Ubiquitin-mediated Proteolytic System Research Articles

Augmentation of the ubiquitin-mediated proteolytic system by F-box and additional motif-containing proteins (Review)

Cells are tightly regulated by sophisticated systems. Among such systems, post-translational modification has been intensively studied, and the ubiquitin-proteasome system is one of the best-known proteolytic mechanisms for the timely destruction of regulatory proteins. As progress has been made in research on the ubiquitin-proteasome system, our understanding of fundamental regulatory circuits, such as the cell cycle and circadian rhythms, has progressed. Here, we focused on the SKP1-CUL1-F-box protein of E3 ubiquitin ligase, especially the F-box and additional characteristic domain-containing proteins, as adaptors for regulatory proteins. Accumulating evidence suggests that the F-box and additional characteristic domain-containing proteins are involved in a wide variety of cellular processes and pathogenesis. This raises the possibility that F-box and additional characteristic domain-containing proteins could be used as biomarkers for certain diseases.

Programmed Cell Death of the Megagametophyte during Post-germinative Growth of White Spruce (Picea glauca) Seeds is Regulated by Reactive Oxygen Species and the Ubiquitin-mediated Proteolytic System

The megagametophyte of white spruce (Picea glauca) seeds undergoes programmed cell death following seed germination. This process is characterized by distinct morphological and biochemical features, such as DNA fragmentation and the induction of proteases. Biphasic production of hydrogen peroxide was detected in the megagametophyte following seed germination. ROS scavengers or inhibitors of ROS production decreased caspase-like protease activity and slowed the progression of cell death. One catalase (CAT) of white spruce reacted with antibodies directed against cotton-seed CAT. The corresponding CAT gene was cloned and compared with the catalase genes of other plant species. The activity of the white spruce CAT enzyme was stimulated by tyrosine phosphorylation. The phosphorylated CAT was subjected to ubiquitination and degraded by the proteasome. Furthermore, the proteasome inhibitor MG132 inhibited the degradation of CAT and delayed cell death. These results suggest that the interplay of CAT and the ubiquitin-mediated proteolytic system is critical in the control of ROS production and subsequent cell death.

Manganese‐induced downregulation of astroglial glutamine transporter SNAT3 involves ubiquitin‐mediated proteolytic system

SNAT3 is a major facilitator of glutamine (Gln) efflux from astrocytes, supplying Gln to neurons for neurotransmitter synthesis. Our previous investigations have shown that, in primary cortical astrocyte cultures, SNAT3 protein is degraded after exposure to manganese (Mn(2+)). The present studies were performed to identify the processes responsible for this effect. One of the well-established mechanisms for protein-level regulation is posttranslational modification via ubiquitination, which leads to the rapid degradation of proteins by the 26S proteasome pathway. Here, we show that astrocytic SNAT3 directly interacts with the ubiquitin ligase, Nedd4-2 (neural precursor cells expressed developmentally downregulated 4-2), and that Mn(2+) increases both Nedd4-2 mRNA and protein levels. Additionally, we have found that Mn(2+) exposure elevates astrocytic ubiquitin B mRNA expression, free ubiquitin protein levels, and total protein ubiquitination. Furthermore, Mn(2+) effectively decreases astrocytic mRNA expression and the phosphorylation of serum and glucocorticoid-inducible kinase, a regulatory protein, which, in the active phosphorylated form, is responsible for the phosphorylation and subsequent inactivation of Nedd4-2. Additional findings establish that Mn(2+) increases astrocytic caspase-like proteolytic proteasome activity and that the Mn(2+)-dependent degradation of SNAT3 protein is blocked by the proteasome inhibitors, N-acetyl-leu-leu-norleucinal and lactacystin. Combined, these results demonstrate that Mn(2+)-induced SNAT3 protein degradation and the dysregulation of Gln homeostasis in primary astrocyte cultures proceeds through the ubiquitin-mediated proteolytic system.

Cachexia in chronic kidney disease: role of inflammation and neuropeptide signaling

This review will update clinicians and basic scientists who are interested in the clinical relevance and molecular mechanism of uremic cachexia. Recent studies that examine the role of cytokines and hypothalamic neuropeptides are emphasized. A current hypothesis of the cause of cachexia in chronic illness is that proinflammatory cytokines, such as tumor necrosis factor-alpha, interleukin-6, and leptin, act on the central nervous system to alter the release and function of several key neurotransmitters, thereby altering both appetite and metabolic rate. Proinflammatory cytokines also activate the transcription factor nuclear factor-kappaB, resulting in decreased protein synthesis, and activate the ubiquitin-mediated proteolytic system, which is the major system involved in increased protein degradation. This review highlights the importance of melanocortin signaling in the pathogenesis of uremia-associated cachexia and the potential of peripheral administration of melanocortin-4 receptor antagonists as a novel therapeutic approach.

