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A Ciechanover

Publications and source records attributed to A Ciechanover.

At least 37 records · Page 2Linked to original sources
PubMed2000

Ubiquitin-mediated proteolysis: biological regulation via destruction.

The ubiquitin proteolytic system plays an important role in a broad array of basic cellular processes. Among these are regulation of cell cycle, modulation of the immune and inflammatory responses, control of signal transduction pathways, development and differentiation. These complex processes are controlled via specific degradation of a single or a subset of proteins. Degradation of a protein by the ubiquitin system involves two successive steps, conjugation of multiple moieties of ubiquitin and degradation of the tagged protein by the 26S proteasome. An important question concerns the identity of the mechanisms that underlie the high degree of specificity of the system. Substrate recognition is governed by a large family ubiquitin ligases that recognize the substrates, bind them and catalyze/facilitate their interaction with ubiquitin.

Animals↗
PubMed1999

Identification of the ubiquitin carrier proteins, E2s, involved in signal-induced conjugation and subsequent degradation of IkappaBalpha.

The last step in the activation of the transcription factor NF-kappaB is signal-induced, ubiquitin- and proteasome-mediated degradation of the inhibitor IkappaBalpha. Although most of the components involved in the activation and degradation pathways have been identified, the ubiquitin carrier proteins (E2) have remained elusive. Here we show that the two highly homologous members of the UBCH5 family, UBCH5b and UBCH5c, and CDC34/UBC3, the mammalian homolog of yeast Cdc34/Ubc3, are the E2 enzymes involved in the process. The conjugation reaction they catalyze in vitro is specific, as they do not recognize the S32A,S36A mutant species of IkappaBalpha that cannot be phosphorylated and conjugated following an extracellular signal. Furthermore, the reaction is specifically inhibited by a doubly phosphorylated peptide that spans the ubiquitin ligase recognition domain of the inhibitor. Cys-to-Ala mutant species of the enzymes that cannot bind ubiquitin inhibit tumor necrosis factor alpha-induced degradation of the inhibitor in vivo. Not surprisingly, they have a similar effect in a cell-free system as well. Although it is clear that the E2 enzymes are not entirely specific to IkappaBalpha, they are also not involved in the conjugation and degradation of the bulk of cellular proteins, thus exhibiting some degree of specificity that is mediated probably via their association with a defined subset of ubiquitin-protein ligases. The mechanisms that underlie the involvement of two different E2 species in IkappaBalpha conjugation are not clear at present. It is possible that different conjugating machineries operate under different physiological conditions or in different cells.

Carrier Proteins↗
PubMed1999

Mutation of the E6-AP ubiquitin ligase reduces nuclear inclusion frequency while accelerating polyglutamine-induced pathology in SCA1 mice.

Mutant ataxin-1, the expanded polyglutamine protein causing spinocerebellar ataxia type 1 (SCA1), aggregates in ubiquitin-positive nuclear inclusions (NI) that alter proteasome distribution in affected SCA1 patient neurons. Here, we observed that ataxin-1 is degraded by the ubiquitin-proteasome pathway. While ataxin-1 [2Q] and mutant ataxin-1 [92Q] are polyubiquitinated equally well in vitro, the mutant form is three times more resistant to degradation. Inhibiting proteasomal degradation promotes ataxin-1 aggregation in transfected cells. And in mice, Purkinje cells that express mutant ataxin-1 but not a ubiquitin-protein ligase have significantly fewer NIs. Nonetheless, the Purkinje cell pathology is markedly worse than that of SCA1 mice. Taken together, NIs are not necessary to induce neurodegeneration, but impaired proteasomal degradation of mutant ataxin-1 may contribute to SCA1 pathogenesis.

Animals↗
PubMed1999

Ubiquitin ligase activity and tyrosine phosphorylation underlie suppression of growth factor signaling by c-Cbl/Sli-1.

