the Nedd8. and purified DEN1 and purified by and that contains we purified the from that to and C-terminal hydrolytic activity with yield of which by of enzyme results with is of of the that from in of the activity of DEN1 by the enzyme with the which the DEN1 the to the 1 and and of the enzyme in to the the by DEN1 of the of the CUL1 C-terminal to the CUL1 the the in a in of concentrations, its to the C-terminal derivatives of Nedd8 and DEN1 the and results suggest that the DEN1 the Lys-720CUL1-Nedd8 the purified the of Nedd8 from CUL1 with low These the efficiency with which the Lys-720CUL1-Nedd8 the DEN1, in able to that protease Nedd8 that through we a hyper-neddylated of CUL1. and that hyperneddylated CUL1 and low of and the of DEN1 a of the the 1 and the to to a of the increasing the of the CUL1 DEN1 the CUL1 the and These results that low concentrations, DEN1 efficiently hyper-neddylated CUL1 to yield the the to to that with the efficiently hyper-neddylated CUL1 that in the of to of the CUL1 hyper-neddylated CUL1 to a to in deconjugating hyper-neddylated CUL1. of DEN1 by of the of DEN1 in the of in the of from protein a in and in and the of the Here we report the isolation, cloning, and characterization of DEN1, a previously uncharacterized protease capable of the of Nedd8 and deconjugating hyper-neddylated to the protease of cysteinyl of the chain of and the the that is by and the of the that the of deconjugating contains a to the C-terminal of DEN1 is by its which a with Nedd8 and the and of the Nedd8 C-terminal we DEN1 Nedd8 and we that DEN1 binds Nedd8 with a of the DEN1 and a of to in the of and the of the of Ulp1 to the DEN1 with its in we suggest that the of DEN1 is its to Nedd8. DEN1 efficiently the Nedd8 to of the C-terminal from we DEN1 to a Nedd8 that is to presumably the Nedd8 is to which DEN1 the that DEN1 able to the CUL1 the elevated the of DEN1 to the Nedd8 the of the Nedd8 hyperneddylated CUL1 to that by the assembly of and from the conjugation of Nedd8 to and CUL1 DEN1 a Nedd8 that is the of a Nedd8 chain is to a CUL1 DEN1 efficiently the and the CUL1 we that the of a Nedd8 to by DEN1 the of the Nedd8 with the to Nedd8 C-terminal hydrolytic activity the Lys-720CUL1-Nedd8 of that is the of and we that the the the protease and a that the cullin Nedd8 by is the of a by from the CUL1 the that and by of the cullin the Nedd8 in a that is of the and CUL1 in in and the in we These of and DEN1 to efficiently hyper-neddylated the results from DEN1 a in the Nedd8 its Nedd8 C-terminal hydrolytic DEN1 in the of the of its conjugation to DEN1 the of in a concentration-dependent manner. At a low concentration, protease Nedd8 that to the cullin in a DEN1 that of At elevated concentrations, DEN1 complete the removal of These activities in of the that efficiently hyper-neddylated CUL1 of with Nedd8 is by the of that the DEN1 is in the of derivatives of Nedd8. C-terminal hydrolytic activity of DEN1 to Nedd8 by in a to the of we that DEN1 Nedd8 from in protease a in the of the Nedd8 is a cullin COP9 cullin COP9 protein that a in and Nedd8 is Nedd8 is of the Nedd8 in results in of Nedd8 is by its activity a protein to of the cullin is of the ubiquitination by the of the of a the of a cullin to the of Nedd8 enzyme of the a Nedd8-specific enzyme of the enzyme and the protein in that Nedd8 activates the ubiquitination of through its conjugation to cullin 1 These by in which CUL1 a that of by Nedd8 Nedd8 to that the protein in These suggest a Nedd8 in the assembly of that conjugation of Nedd8 to CUL1 the of a the to polyubiquitin in a by the enzyme we that the of Nedd8 in polyubiquitin chain assembly is its that Nedd8 to the of with that of cullin in polyubiquitin and Nedd8 conjugation to the and the that the covalent of Nedd8 CUL1 Nedd8 of a is to These a that Nedd8 activates ubiquitination by increasing the of the assembly of polyubiquitin suggest a of in CUL1 and called from and a COP9 in the of Nedd8 from CUL1. a of in a that the Nedd8 activity is the the of that a activities from that Nedd8 from we purified a novel cysteinyl protease we called DEN1 DEN1 selectively binds efficiently processes the of and deconjugates hyper-neddylated CUL1. These suggest a DEN1 in the Nedd8 of DEN1 and protease from its to by and of previously and 1 and to through with the protein by in of the of with a and protein by the by through