The E6AP ubiquitin-protein ligase (E3) mediates the human papillomavirus-induced degradation of the p53 tumor suppressor in cervical cancer and is mutated in Angelman syndrome, a neurological disorder. The crystal structure of the catalytic hect domain of E6AP reveals a bilobal structure with a broad catalytic cleft at the junction of the two lobes. The cleft consists of conserved residues whose mutation interferes with ubiquitin-thioester bond formation and is the site of Angelman syndrome mutations. The crystal structure of the E6AP hect domain bound to the UbcH7 ubiquitin-conjugating enzyme (E2) reveals the determinants of E2-E3 specificity and provides insights into the transfer of ubiquitin from the E2 to the E3.

E6-AP is a 100-kDa cellular protein that interacts with the E6 protein of the cancer-associated human papillomavirus types 16 and 18. The E6/E6-AP complex binds to and targets the p53 tumor-suppressor protein for ubiquitinmediated proteolysis. E6-AP is an E3 ubiquitin-protein ligase which accepts ubiquitin from an E2 ubiquitin-conjugating enzyme in the form of a thioester and then directly transfers the ubiquitin to targeted substrates. The amino acid sequence of E6-AP shows similarity to a number of protein sequences over an -350-aa region corresponding to the carboxyl termini of both E6-AP and the E6-AP-related proteins. Of particular note is a conserved cysteine residue within the last 32-34 aa, which in E6-AP is likely to be the site of ubiquitin thioester formation. Two of the E6-AP-related proteins, a rat 100-kDa protein and a yeast 95-kDa protein (RSP5), both of previously unknown function, are shown here to form thioesters with ubiquitin. Mutation of the conserved cysteine residue of these proteins destroys their ability to accept ubiquitin. These data strongly suggest that the rat 100-kDa protein and RSP5, as well as the other E6-AP-related proteins, belong to a class of functionally related E3 ubiquitin-protein ligases, defined by a domain homologous to the E6-AP carboxyl terminus (hect domain).The hallmark of the ubiquitin-mediated proteolytic pathway is the covalent attachment of the 76-aa ubiquitin polypeptide to target proteins, through isopeptide bond formation between the carboxyl terminus of ubiquitin and the s-amino group of one or more lysine residues on the protein substrate (reviewed in refs. 1 and 2). Additional ubiquitin moieties can be ligated via lysine residues of ubiquitin itself, resulting in the formation of multiubiquitinated proteins, which are then recognized and degraded by the 26S protease complex. While much of the biochemistry of the ubiquitin proteolysis system has been elucidated, a basic question has remained largely unanswered: how are proteins specifically recognized and targeted for ubiquitination?Protein ubiquitination involves three classes of enzymes. Ubiquitin is activated by the El ubiquitin-activating enzyme in an ATP-dependent reaction, resulting in thioester formation between a specific cysteine of the enzyme and the carboxyl terminus of ubiquitin. The activated ubiquitin is transferred to a cysteine residue of one of a number of low molecular weight E2 ubiquitin-conjugating enzymes. The E2 proteins have generally been thought to catalyze the final ubiquitination of the substrate protein, often in conjunction with a third group of proteins, the E3 ubiquitin-protein ligases. E3 activities have been proposed to play a major role in defining the substrate specificity of the ubiqulitin system, perhaps through direct binding of substrates. In the yeast Saccharomyces cerevisiae at least 12 different E2 genes have been identified (1). Only two known E3 genes have been cloned, yeast UBR1 (3) and the human E6-AP gene (4, 5).E6-AP was discovered in the course of c...

