Mechanism of ubiquitin ligation and lysine prioritization by a HECT E3

Kamadurai, Hari; Qiu, Yu; Deng, Alan Y.; Harrison, Joseph S.; MacDonald, Chris; Actis, Marcelo L.; Rodrigues, Patrick; Miller, Darcie J.; Souphron, Judith; Lewis, Steven M.; Kurinov, Igor; Fujii, Naoaki; Hammel, Michal; Piper, Robert; Kuhlman, Brian; Schulman, Brenda A.

doi:10.7554/elife.00828

Cited by 141 publications

(225 citation statements)

References 59 publications

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“…1A)-have been more closely examined for their role in ubiquitination mediated by distinct E2 ubiquitin-conjugating enzymes and E3 enzymes (e.g., refs. [22][23][24][25][26][27][28][29][30][31]. Notably, patch II but not patch I was shown to be required for covalent attachment of ubiquitin to substrate proteins catalyzed by the HECT E3s Rsp5 and NEDD4L (22,30).…”

Section: Resultsmentioning

confidence: 99%

“…[22][23][24][25][26][27][28][29][30][31]. Notably, patch II but not patch I was shown to be required for covalent attachment of ubiquitin to substrate proteins catalyzed by the HECT E3s Rsp5 and NEDD4L (22,30). In addition, the HECT domains of Rsp5, NEDD4L, NEDD4, and SMURF2, which are all members of the NEDD4 subfamily of HECT E3s, harbor a noncovalent binding site for a second ubiquitin molecule (that is not in thioester complex with the HECT domain) (23,24,32,33).…”

Section: Resultsmentioning

confidence: 99%

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Role of ubiquitin and the HPV E6 oncoprotein in E6AP-mediated ubiquitination

Mortensen

Schneider

Barbic

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Deregulation of the ubiquitin ligase E6 associated protein (E6AP) encoded by the UBE3A gene has been associated with three different clinical pictures. Hijacking of E6AP by the E6 oncoprotein of distinct human papillomaviruses (HPV) contributes to the development of cervical cancer, whereas loss of E6AP expression or function is the cause of Angelman syndrome, a neurodevelopmental disorder, and increased expression of E6AP has been involved in autism spectrum disorders. Although these observations indicate that the activity of E6AP has to be tightly controlled, only little is known about how E6AP is regulated at the posttranslational level. Here, we provide evidence that the hydrophobic patch of ubiquitin comprising Leu-8 and Ile-44 is important for E6AP-mediated ubiquitination, whereas it does not affect the catalytic properties of the isolated catalytic HECT domain of E6AP. Furthermore, we show that the HPV E6 oncoprotein rescues the disability of full-length E6AP to use a respective hydrophobic patch mutant of ubiquitin for ubiquitination and that it stimulates E6AP-mediated ubiquitination of Ring1B, a known substrate of E6AP, in vitro and in cells. Based on these data, we propose that E6AP exists in at least two different states, an active and a less active or latent one, and that the activity of E6AP is controlled by noncovalent interactions with ubiquitin and allosteric activators such as the HPV E6 oncoprotein.I n eukaryotes, posttranslational modification of proteins by ubiquitin plays a pivotal role in the regulation of many cellular processes, including cell cycle, DNA metabolism (e.g., DNA repair, transcription), and various signal transduction pathways (1-4). The specificity of the ubiquitin-conjugation system is mainly ensured by E3 ubiquitin ligases, which mediate the recognition of target proteins. Based on the presence of distinct domains and their mode of action, E3 proteins can be grouped into three families, RING/RING-like E3s, RING-in-between-RING (RBR) E3s, and HECT E3s (5-7). All E3s have interaction sites for both substrate proteins and E2 ubiquitinconjugating enzymes. However, whereas in the case of RBR E3s and HECT E3s, ubiquitin is transferred from the E3 to substrates, RING/RING-like E3s function as adaptors between substrates and E2s (i.e., ubiquitin is transferred from the E2 to the substrate).E6AP, the founding member of the HECT E3 family, was originally identified as an interacting protein of the E6 oncoprotein of cancer-associated human papillomaviruses (HPVs) (8, 9). The E6-E6AP complex targets the tumor suppressor p53 and other proteins-which in the absence of E6 are not targeted by E6AP-for ubiquitination and degradation thereby contributing to HPV-induced cervical carcinogenesis (10, 11). In 1997, it was recognized that alterations in the UBE3A gene, which encodes E6AP, resulting in loss of E6AP expression or in the expression of E6AP variants with compromised E3 activity, are the cause of the Angelman syndrome (AS), a neurodevelopmental disorder (12-14). Recently, it was rep...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Role of ubiquitin and the HPV E6 oncoprotein in E6AP-mediated ubiquitination

