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Biomedical subjects

Aaron Ciechanover

Publications and source records attributed to Aaron Ciechanover.

At least 37 records · Page 2Linked to original sources
PubMed2004

Ubiquitin-Proteasome-mediated degradation of Id1 is modulated by MyoD.

Degradation of many short-lived cellular proteins such as the transcription factor MyoD occurs via the ubiquitin-proteasome pathway. MyoD, similar to many rapidly degraded regulatory factors, interacts with several high affinity binding partners, including members of the Id (inhibitors of DNA binding) family. Following transfection to HeLa cells, Id1 is localized to the nucleus and rapidly (t(1/2) approximately 1 h) degraded via the ubiquitin-proteasome system. Mutagenesis of lysine residues within the putative nuclear localization region (amino acids 68-82) directs Id1(NLS) to the cytoplasm yet confers an increased rate of degradation (t(1/2) approximately 0.5 h). Id1 in which all lysine residues were mutagenized to alanine (lysineless Id1) was also rapidly degraded (t(1/2) approximately 0.6 h). Addition of a Myc(6) tag to the N terminus of lysine-less Id1 markedly stabilized Id1 (t(1/2) > 10 h) and suggests degradation via the N terminus-dependent pathway. Co-transfection of MyoD with Id1 or Id1(NLS) increases Id1 or Id1(NLS) within the nucleus and markedly reduces the rate of Id1 or Id1(NLS) degradation. These results thus demonstrate that in vivo MyoD modulates the rate of Id1 degradation and suggest a dynamic interplay of these factors.

Cell Nucleus↗
PubMed2004

Caspase activation inhibits proteasome function during apoptosis.

The ubiquitin/proteasome system regulates protein turnover by degrading polyubiquitinated proteins. To date, all studies on the relationship of apoptosis and the proteasome have emphasized the key role of the proteasome in the regulation of apoptosis, by virtue of its ability to degrade regulatory molecules involved in apoptosis. We now demonstrate how induction of apoptosis may regulate the activity of the proteasome. During apoptosis, caspase activation results in the cleavage of three specific subunits of the 19S regulatory complex of the proteasome: S6' (Rpt5) and S5a (Rpn10), whose role is to recognize polyubiquitinated substrates of the proteasome, and S1 (Rpn2), which with S5a and S2 (Rpn1) holds together the lid and base of the 19S regulatory complex. This caspase-mediated cleavage inhibits the proteasomal degradation of ubiquitin-dependent and -independent cellular substrates, including proapoptotic molecules such as Smac, so facilitating the execution of the apoptotic program by providing a feed-forward amplification loop.

Apoptosis↗
PubMed2004

Mechanism of processing of the NF-kappa B2 p100 precursor: identification of the specific polyubiquitin chain-anchoring lysine residue and analysis of the role of NEDD8-modification on the SCF(beta-TrCP) ubiquitin ligase.

Processing of the NF-kappa B2 precursor p100 to the mature p52 subunit is regulated via a unique pathway. NF-kappa B-inducing kinase (NIK) induces I kappa B kinase alpha (IKK alpha)-mediated phosphorylation of specific serine residues in the C-terminal domain of p100, leading to recruitment of the SCF(beta-TrCP) ubiquitin ligase. We identified a single lysine residue, K855, that serves as the ubiquitin-anchoring residue required for signal-induced processing of p100. In a reconstituted system containing purified components, p100-K855R could not be ubiquitinated. In a crude extract and cells, only residual, signal-independent ubiquitination and processing were retained. Importantly, K855 is located in a site homologous to K22 that serves as an ubiquitination site in I kappa B alpha. This suggests a common recognition mechanism for the two molecules. In contrast, p105, the p100 homologue, lacks a similar Lys residue. We also demonstrate that the NEDD8 pathway is essential for the SCF(beta-TrCP) activity. In a reconstituted system, efficient ubiquitination of p100 required all three components of the pathway - E1, the UBC12 E2 and NEDD8. Experiments in reconstituted systems and in cells demonstrated that SCF(beta-TrCP), which contains a mutant Cul-1 that cannot be NEDDylated, cannot stimulate ubiquitination and processing. Similarly, dominant negative UBC12 inhibits, in a reversible manner, both ubiquitination and processing of p100.

Baculoviridae↗
PubMed2004

Degradation of the Id2 developmental regulator: targeting via N-terminal ubiquitination.

