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A promising approach in cancer therapy is to find ligands that directly bind ubiquitin (Ub) chains. However, finding molecules capable of tightly and specifically binding Ub chains is challenging given the range of Ub polymer lengths and linkages and their subtle structural differences. Here, we use total chemical synthesis of proteins to generate highly homogeneous Ub chains for screening against trillion-member macrocyclic peptide libraries (RaPID system). De novo cyclic peptides were found that can bind tightly and specifically to K48-linked Ub chains, confirmed by NMR studies. These cyclic peptides protected K48-linked Ub chains from deubiquitinating enzymes and prevented proteasomal degradation of Ub-tagged proteins. The cyclic peptides could enter cells, inhibit growth and induce programmed cell death, opening new opportunities for therapeutic intervention. This highly synthetic approach, with both protein target generation and cyclic peptide discovery performed in vitro, will make other elaborate post-translationally modified targets accessible for drug discovery.

Systems analogous to the eukaryotic ubiquitin-proteasome system have been previously identified in Archaea, and Actinobacteria (gram-positive), but not in gram-negative bacteria. Here, we report the bioinformatic identification of a novel prokaryotic ubiquitin-like protein, which we name UBact. The phyletic distribution of UBact covers at least five gram-negative bacterial phyla, including Nitrospirae, Armatimonadetes, Verrucomicroba, Nitrospinae, and Planctomycetes. Additionally, it was identified in seven candidate (uncultured) phyla and one Archaeon. UBact might have been overlooked because only few species in the phyla where it is found have been sequenced. In most of the species where we identified UBact, its neighbors in the genome code for proteins homologous to those involved in conjugation and/or degradation of Pup and Pup-tagged substrates. Among them are PafA-, Dop-, Mpa- and proteasome-homologous proteins. This gene association as well as UBact's size and conserved C-terminal G[E/Q] motif, strongly suggest that UBact is used as a conjugatable tag for degradation. With regard to its C-terminus, UBact differs from ubiquitin and most ubiquitin-like proteins (including the mycobacterial Pup) in that it lacks the characteristic C-terminal di-glycine motif, and it usually ends with the sequence R[T/S]G[E/Q]. The phyla that contain UBact are thought to have diverged over 3000 million years ago, indicating that either this ubiquitin-like conjugation system evolved early in evolution or that its occurrence in distant gram-negative phyla is due to multiple instances of horizontal gene transfer., (Copyright © 2017 Elsevier Inc. All rights reserved.)

FAT10 is a ubiquitin-like protein made of two tandem, head-to-tail, ubiquitin domains. It is known to covalently modify proteins in a mechanism similar, though not identical, to that of other ubiquitin-like proteins. The lack of known physiological substrates covalently conjugated by the protein made it difficult to unravel its biological functions. Here we identify two proteins that are covalently modified by FAT10, the inflammatory mediator LRRFIP2 and the endoplasmic reticulum membrane protein LULL1. LRRFIP2 is involved in NF-κB activation following stimulation of TLR4. It is recruited along with MYD88 to the cytosolic tail of the receptor, and by that mediates activation of the downstream signaling cascade. We show that FATylation of LRRFIP2 occurs on two distinct sites, each being modified by a single FAT10 moiety. Furthermore, the second modification is regulated by the first one. Importantly, FATylation of LRRFIP2 interferes with its recruitment to the membrane by translocating it to the cellular insoluble fraction, thus inhibiting NF-κB activation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

FAT10 is a ubiquitin-like protein modifier that is induced in vertebrates following certain inflammatory stimuli. Its functions and the repertoire of its target substrates have remained elusive. In contrast to ubiquitin, its cellular abundance is tightly controlled by both transcriptional and posttranslational regulation, and it was reported to be rapidly degraded by the proteasome. Here we provide data to indicate that the degradation of FAT10 requires ubiquitination: degradation was inhibited in cells expressing a ubiquitin mutant that cannot be polymerized and in a mutant cell harboring a thermolabile ubiquitin-activating enzyme, E1. Of importance, FAT10 can serve as a degradation signal for otherwise stable proteins, and in this case, too, the targeting to the proteasome requires ubiquitination. Degradation of FAT10 is accelerated after induction of apoptosis, suggesting that it plays a role in prosurvival pathways.

Cellular proteins are in a dynamic state maintained by synthesis and degradation. The ubiquitin proteolytic pathway is responsible for the degradation of the bulk of cellular proteins including short-lived, regulatory, and misfolded/denatured proteins. Ubiquitin-mediated proteolysis involves covalent attachment of multiple ubiquitin molecules to the protein substrate and degradation of the targeted protein by the 26S proteasome. Recent understanding of the molecular mechanisms involved provides a framework to understand a wide variety of human pathophysiological states as well as therapeutic interventions. This review focuses on the response to hypoxia, inflammatory diseases, neurodegenerative diseases, and muscle-wasting disorders, as well as human papillomaviruses, cervical cancer and other malignancies.

Processing of the NF-kappa B2 precursor p100 to the mature p52 subunit is regulated via a unique pathway. NF-kappa B-inducing kinase (NIK) induces I kappa B kinase alpha (IKK alpha)-mediated phosphorylation of specific serine residues in the C-terminal domain of p100, leading to recruitment of the SCF(beta-TrCP) ubiquitin ligase. We identified a single lysine residue, K855, that serves as the ubiquitin-anchoring residue required for signal-induced processing of p100. In a reconstituted system containing purified components, p100-K855R could not be ubiquitinated. In a crude extract and cells, only residual, signal-independent ubiquitination and processing were retained. Importantly, K855 is located in a site homologous to K22 that serves as an ubiquitination site in I kappa B alpha. This suggests a common recognition mechanism for the two molecules. In contrast, p105, the p100 homologue, lacks a similar Lys residue. We also demonstrate that the NEDD8 pathway is essential for the SCF(beta-TrCP) activity. In a reconstituted system, efficient ubiquitination of p100 required all three components of the pathway - E1, the UBC12 E2 and NEDD8. Experiments in reconstituted systems and in cells demonstrated that SCF(beta-TrCP), which contains a mutant Cul-1 that cannot be NEDDylated, cannot stimulate ubiquitination and processing. Similarly, dominant negative UBC12 inhibits, in a reversible manner, both ubiquitination and processing of p100.

The ubiquitin-proteasome system (UPS) is involved in selective targeting of innumerable cellular proteins through a complex pathway that plays important roles in a broad array of processes. An important step in the proteolytic cascade is specific recognition of the substrate by one of many ubiquitin ligases, E3s, which is followed by generation of the polyubiquitin degradation signal. For most substrates, it is believed that the first ubiquitin moiety is conjugated, through its C-terminal Gly76 residue, to an sigma-NH2 group of an internal Lys residue. Recent findings indicate that, for several proteins, the first ubiquitin moiety is fused linearly to the alpha-NH2 group of the N-terminal residue. An important biological question relates to the evolutionary requirement for an alternative mode of ubiquitination.