No abstract

ISG15 is an interferon (IFN)-␣/␤-induced ubiquitin-like protein that is conjugated to cellular proteins during innate immune responses to viral and bacterial infections.A recent proteomics study identified 158 human proteins targeted for ISG15 conjugation, including the ISG15 E1 and E2 enzymes (Ube1L and UbcH8, respectively) and a HECT E3 enzyme, Herc5. Like the genes encoding Ube1L and UbcH8, expression of Herc5 was also induced by IFN-␤, suggesting that Herc5 might be a component of the ISG15 conjugation system. Consistent with this, small interfering RNAs targeting Herc5 had a dramatic effect on overall ISG15 conjugation in human cells, abrogating conjugation to the vast majority of ISG15 target proteins in vivo. In addition, co-transfection of plasmids expressing ISG15, Ube1L, UbcH8, and Herc5 resulted in robust ISG15 conjugation in non-IFN-treated cells, while the active-site cysteine mutant of Herc5 or a mutant lacking the RCC1 repeat region did not support ISG15 conjugation. These results demonstrate that Herc5 is required for conjugation of ISG15 to a broad spectrum of target proteins in human cells.Type 1 interferons play an essential role in innate immunity (1). One of the many genes strongly activated by IFN 4 -␣/␤ encodes ISG15, a 15-kDa ubiquitin-like protein (Ubl) (2, 3). Like ubiquitin (Ub), Ubls are linked to target proteins via isopeptide bonds between their terminal carboxyl group and lysine side chains of target proteins (4). The fact that ISG15 is expressed and conjugated in IFN-␣/␤-stimulated cells and lipopolysaccharide-stimulated cells implies that ISG15 conjugation is likely to mediate an important component of the innate immune response. This is supported by the finding that the influenza B virus NS1B protein specifically blocks ISG15 conjugation (5).The biochemical effect of ISG15 on target proteins is unknown; however, the recent identification of a large number of target proteins (6) provides opportunities for determining both the function of ISG15 and its role in the innate immune response. Also essential for functional studies is the identification of the complete set of enzymes required for ISG15 conjugation. As with Ub conjugation, it is presumed that a cooperating set of E1, E2, and E3 enzymes, in addition to possible accessory factors, will be required for ISG15 conjugation. The ISG15 E1 and E2 enzymes have been identified. Ube1L is a single-subunit enzyme 62% similar to the Ub E1 enzyme (5), and UbcH8 is the major, if not exclusive, E2 enzyme for ISG15 (7,8). The genes encoding both Ube1L and UbcH8 are, like ISG15, transcriptionally activated by IFN-␣/␤ (5, 7-9), suggesting that the entire conjugation system might be coordinately regulated. A candidate E3 enzyme for ISG15 conjugation emerged from mass spectrometry-based identification of ISG15 target proteins (6). Proteomics analyses of SUMO-and Ub-conjugated proteins have shown that enzymatic components of Ub/Ubl conjugation pathways are often auto-conjugated (10, 11), and consistent with this, Ube1L and UbcH8 were identified ...

Ubiquitin-(Ub) like proteins (Ubls) are conjugated to their targets by an enzymatic cascade involving an E1 activating enzyme, an E2 conjugating enzyme, and in some cases an E3 ligase. ISG15 is a Ubl that is conjugated to cellular proteins after IFN-␣͞␤ stimulation. Although the E1 enzyme for ISG15 (Ube1L͞E1 ISG15 ) has been identified, the identities of the downstream components of the ISG15 conjugation cascade have remained elusive. Here we report the purification of an E2 enzyme for ISG15 and demonstrate that it is UbcH8, an E2 that also functions in Ub conjugation. In vitro assays with purified Ub E2 enzymes and in vivo RNA interference assays indicate that UbcH8 is a major E2 enzyme for ISG15 conjugation. These results indicate that the ISG15 conjugation pathway overlaps or converges with the Ub conjugation pathway at the level of a specific E2 enzyme. Furthermore, these results raise the possibility that the ISG15 conjugation pathway might use UbcH8-competent Ub ligases in vivo. As an initial test of this hypothesis, we have shown that a UbcH8-competent Ub ligase conjugates ISG15 to a specific target in vitro. These results challenge the concept that Ub and Ubl conjugation pathways are strictly parallel and nonoverlapping and have important implications for understanding the regulation and function of ISG15 conjugation in the IFN-␣͞␤ response. IFN-␣͞␤ play an essential role in innate immunity and are induced during many types of viral infections (1). Many genes are transcriptionally induced by IFN-␣͞␤, including ISG15 (IFNstimulated gene, 15 kDa) (2, 3). The ISG15 protein is a 15-kDa ubiquitin (Ub)-like protein (Ubl), consisting of two Ub-related domains, Ϸ30% (N-terminal domain) and 36% (C-terminal domain) identical to Ub. ISG15 becomes conjugated to a diverse set of cellular proteins after IFN-␣͞␤ stimulation (4). Although the biochemical consequences of ISG15 conjugation and the fate of the conjugated proteins are not known, it does not appear that ISG15 targets proteins for proteasomal degradation (5, 6).Conjugation of Ub to target proteins requires the cooperative activities of at least three classes of enzymes (7). The ATPdependent E1 enzyme activates Ub by C-terminal adenylation, followed by formation of a high-energy thioester bond between the terminal carboxylate of Ub and the active-site cysteine of E1. Ub is then transferred to the active-site cysteine of one of a number of related E2 enzymes. E3 enzymes then promote transfer of Ub from the E2 to the substrate, resulting in a stable amide bond between -amino groups of lysine side chains and Ub. E3 enzymes are the primary determinants of substrate specificity and can be divided into two classes based on mechanism. HECT E3s accept Ub from the E2 enzyme, again in the form of a thioester adduct, and transfer Ub from their active-site cysteine to the bound substrate (8). RING E3s consist of several subclasses and are either single or multisubunit enzymes that serve as docking proteins for both protein substrates and activated E2 enzymes, with transfe...

