HACE1 (HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1)
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ABSTRACT:
SUBMITTER: Cui F
PROVIDER: S-EPMC8547717 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Sprouty (Spry) proteins are important regulators of receptor tyrosine kinase signaling in development and disease. Alterations in cellular Spry content have been associated with certain forms of cancers and also in cardiovascular diseases. Thus, understanding the mechanisms that regulate cellular Spry levels are important. Herein, we demonstrate that Spry1 and Spry2, but not Spry3 or Spry4, associate with the HECT domain family E3 ubiquitin ligase, Nedd4. The Spry2/Nedd4 association involves the WW domains of Nedd4 and requires phosphorylation of the Mnk2 kinase sites, Ser(112) and Ser(121), on Spry2. The phospho-Ser(112/121) region on Spry2 that binds WW domains of Nedd4 is a novel non-canonical WW domain binding region that does not contain Pro residues after phospho-Ser. Endogenous and overexpressed Nedd4 polyubiquitinate Spry2 via Lys(48) on ubiquitin and decrease its stability. Silencing of endogenous Nedd4 increased the cellular Spry2 content and attenuated fibroblast growth factor-elicited ERK1/2 activation that was reversed when elevations in Spry2 levels were prevented by Spry2-specific small interfering RNA. Mnk2 silencing decreased Spry2-Nedd4 interactions and also augmented the ability of Spry2 to inhibit fibroblast growth factor signaling. This is the first report demonstrating the regulation of cellular Spry content and its ability to modulate receptor tyrosine kinase signaling by a HECT domain-containing E3 ubiquitin ligase.
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Project description: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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Project description:Ubiquitination is one of the most prevalent protein post-translational modifications in eukaryotes, and its malfunction is associated with a variety of human diseases. Despite the significance of this process, the molecular mechanisms that govern the regulation of ubiquitination remain largely unknown. Here we used a combination of yeast proteome chip assays, genetic screening, and in vitro/in vivo biochemical analyses to identify and characterize eight novel in vivo substrates of the ubiquitinating enzyme Rsp5, a homolog of the human ubiquitin-ligating enzyme Nedd4, in yeast. Our analysis of the effects of a deubiquitinating enzyme, Ubp2, demonstrated that an accumulation of Lys-63-linked polyubiquitin chains results in processed forms of two substrates, Sla1 and Ygr068c. Finally we showed that the localization of another newly identified substrate, Rnr2, is Rsp5-dependent. We believe that our approach constitutes a paradigm for the functional dissection of an enzyme with pleiotropic effects.
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Project description:Dishevelled is a pivot in Wnt signal transduction, controlling both ?-catenin-dependent transcription to specify proliferative cell fates, and cell polarity and other non-nuclear events in post-mitotic cells. In response to Wnt signals, or when present at high levels, Dishevelled forms signalosomes by dynamic polymerization. Its levels are controlled by ubiquitylation, mediated by various ubiquitin ligases, including NEDD4 family members that bind to a conserved PPxY motif in Dishevelled (mammalian Dvl1-3). Here, we show that Dvl2 binds to the ubiquitin ligase WWP2 and unlocks its ligase activity from autoinhibition. This disinhibition of WWP2 depends on several features of Dvl2 including its PPxY motif and to a lesser extent its DEP domain, but crucially on the ability of Dvl2 to polymerize, indicating that WWP2 is activated in Wnt signalosomes. We show that Notch intracellular domains are substrates for Dvl-activated WWP2 and their transcriptional activity is consequently reduced, providing a molecular mechanism for cross-talk between Wnt and Notch signalling. These regulatory interactions are conserved in Drosophila whose WWP2 orthologue, Suppressor-of-deltex, downregulates Notch signalling upon activation by Dishevelled in developing wing tissue. Attentuation of Notch signalling by Dishevelled signalosomes could be important during the transition of cells from the proliferative to the post-mitotic state.