The ubiquitin-proteasome system (UPS) is involved in the removal of damaged proteins and the activation of transcription factors, such as nuclear-factor-kappaB. Recent reports, however, questioned the functional activity of the UPS under conditions of increased oxidative stress, such as experimental hypercholesterolemia, which was the objective of our study. Pigs were placed on a normal chow diet (N) or on a hypercholesterolemic diet without (HC) or with vitamin C and E supplementation (HC+VIT) for 12 weeks. Compared with N, plasma concentration of total cholesterol increased in both HC and HC+VIT [76 +/- 21 vs. 400 +/- 148 (P<0.05) and 329 +/- 102 (P<0.05) mg/dL], whereas increase in lipid peroxidation, as assessed by LDL-malondialdehyde plasma concentration, was found in HC but not in HC+VIT [6.6 +/- 0.7 vs. 8.5 +/- 0.3 (P<0.05) and 6.8 +/- 0.7 nmol/mg protein]. In comparison with N, the level of ubiquitin conjugates in the coronary artery, as assessed by immunoblotting, increased by 42% in HC but not in HC+VIT and was localized predominantly to media vascular smooth muscle cells by immunostaining. There was no difference in proteasome proteolytic activity among the study groups. These results demonstrate that the UPS is functionally active in early atherogenesis despite increase in oxidative stress with important repercussions in the pathophysiology and therapy of cardiovascular diseases.

The p50 subunit of NF-kappaB is generated by limited processing of the precursor p105. IkappaB kinase-mediated phosphorylation of the C-terminal domain of p105 recruits the SCF(beta-TrCP) ubiquitin ligase, resulting in rapid ubiquitination and subsequent processing/degradation of p105. NEDD8 is known to activate SCF ligases following modification of their cullin component. Here we show that NEDDylation is required for conjugation and processing of p105 by SCF(beta-TrCP) following phosphorylation of the molecule. In a crude extract, a dominant negative E2 enzyme, UBC12, inhibits both conjugation and processing of p105, and inhibition is alleviated by an excess of WT- UBC12. In a reconstituted cell-free system, ubiquitination of p105 was stimulated only in the presence of all three components of the NEDD8 pathway, E1, E2, and NEDD8. A Cul-1 mutant that cannot be NEDDylated could not stimulate ubiquitination and processing of p105. Similar findings were observed also in cells. It should be noted that NEDDylation is required only for the stimulated but not for basal processing of p105. Although the mechanisms that underlie processing of p105 are largely obscure, it is clear that NEDDylation and the coordinated activity of SCF(beta-TrCP) on both p105 and IkappaBalpha serve as an important regulatory mechanism controlling NF-kappaB activity.

The p53 protein is subject to Mdm2-mediated degradation by the ubiquitin-proteasome pathway. This degradation requires interaction between p53 and Mdm2 and the subsequent ubiquitination and nuclear export of p53. Exposure of cells to DNA damage results in the stabilization of the p53 protein in the nucleus. However, the underlying mechanism of this effect is poorly defined. Here we demonstrate a key role for c-Abl in the nuclear accumulation of endogenous p53 in cells exposed to DNA damage. This effect of c-Abl is achieved by preventing the ubiquitination and nuclear export of p53 by Mdm2, or by human papillomavirus E6. c-Abl null cells fail to accumulate p53 efficiently following DNA damage. Reconstitution of these cells with physiological levels of c-Abl is sufficient to promote the normal response of p53 to DNA damage via nuclear retention. Our results help to explain how p53 is accumulated in the nucleus in response to DNA damage.

Processing of the p105 precursor to generate the p50 subunit of the nuclear factor kappaB transcription factor is an exceptional case in which the ubiquitin system is involved in limited processing rather than in complete destruction of the target substrate. A Gly-rich region "stop" signal in the middle of the molecule along with a neighboring downstream ubiquitination, and probably an E3 anchoring domain, have been demonstrated to be important for processing. In addition, we have shown that IkappaB kinase-mediated phosphorylation of the C-terminal domain leads to recruitment of the SCF(beta)-TrCP ubiquitin ligase with subsequent accelerated ubiquitination and processing/degradation of the precursor (Orian, A., Gonen, H., Bercovich, B., Fajerman, I., Eytan, E., Israël, A., Mercurio, F., Iwai, K., Schwartz, A. L., and Ciechanover, A. (2000) EMBO J. 19, 2580-2591). Here we show that processing of p105 molecules that contain more then four ankyrin repeats, but lack the C-terminal phosphorylation/ubiquitin ligase binding domain, is strongly inhibited by docked p50 subunits. Inhibition is caused by interference with the function of the proteasome, as conjugation is not affected. Inhibition is alleviated after IkappaB kinase phosphorylation of the C-terminal domain leads to accelerated, beta-TrCP-mediated ubiquitination and processing/degradation of p105. We suggest that under basal conditions, slow generation of p50 probably involves the mid-molecule ubiquitination/E3 recognition motif. Following stimulation, the C-terminal domain is involved in rapid processing/degradation of p105 with release of a large amount of the stored subunits that now become transcriptionally active.

Many short-lived nuclear proteins are targeted for degradation by the ubiquitin-proteasome pathway. The role of the nucleus in regulating the turnover of these proteins is not well defined, although many components of the ubiquitin-proteasome system are localized in the nucleus. We have used nucleoplasm from highly purified HeLa nuclei to examine the degradation of a physiological substrate of the ubiquitin-proteasome system (MyoD). In vitro studies using inhibitors of the system demonstrate MyoD is degraded via the ubiquitin-proteasome pathway in HeLa nucleoplasm. Purified nucleoplasm in vitro also supports the generation of high molecular mass MyoD-ubiquitin adducts. In addition, in vivo studies, using leptomycin B to inhibit nuclear export, demonstrate that MyoD is degraded in HeLa cells by the nuclear ubiquitin-proteasome system.