Degradation of cellular proteins via the ubiquitin-proteasome system (UPS) involves: (i) generation of a substrate-anchored polyubiquitin degradation signal and (ii) destruction of the tagged protein by the 26S proteasome with release of free and reusable ubiquitin. For most substrates, it is believed that the first ubiquitin moiety is conjugated to a epsilon-NH(2) group of an internal Lys residue. Recent findings indicate that for several proteins, the first ubiquitin moiety is fused, in a linear manner, to the free alpha-NH(2) group of the protein. Here, we demonstrate that the inhibitor of differentiation (or inhibitor of DNA binding) 2, Id2, that downregulates gene expression in undifferentiated and self-renewing cells, is degraded by the UPS following ubiquitination at its N-terminal residue. Lysine-less (LL) Id2 is degraded efficiently by the proteasome following ubiquitination. Fusion of a Myc tag to the N-terminal but not to the C-terminal residue of Id2 stabilizes the protein. Furthermore, deletion of the first 15 N-terminal residues of Id2 stabilizes the protein, suggesting that this domain serves as a recognition element, possibly for the ubiquitin ligase, E3. The mechanisms and structural motives that govern Id2 stability may have important implications to the regulation of the protein during normal differentiation and malignant transformation.

Adenosine Triphosphate↗
PubMed2004

The ubiquitin-proteasome system in cardiovascular diseases-a hypothesis extended.

During recent years, the ubiquitin-proteasome system has become known as the major pathway of non-lysosomal degradation of intracellular proteins, involving two sequential steps. In the first step, multiple moieties of ubiquitin are covalently bound to target proteins to be recognized and degraded by the multi-enzymatic proteasome complex in the second step. In addition to the elimination of damaged and unneeded proteins, this system fulfills an important function in the regulation of cellular mediators in various biological pathways. Foremost, these biological pathways include inflammation, cell proliferation, and apoptosis, all of which constitute important characteristics of atherosclerosis. Indeed, recent experimental evidence supports a potential involvement of the ubiquitin-proteasome system in the initiation, progression, and complication stage of atherogenesis. This review summarizes recent findings regarding the ubiquitin-proteasome system in cardiovascular diseases and discusses the potential use of proteasome inhibitors in cardiovascular therapy.

Apoptosis↗
PubMed2004

N-terminal ubiquitination: more protein substrates join in.

The ubiquitin-proteasome system (UPS) is involved in selective targeting of innumerable cellular proteins through a complex pathway that plays important roles in a broad array of processes. An important step in the proteolytic cascade is specific recognition of the substrate by one of many ubiquitin ligases, E3s, which is followed by generation of the polyubiquitin degradation signal. For most substrates, it is believed that the first ubiquitin moiety is conjugated, through its C-terminal Gly76 residue, to an sigma-NH2 group of an internal Lys residue. Recent findings indicate that, for several proteins, the first ubiquitin moiety is fused linearly to the alpha-NH2 group of the N-terminal residue. An important biological question relates to the evolutionary requirement for an alternative mode of ubiquitination.

Humans↗
PubMed2004

The ubiquitin system: from basic mechanisms to the patient bed.

In the ubiquitin system, a target substrate is modified by ubiquitin or a ubiquitin-like protein. This modification remodels the surface of the target proteins, affecting, among other properties, their stability, interactions with other proteins, activity, and subcellular localization. At least 10 different modifiers have been described in mammalian cells and conjugation of each modifier to its target may result in a different biological effect. In many cases proteins are modified by multiple moieties of ubiquitin that generate a branched polyubiquitin chain. For most proteins, this modification leads to their degradation by the 26S proteasome. Yet, dependent on the character of the internal linkage between the ubiquitin moieties, it can also lead to activation of transcriptional regulators. Modification by a single moiety of ubiquitin can target proteins for degradation in the lysosome/vacuole. Conjugation of ubiquitin or ubiquitin-like proteins can serve a variety of non-proteolytic functions, such as activation of enzymes, modulation of membrane dynamics, or routing of the tagged proteins to their sub-cellular destination. Ubiquitination of cellular proteins is a highly complex, temporally controlled, and tightly regulated process that targets, in a specific manner, thousands of cellular proteins. It is carried out by a modular cascade of enzymes with high specificity towards defined structural motifs in the target proteins. It has emerged as a critically important post-translational modification that plays major roles in regulating a broad array of basic cellular processes, such as cell division, differentiation, signal transduction, trafficking, and quality control. Not surprisingly, aberrations in the system have been implicated in the pathogenesis of many diseases, certain malignancies, neurodegenerative disorders and pathologies of the inflammatory and immune response among them. Understanding of the underlying mechanisms involved is important for the development of novel, mechanism-based drugs.

