The degradation of many proteins involves the sequential ligation of ubiquitin molecules to the substrate to form a multiubiquitin chain linked through Lys-48 of ubiquitin. To test for the existence of alternate forms of multiubiquitin chains, we examined the effects of individually substituting each of six other Lys residues in ubiquitin with Arg. Substitution of Lys-63 resulted in the disappearance of a family of abundant multiubiquitin-protein conjugates. The UbK63R mutants were not generally impaired in ubiquitination, because they grew at a wild-type rate, were fully proficient in the turnover of a variety of short-lived proteins, and exhibited normal levels of many ubiquitin-protein conjugates. The UbK63R mutation also conferred sensitivity to the DNA-damaging agents methyl methanesulfonate and UV as well as a deficiency in DNA damage-induced mutagenesis. Induced mutagenesis is mediated by a repair pathway that requires Rad6 (Ubc2), a ubiquitinconjugating enzyme. Thus, the UbK63R mutant appears to be deficient in the Rad6 pathway of DNA repair. However, the UbK63R mutation behaves as a partial suppressor of a rad6 deletion mutation, indicating that an effect of UbK63R on repair can be manifest in the absence of the Rad6 gene product. The UbK63R mutation may therefore define a new role of ubiquitin in DNA repair. The results of this study suggest that Lys-63 is used as a linkage site in the formation of novel multiubiquitin chain structures that play an important role in DNA repair.

Proteasome-dependent degradation of ubiquitinated proteins plays a key role in many important cellular processes. Ubiquitination requires the E1 ubiquitin activating enzyme, an E2 ubiquitin conjugating enzyme, and frequently a substrate-specific ubiquitin protein ligase (E3). One class of E3 ubiquitin ligases has been shown to contain a common zinc-binding RING finger motif. We have previously shown that herpes simplex virus type 1 ICP0, itself a RING finger protein, induces the proteasome-dependent degradation of several cellular proteins and induces the accumulation of colocalizing conjugated ubiquitin in vivo. We now report that both full-length ICP0 and its isolated RING finger domain induce the accumulation of polyubiquitin chains in vitro in the presence of E1 and the E2 enzymes UbcH5a and UbcH6. Mutations within the RING finger region that abolish the in vitro ubiquitination activity also cause severe reductions in ICP0 activity in other assays. We conclude that ICP0 has the potential to act as an E3 ubiquitin ligase during viral infection and to target specific cellular proteins for destruction by the 26S proteasome.Herpes simplex virus type 1 (HSV-1) is a significant human pathogen whose biological and clinical importance is emphasized by its ability to attain and reactivate from a latent state in sensory neurons (reviewed in reference 16). The mechanisms that control the balance between the lytic and latent states are of considerable interest yet are incompletely understood. Our past studies have concentrated on the functions and mechanisms of action of HSV-1 immediate-early regulatory protein ICP0, which is required for the efficient initiation of lytic cycle gene expression, reactivation of quiescent virus in cultured cells, and reactivation of latent virus in mouse models (for reviews, see references 5 and 15; see also references 18 and 19).ICP0 is being actively studied in a number of laboratories, and a wide spectrum of possible functions, interactions, and mechanisms of action are being revealed. Early transfection studies showed that ICP0 is able to increase the expression of a wide variety of genes in cotransfected cells, and this effect does not depend on specific promoter sequences. Since ICP0 does not bind directly to DNA (13), it is likely that it functions via interactions with other proteins. Recent studies have proposed a number of possible interactions, including USP7 (a ubiquitin-specific protease) (11), cyclin D3 (25), elongation factor EF-1␦ (23), the transcription factor BMAL1 (24), and the major HSV-1 transcriptional regulator ICP4 (42). ICP0 has also been suggested to activate cdk4 and to stabilize both cyclin D1 and cyclin D3 (40). Whatever the significance of these varied observations, it is now generally accepted that a major biological activity of ICP0 causes the disruption of specific nuclear structures known as ND10 or promyelocytic leukemia (PML) nuclear bodies in a process which correlates with the ability of ICP0 to stimulate viral infection and reactivation from quiescen...