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Project description:Candida albicans is the most common cause of fungal infections in humans, and disseminated candidiasis has become one of the leading causes of hospital-acquired bloodstream infections with a high mortality rate. However, little is known about the host-pathogen interactions and the mechanisms of antifungal immunity. Here, we report that Nedd4 (neuronal precursor cell-expressed developmentally downregulated 4) is essential for signaling through Dectin-1 and Dectin-2/3. We showed that mice that lack Nedd4 globally or only in the myeloid compartment are highly susceptible to systemic C. albicans infection, which correlates with heightened organ fungal burden, defective inflammatory response, impaired leukocyte recruitment to the kidneys, and defective reactive oxygen species expression by granulocytes. At the molecular level, Nedd4 -/- macrophages displayed impaired activation of TGF-β-activating kinase-1 and NF-κB, but normal activation of spleen tyrosine kinase and protein kinase C-δ on C. albicans yeast and hyphal infections. These data suggest that Nedd4 regulates signaling events downstream of protein kinase C-δ but upstream of or at TGF-β-activating kinase-1.
| S-EPMC8324540 | biostudies-literature
Project description:Here we describe a systematic structure-function analysis of the human ubiquitin (Ub) E2 conjugating proteins, consisting of the determination of 15 new high-resolution three-dimensional structures of E2 catalytic domains, and autoubiquitylation assays for 26 Ub-loading E2s screened against a panel of nine different HECT (homologous to E6-AP carboxyl terminus) E3 ligase domains. Integration of our structural and biochemical data revealed several E2 surface properties associated with Ub chain building activity; (1) net positive or neutral E2 charge, (2) an "acidic trough" located near the catalytic Cys, surrounded by an extensive basic region, and (3) similarity to the previously described HECT binding signature in UBE2L3 (UbcH7). Mass spectrometry was used to characterize the autoubiquitylation products of a number of functional E2-HECT pairs, and demonstrated that HECT domains from different subfamilies catalyze the formation of very different types of Ub chains, largely independent of the E2 in the reaction. Our data set represents the first comprehensive analysis of E2-HECT E3 interactions, and thus provides a framework for better understanding the molecular mechanisms of ubiquitylation.
| S-EPMC3412965 | biostudies-literature
Project description:UBR5 ubiquitin ligase (also known as EDD, Rat100 or hHYD) is a member of the E3 protein family of HECT (homologous to E6-AP C-terminus) ligases as it contains a C-terminal HECT domain. In ubiquitination cascades involving E3s of the HECT class, ubiquitin is transferred from an associated E2 ubiquitin-conjugating enzyme to the acceptor cysteine of the HECT domain, which consists of structurally distinct N- and C-lobes connected by a flexible linker. Here, the high-resolution crystal structure of the C-lobe of the HECT domain of human UBR5 is presented. The structure reveals important features that are unique compared with other HECT domains. In particular, a distinct four-residue insert in the second helix elongates this helix, resulting in a strikingly different orientation of the preceding loop. This protruding loop is likely to contribute to specificity towards the E2 ubiquitin-conjugating enzyme UBCH4, which is an important functional partner of UBR5. Ubiquitination assays showed that the C-lobe of UBR5 is able to form a thioester-linked E3-ubiquitin complex, although it does not physically interact with UBCH4 in NMR experiments. This study contributes to a better understanding of UBR5 ubiquitination activity.
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Project description:Homologous to E6AP C-terminal (HECT) ubiquitin (Ub) ligases (E3s) are a large class of enzymes that bind to their substrates and catalyze ubiquitination through the formation of a Ub thioester intermediate. The mechanisms by which these E3s assemble polyubiquitin chains on their substrates remain poorly defined. We report here that the Nedd4 family HECT E3, WWP1, assembles substrate-linked Ub chains containing Lys-63, Lys-48, and Lys-11 linkages (Lys-63 > Lys-48 > Lys-11). Our results demonstrate that WWP1 catalyzes the formation of Ub chains through a sequential addition mechanism, in which Ub monomers are transferred in a successive fashion to the substrate, and that ubiquitination by WWP1 requires the presence of a low-affinity, noncovalent Ub-binding site within the HECT domain. Unexpectedly, we find that the formation of Ub chains by WWP1 occurs in two distinct phases. In the first phase, chains are synthesized in a unidirectional manner and are linked exclusively through Lys-63 of Ub. In the second phase, chains are elongated in a multidirectional fashion characterized by the formation of mixed Ub linkages and branched structures. Our results provide new insight into the mechanism of Ub chain formation employed by Nedd4 family HECT E3s and suggest a framework for understanding how this family of E3s generates Ub signals that function in proteasome-independent and proteasome-dependent pathways.