Cystic Fibrosis↗
PubMed2004

Dual effects of IkappaB kinase beta-mediated phosphorylation on p105 Fate: SCF(beta-TrCP)-dependent degradation and SCF(beta-TrCP)-independent processing.

Processing of the p105 NF-kappaB precursor to yield the p50 active subunit is a unique and rare case in which the ubiquitin system is involved in limited processing rather than in complete destruction of its target. The mechanisms involved in this process are largely unknown, although a glycine repeat in the middle of p105 has been identified as a processing stop signal. IkappaB kinase (IKK)beta-mediated phosphorylation at the C-terminal domain with subsequent recruitment of the SCF(beta-TrCP) ubiquitin ligase leads to accelerated processing and degradation of the precursor, yet the roles that the kinase and ligase play in each of these two processes have not been elucidated. Here we demonstrate that IKKbeta has two distinct functions: (i) stimulation of degradation and (ii) stimulation of processing. IKKbeta-induced degradation is dependent on SCF(beta-TrCP), which acts through multiple lysine residues in the IkappaBgamma domain. In contrast, IKKbeta-induced processing of p105 is beta-transduction repeat-containing protein (beta-TrCP) independent, as it is not affected by expression of a dominant-negative beta-TrCP or following its silencing by small inhibitory RNA. Furthermore, removal of all 30 lysine residues from IkappaBgamma results in complete inhibition of IKK-dependent degradation but has no effect on IKK-dependent processing. Yet processing still requires the activity of the ubiquitin system, as it is inhibited by dominant-negative UbcH5a. We suggest that IKKbeta mediates its two distinct effects by affecting, directly and indirectly, two different E3s.

Antigens, Nuclear↗
PubMed2003

The ubiquitin proteasome system in neurodegenerative diseases: sometimes the chicken, sometimes the egg.

The ubiquitin-proteasome system targets numerous cellular proteins for degradation. In addition, modifications by ubiquitin-like proteins as well as proteins containing ubiquitin-interacting and -associated motifs modulate many others. This tightly controlled process involves multiple specific and general enzymes of the system as well as many modifying and ancillary proteins. Thus, it is not surprising that ubiquitin-mediated degradation/processing/modification regulates a broad array of basic cellular processes. Moreover, aberrations in the system have been implicated, either as a primary cause or secondary consequence, in the pathogenesis of both inherited and acquired neurodegenerative diseases. Recent findings indicate that the system is involved in the pathogenesis of Parkinson's, Alzheimer's, Huntington's, and Prion diseases as well as amyotrophic lateral sclerosis. This raises hopes for a better understanding of the pathogenetic mechanisms involved in these diseases and for the development of novel, mechanism-based therapeutic modalities.

Animals↗
PubMed2003

HdmX stimulates Hdm2-mediated ubiquitination and degradation of p53.

The RING finger proteins HdmX and Hdm2 share significant structural and functional similarity. Hdm2 is a member of the RING finger family of ubiquitin-protein ligases E3 and targets the tumor suppressor protein p53 for degradation. Although HdmX also binds to p53, HdmX does not induce p53 degradation. Moreover, HdmX has been reported to interfere with p53 degradation in overexpression experiments. To obtain insight into the mechanism by which HdmX interferes with p53 degradation, we studied the effect of HdmX on the E3 activity of Hdm2 in vitro. Surprisingly, this revealed that HdmX stimulates Hdm2-mediated ubiquitination of p53 and that HdmX facilitates ubiquitination of Hdm2 and vice versa. In addition, down-regulation of HdmX expression within cells results in the accumulation of both p53 and Hdm2. Because HdmX alone does not have appreciable E3 activity, these data indicate that HdmX acts as a stimulator, rather than as an inhibitor, of the E3 activity of Hdm2 and that, at least under certain conditions, HdmX is actively involved in the degradation of both p53 and Hdm2.

Amino Acid Sequence↗
PubMed2003

Oxidative stress-related increase in ubiquitination in early coronary atherogenesis.

