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1.  Proteomic snapshot of the EGF-induced ubiquitin network 
In this work, the authors report the first proteome-wide analysis of EGF-regulated ubiquitination, revealing surprisingly pervasive growth factor-induced ubiquitination across a broad range of cellular systems and signaling pathways.
Epidermal growth factor (EGF) triggers a novel ubiquitin (Ub)-based signaling cascade that appears to intersect both housekeeping and regulatory circuitries of cellular physiology.The EGF-regulated Ubiproteome includes scores ubiquitinating and deubiquitinating enzymes, suggesting that the Ub signal might be rapidly transmitted and amplified through the Ub machinery.The EGF-Ubiproteome overlaps significantly with the EGF-phosphotyrosine proteome, pointing to a possible crosstalk between these two signaling mechanisms.The significant number of biological insights uncovered in our study (among which EphA2 as a novel, downstream ubiquitinated target of EGF receptor) illustrates the general relevance of such proteomic screens and calls for further analysis of the dynamics of the Ubiproteome.
Ubiquitination is a process by which one or more ubiquitin (Ub) monomers or chains are covalently attached to target proteins by E3 ligases. Deubiquitinating enzymes (DUBs) revert Ub conjugation, thus ensuring a dynamic equilibrium between pools of ubiquitinated and deubiquitinated proteins (Amerik and Hochstrasser, 2004). Traditionally, ubiquitination has been associated with protein degradation; however, it is now becoming apparent that this post-translation modification is an important signaling mechanism that can modulate the function, localization and protein/protein interaction abilities of targets (Mukhopadhyay and Riezman, 2007; Ravid and Hochstrasser, 2008).
One of the best-characterized signaling pathways involving ubiquitination is the epidermal growth factor (EGF)-induced pathway. Upon EGF stimulation, a variety of proteins are subject to Ub modification. These include the EGF receptor (EGFR), which undergoes both multiple monoubiquitination (Haglund et al, 2003) and K63-linked polyubiquitination (Huang et al, 2006), as well as components of the downstream endocytic machinery, which are modified by monoubiquitination (Polo et al, 2002; Mukhopadhyay and Riezman, 2007). Ubiquitination of the EGFR has been shown to have an impact on receptor internalization, intracellular sorting and metabolic fate (Acconcia et al, 2009). However, little is known about the wider impact of EGF-induced ubiquitination on cellular homeostasis and on the pleiotropic biological functions of the EGFR. In this paper, we attempt to address this issue by characterizing the repertoire of proteins that are ubiquitinated upon EGF stimulation, i.e., the EGF-Ubiproteome.
To achieve this, we employed two different purification procedures (endogenous—based on the purification of proteins modified by endogenous Ub from human cells; tandem affinity purification (TAP)—based on the purification of proteins modified by an ectopically expressed tagged-Ub from mouse cells) with stable isotope labeling with amino acids in cell culture-based MS to obtain both steady-state Ubiproteomes and EGF-induced Ubiproteomes. The steady-state Ubiproteomes consist of 1175 and 582 unambiguously identified proteins for the endogenous and TAP approaches, respectively, which we largely validated. Approximately 15% of the steady-state Ubiproteome was EGF-regulated at 10 min after stimulation; 176 of 1175 in the endogenous approach and 105 of 582 in the TAP approach. Both hyper- and hypoubiquitinated proteins were detected, indicating that EGFR-mediated signaling can modulate the ubiquitin network in both directions. Interestingly, many E2, E3 and DUBs were present in the EGF-Ubiproteome, suggesting that the Ub signal might be rapidly transmitted and amplified through the Ub machinery. Moreover, analysis of Ub-chain topology, performed using mass spectrometry and specific abs, suggested that the K63-linkage was the major Ub-based signal in the EGF-induced pathway.
To obtain a higher-resolution molecular picture of the EGF-regulated Ub network, we performed a network analysis on the non-redundant EGF-Ubiproteome (265 proteins). This analysis revealed that in addition to well-established liaisons with endocytosis-related pathways, the EGF-Ubiproteome intersects many circuitries of intracellular signaling involved in, e.g., DNA damage checkpoint regulation, cell-to-cell adhesion mechanisms and actin remodeling (Figure 5A).
Moreover, the EGF-Ubiproteome was enriched in hubs, proteins that can establish multiple protein/protein interaction and thereby regulate the organization of networks. These results are indicative of a crosstalk between EGFR-activated pathways and other signaling pathways through the Ub-network.
As EGF binding to its receptor also triggers a series of phosphorylation events, we examined whether there was any overlap between our EGF-Ubiproteome and published EGF-induced phosphotyrosine (pY) proteomes (Blagoev et al, 2004; Oyama et al, 2009; Hammond et al, 2010). We observed a significant overlap between ubiquitinated and pY proteins: 23% (61 of 265) of the EGF-Ubiproteome proteins were also tyrosine phosphorylated. Pathway analysis of these 61 Ub/pY-containing proteins revealed a significant enrichment in endocytic and signal-transduction pathways, while ‘hub analysis' revealed that Ub/pY-containing proteins are enriched in highly connected proteins to an even greater extent than Ub-containing proteins alone. These data point to a complex interplay between the Ub and pY networks and suggest that the flow of information from the receptor to downstream signaling molecules is driven by two complementary and interlinked enzymatic cascades: kinases/phosphatases and E3 ligases/DUBs.
Finally, we provided a proof of principle of the biological relevance of our EGF-Ubiproteome. We focused on EphA2, a receptor tyrosine kinase, which is involved in development and is often overexpressed in cancer (Pasquale, 2008). We started from the observation that EphA2 is present in the EGF-Ubiproteome and that proteins of the EGF-Ubiproteome are enriched in the Ephrin receptor signaling pathway(s). We confirmed the MS data by demonstrating that the EphA2 is ubiquitinated upon EGF stimulation. Moreover, EphA2 also undergoes tyrosine phosphorylation, indicating crosstalk between the two receptors. The EGFR kinase domain was essential for these modifications of EphA2, and a partial co-internalization with EGFR upon EGF activation was clearly detectable. Finally, we demonstrated by knockdown of EphA2 in MCF10A cells that this receptor is critically involved in EGFR biological outcomes, such as proliferation and migration (Figure 7).
Overall, our results unveil the complex impact of growth factor signaling on Ub-based intracellular networks to levels that extend well beyond what might have been expected and highlight the ‘resource' feature of our EGF-Ubiproteome.
The activity, localization and fate of many cellular proteins are regulated through ubiquitination, a process whereby one or more ubiquitin (Ub) monomers or chains are covalently attached to target proteins. While Ub-conjugated and Ub-associated proteomes have been described, we lack a high-resolution picture of the dynamics of ubiquitination in response to signaling. In this study, we describe the epidermal growth factor (EGF)-regulated Ubiproteome, as obtained by two complementary purification strategies coupled to quantitative proteomics. Our results unveil the complex impact of growth factor signaling on Ub-based intracellular networks to levels that extend well beyond what might have been expected. In addition to endocytic proteins, the EGF-regulated Ubiproteome includes a large number of signaling proteins, ubiquitinating and deubiquitinating enzymes, transporters and proteins involved in translation and transcription. The Ub-based signaling network appears to intersect both housekeeping and regulatory circuitries of cellular physiology. Finally, as proof of principle of the biological relevance of the EGF-Ubiproteome, we demonstrated that EphA2 is a novel, downstream ubiquitinated target of epidermal growth factor receptor (EGFR), critically involved in EGFR biological responses.
doi:10.1038/msb.2010.118
PMCID: PMC3049407  PMID: 21245847
EGF; network; proteomics; signaling; ubiquitin
2.  Mechanism of ubiquitin ligation and lysine prioritization by a HECT E3 
eLife  2013;2:e00828.