Involvement of the Ubiquitin-Mediated Proteolytic System in the Signaling Pathway Induced by Ceramide in Primary Astrocyte Cultures

The selective degradation of abnormal or short half-life proteins in eukaryotic cells proceeds through the ubiquitin-mediated proteolytic system (UbPS). The signals that tag the proteins for their ubiquitination are well known. In the present study, our aim was to investigate the relationship between the action of ceramide and the changes in the expression of certain mRNAs of the Ub pathway and in the activation of the UbPS in cultured astrocytes (ASTs). Changes in the expression of components that are known to be substrates of the UbPS and that participate in the regulation of the cell death process were also studied. Addition of different concentrations of C₂ ceramide to cultured ASTs produced an increase in the expression of the Ub gene and in the gene that encodes E₁, one of the enzymes involved in the ubiquitination process, without any changes on cell viability. Immunocytochemical studies showed an increase in the expression of Bcl-2 with no changes in cytochrome c. Also, there was an increase in the nuclear reactivity of NFĸB, suggesting a translocation of this factor towards the nucleus. Western blots showed a decrease in IĸB and its phosphorylated form as well as an increase in Bcl-2 with no changes in cytochrome c. All of these compounds appear to be acting as possible modulators of AST responses to C₂ ceramide. Our results suggest that in AST primary cultures, C₂ ceramide, at the concentrations used in this study, does not produce apoptosis. However, it induces an activation of the UbPS, probably as a consequence of an activation of phosphatases and kinases, or through the generation of reactive oxygen species, which act as triggering signals of the UbPS. The fundamental role of NFĸB and Bcl-2 as antiapoptotic factors is discussed.

Signals in hepatitis A virus P3 region proteins recognized by the ubiquitin-mediated proteolytic system

The hepatitis A virus 3C protease and 3D RNA polymerase are present in low concentrations in infected cells. The 3C protease was previously shown to be rapidly degraded by the ubiquitin/26S proteasome system and we present evidence here that the 3D polymerase is also subject to ubiquitination-mediated proteolysis. Our results show that the sequence 32LGVKDDWLLV 41 in the 3C protease serves as a protein destruction signal recognized by the ubiquitin-protein ligase E3α and that the destruction signal for the RNA polymerase does not require the carboxyl-terminal 137 amino acids. Both the viral 3ABCD polyprotein and the 3CD diprotein were also found to be substrates for ubiquitin-mediated proteolysis. Attempts to determine if the 3C protease or the 3D polymerase destruction signals trigger the ubiquitination and degradation of these precursors yielded evidence suggesting, but not unequivocally proving, that the recognition of the 3D polymerase by the ubiquitin system is responsible.

Sensitivity of Mammalian Cells Expressing Mutant Ubiquitin to Protein-damaging Agents

There is convincing evidence from studies in yeast that a functional ubiquitin/proteasome pathway is required to degrade misfolded or oxidatively damaged proteins but for technical reasons, it has been difficult to perform comparable studies in mammalian cells. To investigate the possibility that the ubiquitin/proteasome pathway is cytoprotective for mammalian cells, we have introduced epitope-tagged wild-type ubiquitin or dominant-negative mutant versions of ubiquitin into mouse HT4 neuroblastoma cells. Cells expressing mutant versions of ubiquitin were found to be sensitive to cadmium, an agent that causes oxidative damage to cellular components, and to canavanine, an amino acid analog that generates misfolded proteins. The greatest sensitivity to canavanine was observed in cells expressing a mutant version of ubiquitin unable to support the formation of Lys(48) linkages. Substrates of the proteasome were found to accumulate in these cells, suggesting a general deficit in proteolysis. Our data suggest that defects in the ubiquitin-mediated proteolytic system predispose mammalian cells to the toxic effects of abnormal protein.

