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...

Most human cancer cells display increased telomerase activity that appears to be critical for continued cell proliferation and tumor formation. The E6 protein of malignancy-associated human papillomaviruses increases cellular telomerase in primary human keratinocytes at least partly via transcriptional activation of the telomerase catalytic subunit, hTERT. In the present study, we investigated whether E6AP, a ubiquitin ligase well known for binding and mediating some of the activities of the E6 oncoprotein, participated in the transactivation of the hTERT promoter. Our results demonstrate that E6 mutants that fail to bind E6AP are also defective for increasing telomerase activity and transactivating the hTERT promoter. More importantly, E6AP knock-out mouse cells and small interfering RNA techniques demonstrated that E6AP was required for hTERT promoter transactivation in both mouse and human cells. Neither E6 nor E6AP bound to the hTERT promoter or activated the promoter in the absence of the partner protein. With all transactivation-competent E6 proteins, induction of the hTERT promoter was dependent upon E box elements in the core promoter. It appears, therefore, that E6-mediated activation of the hTERT promoter requires a complex of E6-E6AP to engage the hTERT promoter and that activation is dependent upon Myc binding sites in the promoter. The recruitment of a cellular ubiquitin ligase to the hTERT promoter during E6-mediated transcriptional activation suggests a role for the local ubiquitination (and potential degradation) of promoter-associated regulatory proteins, including the Myc protein.

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The function of the human papillomavirus (HPV) E6 protein that is most clearly linked to carcinogenesis is the targeted degradation of p53, which is dependent on the E6AP ubiquitin ligase. Additional functions have been attributed to E6, including the stimulation of telomerase activity and the targeted degradation of other cellular proteins, but in most cases it is unclear whether these activities are also E6AP dependent. While E6 clearly influences the transcriptional program of HPV-positive cell lines through the inactivation of p53, it has been shown that at least a subset of its p53-independent functions are also reflected in the transcriptional program. For this study, we have determined the extent to which E6AP is involved in mediating the set of E6 functions that impact on the global transcriptional program of HPV-positive cell lines. The transcriptional profiles of ϳ31,000 genes were characterized for three cell lines (HeLa, Caski, and SiHa cells) after small interfering RNA (siRNA)-mediated silencing of E6 or E6AP. We found that E6 and E6AP siRNAs elicited nearly identical alterations in the transcriptional profile of each cell line. Some of the expression alterations were apparent secondary effects of p53 stabilization, while the basis of most other changes was not reconcilable with previously proposed E6 functions. While expression changes of the TERT gene (telomerase catalytic subunit) were not revealed by the array, telomerase repeat amplification protocol assays showed that both E6 and E6AP knockouts resulted in a suppression of telomerase activity. Together, these results suggest that E6AP mediates a broad spectrum of E6 functions, including virtually all functions that impact on the transcriptional program of HPV-positive cell lines.Human papillomaviruses (HPVs) are small DNA tumor viruses that infect cutaneous or mucosal epithelial cells. Approximately 30 HPV types are sexually transmitted and can be further categorized according to their associations with malignancies (65). Low-risk HPV types (e.g., types 6 and 11) are associated with benign lesions, while DNAs of high-risk HPV types (e.g., types 16, 18, 33, and 39) are found in Ͼ95% of all cervical cancers, strongly suggesting that HPV is a causative agent of this disease (65). The only two viral proteins that are generally expressed in HPV-positive cervical cancers, E6 and E7, have been shown to be necessary and sufficient to immortalize human foreskin keratinocytes, further suggesting that they are cooperating oncoproteins (21, 41). HPV E7 interacts with and inactivates the retinoblastoma protein (pRb) and the pRb-related proteins p107 and p130 (40). E7 inactivation of pRb activates E2F transcription factors, which results in increased transcription of E2F-responsive genes and S-phase cell cycle progression. While several other E7-interacting proteins have been identified (including histone deacetylase [38], p21, TATA-binding protein, and TAF110 [40]), results from animal studies indicate that the major biologic activities of E7 are related ...