Oligomerization of the HECT ubiquitin ligase NEDD4-2/NEDD4L is essential for polyubiquitin chain assembly

Todaro, Dustin R.; Augustus‐Wallace, Allison; Klein, Jennifer M.; Haas, Arthur

doi:10.1074/jbc.ra118.003716

Cited by 10 publications

(6 citation statements)

References 60 publications

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“…To unravel the precise role of the exosite during ubiquitin chain formation by E6AP will require further investigation. On a broader scope, understanding how the exosite promotes catalysis in different HECT ligases will likely also help to clarify the directionality of ubiquitin chain elongation, for which different models have been presented (9, 26, 32, 51, 54, 75, 76), as well as the functional consequences of oligomerization, which has emerged as an important regulatory theme and has been associated with the exosite in certain cases (39, 69, 77–79).…”

Section: Discussionmentioning

confidence: 99%

Analysis of ubiquitin recognition by the HECT ligase E6AP provides insight into its linkage specificity

Ries

Sander

Deol³

et al. 2019

Journal of Biological Chemistry

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Deregulation of the HECT-type ubiquitin ligase E6AP (UBE3A) is implicated in human papilloma virus-induced cervical tumorigenesis and several neurodevelopmental disorders. Yet the structural underpinnings of activity and specificity in this crucial ligase are incompletely understood. Here, we unravel the determinants of ubiquitin recognition by the catalytic domain of E6AP and assign them to particular steps in the catalytic cycle. We identify a functionally critical interface that is specifically required during the initial formation of a thioester-linked intermediate between the C terminus of ubiquitin and the ligase-active site. This interface resembles the one utilized by NEDD4-type enzymes, indicating that it is widely conserved across HECT ligases, independent of their linkage specificities. Moreover, we uncover surface regions in ubiquitin and E6AP, both in the N- and C-terminal portions of the catalytic domain, that are important for the subsequent reaction step of isopeptide bond formation between two ubiquitin molecules. We decipher key elements of linkage specificity, including the C-terminal tail of E6AP and a hydrophilic surface region of ubiquitin in proximity to the acceptor site Lys-48. Intriguingly, mutation of Glu-51, a single residue within this region, permits formation of alternative chain types, thus pointing to a key role of ubiquitin in conferring linkage specificity to E6AP. We speculate that substrate-assisted catalysis, as described previously for certain RING-associated ubiquitin–conjugating enzymes, constitutes a common principle during linkage-specific ubiquitin chain assembly by diverse classes of ubiquitination enzymes, including HECT ligases.

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Section: Discussionmentioning

confidence: 99%

Analysis of ubiquitin recognition by the HECT ligase E6AP provides insight into its linkage specificity

Ries

Sander

Deol³

et al. 2019

Journal of Biological Chemistry

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“…Our data indicate that ITCH interacts with GRA35 via its C2 domain, which is known for membrane binding as well as mediating protein oligomerization (43). Thus, one possibility is that GRA35 could mediate ITCH oligomerization, which further enhances the ubiquitin ligase activity (44) and promotes its binding to the substrates, potentially including proteins that modulate LEW rat NLRP1 stability. Alternatively, this binding event could initiate broader perturbations to host cell homeostasis that are ultimately integrated through the inflammasome response.…”

Section: Discussionmentioning

confidence: 99%

Host E3 ubiquitin ligase ITCH mediatesToxoplasma gondiieffector GRA35-triggered NLRP1 inflammasome activation and cell-autonomous immunity