Between the 1960s and 1980s, the main focus of biological research was nucleic acids and the translation of the coded information into proteins. Protein degradation was a neglected area and regarded by many as a scavenger, non-specific and end process. While it was known that proteins are turning over, the large extent and high specificity of the process--where distinct proteins have half-lives that range from a few minutes to several days--have not been appreciated. The discovery of the lysosome by Dr. Christian de Duve did not change this view significantly, as this organelle is involved mostly in the degradation of extra- and not intracellular proteins, and it was clear that lysosomal proteases, similar to those of the gastrointestinal tract, cannot be substrate specific. The discovery of the complex cascade of the ubiquitin pathway has changed this view dramatically. It is now clear that degradation of cellular proteins is a highly complex, temporally controlled, and tightly regulated process that plays major roles in a broad array of basic pathways during cell life and death. With the multitude of substrates targeted and processes involved, it is not surprising that aberrations in the pathway have been recently implicated in the pathogenesis of many diseases, certain malignancies and neurodegeneration among them. Degradation of a protein via the ubiquitin pathway involves two successive steps: a) conjugation of multiple ubiquitin moieties to the substrate, and b) degradation of the tagged protein by the downstream 26S proteasome complex with release of free and re-utilizable ubiquitin. Despite intensive research, the unknown still exceeds what we currently know on intracellular protein degradation and major key problems remain unsolved. Among these are the modes of specific and timed recognition of the myriad substrates of the system and the nature of the mechanisms that underlie aberrations in the system and pathogenesis of diseases.

In most cases, target proteins of the ubiquitin system are completely degraded. In several exceptions, such as the first step in the activation of the transcriptional regulator NF-kappaB, the substrate, the precursor protein p105, is processed in a limited manner to yield the active subunit p50. p50 is derived from the N-terminal domain of p105, whereas the C-terminal domain is degraded. The mechanisms involved in this unique process have remained elusive. We have shown that a Gly-rich region (GRR) at the C-terminal domain of p50 is one important processing signal and that it interferes with processing of the ubiquitinated precursor by the 26S proteasome. Also, amino acid residues 441-454 are important for processing under non-stimulated conditions. Lys 441 and 442 serve as ubiquitination targets, whereas residues 446-454 may serve as a ligase recognition motif. Following IkappaB kinase (IKK)-mediated phosphorylation, the C-terminal domain of p105, residues 918-934, recruits the SCF(beta-TrCP) ubiquitin ligase, and ubiquitination by this complex leads to accelerated processing. The two sites appear to be recognized under different physiological conditions by two different ligases, targeting two distinct recognition motifs. We have shown that ubiquitin conjugation and processing of a series of precursors of p105 that lack the C-terminal IKK phosphorylation/TrCP binding domain, is progressively inhibited with increasing number of ankyrin repeats. Inhibition is due to docking of active NF-kappaB subunits to the ankyrin repeat domain in the C-terminal half of p105 (IkappaBgamma). Inhibition is alleviated by phosphorylation of the C-terminal domain that leads to ubiquitin-mediated degradation of the ankyrin repeat domain and release of the anchored subunits. We propose a model that may explain the requirement for two sites: a) a basal site that may be involved in co-translational processing prior to the synthesis of the ankyrin repeat domain; and b) a signal-induced site that is involved in processing/degradation of the complete molecule following cell activation, with rapid release of stored, transcriptionally active subunits.

The E7 oncoprotein of the high risk human papillomavirus type 16 (HPV-16), which is etiologically associated with uterine cervical cancer, is a potent immortalizing and transforming agent. It probably exerts its oncogenic functions by interacting and altering the normal activity of cell cycle control proteins such as p21WAF1, p27KIP1 and pRb, transcriptional activators such as TBP and AP-1, and metabolic regulators such as M2-pyruvate kinase (M2-PK). Here we show that E7 is a short-lived protein and its degradation both in vitro and in vivo is mediated by the ubiquitin-proteasome pathway. Interestingly, ubiquitin does not attach to any of the two internal Lysine residues of E7. Substitution of these residues with Arg does not affect the ability of the protein to be conjugated and degraded; in contrast, addition of a Myc tag to the N-terminal but not to the C-terminal residue, stabilizes the protein. Also, deletion of the first 11 amino acid residues stabilizes the protein in cells. Taken together, these findings strongly suggest that, like MyoD and the Epstein Barr Virus (EBV) transforming Latent Membrane Protein 1 (LMPI), the first ubiquitin moiety is attached linearly to the free N-terminal residue of E7. Additional ubiquitin moieties are then attached to an internal Lys residue of the previously conjugated molecule. The involvement of E7 in many diverse and apparently unrelated processes requires tight regulation of its function and cellular level, which is controlled in this case by ubiquitin-mediated proteolysis.

The latent membrane protein 1 (LMP1) of the Epstein-Barr virus is a constitutively active receptor essential for B lymphocyte transformation by the Epstein-Barr virus. It is a short-lived protein, but the proteolytic pathway involved in its degradation is not known. The ubiquitin pathway is a major system for specific protein degradation in eukaryotes. Most plasma membrane substrates of the pathway are internalized upon ubiquitination and delivered for degradation in the lysosome/vacuole. Here we show that LMP1 is a substrate of the ubiquitin pathway and is ubiquitinated both in vitro and in vivo. However, in contrast to other plasma membrane substrates of the ubiquitin system, it is degraded mostly by the proteasome and not by lysosomes. Degradation is independent of the single Lys residue of the protein; a lysine-less mutant LMP1 is degraded in a ubiquitin- and proteasome-dependent manner similar to the wild type protein. Degradation of both wild type and lysine-less protein is sensitive to fusion of a Myc tag to the N terminus of LMP1. In addition, deletion of as few as 12 N-terminal amino acid residues stabilizes the protein. These findings suggest that the first event in LMP1 degradation is attachment of ubiquitin to the N-terminal residue of the protein. We present evidence suggesting that phosphorylation is also required for degradation of LMP1.

The ubiquitin proteolytic system plays an important role in a broad array of basic cellular processes. Among these are regulation of cell cycle, modulation of the immune and inflammatory responses, control of signal transduction pathways, development and differentiation. These complex processes are controlled via specific degradation of a single or a subset of proteins. Degradation of a protein by the ubiquitin system involves two successive steps, conjugation of multiple moieties of ubiquitin and degradation of the tagged protein by the 26S proteasome. An important question concerns the identity of the mechanisms that underlie the high degree of specificity of the system. Substrate recognition is governed by a large family ubiquitin ligases that recognize the substrates, bind them and catalyze/facilitate their interaction with ubiquitin.