a and a protein with and of to 1 and the a of in and 1 1 and to through a with a of in of and a by protein a DEN1, the from the and by by and previously with the to purified from that and a previously of and the purified a the to the protein with in 1 and a of and by from a human from of the a protease and the the the and by DEN1 a protein in the and and previously that and from the previously with that protease DEN1, with of and by the through and DEN1 purified by protein the of DEN1 of DEN1 the with that with purified of protein of with by with by a the and the Nedd8 from the and the Nedd8 in the through to with a by from a human the and a the the by with and and a protein in and purified the protein by with by with CUL1 of and and previously purified through and Nedd8 in a that of and of to a to of and of of the the to and the protein by in 1 and hyper-neddylated purified in a and Nedd8 the and the with in 1 and by and hyper-neddylated in deconjugates hyper-neddylated CUL1. Nedd8 1 of hyper-neddylated and DEN1 in by and by a of 1 of hyper-neddylated of by and and that with and by with and with a that the by the by and in and purified the 1 purified in and DEN1 by and by 1 and DEN1 in by and by of DEN1 and protease from its to by and of previously and 1 and to through with the protein by in of the of with a and protein by the by through a and a protein with and of to 1 and the a of in and 1 1 and to through a with a of in of and a by protein a DEN1, the from the and by by and previously with the to purified from that and a previously of and the purified a the to the protein with in 1 and a of protease from its to by and of previously and 1 and to through with the protein by in of the of with a and protein by the by through a and a protein with and of to 1 and the a of in and 1 1 and to through a with a of in of and a by protein a DEN1, the from the and by by and previously with the to purified from that and a previously of and the purified a the to the protein with in 1 and a of DEN1 and by from a human from of the a protease and the the the and by DEN1 a protein in the and and previously that and from the previously with that protease DEN1, with of and by the through and DEN1 purified by protein the of DEN1 of DEN1 the with that with purified of protein of with by with DEN1 by from a human from of the a protease and the the the and by Recombinant DEN1 a protein in the and and previously that and from the previously with that protease DEN1, with of and by the through and DEN1 purified by protein the of DEN1 of DEN1 the with that with purified of protein of with by with by a the and the Nedd8 from the and the Nedd8 in the through to with a by from a human the and a the the by with and and a protein in and purified the protein by with by with CUL1 of and and previously purified through and Nedd8 in a that of and of to a to of and of of the the to and the protein by in 1 and hyper-neddylated purified in a and Nedd8 the and the with in 1 and by and hyper-neddylated in by a the and the Nedd8 from the and the Nedd8 in the through to with a by from a human the and a the the by with and and a protein in and purified the protein by with by with CUL1 of and and previously purified through and Nedd8 in a that of and of to a to of and of of the the to and the protein by in 1 and hyper-neddylated purified in a and Nedd8 the and the with in 1 and by and hyper-neddylated in of by and and that with and by with and with a that the by the by and in and purified the by and and that with and by with and with a that the by the by and in and purified the Nedd8 1 purified in and DEN1 by and by 1 and DEN1 in by and by 1 purified in and DEN1 by and by Nedd8 1 and DEN1 in by and by of a Human Nedd8 protease activities capable of deconjugating Nedd8 from a protein we a a Nedd8 that to a CUL1 by the Nedd8 to the purified of CUL1 C-terminal results with CUL1 by the purified the and a of with that in a of with Nedd8 the conjugation of Nedd8 to is by the assembly of a chain to by the conjugation of Nedd8 to and CUL1 C-terminal the we and purified a Nedd8 protease activity from of that we DEN1 of the with a purified DEN1 in of the of and of the Nedd8 1 and of by DEN1 of the low of enzyme in the of the DEN1 by a of Nedd8 activity to that DEN1 is a its the in the DEN1 activity by and by results that the protein of a of that to 1 and a previously