Homologous To E6-AP Carboxyl Terminus Research Articles

E2UbcH5B-derived peptide ligands target HECT E3-E2 binding site and block the Ub-dependent SARS-CoV-2 egression: A computational study

Homologous to E6AP carboxyl-terminus (HECT)-type E3 ligase performs ubiquitin (Ub)-proteasomal protein degradation via forming a complex with E2∼Ub. Enveloped viruses including SARS-CoV-2 escape from the infected cells by harnessing the E-class vacuolar protein-sorting (ESCRT) machinery and mimic the cellular system through PPAY motif-based linking to HECT Ub ligase activity. In the present study, we have characterized the binding pattern of E2UbcH5B to HECT domains of NEDD4L, WWP1, WWP2, HECW1, and HECW2 through in silico analysis to isolate the E2UbcH5B-specific peptide inhibitors that may target SARS-CoV-2 viral egression. Molecular dynamics analysis revealed more opening of E2UbcH5B-binding pocket upon binding to HECTNEDD4L, HECTWWP1, HECTWWP2, HECTHECW1, and HECTHECW2. We observed similar binding pattern for E2UbcH5B and mentioned HECT domains as previously reported for HECTNEDD4L where Trp762, Trp709, and Trp657 residues of HECTNEDD4L, HECTWWP1, and HECTWWP2 are involved in making contacts with Ser94 residue of E2UbcH5B. Similarly, corresponding to HECTNEDD4L Tyr756 residue, HECTWWP1, HECTWWP2, HECTHECW1, and HECTHECW2-specific Phe703, Phe651, Phe1387, and Phe1353 residues execute interaction with E2UbcH5B. Our analysis suggests that corresponding to Cys942 of HECTNEDD4L, Cys890, Cys838, Cys1574, and Cys1540 residues of HECTWWP1, HECTWWP2, HECTHECW1, and HECTHECW2, respectively are involved in E2-to-E3 Ub transfer. Furthermore, MM-PBSA free energy calculations revealed favorable energy values for E2UbcH5B-HECT complexes along with the individual residue contributions. Subsequently, two E2UbcH5B-derived peptides (His55-Phe69 and Asn81-Ala96) were tested for their binding abilities against HECT domains of NEDD4L, WWP1, WWP2, HECW1, and HECW2. Their binding was validated through substitution of Phe62, Pro65, Ile84, and Cys85 residues into Ala, which revealed an impaired binding, suggesting that the proposed peptide ligands may selectively target E2-HECT binding and Ub-transfer. Collectively, we propose that peptide-driven blocking of E2-to-HECT Ub loading may limit SARS-CoV-2 egression and spread in the host cells.

RSP5 Positively Regulates the Osteogenic Differentiation of Mesenchymal Stem Cells by Activating the K63-Linked Ubiquitination of Akt.

Mesenchymal stem cells (MSCs) are multipotent stem cells that have a strong osteogenic differentiation capacity. However, the molecular mechanism underlying the osteogenic differentiation of MSCs remains largely unknown and thus hinders further development of MSC-based cell therapies for bone repair in the clinic. RSP5, also called NEDD4L (NEDD4-like E3 ubiquitin protein ligase), belongs to the HECT (homologous to E6-AP carboxyl terminus) domain-containing E3 ligase family. Nevertheless, although many studies have been conducted to elucidate the role of RSP5 in various biological processes, its effect on osteogenesis remains elusive. In this study, we demonstrated that the expression of RSP5 was elevated during the osteogenesis of MSCs and positively regulated the osteogenic capacity of MSCs by inducing K63-linked polyubiquitination and activation of the Akt pathway. Taken together, our findings suggest that RSP5 may be a promising target to improve therapeutic efficiency by using MSCs for bone regeneration and repair.