Ubiquitination by HECT E3 enzymes regulates myriad processes, including tumor suppression, transcription, protein trafficking, and degradation. HECT E3s use a two-step mechanism to ligate ubiquitin to target proteins. The first step is guided by interactions between the catalytic HECT domain and the E2∼ubiquitin intermediate, which promote formation of a transient, thioester-bonded HECT∼ubiquitin intermediate. Here we report that the second step of ligation is mediated by a distinct catalytic architecture established by both the HECT E3 and its covalently linked ubiquitin. The structure of a chemically trapped proxy for an E3∼ubiquitin-substrate intermediate reveals three-way interactions between ubiquitin and the bilobal HECT domain orienting the E3∼ubiquitin thioester bond for ligation, and restricting the location of the substrate-binding domain to prioritize target lysines for ubiquitination. The data allow visualization of an E2-to-E3-to-substrate ubiquitin transfer cascade, and show how HECT-specific ubiquitin interactions driving multiple reactions are repurposed by a major E3 conformational change to promote ligation.
DOI: http://dx.doi.org/10.7554/eLife.00828.001
eLife digest
Ubiquitin is a small protein that can be covalently linked to other, ‘target’, proteins in a cell to influence their behavior. Ubiquitin can be linked to its targets either as single copies or as polyubiquitin chains in which several ubiquitin molecules are bound end-on-end to each other, with one end of the chain attached to the target protein. A multi-step cascade involving enzymes known as E1, E2, and E3 adds ubiquitin to its targets. These enzymes function in a manner like runners in a relay, with ubiquitin a baton that is passed from E1 to E2 to E3 to the target.
The E3 enzyme is a ligase that catalyzes the formation of a new chemical bond between a ubiquitin and its target. There are approximately 600 different E3 enzymes in human cells that regulate a wide variety of target proteins. A major class of E3 enzymes, called HECT E3s, attaches ubiquitin to its targets in a unique two-step mechanism: the E2 enzymes covalently link a ubiquitin to a HECT E3 to form a complex that subsequently transfers the ubiquitin to its target protein. The ubiquitin is typically added to a particular amino acid, lysine, on the target protein, but the details of how HECT E3s execute this transfer are not well understood. To address this issue, Kamadurai et al. investigate how Rsp5, a HECT E3 ligase in yeast, attaches ubiquitin to a target protein called Sna3.
All HECT E3s have a domain—the HECT domain—that catalyzes the transfer of ubiquitin to its target protein. This domain consists of two sub-structures: the C-lobe, which can receive ubiquitin from E2 and then itself become linked to ubiquitin, and the N-lobe. These lobes were previously thought to adopt various orientations relative to each other to deliver ubiquitin to sites on different target proteins (including to multiple lysines on a single target protein). Unexpectedly, Kamadurai et al. find that in order to transfer the ubiquitin to Sna3, Rsp5 adopts a discrete HECT domain architecture that creates an active site in which parts of the C-lobe and the N-lobe, which are normally separated, are brought together with a ubiquitin molecule. This architecture also provides a mechanism that dictates which substrate lysines can be ubiquitinated based on how accessible they are to this active site.
The same regions of Rsp5 transfer ubiquitin to targets other than Sna3, suggesting that a uniform mechanism—which Kamadurai et al. show is conserved in two related human HECT E3 ligases—might transfer ubiquitin to all its targets. These studies therefore represent a significant step toward understanding how a major class of E3 enzymes modulates the functions of their targets.
DOI: http://dx.doi.org/10.7554/eLife.00828.002
doi:10.7554/eLife.00828
PMCID: PMC3738095  PMID: 23936628
ubiquitin; HECT; E3 ligase; E2 conjugating enzyme; NEDD4; Rsp5; S. cerevisiae
3.  Hedgehog-Regulated Ubiquitination Controls Smoothened Trafficking and Cell Surface Expression in Drosophila 
PLoS Biology  2012;10(1):e1001239.
Hedgehog transduces signal by promoting cell surface expression of the seven-transmembrane protein Smoothened (Smo) in Drosophila, but the underlying mechanism remains unknown. Here we demonstrate that Smo is downregulated by ubiquitin-mediated endocytosis and degradation, and that Hh increases Smo cell surface expression by inhibiting its ubiquitination. We find that Smo is ubiquitinated at multiple Lysine residues including those in its autoinhibitory domain (SAID), leading to endocytosis and degradation of Smo by both lysosome- and proteasome-dependent mechanisms. Hh inhibits Smo ubiquitination via PKA/CK1-mediated phosphorylation of SAID, leading to Smo cell surface accumulation. Inactivation of the ubiquitin activating enzyme Uba1 or perturbation of multiple components of the endocytic machinery leads to Smo accumulation and Hh pathway activation. In addition, we find that the non-visual β-arrestin Kurtz (Krz) interacts with Smo and acts in parallel with ubiquitination to downregulate Smo. Finally, we show that Smo ubiquitination is counteracted by the deubiquitinating enzyme UBPY/USP8. Gain and loss of UBPY lead to reciprocal changes in Smo cell surface expression. Taken together, our results suggest that ubiquitination plays a key role in the downregulation of Smo to keep Hh pathway activity off in the absence of the ligand, and that Hh-induced phosphorylation promotes Smo cell surface accumulation by inhibiting its ubiquitination, which contributes to Hh pathway activation.
Author Summary
The Hedgehog (Hh) family of secreted proteins governs cell growth and patterning in diverse species ranging from Drosophila to human. Hh signals across the cell surface membrane by regulating the subcellular location and conformation of a membrane protein called Smoothened (Smo). In Drosophila, Smo accumulates on the cell surface in response to Hh, whereas in the absence of Hh it is internalized and degraded. The molecular mechanisms that control this intracellular trafficking and degradation of Smo were unknown, but here we show that Smo is modified by attachment of several molecules of a small protein called ubiquitin, which tags it for internalization and degradation within the cell. Hh inhibits this ubiquitination of Smo by inducing another modification, phosphorylation, of its intracellular tail by two types of protein kinase enzymes. This loss of ubiquitination and gain of phosphorylation causes the accumulation of Smo at the cell surface. What's more, we find that another protein called Kurtz interacts with Smo and acts in parallel with the ubiquitination process to promote internalization of Smo, and that the deubiquitinating enzyme UBPY/USP8 counteracts ubiquitination of Smo to promote its cell surface accumulation. Our study demonstrates that reversible ubiquitination plays a key role in regulating Smo trafficking to and from the cell surface and thus it provides novel insights into the mechanism of Hh signaling from the outside to the inside of the cell.
doi:10.1371/journal.pbio.1001239
PMCID: PMC3254653  PMID: 22253574
4.  A human polymorphism affects NEDD4L subcellular targeting by leading to two isoforms that contain or lack a C2 domain 
BMC Cell Biology  2009;10:26.