Receptor desensitization is accomplished by accelerated endocytosis and degradation of ligand-receptor complexes. An in vitro reconstituted system indicates that Cbl adaptor proteins directly control downregulation of the receptor for the epidermal growth factor (EGFR) by recruiting ubiquitin-activating and -conjugating enzymes. We infer a sequential process initiated by autophosphorylation of EGFR at a previously identified lysosome-targeting motif that subsequently recruits Cbl. This is followed by tyrosine phosphorylation of c-Cbl at a site flanking its RING finger, which enables receptor ubiquitination and degradation. Whereas all three members of the Cbl family can enhance ubiquitination, two oncogenic Cbl variants, whose RING fingers are defective and phosphorylation sites are missing, are unable to desensitize EGFR. Our study identifies Cbl proteins as components of the ubiquitin ligation machinery and implies that they similarly suppress many other signaling pathways.

Caenorhabditis elegans Proteins↗
PubMed1999

Degradation of MyoD by the ubiquitin pathway: regulation by specific DNA-binding and identification of a novel site for ubiquitination.

MyoD is a tissue-specific transcriptional activator involvd in skeletal muscle differentiation. It is induced during transition from proliferating, non-differentiated myoblasts to the resting and well differentiated myotubes. Like many other transcriptional regulators, it is short-lived, however, the targeting proteolytic pathway and the underlying regulatory mechanisms involved have remained obscure. Here we show that MyoD is degraded by the ubiquitin system both in vivo and in vitro. In cells, degradation is inhibited by lactacystin, a specific inhibitor of the 20S proteasome. Inhibition is accompanied by accumulation of MyoD-ubiquitin conjugates. In a cell free system, the proteolytic process requires both ATP and ubiquitin and is preceded by formation of MyoD-ubiquitin adducts. Interestingly, the process is inhibited by the specific DNA sequence to which MyoD binds. Analysis of the ubiquitination site has revealed that the N-terminal residue of MyoD is sufficient and essential to promote conjugation and subsequent degradation of the protein: conjugation to internal Lys residues is not necessary. Substitution of all Lys residues did not affect significantly its degradation either in intact cells or in a reconstituted cell free system. Degradation was inhibited by specific proteasome inhibitors and was accompanied by accumulation of ubiquitinated species of the protein. We concluded that the first ubiquitin moiety is attached via its C-terminal Gly to the N-terminal residue of MyoD, and the polyubiquitin chain is then synthesized on Lys48 of this moiety.

Adenosine Triphosphate↗
PubMed1999

Structural motifs involved in ubiquitin-mediated processing of the NF-kappaB precursor p105: roles of the glycine-rich region and a downstream ubiquitination domain.

The ubiquitin proteolytic system plays a major role in a variety of basic cellular processes. In the majority of these processes, the target proteins are completely degraded. In one exceptional case, generation of the p50 subunit of the transcriptional regulator NF-kappaB, the precursor protein p105 is processed in a limited manner: the N-terminal domain yields the p50 subunit, whereas the C-terminal domain is degraded. The identity of the mechanisms involved in this unique process have remained elusive. It has been shown that a Gly-rich region (GRR) at the C-terminal domain of p50 is an important processing signal. Here we show that the GRR does not interfere with conjugation of ubiquitin to p105 but probably does interfere with the processing of the ubiquitin-tagged precursor by the 26S proteasome. Structural analysis reveals that a short sequence containing a few Gly residues and a single essential Ala is sufficient to generate p50. Mechanistically, the presence of the GRR appears to stop further degradation of p50 and to stabilize the molecule. It appears that the localization of the GRR within p105 plays an important role in directing processing: transfer of the GRR within p105 or insertion of the GRR into homologous or heterologous proteins is not sufficient to promote processing in most cases, which is probably due to the requirement for an additional specific ubiquitination and/or recognition domain(s). Indeed, we have shown that amino acid residues 441 to 454 are important for processing. In particular, both Lys 441 and Lys 442 appear to serve as major ubiquitination targets, while residues 446 to 454 are independently important for processing and may serve as the ubiquitin ligase recognition motif.

Amino Acid Sequence↗
PubMed1999

The ubiquitin-proteasome pathway and pathogenesis of human diseases.