The E3 ubiquitin-protein ligases play an important role in controlling substrate specificity of the ubiquitin proteolysis system. A biochemical approach was taken to identify substrates of Rsp5, an essential hect (homologous to E6-AP carboxyl terminus) E3 of Saccharomyces cerevisiae. We show here that Rsp5 binds and ubiquitinates the largest subunit of RNA polymerase II (Rpb1) in vitro. The ubiquitin-dependent proteolysis system is characterized by the covalent ligation of multiple ubiquitin peptides to target proteins, which serves as a signal for recognition and degradation by the 26S proteasome (1, 2). Ubiquitination may in some cases serve a regulatory function independent of proteolysis (2). Three classes of enzymes are known to be involved in ligation of ubiquitin to protein substrates: the E1 ubiquitinactivating enzyme, the E2 ubiquitin-conjugating enzymes, and the E3 ubiquitin-protein ligases. The E3 proteins are thought to play the major role in determining the substrate specificity of the system. Characterization of the human papillomavirus E6-mediated degradation of p53 led to the discovery of the E6-AP ubiquitin-protein ligase, which forms a stable ternary complex with E6 and p53, then directly catalyzes the ubiquitination of p53 (3-6). Ubiquitination is dependent on a ''thioester cascade,'' in which ubiquitin is transferred from the active site cysteine of the E1 enzyme, to the active site cysteine of an E2 enzyme, then finally to the active site cysteine of E6-AP, which catalyzes isopeptide bond formation between ubiquitin and one or more lysine residues of the substrate.The carboxyl-terminal domain of E6-AP [the hect domain (homologous to E6-AP carboxyl terminus)], consisting of Ϸ350 amino acids, is similar to that found in at least 30 other eukaryotic proteins (7). The active-site cysteine of E6-AP is within the hect domain and is absolutely conserved among all of the E6-AP-related proteins. Several of these proteins have been shown to also form ubiquitin-thioesters with the same requirements as E6-AP, strongly suggesting that these proteins represent a family of E3 ubiquitin-protein ligases. While other E3 proteins and activities have been identified (8-10), the hect E3s are so far the only known family of related E3 proteins.The yeast Saccharomyces cerevisiae has five genes that encode hect E3 proteins, including the essential RSP5 gene, also known as NPI1 (7, 11). RSP5 was first identified in a search for suppressors of mutations in SPT3 (B. Berg, A. Happel, and F. Winston, cited in ref. 7), which encodes a protein that interacts with the TATA-binding protein (the SPT15 gene product) (12). We report here the results of a biochemical approach aimed at identifying substrates of Rsp5. This approach was based on our prior characterization of E6-AP, which suggested two criteria for identification of putative substrates: stable complex formation between the E3 and its substrates, and direct catalysis of substrate ubiquitination by the E3, with the latter being dependent on ubiquitin thioeste...

