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Results for “NEDD8 Protein”

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At least 19 recordsLinked to original sources

PubMed2004

A unique E1-E2 interaction required for optimal conjugation of the ubiquitin-like protein NEDD8.

Ubiquitin-like proteins (UBLs) such as NEDD8 are transferred to their targets by distinct, parallel, multienzyme cascades that involve the sequential action of E1, E2 and E3 enzymes. How do enzymes within a particular UBL conjugation cascade interact with each other? We report here that the unique N-terminal sequence of NEDD8's E2, Ubc12, selectively recruits NEDD8's E1 to promote thioester formation between Ubc12 and NEDD8. A peptide corresponding to Ubc12's N terminus (Ubc12N26) specifically binds and inhibits NEDD8's E1, the heterodimeric APPBP1-UBA3 complex. The structure of APPBP1-UBA3- Ubc12N26 reveals conserved Ubc12 residues docking in a groove generated by loops conserved in UBA3s but not other E1s. These data explain why the Ubc12-UBA3 interaction is unique to the NEDD8 pathway. These studies define a novel mechanism for E1-E2 interaction and show how enzymes within a particular UBL conjugation cascade can be tethered together by unique protein-protein interactions emanating from their common structural scaffolds.

Amino Acid Sequence↗

PubMed1998

Crystal structure of the human ubiquitin-like protein NEDD8 and interactions with ubiquitin pathway enzymes.

The NEDD8/Rub1 class of ubiquitin-like proteins has been implicated in progression of the cell cycle from G1 into S phase. These molecules undergo a metabolism that parallels that of ubiquitin and involves specific interactions with many different proteins. We report here the crystal structure of recombinant human NEDD8 refined at 1.6-A resolution to an R factor of 21.9%. As expected from the high sequence similarity (57% identical), the NEDD8 structure closely resembles that reported previously for ubiquitin. We also show that recombinant human NEDD8 protein is activated, albeit inefficiently, by the ubiquitin-activating (E1) enzyme and that NEDD8 can be transferred from E1 to the ubiquitin conjugating enzyme E2-25K. E2-25K adds NEDD8 to a polyubiquitin chain with an efficiency similar to that of ubiquitin. A chimeric tetramer composed of three ubiquitins and one histidine-tagged NEDD8 binds to the 26 S proteasome with an affinity similar to that of tetraubiquitin. Seven residues that differ from the corresponding residues in ubiquitin, but are conserved between NEDD8 orthologs, are candidates for mediating interactions with NEDD8-specific partners. One such residue, Ala-72 (Arg in ubiquitin), is shown to perform a key role in selecting against reaction with the ubiquitin E1 enzyme, thereby acting to prevent the inappropriate diversion of NEDD8 into ubiquitin-specific pathways.

Amino Acid Sequence↗

PubMed2003

NEDD8 protein is involved in ubiquitinated inclusion bodies.

Proteolysis by the ubiquitin-proteasome system is considered to play a pathological role in several degenerative diseases that involve ubiquitinated inclusion bodies. In recent years, several ubiquitin-like proteins have been isolated, but it is uncertain whether their roles are associated with protein degradation through the ubiquitin-proteasome system. NEDD8 (neural precursor cell-expressed and developmentally down-regulated gene), which consists of 81 amino acid residues, possesses the highest sequence similarity to ubiquitin. Recent studies have indicated that NEDD8 is covalently ligated to cullin family proteins, which are components of certain ubiquitin E3 ligases, by a pathway analogous to that of ubiquitin. Thus, by focusing on the structural and functional association between NEDD8 and ubiquitin, it would be of interest to know whether the NEDD8 system is involved in pathological disorders of the ubiquitin-proteasome system. This study has examined the immunohistochemical distribution of NEDD8 protein by using a highly purified antibody in normal tissues and in tissues known to contain ubiquitinated inclusions. NEDD8 protein expression was widely observed in most types of tissues. Furthermore, accumulation of the NEDD8 protein was commonly observed in ubiquitinated inclusion bodies, including Lewy bodies in Parkinson's disease, Mallory bodies in alcoholic liver disease, and Rosenthal fibres in astrocytoma. Two of ten cases of neurofibrillary tangles and senile plaques from patients with Alzheimer's disease showed intense staining for NEDD8 as well as for ubiquitin. These findings suggest the possibility that the NEDD8 system is involved in the metabolism of these inclusion bodies via the ubiquitin-proteasome system.