Mortensen

Schneider

Barbic

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…The answer will probably require crystallographic and kinetic analyses of neddylated CRLs bound to charged E2s and substrate. Methods developed recently to examine the structure of a HECT E3-activated ubiquitin-substrate complex that mimics the ubiquitin transfer step could provide a route towards addressing this question [69]. Although the activation of the intrinsic activity of a CRL is one mode of regulation by neddylation, it is not the only one.…”

Section: Consequences Of Cullin Neddylation For Crl Activitymentioning

confidence: 99%

Building and remodelling Cullin–RING E3 ubiquitin ligases

2013

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Cullin-RING E3 ubiquitin ligases (CRLs) control a plethora of biological pathways through targeted ubiquitylation of signalling proteins. These modular assemblies use substrate receptor modules to recruit specific targets. Recent efforts have focused on understanding the mechanisms that control the activity state of CRLs through dynamic alterations in CRL architecture. Central to these processes are cycles of cullin neddylation and deneddylation, as well as exchange of substrate receptor modules to re-sculpt the CRL landscape, thereby responding to the cellular requirements to turn over distinct proteins in different contexts. This review is focused on how CRLs are dynamically controlled with an emphasis on how c ullin neddylation cycles are integrated with receptor exchange. Keywords: cullin; ubiquitin; Nedd8; COP9 signalosome; E3 ligase; CAND1 EMBO reports (2013) 14, 1050-1061 published online 15 November 2013; doi:10.1038/embor.2013 See the Glossary for abbreviations used in this article. IntroductionThe proteome is constantly remodelled to suit the needs of the cell, through both synthesis and turnover. Protein turnover is controlled through two main systems: the ubiquitin-proteasome system (UPS) and lysosomal degradation system, including autophagy. Although selective autophagy can provide a means by which to control the abundance of particular organelles and even single proteins, it is best understood as a means to control bulk turnover of cellular components [1]. Conversely, the UPS is a particularly versatile and highly regulated system [2] that allows selective turnover of individual regulatory proteins, even when they are incorporated into higher-order multiprotein assemblies. On the one hand, the entire population of a given protein targeted by the UPS might be subject to rapid tagging with ubiquitin and degradation by the proteasome. On the other hand, the UPS might control the degradation of only a small pool of a particular target protein, for example the population of a protein that has been phosphorylated by an upstream pathway. Thus, the UPS functions in space and time to sculpt the proteome and to control the abundance of active or inactive forms of signalling complexes and cellular machines. As illustrated in this Review, the UPS machinery that controls turnover of a wide swathe of the p roteome is itself dynamically regulated through many mechanisms.The tagging of proteins with particular types of ubiquitin chains serves to mark them for proteasomal degradation. This process occurs through an E1 (ubiquitin-activating enzyme)-E2 (ubiquitinconjugating enzyme)-E3 (ubiquitin ligase) cascade [3][4][5][6][7]. E3 plays a central role in substrate targeting by binding directly to the substrate and presenting target lysine residues to receive ubiquitin from the E2, in the case of RING E3s, or from a ubiquitin-charged cysteine residue in HECT and RBR E3s [6,[8][9][10]. Cullin-RING E3 ubiquitin ligases (CRLs), which were first discovered almost two decades ago [11][12][13], are a superfamily of RING E3...

