The ubiquitin E3 ligase POSH regulates calcium homeostasis through spatial control of Herp (original) (raw)
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Neuralized functions as an E3 ubiquitin ligase during Drosophila development
Current Biology, 2001
The Notch pathway is a widely studied means of to the protein target via the E3 ubiquitin ligase. The E3 ubiquitin ligases are responsible for conferring target or intercellular signaling responsible for the determination of cell fate, cell differentiation, and substrate specificity. boundary formation (reviewed in [1, 2]). The main effectors of this pathway, Notch (N) and Delta (Dl), Several recent studies have implicated ubiquitination as have been shown to function as a receptor and a potential regulatory mechanism involved in N signaling. ligand, respectively. Genetic and phenotypic studies The molecular characterization of Suppressor of deltex, Su suggest that Neuralized (Neu), a RING finger (dx), which has been shown to genetically interact with protein, also plays a role within the N-Dl pathway, N, revealed that it encodes a HECT domain found in although its biochemical function is unknown. proteins that possess E3 ubiquitin ligase activity [6]. Al-Here, we show that Neu is required at the plasma though a direct role for Su (dx) in the ubiquitination of membrane for functional activity and that its RING Notch (N) has not yet been shown, a related mammalian finger domain acts as an E3 ubiquitin ligase. These protein called Itch has been shown to interact with and data suggest that the role of Neu is to target ubiquitinate N in vitro and in human Jurkat cells [7]. components of the N-Dl pathway for ubiquitination, Further genetic evidence implicating ubiquitination in N allowing for propagation and/or regulation of the signaling comes from observations that mutations in the signal. 2 and 6 subunits of the 20S proteasome cause N gainof-function phenotypes during sense organ development Addresses: *Program in Developmental Biology, † Program in Cell in Drosophila [8]. These mutations also increase the stabil-Biology,
HERCing: Structural and Functional Relevance of the Large HERC Ubiquitin Ligases
Frontiers in Physiology, 2019
Homologous to the E6AP carboxyl terminus (HECT) and regulator of chromosome condensation 1 (RCC1)-like domain-containing proteins (HERCs) belong to the superfamily of ubiquitin ligases. HERC proteins are divided into two subfamilies, Large and Small HERCs. Despite their similarities in terms of both structure and domains, these subfamilies are evolutionarily very distant and result from a convergence phenomenon rather than from a common origin. Large HERC genes, HERC1 and HERC2, are present in most metazoan taxa. They encode very large proteins (approximately 5,000 amino acid residues in a single polypeptide chain) that contain more than one RCC1-like domain as a structural characteristic. Accumulating evidences show that these unusually large proteins play key roles in a wide range of cellular functions which include neurodevelopment, DNA damage repair, and cell proliferation. To better understand the origin, evolution, and function of the Large HERC family, this minireview provides with an integrated overview of their structure and function and details their physiological implications. This study also highlights and discusses how dysregulation of these proteins is associated with severe human diseases such as neurological disorders and cancer.
Distinct activation of an E2 ubiquitin-conjugating enzyme by its cognate E3 ligases
Proceedings of the National Academy of Sciences of the United States of America, 2015
A significant portion of ubiquitin (Ub)-dependent cellular protein quality control takes place at the endoplasmic reticulum (ER) in a process termed "ER-associated degradation" (ERAD). Yeast ERAD employs two integral ER membrane E3 Ub ligases: Hrd1 (also termed "Der3") and Doa10, which recognize a distinct set of substrates. However, both E3s bind to and activate a common E2-conjugating enzyme, Ubc7. Here we describe a novel feature of the ERAD system that entails differential activation of Ubc7 by its cognate E3s. We found that residues within helix α2 of Ubc7 that interact with donor Ub were essential for polyUb conjugation. Mutagenesis of these residues inhibited the in vitro activity of Ubc7 by preventing the conjugation of donor Ub to the acceptor. Unexpectedly, Ub chain formation by mutant Ubc7 was restored selectively by the Hrd1 RING domain but not by the Doa10 RING domain. In agreement with the in vitro data, Ubc7 α2 helix mutations selectively impaired ...