Alzheimer Disease↗

PubMed1999

Conjugation of the ubiquitin-like protein NEDD8 to cullin-2 is linked to von Hippel-Lindau tumor suppressor function.

The von Hippel-Lindau tumor suppressor protein pVHL assembles with cullin-2 (hCUL-2) and elongin B/C forming a protein complex, CBCVHL, that resembles SKP1-CDC53-F-box protein ubiquitin ligases. Here, we show that hCUL-2 is modified by the conserved ubiquitin-like protein NEDD8 and that NEDD8-hCUL-2 conjugates are part of CBCVHL complexes in vivo. Remarkably, the formation of these conjugates is stimulated by the pVHL tumor suppressor. A tumorigenic pVHL variant, however, is essentially deficient in this activity. Thus, ligation of NEDD8 to hCUL-2 is linked to pVHL activity and may be important for pVHL tumor suppressor function.

Carrier Proteins↗

PubMed2000

Modification of cullin-1 by ubiquitin-like protein Nedd8 enhances the activity of SCF(skp2) toward p27(kip1).

The periodic expression of cell cycle proteins is important for the regulation of cell cycle progression. The amount of CDK inhibitor, p27(kip1), one such protein, seems to be regulated by the ubiquitin-proteasome system. The ubiquitin ligase (E3) toward p27(kip1) is thought to be SCF(skp2). The activity of SCF(skp2) was increased by the addition of Roc1 protein to the complex. Furthermore, the ubiquitination of p27(kip1) seemed to be dependent on the phosphorylation of T187 of p27(kip1) because the mutant T187A was not ubiquitinated at all in an in vitro ubiquitination system. Cullin-1, a component of SCF, is modified by ubiquitin-like protein Nedd8. The modification site of cullin-1 was shown to be K696 because the K696R mutant was not modified. When the effect of the Nedd8 modification on the SCF(skp2) activity toward p27(kip1) was investigated, the activity was markedly decreased by using the Nedd8-unmodified mutant cullin-1 (K696R), indicating that the modification may play an important role on the SCF(skp2) activity toward p27(kip1).

Amino Acid Sequence↗

PubMed1997

Characterization of NEDD8, a developmentally down-regulated ubiquitin-like protein.

NEDD8 is a novel 81 amino acid polypeptide which is 60% identical and 80% homologous to ubiquitin. Northern blot analysis showed that the NEDD8 message was developmentally down-regulated. In adult tissues, NEDD8 expression was mostly restricted to the heart and skeletal muscle. Antiserum specific for NEDD8 detected a 6-kDa monomer in SK-N-SH, BJAB, and HL60 cell lysates. A 14-kDa band was also detected in BJAB, HL60, and SK-MEL28 but not in SK-N-SH and K562 cell lysates. An approximately 90-kDa band was detected in all cell lines tested. Thus, NEDD8 is likely to be conjugated to other proteins in a manner analogous to ubiquitination. However, the conjugation pattern of NEDD8 is entirely different from that of ubiquitin in all cell lines tested. To study NEDD8 conjugation in more detail, hemagglutinin-epitope-tagged NEDD8 was expressed in COS cells. Western blot analysis revealed an NEDD8 monomer and a series of higher molecular weight NEDD8-conjugated proteins or NEDD8 multimers. Immunocytochemical analysis showed that NEDD8 expression was highly enriched in the nucleus and was much weaker in the cytosol. In contrast, ubiquitin expression was detectable equally well in the nucleus and cytosol. Mutational analysis showed that the C terminus of NEDD8 was efficiently cleaved and that Gly-76 was required for conjugation of NEDD8 to other proteins. Taken together, NEDD8 provides another substrate for covalent protein modification and may play a unique role during development.

