The ubiquitin-like protein NEDD8 is essential for activity of SCF-like ubiquitin ligase complexes. Here we identify and characterize NEDP1, a human NEDD8-specific protease. NEDP1 is highly conserved throughout evolution and equivalent proteins are present in yeast, plants, insects, and mammals. Bacterially expressed NEDP1 is capable of processing NEDD8 in vitro to expose the diglycine motif required for conjugation and can deconjugate NEDD8 from modified substrates. NEDP1 appears to be specific for NEDD8 as neither ubiquitin nor SUMO bearing COOH-terminal extensions are utilized as substrates. Inhibition studies and mutagenesis indicate that NEDP1 is a cysteine protease with sequence similarities to SUMO-specific proteases and the class of viral proteases typified by the adenovirus protease. In vivo NEDP1 deconjugates NEDD8 from a wide variety of substrates including the cullin component of SCF-like complexes. Thus NEDP1 is likely to play an important role in ubiquitin-mediated proteolysis by controlling the activity of SCF complexes.Ubiquitin and ubiquitin-like proteins are conjugated to acceptor lysine residues on target proteins and have diverse effects on the modified proteins. Whereas conjugation of multiple copies of ubiquitin targets proteins for degradation via the proteasome, addition of SUMO or NEDD8 can alter the function of the conjugated protein (1). Formation of the isopeptide bond between the COOH-terminal glycine of the Ulp and the ⑀-amino group of lysine in the modified protein is accomplished by an enzymatic cascade that typically involves three enzymes, E1 (activating enzyme), E2 (conjugating enzyme), and E3 (ligase). In the case of NEDD8, or its yeast equivalent Rub1, the ubiquitin-like protein is activated by a heterodimeric complex of APP-BP1 and Uba3, and is conjugated to substrates by the conjugating enzyme Ubc12 (2, 3). Although an E3 ligase specific for NEDD8 has yet to be identified, the parallels with the ubiquitin and SUMO systems indicate that it is likely such an activity will exist. To date the only targets for NEDD8 modification that have been described are members of the Cullin family of proteins (2-8). Cullins are important components of multiple ubiquitin ligase complexes that also contain Rbx1, Skp1 (or homologue), and a substrate receptor protein that contains an F-box motif. These SCF-like complexes are responsible for the ubiquitination of proteins such as phosphorylated IB␣ and hydroxylated HIF1␣. Genetic experiments in yeast and plants indicate that Rub1 (NEDD8) modification is important for SCF ubiquitin ligase activity (3, 9 -11), whereas biochemical experiments demonstrated that NEDD8 modification of Cul-1 was responsible for recruitment of the Ubc4-ubiquitin thioester to the SCF complex (12). It has been demonstrated that the Rbx1 component of SCF complexes activates Ubc12-mediated NEDD8 modification of Cdc53 and Cul-2 (5). Whereas a complete Rub1 (NEDD8) modification pathway is not required for the viability of Saccharomyces cerevisiae (3, 9) it is required for viab...

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Apoptosis as a form of programmed cell death (PCD) in multicellular organisms is a well-established genetically controlled process that leads to elimination of unnecessary or damaged cells. Recently, PCD has also been described for unicellular organisms as a process for the socially advantageous regulation of cell survival. The human Bcl-2 family member Bak induces apoptosis in mammalian cells which is counteracted by the Bcl-x L protein. We show that Bak also kills the unicellular fission yeast Schizosaccharomyces pombe and that this is inhibited by coexpression of human Bcl-x L . Moreover, the same critical BH3 domain of Bak that is required for induction of apoptosis in mammalian cells is also required for inducing death in yeast. This suggests that Bak kills mammalian and yeast cells by similar mechanisms. The phenotype of the Bak-induced death in yeast involves condensation and fragmentation of the chromatin as well as dissolution of the nuclear envelope, all of which are features of mammalian apoptosis. These data suggest that the evolutionarily conserved metazoan PCD pathway is also present in unicellular yeast.Programmed cell death (PCD) in metazoans is an essential homeostatic mechanism permitting the removal of surplus cells during morphogenesis and tissue maintenance and the deletion of cells that present a risk to the organism because they are mutated or infected (10,19,35,36,40). For vertebrates, the descriptive name commonly given to the process of PCD is apoptosis. Classical apoptosis is characterized by membrane blebbing, cell shrinkage, chromatin condensation, and nuclear and cellular fragmentation, and it results from the activation of an intrinsic suicide program (47). Recent studies implicate the dysregulation of PCD in the pathophysiology of several human diseases, including AIDS (12, 28), neurodegenerative disease (25,37,44), and cancer (for a review, see reference 42).The basal machinery responsible for metazoan PCD is highly evolutionarily conserved and, at its execution level, involves the action of a discrete class of cysteine proteases, of which the prototypes are the interleukin-1␤-converting enzyme in humans and Ced-3 in the nematode (49). Also conserved are key regulators of apoptosis: in Caenorhabditis elegans the Ced-9 protein and in humans the Bcl-2 protein family (18, 46), which comprises both suppressors (e.g., Bcl-2 and Bcl-x L ) and promoters (e.g., Bax and Bak) of PCD (17,30,46).Recently, there have been several reports describing apparent PCD in the unicellular eukaryotes Tetrahymena thermophila, Dictyostelium discoideum, Trypanosoma brucei rhodsiense, and Trypanosoma cruzi and even in bacteria (2, 6, 45, 48; for an overview, see reference 1). PCD in unicellular organisms might facilitate constant selection for the fittest cell in the colony or optimal adaptation of cell numbers to the environment or might serve as a means for altruistic cell death to prevent the spread of virus in the event of infection.It has been shown that expression of the mammalian Bax protein in th...