Background
Ubiquitination serves multiple cellular functions, including proteasomal degradation and the control of stability, function, and intracellular localization of a wide variety of proteins. NEDD4L is a member of the HECT class of E3 ubiquitin ligases. A defining feature of NEDD4L protein isoforms is the presence or absence of an amino-terminal C2 domain, a class of subcellular, calcium-dependent targeting domains. We previously identified a common variant in human NEDD4L that generates isoforms that contain or lack a C2 domain.
Results
To address the potential functional significance of the NEDD4L common variant on NEDD4L subcellular localization, NEDD4L isoforms that either contained or lacked a C2 domain were tagged with enhanced green fluorescent protein, transfected into Xenopus laevis kidney epithelial cells, and imaged by performing confocal microscopy on live cells. We report that the presence or absence of this C2 domain exerts differential effects on the subcellular distribution of NEDD4L, the ability of C2 containing and lacking NEDD4L isoforms to mobilize in response to a calcium stimulus, and the intracellular transport of subunits of the NEDD4L substrate, ENaC. Furthermore, the ability of the C2-containing isoform to influence β-ENaC mobilization from intracellular pools involves the NEDD4L active site for ubiquitination. We propose a model to account for the potential impact of this common genetic variant on protein function at the cellular level.
Conclusion
NEDD4L isoforms that contain or lack a C2 domain target different intracellular locations. Additionally, whereas the C2-containing NEDD4L isoform is capable of shuttling between the plasma membrane and intracellular compartments in response to calcium stimulus the C2-lacking isoform can not. The C2-containing isoform differentially affects the mobilization of ENaC subunits from intracellular pools and this trafficking step requires NEDD4L ubiquitin ligase activity. This observation suggests a new mechanism for the requirement for the PY motif in cAMP-mediated exocytosis of ENaC. We have elucidated how a common genetic variant can underlie significant functional diversity in NEDD4L at the cellular level. We propose a model that describes how that functional variation may influence blood pressure. Moreover, our observations regarding differential function of the NEDD4L isoforms may impact other aspects of physiology that involve this ubiquitin ligase.
doi:10.1186/1471-2121-10-26
PMCID: PMC2678989  PMID: 19364400
5.  Transport of LAPTM5 to lysosomes requires association with the ubiquitin ligase Nedd4, but not LAPTM5 ubiquitination 
The Journal of Cell Biology  2006;175(4):631-645.
LAPTM5 is a lysosomal transmembrane protein expressed in immune cells. We show that LAPTM5 binds the ubiquitin-ligase Nedd4 and GGA3 to promote LAPTM5 sorting from the Golgi to the lysosome, an event that is independent of LAPTM5 ubiquitination. LAPTM5 contains three PY motifs (L/PPxY), which bind Nedd4-WW domains, and a ubiquitin-interacting motif (UIM) motif. The Nedd4–LAPTM5 complex recruits ubiquitinated GGA3, which binds the LAPTM5-UIM; this interaction does not require the GGA3-GAT domain. LAPTM5 mutated in its Nedd4-binding sites (PY motifs) or its UIM is retained in the Golgi, as is LAPTM5 expressed in cells in which Nedd4 or GGA3 is knocked-down with RNAi. However, ubiquitination-impaired LAPTM5 can still traffic to the lysosome, suggesting that Nedd4 binding to LAPTM5, not LAPTM5 ubiquitination, is required for targeting. Interestingly, Nedd4 is also able to ubiquitinate GGA3. These results demonstrate a novel mechanism by which the ubiquitin-ligase Nedd4, via interactions with GGA3 and cargo (LAPTM5), regulates cargo trafficking to the lysosome without requiring cargo ubiquitination.
doi:10.1083/jcb.200603001
PMCID: PMC2064599  PMID: 17116753
6.  Proteomic analysis reveals Hrs UIM-mediated ubiquitin signaling in multiple cellular processes 
The FEBS journal  2009;276(1):118-131.
Summary
Despite the critical importance of protein ubiquitination in regulation of diverse cellular processes, the molecular mechanisms by which cells recognize and transmit ubiquitin signals remain poorly understood. The endosomal sorting machinery component hepatocyte growth factor regulated tyrosine kinase substrate (Hrs) contains an ubiquitin-interacting motif (UIM), which is believed to bind ubiquitinated membrane cargo proteins and mediate their sorting to the lysosomal degradation pathway. To gain insight into the role of Hrs UIM-mediated ubiquitin signaling in cells, we performed a proteomic screen for Hrs UIM-interacting ubiquitinated proteins in human brain by using an in vitro expression cloning (IVEC) screening approach. We have identified 48 ubiquitinated proteins that are specifically recognized by the UIM domain of Hrs. Among them, 12 are membrane proteins which are likely to be Hrs cargo proteins, and 4 are membrane protein-associated adaptor proteins whose ubiquitination may act as a signal to target their associated membrane cargo for Hrs-mediated endosomal sorting. Other classes of the identified proteins include components of the vesicular trafficking machinery, cell signaling molecules, proteins associated with the cytoskeleton and cytoskeleton-dependent transport, and enzymes involved in ubiquitination and metabolism, suggesting the involvement of Hrs UIM-mediated ubiquitin signaling in regulation of multiple cellular processes. We have characterized the ubiquitination of two identified proteins, Munc18-1 and Hsc70, and their interaction with Hrs UIM and provided functional evidence supporting a role for Hsc70 in regulation of Hrs-mediated endosome-to-lysosome trafficking.
doi:10.1111/j.1742-4658.2008.06760.x
PMCID: PMC2647816  PMID: 19019082
Ubiquitination; ubiquitin-interacting motif; Hrs; endocytic trafficking; in vitro expression cloning
7.  Stabilization of the E3 Ubiquitin Ligase Nrdp1 by the Deubiquitinating Enzyme USP8 
Molecular and Cellular Biology  2004;24(17):7748-7757.
Nrdp1 is a RING finger-containing E3 ubiquitin ligase that physically interacts with and regulates steady-state cellular levels of the ErbB3 and ErbB4 receptor tyrosine kinases and has been implicated in the degradation of the inhibitor-of-apoptosis protein BRUCE. Here we demonstrate that the Nrdp1 protein undergoes efficient proteasome-dependent degradation and that mutations in its RING finger domain that disrupt ubiquitin ligase activity enhance stability. These observations suggest that Nrdp1 self-ubiquitination and stability could play an important role in regulating the activity of this protein. Using affinity chromatography, we identified the deubiquitinating enzyme USP8 (also called Ubpy) as a protein that physically interacts with Nrdp1. Nrdp1 and USP8 could be coimmunoprecipitated, and in transfected cells USP8 specifically bound to Nrdp1 but not cbl, a RING finger E3 ligase involved in ligand-stimulated epidermal growth factor receptor down-regulation. The USP8 rhodanese and catalytic domains mediated Nrdp1 binding. USP8 markedly enhanced the stability of Nrdp1, and a point mutant that disrupts USP8 catalytic activity destabilized endogenous Nrdp1. Our results indicate that Nrdp1 is a specific target for the USP8 deubiquitinating enzyme and are consistent with a model where USP8 augments Nrdp1 activity by mediating its stabilization.
doi:10.1128/MCB.24.17.7748-7757.2004
PMCID: PMC506982  PMID: 15314180
8.  Structural Basis for the Ubiquitin-Linkage Specificity and deISGylating Activity of SARS-CoV Papain-Like Protease 
PLoS Pathogens  2014;10(5):e1004113.