The ubiquitin-proteasome system (UPS) is involved in the removal of damaged proteins and the activation of transcription factors, such as nuclear-factor-kappaB. Recent reports, however, questioned the functional activity of the UPS under conditions of increased oxidative stress, such as experimental hypercholesterolemia, which was the objective of our study. Pigs were placed on a normal chow diet (N) or on a hypercholesterolemic diet without (HC) or with vitamin C and E supplementation (HC+VIT) for 12 weeks. Compared with N, plasma concentration of total cholesterol increased in both HC and HC+VIT [76 +/- 21 vs. 400 +/- 148 (P<0.05) and 329 +/- 102 (P<0.05) mg/dL], whereas increase in lipid peroxidation, as assessed by LDL-malondialdehyde plasma concentration, was found in HC but not in HC+VIT [6.6 +/- 0.7 vs. 8.5 +/- 0.3 (P<0.05) and 6.8 +/- 0.7 nmol/mg protein]. In comparison with N, the level of ubiquitin conjugates in the coronary artery, as assessed by immunoblotting, increased by 42% in HC but not in HC+VIT and was localized predominantly to media vascular smooth muscle cells by immunostaining. There was no difference in proteasome proteolytic activity among the study groups. These results demonstrate that the UPS is functionally active in early atherogenesis despite increase in oxidative stress with important repercussions in the pathophysiology and therapy of cardiovascular diseases.

Animals↗
PubMed2003

A novel mammalian endoplasmic reticulum ubiquitin ligase homologous to the yeast Hrd1.

The yeast hHrd1 is a ubiquitin-protein ligase (E3) involved in ER-associated degradation. It was originally identified by genetic methods as an E3 of the yeast cholesterol biosynthetic enzyme HMG-CoA reductase (HMGR). We report the identification and cloning of a human homologue of Hrd1 (hHrd1). Immunofluorescence imaging confirms that the endogenous hHrd1 resides in the ER and in vitro assay demonstrates that it has a ubiquitin-ligase activity. However, the homology between the human and yeast Hrd1 is limited to the N-terminal domain of the proteins, and hHrd1 does not appear to be involved in the degradation of mammalian HMGR.

Amino Acid Sequence↗
PubMed2003

Inverse relationship between Skp2 ubiquitin ligase and the cyclin dependent kinase inhibitor p27Kip1 in prostate cancer.

PURPOSE: Prostate carcinomas show a low level of the cell cycle inhibitor p27, which correlates with tumor aggressiveness. In tumors p27 is of the WT species and its deregulation is due to aberrant ubiquitin mediated degradation. The p27 is degraded following phosphorylation and subsequent recognition by SCFSkp2 ubiquitin ligase. We examined the relationship between p27 and its specific ligase Skp2 in normal and malignant prostate tissues. A possible correlation among the levels of these proteins, tumor grading and clinical state was also investigated. MATERIALS AND METHODS: Using immunohistochemistry immunofluorescence microscopy and Western blot analysis 51 samples from needle biopsies, transurethral resection and radical prostatectomy were analyzed for p27 and Skp2 expression. Correlation with tumor grading (Gleason) was performed. In 22 proven metastatic or organ confined cases correlation was also done with the Ki67 proliferative marker. RESULTS: Skp2 expression demonstrated a significant and direct correlation with malignancy (p <0.0001). Furthermore, a significant correlation was found between Skp2 level and tumor aggressiveness graded by Gleason score (p <0.0002) and prostate specific antigen. Patients with metastases had significantly higher Skp2 and Ki67 expression than those with organ confined disease (p <0.0001). In addition, Skp2 levels significantly correlated with Ki67 (r = 0.73, p <0.0001). An inverse correlation was found between p27 and Skp2 ligase. CONCLUSIONS: Skp2 expression in prostate biopsies may be used as an additional marker for tumor aggressiveness. The results also suggest a role for Skp2 in the pathogenesis of prostate malignancy.

Aged↗
PubMed2002

Increased ubiquitin immunoreactivity in unstable atherosclerotic plaques associated with acute coronary syndromes.

OBJECTIVES: The current study was designed to examine whether ubiquitin expression is higher in unstable than in stable lesions of patients with acute coronary syndrome (ACS). BACKGROUND: The ubiquitin system has been identified as the nonlysosomal pathway of protein degradation; it is involved in a number of biologic processes crucial to cell and tissue integrity and therefore, might be potentially involved in the rupture of unstable coronary plaques. METHODS: We conducted an autopsy-based study of 25 consecutive patients with fatal ACS. Lesions of both infarct-related and noninfarct-related segments from the same patients were examined for the expression and localization of ubiquitin by use of immunohistochemistry and a semiquantitative grading scale. RESULTS: Ubiquitin immunoreactivity was higher in infarct-related than in noninfarct-related lesions (1.4 +/- 0.5 vs. 1.1 +/- 0.6, p = 0.03). Compared with areas adjacent to the plaque (0.6 +/- 0.7), ubiquitin immunoreactivity was higher in areas around the lipid core (2.5 +/- 0.8, p < 0.001), plaque shoulders (1.6 +/- 1.1, p < 0.001), and fibrous cap regions (1.6 +/- 1.1, p < 0.001). Within the plaque area, co-localization of ubiquitin immunoreactivity with T cells and macrophages was found. In areas adjacent to the plaque, ubiquitin immunoreactivity co-localized with neointima cells and media smooth muscle cells. CONCLUSIONS: In patients with ACS, ubiquitin immunoreactivity is enhanced in unstable, infarct-related lesions, predominantly in plaque regions of tissue degradation. Based on these findings, this study suggests a role for the ubiquitin system in the destabilization and rupture of coronary atherosclerotic plaques in humans.