Several recombinant cowpox viruses were constructed and used to identify a viral gene that controls the production of hemorrhage in lesions caused by the Brighton Red strain of cowpox virus (CPV-BR). This gene is located in the KpnD fragment of CPV-BR DNA, between 31 and 32 kilobases from the end of the genome. This position corresponds well with that predicted from analyses of the DNA structures of spontaneously generated deletion mutants. The gene responsible for hemorrhage encodes a 38-kDa protein that is one of the most abundant early gene products. The 11-basepair sequence GAAAATATATT present 84 base pairs upstream of its coding region is also present upstream of three other early genes of vaccinia virus; therefore, this sequence may be involved in the regulation of transcription. There is extensive similarity between the predicted amino acid sequence of the 38-kDa protein and the amino acid sequences of several plasma proteins that are inhibitors of various serine proteases involved in blood coagulation pathways. This suggests that the viral protein may possess a similar biological activity, which may enable it to effect hemorrhage by inhibiting one or more of the serine proteases involved in the host's normal processes of blood coagulation and wound containment.The inoculation of cowpox virus (CPV) into the skin of various mammals (guinea pigs, rabbits, humans) results in lesions that exhibit edema, hypertrophy ofthe epidermis, and hemorrhage. Similar effects are produced in CPV lesions (pocks) in the chorioallantoic membrane of developing chicken embryos; there is extensive proliferation of ectodermal and mesodermal cells, edema, and localized hemorrhage that gives the pocks a deep red color (1).CPV variants that do not produce hemorrhage have been isolated from the white pocks that usually comprise up to 1% of the pocks present on chorioallantoic membranes infected with wild-type CPV (2, 3). The existence of these white-pock variants demonstrates that a viral function controls the production of the hemorrhage.Studies of the structures of the DNAs of these white-pock variants have shown that their genomes are generated from the genome of the wild-type virus by large deletions and duplications of sequences (4, 5). Similar results have been obtained from studies of the DNAs of white-pock variants of rabbitpox virus and monkeypox virus (6-8). The rearrangements of DNA that generate the white-pock variants of CPV typically involve the replacement of up to 39 kilobases (kb) of one end of the genome with an inverted copy of up to 50 kb of DNA from the other end (4, 5). The mechanisms by which these rearrangements proceed are unknown; but it is clear that there are no identifiable homologous sequence elements at the sites of the duplication/deletion end points (5). This result suggests that it is the location of essential genes rather than DNA sequence content that limits where rearrangements may occur. The position ofgenes that encode markers, such as production of hemorrhage, will place a further cons...

The small ubiquitin-like modifier (SUMO)-1 is an important posttranslational regulator of different signaling pathways and involved in the formation of promyelocytic leukemia (PML) protein nuclear bodies (NBs). Overexpression of SUMO-1 has been associated with alterations in apoptosis, but the underlying mechanisms and their relevance for human diseases are not clear. Here, we show that the increased expression of SUMO-1 in rheumatoid arthritis (RA) synovial fibroblasts (SFs) contributes to the resistance of these cells against Fas-induced apoptosis through increased SUMOylation of nuclear PML protein and increased recruitment of the transcriptional repressor DAXX to PML NBs. We also show that the nuclear SUMO-protease SENP1, which is found at lower levels in RA SFs, can revert the apoptosis-inhibiting effects of SUMO-1 by releasing DAXX from PML NBs. Our findings indicate that in RA SFs overexpression of SENP1 can alter the SUMO-1-mediated recruitment of DAXX to PML NBs, thus influencing the proapoptotic effects of DAXX. Accumulation of DAXX in PML NBs by SUMO-1 may, therefore, contribute to the pathogenesis of inflammatory disorders.inflammation ͉ autoimmunity ͉ DAXX ͉ SENP

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Examination of the genomes of 10 white-pock variants of cowpox virus strain Brighton red (CPV-BR) revealed that 9 of them had lost 32 to 38 kilobase pairs (kbp) from their right-hand ends and that the deleted sequences had been replaced by inverted copies of regions from 21 to 50 kbp long from the left-hand end of the genome. These variants thus possess inverted terminal repeats (ITRs) from 21 to 50 kbp long; all are longer than the ITRs of CPV-BR (10 kbp). The 10th variant is a simple deletion mutant that has lost the sequences between 32 and 12 kbp from the right-hand end of the genome. The limits of the inner ends of the observed deletions (between 32 and 38 kbp from the right-hand end of the CPV-BR genome) appear to be defined by the location of the nearest essential gene on the one hand and the location of the gene that encodes "pock redness" on the other. The genomes of the deletion/duplication white-pock variants appear to have been generated either by single crossover recombinational events between two CPV-BR genomes aligned in opposite directions or by the nonreciprocal transfer of genetic information. The sites where such recombination/transfer occurred were sequenced in four variants. In all of them, the sequences adjacent to such sites show no sequence homology or any other unusual strutural feature. The analogous sites at the internal ends of the two ITRs of CPV-BR also were sequenced and also show no unusual features. It is likely that the ITRs of CPV-BR and of its white-pock variants, and probably those of other orthopoxvirus genomes, arise as a result of nonhomologous recombination or by random nonreciprocal transfer of genetic information.