Severe acute respiratory syndrome coronavirus (SARS-CoV) encodes a papain-like protease (PLpro) with both deubiquitinating (DUB) and deISGylating activities that are proposed to counteract the post-translational modification of signaling molecules that activate the innate immune response. Here we examine the structural basis for PLpro's ubiquitin chain and interferon stimulated gene 15 (ISG15) specificity. We present the X-ray crystal structure of PLpro in complex with ubiquitin-aldehyde and model the interaction of PLpro with other ubiquitin-chain and ISG15 substrates. We show that PLpro greatly prefers K48- to K63-linked ubiquitin chains, and ISG15-based substrates to those that are mono-ubiquitinated. We propose that PLpro's higher affinity for K48-linked ubiquitin chains and ISG15 stems from a bivalent mechanism of binding, where two ubiquitin-like domains prefer to bind in the palm domain of PLpro with the most distal ubiquitin domain interacting with a “ridge” region of the thumb domain. Mutagenesis of residues within this ridge region revealed that these mutants retain viral protease activity and the ability to catalyze hydrolysis of mono-ubiquitin. However, a select number of these mutants have a significantly reduced ability to hydrolyze the substrate ISG15-AMC, or be inhibited by K48-linked diubuiquitin. For these latter residues, we found that PLpro antagonism of the nuclear factor kappa-light-chain-enhancer of activated B-cells (NFκB) signaling pathway is abrogated. This identification of key and unique sites in PLpro required for recognition and processing of diubiquitin and ISG15 versus mono-ubiquitin and protease activity provides new insight into ubiquitin-chain and ISG15 recognition and highlights a role for PLpro DUB and deISGylase activity in antagonism of the innate immune response.
Author Summary
All coronaviruses such as the SARS virus and the recently identified Middle East Respiratory Syndrome (MERS) virus encode in their genomes at least one papain-like protease (PLpro) enzyme that has two distinct functions in viral pathogenesis. The first function is to process the viral polyprotein into individual proteins that are essential for viral replication. The second function is to remove ubiquitin and ISG15 proteins from host cell proteins, which likely helps coronaviruses short circuit the host's innate immune response. The 3-dimensional structure of SARS virus PLpro in complex with a human ubiquitin analog was determined and reveals how coronavirus PLpro enzymes strip ubiquitin and ISG15 from host cell proteins at the molecular level. A series of amino acid residues involved in interactions between PLpro and ubiquitin were mutated to identify which interactions are important only for the recognition of ubiquitin and ISG15 modified proteins by PLpro and not for recognition and cleaving of the viral polyprotein. The 3D structure of SARS PLpro with ubiquitin-aldehyde sheds significant new light into how PLpro interacts with ubiquitin-like molecules and provides a molecular road map for performing similar studies on other deadly coronaviruses such as MERS.
doi:10.1371/journal.ppat.1004113
PMCID: PMC4031219  PMID: 24854014
9.  Hse1, a Component of the Yeast Hrs-STAM Ubiquitin-sorting Complex, Associates with Ubiquitin Peptidases and a Ligase to Control Sorting Efficiency into Multivesicular Bodies 
Molecular Biology of the Cell  2007;18(1):324-335.
Ubiquitinated integral membrane proteins are delivered to the interior of the lysosome/vacuole for degradation. This process relies on specific ubiquitination of potential cargo and recognition of that Ub-cargo by sorting receptors at multiple compartments. We show that the endosomal Hse1-Vps27 sorting receptor binds to ubiquitin peptidases and the ubiquitin ligase Rsp5. Hse1 is linked to Rsp5 directly via a PY element within its C-terminus and through a novel protein Hua1, which recruits a complex of Rsp5, Rup1, and Ubp2. The SH3 domain of Hse1 also binds to the deubiquitinating protein Ubp7. Functional analysis shows that when both modes of Rsp5 association with Hse1 are altered, sorting of cargo that requires efficient ubiquitination for entry into the MVB is blocked, whereas sorting of cargo containing an in-frame addition of ubiquitin is normal. Further deletion of Ubp7 restores sorting of cargo when the Rsp5:Hse1 interaction is compromised suggesting that both ubiquitin ligases and peptidases associate with the Hse1-Vps27 sorting complex to control the ubiquitination status and sorting efficiency of cargo proteins. Additionally, we find that disruption of UBP2 and RUP1 inhibits MVB sorting of some cargos suggesting that Rsp5 requires association with Ubp2 to properly ubiquitinate cargo for efficient MVB sorting.
doi:10.1091/mbc.E06-06-0557
PMCID: PMC1751313  PMID: 17079730
10.  Multiple Interactions between the ESCRT Machinery and Arrestin-Related Proteins: Implications for PPXY-Dependent Budding ▿  
Journal of Virology  2010;85(7):3546-3556.
Late domains are short peptide sequences encoded by enveloped viruses to promote the final separation of the nascent virus from the infected cell. These amino acid motifs facilitate viral egress by interacting with components of the ESCRT (endosomal sorting complex required for transport) machinery, ultimately leading to membrane scission by recruiting ESCRT-III to the site of viral budding. PPXY late (L) domains present in viruses such as murine leukemia virus (MLV) or human T-cell leukemia virus type 1 (HTLV-1) access the ESCRT pathway via interaction with HECT ubiquitin ligases (WWP1, WWP2, and Itch). However, the mechanism of ESCRT-III recruitment in this context remains elusive. In this study, we tested the arrestin-related trafficking (ART) proteins, namely, ARRDC1 (arrestin domain-containing protein 1) to ARRDC4 and TXNIP (thioredoxin-interacting protein), for their ability to function as adaptors between HECT ubiquitin ligases and the core ESCRT machinery in PPXY-dependent budding. We present several lines of evidence in support of such a role: ARTs interact with HECT ubiquitin ligases, and they also exhibit multiple interactions with components of the ESCRT pathway, namely, ALIX and Tsg101, and perhaps with an as yet unidentified factor. Additionally, the ARTs can be recruited to the site of viral budding, and their overexpression results in a PPXY-specific inhibition of MLV budding. Lastly, we show that WWP1 changes the ubiquitination status of ARRDC1, suggesting that the ARTs may provide a platform for ubiquitination in PPXY-dependent budding. Taken together, our results support a model whereby ARTs are involved in PPXY-mediated budding by interacting with HECT ubiquitin ligases and providing several alternative routes for ESCRT-III recruitment.