The HIV-1 Vpu Protein Induces Apoptosis in Drosophila via Activation of JNK Signaling
PLoS ONE, 2012
The genome of the human immunodeficiency virus type 1 (HIV-1) encodes the canonical retroviral proteins, as well as additional accessory proteins that enhance the expression of viral genes, the infectivity of the virus and the production of virions. The accessory Viral Protein U (Vpu), in particular, enhances viral particle production, while also promoting apoptosis of HIV-infected human T lymphocytes. Some Vpu effects rely on its interaction with the ubiquitin-proteasome protein degradation system, but the mechanisms responsible for its pro-apoptotic effects in vivo are complex and remain largely to be elucidated. We took advantage of the Drosophila model to study the effects of Vpu activity in vivo. Expression of Vpu in the developing Drosophila wing provoked tissue loss due to caspase-dependent apoptosis. Moreover, Vpu induced expression of the pro-apoptotic gene reaper, known to down-regulate Inhibitor of Apoptosis Proteins (IAPs) which are caspase-antagonizing E3 ubiquitin ligases. Indeed, Vpu also reduced accumulation of Drosophila IAP1 (DIAP1). Though our results demonstrate a physical interaction between Vpu and the proteasome-addressing SLIMB/b-TrCP protein, as in mammals, both SLIMB/bTrCP-dependent and-independent Vpu effects were observed in the Drosophila wing. Lastly, the pro-apoptotic effect of Vpu in this tissue was abrogated upon inactivation of the c-Jun N-terminal Kinase (JNK) pathway. Our results in the fly thus provide the first functional evidence linking Vpu pro-apoptotic effects to activation of the conserved JNK pathway.
Occurrence of a putative SCF ubiquitin ligase complex in Drosophila
Biochemical and …, 2001
Many proteins are targeted to proteasome degradation by a family of E3 ubiquitin ligases, termed SCF complexes, that link substrate proteins to an E2 ubiquitin-conjugating enzyme. SCFs are composed of three core proteins-Skp1, Cdc53/Cull, Rbx1/Hrt1-and a substrate specific F-box protein. We have identified in Drosophila melanogaster the closest homologues to the human components of the SCF TrCP complex and the E2 ubiquitin-conjugating enzyme UbcH5. We show that putative Drosophila SCF core subunits dSkpA and dRbx1 both interact directly with dCu11 and the F-box protein Slmb. We also describe the direct interaction of the UbcH5 related protein UbcD1 with dCul1 and Slmb. In addition, a functional complementation test performed on a Saccharomyces cerevisiae Hrt1pdeficient mutant showed that Drosophila Rbx1 is able to restore the yeast cells viability. Our results suggest that dRbx1, dSkpA, dCullin1, and Slimb proteins are components of a Drosophila SCF complex that functions in combination with the ubiquitin conjugating enzyme UbcD1.
RAS-induced MAPK signaling is a central driver of the cell proliferation apparatus. Disruption of this pathway is widely observed in cancer and other pathologies. Consequently, considerable effort has been devoted to understanding the mechanistic aspects of RAS-MAPK signal transmission and regulation. While much information has been garnered on the steps leading up to the activation and inactivation of core pathway components, comparatively little is known on the mechanisms controlling their expression and turnover. We recently identified several factors that dictate Drosophila MAPK levels. Here, we describe the function of one of these, the deubiquitinase (DUB) USP47. We found that USP47 acts post-translation-ally to counteract a proteasome-mediated event that reduces MAPK half-life and thereby dampens signaling output. Using an RNAi-based genetic interaction screening strategy, we identified UBC6, POE/UBR4, and UFD4, respectively, as E2 and E3 enzymes that oppose USP47 activity. Further characterization of POE-associated factors uncovered KCMF1 as another key component modulating MAPK levels. Together, these results identify a novel protein degradation module that governs MAPK levels. Given the role of UBR4 as an N-recognin ubiquitin ligase, our findings suggest that RAS-MAPK signaling in Drosophila is controlled by the N-end rule pathway and that USP47 counteracts its activity. The RAS-MAPK pathway plays a central role in the development of multicellular organisms , predominantly by regulating cell proliferation and differentiation. At its core, the pathway includes the RAS GTPase and three kinases (RAF, MEK, and MAPK) that transmit RAS signals through a phosphorylation cascade. Several factors have been discovered over the years that modulate signal transmission by altering the kinetics of phosphoryla-tion/dephosphorylation of the core pathway components. In contrast, scant information is available on the mechanisms governing their expression and steady-state levels. Here, we
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Herp coordinates compartmentalization and recruitment of HRD1 and misfolded proteins for ERAD
Molecular Biology of the Cell, 2014
A functional unfolded protein response (UPR) is essential for endoplasmic reticulum (ER)-associated degradation (ERAD) of misfolded secretory proteins, reflecting the fact that some level of UPR activation must exist under normal physiological conditions. A coordinator of the UPR and ERAD processes has long been sought. We previously showed that the PKR-like, ER-localized eukaryotic translation initiation factor 2α kinase branch of the UPR is required for the recruitment of misfolded proteins and the ubiquitin ligase HRD1 to the ERderived quality control compartment (ERQC), a staging ground for ERAD. Here we show that homocysteine-induced ER protein (Herp), a protein highly upregulated by this UPR branch, is responsible for this compartmentalization. Herp localizes to the ERQC, and our results suggest that it recruits HRD1, which targets to ERAD the substrate presented by the OS-9 lectin at the ERQC. Predicted overall structural similarity of Herp to the ubiquitin-proteasome shuttle hHR23, but including a transmembrane hairpin, suggests that Herp may function as a hub for membrane association of ERAD machinery components, a key organizer of the ERAD complex.