The ubiquitin-proteasome pathway plays a pivotal role in the degradation of short-lived and regulatory proteins important in a variety of basic cellular processes, including regulation of the cell cycle, modulation of cell surface receptors and ion channels, and antigen presentation. The pathway involves an enzymatic cascade through which multiple ubiquitin molecules are covalently attached to the protein substrate, which is then degraded by the 26S proteasome complex. The pathway has been implicated in several forms of malignancy, in the pathogenesis of several genetic diseases (including cystic fibrosis, Angelman's syndrome, and Liddle syndrome), in immune surveillance/viral pathogenesis, and in the pathology of muscle wasting. The molecular mechanisms that underlie these processes are being unraveled at present.

Antigen Presentation↗
PubMed1998

A novel site for ubiquitination: the N-terminal residue, and not internal lysines of MyoD, is essential for conjugation and degradation of the protein.

The ubiquitin proteolytic pathway is a major system for selective protein degradation in eukaryotic cells. One of the first steps in the degradation of a protein via this pathway involves selective modification of epsilon-NH2 groups of internal lysine residues by ubiquitination. To date, this amino group has been the only known target for ubiquitination. Here we report that the N-terminal residue of MyoD is sufficient and necessary for promotion of conjugation and subsequent degradation of the protein. Substitution of all lysine residues in the protein did not affect significantly its conjugation and degradation either in vivo or in vitro. In cells, degradation of the lysine-less protein is inhibited by the proteasome inhibitors MG132 and lactacystin. Inhibition is accompanied by accumulation of high molecular mass ubiquitinated forms of the modified MyoD. In striking contrast, wild-type MyoD, in which all the internal Lys residues have been retained but the N-terminus has been extended by fusion of a short peptide, is stable both in vivo and in vitro. In a cell-free system, ATP and multiple ubiquitination are essential for degradation of the lysine-less protein. Specific chemical modifications have yielded similar results. Selective blocking of the alpha-NH2 group of wild-type protein renders it stable, while modification of the internal Lys residues with preservation of the free N-terminal group left the protein susceptible to degradation. Our data suggest that conjugation of MyoD occurs via a novel modification involving attachment of ubiquitin to the N-terminal residue. The polyubiquitin chain is then synthesized on an internal Lys residue of the linearly attached first ubiquitin moiety.

Adenosine Triphosphate↗
PubMed1998

Basal and human papillomavirus E6 oncoprotein-induced degradation of Myc proteins by the ubiquitin pathway.

We have previously shown that the degradation of c-myc and N-myc in vitro is mediated by the ubiquitin system. However, the role of the system in targeting the myc proteins in vivo and the identity of the conjugating enzymes and possible ancillary proteins involved has remained obscure. Here we report that the degradation of the myc proteins in cells is inhibited by lactacystin and MG132, two inhibitors of the 20S proteasome. Inhibition is accompanied by accumulation of myc-ubiquitin conjugates. Dissection of the ancillary proteins involved revealed that the high-risk human papillomavirus oncoprotein E6-16 stimulates conjugation and subsequent degradation of the myc proteins in vitro. Expression of E6-16 in cells results in significant shortening of the t1/2 of the myc proteins with subsequent decrease in their cellular level. Analysis of the conjugating enzymes revealed that under basal conditions the proteins can be conjugated by two pairs of E2s and E3s-E2-14 kDa and E3alpha involved in the "N-end rule" pathway, and E2-F1 (UbcH7) and E3-Fos involved also in conjugation of c-Fos. In the presence of E6-16, a third pair, E2-F1 and E6-AP mediate conjugation of myc by means of a mechanism that appears to be similar to that involved in the targeting of p53, formation of a myc. E6.E6-AP targeting complex. It is possible that in certain cells E6-mediated targeting of myc prevents myc-induced apoptosis and thus ensures maintenance of viral infection.

Acetylcysteine↗
PubMed1998

Role of UEV-1, an inactive variant of the E2 ubiquitin-conjugating enzymes, in in vitro differentiation and cell cycle behavior of HT-29-M6 intestinal mucosecretory cells.