doi:10.1128/JVI.02045-10
PMCID: PMC3067843  PMID: 21191027
11.  Regulation of Epithelial Sodium Channel Trafficking by Ubiquitination 
Amiloride-sensitive epithelial sodium (Na+) channels (ENaC) play a crucial role in Na+ transport and fluid reabsorption in the kidney, lung, and colon. The magnitude of ENaC-mediated Na+ transport in epithelial cells depends on the average open probability of the channels and the number of channels on the apical surface of epithelial cells. The number of channels in the apical membrane, in turn, depends upon a balance between the rate of ENaC insertion and the rate of removal from the apical membrane. ENaC is made up of three homologous subunits, α, β, and γ. The C-terminal domain of all three subunits is intracellular and contains a proline rich motif (PPxY). Mutations or deletion of this PPxY motif in the β and γ subunits prevent the binding of one isoform of a specific ubiquitin ligase, neural precursor cell expressed developmentally down-regulated protein (Nedd4-2) to the channel in vitro and in transfected cell systems, thereby impeding ubiquitin conjugation of the channel subunits. Ubiquitin conjugation would seem to imply that ENaC turnover is determined by the ubiquitin-proteasome system, but when MDCK cells are transfected with ENaC, ubiquitin conjugation apparently leads to lysosomal degradation. However, in untransfected epithelial cells (A6) expressing endogenous ENaC, ENaC appears to be degraded by the ubiquitin-proteasome system. Nonetheless, in both transfected and untransfected cells, the rate of ENaC degradation is apparently controlled by the rate of Nedd4-2–mediated ENaC ubiquitination. Controlling the rate of degradation is apparently important enough to have multiple, redundant pathways to control Nedd4-2 and ENaC ubiquitination.
doi:10.1513/pats.200909-096JS
PMCID: PMC3137150  PMID: 20160149
ENaC; degradation; trafficking; proteasome
12.  Regulation of Proteolysis by Human Deubiquitinating Enzymes 
Biochimica et biophysica acta  2013;1843(1):10.1016/j.bbamcr.2013.06.027.
The post-translational attachment of one or several ubiquitin molecules to a protein generates a variety of targeting signals that are used in many different ways in the cell. Ubiquitination can alter the activity, localization, protein-protein interactions or stability of the targeted protein. Further, a very large number of proteins are subject to regulation by ubiquitin-dependent processes, meaning that virtually all cellular functions are impacted by these pathways. Nearly a hundred enzymes from five different gene families (the deubiquitinating enzymes or DUBs), reverse this modification by hydrolyzing the (iso)peptide bond tethering ubiquitin to itself or the target protein. Four of these families are thiol proteases and one is a metalloprotease. DUBs of the Ubiquitin C-terminal Hydrolase (UCH) family act on small molecule adducts of ubiquitin, process the ubiquitin proprotein, and trim ubiquitin from the distal end of a polyubiquitin chain. Ubiquitin Specific Proteases (USP) tend to recognize and encounter their substrates by interaction of the variable regions of their sequence with the substrate protein directly, or with scaffolds or substrate adapters in multiprotein complexes. Ovarian Tumor (OTU) domain DUBs show remarkable specificity for different Ub chain linkages and may have evolved to recognize substrates on the basis of those linkages. The Josephin family of DUBs may specialize in distinguishing between polyubiquitin chains of different lengths. Finally, the JAB1/MPN+/MOV34 (JAMM) domain metalloproteases cleave the isopeptide bond near the attachment point of polyubiquitin and substrate, as well as being highly specific for the K63 poly-Ub linkage. These DUBs regulate proteolysis by: directly interacting with and co-regulating E3 ligases; altering the level of substrate ubiquitination; hydrolyzing or remodeling ubiquitinated and poly-ubiquitinated substrates; acting in specific locations in the cell and altering the localization of the target protein; and acting on proteasome bound substrates to facilitate or inhibit proteolysis. Thus, the scope and regulation of the ubiquitin pathway is very similar to that of phosphorylation, with the DUBs serving the same functions as the phosphatase.
doi:10.1016/j.bbamcr.2013.06.027
PMCID: PMC3833951  PMID: 23845989
Deubiquitinating enzyme; Ubiquitin; Poly-Ubiquitin; Proteolysis; Regulation
13.  Neuregulin-Induced ErbB3 Downregulation Is Mediated by a Protein Stability Cascade Involving the E3 Ubiquitin Ligase Nrdp1▿  
Molecular and Cellular Biology  2007;27(6):2180-2188.
The molecular mechanisms underlying epidermal growth factor (EGF) receptor tyrosine kinase down-regulation in response to growth factor binding are coming into focus and involve cbl-mediated receptor ubiquitination followed by lysosomal degradation. However, mechanisms underlying the ligand-stimulated degradation of the related receptor tyrosine kinases of the ErbB family do not involve cbl and remain unexplored. Previous studies have demonstrated that the E3 ubiquitin ligase Nrdp1 contributes to the maintenance of steady-state ErbB3 levels by mediating its growth factor-independent degradation. Here we demonstrate that treatment of cells with the ErbB3 ligand neuregulin-1 (NRG1) stabilizes the deubiquitinating enzyme USP8, which in turn stabilizes Nrdp1. The catalytic activity of USP8 is required for NRG1-induced Nrdp1 stabilization. We provide evidence that Akt-mediated phosphorylation of USP8 threonine residue T907 contributes to USP8 stability. Finally, we demonstrate that Nrdp1 or USP8 knockdown suppresses NRG1-induced ErbB3 ubiquitination and degradation in MCF7 breast cancer cells. We conclude that an NRG1-induced protein stability cascade involving USP8 and Nrdp1 mediates the down-regulation of ErbB3. Our observations raise the possibility that the ligand-induced augmentation of pathways involved in the maintenance of basal levels of receptor tyrosine kinases can contribute to ligand-stimulated down-regulation.
doi:10.1128/MCB.01245-06
PMCID: PMC1820496  PMID: 17210635
14.  Stabilization of an unusual salt bridge in ubiquitin by the extra C-terminal domain of the proteasome-associated deubiquitinase UCH37 as a mechanism of its exo specificity 
Biochemistry  2013;52(20):3564-3578.
Ubiquitination is countered by a group of enzymes collectively called deubiquitinases (DUBs) - about 100 of them can be found in the human genome. One of the most interesting aspects of these enzymes is the ability of some members to selectively recognize specific linkage types between ubiquitin in polyubiquitin chains and their endo and exo specificity. The structural basis of exo-specific deubiquitination catalyzed by a DUB is poorly understood. UCH37, a cysteine DUB conserved from fungi to humans, is a proteasome-associated factor that regulates the proteasome by sequentially cleaving polyubiquitin chains from their distal ends, i.e., by exo-specific deubiquitination. In addition to the catalytic domain, the DUB features a functionally uncharacterized UCH37-like domain (ULD), presumed to keep the enzyme in an inhibited state in its proteasome-free form. Herein we report the crystal structure of two constructs of UCH37 from Trichinella spiralis in complex with a ubiquitin-based suicide inhibitor, ubiquitin vinyl methyl ester (UbVME). These structures show that the ULD makes direct contact with ubiquitin stabilizing a highly unusual intra-molecular salt bridge between Lys48 and Glu51 of ubiquitin, an interaction that would be favored only with the distal ubiquitin but not with the internal ones in a Lys48-linked polyubiquitin chain. An inspection of 39 DUB-ubiquitin structures in the protein data bank reveals the uniqueness of the salt bridge in ubiquitin bound to UCH37, an interaction that disappears when the ULD is deleted, as revealed in the structure of the catalytic domain alone bound to UbVME. The structural data are consistent with previously reported mutational data on the mammalian enzyme, which, together with the fact that the ULD residues that bind to ubiquitin are conserved, points to a similar mechanism behind the exo specificity of the human enzyme. To the best of our knowledge, these data provide the only structural example so far of how the exo specificity of a DUB can be determined by its non-catalytic domain. Thus, our data show that, contrary to its proposed inhibitory role, the ULD actually contributes to substrate recognition and could be a major determinant of proteasome-associated function of UCH37. Moreover, our structures show that the unproductively oriented catalytic cysteine in the free enzyme is aligned correctly when ubiquitin binds, suggesting a mechanism for ubiquitin selectivity.
doi:10.1021/bi4003106
PMCID: PMC3898853  PMID: 23617878
15.  Functional Interchangeability of Late Domains, Late Domain Cofactors and Ubiquitin in Viral Budding 
PLoS Pathogens  2010;6(10):e1001153.