Repression of GCN4 mRNA Translation by Nitrogen Starvation in Saccharomyces cerevisiae

Saccharomyces cerevisiae activates a regulatory network called "general control" that provides the cell with sufficient amounts of protein precursors during amino acid starvation. We investigated how starvation for nitrogen affects the general control regulatory system, because amino acid biosynthesis is part of nitrogen metabolism. Amino acid limitation results in the synthesis of the central transcription factor Gcn4p, which binds to specific DNA-binding motif sequences called Gcn4-protein-responsive elements (GCREs) that are present in the promoter regions of its target genes. Nitrogen starvation increases GCN4 transcription but efficiently represses expression of both a synthetic GCRE6::lacZ reporter gene and the natural amino acid biosynthetic gene ARO4. Repression of Gcn4p-regulated transcription by nitrogen starvation is independent of the ammonium sensing systems that include Mep2p and Gpa2p or Ure2p and Gln3p but depends on the four upstream open reading frames in the GCN4 mRNA leader sequence. Efficient translation of GCN4 mRNA is completely blocked by nitrogen starvation, even when cells are simultaneously starved for amino acids and eukaryotic initiation factor-2 alpha is fully phosphorylated by Gcn2p. Our data suggest that nitrogen starvation regulates translation of GCN4 by a novel mechanism that involves the four upstream open reading frames but that still acts independently of eukaryotic initiation factor-2 alpha phosphorylation by Gcn2p.

Bryostatin 1 induces ubiquitination and proteasome degradation of Bcl-2 in the human acute lymphoblastic leukemia cell line, Reh.

The ubiquitin-mediated proteolytic system has been implicated in the turnover of a number of intracellular proteins. In the present study, we investigated the novelty and potential role of bryostatin 1, a macrocyclic lactone isolated from the marine bryozoan, Bugula neritina, in inducing the ubiquitin-mediated proteolysis of the oncoprotein Bcl-2. Immunoprecipitation and immunoblotting analyses revealed that Bcl-2 is ubiquitinated following exposure of the acute lymphoblastic leukemia (ALL) cell line Reh to 1 nM bryostatin 1. Bcl-2 protein rapidly decreases to 50% of that recorded in the control after 24 h of bryostatin 1 treatment. In the subsequent 24 h, Bcl-2 protein again rapidly decreases to 6% of its pre-bryostatin 1 level at which time a plateau is reached and maintained for another 72 h. Furthermore, ubiquitin-Bcl-2 conjugates are detected in untreated as well as bryostatin 1 treated cells, indicating that ubiquitin-dependent proteolysis plays a role in the normal turnover of Bcl-2. However, ubiquitin-Bcl-2 conjugates increase in a time-dependent manner following bryostatin 1 treatment. Lactacystin, which inhibits the proteinase activities of the proteasome, inhibited the bryostatin 1-induced decrease of Bcl-2 protein. The effect of bryostatin 1 on the proteolytic efficiency of the 26S proteasome in Reh cell extracts was also investigated and shown to increase following 1 h of bryostatin 1 treatment. Proteolytic activity reached its highest point by 3 h, and subsequently returned to control levels by 12 h, post-bryostatin 1 treatment. In addition, bryostatin 1 treatment of the Reh cell line decreased expression of bcl-2 mRNA within 3 h. However, bcl-2 mRNA expression returned after 24 h. We speculate that this decrease in mRNA together with increased 26S proteolytic activity accounts for the initial rapid decrease recorded in Bcl-2 protein. These findings indicate that bryostatin 1 treatment of Reh ALL cells decreases Bcl-2 expression through two processes: a) enhanced Bcl-2 protein degradation through the activation of the ubiquitin-proteasome pathway and b) decreased bcl-2 mRNA expression.

Multiubiquitin chain binding subunit MCB1 (RPN10) of the 26S proteasome is essential for developmental progression in Physcomitrella patens.

The 26S proteasome, a multisubunit complex, is the primary protease of the ubiquitin-mediated proteolytic system in eukaryotes. We have recently characterized MCB1 (RPN10), a subunit of the 26S complex that has affinity for multiubiquitin chains in vitro and as a result may function as a receptor for ubiquitinated substrates. To define the role of MCB1 further, we analyzed its function in Physcomitrella patens by generating MCB1 gene disruptions using homologous recombination. PpMCB1, which is 50 to 75% similar to orthologs from other eukaryotes, is present in the 26S proteasome complex and has a similar affinity for multiubiquitin chains, using a conserved hydrophobic domain within the C-terminal half of the polypeptide. Unlike yeast Deltamcb1 strains, which grow normally, P. patens Deltamcb1 strains are viable but are under developmental arrest, generating abnormal caulonema that are unable to form buds and gametophores. Treatment with auxin and cytokinin restored bud formation and subsequent partial development of gametophores. Complementation of a Deltamcb1 strain with mutated versions of PpMCB1 revealed that the multiubiquitin chain binding site is not essential for the wild-type phenotype. These results show that MCB1 has an important function in the 26S proteasome of higher order eukaryotes in addition to its ability to bind multiubiquitin chains, and they provide further support for a role of the ubiquitin/26S proteasome proteolytic pathway in plant developmental processes triggered by hormones.

