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A Ciechanover

Publications and source records attributed to A Ciechanover.

At least 109 records · Page 6Linked to original sources
PubMed1987

Regulation of the ubiquitin-mediated proteolytic pathway: role of the substrate alpha-NH2 group and of transfer RNA.

Degradation of intracellular proteins via the ubiquitin pathway involves several steps. In the initial event, ubiquitin becomes covalently linked to the protein substrate in an ATP-requiring reaction. Following ubiquitin conjugation, the protein moiety of the adduct is selectively degraded with the release of free and reusable ubiquitin. Ubiquitin modification of a variety of protein targets in the cell plays a role in basic cellular functions. Modification of core nucleosomal histones is probably involved in regulation of gene expression at the level of chromatin structure. Ubiquitin attachment to cell surface proteins may play roles in processes of cell-cell interaction and adhesion, and conjugation of ubiquitin to other yet to be identified protein(s) could be involved in the progression of cells through the cell cycle. Despite the considerable progress that has been made in the elucidation of the mode of action and cellular roles of the ubiquitin pathway, many major problems remain unsolved. A problem of central importance is the specificity in the ubiquitin ligation system. Why are certain proteins conjugated and committed for degradation, whereas other proteins are not? A free alpha-NH2 group is an important feature of the protein structure recognized by the ubiquitin conjugation system, and tRNA is required for the conjugation of ubiquitin to selective proteolytic substrates and for their subsequent degradation. These findings can shed light on some of the features of a substrate that render it susceptible to ubiquitin-mediated degradation.

Amines↗
PubMed1986

Antibody-induced receptor loss. Different fates for asialoglycoproteins and the asialoglycoprotein receptor in HepG2 cells.

The human asialoglycoprotein receptor (ASGP-R) is a membrane glycoprotein which participates in receptor-mediated endocytosis and delivery of its ligands to lysosomes for degradation. In order to examine the pathways and mechanisms responsible for the turnover and degradation of the ASGP-R we have followed the fate of the ASGP-R in HepG2 cells during exposure to anti-receptor antibody as well as inhibitors of lysosomal processing and receptor recycling. Incubation of cells at 37 degrees C with anti-ASGP-R antibody results in the rapid (t 1/2 30 min) loss of mature 46,000-Da ASGP-R (control, t 1/2 20 h). This process requires whole IgG, since Fab fragments do not induce loss of receptor. Furthermore, this antibody-induced loss is specific, since incubation with antibody to the transferrin receptor does not alter cellular ASGP-R content. Of note, weak bases (e.g. primaquine) abrogate this antibody-induced loss of ASGP-R. Inhibitors of lysosomal proteases (EC64 and leupeptin) do not alter this antibody-mediated loss. Furthermore, this effect occurs at 18 degrees C, a temperature at which delivery of ligand to the lysosome is blocked. Thus, the present observations suggest a unique pathway for antibody-induced ASGP-R loss which is distinct from the pathway of lysosomal delivery of ligand.

Antigen-Antibody Complex↗
PubMed1986

The eye lens has an active ubiquitin-protein conjugation system.

Using exogenous 125I-ubiquitin, ubiquitin-lens protein conjugation was observed with supernatants of cultured rabbit lens epithelial cells and lens cortex tissue. Conjugation was ATP-dependent with the greatest variety and amount of conjugates larger than 150 kDa. In vivo production of ubiquitin-protein conjugates in cultured rabbit and beef lens epithelial cells and rabbit lens tissues of different developmental age was established using immunological detection. There were limited similarities between conjugates found in youngest as opposed to oldest tissue. Cultured rabbit cells contained 27 pmol/mg free ubiquitin and 18 pmol/mg conjugated ubiquitin. Levels of free ubiquitin in lens tissue epithelium, cortex, and core were 36, 5, and 5 pmol/mg, respectively. There were only 2 pmol/mg conjugated ubiquitin in each of these tissues. Hydrolysis of 125I-ubiquitin was catalyzed by supernatants of cultured lens cells, beef and human lens tissues, and reticulocytes. Degradation was greatest in epithelial tissues, and least in core. This corroborates studies which show that proteolytic capabilities are attenuated in older tissue. Decreased initiation of proteolysis by ubiquitination as well as diminished proteolysis in older lens tissue may be related to the accumulation of damaged proteins in aging lens tissue.