Implementation of highly sophisticated technologies, such as next-generation sequencing (NGS), into routine clinical practice requires compatibility with common tumor biopsy types, such as formalin-fixed, paraffin-embedded (FFPE) and fine-needle aspiration specimens, and validation metrics for platforms, controls, and data analysis pipelines. In this study, a two-step PCR enrichment workflow was used to assess 540 known cancer-relevant variants in 16 oncogenes for high-depth sequencing in tumor samples on either mature (Illumina GAIIx) or emerging (Ion Torrent PGM) NGS platforms. The results revealed that the background noise of variant detection was elevated approximately twofold in FFPE compared with cell line DNA. Bioinformatic algorithms were optimized to accommodate this background. Variant calls from 38 residual clinical colorectal cancer FFPE specimens and 10 thyroid fine-needle aspiration specimens were compared across multiple cancer genes, resulting in an accuracy of 96.1% (95% CI, 96.1% to 99.3%) compared with Sanger sequencing, and 99.6% (95% CI, 97.9% to 99.9%) compared with an alternative method with an analytical sensitivity of 1% mutation detection. A total of 45 of 48 samples were concordant between NGS platforms across all matched regions, with the three discordant calls each represented at <10% of reads. Consequently, NGS of targeted oncogenes in real-life tumor specimens using distinct platforms addresses unmet needs for unbiased and highly sensitive mutation detection and can accelerate both basic and clinical cancer research.

Rsp5 is an E3 ubiquitin-protein ligase of Saccharomyces cerevisiae that belongs to the hect domain family of E3 proteins. We have previously shown that Rsp5 binds and ubiquitinates the largest subunit of RNA polymerase II, Rpb1, in vitro. We show here that Rpb1 ubiquitination and degradation are induced in vivo by UV irradiation and by the UV-mimetic compound 4-nitroquinoline-1-oxide (4-NQO) and that a functional RSP5 gene product is required for this effect. The 26S proteasome is also required; a mutation of SEN3/RPN2 (sen3-1), which encodes an essential regulatory subunit of the 26S proteasome, partially blocks 4-NQO-induced degradation of Rpb1. These results suggest that Rsp5-mediated ubiquitination and degradation of Rpb1 are components of the response to DNA damage. A human WW domain-containing hect (WW-hect) E3 protein closely related to Rsp5, Rpf1/hNedd4, also binds and ubiquitinates both yeast and human Rpb1 in vitro, suggesting that Rpf1 and/or another WW-hect E3 protein mediates UV-induced degradation of the large subunit of polymerase II in human cells.Ubiquitin-dependent proteolysis involves the covalent ligation of ubiquitin to substrate proteins, which are then recognized and degraded by the 26S proteasome. While many of the components involved in catalyzing protein ubiquitination have been identified and characterized biochemically, we are only beginning to understand how the system specifically recognizes appropriate substrates. At least three classes of activities, known as E1 (ubiquitin-activating), E2 (ubiquitin-conjugating), and E3 (ubiquitin-protein ligase) enzymes, cooperate in catalyzing protein ubiquitination (34). The enzymatic mechanisms and functions of the E1 and E2 proteins have been well characterized. In contrast, the E3 enzymes are a diverse and less-well-characterized group of activities, and many lines of evidence indicate that E3 activities play a major role in determining the substrate specificity of the ubiquitination pathway (14,28,34).The hect (homologous to E6-AP carboxyl terminus) domain defines a family of E3 proteins that were discovered through the characterization of human E6-AP (17). The interaction of E6-AP with the E6 protein of the cervical cancer-associated human papillomavirus types causes E6-AP to associate with and ubiquitinate p53, suggesting that E6 functions in promoting cellular immortalization by, at least in part, stimulating the destruction of this important tumor suppressor protein (16). The hect E3 molecular masses range from 92 to over 500 kDa, with the hect domain comprising the approximately 350 carboxyl-terminal amino acids (17, 34). Exactly five hect E3s are encoded by the Saccharomyces cerevisiae genome, and over 30 have been identified so far in mammalian species. An obligatory intermediate in the ubiquitination reactions catalyzed by hect E3s is a ubiquitin-thioester formed between the thiol group of an absolutely conserved cysteine within the hect domain and the terminal carboxyl group of ubiquitin (33). E3 becomes "charged" with ubiquitin vi...