uncharacterized and the a protein that shares a homology with the Ulp1 of the a of that with the DEN1 activity These with the of Nedd8 protease activities by the protein that DEN1 is encoded by by DEN1 contains a the a of cysteinyl protease and the of the of DEN1 in and the of which and amino with the human suggest that DEN1 is cysteinyl Nedd8 and the of Nedd8 DEN1 with Nedd8. DEN1 and purified a protein from with by of to the protein from the through and by results that but a Nedd8 we Nedd8 with to with by that of the Nedd8 of DEN1 binds Nedd8 with Nedd8. but binds Nedd8. with increasing of with a and and with by the with and of the by to 1 of the DEN1 with Nedd8 but with with of and and and the is in and previously by in a to that the of we the of DEN1 to the of Nedd8. Nedd8 is a which is the form, by a C-terminal hydrolytic the Nedd8 is to cullin which to the of the Nedd8 C-terminal hydrolytic the DEN1 with a Nedd8 protein that the by DEN1 the of a of the to the Nedd8 1 of 1 and of hydrolytic activity of results we that DEN1 hydrolyzes C-terminal derivatives of Nedd8 is a Nedd8 C-terminal DEN1 but the Nedd8 in a and concentration-dependent manner. contains of the Nedd8. and purified DEN1 and purified by and that contains we purified the from that to and C-terminal hydrolytic activity with yield of which by of enzyme results with is of of the that from in of the activity of DEN1 by the enzyme with the which the DEN1 the to the 1 and and of the enzyme in to the the by DEN1 of the of the CUL1 C-terminal to the CUL1 the the in a in of concentrations, its to the C-terminal derivatives of Nedd8 and DEN1 the and results suggest that the DEN1 the Lys-720CUL1-Nedd8 the purified the of Nedd8 from CUL1 with low These the efficiency with which the Lys-720CUL1-Nedd8 the DEN1, in able to that protease Nedd8 that through we a hyper-neddylated of CUL1. and that hyperneddylated CUL1 and low of and the of DEN1 a of the the 1 and the to to a of the increasing the of the CUL1 DEN1 the CUL1 the and These results that low concentrations, DEN1 efficiently hyper-neddylated CUL1 to yield the the to to that with the efficiently hyper-neddylated CUL1 that in the of to of the CUL1 hyper-neddylated CUL1 to a to in deconjugating hyper-neddylated CUL1. of DEN1 by of the of DEN1 in the of in the of a Human Nedd8 protease activities capable of deconjugating Nedd8 from a protein we a a Nedd8 that to a CUL1 by the Nedd8 to the purified of CUL1 C-terminal results with CUL1 by the purified the and a of with that in a of with Nedd8 the conjugation of Nedd8 to is by the assembly of a chain to by the conjugation of Nedd8 to and CUL1 C-terminal the we and purified a Nedd8 protease activity from of that we DEN1 of the with a purified DEN1 in of the of and of the Nedd8 1 and of by DEN1 of the low of enzyme in the of the DEN1 by a of Nedd8 activity to that DEN1 is a its the in the DEN1 activity by and by results that the protein of a of that to 1 and a previously uncharacterized and the a protein that shares a homology with the Ulp1 of the a of that with the DEN1 activity These with the of Nedd8 protease activities by the protein that DEN1 is encoded by by DEN1 contains a the a of cysteinyl protease and the of the of DEN1 in and the of which and amino with the human suggest that DEN1 is cysteinyl DEN1 Nedd8 and the of Nedd8 DEN1 with Nedd8. DEN1 and purified a protein from with by of to the protein from the through and by results that but a Nedd8 we Nedd8 with to with by that of the Nedd8 of DEN1 binds Nedd8 we the of DEN1 to the of Nedd8. Nedd8 is a which is the form, by a C-terminal hydrolytic the Nedd8 is to cullin which to the of the Nedd8 C-terminal hydrolytic the DEN1 with a Nedd8 protein that the by DEN1 the of a of the to the Nedd8 1 of 1 and of hydrolytic activity of results we that DEN1 hydrolyzes C-terminal derivatives of Nedd8 that contains we purified the from that to and C-terminal hydrolytic activity with yield of which by of enzyme results with is of of the that from in DEN1 of the activity of DEN1 by the enzyme with the which the DEN1 the to the 1 and and of the enzyme in to the the by DEN1 of the of the CUL1 C-terminal to the CUL1 the the in a in of concentrations, its to the C-terminal derivatives of Nedd8 and DEN1 the and results suggest that the DEN1 the Lys-720CUL1-Nedd8 the purified the of Nedd8 from CUL1 with low These the efficiency with which