Lysine 33 polyubiquitin chain formation by the HECT Ubiquitin E3 Ligase AREL1

Apoptosis Resistant E3 Ligase 1 (AREL1) is a member of the Homologous to E6AP Carboxyl Terminus (HECT) E3 ubiquitin ligase family. All HECT E3 ubiquitin ligases contain a conserved HECT domain that facilitate the covalent attachment of ubiquitin onto their intracellular substrates. Ubiquitylation is involved in numerous cell signaling pathways that can result in proteasomal degradation, DNA damage repair, and cell trafficking, among others. The biological function of AREL1 is to inhibit intrinsic apoptosis by ubiquitylation of pro‐apoptotic inhibitor of apoptosis (IAP) antagonist proteins, SMAC, ARTS, and HtrA2, for proteasomal degradation. Interestingly, previous studies have found that AREL1 is the only HECT E3 ubiquitin ligase identified to date that is capable of building polyubiquitin chains via lysine 33 linkages, and the biological role of this linkage remains unclear. Using circular dichroism (CD), in vitro ubiquitin activity assays, disulfide complex formation, and multidimensional heteronuclear NMR spectroscopy, we are currently deciphering the unique molecular mechanism used by AREL1 to build the lysine 33 poly‐ubiquitin chains.Support or Funding InformationNIH NIGMS Maurine H. Milburn Fund Clark University Carlson School of Biochemistry and Chemistry

Hell's BELs: bacterial E3 ligases that exploit the eukaryotic ubiquitin machinery.

A common post-translational modification in eukaryotes is the covalent attachment of ubiquitin, a 76 amino acid protein, to specific proteins. Most commonly, ubiquitin is conjugated to e– amino groups of lysine residues, and in unusual cases it can be conjugated to serine and cysteine residues or the terminal amino group of a protein. This process, referred to as ubiquitination (or ubiquitylation), can result in a variety of outcomes for a protein, depending upon how many Ub molecules are attached, whether a polyubiquitin chain is formed, and the nature of the chain. Monoubiquitination can result in relocalization of proteins, while most polyubiquitin chains (e.g., K48 and K11-linked chains) direct proteins for proteasomal degradation. Linkages of Ub formed using Lys 63 or by end-to-end linkages (also known as Met Ub) [1] are not directed to the proteasome and can mediate protein trafficking, scaffolding of protein complexes, or enzyme activation. Ub chains are also used for targeting invading microbes for clearance via xenophagy [2]. Ubiquitination is carried out by a series of enzymes. First, a ubiquitin activating enzyme (E1) forms a thioester with the Cterminus of Ub. The activated Ub is then transferred to one of many (,40 human) ubiquitin conjugating enzymes (E2s). Finally, the E3 enzymes (perhaps over 500 human E3s) direct the transfer of Ub to specific substrates. In eukaryotic cells there are two general classes of E3 ubiquitin ligases. The HECT (Homologous to E6-AP Carboxyl Terminus) and Ring Between Ring (RBR) domain E3s possess an invariant catalytic Cys residue that accepts Ub from a charged E2 before catalyzing transfer of Ub to substrates. Other E3s contain a Really Interesting New Gene (RING) or RING-like domain (U-box) that recruits a charged E2, as well as a domain that recruits substrates. Ub is then transferred from the E2 to the substrate, with the E3 serving primarily as a scaffold. Some RING E3s are single polypeptides, while the cullinRING Ligases (CRLs) are modular multisubunit complexes [3]. Mammalian CRLs are nucleated by one of seven cullin family members, with a RING domain protein that binds to its Cterminus. The N-terminal region of the cullin binds specific cullin adaptor proteins that engage substrate receptor proteins; the most studied class of substrate receptors are the F-Box proteins. CRLs are subject to an additional level of control by a ubiquitin-like modifier protein, Nedd8. E3 enzymes have two important roles. First, they recognize substrates and position them for ubiquitination. Second, E3s dictate the nature of the Ub linkage(s), which will determine the substrate’s fate. For HECT E3s, the Ub chain type is dictated by the C-terminal lobe of the HECT domain [4]. By contrast, RINGtype E3s direct Ub chain type based upon the charged E2 that they recruit [5]. Successful pathogens use proteins that interfere with host cell function that are delivered into eukaryotic host cells via specialized secretion systems and collectively referred to as ‘‘effectors.’’ Remarkably, although ubiquitin is restricted to eukaryotic cells, the past decade has revealed that both bacterial and viral pathogens use effectors to interfere with or manipulate the ubiquitination system [6]. This involves a large number of Bacterially encoded E3 ubiquitin Ligases (BELs). There are multiple RING-type BELs, HECT-like BELs, and even BELs that bear no resemblance to known eukaryotic ubiquitin ligases (Figure 1A).