Amino Acid Sequence↗

PubMed1999

Covalent modification of all members of human cullin family proteins by NEDD8.

Recently we found that NEDD8, a ubiquitin-like protein, was linked covalently to human cullin-4A (abbreviated Cul-4A) by a new ubiquitin-related pathway that is analogous to but distinct from the ligating system for SUMO1, another ubiquitin-like protein. However, it remained unknown whether the other five members of the family of human cullin/Cdc53 proteins are modified by NEDD8. Here we report that all Hs-Cul family proteins, such as Cul-1, Cul-2, Cul-3, Cul-4B, and Cul-5, in addition to Cul-4A, were modified by covalent attachment of NEDD8 in rabbit reticulocyte lysates. Moreover, by comprehensive Northern-blot analyses, we examined multiple tissue distributions of the messages for all Cul-family proteins, NEDD8, and the NEDD8-ligating system consisting of APP-BP1/hUba3, and hUbc12, which function as E1- and E2-like enzymes, respectively. The expressions of Cul-1, Cul-2, and Cul-3 resembled each other and were apparently correlated to those of NEDD8 and the NEDD8-ligating system in various human cells and tissues. However, the mRNA levels of Cul-4A, Cul-4B, and Cul-5 differed considerably from each other as well as from other Cul-family proteins. The enhanced expression of all Cul-family proteins except Cul-5 was observed in a variety of tumor cell lines.

Cell Cycle Proteins↗

PubMed2001

NUB1, a NEDD8-interacting protein, is induced by interferon and down-regulates the NEDD8 expression.

NEDD8, a ubiquitin-like protein, covalently conjugates to cullin family members. It appears to control vital biological events through its conjugation to cullins. To study how this conjugation pathway is regulated, we performed yeast two-hybrid screening by using NEDD8 as a bait and isolated a cDNA fragment encoding a potent down-regulator of the NEDD8 expression. Here, we report this novel regulator, NUB1 (NEDD8 Ultimate Buster-1). NUB1 is composed of 601 residues with a calculated 69.1-kDa molecular mass. It is an interferon-inducible protein and predominantly localized in the nucleus. The NUB1 message is specifically expressed in adult human testis, ovary, heart, and skeletal muscle tissues and is developmentally down-regulated in mouse embryos. In biochemical analysis, we found that NUB1 overexpression leads to severe reduction of NEDD8 monomer and NEDD8 conjugates in cells. This reduction is not due to down-regulation of NEDD8 transcription, but due to post-transcriptional mechanism. As expected from this activity, overexpression of NUB1 had a profound growth-inhibitory effect on U2OS cells. Thus, NUB1 is a strong down-regulator of the NEDD8 expression and appears to play critical roles in regulating biological events, including cell growth.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Promotion of NEDD-CUL1 conjugate cleavage by COP9 signalosome.

SCF ubiquitin ligases control various processes by marking regulatory proteins for ubiquitin-dependent proteolysis. To illuminate how SCF complexes are regulated, we sought proteins that interact with the human SCF component CUL1. The COP9 signalosome (CSN), a suppressor of plant photomorphogenesis, associated with multiple cullins and promoted cleavage of the ubiquitin-like protein NEDD8 from Schizosaccharomyces pombe CUL1 in vivo and in vitro. Multiple NEDD8-modified proteins uniquely accumulated in CSN-deficient S. pombe cells. We propose that the broad spectrum of activities previously attributed to CSN subunits--including repression of photomorphogenesis, activation of JUN, and activation of p27 nuclear export--underscores the importance of dynamic cycles of NEDD8 attachment and removal in biological regulation.