By means of differential RNA display, we have isolated a cDNA corresponding to transcripts that are down-regulated upon differentiation of the goblet cell-like HT-29-M6 human colon carcinoma cell line. These transcripts encode proteins originally identified as CROC-1 on the basis of their capacity to activate transcription of c-fos. We show that these proteins are similar in sequence, and in predicted secondary and tertiary structure, to the ubiquitin-conjugating enzymes, also known as E2. Despite the similarities, these proteins lack a critical cysteine residue essential for the catalytic activity of E2 enzymes and, in vitro, they do not conjugate or transfer ubiquitin to protein substrates. These proteins constitute a distinct subfamily within the E2 protein family and are highly conserved in phylogeny from yeasts to mammals. Therefore, we have designated them UEV (ubiquitin-conjugating E2 enzyme variant) proteins, defined as proteins similar in sequence and structure to the E2 ubiquitin-conjugating enzymes but lacking their enzymatic activity (HW/GDB-approved gene symbol, UBE2V). At least two human genes code for UEV proteins, and one of them, located on chromosome 20q13.2, is expressed as at least four isoforms, generated by alternative splicing. All human cell types analyzed expressed at least one of these isoforms. Constitutive expression of exogenous human UEV in HT-29-M6 cells inhibited their capacity to differentiate upon confluence and caused both the entry of a larger proportion of cells in the division cycle and an accumulation in G2-M. This was accompanied with a profound inhibition of the mitotic kinase, cdk1. These results suggest that UEV proteins are involved in the control of differentiation and could exert their effects by altering cell cycle distribution.

Amino Acid Sequence↗
PubMed1998

Degradation of myogenic transcription factor MyoD by the ubiquitin pathway in vivo and in vitro: regulation by specific DNA binding.

MyoD is a tissue-specific transcriptional activator that acts as a master switch for skeletal muscle differentiation. Its activity is induced during the transition from proliferating, nondifferentiated myoblasts to resting, well-differentiated myotubes. Like many other transcriptional regulators, it is a short-lived protein; however, the targeting proteolytic pathway and the underlying regulatory mechanisms involved in the process have remained obscure. It has recently been shown that many short-lived regulatory proteins are degraded by the ubiquitin system. Degradation of a protein by the ubiquitin system proceeds via two distinct and successive steps, conjugation of multiple molecules of ubiquitin to the target protein and degradation of the tagged substrate by the 26S proteasome. Here we show that MyoD is degraded by the ubiquitin system both in vivo and in vitro. In intact cells, the degradation is inhibited by lactacystin, a specific inhibitor of the 26S proteasome. Inhibition is accompanied by accumulation of high-molecular-mass MyoD-ubiquitin conjugates. In a cell-free system, the proteolytic process requires both ATP and ubiquitin and, like the in vivo process, is preceded by formation of ubiquitin conjugates of the transcription factor. Interestingly, the process is inhibited by the specific DNA sequence to which MyoD binds: conjugation and degradation of a MyoD mutant protein which lacks the DNA-binding domain are not inhibited. The inhibitory effect of the DNA requires the formation of a complex between the DNA and the MyoD protein. Id1, which inhibits the binding of MyoD complexes to DNA, abrogates the effect of DNA on stabilization of the protein.

Animals↗
PubMed1998

The ubiquitin system.

The selective degradation of many short-lived proteins in eukaryotic cells is carried out by the ubiquitin system. In this pathway, proteins are targeted for degradation by covalent ligation to ubiquitin, a highly conserved small protein. Ubiquitin-mediated degradation of regulatory proteins plays important roles in the control of numerous processes, including cell-cycle progression, signal transduction, transcriptional regulation, receptor down-regulation, and endocytosis. The ubiquitin system has been implicated in the immune response, development, and programmed cell death. Abnormalities in ubiquitin-mediated processes have been shown to cause pathological conditions, including malignant transformation. In this review we discuss recent information on functions and mechanisms of the ubiquitin system. Since the selectivity of protein degradation is determined mainly at the stage of ligation to ubiquitin, special attention is focused on what we know, and would like to know, about the mode of action of ubiquitin-protein ligation systems and about signals in proteins recognized by these systems.

Carrier Proteins↗
PubMed1997

Inhibition of ubiquitin/proteasome-dependent protein degradation by the Gly-Ala repeat domain of the Epstein-Barr virus nuclear antigen 1.