Identification of a Novel Anti-apoptotic E3 Ubiquitin Ligase That Ubiquitinates Antagonists of Inhibitor of Apoptosis Proteins SMAC, HtrA2, and ARTS

Identification of new anti-apoptotic genes is important for understanding the molecular mechanisms underlying apoptosis and tumorigenesis. The present study identified a novel anti-apoptotic gene named AREL1, which encodes a HECT (homologous to E6-AP carboxyl terminus) family E3 ubiquitin ligase. AREL1 interacted with and ubiquitinated IAP antagonists such as SMAC, HtrA2, and ARTS. However, AREL1 was cytosolic and did not localize to nuclei or mitochondria. The interactions between AREL1 and the IAP antagonists were specific for apoptosis-stimulated cells, in which the IAP antagonists were released into the cytosol from mitochondria. Furthermore, the ubiquitination and degradation of SMAC, HtrA2, and ARTS were significantly enhanced in AREL1-expressing cells following apoptotic stimulation, indicating that AREL1 binds to and ubiquitinates cytosolic but not mitochondria-associated forms of IAP antagonists. Furthermore, the anti-apoptotic role of AREL1-mediated degradation of SMAC, HtrA2, and ARTS was shown by simultaneous knockdown of three IAP antagonists, which caused the inhibition of caspase-3 cleavage, XIAP degradation, and induction of apoptosis. Therefore, the present study suggests that AREL1-mediated ubiquitination and degradation of cytosolic forms of three IAP antagonists plays an important role in the regulation of apoptosis.

Molecular cloning, characterization and expression analysis of ubiquitin protein ligase gene (As-ubpl) from Artemia sinica

Ubiquitylation is an important protein post-translational regulation pathway, which is involved in controlling protein degradation, tumor occurrence and cell cycle regulation. E3 ubiquitin protein ligase (UBPL) plays a crucial role of the conjugation of activated ubiquitin to protein substrates and leads to targeting proteins for degradation by the proteasome. We amplified one full-length cDNA of the A. sinica UBPL (As-ubpl) gene by RACE technology. The full-length cDNA of As-ubpl is composed of 2931 bp, with a 2571 bp open reading frame (ORF) that encodes a polypeptide of 856 amino acids with a C2 domain, two domains with two conserved Trp (W) residues (WW) and a homologous to E6-AP Carboxyl Terminus (HECT) domain. The amount of As-ubpl showed from real-time PCR indicates that a high expression levels of As-ubpl at 20 h, 40 h and 3 days of embryo development, with highest expression levels appearing in the larval stage (40 h). Furthermore, As-ubpl transcripts were highly up-regulated under salinity (50‰) and low temperature stress (15 °C). These results indicate that As-ubpl is involved in protein regulation of the postdiapause development and in responses to salinity and low temperature stress.

Promiscuous Interactions of gp78 E3 Ligase CUE Domain with Polyubiquitin Chains

Recognition of ubiquitin and polyubiquitin chains by ubiquitin-binding domains (UBDs) is vital for ubiquitin-mediated signaling pathways. The endoplasmic reticulum resident RING finger ubiquitin ligase (E3) gp78 regulates critical proteins via the ubiquitin-proteasome system to maintain cellular homeostasis and includes a UBD known as the CUE domain, which is essential for function. A probable role of this domain is to recognize ubiquitin-modified substrates, enabling gp78 to assemble polyubiquitin chains on these substrates and mark them for degradation. Here, we report the molecular details of the interaction of gp78CUE domain with ubiquitin and diubiquitin. The gp78CUE domain exhibits a well-defined set of interactions with ubiquitin anda dynamic, promiscuous interaction with diubiquitin chains. This leads to a model in which the CUE domain functions to both facilitate substrate binding and enable switching between adjacent ubiquitin molecules of a growing chain to enable processivity in ubiquitination.

A Human Ubiquitin Conjugating Enzyme (E2)-HECT E3 Ligase Structure-function Screen

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.

EDD Inhibits ATM-mediated Phosphorylation of p53

The EDD (E3 identified by differential display) gene, first identified as a progestin-induced gene in T-47D breast cancer cells, encodes an E3 ubiquitin ligase with a HECT domain. It was reported that EDD is involved in the G(2)/M progression through ubiquitination of phospho-katanin p60. Previous study has also shown that EDD can act as a transcription cofactor independently of its E3 ligase activity. In this study, we uncover a new role for EDD during cell cycle progression in an E3 ligase-independent manner. We demonstrate that EDD can physically interact with p53 and that this interaction blocks the phosphorylation of p53 by ataxia telangiectasia mutated (ATM). Silencing of EDD induces phosphorylation of p53 at Ser(15) and activates p53 target genes in fibroblasts and some transformed cells without activation of DNA damage response. The G(1)/S arrest induced by EDD depletion depends on p53. On the other hand, overexpression of EDD inhibits p53-Ser(15) phosphorylation and suppresses the induction of p53 target genes during DNA damage, and this effect does not require its E3 ligase activity. Thus, through binding to p53, EDD actively inhibits p53 phosphorylation by ATM and plays a role in ensuring smooth G(1)/S progression.