Identification and Characterization of a Protein Destruction Signal in the Encephalomyocarditis Virus 3C Protease

The amino acid sequence LLVRGRTLVV, which is probably located in a strand-turn-strand structure, has been identified as a protein destruction signal in the rapidly degraded encephalomyocarditis virus 3C protease. Mutations within this sequence reduced the susceptibility of the 3C protease toward ubiquitination and degradation in rabbit reticulocyte lysate. This signal is transferable, since poliovirus 3C protease, which is a poor ubiquitin-mediated proteolytic system substrate, was found to be ubiquitinated and degraded when the signal sequence was either generated at an internal location in the protein or fused to the N terminus. An evaluation of the behavior of 3C protease proteins containing mutations in the signal region indicates that considerable variability in the primary structure is tolerated, although the conservation of certain features appears to be required for signal function. Two E3 ubiquitin-protein ligases that recognize the encephalomyocarditis virus 3C protease as a substrate were also partially purified. One of these was identified as the previously described E3alpha, and this was shown to require the destruction signal sequence to catalyze efficiently the ubiquitination of the 3C protease. The other is a Ubc5-dependent E3 that appears to recognize a different, unidentified feature of the 3C protease.

Evaluation of the Susceptibility of the 3C Proteases of Hepatitis A Virus and Poliovirus to Degradation by the Ubiquitin-Mediated Proteolytic System

The picornavirus 3C proteases are required for the processing of viral polyproteins during infections of host cells. Here we report that the 3C protease of the hepatitis A virus, like that of the encephalomyocarditis virus, is a substrate for rapid, ubiquitin-mediated degradationin vitro.Ubiquitin was shown to stimulate the turnover of the hepatitis virus 3C protease, and labeled protease was found to become incorporated into a mixture of high molecular weight species, which is characteristic of conjugation with polyubiquitin chains. In the presence of methylated ubiquitin, a new 33 kDa species formed, consistent with the generation of a monoubiquitin-3C protease conjugate. The rate of degradation of the 3C protease was reduced by inhibitors of the 26S proteasome. A similar evaluation of the 3C protease of poliovirus revealed that it is stable protein and is not conjugated with ubiquitin. It was also determined that the hepatitis A and encephalomyocarditis virus 3C proteases compete with each other for conjugation with ubiquitin and for degradation. This suggests that the two 3C proteases are both recognized by the same ubiquitin system enzyme, or enzymes, responsible for selecting them as targets for destruction.

The Ubiquitin-mediated Proteolytic System: Involvement of Molecular Chaperones, Degradation of Oncoproteins, and Activation of Transcriptional Regulators

The Ubiquitin-mediated Proteolytic System: Involvement of Molecular Chaperones, Degradation of Oncoproteins, and Activation of Transcriptional Regulators

The encephalomyocarditis virus 3C protease is a substrate for the ubiquitin-mediated proteolytic system.

The encephalomyocarditis virus 3C protease has been shown to be rapidly degraded in infected cells and in vitro in rabbit reticulocyte lysate. The in vitro degradation, at least, is accomplished by a virus-independent, ATP-dependent proteolytic system. Here we identify this proteolytic system as the ubiquitin-mediated system. Incubation of the 3C protease in rabbit reticulocyte or cultured mouse cell lysate preparations, alone or in the presence of added ubiquitin or methylated ubiquitin, resulted in the generation of new higher molecular weight species. These new products were shown to be 3C protease-ubiquitin conjugates by their ability to bind antibodies against both the 3C protease and ubiquitin. Supplemental ubiquitin also stimulated the degradation of the 3C protease in these preparations. Large 3C protease-polyubiquitin conjugates were observed to accumulate in reticulocyte lysate in the presence of adenosine 5'-O-(3-thiotriphosphate), an inhibitor of the 26 S multicatalytic protease. This, combined with the fact that the proteolytic activity could be removed from the lysate by sedimentation, implicates the multicatalytic protease in the degradation of the 3C protease-ubiquitin conjugates. It was also found that the slow rate of degradation of a model polyprotein, which resembles the stable viral 3CD diprotein produced in vivo, is likely due to the fact that the polyprotein is a poor substrate for the ubiquitin-conjugating system.