A new role for HERPUD1 and ERAD activation in osteoblast differentiation and mineralization
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2018
Bone integrity depends on a finely tuned balance between bone synthesis by osteoblasts and resorption by osteoclasts. The secretion capacity of mature osteoblasts requires strict control of proteostasis. Endoplasmic reticulum-associated degradation (ERAD) prevents the accumulation of unfolded ER proteins via dislocation to the cytosol and degradation by the proteasome. The ER membrane protein, homocysteine-inducible endoplasmic reticulum protein with ubiquitin-like domain 1 (HERPUD1), is a key component of the ERAD multiprotein complex which helps to stabilize the complex and facilitate the efficient degradation of unfolded proteins. HERPUD1 expression is strongly up-regulated by the unfolded protein response and cellular stress. The aim of the current study was to establish whether HERPUD1 and ERAD play roles in osteoblast differentiation and maturation. We evaluated preosteoblastic MC3T3-E1 cell and primary rat osteoblast differentiation by measuring calcium deposit levels, alkali...
Protein interaction screening identifies SH3RF1 as a new regulator of FAT1 protein levels
FEBS letters, 2017
Mutations and ectopic FAT1 cadherin expression are implicated in a broad spectrum of diseases ranging from developmental disorders to cancer. The regulation of FAT1 and its downstream signalling pathways remain incompletely understood. We hypothesized that identification of additional proteins interacting with the FAT1 cytoplasmic tail would further delineate its regulation and function. A yeast two-hybrid library screen carried out against the juxtamembrane region of the cytoplasmic tail of FAT1 identified the E3 ubiquitin-protein ligase SH3RF1 as the most frequently recovered protein-binding partner. Ablating SH3RF1 using siRNA increased cellular FAT1 protein levels and stabilized expression at the cell surface, while overexpression of SH3RF1 reduced FAT1 levels. We conclude that SH3RF1 acts as a negative post-translational regulator of FAT1 levels. This article is protected by copyright. All rights reserved.
Journal of Biological Chemistry, 2009
Clearance of misfolded proteins by endoplasmic reticulum (ER)-associated degradation (ERAD) requires concerted activity of chaperones, adaptor proteins, ubiquitin ligases, and proteasomes. RNF5 is a ubiquitin ligase anchored to the ER membrane implicated in ERAD via ubiquitination of misfolded proteins. Among RNF5-associated proteins is JNK-associated membrane protein (JAMP), a 7-transmembrane protein located within the ER membrane that facilitates degradation of misfolded proteins through recruitment of proteasomes and ERAD regulatory components. Here we demonstrate that RNF5 associates with JAMP in the ER membrane. This association results in Ubc13-dependent RNF5-mediated noncanonical ubiquitination of JAMP. This ubiquitination does not alter JAMP stability but rather inhibits its association with Rpt5 and p97. Consequently, clearance of misfolded proteins, such as CFTR⌬508 and T cell receptor ␣, is less efficient, resulting in their greater accumulation. Significantly, the RNF5 effect on JAMP is seen prior to and after ER stress response, thereby highlighting a novel mechanism to limit ERAD and proteasome assembly at the ER, to the actual ER stress response.