Adenosine Triphosphate↗
PubMed1986

Transfer RNA is required for conjugation of ubiquitin to selective substrates of the ubiquitin- and ATP-dependent proteolytic system.

Degradation of intracellular proteins via the ubiquitin- and ATP-dependent proteolytic pathway involves several steps. In the initial event, ubiquitin, an abundant 76-residue polypeptide is covalently linked to the protein substrate in an ATP-requiring reaction. Proteins marked by ubiquitin are selectively proteolyzed in a reaction that also requires ATP. Ubiquitin conjugation to proteins appears also to be involved in regulation of cell cycle and cell division, and probably in the regulation of gene expression at the level of chromatin structure. We have previously shown (Ciechanover, A., Wolin, S. L., Steitz, J. A., and Lodish, H. F. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1341-1345) that transfer RNA is an essential component of the ubiquitin pathway. Ribonucleases strongly and specifically inhibited the degradation of 125I-labeled bovine serum albumin, while tRNA purified from reticulocyte extract could restore the proteolytic activity. Specifically, pure tRNAHis isolated by immunoprecipitation with human autoimmune serum could restore the proteolytic activity. Here we demonstrate that tRNA is required for conjugation of ubiquitin to some but not all proteolytic substrates of the ubiquitin mediated pathway. Conjugation of 125I-labeled ubiquitin to reduced carboxymethylated bovine serum albumin, alpha-lactalbumin, and soybean trypsin inhibitor was strongly and specifically inhibited by ribonucleases. Consequently, the ATP-dependent degradation of these substrates in the cell-free ubiquitin-dependent reticulocyte system was inhibited as well. Addition of tRNA to the ribonuclease inhibited system (following inhibition of the ribonuclease) restored both the conjugation activity and the ubiquitin- and ATP-dependent degradation of these substrates. Conjugation of ubiquitin to some endogenous reticulocyte proteins was also inhibited by ribonucleases and could be restored by the addition of tRNA. In striking contrast, the conjugation of radiolabeled ubiquitin to lysozyme, oxidized RNase A, alpha-casein, and beta-lactoglobulin was not affected by the ribonuclease treatment, and the degradation of these substrates was significantly accelerated by the ribonucleases. These findings indicate that there are at least two distinct ubiquitin conjugation systems. One requires tRNA, and the other is tRNA independent. These pathways, however, must share some common component(s) of the system, since the inhibition of one system accelerates the other. The possible function of tRNA in the selective conjugation reaction and the possible role of the two distinct ubiquitin marking mechanisms are discussed.

Adenosine Triphosphate↗
PubMed1985

Mammalian reticulocytes lose adhesion to fibronectin during maturation to erythrocytes.

We describe three situations in which a large fraction of circulating red blood cells attach tightly and specifically to fibronectin: (i) rabbits made anemic by repeated bleeding, (ii) patients with hemolytic anemia and functional asplenia and splenectomized normal humans, and (iii) splenectomized mice. Upon induction of anemia in rabbits, the proportion of circulating red blood cells capable of specifically attaching to fibronectin-coated plastic increased in parallel with the number of reticulocytes. Fibronectin-adherent red cells were barely detectable when the rabbit had recovered from the anemia. Attachment of reticulocytes to fibronectin was specific; cells did not attach to dishes coated with albumin, laminin, or collagen. None of these proteins promoted the attachment of normal erythrocytes. About 75% of the erythrocytes from splenectomized mice (but not control mice) also attached specifically to fibronectin 40 days after surgery. The effect of splenectomy was incomplete and transient; adherent cells were not detectable 8 weeks after splenectomy. As judged by labeling studies with [35S]methionine, newly emergent reticulocytes preferentially attached to fibronectin. We suggest that about half of the reticulocytes in erythropoietically unstressed mice lose their ability to attach to fibronectin, possibly due to loss of fibronectin-adhesive components, during passage through the spleen. The others lose their ability to interact with fibronectin before release, in the bone marrow, or in some extrasplenic site.