The membrane scission event that separates nascent enveloped virions from host cell membranes often requires the ESCRT pathway, which can be engaged through the action of peptide motifs, termed late (L-) domains, in viral proteins. Viral PTAP and YPDL-like L-domains bind directly to the ESCRT-I and ALIX components of the ESCRT pathway, while PPxY motifs bind Nedd4-like, HECT-domain containing, ubiquitin ligases (e.g. WWP1). It has been unclear precisely how ubiquitin ligase recruitment ultimately leads to particle release. Here, using a lysine-free viral Gag protein derived from the prototypic foamy virus (PFV), where attachment of ubiquitin to Gag can be controlled, we show that several different HECT domains can replace the WWP1 HECT domain in chimeric ubiquitin ligases and drive budding. Moreover, artificial recruitment of isolated HECT domains to Gag is sufficient to stimulate budding. Conversely, the HECT domain becomes dispensable if the other domains of WWP1 are directly fused to an ESCRT-1 protein. In each case where budding is driven by a HECT domain, its catalytic activity is essential, but Gag ubiquitination is dispensable, suggesting that ubiquitin ligation to trans-acting proteins drives budding. Paradoxically, however, we also demonstrate that direct fusion of a ubiquitin moiety to the C-terminus of PFV Gag can also promote budding, suggesting that ubiquitination of Gag can substitute for ubiquitination of trans-acting proteins. Depletion of Tsg101 and ALIX inhibits budding that is dependent on ubiquitin that is fused to Gag, or ligated to trans-acting proteins through the action of a PPxY motif. These studies underscore the flexibility in the ways that the ESCRT pathway can be engaged, and suggest a model in which the identity of the protein to which ubiquitin is attached is not critical for subsequent recruitment of ubiquitin-binding components of the ESCRT pathway and viral budding to proceed.
Author Summary
The release of an enveloped virus particle from an infected cell requires the separation of the viral and cell membranes. Many enveloped viruses accomplish this by parasitizing a set of cellular proteins, termed the ESCRT pathway, that normally separates cellular membranes from each other. In some cases, viral structural proteins encode peptides motifs that bind directly to, and thereby recruit, the ESCRT machinery. Alternatively, viruses can recruit enzymes, termed ubiquitin ligases, that bind to other proteins, and catalyze the addition of ubiquitin to them. It has, heretofore, been somewhat unclear precisely how the recruitment of ubiquitin ligases leads to the engagement of the ESCRT machinery. We show that the simple recruitment of a fragment of a ubiquitin ligase that is responsible for the addition of ubiquitin to other proteins is sufficient to drive virus particle release, even when it is not possible to attach ubiquitin to viral proteins. Paradoxically, we also found that simple attachment of ubiquitin to the same viral protein can also drive particle release. These results show that there is flexibility in the ways in which the ESCRT machinery can be recruited and how ubiquitin can be co-opted to enable this.
doi:10.1371/journal.ppat.1001153
PMCID: PMC2958808  PMID: 20975941
16.  A single ubiquitin is sufficient for cargo protein entry into MVBs in the absence of ESCRT ubiquitination 
The Journal of Cell Biology  2011;192(2):229-242.
While ESCRT-0 is ubiquitinated by the Rsp5 E3 ligase, loss of Rsp5 does not disrupt monoubiquitin-dependent sorting into multivesicular bodies.
ESCRTs (endosomal sorting complexes required for transport) bind and sequester ubiquitinated membrane proteins and usher them into multivesicular bodies (MVBs). As Ubiquitin (Ub)-binding proteins, ESCRTs themselves become ubiquitinated. However, it is unclear whether this regulates a critical aspect of their function or is a nonspecific consequence of their association with the Ub system. We investigated whether ubiquitination of the ESCRTs was required for their ability to sort cargo into the MVB lumen. Although we found that Rsp5 was the main Ub ligase responsible for ubiquitination of ESCRT-0, elimination of Rsp5 or elimination of the ubiquitinatable lysines within ESCRT-0 did not affect MVB sorting. Moreover, by fusing the catalytic domain of deubiquitinating peptidases onto ESCRTs, we could block ESCRT ubiquitination and the sorting of proteins that undergo Rsp5-dependent ubiquitination. Yet, proteins fused to a single Ub moiety were efficiently delivered to the MVB lumen, which strongly indicates that a single Ub is sufficient in sorting MVBs in the absence of ESCRT ubiquitination.
doi:10.1083/jcb.201008121
PMCID: PMC3172180  PMID: 21242292
17.  Structure of the HHARI Catalytic Domain Shows Glimpses of a HECT E3 Ligase 
PLoS ONE  2013;8(8):e74047.
The ubiquitin-signaling pathway utilizes E1 activating, E2 conjugating, and E3 ligase enzymes to sequentially transfer the small modifier protein ubiquitin to a substrate protein. During the last step of this cascade different types of E3 ligases either act as scaffolds to recruit an E2 enzyme and substrate (RING), or form an ubiquitin-thioester intermediate prior to transferring ubiquitin to a substrate (HECT). The RING-inBetweenRING-RING (RBR) proteins constitute a unique group of E3 ubiquitin ligases that includes the Human Homologue of Drosophila Ariadne (HHARI). These E3 ligases are proposed to use a hybrid RING/HECT mechanism whereby the enzyme uses facets of both the RING and HECT enzymes to transfer ubiquitin to a substrate. We now present the solution structure of the HHARI RING2 domain, the key portion of this E3 ligase required for the RING/HECT hybrid mechanism. The structure shows the domain possesses two Zn2+-binding sites and a single exposed cysteine used for ubiquitin catalysis. A structural comparison of the RING2 domain with the HECT E3 ligase NEDD4 reveals a near mirror image of the cysteine and histidine residues in the catalytic site. Further, a tandem pair of aromatic residues exists near the C-terminus of the HHARI RING2 domain that is conserved in other RBR E3 ligases. One of these aromatic residues is remotely located from the catalytic site that is reminiscent of the location found in HECT E3 enzymes where it is used for ubiquitin catalysis. These observations provide an initial structural rationale for the RING/HECT hybrid mechanism for ubiquitination used by the RBR E3 ligases.
doi:10.1371/journal.pone.0074047
PMCID: PMC3772753  PMID: 24058416
18.  Structure of the HECT:ubiquitin complex and its role in ubiquitin chain elongation 
EMBO Reports  2011;12(4):342-349.