Beta-catenin and plakoglobin are closely related armadillo family proteins with shared and distinct properties; Both are associated with cadherins in actin-containing adherens junctions. Plakoglobin is also found in desmosomes where it anchors intermediate filaments to the desmosomal plaques. Beta-catenin, on the other hand, is a component of the Wnt signaling pathway, which is involved in embryonic morphogenesis and tumorigenesis. A key step in the regulation of this pathway involves modulation of beta-catenin stability. A multiprotein complex, regulated by Wnt, directs the phosphorylation of beta-catenin and its degradation by the ubiquitin-proteasome system. Plakoglobin can also associate with members of this complex, but inhibition of proteasomal degradation has little effect on its levels while dramatically increasing the levels of beta-catenin. Beta-TrCP, an F-box protein of the SCF E3 ubiquitin ligase complex, was recently shown to play a role in the turnover of beta-catenin. To elucidate the basis for the apparent differences in the turnover of beta-catenin and plakoglobin we compared the handling of these two proteins by the ubiquitin-proteasome system. We show here that a deletion mutant of beta-TrCP, lacking the F-box, can stabilize the endogenous beta-catenin leading to its nuclear translocation and induction of beta-catenin/LEF-1-directed transcription, without affecting the levels of plakoglobin. However, when plakoglobin was overexpressed, it readily associated with beta-TrCP, efficiently competed with beta-catenin for binding to beta-TrCP and became polyubiquitinated. Fractionation studies revealed that about 85% of plakoglobin in 293 cells, is Triton X-100-insoluble compared to 50% of beta-catenin. These results suggest that while both plakoglobin and beta-catenin can comparably interact with beta-TrCP and the ubiquitination system, the sequestration of plakoglobin by the membrane-cytoskeleton system renders it inaccessible to the proteolytic machinery and stabilizes it.

Proteolysis via the ubiquitin system plays important roles in a variety of basic cellular processes. Among these are regulation of cell cycle and division, modulation of the immune and inflammatory responses, and development and differentiation. In all cases studied, these complex processes are mediated via degradation or processing of a single or a subset of specific proteins. Ubiquitin-mediated degradation of a protein involves two discrete and successive steps: (1) conjugation of multiple moieties of ubiquitin to the protein, and (2) degradation of the conjugated protein by the 26S proteasome complex with the release of free and reutilizable ubiquitin. In a few cases, it has been reported that ubiquitination targets membrane-anchored proteins to degradation in the lysosome/vacuole. An important yet largely unresolved problem involves the mechanisms that endow the system with the high degree specificity and selectivity toward its many substrates. These are determined by a large family of ubiquitin-protein ligases that recognize different primary and/or secondary/post-translational motifs in the different substrates and by a wide array of modifying enzymes, such as protein kinases, and ancillary proteins, such as molecular chaperones, that render them susceptible for recognition by the ligases via modification or association with protein substrates. With the broad spectrum of protein substrates and the complex enzymatic machinery involved in targeting them, it is not surprising that the system was recently implicated in the pathogenesis of several important diseases. In addition, genetic studies in animals underscore the role of the system in normal development. We briefly review the enzymatic cascade involved in ubiquitin-mediated degradation, describe some of the structural motifs identified by the conjugating machinery, and summarize recent developments in the involvement of the system in the pathogenesis of selected disease states.

The ubiquitin-proteasome pathway is responsible for the major portion of specific cellular protein degradation. Ubiquitin-mediated degradation is involved in physiological regulation of many cellular processes, including cell cycle progression, differentiation, and signal transduction. Here, we review the basic mechanisms of the ubiquitin system and the various ways in which ubiquitin-mediated degradation can be modulated by physiological signals., (Copyright 2000 Wiley-Liss, Inc.)

The last step in the activation of the transcription factor NF-kappaB is signal-induced, ubiquitin- and proteasome-mediated degradation of the inhibitor IkappaBalpha. Although most of the components involved in the activation and degradation pathways have been identified, the ubiquitin carrier proteins (E2) have remained elusive. Here we show that the two highly homologous members of the UBCH5 family, UBCH5b and UBCH5c, and CDC34/UBC3, the mammalian homolog of yeast Cdc34/Ubc3, are the E2 enzymes involved in the process. The conjugation reaction they catalyze in vitro is specific, as they do not recognize the S32A,S36A mutant species of IkappaBalpha that cannot be phosphorylated and conjugated following an extracellular signal. Furthermore, the reaction is specifically inhibited by a doubly phosphorylated peptide that spans the ubiquitin ligase recognition domain of the inhibitor. Cys-to-Ala mutant species of the enzymes that cannot bind ubiquitin inhibit tumor necrosis factor alpha-induced degradation of the inhibitor in vivo. Not surprisingly, they have a similar effect in a cell-free system as well. Although it is clear that the E2 enzymes are not entirely specific to IkappaBalpha, they are also not involved in the conjugation and degradation of the bulk of cellular proteins, thus exhibiting some degree of specificity that is mediated probably via their association with a defined subset of ubiquitin-protein ligases. The mechanisms that underlie the involvement of two different E2 species in IkappaBalpha conjugation are not clear at present. It is possible that different conjugating machineries operate under different physiological conditions or in different cells.

The ubiquitin proteolytic system plays a major role in a variety of basic cellular processes. In the majority of these processes, the target proteins are completely degraded. In one exceptional case, generation of the p50 subunit of the transcriptional regulator NF-kappaB, the precursor protein p105 is processed in a limited manner: the N-terminal domain yields the p50 subunit, whereas the C-terminal domain is degraded. The identity of the mechanisms involved in this unique process have remained elusive. It has been shown that a Gly-rich region (GRR) at the C-terminal domain of p50 is an important processing signal. Here we show that the GRR does not interfere with conjugation of ubiquitin to p105 but probably does interfere with the processing of the ubiquitin-tagged precursor by the 26S proteasome. Structural analysis reveals that a short sequence containing a few Gly residues and a single essential Ala is sufficient to generate p50. Mechanistically, the presence of the GRR appears to stop further degradation of p50 and to stabilize the molecule. It appears that the localization of the GRR within p105 plays an important role in directing processing: transfer of the GRR within p105 or insertion of the GRR into homologous or heterologous proteins is not sufficient to promote processing in most cases, which is probably due to the requirement for an additional specific ubiquitination and/or recognition domain(s). Indeed, we have shown that amino acid residues 441 to 454 are important for processing. In particular, both Lys 441 and Lys 442 appear to serve as major ubiquitination targets, while residues 446 to 454 are independently important for processing and may serve as the ubiquitin ligase recognition motif.

MyoD is a tissue-specific transcriptional activator involvd in skeletal muscle differentiation. It is induced during transition from proliferating, non-differentiated myoblasts to the resting and well differentiated myotubes. Like many other transcriptional regulators, it is short-lived, however, the targeting proteolytic pathway and the underlying regulatory mechanisms involved have remained obscure. Here we show that MyoD is degraded by the ubiquitin system both in vivo and in vitro. In cells, degradation is inhibited by lactacystin, a specific inhibitor of the 20S proteasome. Inhibition is accompanied by accumulation of MyoD-ubiquitin conjugates. In a cell free system, the proteolytic process requires both ATP and ubiquitin and is preceded by formation of MyoD-ubiquitin adducts. Interestingly, the process is inhibited by the specific DNA sequence to which MyoD binds. Analysis of the ubiquitination site has revealed that the N-terminal residue of MyoD is sufficient and essential to promote conjugation and subsequent degradation of the protein: conjugation to internal Lys residues is not necessary. Substitution of all Lys residues did not affect significantly its degradation either in intact cells or in a reconstituted cell free system. Degradation was inhibited by specific proteasome inhibitors and was accompanied by accumulation of ubiquitinated species of the protein. We concluded that the first ubiquitin moiety is attached via its C-terminal Gly to the N-terminal residue of MyoD, and the polyubiquitin chain is then synthesized on Lys48 of this moiety.