Anemia, Sickle Cell↗
PubMed1985

Transfer RNA is an essential component of the ubiquitin- and ATP-dependent proteolytic system.

Protein degradation via the nonlysosomal ATP-dependent pathway in rabbit reticulocytes involves a number of components. In the initial event, ubiquitin, an abundant 76-residue polypeptide, becomes covalently linked to the protein substrate in an ATP-requiring reaction. Once marked in this way, the conjugated protein is proteolyzed in a reaction that also requires ATP. Ubiquitin-marking appears to be important to the progression of cells from one stage to another of the cell cycle; it may also be involved in gene activation. Here we show that tRNA is another essential component of the system. Ribonucleases strongly inhibit the ubiquitin- and ATP-dependent degradation of 125I-labeled bovine serum albumin in the reticulocyte system in vitro. RNAs extracted from fractions of the reticulocyte extract or from mouse cells restore proteolytic activity. When the RNA is fractionated by gel electrophoresis, only the tRNA fraction is active in restoring proteolysis. Furthermore, pure mouse tRNAHis, isolated by immunoprecipitation with patient autoimmune sera, restores the proteolytic activity. The possibility that the level of uncharged tRNA in mammalian cells regulates the ubiquitin- and ATP-dependent proteolytic system is discussed.

Adenosine Triphosphate↗
PubMed1985

Intracellular transport of transferrin- and asialoorosomucoid-colloidal gold conjugates to lysosomes after receptor-mediated endocytosis.

Proteins coupled to colloidal gold particles have been widely used to visualize the uptake and intracellular transport of specific ligands by receptor-mediated endocytosis. The intracellular route of lysosome-directed ligands such as asialoglycoproteins (ASGP) are apparently unaltered by conjugation to gold, but the pathway of transferrin, a ligand that normally recycles to the cell surface, was reported to be altered by conjugation to 15-20 nm gold. In this study, we sought to determine whether a smaller transferrin-gold probe would recycle, and whether it might enter the same endosomal and lysosomal compartments as does a larger, lysosome-directed ASGP gold probe by visualizing their simultaneous uptake in human hepatoma (HepG2) cells. In the same cells, endocytosis of fluid-phase protein was followed using the soluble tracer native ferritin; lysosomal compartments were identified by acid phosphatase cytochemistry; and cell surfaces were labeled with ruthenium red or cationized ferritin. During the first 10 min of uptake at 37 degrees C, specific receptor-bound ferrotransferrin (FeTf)-8 nm gold and asialoorosomucoid (ASOR)-20 nm gold were clustered together in coated pits and entered the same coated vesicles, smooth vesicles, and tubules in the peripheral cytoplasm. At later times, however, transferrin-gold did not return to the cell surface; unlike native transferrin, this gold probe accompanied ASOR-gold into multivesicular bodies (MVB). The MVBs that contained probes were at first devoid of acid phosphatase activity, but at 30 min, enzyme activity was detected in a few MVBs. Native ferritin was present, along with gold probes, in all compartments of the endocytic pathway. We conclude that the normal intracellular pathway of transferrin is altered by its association with a colloidal gold particle.

Asialoglycoproteins↗
PubMed1985

Mammalian cell cycle mutant defective in intracellular protein degradation and ubiquitin-protein conjugation.