Smurf1 ubiquitin ligase targets Kruppel-like factor KLF2 for ubiquitination and degradation in human lung cancer H1299 cells

Krüppel-like factor 2 (KLF2) has been demonstrated to be essential for normal lung development, erythroid differentiation, T-cell differentiation, migration and homing. However, the mechanisms underlying the regulation of KLF2, in particular its responsible E3 ligase is still unclear. Here we show that the homologous to E6AP carboxyl terminus (HECT)-type ubiquitin ligase Smad ubiquitination regulatory factor 1 (Smurf1) interacts with and targets KLF2 for poly-ubiquitination and proteasomal degradation specifically in lung cancer H1299 cells. The catalytic ligase activity of Smurf1 is required for it to regulate KLF2. Consequently, Smurf1 represses the transcriptional factor activity of KLF2 and regulates the expression its downstream genes such as CD62L and Wee1. This study provided the first evidence that Smurf1 functions as an E3 ligase to promote the ubiquitination and proteasomal degradation of KLF2.

Absence of Association between Two HECTD2 Polymorphisms and Sporadic Creutzfeldt-Jakob Disease

Background: HECT (homologous to E6-AP carboxyl terminus) E3 ubiquitin ligases are fundamental components of the eukaryotic ubiquitin-proteasome system and are involved in the pathogenesis of several human diseases, including polyglutamine diseases. HECTD2, an E3 ubiquitin ligase, has been linked to the incubation time of prion disease in mice, and its polymorphisms have been associated with sporadic Creutzfeldt-Jakob disease (CJD) in the British population. Objective: To investigate whether 2 HECTD2 polymorphisms, –247G→A (rs7081363) and +16066T→A (rs12249854), are associated with sporadic CJD in the Korean population. Methods: We compared the genotype, allele and haplotype frequencies of the 2 HECTD2 polymorphisms in 205 sporadic CJD patients to those of 208 healthy Koreans. Results and Conclusion: Our study does not show significant differences in the genotype and allele frequencies of these 2 polymorphisms between sporadic CJD and normal controls. Significant differences in the haplotype frequencies of these 2 polymorphisms were not observed between sporadic CJD and normal controls either. Our results indicate that these 2 HECTD2 polymorphisms are not associated with genetic susceptibility to sporadic CJD in a Korean population. This is the first genetic association study of HECTD2 with sporadic CJD in an Asian population.

HECT ubiquitin ligase Smurf1 targets the tumor suppressor ING2 for ubiquitination and degradation

Inhibitor of growth 2 (ING2) gene encodes a candidate tumor suppressor and is frequently reduced in many tumors. However, the mechanisms underlying the regulation of ING2, in particular its protein stability, are still unclear. Here we show that the homologous to E6AP carboxyl terminus (HECT)-type ubiquitin ligase Smad ubiquitination regulatory factor 1 (Smurf1) interacts with and targets ING2 for poly-ubiquitination and proteasomal degradation. Intriguingly, the ING2 binding domain in Smurf1 was mapped to the catalytic HECT domain. Furthermore, the C-terminal PHD domain of ING2 was required for Smurf1-mediated degradation. This study provided the first evidence that the stability of ING2 could be regulated by ubiquitin-mediated degradation. Structured summary MINT- 7894271: ING2 (uniprotkb: Q9H160) binds (MI: 0407) to Smurf1 (uniprotkb: Q9HCE7) by pull-down (MI: 0096) MINT- 7894319, MINT- 7894339: ING2 (uniprotkb: Q9H160) physically interacts (MI: 0915) with Smurf1 (uniprotkb: Q9HCE7) by anti tag co-immunoprecipitation (MI: 0007) MINT- 7894301: Smurf1 (uniprotkb: Q9HCE7) physically interacts (MI: 0915) with ING2 (uniprotkb: Q9H160) by anti bait co-immunoprecipitation (MI: 0006) MINT- 7894358: ING1b (uniprotkb: Q9UK53-2) physically interacts (MI: 0915) with Smurf1 (uniprotkb: Q9HCE7) by anti tag co-immunoprecipitation (MI: 0007) MINT- 7894249: ING2 (uniprotkb: Q9H160) physically interacts (MI: 0915) with ubiquitin (uniprotkb: P62988) by anti tag co-immunoprecipitation (MI: 0007)