Objective. To compare the efficacy, safety, and tolerability of 5 doses of oral tofacitinib (CP-690,550) or adalimumab monotherapy with placebo for the treatment of active rheumatoid arthritis (RA) in patients with an inadequate response to disease-modifying antirheumatic drugs.Methods. In this 24-week, double-blind, phase IIb study, patients with RA (n ‫؍‬ 384) were randomized to receive placebo, tofacitinib at 1, 3, 5, 10, or 15 mg administered orally twice a day, or adalimumab at 40 mg injected subcutaneously every 2 weeks (total of 6 injections) followed by oral tofacitinib at 5 mg twice a day for 12 weeks. The primary end point was the responder rate according to the American College of Rheumatology 20% improvement criteria (ACR20) at week 12.Results. Treatment with tofacitinib at a dose of >3 mg twice a day resulted in a rapid response with significant efficacy when compared to placebo, as indicated by the primary end point (ACR20 response at week 12), achieved in 39.2% (3 mg; P < 0.05), 59.2% (5 mg; P < 0.0001), 70.5% (10 mg; P < 0.0001), and 71.9% (15 mg; P < 0.0001) in the tofacitinib group and 35.9% of patients in the adalimumab group (P ‫؍‬ 0.105), compared with 22.0% of patients receiving placebo. Improvements were sustained at week 24, according to the ACR20, ACR50, and ACR70 response rates as well as classifications of remission according to the 3-variable Disease Activity Score in 28 joints (DAS28) using C-reactive protein and the 4-variable DAS28 using the erythrocyte sedimentation rate. The most common

The degradation of many proteins requires their prior attachment to ubiquitin. Proteolytic substrates are characteristically multiubiquitinated through the formation of ubiquitin-ubiquitin linkages. Lys-48 of ubiquitin can serve as a linkage site in the formation of such chains and is required for the degradation of some substrates of this pathway in vitro. We have characterized the recessive and dominant effects of a Lys-48-to-Arg mutant of ubiquitin (UbK48R) The degradation of short-lived proteins is a complex and highly regulated process (21,29). In eukaryotes, the primary mechanism of selective degradation involves ligation of the C terminus of ubiquitin to E-amino groups of lysine residues within the proteolytic substrate. Coupling of ubiquitin to proteolytic substrates is mediated by a family of ubiquitinconjugating enzymes, which has at least 12 distinct members in Saccharomyces cerevisiae (35,49). These enzymes are required for a wide range of cellular functions, including cell cycle control (35), DNA repair (35), peroxisome biogenesis (57), and heavy metal resistance (37). In the case of higher eukaryotes, ubiquitination has been implicated in the degradation of mitotic cyclins (25), p53 (50), c-mos, c-fos, and other regulators of growth and the cell cycle (9, 41).Ubiquitination targets proteins for degradation by the 26S protease. This particle contains over 20 distinct subunits, including multiple peptidase and ATPase activities and a deubiquitinating activity (19,24,45). The mechanisms by which these activities are coordinated and controlled by substrate ubiquitination are unknown. However, in vitro experiments have indicated that the efficacy of ubiquitination in signaling degradation is a function of the multiplicity and arrangement of ubiquitin groups bound to the substrate (8,21,26). Substrates of the N-end rule pathway, such as Arg-3-galactosidase (P-Gal), are modified in rabbit reticulocyte extracts by a multiubiquitin chain with ubiquitin-ubiquitin linkages at 54). Both multiubiquitination and degradation of Arg-3-Gal were blocked when UbK48R was substituted for wild-type ubiquitin, suggesting that degradation requires multiubiquitin chain formation. However, there may be substrate-to-substrate differences in the degree to which * Corresponding author. Phone: (617) 432-1144. degradation is inhibited by preventing Lys-48 chain synthesis (19,28,31).One possible basis for this variation is the existence of alternative forms of multiubiquitin chains. Lys-63 of ubiquitin can apparently be used as a multiubiquitination site on endogenous substrates in vivo (52). In the present work, a failure to form Lys-48 chains was seen to inhibit various processes despite the continued presence of Lys-63 as a potential compensatory attachment site. The phenotypic effects of preventing Lys-48 chain synthesis are more dramatic than those seen with Lys-63, consistent with the view that obligatory Lys-48 chain formation is a dominant mode of degradative signaling in the ubiquitin pathway. MATERUILS AND METHODS...

The 26S proteasome is an essential proteolytic complex that is responsible for degrading proteins conjugated with ubiquitin. It has been proposed that the recognition of substrates by the 26S proteasome is mediated by a multiubiquitin-chain-binding protein that has previously been characterized in both plants and animals. In this study, we identified a Saccharomyces cerevisiae homolog of this protein, designated Mcb1. Mcb1 copurified with the 26S proteasome in both conventional and nickel chelate chromatography. In addition, a significant fraction of Mcb1 in cell extracts was present in a low-molecular-mass form free of the 26S complex. Recombinant Mcb1 protein bound multiubiquitin chains in vitro and, like its plant and animal counterparts, exhibited a binding preference for longer chains. Surprisingly, (delta)mcb1 deletion mutants were viable, grew at near-wild-type rates, degraded the bulk of short-lived proteins normally, and were not sensitive to UV radiation or heat stress. These data indicate that Mcb1 is not an essential component of the ubiquitin-proteasome pathway in S.cerevisiae. However, the (delta)mcb1 mutant exhibited a modest sensitivity to amino acid analogs and had increased steady-state levels of ubiquitin-protein conjugates. Whereas the N-end rule substrate, Arg-beta-galactosidase, was degraded at the wild-type rate in the (delta)mcb1 strain, the ubiquitin fusion degradation pathway substrate, ubiquitin-Pro-beta-galactosidase, was markedly stabilized. Collectively, these data suggest that Mcb1 is not the sole factor involved in ubiquitin recognition by the 26S proteasome and that Mcb1 may interact with only a subset of ubiquitinated substrates.