3T3 Cells↗

PubMed2000

Identification of a novel isopeptidase with dual specificity for ubiquitin- and NEDD8-conjugated proteins.

Covalent conjugation of proteins by ubiquitin or ubiquitin-like molecules is an important form of post-translational modification and plays a critical role in many cellular processes. Similar to the concept of phosphorylation and dephosphorylation, these conjugates are regulated by a large number of deconjugating enzymes. Here, we report the cloning of a 2,141-base pair DNA fragment from human placenta cDNA library by a strategy that involves expressed sequence tag data base searching, polymerase chain reaction, and rapid amplification of cDNA ends. Nucleotide sequence analysis revealed that the cloned cDNA contains an open reading frame of 1,143 base pairs encoding a novel protease, USP21, which is composed of 381 residues with a calculated molecular mass of 43 kDa. The human USP21 gene is located on chromosome 1q21 and encodes a member of the ubiquitin-specific protease family with highly conserved Cys and His domains. The activity and specificity of USP21 were determined by using a COS cell expression system in vivo. We showed that USP21 is capable of removing ubiquitin from ubiquitinated proteins as expected. Furthermore, USP21 is capable of removing NEDD8 from NEDD8 conjugates but has no effect on Sentrin-1 conjugates. As expected from its biochemical activity, overexpression of USP21 has a profound growth inhibitory effect on U2OS cells. Thus, USP21 is the first ubiquitin-specific protease shown to have dual specificity for both ubiquitin and NEDD8 and may play an important role in the regulation of cell growth.

Amino Acid Sequence↗

PubMed2003

APP-BP1 mediates APP-induced apoptosis and DNA synthesis and is increased in Alzheimer's disease brain.

APP-BP1, first identified as an amyloid precursor protein (APP) binding protein, is the regulatory subunit of the activating enzyme for the small ubiquitin-like protein NEDD8. We have shown that APP-BP1 drives the S- to M-phase transition in dividing cells, and causes apoptosis in neurons. We now demonstrate that APP-BP1 binds to the COOH-terminal 31 amino acids of APP (C31) and colocalizes with APP in a lipid-enriched fraction called lipid rafts. We show that coexpression of a peptide representing the domain of APP-BP1 that binds to APP, abolishes the ability of overexpressed APP or the V642I mutant of APP to cause neuronal apoptosis and DNA synthesis. A dominant negative mutant of the NEDD8 conjugating enzyme hUbc12, which participates in the ubiquitin-like pathway initiated by APP-BP1, blocks neuronal apoptosis caused by APP, APP(V642I), C31, or overexpression of APP-BP1. Neurons overexpressing APP or APP(V642I) show increased APP-BP1 protein levels in lipid rafts. A similar increase in APP-BP1 in lipid rafts is observed in the Alzheimer's disease brain hippocampus, but not in less-affected areas of Alzheimer's disease brain. This translocation of APP-BP1 to lipid rafts is accompanied by a change in the subcellular localization of the ubiquitin-like protein NEDD8, which is activated by APP-BP1.

Alzheimer Disease↗

PubMed2005

Interaction of NUB1 with the proteasome subunit S5a.

NUB1 interacts with a ubiquitin-like protein NEDD8 to target the NEDD8 monomer and neddylated proteins to the proteasome for degradation. Therefore, NUB1 is thought to be a potent downregulator of NEDD8 conjugation system. Since NUB1 possesses a UBL domain, which was previously shown to be an S5a-interacting motif in RAD23/HHR23, we initially hypothesized that NUB1 interacts with the S5a subunit of the proteasome through its UBL domain. To examine this, we performed an in vitro GST pull-down assay and a yeast two-hybrid assay. Unexpectedly, our studies revealed that NUB1 directly interacts with the S5a subunit through its C-terminal region between amino acid residues 536 and 584, not through its UBL domain. Although the UBL domain was not an S5a-interacting motif in NUB1, our further studies revealed that the UBL domain is required for the function of NUB1.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Regulation of neddylation and deneddylation of cullin1 in SCFSkp2 ubiquitin ligase by F-box protein and substrate.