Ubiquitin ligases join the field of dietary restriction in C.elegans

Ubiquitin ligases join the field of dietary restriction in C.elegans

EDD Mediates DNA Damage-induced Activation of CHK2

EDD, the human orthologue of Drosophila melanogaster "hyperplastic discs," is overexpressed or mutated in a number of common human cancers. Although EDD has been implicated in DNA damage signaling, a definitive role has yet to be demonstrated. Here we report a novel interaction between EDD and the DNA damage checkpoint kinase CHK2. EDD and CHK2 associate through a phospho-dependent interaction involving the CHK2 Forkhead-associated domain and a region of EDD spanning a number of putative Forkhead-associated domain-binding threonines. Using RNA interference, we demonstrate a critical role for EDD upstream of CHK2 in the DNA damage signaling pathway. EDD is necessary for the efficient activating phosphorylation of CHK2 in response to DNA damage following exposure to ionizing radiation or the radiomimetic, phleomycin. Cells depleted of EDD display impaired CHK2 kinase activity and an inability to respond to DNA damage. These results identify EDD as a novel mediator in DNA damage signal transduction via CHK2 and emphasize the potential importance of EDD in cancer.

The HECT ubiquitin ligase AIP4 regulates the cell surface expression of select TRP channels

TRPV4 is a widely expressed member of the transient receptor potential (TRP) family that facilitates Ca(2+) entry into nonexcitable cells. TRPV4 is activated by several stimuli, but it is largely unknown how the activity of this channel is terminated. Here, we show that ubiquitination represents an important mechanism to control the presence of TRPV4 at the plasma membrane. Ubiquitination of TRPV4 is dramatically increased by the HECT (homologous to E6-AP carboxyl terminus)-family ubiquitin ligase AIP4 without inducing degradation of this channel. Instead, AIP4 promotes the endocytosis of TRPV4 and decreases its amount at the plasma membrane. Consequently, the basal activity of TRPV4 is reduced despite an overall increase in TRPV4 levels. This mode of regulation is not limited to TRPV4. TRPC4, another member of the TRP channel family, is also strongly ubiquitinated in the presence of AIP4, leading to the increased intracellular localization of TRPC4 and the reduction of its basal activity. However, ubiquitination of several other TRP channels is not affected by AIP4, demonstrating that AIP4-mediated regulation is a unique property of select TRP channels.

Itch/AIP4 mediates Deltex degradation through the formation of K29‐linked polyubiquitin chains

Deltex (DTX) and AIP4 are the human orthologues of the Drosophila deltex and Suppressor of deltex, which have been genetically described as being antagonistically involved in the Notch signalling pathway. Both genes encode E3 ubiquitin ligases of the RING (Really interesting new gene)-H2 and HECT (Homologous to E6AP carboxyl terminus) families, respectively. In an attempt to understand the molecular basis of their genetic interactions, we studied the relationship between DTX and AIP4 in the absence of activation of the Notch pathway. We show here that both molecules interact and partially colocalize to endocytic vesicles, and that AIP4 targets DTX for lysosomal degradation. Furthermore, AIP4-generated polyubiquitin chains are mainly conjugated through lysine 29 of ubiquitin in vivo, indicating a link between this type of chain and lysosomal degradation.

NARF, an Nemo-like Kinase (NLK)-associated Ring Finger Protein Regulates the Ubiquitylation and Degradation of T Cell Factor/Lymphoid Enhancer Factor (TCF/LEF)

beta-Catenin is a key player in the Wnt signaling pathway, and interacts with cofactor T cell factor/lymphoid enhancer factor (TCF/LEF) to generate a transcription activator complex that activates Wnt-induced genes. We previously reported that Nemo-like kinase (NLK) negatively regulates Wnt signaling via phosphorylation of TCF/LEF. To further evaluate the physiological roles of NLK, we performed yeast two-hybrid screening to identify NLK-interacting proteins. From this screen, we isolated a novel RING finger protein that we term NARF (NLK associated RING finger protein). Here, we show that NARF induces the ubiquitylation of TCF/LEF in vitro and in vivo, and functions as an E3 ubiquitin-ligase that specifically cooperates with the E2 conjugating enzyme E2-25K. We found that NLK augmented NARF binding and ubiquitylation of TCF/LEF, and this required NLK kinase activity. The ubiquitylated TCF/LEF was subsequently degraded by the proteasome. Furthermore, NARF inhibited formation of the secondary axis induced by the ectopic expression of beta-catenin in Xenopus embryos. Collectively, our findings raise the possibility that NARF functions as a novel ubiquitin-ligase to suppress the Wnt-beta-catenin signaling.