The ubiquitin-proteasome pathway plays a pivotal role in the degradation of short-lived and regulatory proteins important in a variety of basic cellular processes, including regulation of the cell cycle, modulation of cell surface receptors and ion channels, and antigen presentation. The pathway involves an enzymatic cascade through which multiple ubiquitin molecules are covalently attached to the protein substrate, which is then degraded by the 26S proteasome complex. The pathway has been implicated in several forms of malignancy, in the pathogenesis of several genetic diseases (including cystic fibrosis, Angelman's syndrome, and Liddle syndrome), in immune surveillance/viral pathogenesis, and in the pathology of muscle wasting. The molecular mechanisms that underlie these processes are being unraveled at present.

The ubiquitin proteolytic pathway is a major system for selective protein degradation in eukaryotic cells. One of the first steps in the degradation of a protein via this pathway involves selective modification of epsilon-NH2 groups of internal lysine residues by ubiquitination. To date, this amino group has been the only known target for ubiquitination. Here we report that the N-terminal residue of MyoD is sufficient and necessary for promotion of conjugation and subsequent degradation of the protein. Substitution of all lysine residues in the protein did not affect significantly its conjugation and degradation either in vivo or in vitro. In cells, degradation of the lysine-less protein is inhibited by the proteasome inhibitors MG132 and lactacystin. Inhibition is accompanied by accumulation of high molecular mass ubiquitinated forms of the modified MyoD. In striking contrast, wild-type MyoD, in which all the internal Lys residues have been retained but the N-terminus has been extended by fusion of a short peptide, is stable both in vivo and in vitro. In a cell-free system, ATP and multiple ubiquitination are essential for degradation of the lysine-less protein. Specific chemical modifications have yielded similar results. Selective blocking of the alpha-NH2 group of wild-type protein renders it stable, while modification of the internal Lys residues with preservation of the free N-terminal group left the protein susceptible to degradation. Our data suggest that conjugation of MyoD occurs via a novel modification involving attachment of ubiquitin to the N-terminal residue. The polyubiquitin chain is then synthesized on an internal Lys residue of the linearly attached first ubiquitin moiety.

MyoD is a tissue-specific transcriptional activator that acts as a master switch for skeletal muscle differentiation. Its activity is induced during the transition from proliferating, nondifferentiated myoblasts to resting, well-differentiated myotubes. Like many other transcriptional regulators, it is a short-lived protein; however, the targeting proteolytic pathway and the underlying regulatory mechanisms involved in the process have remained obscure. It has recently been shown that many short-lived regulatory proteins are degraded by the ubiquitin system. Degradation of a protein by the ubiquitin system proceeds via two distinct and successive steps, conjugation of multiple molecules of ubiquitin to the target protein and degradation of the tagged substrate by the 26S proteasome. Here we show that MyoD is degraded by the ubiquitin system both in vivo and in vitro. In intact cells, the degradation is inhibited by lactacystin, a specific inhibitor of the 26S proteasome. Inhibition is accompanied by accumulation of high-molecular-mass MyoD-ubiquitin conjugates. In a cell-free system, the proteolytic process requires both ATP and ubiquitin and, like the in vivo process, is preceded by formation of ubiquitin conjugates of the transcription factor. Interestingly, the process is inhibited by the specific DNA sequence to which MyoD binds: conjugation and degradation of a MyoD mutant protein which lacks the DNA-binding domain are not inhibited. The inhibitory effect of the DNA requires the formation of a complex between the DNA and the MyoD protein. Id1, which inhibits the binding of MyoD complexes to DNA, abrogates the effect of DNA on stabilization of the protein.

We have previously shown that the degradation of c-myc and N-myc in vitro is mediated by the ubiquitin system. However, the role of the system in targeting the myc proteins in vivo and the identity of the conjugating enzymes and possible ancillary proteins involved has remained obscure. Here we report that the degradation of the myc proteins in cells is inhibited by lactacystin and MG132, two inhibitors of the 20S proteasome. Inhibition is accompanied by accumulation of myc-ubiquitin conjugates. Dissection of the ancillary proteins involved revealed that the high-risk human papillomavirus oncoprotein E6-16 stimulates conjugation and subsequent degradation of the myc proteins in vitro. Expression of E6-16 in cells results in significant shortening of the t1/2 of the myc proteins with subsequent decrease in their cellular level. Analysis of the conjugating enzymes revealed that under basal conditions the proteins can be conjugated by two pairs of E2s and E3s-E2-14 kDa and E3alpha involved in the "N-end rule" pathway, and E2-F1 (UbcH7) and E3-Fos involved also in conjugation of c-Fos. In the presence of E6-16, a third pair, E2-F1 and E6-AP mediate conjugation of myc by means of a mechanism that appears to be similar to that involved in the targeting of p53, formation of a myc. E6.E6-AP targeting complex. It is possible that in certain cells E6-mediated targeting of myc prevents myc-induced apoptosis and thus ensures maintenance of viral infection.

The selective degradation of many short-lived proteins in eukaryotic cells is carried out by the ubiquitin system. In this pathway, proteins are targeted for degradation by covalent ligation to ubiquitin, a highly conserved small protein. Ubiquitin-mediated degradation of regulatory proteins plays important roles in the control of numerous processes, including cell-cycle progression, signal transduction, transcriptional regulation, receptor down-regulation, and endocytosis. The ubiquitin system has been implicated in the immune response, development, and programmed cell death. Abnormalities in ubiquitin-mediated processes have been shown to cause pathological conditions, including malignant transformation. In this review we discuss recent information on functions and mechanisms of the ubiquitin system. Since the selectivity of protein degradation is determined mainly at the stage of ligation to ubiquitin, special attention is focused on what we know, and would like to know, about the mode of action of ubiquitin-protein ligation systems and about signals in proteins recognized by these systems.