Structure of the HECT:ubiquitin complex and its role in ubiquitin chain elongation
Analysis of ubiquitin binding to the HECT domain of Nedd4 suggests that the ubiquitin chain being elongated is kept close to the catalytic cysteine to promote processivity. Together with the accompanying paper by the Huibregtse group, this study shows the catalysis of polyubiquitin chains by HECT E3 ligases.
Several mechanisms have been proposed for the synthesis of substrate-linked ubiquitin chains. HECT ligases directly catalyse protein ubiquitination and have been found to non-covalently interact with ubiquitin. We report crystal structures of the Nedd4 HECT domain, alone and in complex with ubiquitin, which show a new binding mode involving two surfaces on ubiquitin and both subdomains of the HECT N-lobe. The structures suggest a model for HECT-to-substrate ubiquitin transfer, in which the growing chain on the substrate is kept close to the catalytic cysteine to promote processivity. Mutational analysis highlights differences between the processes of substrate polyubiquitination and self-ubiquitination.
doi:10.1038/embor.2011.21
PMCID: PMC3077247  PMID: 21399620
catalysis; E3 ligase; polyubiquitination; structure; ubiquitin
19.  Regulation and Cellular Roles of Ubiquitin-specific Deubiquitinating Enzymes 
Annual review of biochemistry  2009;78:363-397.
Deubiquitinating enzymes (DUBs) are proteases that process ubiquitin or ubiquitin-like gene products, reverse the modification of proteins by a single ubiquitin (or ubiquitin-like protein), and remodel polyubiquitin (or ubiquitin-like) chains on target proteins. The human genome encodes nearly 100 DUBs with specificity for ubiquitin in five families: the UCH, USP, OTU, Josephin, and JAMM families. Four families are cysteine proteases, while the later is a family of metalloproteases. Most DUB activity is cryptic and active site rearrangements often occur during the binding of ubiquitin and/or scaffold proteins. DUBs with specificity for ubiquitin contain multiple domains with insertions and extensions modulating DUB substrate specificity, protein-protein interactions, and cellular localization. Binding partners and multi-protein complexes with which DUBs associate modulate DUB activity and substrate specificity. Quantitative studies of activity and protein-protein interactions, together with genetic studies and the advent of RNAi, have lead to new insights into the function of yeast and human DUBs. This review will discuss ubiquitin-specific DUBs, some of the generalizations emerging from recent studies of the regulation of DUB activity, and their roles in various cellular processes. Specific examples are drawn from studies of protein degradation, DNA repair, chromatin remodeling, cell cycle regulation, endocytosis, and modulation of signaling kinases.
doi:10.1146/annurev.biochem.78.082307.091526
PMCID: PMC2734102  PMID: 19489724
Ubiquitin; Proteasome; Histone; Cell cycle; Endocytosis; DNA Damage; Signal Transduction
20.  Regulation of Epidermal Growth Factor Receptor Down-Regulation by UBPY-mediated Deubiquitination at EndosomesD⃞ 
Molecular Biology of the Cell  2005;16(11):5163-5174.
Ligand-activated receptor tyrosine kinases undergo endocytosis and are transported via endosomes to lysosomes for degradation. This “receptor down-regulation” process is crucial to terminate the cell proliferation signals produced by activated receptors. During the process, ubiquitination of the receptors serves as a sorting signal for their trafficking from endosomes to lysosomes. Here, we describe the role of a deubiquitinating enzyme UBPY/USP8 in the down-regulation of epidermal growth factor (EGF) receptor (EGFR). Overexpression of UBPY reduced the ubiquitination level of EGFR and delayed its degradation in EGF-stimulated cells. Immunopurified UBPY deubiquitinated EGFR in vitro. In EGF-stimulated cells, UBPY underwent ubiquitination and bound to EGFR. Overexpression of Hrs or a dominant-negative mutant of SKD1, proteins that play roles in the endosomal sorting of ubiquitinated receptors, caused the accumulation of endogenous UBPY on exaggerated endosomes. A catalytically inactive UBPY mutant clearly localized on endosomes, where it overlapped with EGFR when cells were stimulated with EGF. Finally, depletion of endogenous UBPY by RNA interference resulted in elevated ubiquitination and accelerated degradation of EGF-activated EGFR. We conclude that UBPY negatively regulates the rate of EGFR down-regulation by deubiquitinating EGFR on endosomes.
doi:10.1091/mbc.E05-06-0560
PMCID: PMC1266416  PMID: 16120644
21.  Direct Binding to Rsp5p Regulates Ubiquitination-independent Vacuolar Transport of Sna3p 
Molecular Biology of the Cell  2007;18(5):1781-1789.
The sorting of integral membrane proteins such as carboxypeptidase S (Cps1p) into the luminal vesicles of multivesicular bodies (MVBs) in Saccharomyces cerevisiae requires ubiquitination of their cytosolic domains by the ubiquitin ligases Rsp5p and/or Tul1p. An exception is Sna3p, which does not require ubiquitination for entry into MVBs. The mechanism underlying this ubiquitination-independent MVB sorting pathway has not yet been characterized. Here, we show that Sna3p sorting into the MVB pathway depends on a direct interaction between a PPAY motif within its C-terminal cytosolic tail and the WW domains of Rsp5p. Disruption of this interaction inhibits vacuolar targeting of Sna3p and causes its accumulation in a compartment that overlaps only partially with MVBs. Surprisingly, Sna3p does require a functional ubiquitin-ligase HECT domain within Rsp5p; however, the dependence of Sna3p on HECT domain activity is distinct from that of Cps1p. Last, we show that Sna3p requires neither Tul1p nor the transmembrane adaptor protein Bsd2p for its MVB sorting. Our data demonstrate that Sna3p follows a novel ubiquitination-independent, but Rsp5p-mediated, sorting pathway to the vacuole.
doi:10.1091/mbc.E06-10-0887
PMCID: PMC1855027  PMID: 17332499
22.  The Vps27/Hse1 complex is a GAT domain-based scaffold for ubiquitin-dependent sorting 
Developmental cell  2007;12(6):973-986.
Summary
The yeast Vps27/Hse1 complex and homologous mammalian Hrs/STAM complex deliver ubiquitinated transmembrane proteins to the ESCRT endosomal sorting pathway. The Vps27/Hse1 complex directly binds to ubiquitinated transmembrane proteins and recruits both ubiquitin ligases and deubiquitinating enzymes. We have solved the crystal structure of the core responsible for the assembly of the Vps27/Hse1 complex at 3.0Å resolution. The structure consists of two intertwined GAT domains, each consisting of two helices from one subunit and one from the other. The two GAT domains are connected by an antiparallel coiled-coil to form a 90 Å-long barbell-like structure. This structure places the domains of Vps27 and Hse1 that recruit ubiquitinated cargo and deubiquitinating enzymes close to each other. Coarse-grained Monte Carlo simulations of the Vps27/Hse1 complex on a membrane show how the complex binds cooperatively to lipids and ubiquitinated membrane proteins and acts as a scaffold for ubiquitination reactions.
doi:10.1016/j.devcel.2007.04.013
PMCID: PMC2292400  PMID: 17543868
23.  The Translation Initiation Factor 3f (eIF3f) Exhibits a Deubiquitinase Activity Regulating Notch Activation 
PLoS Biology  2010;8(11):e1000545.