2019
Perturbation of endoplasmic reticulum (ER) functions can have critical consequences for cellular homeostasis. An elaborate surveillance system known as ER quality control (ERQC) ensures that only correctly assembled proteins reach their destination. Persistence of misfolded or improperly matured proteins upregulates the unfolded protein response (UPR) to cope with stress, activates ER associated degradation (ERAD) for delivery to proteasomes for degradation. We have identified a Brucella abortus type IV secretion system effector called BspL that targets Herp, a key component of ERQC and is able to augment ERAD. Modulation of ERQC by BspL results in tight control of the kinetics of autophagic Brucella-containing vacuole formation, preventing premature bacterial egress from infected cells. This study highlights how bacterial pathogens may hijack ERAD components for fine regulation of their intracellular trafficking.
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Developmental Cell, 2001
other proteins that function as part of multiprotein complexes, suggesting that Neur may as well. Second, we 2 Salk Institute for Biological Studies San Diego, California 92186 observed that Neur deleted for the RING finger (Neur⌬RF) behaved as a strong dominant-negative protein, indicating that this domain is essential for normal Neur function. Third, we observed that ectopic Neur and Neur⌬RF af-Summary fected multiple neur-independent, N pathway-regulated processes, suggesting that Neur regulates the activity The Drosophila gene neuralized (neur) has long been recognized to be essential for the proper execution of of a core member of the N pathway. Finally, we determined that epitope-tagged Neur associates primarily a wide variety of processes mediated by the Notch (N) pathway, but its role in the pathway has been elusive.
BioE3 identifies specific substrates of ubiquitin E3 ligases
Nature Communications, 2023
Hundreds of E3 ligases play a critical role in recognizing specific substrates for modification by ubiquitin (Ub). Separating genuine targets of E3s from E3-interactors remains a challenge. We present BioE3, a powerful approach for matching substrates to Ub E3 ligases of interest. Using BirA-E3 ligase fusions and bioUb, site-specific biotinylation of Ub-modified substrates of particular E3s facilitates proteomic identification. We show that BioE3 identifies both known and new targets of two RING-type E3 ligases: RNF4 (DNA damage response, PML bodies), and MIB1 (endocytosis, autophagy, centrosome dynamics). Versatile BioE3 identifies targets of an organellespecific E3 (MARCH5) and a relatively uncharacterized E3 (RNF214). Furthermore, BioE3 works with NEDD4, a HECT-type E3, identifying new targets linked to vesicular trafficking. BioE3 detects altered specificity in response to chemicals, opening avenues for targeted protein degradation, and may be applicable for other Ub-likes (UbLs, e.g., SUMO) and E3 types. BioE3 applications shed light on cellular regulation by the complex UbL network. Protein ubiquitination is conserved in all eukaryotes and plays crucial roles in almost all cellular processes. Ubiquitin (Ub) conjugation is coordinated by a three-step enzymatic cascade, which can be reversed by the action of deubiquitinating enzymes (DUBs). This cycle is conserved among the different ubiquitin-like proteins (UbLs), each using their own set of enzymes, often depicted as E1 (activating), E2 (conjugating), E3 (ligating), and DUBs. Specificity of ubiquitin toward particular targets is achieved as the cycle progresses. In humans, two Ub E1 enzymes, around 40 E2s and about 700 E3 ligases cooperate to selectively target thousands of substrates 1. The question of how substrate specificity is achieved might benefit from a compendium of targets for specific E3 ligases. Ub E3 ligases are subdivided into categories, according to shared domains and modes of action for substrate modification 2. The main family covers more than 600 RING (Really Interesting New Gene) type Ub E3 ligases. The RING domain allows the direct transfer of Ub from the E2 to the target protein by placing them in close proximity 3. To function, some RING E3 ligases (e.g., RNF4; RING Finger protein 4), dimerize through their RING domain 4 , or create multi-subunit complexes, (e.g., Cullin RING Ligases). CRLs can recognize diverse targets with specificity by forming complexes with >300 different substrate receptors 2. In the case of HECT (Homology to E6AP C Terminus) and RBR (RING-Between-RING) E3 ligases, a covalent E3~Ub thioester
Cell regulation by phosphotyrosine-targeted ubiquitin ligases
Molecular and cellular biology, 2015
Three classes of E3 ubiquitin ligases, members of the Cbl, Hakai and SOCS families, stimulate the ubiquitination of phosphotyrosine-containing proteins, including receptor and non-receptor tyrosine kinases and their phosphorylated substrates. Because ubiquitination frequently routes proteins for degradation by the lysosome or proteasome, these E3 ligases are able to potently inhibit tyrosine kinase signaling. Their loss or mutational inactivation can contribute to cancer, autoimmunity or endocrine disorders such as diabetes. However, these ligases also have biological functions that are independent of their ubiquitination activity. Here we review relevant literature and then focus on more recent developments in understanding the structures, substrates and pathways through which the phosphotyrosine-specific ubiquitin ligases regulate diverse aspects of cell biology.