Degradation of the tumor suppressor protein p53 by the ubiquitin-mediated proteolytic system requires a novel species of ubiquitin-carrier protein, E2

The tumor suppressor protein p53 is extremely unstable in most cell lines. In contrast, many mutant and oncogenic species of the protein are stable. The degradation of p53 in vivo requires metabolic energy; however, the proteolytic system(s) involved have not been identified. The ubiquitin system has been implicated in the degradation of p53 in vitro. The degradation is stimulated significantly by the human papillomavirus (HPV) oncoprotein E6 that associates with p53 and facilitates conjugate formation and subsequent degradation. Complex formation between E6 and p53 is promoted by a cellular protein designated E6-associated protein (E6-AP). Initial dissection of the conjugation process have demonstrated a role for the ubiquitin-activating enzyme, E1, but the ubiquitin-carrier protein (E2, UBC) and the ubiquitin protein ligase, E3, have not been identified. In this study, we report that a novel species of ubiquitin-carrier protein designated E2-F1 (Blumenfeld, N., Gonen, H., Mayer, A., Smith, C., Siegel, N.R., Schwartz, A.L., and Ciechanover, A. (1994) J. Biol. Chem. 269, 9574-9581) is involved in the conjugation and degradation of p53. This E2 enzyme recognizes non-"N-end rule" protein substrates and appears to mediate their conjugation via a novel species of E3. The process of recognition appears to be selective; E2-F1 is not required for the conjugation and degradation of human N-myc. The involvement of E2-F1 in the in vitro process appears to be physiologically meaningful and to reproduce the in vivo process; mutant species of p53 that do not interact with E6 and are stable in vivo are not recognized by the cell free system.

Degradation of nuclear oncoproteins by the ubiquitin system in vitro.

Nuclear oncoproteins are among the most rapidly degraded intracellular proteins. Previous work has implicated the ubiquitin-mediated proteolytic system in the turnover of short-lived intracellular proteins. In the present study, we have evaluated the potential role of the ubiquitin system in the degradation of the specific nuclear oncoproteins encoded by the N-myc, c-myc, c-fos, p53 and E1A genes. Each of these nuclear oncoproteins was synthesized in vitro by transcription of the appropriate cDNA and translation of the resulting mRNA in the presence of [35S]methionine. Degradation of labeled proteins was monitored in the ubiquitin cell-free system. ATP stimulated the degradation of all the proteins between 3- and 10-fold. The degradation was completely inhibited by neutralizing antibody directed against the ubiquitin-activating enzyme, E1, the first enzyme in the ubiquitin-mediated proteolytic cascade. Moreover, degradation in E1-depleted lysates could be restored in each case by the addition of affinity-purified E1. These data suggest that the ubiquitin system mediates the degradation of these oncoproteins in vitro. Degradation of other proteins, such as superoxide dismutase, cytochrome c, enolase, RNase A, and ornithine decarboxylase, is not mediated by the ubiquitin cell-free system. This suggests that the nuclear oncoproteins studied here possess specific signals that target them for rapid turnover by this proteolytic pathway. Furthermore, the relative sensitivity to degradation of various E1A mutants in vivo is also maintained in the cell-free system, suggesting that the ubiquitin pathway may play a role in the cellular degradation of these proteins as well.

Alterations in components of the ubiquitin-protein ligase system following maturation of reticulocytes to erythrocytes

Previous studies have shown that the activity of the ubiquitin-mediated proteolytic system declines markedly following reticulocyte maturation, but the specific alterations responsible for this phenomenon have not been defined. We find that the rate of ATP-dependent degradation of 125I-albumin is reduced 20-fold in lysates of rabbit erythrocytes, as compared to reticulocyte lysates. The activity of the proteolytic system in erythrocyte extracts can be restored by supplementation with components of the ubiquitin-protein ligase system purified from reticulocytes by affinity chromatography. These components are the ubiquitin-carrier protein E 2, the activity of which is nearly completely absent, and the ligase E 3, the activity of which is partially reduced in erythrocytes. Erythrocyte extracts contain other ligases which attach a single, or a few ubiquitin molecules to proteins; these products are different from the multi-ubiquitin derivatives which are formed by the ligase system of protein breakdown. Mature red cells may thus serve to distinguish between different ubiquitin-protein ligase systems with presumably different functions.