A Novel UbcH10-Binding Protein Facilitates the Ubiquitinylation of Cyclin B In Vitro

UbcH10 is known to act as a ubiquitin-conjugating enzyme (E2) for anaphase-promoting complex/cyclosome. Since some E2s support different ubiquitin ligases (E3), it is possible that UbcH10 interacts with other proteins. We cloned a novel protein named H10BH by using a yeast two-hybrid screening method with UbcH10 as bait. The carboxyl terminus of H10BH showed a weak homology to the HECT (homologous to E6-AP carboxyl terminus) domain, which is conserved in one of the families of E3. H10BH bound UbcH10, and the amino acid sequence between 235 and 257 was necessary for this binding. H10BH showed a self-ubiquitinylation activity in a HECT-like sequence-dependent manner. The carboxyl terminal half (amino acids 188-389) showed stronger activity than the full-length H10BH. Furthermore, the carboxyl terminal half of H10BH was able to bind cyclin B and ubiquitinylate cyclin B in vitro. These results suggest that H10BH functions as an E3 using UbcH10 for its E2.

NEDL1, a Novel Ubiquitin-protein Isopeptide Ligase for Dishevelled-1, Targets Mutant Superoxide Dismutase-1

Approximately 20% of familial amyotrophic lateral sclerosis (FALS) arises from germ-line mutations in the superoxide dismutase-1 (SOD1) gene. However, the molecular mechanisms underlying the process have been elusive. Here, we show that a neuronal homologous to E6AP carboxyl terminus (HECT)-type ubiquitin-protein isopeptide ligase (NEDL1) physically binds translocon-associated protein-delta and also binds and ubiquitinates mutant (but not wild-type) SOD1 proportionately to the disease severity caused by that particular mutant. Immunohistochemically, NEDL1 is present in the central region of the Lewy body-like hyaline inclusions in the spinal cord ventral horn motor neurons of both FALS patients and mutant SOD1 transgenic mice. Two-hybrid screening for the physiological targets of NEDL1 has identified Dishevelled-1, one of the key transducers in the Wnt signaling pathway. Mutant SOD1 also interacted with Dishevelled-1 in the presence of NEDL1 and caused its dysfunction. Thus, our results suggest that an adverse interaction among misfolded SOD1, NEDL1, translocon-associated protein-delta, and Dishevelled-1 forms a ubiquitinated protein complex that is included in potentially cytotoxic protein aggregates and that mutually affects their functions, leading to motor neuron death in FALS.

EDD, the Human Hyperplastic Discs Protein, Has a Role in Progesterone Receptor Coactivation and Potential Involvement in DNA Damage Response

The ubiquitin-protein ligase EDD encodes an orthologue of the hyperplastic discs tumor suppressor gene, which has a critical role in Drosophila development. Frequent allelic imbalance at the EDD chromosomal locus in human cancers suggests a role in tumorigenesis. In addition to a HECT (homologous to E6-AP carboxyl terminus) domain, the EDD protein contains a UBR1 zinc finger motif and ubiquitin-associated domain, each of which indicates involvement in ubiquitinylation pathways. This study shows that EDD interacts with importin alpha 5 through consensus basic nuclear localization signals and is localized in cell nuclei. EDD also binds progesterone receptor (PR) and potentiates progestin-mediated gene transactivation. This activity is comparable with that of the coactivator SRC-1, but, in contrast, the interaction between EDD and PR does not appear to involve an LXXLL receptor-binding motif. EDD also binds calcium- and integrin-binding protein/DNA-dependent protein kinase-interacting protein, a potential target of ubiquitin-mediated proteolysis, and an altered association is found between EDD and calcium- and integrin-binding protein/DNA-dependent protein kinase-interacting protein in response to DNA damage. The data presented here demonstrate a role for EDD in PR signaling but also suggest a link to cancer through DNA damage response pathways.