The Epstein-Barr virus (EBV) encoded nuclear antigen (EBNA) 1 is expressed in latently infected B lymphocytes that persist for life in healthy virus carriers and is the only viral protein regularly detected in all EBV associated malignancies. The Gly-Ala repeat domain of EBNA1 was shown to inhibit in cis the presentation of major histocompatibility complex (MHC) class I restricted cytotoxic T cell epitopes from EBNA4. It appears that the majority of antigens presented via the MHC I pathway are subject to ATP-dependent ubiquitination and degradation by the proteasome. We have investigated the influence of the repeat on this process by comparing the degradation of EBNA1, EBNA4, and Gly-Ala containing EBNA4 chimeras in a cell-free system. EBNA4 was efficiently degraded in an ATP/ubiquitin/proteasome-dependent fashion whereas EBNA1 was resistant to degradation. Processing of EBNA1 was restored by deletion of the Gly-Ala domain whereas insertion of Gly-Ala repeats of various lengths and in different positions prevented the degradation of EBNA4 without appreciable effect on ubiquitination. Inhibition was also achieved by insertion of a Pro-Ala coding sequence. The results suggest that the repeat may affect MHC I restricted responses by inhibiting antigen processing via the ubiquitin/proteasome pathway. The presence of regularly interspersed Ala residues appears to be important for the effect.

Animals↗
PubMed1997

Regulation of stability and function of the epithelial Na+ channel (ENaC) by ubiquitination.

The epithelial Na+ channel (ENaC), composed of three subunits (alpha beta gamma), plays a critical role in salt and fluid homeostasis. Abnormalities in channel opening and numbers have been linked to several genetic disorders, including cystic fibrosis, pseudohypoaldosteronism type I and Liddle syndrome. We have recently identified the ubiquitin-protein ligase Nedd4 as an interacting protein of ENaC. Here we show that ENaC is a short-lived protein (t1/2 approximately 1 h) that is ubiquitinated in vivo on the alpha and gamma (but not beta) subunits. Mutation of a cluster of Lys residues (to Arg) at the N-terminus of gamma ENaC leads to both inhibition of ubiquitination and increased channel activity, an effect augmented by N-terminal Lys to Arg mutations in alpha ENaC, but not in beta ENaC. This elevated channel activity is caused by an increase in the number of channels present at the plasma membrane; it represents increases in both cell-surface retention or recycling of ENaC and incorporation of new channels at the plasma membrane, as determined by Brefeldin A treatment. In addition, we find that the rapid turnover of the total pool of cellular ENaC is attenuated by inhibitors of both the proteasome and the lysosomal/endosomal degradation systems, and propose that whereas the unassembled subunits are degraded by the proteasome, the assembled alpha beta gamma ENaC complex is targeted for lysosomal degradation. Our results suggest that ENaC function is regulated by ubiquitination, and propose a paradigm for ubiquitination-mediated regulation of ion channels.

Acetylcysteine↗
PubMed1997

Inhibition of NF-kappa-B cellular function via specific targeting of the I-kappa-B-ubiquitin ligase.

Activation of the transcription factor NF-kappa B is a paradigm for signal transduction through the ubiquitin-proteasome pathway: ubiquitin-dependent degradation of the transcriptional inhibitor I kappa B in response to cell stimulation. A major issue in this context is the nature of the recognition signal and the targeting enzyme involved in the proteolytic process. Here we show that following a stimulus-dependent phosphorylation, and while associated with NF-kappa B, I kappa B is targeted by a specific ubiquitin-ligase via direct recognition of the signal-dependent phosphorylation site; phosphopeptides corresponding to this site specifically inhibit ubiquitin conjugation of I kappa B and its subsequent degradation. The ligase recognition signal is functionally conserved between I kappa B alpha and I kappa B beta, and does not involve the nearby ubiquitination site. Microinjection of the inhibitory peptides into stimulated cells abolished NF-kappa B activation in response to TNF alpha and the consequent expression of E-selectin, an NF-kappa B-dependent cell-adhesion molecule. Inhibition of NF-kappa B function by specific blocking of ubiquitin ligase activity provides a novel approach for intervening in cellular processes via regulation of unique proteolytic events.

Amino Acid Sequence↗