Ubiquitin, a 76 residue protein, occurs in eukaryotic cells either free or covalently joined via its carboxyl terminus to epsilon-amino groups of lysine residues in a wide variety of protein species. Previous work has shown that ubiquitin-protein conjugates are preferred substrates in vitro for a non-lysosomal ATP-dependent proteolytic pathway, suggesting that ubiquitin may function as a signal for attack by proteinases specific for ubiquitin-protein conjugates. One strategy to define the potential significance of the ubiquitin-dependent proteolytic pathway is to identify conditional mutants in the pathway. ts85 is a mouse derived cell-cycle mutant which has been shown to lose uH2A, a specific ubiquitin-histone H2A conjugate, at the nonpermissive temperature. We show that the loss of uH2A from ts85 cells is due to reduced ubiquitin-protein conjugation. We further show that the reduced conjugation is due to the specific thermolability of ubiquitin activating enzyme, E1, one of the three enzymic components of the ubiquitin-protein ligase system. We therefore proceeded to test whether the degradation of short-lived proteins is also temperature-sensitive in ts85 cells. Indeed, while more than 70% of the prelabeled abnormal (amino acid analog-containing) proteins or puromycyl peptides are degraded within 4 hours at the permissive temperature in the mutant (ts85), wild type (FM3A), and revertant (ts85R-MN3) cells, less than 15% of these proteins are degraded in ts85 cells at the nonpermissive temperature. In contrast, the rate of degradation of these proteins does not change significantly in either wild-type or revertant cells between permissive and nonpermissive temperatures. Degradation of normal short-lived proteins is also specifically temperature-sensitive in ts85 cells. Immunochemical analysis shows a strong and specific reduction in ubiquitin-protein conjugate levels in vivo at the nonpermissive temperature in ts85 cells. Taken together, our in vitro and in vivo findings with ts85 cells demonstrate that the degradation of the bulk of short-lived proteins in this higher eukaryotic cell is accomplished through a ubiquitin-mediated pathway.

Adenosine Triphosphate↗
PubMed1984

The ubiquitin-mediated proteolytic pathway and mechanisms of energy-dependent intracellular protein degradation.

In this review we briefly describe the lysosomal system, consider the evidence for multiplicity of protein degradation pathways in vivo, discuss in detail the ubiquitin-mediated pathway of intracellular ATP-dependent protein degradation, and also the possible significance of ubiquitin-histone conjugates in chromatin. For detailed discussions of the various characteristics and physiological roles of intracellular protein breakdown, the reader is referred to earlier reviews [1-7] and reports of recent symposia [8-10]. Information on the ubiquitin system prior to 1981 was described in an earlier review [11]. Hershko has briefly reviewed more recent information [12].

Adenosine Triphosphate↗
PubMed1984

Thermolability of ubiquitin-activating enzyme from the mammalian cell cycle mutant ts85.

Ubiquitin, a 76 residue protein, occurs in eucaryotic cells either free or covalently joined to a variety of protein species. Previous work suggested that ubiquitin may function as a signal for attack by proteinases specific for ubiquitin-protein conjugates. We show that the mouse cell line ts85 , a previously isolated cell cycle mutant, is temperature-sensitive in ubiquitin-protein conjugation, and that this effect is due to the specific thermolability of the ts85 ubiquitin-activating enzyme (E1). From E1 thermoinactivation kinetics in mixed (wild-type plus ts85 ) extracts, and from copurification of the determinant of E1 thermolability with E1 in ubiquitin-affinity chromatography, we conclude that the determinant of E1 thermolability is contained within the E1 polypeptide. ts85 cells fail to degrade otherwise short-lived intracellular proteins at the nonpermissive temperature (accompanying paper), demonstrating that degradation of the bulk of short-lived proteins in this higher eucaryotic cell proceeds through a ubiquitin-dependent pathway. We discuss possible roles of ubiquitin-dependent pathways in DNA transactions, the cell cycle, and the heat shock response.

Animals↗
PubMed1984

Ubiquitin dependence of selective protein degradation demonstrated in the mammalian cell cycle mutant ts85.