The Epstein-Barr virus (EBV) encoded nuclear antigen (EBNA) 1 is expressed in latently infected B lymphocytes that persist for life in healthy virus carriers and is the only viral protein regularly detected in all EBV associated malignancies. The Gly-Ala repeat domain of EBNA1 was shown to inhibit in cis the presentation of major histocompatibility complex (MHC) class I restricted cytotoxic T cell epitopes from EBNA4. It appears that the majority of antigens presented via the MHC I pathway are subject to ATP-dependent ubiquitination and degradation by the proteasome. We have investigated the influence of the repeat on this process by comparing the degradation of EBNA1, EBNA4, and Gly-Ala containing EBNA4 chimeras in a cell-free system. EBNA4 was efficiently degraded in an ATP/ubiquitin/proteasome-dependent fashion whereas EBNA1 was resistant to degradation. Processing of EBNA1 was restored by deletion of the Gly-Ala domain whereas insertion of Gly-Ala repeats of various lengths and in different positions prevented the degradation of EBNA4 without appreciable effect on ubiquitination. Inhibition was also achieved by insertion of a Pro-Ala coding sequence. The results suggest that the repeat may affect MHC I restricted responses by inhibiting antigen processing via the ubiquitin/proteasome pathway. The presence of regularly interspersed Ala residues appears to be important for the effect.

Activation of the transcription factor NF-kappa B is a paradigm for signal transduction through the ubiquitin-proteasome pathway: ubiquitin-dependent degradation of the transcriptional inhibitor I kappa B in response to cell stimulation. A major issue in this context is the nature of the recognition signal and the targeting enzyme involved in the proteolytic process. Here we show that following a stimulus-dependent phosphorylation, and while associated with NF-kappa B, I kappa B is targeted by a specific ubiquitin-ligase via direct recognition of the signal-dependent phosphorylation site; phosphopeptides corresponding to this site specifically inhibit ubiquitin conjugation of I kappa B and its subsequent degradation. The ligase recognition signal is functionally conserved between I kappa B alpha and I kappa B beta, and does not involve the nearby ubiquitination site. Microinjection of the inhibitory peptides into stimulated cells abolished NF-kappa B activation in response to TNF alpha and the consequent expression of E-selectin, an NF-kappa B-dependent cell-adhesion molecule. Inhibition of NF-kappa B function by specific blocking of ubiquitin ligase activity provides a novel approach for intervening in cellular processes via regulation of unique proteolytic events.

The epithelial Na+ channel (ENaC), composed of three subunits (alpha beta gamma), plays a critical role in salt and fluid homeostasis. Abnormalities in channel opening and numbers have been linked to several genetic disorders, including cystic fibrosis, pseudohypoaldosteronism type I and Liddle syndrome. We have recently identified the ubiquitin-protein ligase Nedd4 as an interacting protein of ENaC. Here we show that ENaC is a short-lived protein (t1/2 approximately 1 h) that is ubiquitinated in vivo on the alpha and gamma (but not beta) subunits. Mutation of a cluster of Lys residues (to Arg) at the N-terminus of gamma ENaC leads to both inhibition of ubiquitination and increased channel activity, an effect augmented by N-terminal Lys to Arg mutations in alpha ENaC, but not in beta ENaC. This elevated channel activity is caused by an increase in the number of channels present at the plasma membrane; it represents increases in both cell-surface retention or recycling of ENaC and incorporation of new channels at the plasma membrane, as determined by Brefeldin A treatment. In addition, we find that the rapid turnover of the total pool of cellular ENaC is attenuated by inhibitors of both the proteasome and the lysosomal/endosomal degradation systems, and propose that whereas the unassembled subunits are degraded by the proteasome, the assembled alpha beta gamma ENaC complex is targeted for lysosomal degradation. Our results suggest that ENaC function is regulated by ubiquitination, and propose a paradigm for ubiquitination-mediated regulation of ion channels.

Degradation of a protein via the ubiquitin system involves two discrete steps, signaling by covalent conjugation of multiple moieties of ubiquitin and degradation of the tagged substrate. Conjugation is catalyzed via a three-step mechanism that involves three distinct enzymes that act successively: E1, E2, and E3. The first two enzymes catalyze activation of ubiquitin and transfer of the activated moiety to E3, respectively. E3, to which the substrate is specifically bound, catalyzes formation of a polyubiquitin chain that is anchored to the targeted protein. The polyubiquitin-tagged protein is degraded by the 26 S proteasome, and free and reutilizable ubiquitin is released. In addition to the three conjugating enzymes, targeting of certain proteins requires association with ancillary proteins and/or post-translational modification(s). Using a specific antibody to deplete cell extract from the molecular chaperone Hsc70, we demonstrate that this protein is required for the degradation of actin, alpha-crystallin, glyceraldehyde-3-phosphate dehydrogenase, alpha-lactalbumin, and histone H2A. In contrast, the degradation of bovine serum albumin, lysozyme, and oxidized RNase A is Hsc70-independent. Mechanistic analysis revealed that the chaperone is required for the conjugation reaction; however, it does not substitute for E3. Involvement of the chaperone in the proteolytic process requires complex formation with the substrate. Formation of this complex appears to be essential in the proteolytic process. In addition, the proper function of the chaperone in the proteolytic process requires the presence of K+, which allows rapid cycles of dissociation and association of the complex. The chaperone may act by binding to the substrate and unfolding it to expose a ubiquitin ligase-binding site. In addition, it can also act directly on the ubiquitination machinery.

Most of the known cellular substrates of the ubiquitin system are short-lived growth regulators and transcriptional activators. Very few enzymes involved in intermediary metabolism have been shown to be targeted by the system. In a reconstituted cell-free system, we show that tyrosine aminotransferase (TAT), a key enzyme involved in amino acid metabolism, is conjugated and degraded in an ATP- and ubiquitin-dependent manner. Degradation of ubiquitin-TAT adducts requires, in addition to the 26S proteasome, a novel, yet unidentified, factor. TAT can be protected from degradation by association with its coenzyme pyridoxal phosphate. To examine the potential role of the ubiquitin system in regulating the stability of the enzyme in vivo, we show that cell extracts derived from livers of animals in which TAT was induced, display a corollary increase in the formation of specific TAT-ubiquitin adducts.

Degradation of a protein via the ubiquitin proteolytic pathway involves two successive steps. Covalent attachment of ubiquitin to the target protein and degradation of the tagged substrate by the 26S proteasome. Most native cellular proteins that are targeted by the ubiquitin system are short-lived transcriptional activators and growth and cell cycle regulators, as well as unstable membrane proteins. In the present study we demonstrate the involvement of the system in the degradation of tyrosine aminotransferase (TAT), a key enzyme in intermediary metabolism. In vitro, we have shown that the native enzyme is conjugated and degraded in a system that requires ATP and ubiquitin. Degradation was monitored by following the decrease of catalytic activity as well as disappearance of the protein molecule. The enzyme could be protected from degradation by association with its specific cofactor, pyridoxal phosphate (PLP). In vivo, we prepared cell extracts from livers of animals in which TAT was induced by starvation and corticosteroid administration. The dramatic increase in the level of the enzyme was accompanied by a concomitant increase in the level of specific TAT-ubiquitin adducts.