Expression of the Rho-GEF Pbl/ECT2 is regulated by the UBE3A E3 ubiquitin ligase
Human Molecular Genetics, 2006
We applied genetic tools available in Drosophila to identify candidate substrates of the UBE3A ubiquitin ligase, the gene responsible for Angelman syndrome (AS). Human UBE3A was expressed in Drosophila heads to identify proteins differentially regulated in UBE3A-expressing versus wild-type extracts. Using twodimensional gel and MALDI-TOF analysis, we detected 20 proteins that were differentially regulated by overexpression of human UBE3A in Drosophila heads. One protein responsive to UBE3A was the Rho-GEF pebble ( pbl ). Here, we present three lines of evidence suggesting that UBE3A regulates Pbl. First, we show genetic evidence that UBE3A and the Drosophila de-ubiquitinase fat facets (faf) exert opposing effects on Pbl function. Secondly, we find that both Pbl and ECT2, the mammalian orthologue of Pbl called epithelial cell transforming sequence 2 oncogene, physically interact with their respective ubiquitin E3 ligases. Finally, we show that Ect2 expression is regulated by Ube3a in mouse neurons as the pattern of Ect2 expression is dramatically altered in the hippocampus and cerebellum of Ube3a null mice. These results suggest that an orthologous UBE3A post-translational regulatory pathway regulates neuronal outgrowth in the mammalian brain and that dysregulation of this pathway may result in neurological phenotypes including AS and possibly other autism spectrum disorders.
Molecular cell, 2017
RING and U-box E3 ubiquitin ligases regulate diverse eukaryotic processes and have been implicated in numerous diseases, but targeting these enzymes remains a major challenge. We report the development of three ubiquitin variants (UbVs), each binding selectively to the RING or U-box domain of a distinct E3 ligase: monomeric UBE4B, phosphorylated active CBL, or dimeric XIAP. Structural and biochemical analyses revealed that UbVs specifically inhibited the activity of UBE4B or phosphorylated CBL by blocking the E2∼Ub binding site. Surprisingly, the UbV selective for dimeric XIAP formed a dimer to stimulate E3 activity by stabilizing the closed E2∼Ub conformation. We further verified the inhibitory and stimulatory functions of UbVs in cells. Our work provides a general strategy to inhibit or activate RING/U-box E3 ligases and provides a resource for the research community to modulate these enzymes.
Development, 2007
Ubiquitination is an essential process regulating turnover of proteins for basic cellular processes such as the cell cycle and cell death (apoptosis). Ubiquitination is initiated by ubiquitin-activating enzymes (E1), which activate and transfer ubiquitin to ubiquitin-conjugating enzymes (E2). Conjugation of target proteins with ubiquitin is then mediated by ubiquitin ligases (E3). Ubiquitination has been well characterized using mammalian cell lines and yeast genetics. However, the consequences of partial or complete loss of ubiquitin conjugation in a multi-cellular organism are not well understood. Here, we report the characterization of Uba1, the only E1 in Drosophila. We found that weak and strong Uba1 alleles behave genetically differently with sometimes opposing phenotypes. Whereas weak Uba1 alleles protect cells from cell death, clones of strong Uba1 alleles are highly apoptotic. Strong Uba1 alleles cause cell cycle arrest which correlates with failure to reduce cyclin levels. Surprisingly, clones of strong Uba1 mutants stimulate neighboring wild-type tissue to undergo cell division in a non-autonomous manner giving rise to overgrowth phenotypes of the mosaic fly. We demonstrate that the non-autonomous overgrowth is caused by failure to downregulate Notch signaling in Uba1 mutant clones. In summary, the phenotypic analysis of Uba1 demonstrates that impaired ubiquitin conjugation has significant consequences for the organism, and may implicate Uba1 as a tumor suppressor gene.
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