Cln3 cyclin of the budding yeast Saccharomyces cerevisiae is a key regulator of Start, a cell cycle event in G1 phase at which cells become committed to division. The time of Start is sensitive to Cln3 levels, which in turn depend on the balance between synthesis and rapid degradation. Here we report that the breakdown of Cln3 is ubiquitin dependent and involves the ubiquitin-conjugating enzyme Cdc34 (Ubc3). The C-terminal tail of Cln3 functions as a transferable signal for degradation. Sequences important for Cln3 degradation are spread throughout the tail and consist largely of PEST elements, which have been previously suggested to target certain proteins for rapid turnover. The Cln3 tail also appears to contain multiple phosphorylation sites, and both phosphorylation and degradation of Cln3 are deficient in a cdc28ts mutant at the nonpermissive temperature. A point mutation at Ser-468, which lies within a Cdc28 kinase consensus site, causes approximately fivefold stabilization of a Cln3-beta-galactosidase fusion protein that contains a portion of the Cln3 tail and strongly reduces the phosphorylation of this protein. These data indicate that the degradation of Cln3 involves CDC28-dependent phosphorylation events.

The 26 S proteasome is a multisubunit proteolytic complex responsible for degrading eukaryotic proteins targeted by ubiquitin modification. Substrate recognition by the complex is presumed to be mediated by one or more common receptor(s) with affinity for multiubiquitin chains, especially those internally linked through lysine 48. We have identified previously a candidate for one such receptor from diverse species, designated here as The ubiquitin/26 S proteasome pathway is a major route for the selective degradation of eukaryotic proteins. Through the removal of key regulatory components, the pathway helps control many aspects of cell homeostasis, growth, and development (1-4). Examples include cell cycle progression, maintenance of chromatin structure, DNA repair, enzymatic regulation, transcription, signal transduction, and programmed cell death. In addition, the ubiquitin pathway participates in cellular housekeeping and the stress response by removing abnormal and denatured proteins.In the ubiquitin pathway, proteins are first enzymatically tagged for breakdown by the covalent attachment of one or more chains of ubiquitin monomers. This process is catalyzed by an enzymatic cascade, involving ubiquitin-activating enzymes (E1s), 1 ubiquitin-conjugating enzymes (E2s), and ubiquitin-protein ligases (E3s), that couples ATP hydrolysis to ubiquitin ligation (1-3). Attachment is via an isopeptide bond between the C-terminal glycine of ubiquitin and free lysines either in the target or in the preceding ubiquitin in the chain. Within the multiubiquitin chain, Lys 48 appears to be the preferred intermolecular linkage site (5, 6) but genetic evidence has implicated several other lysines as well (e.g. Lys 29 and Lys 63 ) (7-9). Once assembled, the multiubiquitin chain functions as a recognition signal for degradation of the substrate by the 26 S proteasome, a multisubunit complex specific for multiubiquitinated proteins (10). The tagged proteins are broken down into short peptides, while the ubiquitin moieties are released intact for reuse.Specificity within the ubiquitin pathway is achieved by at least three mechanisms. The most important determines which proteins should be ubiquitinated by the E1/E2/E3 cascade of reactions. Here, substrate specificity is primarily regulated by

Although the biochemical targets of most drugs are known, the biological consequences of their actions are typically less well understood. In this study, we have used two whole-genome technologies in Saccharomyces cerevisiae to determine the cellular impact of the proteasome inhibitor PS-341. By combining population genomics, the screening of a comprehensive panel of bar-coded mutant strains, and transcript profiling, we have identified the genes and pathways most affected by proteasome inhibition. Many of these function in regulated protein degradation or a subset of mitotic activities. In addition, we identified Rpn4p as the transcription factor most responsible for the cell's ability to compensate for proteasome inhibition. Used together, these complementary technologies provide a general and powerful means to elucidate the cellular ramifications of drug treatment.