The activity of cullin-containing ubiquitin protein ligase complexes is stimulated by linkage to cullin of the ubiquitin-like protein Nedd8 ("neddylation"). Neddylation is inhibited by the tight binding of cullins to CAND1 (cullin-associated and neddylation-dissociated 1) protein, and Nedd8 is removed from cullins by specific isopeptidase activity of the COP9/signalosome (CSN) complex. The mechanisms that regulate neddylation and deneddylation of cullins were unknown. We examined this problem for the case of SCF(Skp2), a cullin1 (Cul1)-containing ubiquitin ligase complex that contains the S phase-associated protein Skp2 as the substrate-binding F-box protein subunit. SCF(Skp2) targets for degradation the cyclin-dependent kinase (cdk) inhibitor p27 in the G(1)-to-S phase transition, a process that requires its phosphorylation and binding to cdk2-cyclin E. Because levels of Skp2, cyclin E, and the accessory protein Cks1 (cyclin kinase subunit 1) all rise at the end of G(1) phase, it seemed possible that the neddylation of Cul1 in SCF(Skp2) is regulated by the availability of the F-box protein and/or the substrate. We found that the supplementation of Skp2-Skp1 and substrate (along with further components necessary for substrate presentation to the ubiquitin ligase) to extracts of HeLa cells synergistically increased levels of neddylated Cul1. Skp2-Skp1 abrogates the inhibitory influence of CAND1 on the neddylation of Cul1 by promoting the dissociation of the cullin-CAND1 complex, whereas substrate, together with substrate-presenting components, prevents the action of CSN to deneddylate cullin. We propose a sequence of events in which the increased availability of Skp2 and substrate in the transition of cells to S phase promotes the neddylation and assembly of the SCF(Skp2) ubiquitin ligase complex.

Cell Cycle Proteins↗

PubMed2002

Role of predicted metalloprotease motif of Jab1/Csn5 in cleavage of Nedd8 from Cul1.

COP9 signalosome (CSN) cleaves the ubiquitin-like protein Nedd8 from the Cul1 subunit of SCF ubiquitin ligases. The Jab1/MPN domain metalloenzyme (JAMM) motif in the Jab1/Csn5 subunit was found to underlie CSN's Nedd8 isopeptidase activity. JAMM is found in proteins from archaea, bacteria, and eukaryotes, including the Rpn11 subunit of the 26S proteasome. Metal chelators and point mutations within JAMM abolished CSN-dependent cleavage of Nedd8 from Cul1, yet had little effect on CSN complex assembly. Optimal SCF activity in yeast and both viability and proper photoreceptor cell (R cell) development in Drosophila melanogaster required an intact Csn5 JAMM domain. We propose that JAMM isopeptidases play important roles in a variety of physiological pathways.

Amino Acid Motifs↗

PubMed2000

Ubiquitin-like proteins: new wines in new bottles.

Ubiquitin is a small polypeptide that covalently modifies other cellular proteins and targets them to the proteasome for degradation. In recent years, ubiquitin-dependent proteolysis has been demonstrated to play a critical role in the regulation of many cellular processes, such as cell cycle progression, cell signaling, and immune recognition. The recent discovery of three new ubiquitin-like proteins, NEDD8, Sentrin/SUMO, and Apg12, has further broadened the horizon of this type of post-translational protein modification. This review will focus on the biology and biochemistry of the Sentrin/SUMO and NEDD8 modification pathways, which are clearly distinct from the ubiquitination pathway and have unique biological functions.

Amino Acid Sequence↗

PubMed2002

Distinct protein degradation mechanisms mediated by Cul1 and Cul3 controlling Ci stability in Drosophila eye development.