We have shown that covalent conjugation of ubiquitin to proteins is temperature-sensitive in the mouse cell cycle mutant ts85 due to a specifically thermolabile ubiquitin-activating enzyme (accompanying paper). We show here that degradation of short-lived proteins is also temperature sensitive in ts85 , in contrast to wild-type and revertant cells. While more than 70% of the prelabeled abnormal proteins (containing amino acid analogs) or puromycyl peptides are degraded within 4 hr at the permissive temperature in both ts85 and wild-type cells, less than 15% are degraded in ts85 cells at the nonpermissive temperature. Degradation of abnormal proteins and puromycyl peptides in both ts85 cells and wild-type cells is nonlysosomal and ATP-dependent. Immunochemical analysis shows a strong and specific reduction in the levels of in vivo labeled ubiquitin-protein conjugates at the nonpermissive temperature in ts85 cells. Degradation of normal, short-lived proteins is also specifically temperature sensitive in ts85 . We suggest that the contribution of ubiquitin-independent pathways to the degradation of short-lived proteins in this higher eucaryotic cell is no more than 10%, and possibly less.

Adenosine Triphosphate↗
PubMed1983

Kinetics of internalization and recycling of transferrin and the transferrin receptor in a human hepatoma cell line. Effect of lysosomotropic agents.

Growing HepG2 cells contain 50,000 functional surface transferrin-binding sites (Ciechanover, A., Schwartz, A.L., and Lodish, H.F. (1983) Cell 32,267-275) and 100,000 intracellular sites. At saturating concentrations of [59Fe]transferrin, and under conditions in which protein synthesis is blocked, iron uptake is linear for several hours at a rate of 9,500 transferrin molecules/cell/min. Thus, each receptor must recycle a ligand, on the average, each 15.8 min. Surface-bound transferrin is rapidly endocytosed (t1/2 = 3.5 min). All of the iron remains within the cell, while the apotransferrin is rapidly (t1/2 = 5.0 min) secreted into the medium. Previously, we showed (Dautry-Varsat, A., Ciechanover, A., and Lodish, H.F. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2258-2262) that exposure of a ferrotransferrin-receptor complex to medium of pH less than 5.0 results in dissociation of iron, but that apotransferrin remains bound to its receptor. If the pH is raised to 7.0, such as would occur when an acidic intracellular vesicle fuses with the plasma membrane, apotransferrin is very rapidly dissociated (t1/2 = 17 s at 37 degrees C) from its receptor. Taken together, these results indicate that transferrin remains bound to its receptor throughout the endocytic cycle. In the present study, we have directly measured all the kinetic parameters involved in the transferrin receptor cycle. They are similar to those of the asialoglycoprotein receptor in the same cell line, and can be described by a simple kinetic model. In the presence of lysosomotropic agents, ferrotransferrin binds to its surface receptor and is internalized normally. However, iron is not dissociated from transferrin, and ferrotransferrin recycles back to the cell surface and is secreted into the medium. We conclude that the low pH in endocytic vesicles is essential for the dissociation of iron from transferrin and its delivery to the cell, but is not required for recycling of transferrin, and presumably of its receptor.

Carcinoma, Hepatocellular↗
PubMed1983

Components of ubiquitin-protein ligase system. Resolution, affinity purification, and role in protein breakdown.

By affinity chromatography of a crude reticulocyte extract on ubiquitin-Sepharose, three enzymes required for the conjugation of ubiquitin with proteins have been isolated. One is the ubiquitin-activating enzyme (E1), which is covalently linked to the affinity column in the presence of ATP and can be specifically eluted with AMP and pyrophosphate (Ciechanover, A., Elias, S., Heller, H., and Hershko, A. (1982) J. Biol. Chem. 257, 2537-2542). A second enzyme, designated E2, is bound to the ubiquitin column when E1 and ATP are present, and is eluted with a thiol compound at high concentration. The third enzyme, designated E3, is adsorbed to the affinity column by noncovalent interactions and can be eluted with high salt or increased pH. The presence of all three enzymes is absolutely required for the conjugation of 125I-ubiquitin with proteins. All three affinity-purified enzymes are also required for the breakdown of 125I-albumin to acid-soluble material in the presence of ubiquitin, ATP, and the unadsorbed fraction of the affinity column. The following observations indicate that the function of E2 is the transfer of activated ubiquitin to the site of conjugation in the form of an E2-ubiquitin thiol ester intermediate. (a) E2 is rapidly inactivated by iodoacetamide, but can be protected against inactivation by a prior incubation with E1, ATP, and ubiquitin. This suggests an E1-mediated transfer of activated ubiquitin to an iodoacetamide-sensitive thiol site of E2. (b) The requirements for the binding of E2 to the ubiquitin column and the mode of its elution, cited above, are consistent with the notion that a covalent linkage is formed between E2 and Sepharose-bound ubiquitin. (c) Upon the incubation of 125I-ubiquitin with E1 and ATP, followed by the addition of purified E2, activated ubiquitin is transferred from E1 to several low molecular weight forms of E2, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The linkage of ubiquitin to all these forms has the characteristics of a thiol ester bond. In a further incubation with E3 and a protein substrate for conjugation, activated ubiquitin was transferred from the different forms of E2-ubiquitin to stable ubiquitin-protein conjugates. Thus, E3 is involved in the last step of the ligase system.