We have investigated the genomic organization of Entamoeba histolytica ubiquitin and looked for the occurrence of a ubiquitin-conjugating system in this organism. Southern blots indicated the presence of > or = 5 ubiquitin-coding regions. One of these, EhUBI1, was cloned and sequenced and found to correspond to a monoubiquitin gene; as shown by a polymerase chain reaction, E. histolytica lacked polyubiquitin genes altogether. Blots of poly(A)+ RNA from exponentially-growing trophozoite cultures exhibited five ubiquitin transcripts, the most prominent and smallest of which corresponded to EhUBI1 mRNA. Expression of the ubiquitin genes was not influenced by heat shock. Although the predicted amino acid sequence of the ubiquitin from E. histolytica differs significantly (in 7-9 amino acid residues) from that of yeast and animals, expression of the coding sequence of EhUBI1 suppressed the heat-sensitive phenotype of a polyubiquitin gene-deficient yeast mutant. In correlation, trophozoite extract catalyzed an ATP-dependent conjugation of radioiodinated bovine ubiquitin to trophozoite proteins. The latter data indicate that E. histolytica contains a functional ubiquitin-conjugating system.

The ubiquitin-dependent protein degradation system has recently been implicated in downregulation of signal transducing receptors. Growth hormone receptor (GHR) cDNA was transfected into Chinese hamster ovary cells, which exhibit a temperature-sensitive defect in ubiquitin conjugation (CHO-ts20), as well as into wild-type cells (CHO-E36). Upon binding of growth hormone (GH), two GHR polypeptides dimerize and initiate signal transduction. In CHO-E36 and in CHO-ts20 at the permissive temperature the GHR was ubiquitinated and degraded in a GH-dependent fashion. However, at the non-permissive temperature in CHO-ts20 cells, neither GH-dependent uptake nor degradation of the GHR was observed, while in CHO-E36 cells both GHR uptake and degradation were accelerated. Incubation of CHO-E36 cells with inhibitors of endosomal/lysosomal function (NH4Cl, bafilomycin A1) markedly reduced ligand-induced GHR degradation. Our results indicate that a functional ubiquitin conjugating system is required for GH-induced endocytosis and that degradation of both the exoplasmic and cytoplasmic portions of the GHR occurs within the endosomal/lysosomal compartment.

Targeting of different cellular proteins for conjugation and subsequent degradation via the ubiquitin pathway involves diverse recognition signals and distinct enzymatic factors. A few proteins are recognized via their N-terminal amino acid residue and conjugated by a ubiquitin-protein ligase that recognizes this residue. However, most substrates, including N-alpha-acetylated proteins that constitute the vast majority of cellular proteins, are targeted by different signals and are recognized by yet unknown ligases. In addition to the ligases, other factors may also be specific for the recognition of this subset of proteins. We have previously shown that degradation of N-terminally blocked proteins require a specific factor, designated FH, and that the factor acts along with the 26S protease complex to degrade ubiquitin-conjugated proteins (Gonen et al., 1991). Further studies have shown that FH is identical to the protein synthesis elongation factor EF-1 alpha, and that it can be substituted by the bacterial elongation factor EF-Tu (Gonen et al., 1994). This, rather surprising, finding raises two important and interesting problems. The first involves the mechanism of action of the factor and the second the possibility that protein synthesis and degradation may be regulated by a commonly shared factor. Here, we demonstrate that EF-1 alpha is a ubiquitin C-terminal hydrolase (isopeptidase) that is probably involved in trimming the conjugates to lower molecular weight forms recognized by the 26S proteasome complex. Additional findings demonstrate that its activity is inhibited specifically by tRNA. This finding raises the possibility that under anabolic conditions, when the factor is associated with AA.tRNA and GTP, it is active in protein synthesis but inactive in proteolysis. Under catabolic conditions, when the factor is predominantly found in its apo form, it is active in proteolysis.

The nuclear translocation of NF-kappa B follows the degradation of its inhibitor, I kappa B alpha, an event coupled with stimulation-dependent inhibitor phosphorylation. Prevention of the stimulation-dependent phosphorylation of I kappa B alpha, either by treating cells with various reagents or by mutagenesis of certain putative I kappa B alpha phosphorylation sites, abolishes the inducible degradation of I kappa B alpha. Yet, the mechanism coupling the stimulation-induced phosphorylation with the degradation has not been resolved. Recent reports suggest a role for the proteasome in I kappa B alpha degradation, but the mode of substrate recognition and the involvement of ubiquitin conjugation as a targeting signal have not been addressed. We show that of the two forms of I kappa B alpha recovered from stimulated cells in a complex with RelA and p50, only the newly phosphorylated form, pI kappa B alpha, is a substrate for an in vitro reconstituted ubiquitin-proteasome system. Proteolysis requires ATP, ubiquitin, a specific ubiquitin-conjugating enzyme, and other ubiquitin-proteasome components. In vivo, inducible I kappa B alpha degradation requires a functional ubiquitin-activating enzyme and is associated with the appearance of high molecular weight adducts of I kappa B alpha. Ubiquitin-mediated protein degradation may, therefore, constitute an integral step of a signal transduction process.

In most cases, the transcriptional factor NF-kappa B is a heterodimer consisting of two subunits, p50 and p65, which are encoded by two distinct genes of the Rel family. p50 is translated as a precursor of 105 kDa. The C-terminal domain of the precursor is rapidly degraded, forming the mature p50 subunit consisted of the N-terminal region of the molecule. The mechanism of generation of p50 is not known. It has been suggested that the ubiquitin-proteasome system is involved in the process; however, the specific enzymes involved and the mechanism of limited proteolysis, in which half of the molecule is spared, have been obscure. Palombella and colleagues (Palombella, V. J., Rando, O. J., Goldberg, A. L., and Maniatis, T. (1994) Cell 78, 773-785) have shown that ubiquitin is required for the processing in a cell-free system of a truncated, artificially constructed, 60-kDa precursor. They have also shown that proteasome inhibitors block the processing both in vitro and in vivo. In this study, we demonstrate reconstitution of a cell-free processing system and demonstrate directly that: (a) the ubiquitin-proteasome system is involved in processing of the intact p105 precursor, (b) conjugation of ubiquitin to the precursor is an essential intermediate step in the processing, (c) the recently discovered novel species of the ubiquitin-carrier protein, E2-F1, that is involved in the conjugation and degradation of p53, is also required for the limited processing of the p105 precursor, and (d) a novel, approximately 320-kDa species of ubiquitin-protein ligase, is involved in the process. This novel enzyme is distinct from E6-AP, the p53-conjugating ligase, and from E3 alpha, the "N-end rule" ligase.