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Project description:HACE1 is an ankyrin repeat (AKR) containing HECT-type E3 ubiquitin ligase that interacts with and ubiquitinates multiple substrates. While HACE1 is a well-known tumor suppressor, its structure and mode of ubiquitination are not understood. The authors present the cryo-EM structures of human HACE1 along with in vitro functional studies that provide insights into how the enzymatic activity of HACE1 is regulated. HACE1 comprises of an N-terminal AKR domain, a middle (MID) domain, and a C-terminal HECT domain. Its unique G-shaped architecture interacts as a homodimer, with monomers arranged in an antiparallel manner. In this dimeric arrangement, HACE1 ubiquitination activity is hampered, as the N-terminal helix of one monomer restricts access to the C-terminal domain of the other. The in vitro ubiquitination assays, hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis, mutagenesis, and in silico modeling suggest that the HACE1 MID domain plays a crucial role along with the AKRs in RAC1 substrate recognition.
| S-EPMC10520629 | biostudies-literature
Project description:UnlabelledThe ubiquitin-proteasome system is targeted by many viruses that have evolved strategies to redirect host ubiquitination machinery. Members of the genus Chlorovirus are proposed to share an ancestral lineage with a broader group of related viruses, nucleo-cytoplasmic large DNA viruses (NCLDV). Chloroviruses encode an Skp1 homolog and ankyrin repeat (ANK) proteins. Several chlorovirus-encoded ANK repeats contain C-terminal domains characteristic of cellular F-boxes or related NCLDV chordopox PRANC (pox protein repeats of ankyrin at C-terminal) domains. These observations suggested that this unique combination of Skp1 and ANK repeat proteins might form complexes analogous to the cellular Skp1-Cul1-F-box (SCF) ubiquitin ligase complex. We identified two ANK proteins from the prototypic chlorovirus Paramecium bursaria chlorella virus-1 (PBCV-1) that functioned as binding partners for the virus-encoded Skp1, proteins A682L and A607R. These ANK proteins had a C-terminal Skp1 interactional motif that functioned similarly to cellular F-box domains. A C-terminal motif of ANK protein A682L binds Skp1 proteins from widely divergent species. Yeast two-hybrid analyses using serial domain deletion constructs confirmed the C-terminal localization of the Skp1 interactional motif in PBCV-1 A682L. ANK protein A607R represents an ANK family with one member present in all 41 sequenced chloroviruses. A comprehensive phylogenetic analysis of these related ANK and viral Skp1 proteins suggested partnered function tailored to the host alga or common ancestral heritage. Here, we show protein-protein interaction between corresponding family clusters of virus-encoded ANK and Skp1 proteins from three chlorovirus types. Collectively, our results indicate that chloroviruses have evolved complementing Skp1 and ANK proteins that mimic cellular SCF-associated proteins.ImportanceViruses have evolved ways to direct ubiquitination events in order to create environments conducive to their replication. As reported in the manuscript, the large chloroviruses encode several components involved in the SCF ubiquitin ligase complex including a viral Skp1 homolog. Studies on how chloroviruses manipulate their host algal ubiquitination system will provide insights toward viral protein mimicry, substrate recognition, and key interactive domains controlling selective protein degradation. These findings may also further understanding of the evolution of other large DNA viruses, like poxviruses, that are reported to share the same monophyly lineage as chloroviruses.
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