Adenosine Triphosphate↗
PubMed1983

Sorting and recycling of cell surface receptors and endocytosed ligands: the asialoglycoprotein and transferrin receptors.

With few exceptions, receptor-mediated endocytosis of specific ligands is mediated through clustering of receptor-ligand complexes in coated pits on the cell surface, followed by internalization of the complex into endocytic vesicles. During this process, ligand-receptor dissociation occurs, most probably in a low pH prelysosomal compartment. In most cases the ligand is ultimately directed to the lysosomes, wherein it is degraded, while the receptor recycles to the cell surface. We have studied the kinetics of internalization and recycling of both the asialoglycoprotein receptor and the transferrin receptor in a human hepatoma cell line. By employing both biochemical and morphological/immunocytochemical approaches, we have gained some insight into the complex mechanisms which govern receptor recycling as well as ligand sorting and targeting. We can, in particular, explain why transferrin is exocytosed intact from the cells, while asialoglycoproteins are degraded in lysosomes. We have also localized the intracellular site at which endocytosed receptor and ligand dissociate.

Animals↗
PubMed1983

The asialoglycoprotein receptor internalizes and recycles independently of the transferrin and insulin receptors.

Receptor-mediated endocytosis of specific ligands is mediated through clustering of receptor-ligand complexes in coated pits on the cell surface, followed by internalization of the complex into endocytic vesicles. We show that internalization of asialoglycoprotein by HepG2 hepatoma cells is accompanied by a rapid (t1/2 = 0.5-1 min) depletion of surface asialoglycoprotein receptors. This is followed by a rapid (t1/2 = 2-4 min) reappearance of surface receptors; most of these originate from endocytosed cell-surface receptors. The loss and reappearance of asialoglycoprotein receptors is specific, and depends on prebinding of ligand to its receptor. HepG2 cells also contain abundant receptors for both insulin and transferrin. Endocytosis of asialoglycoprotein and its receptor has no effect on the number of surface binding sites for transferrin or insulin. We conclude that binding of asialoglycoprotein to its surface receptor triggers a rapid and specific endocytosis of the receptor-ligand complex, probably due to a clustering in clathrin-coated pits or vesicles.

Animals↗
PubMed1983

pH and the recycling of transferrin during receptor-mediated endocytosis.

At pH 5.4 apotransferrin (iron-free transferrin) binds to cell-surface transferrin receptors to the same extent and with the same affinity as does diferric transferrin at pH 7.0. Apotransferrin is quickly dissociated from its receptor when the pH is raised to 7.0. These and other results strongly support a simple model that explains the cycling of transferrin during a single cycle of receptor-mediated endocytosis. Diferric transferrin binds to cell-surface receptors, and the transferrin-receptor complex is endocytosed. The pH of the endocytic vesicle is lowered to 5.5 or below; this causes dissociation of iron from the transferrin-receptor complex, but apotransferrin remains bound to its receptor. The iron remains within the cell, and the apotransferrin-receptor complex is recycled to the cell surface. Upon encountering the neutral pH of the medium, apotransferrin is dissociated from the cell.

Animals↗