Wang,

Hollingsworth,

Sangaré

et al. 2023

Preprint

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Toxoplasma gondiiis an intracellular parasite that can activate the NLRP1 inflammasome leading to macrophage pyroptosis in Lewis rats, but the underlying mechanism is not well understood. In this study, we performed a genome-wide CRISPR screen and identified the dense granule proteins GRA35, GRA42, and GRA43 as theToxoplasmaeffectors mediating cell death in Lewis rat macrophages. GRA35 localizes on the parasitophorous vacuole membrane, where it interacts with the host E3 ubiquitin ligase ITCH. Inhibition of proteasome activity or ITCH knockout prevented pyroptosis inToxoplasma-infected Lewis rat macrophages, consistent with the “NLRP1 functional degradation model”. However, there was no evidence that ITCH directly ubiquitinates or interacts with rat NLRP1. We also found that GRA35-ITCH interaction affectedToxoplasmafitness in IFNγ-activated human fibroblasts, likely due to ITCH’s role in recruiting ubiquitin and the parasite-restriction factor RNF213 to the parasitophorous vacuole membrane. These findings identify a new role of host E3 ubiquitin ligase ITCH in mediating effector-triggered immunity, a critical concept that involves recognizing intracellular pathogens and initiating host innate immune responses.IMPORTANCEEffector-triggered immunity represents an innate immune defense mechanism that plays a crucial role in sensing and controlling intracellular pathogen infection. The NLRP1 inflammasome in the Lewis rats can detectToxoplasmainfection, which triggers proptosis in infected macrophages and eliminates the parasite’s replication niche. The work reported here revealed that host E3 ubiquitin ligase ITCH is able to recognize and interact withToxoplasmaeffector protein GRA35 localized on the parasite-host interface, leading to NLRP1 inflammasome activation in Lewis rat macrophages. Furthermore, ITCH-GRA35 interaction contributes to the restriction ofToxoplasmain human fibroblasts stimulated by IFNγ. Thus, this research provides valuable insights into understanding pathogen recognition and restriction mediated by host E3 ubiquitin ligase.

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“…While it was found that this N-terminal extension also induces HECT-dependent oligomerization, the exact structural mechanisms used by many of the members in the HECT E3 ubiquitin ligase family that employ this modification remain only partially understood. Previous studies have also pointed out that the inclusion of the extended α-helices in-front of the HECT domain are necessary to perform structural analysis of the HECT domain that coincidentally also include highly conserved, essential residues that mediate HECT-dependent oligomerization [ 3 , 8 , 11 , 31 , 43 , 45 ]. Future studies are essential to examine the oligomerization and/or autoinhibition potential for the currently illusive members of the HECT E3 ubiquitin ligases.…”

Section: Discussionmentioning

confidence: 99%

Redefining the catalytic HECT domain boundaries for the HECT E3 ubiquitin ligase family

et al. 2022

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There are 28 unique human members of the homologous to E6AP C-terminus (HECT) E3 ubiquitin ligase family. Each member of the HECT E3 ubiquitin ligases contains a conserved bilobal HECT domain of approximately 350 residues found near their C-termini that is responsible for their respective ubiquitylation activities. Recent studies have begun to elucidate specific roles that each HECT E3 ubiquitin ligase has in various cancers, age-induced neurodegeneration, and neurological disorders. New structural models have been recently released for some of the HECT E3 ubiquitin ligases, but many HECT domain structures have yet to be examined due to chronic insolubility and/or protein folding issues. Building on these recently published structural studies coupled with our in-house experiments discussed in the present study, we suggest that the addition of ∼50 conserved residues preceding the N-terminal to the current UniProt defined boundaries of the HECT domain are required for isolating soluble, stable, and active HECT domains. We show using in silico bioinformatic analyses coupled with secondary structural prediction software that this predicted N-terminal α-helix found in all 28 human HECT E3 ubiquitin ligases forms an obligate amphipathic α-helix that binds to a hydrophobic pocket found within the HECT N-terminal lobe. The present study brings forth the proposal to redefine the residue boundaries of the HECT domain to include this N-terminal extension that will likely be critical for future biochemical, structural, and therapeutic studies on the HECT E3 ubiquitin ligase family.

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Oligomerization of the HECT ubiquitin ligase NEDD4-2/NEDD4L is essential for polyubiquitin chain assembly

Cited by 10 publications

References 60 publications

Analysis of ubiquitin recognition by the HECT ligase E6AP provides insight into its linkage specificity

Analysis of ubiquitin recognition by the HECT ligase E6AP provides insight into its linkage specificity

Host E3 ubiquitin ligase ITCH mediatesToxoplasma gondiieffector GRA35-triggered NLRP1 inflammasome activation and cell-autonomous immunity

Redefining the catalytic HECT domain boundaries for the HECT E3 ubiquitin ligase family

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