Acute Disease↗
PubMed2002

Determinants of nuclear and cytoplasmic ubiquitin-mediated degradation of MyoD.

The ubiquitin-proteasome system is responsible for the regulation and turnover of many short-lived proteins both in the cytoplasm and in the nucleus. Degradation can occur via two distinct pathways, an N terminus-dependent pathway and a lysine-dependent pathway. The pathways are characterized by the site of initial ubiquitination of the protein, the N terminus or an internal lysine, respectively. MyoD, a basic helix-loop-helix transcription factor, is a substrate for the ubiquitin-proteasome pathway and is degraded in the nucleus. It is preferentially tagged for degradation on the N terminus and thus is degraded by the N terminus-dependent pathway. Addition of a 6x Myc tag to the N terminus of MyoD can force degradation through the lysine-dependent pathway by preventing ubiquitination at the N-terminal site. Modifications of the nuclear localization signal and nuclear export signal of MyoD restrict ubiquitination and degradation to the cytoplasm or the nucleus. Using these mutants, we determined which degradation pathway is dominant in the cytoplasm and the nucleus. Our results suggest that the lysine-dependent pathway is the more active pathway within the cytoplasm, whereas in the nucleus the two pathways are both active in protein degradation.

Cell Nucleus↗
PubMed2002

The NEDD8 pathway is essential for SCF(beta -TrCP)-mediated ubiquitination and processing of the NF-kappa B precursor p105.

The p50 subunit of NF-kappaB is generated by limited processing of the precursor p105. IkappaB kinase-mediated phosphorylation of the C-terminal domain of p105 recruits the SCF(beta-TrCP) ubiquitin ligase, resulting in rapid ubiquitination and subsequent processing/degradation of p105. NEDD8 is known to activate SCF ligases following modification of their cullin component. Here we show that NEDDylation is required for conjugation and processing of p105 by SCF(beta-TrCP) following phosphorylation of the molecule. In a crude extract, a dominant negative E2 enzyme, UBC12, inhibits both conjugation and processing of p105, and inhibition is alleviated by an excess of WT- UBC12. In a reconstituted cell-free system, ubiquitination of p105 was stimulated only in the presence of all three components of the NEDD8 pathway, E1, E2, and NEDD8. A Cul-1 mutant that cannot be NEDDylated could not stimulate ubiquitination and processing of p105. Similar findings were observed also in cells. It should be noted that NEDDylation is required only for the stimulated but not for basal processing of p105. Although the mechanisms that underlie processing of p105 are largely obscure, it is clear that NEDDylation and the coordinated activity of SCF(beta-TrCP) on both p105 and IkappaBalpha serve as an important regulatory mechanism controlling NF-kappaB activity.

Animals↗
PubMed2002

Regulation of Drosophila IAP1 degradation and apoptosis by reaper and ubcD1.

Cell death in higher organisms is negatively regulated by Inhibitor of Apoptosis Proteins (IAPs), which contain a ubiquitin ligase motif, but how ubiquitin-mediated protein degradation is regulated during apoptosis is poorly understood. Here, we report that Drosophila melanogaster IAP1 (DIAP1) auto-ubiquitination and degradation is actively regulated by Reaper (Rpr) and UBCD1. We show that Rpr, but not Hid (head involution defective), promotes significant DIAP1 degradation. Rpr-mediated DIAP1 degradation requires an intact DIAP1 RING domain. Among the mutations affecting ubiquitination, we found ubcD1, which suppresses rpr-induced apoptosis. UBCD1 and Rpr specifically bind to DIAP1 and stimulate DIAP1 auto-ubiquitination in vitro. Our results identify a novel function of Rpr in stimulating DIAP1 auto-ubiquitination through UBCD1, thereby promoting its degradation.

Age Factors↗