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“…6A). The E3-Ub thioester is normally a transient intermediate, but certain point mutations in the yeast HECT ligase Rsp5 have been shown to prevent transfer of Ub to substrate lysines, thus allowing the thioester to accumulate (32). An equivalent mutation in Smurf2 (D433A, corresponding to Rsp5 D495A) allowed detection of the thioester and confirmation that heclin prevented its appearance (Fig.…”

Section: Significancementioning

confidence: 99%

“…5). The HECT C-lobe is very flexible in position, and its function requires oscillation between different orientations (32). One places the active site cysteine close to the E2 site, where it can receive ubiquitin (as exemplified by the crystal structure of WWP1) (33), and another places it far away, in a conformation that allows transfer of ubiquitin to a substrate lysine (as seen in the Smurf2 crystal structure) (21).…”

Section: Significancementioning

confidence: 99%

Peptide and small molecule inhibitors of HECT-type ubiquitin ligases

Mund

Lewis

Maslen

2014

Proc. Natl. Acad. Sci. U.S.A.

125

133

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The human genome encodes several hundred E3 ubiquitin ligases containing RING domains, and around 28 containing HECT domains. These enzymes catalyze the transfer of ubiquitin from E2 enzyme thioesters to a huge range of substrates and play crucial roles in many cellular functions. This makes them attractive potential therapeutic targets. However, they have proven difficult to inhibit: very few good inhibitors exist for RING domain ligases, and none have been described for HECT ligases. Here we show that bicyclic peptides isolated by phage display [Heinis C, Rutherford T, Freund S, Winter G (2009) Nat Chem Biol. 5(7):502-507] can target the E2 binding sites on the HECT domains of Smurf2, Nedd4, Mule/ Huwe1, and WWP1, and thus act as specific inhibitors of these enzymes in vitro. By screening for displacement of one of these peptides from Smurf2, we were able to identify a small molecule, heclin (HECT ligase inhibitor), which inhibits several HECT ligases in tissue culture cells. In vitro, heclin does not block E2 binding but causes a conformational change that results in oxidation of the active site Cys. This demonstrates that HECT domains are potentially druggable and provides molecules that may be of experimental use. Heclin kills HEK293 cells growing in culture, consistent with an essential role for HECT ligase activity in mammalian cells.inhibitor | ubiquitin | HECT ligase | phage display U biquitin ligases are involved in almost every important function of cells, including the removal of misfolded proteins, the regulation of signaling pathways, DNA repair, the cell cycle, and apoptosis (1-4). This broad range of functions is mediated by a large number of E3 ubiquitin ligases, which recognize a similarly large range of substrates. Ubiquitination begins with the ATP-dependent formation of a ubiquitin (Ub)-E2 enzyme thioester intermediate by the E1 enzyme. The E3 enzymes then transfer ubiquitin from this intermediate to a lysine residue on the substrate (3). There are two main classes of E3: the RING domain ligases and their relatives, of which there are several hundred in humans, and the HECT domain ligases, which number around 28 (5-7). Because of their diversity and numerous functions and the fact that some ligases are frequently overexpressed in cancer cells, ubiquitin ligases have attracted much attention as possible targets for therapeutic intervention (2,5,6,8).Despite this interest, it has proven challenging to make small molecule inhibitors of ubiquitin ligases. The action of these enzymes involves primarily protein-protein interactions among them, the Ub-E2 conjugate, and the substrate. For the RING enzymes, this is simultaneous, and the Ub moiety is transferred directly from E2 to substrate; for the HECT ligases, the interaction is sequential, with a thioester-linked Ub-E3 intermediate forming transiently. Such protein-protein interactions are considered difficult to block with small molecules. Added to this difficulty has been the complexity of the ubiquitin system, with ligases having many substrat...

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Mechanism of ubiquitin ligation and lysine prioritization by a HECT E3

Cited by 141 publications

References 59 publications

Role of ubiquitin and the HPV E6 oncoprotein in E6AP-mediated ubiquitination

Role of ubiquitin and the HPV E6 oncoprotein in E6AP-mediated ubiquitination

Building and remodelling Cullin–RING E3 ubiquitin ligases

Peptide and small molecule inhibitors of HECT-type ubiquitin ligases

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