The translation initiation factor complex eIF3f has an intrinsic deubiquitinase activity and regulates the Notch signaling pathway.
Activation of the mammalian Notch receptor after ligand binding relies on a succession of events including metalloprotease-cleavage, endocytosis, monoubiquitination, and eventually processing by the gamma-secretase, giving rise to a soluble, transcriptionally active molecule. The Notch1 receptor was proposed to be monoubiquitinated before its gamma-secretase cleavage; the targeted lysine has been localized to its submembrane domain. Investigating how this step might be regulated by a deubiquitinase (DUB) activity will provide new insight for understanding Notch receptor activation and downstream signaling. An immunofluorescence-based screening of an shRNA library allowed us to identify eIF3f, previously known as one of the subunits of the translation initiation factor eIF3, as a DUB targeting the activated Notch receptor. We show that eIF3f has an intrinsic DUB activity. Knocking down eIF3f leads to an accumulation of monoubiquitinated forms of activated Notch, an effect counteracted by murine WT eIF3f but not by a catalytically inactive mutant. We also show that eIF3f is recruited to activated Notch on endocytic vesicles by the putative E3 ubiquitin ligase Deltex1, which serves as a bridging factor. Finally, catalytically inactive forms of eIF3f as well as shRNAs targeting eIF3f repress Notch activation in a coculture assay, showing that eIF3f is a new positive regulator of the Notch pathway. Our results support two new and provocative conclusions: (1) The activated form of Notch needs to be deubiquitinated before being processed by the gamma-secretase activity and entering the nucleus, where it fulfills its transcriptional function. (2) The enzyme accounting for this deubiquitinase activity is eIF3f, known so far as a translation initiation factor. These data improve our knowledge of Notch signaling but also open new avenues of research on the Zomes family and the translation initiation factors.
Author Summary
The highly conserved signaling pathway involving the transmembrane receptor Notch is essential for development, and misregulation of this pathway is linked to many diseases. We previously proposed that the Notch1 receptor is monoubiquitinated during its activation. With the aim of identifying a deubiquinating enzyme that could regulate Notch activation, we demonstrated that eIF3f, known previously as part of the multiprotein translation initiation factor eIF3 complex, harbors an enzymatic activity that acts on Notch. The activated form of Notch is able to interact with eIF3f only in the presence of the E3 ubiquitin ligase Deltex, and Notch needs to be deubiquitinated before it can be cleared and its intracellular domain can enter the nucleus and fulfill its transcriptional function. Our results further decipher the molecular mechanisms of Notch signaling activation, showing that ubiquitination and deubiquitination events are required. Additionally, we show that beyond acting as a translation initiation factor, eIF3f fulfills other functions and has an intrinsic enzymatic activity.
doi:10.1371/journal.pbio.1000545
PMCID: PMC2990700  PMID: 21124883
24.  Mechanism of Recruitment and Activation of the Endosome-associated Deubiquitinase AMSH 
Biochemistry  2013;52(44):7818-7829.
AMSH, a deubiquitinating enzyme (DUB) with exquisite specificity for Lys63-linked polyubiquitin chains, is an endosome-associated DUB that regulates sorting of activated cell-surface signaling receptors to lysosome, a process mediated by the members of the endosomal sorting complexes required for transport (ESCRT) machinery. Whole-exome sequencing of DNA samples from children with microcephaly capillary malformation (MIC-CAP) syndrome identified recessive mutations encoded in the AMSH gene causatively linked to the disease. Herein, we report a number of important observations that significantly advance our understanding of AMSH within the context of the ESCRT machinery. First, we performed mutational and kinetic analysis of the putative residues involved in diubiquitin recognition and catalysis with a view to better understanding the catalytic mechanism of AMSH. Our mutational and kinetic analysis reveals that recognition of the proximal ubiquitin is imperative for the linkage specificity and catalytic efficiency of the enzyme. The MIC-CAP disease mutation, Thr313Ile, shows a substantial loss of catalytic activity without any significant change in thermodynamic stability of the protein, indicating that its perturbed catalytic activity is the basis of the disease. The catalytic activity of AMSH is stimulated upon binding to the ESCRT-0 member STAM, however, the precise mechanism and its significance are not known. Based on a number of biochemical and biophysical analysis, we are able to propose a model for activation according to which activation of AMSH is enabled by facile, simultaneous binding to two ubiquitin groups in a polyubiquitin substrate, one by the catalytic domain of the DUB (binding to the distal ubiquitin) and the other (the proximal ubiquitin) by the ubiquitin interacting motif (UIM) from STAM. Such a mode of binding would stabilize the ubiquitin chain in a productive orientation, resulting in an enhancement of the activity of the enzyme. These data together provide a mechanism for understanding the recruitment and activation of AMSH at ESCRT-0, providing biochemical and biophysical evidence in support of a role for AMSH when it is recruited to the initial ESCRT complex: it functions to facilitate transfer of ubiquitinated receptors (cargo) from one ESCRT member to the next by disassembling the polyubiquitin chain while leaving some ubiquitin groups still attached to the cargo.
doi:10.1021/bi401106b
PMCID: PMC3972757  PMID: 24151880
25.  Split-Ubiquitin Two-Hybrid Assay To Analyze Protein-Protein Interactions at the Endosome: Application to Saccharomyces cerevisiae Bro1 Interacting with ESCRT Complexes, the Doa4 Ubiquitin Hydrolase, and the Rsp5 Ubiquitin Ligase▿ † 
Eukaryotic Cell  2007;6(8):1266-1277.
Targeting of membrane proteins into the lysosomal/vacuolar lumen for degradation requires their prior sorting into multivesicular bodies (MVB). The MVB sorting pathway depends on ESCRT-0, -I, -II, and -III protein complexes functioning on the endosomal membrane and on additional factors, such as Bro1/Alix and the ubiquitin ligase Rsp5/Nedd4. We used the split-ubiquitin two-hybrid assay to analyze the interaction partners of yeast Bro1 at its natural cellular location. We show that Bro1 interacts with ESCRT-I and -III components, including Vps23, the Saccharomyces cerevisiae homologue of human Tsg101. These interactions do not require the C-terminal proline-rich domain (PRD) of Bro1. Rather, this PRD interacts with the Doa4 deubiquitinating enzyme to recruit it to the endosome. This interaction is disrupted by a single amino acid substitution in the conserved ELC box motif in Doa4. The PRD of Bro1 also mediates an association with Rsp5, and this interaction appears to be conserved, as Alix, the human homologue of Bro1, coimmunoprecipitates with Nedd4 in yeast lysates. We further show that the Bro1 PRD domain is essential to MVB sorting of only cargo proteins whose sorting to the vacuolar lumen is dependent on their own ubiquitination and Doa4. The Bro1 region preceding the PRD, however, is required for MVB sorting of proteins irrespective of whether their targeting to the vacuole is dependent on their ubiquitination and Doa4. Our data indicate that Bro1 interacts with several ESCRT components and contributes via its PRD to associating ubiquitinating and deubiquitinating enzymes with the MVB sorting machinery.
doi:10.1128/EC.00024-07
PMCID: PMC1951119  PMID: 17513562

Results 1-25 (942087)