Ubiquitin modification of many protein targets within cells plays important roles in a variety of biological processes. Among these are regulation of gene expression, regulation of cell cycle and division, involvement in the cellular stress response, modification of cell surface receptors, DNA repair, import of proteins into mitochondria, uptake of precursors of neurotransmitters into synaptosomes, biogenesis of peroxisomes, assembly of ribosomes, and programmed cell death. The mechanisms that underlie these complex processes are poorly understood. The best studied modification occurs in the ubiquitin-mediated proteolytic pathway. Recent experimental evidence indicates that the ubiquitin system is involved in the degradation of mitotic cyclins, oncoproteins and tumor suppressors, in the removal of abnormal and otherwise damaged proteins, and in processing of antigens restricted to class I MHC molecules. Degradation of a protein via the ubiquitin system involves two discrete steps. Initially, multiple ubiquitin molecules are covalently linked in an ATP-dependent mode to the protein substrate. The targeted protein is then degraded by a specific, energy-dependent and high molecular mass protease complex into peptides and free amino acids, and free and reutilizable ubiquitin is released. In addition, stable mono-ubiquitin adducts are also found in the cell, for example, those involving nucleosomal histones. Despite the considerable progress that has been made in elucidating the mode of action and roles of the ubiquitin system, many problems remain unsolved. For example, little is known on the signals that target proteins for degradation. While a few proteins are targeted for degradation following recognition of their N-terminal amino acid residue, the vast majority of cellular proteins are targeted by other signals. The identity of the native cellular substrates of the system is another important, yet unresolved problem: only a few proteins have been recognized so far as substrates of the system in vivo. The scope of this review is to discuss the mechanisms involved in ubiquitin activation, selection of substrates for conjugation, and degradation of ubiquitin-conjugated proteins in the cell-free system. In addition, we shall summarize what is currently known of the physiological roles of ubiquitin-mediated proteolysis in vivo.

Targeting of different cellular proteins for conjugation and subsequent degradation via the ubiquitin pathway involves diverse recognition signals and distinct enzymatic factors. A few proteins are recognized via their N-terminal amino acid residue and conjugated by a ubiquitin-protein ligase that recognizes this residue. Most substrates, including the N alpha-acetylated proteins that constitute the vast majority of cellular proteins, are targeted by different signals and are recognized by yet unknown ligases. We have previously shown that degradation of N-terminally blocked proteins requires a specific factor, designated FH, and that the factor acts along with the 26S protease complex to degrade ubiquitin-conjugated proteins. Here, we demonstrate that FH is the protein synthesis elongation factor EF-1 alpha. (a) Partial sequence analysis reveals 100% identity to EF-1 alpha. (b) Like EF-1 alpha, FH binds to immobilized GTP (or GDP) and can be purified in one step using the corresponding nucleotide for elution. (c) Guanine nucleotides that bind to EF-1 alpha protect the ubiquitin system-related activity of FH from heat inactivation, and nucleotides that do not bind do not exert this effect. (d) EF-Tu, the homologous bacterial elongation factor, can substitute for FH/EF-1 alpha in the proteolytic system. This last finding is of particular interest since the ubiquitin system has not been identified in prokaryotes. The activities of both EF-1 alpha and EF-Tu are strongly and specifically inhibited by ubiquitin-aldehyde, a specific inhibitor of ubiquitin isopeptidases. It appears, therefore, that EF-1 alpha may be involved in releasing ubiquitin from multiubiquitin chains, thus rendering the conjugates susceptible to the action of the 26S protease complex.

The wild-type tumor suppressor protein p53 is a short-lived protein that plays important roles in regulation of cell cycle, differentiation, and survival. Mutations that inactivate or alter the tumor suppressor activity of the protein seem to be the most common genetic change in human cancer and are frequently associated with changes in its stability. The ubiquitin system has been implicated in the degradation of p53 both in vivo and in vitro. A mutant cell line that harbors a thermolabile ubiquitin-activating enzyme, E1, fails to degrade p53 at the nonpermissive temperature. Studies in cell-free extracts have shown that covalent attachment of ubiquitin to the protein requires the three conjugating enzymes: E1, a novel species of ubiquitin-carrier protein (ubiquitin-conjugating enzyme; UBC),E2-F1, and an ubiquitin-protein ligase, E3. Recognition of p53 by the ligase is facilitated by formation of a complex between the protein and the human papillomavirus (HPV) oncoprotein E6. Therefore, the ligase has been designated E6-associated protein (E6-AP). However, these in vitro studies have not demonstrated that the conjugates serve as essential intermediates in the proteolytic process. In fact, in many cases, conjugation of ubiquitin to the target protein does not signal its degradation. Thus, it is essential to demonstrate that p53-ubiquitin adducts serve as essential proteolytic intermediates and are recognized and degraded by the 26S protease complex, the proteolytic arm of the ubiquitin pathway. In this study, we demonstrate that conjugates of p53 generated in the presence of purified, E1, E2, E6-AP, E6, ubiquitin and ATP, are specifically recognized by the 26S protease complex and degraded. In contrast, unconjugated p53 remains stable. The ability to reconstitute the system from purified components will enable detailed analysis of the recognition process and the structural motifs involved in targeting the protein for degradation.

The mechanisms that regulate ubiquitin-mediated degradation of proteins such as the mitotic cyclins at defined stages of the cell cycle are poorly understood. The initial step in the conjugation of ubiquitin to substrate proteins involves the activation of ubiquitin by the ubiquitin-activating enzyme, E1. Previously we have described the subcellular localization of this enzyme to both nuclear and cytoplasmic compartments. In the present study, we have used the 1C5 anti-E1 monoclonal antibody in immunofluorescent-microscopy and subcellular-fractionation techniques to examine the distribution of E1 during the HeLa cell cycle. E1 is both cytoskeletal and nuclear during the G1-phase. As the cells progress into S-phase, E1 is exclusively cytoskeletal and has a perinuclear distribution. During G2-phase, E1 reappears in the nucleus before breakdown of the nuclear envelope. In mitotic cells, E1 localizes to both the mitotic spindle and the cytosol, but is absent from the chromosomes. Immunoblot analysis reveals multiple forms of E1 in HeLa whole cell extract. This heterogeneity is not a result of polyubiquitination and may represent inactive pools of E1. Only the characteristic E1 doublet is able to activate ubiquitin. Cell-fractionation studies reveal a differential distribution of specific E1 isoforms throughout the cell cycle. Therefore we propose that the subcellular localization of E1 may play a role in regulating cell-cycle-dependent conjugation of ubiquitin to target proteins.