the Lys-720CUL1-Nedd8 the DEN1, in able to that protease Nedd8 that through we a hyper-neddylated of CUL1. and that hyperneddylated CUL1 and low of and the of DEN1 a of the the 1 and the to to a of the increasing the of the CUL1 DEN1 the CUL1 the and These results that low concentrations, DEN1 efficiently hyper-neddylated CUL1 to yield the the to to that with the efficiently hyper-neddylated CUL1 that in the of to of the CUL1 hyper-neddylated CUL1 to a to in deconjugating hyper-neddylated CUL1. of DEN1 by of the of DEN1 in the of in the of from protein a in and in and the of the Here we report the isolation, cloning, and characterization of DEN1, a previously uncharacterized protease capable of the of Nedd8 and deconjugating hyper-neddylated to the protease of cysteinyl of the chain of and the the that is by and the of the that the of deconjugating contains a to the C-terminal of DEN1 is by its which a with Nedd8 and the and of the Nedd8 C-terminal we DEN1 Nedd8 and we that DEN1 binds Nedd8 with a of the DEN1 and a of to in the of and the of the of Ulp1 to the DEN1 with its in we suggest that the of DEN1 is its to Nedd8. DEN1 efficiently the Nedd8 to of the C-terminal from we DEN1 to a Nedd8 that is to presumably the Nedd8 is to which DEN1 the that DEN1 able to the CUL1 the elevated the of DEN1 to the Nedd8 the of the Nedd8 hyperneddylated CUL1 to that by the assembly of and from the conjugation of Nedd8 to and CUL1 DEN1 a Nedd8 that is the of a Nedd8 chain is to a CUL1 DEN1 efficiently the and the CUL1 we that the of a Nedd8 to by DEN1 the of the Nedd8 with the to Nedd8 C-terminal hydrolytic activity the Lys-720CUL1-Nedd8 of that is the of and we that the the the protease and a that the cullin Nedd8 by is the of a by from the CUL1 the that and by of the cullin the Nedd8 in a that is of the and CUL1 in in and the in we These of and DEN1 to efficiently hyper-neddylated the results from DEN1 a in the Nedd8 its Nedd8 C-terminal hydrolytic DEN1 in the of the of its conjugation to DEN1 the of in a concentration-dependent manner. At a low concentration, protease Nedd8 that to the cullin in a DEN1 that of At elevated concentrations, DEN1 complete the removal of These activities in of the that efficiently hyper-neddylated CUL1 of with Nedd8 is by the of that the DEN1 is in the of derivatives of Nedd8. C-terminal hydrolytic activity of DEN1 to Nedd8 by in a to the of we that DEN1 Nedd8 from in protease a in the of the of from protein a in and in and the of the Here we report the isolation, cloning, and characterization of DEN1, a previously uncharacterized protease capable of the of Nedd8 and deconjugating hyper-neddylated CUL1. DEN1 to the protease of cysteinyl of the chain of and the the that is by and the of the that the of deconjugating contains a to the C-terminal of DEN1 is by its which a with Nedd8 and the and of the Nedd8 C-terminal we DEN1 Nedd8 and we that DEN1 binds Nedd8 with a of the DEN1 and a of to in the of and the of the of Ulp1 to the DEN1 with Nedd8. its in we suggest that the of DEN1 is its to Nedd8. DEN1 efficiently the Nedd8 to of the C-terminal from we DEN1 to a Nedd8 that is to presumably the Nedd8 is to which DEN1 the that DEN1 able to the CUL1 the elevated the of DEN1 to the Nedd8 the of the Nedd8 hyperneddylated CUL1 to that by the assembly of and from the conjugation of Nedd8 to and CUL1 DEN1 a Nedd8 that is the of a Nedd8 chain is to a CUL1 DEN1 efficiently the and the CUL1 we that the of a Nedd8 to by DEN1 the of the Nedd8 with the to Nedd8 C-terminal hydrolytic activity the Lys-720CUL1-Nedd8 of that is the of and we that the the the protease and a that the cullin Nedd8 by is the of a by from the CUL1 the that and by of the cullin the Nedd8 in a that is of the and CUL1 in in and the in we These of and DEN1 to efficiently hyper-neddylated CUL1. the results from DEN1 a in the Nedd8 its Nedd8 C-terminal hydrolytic DEN1 in the of the of its conjugation to DEN1 the of in a concentration-dependent manner. At a low concentration, protease Nedd8 that to the cullin in a DEN1 that of At elevated concentrations, DEN1 complete the removal of These activities in of the that efficiently hyper-neddylated CUL1 of with Nedd8 is by the of that the DEN1 is in the of derivatives of Nedd8. C-terminal hydrolytic activity of DEN1 to Nedd8 by in a to the of we that DEN1 Nedd8 from in protease a in the of the and and of the