The ubiquitin-like protein, Nedd8, covalently modifies members of the Cullin family. Cullins are the major components of a series of ubiquitin ligases that control the degradation of a broad range of proteins. We found that Nedd8 modifies Cul1 in Drosophila. In Drosophila Nedd8 and Cul1 mutants, protein levels of the signal transduction effectors, Cubitus interruptus (Ci) and Armadillo (Arm), and the cell cycle regulator, Cyclin E (CycE), are highly accumulated, suggesting that the Cul1-based SCF complex requires Nedd8 modification for the degradation processes of Ci, Arm, and CycE in vivo. We further show that two distinct degradation mechanisms modulating Ci stability in the developing eye disc are separated by the morphogenetic furrow (MF) in which retinal differentiation is initiated. In cells anterior to the MF, Ci proteolytic processing promoted by PKA requires the activity of the Nedd8-modified Cul1-based SCF(Slimb) complex. In posterior cells, Ci degradation is controlled by a mechanism that requires the activity of Cul3, another member of the Cullin family. This posterior Ci degradation mechanism, which partially requires Nedd8 modification, is activated by Hedgehog (Hh) signaling and is PKA-independent.

Amino Acid Sequence↗

PubMed2004

Cullin-based ubiquitin ligase and its control by NEDD8-conjugating system.

Several studies have examined the importance of ubiquitin-like posttranslational modifiers (which consist of an unexpectedly large family). Of these, NEDD8 (also called Rub1, related to ubiquitin 1) with a high homology to ubiquitin is covalently linked to all members of cullin (Cul)-family proteins through an enzymatic cascade analogous to ubiquitylation. Cul-family proteins are scaffold proteins for a wide series of ubiquitin-protein ligase complexes, such as SCFs (Skp1, Cul-1, Roc1, and F-box proteins), which regulate the degradation of broad range of cellular proteins. Unlike ubiquitin, which mostly acts as a degradation signal for the target proteins, NEDD8 acts as an activation signal for Cul-family proteins; i.e., Cul-based ubiquitin-protein ligases. Accordingly, the NEDD8 conjugation pathway regulating Cul-protein function is responsible for a diverse array of biologically important processes, such as the cell cycle progression, signalling cascades and developmental programs. Furthermore, recent studies have revealed that the COP9/Signalosome complex interacts physically and genetically with Cul-family proteins, and catalyzes deconjugation of NEDD8 ligated to Cul-family proteins. This review summarizes recent advances in biochemical and genetic studies on how the NEDD8-modifying system regulates Cul-family proteins and their physiology.

Animals↗

PubMedDate not supplied

Control of IkappaBalpha proteolysis by the ubiquitin-proteasome pathway.

It has recently been determined that the proteolytic destruction of IkappaB (inhibitor of NF-kappaB) by the ubiquitin-proteasome system plays a key role in the immediate elimination of IkappaB from the IkappaB-(NF-kappaB) complex which allows nuclear translocation of free NF-kappaB, thus leading to activation of a multitude of target genes. The SCF(Fbw1) (composed of Skp1, Cul-1, Roc1, and Fbw1) complex, identified as an IkappaBalpha-E3 ligase, binds and ubiquitylates IkappaBalpha phosphorylated by IkappaB kinase that has been activated in response to extracellular signals. The generating poly-ubiquitin chain is finally recognized by the 26S proteasome for ultimate degradation. In this NF-kappaB signalling pathway, it becomes clear that the SCF(Fbw1) activity is enhanced by a ubiquitin-like protein NEDD8 (equivalent to Rub1) that modifies Cul-1 in a manner analogous to ubiquitylation, and consequently, IkappaBalpha proteolysis is induced. NEDD8 is a new regulator of the SCF ubiquitin-ligase, functioning as a covalent modifier for proteolytic targeting at a physiological level.

Animals↗