Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.25.1 (proteasome)
28,817 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We report the successful combination of mRNA differential-display reverse-transcription PCR (DDRT-PCR) and 5'-rapid amplification of cDNA ends (5'-RACE) in order to isolate full-length cDNAs corresponding to genes activated in tobacco cells treated with cryptogein within 60 min. Cloning and sequencing of two cDNAs, called 'tcI 7' and 'tcI 14' (for tobacco cryptogein-induced), allowed the identification of open reading frames. Deduced amino-acid sequences of 'tcI 7' and 'tcI 14' showed significant homologies with a beta-type proteasome subunit and a transformer-2-like serine/arginine-rich (SR) ribonucleoprotein, respectively. The accumulation of mRNAs corresponding to 'tcI 7' started 30 min after the addition of cryptogein to tobacco cell suspensions and continued up to 180 min, whereas the accumulation of 'tcI 14' corresponding mRNAs was transitory between 30 and 60 min. These results indicated a transcriptional activation of the corresponding genes early after elicitation of tobacco cells by cryptogein. The biological significance of this activation remains to be elucidated.
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PMID:Cloning of two plant cDNAs encoding a beta-type proteasome subunit and a transformer-2-like SR-related protein: early induction of the corresponding genes in tobacco cells treated with cryptogein. 934 50

The evolutionarily conserved multisubunit complex known as the cyclosome or anaphase-promoting complex is involved in catalyzing the ubiquitination of diverse substrates in M phase, allowing their destruction by the 26 S proteasome and the completion of mitosis. Three of the eight subunits of the anaphase-promoting complex (CDC16, CDC23, and CDC27) have been shown to be phosphorylated in M phase, and their phosphorylation is required for the anaphase-promoting complex to be active as a ubiquitin ligase. Several subunits of the anaphase-promoting complex contain tetratricopeptide repeats, a protein motif involved in protein/protein interactions. PP5 is a serine/threonine phosphatase that also contains four copies of the tetratricopeptide repeats motif. Here we show by a combination of two-hybrid analysis and in vitro binding that PP5 interacts with CDC16 and CDC27, two subunits of the anaphase-promoting complex. Only the NH2-terminal domain of PP5, containing all four tetratricopeptide repeats, is required for this physical interaction. Deletion analysis suggests that the site of binding to PP5 is localized to the COOH-terminal block of tetratricopeptide repeats in CDC16 and CDC27. In addition, indirect immunofluorescence showed that PP5 localizes to the mitotic spindle apparatus. The direct interaction of PP5 with CDC16 and CDC27, as well as its overlapping spindle localization in mitosis, suggests that PP5 may be involved in the regulation of the activity of the anaphase-promoting complex.
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PMID:The serine/threonine phosphatase PP5 interacts with CDC16 and CDC27, two tetratricopeptide repeat-containing subunits of the anaphase-promoting complex. 940 94

The transcription factor NF-kappaB is normally sequestered in the cytoplasm by members of the IkappaB family, including IkappaB alpha, IkappaB beta, and the recently cloned IkappaB epsilon. Upon cellular activation, these inhibitors are rapidly phosphorylated on two amino-terminal serines, ubiquitinated, and degraded by the 26S proteasome, releasing a functional NF-kappaB. To determine the importance of IkappaB beta in NF-kappaB regulation in T cells, we generated transgenic mice expressing a constitutively active IkappaB beta mutant (mIkappaB beta) under the control of the lck promoter. The transgene contains the two critical N-terminal serine residues mutated to alanines and therefore no longer susceptible to degradation upon cell activation. mIkappaB beta is unable to totally displace IkappaB alpha from RelA-containing complexes, thus allowing a transient activation of NF-kappaB upon T-cell stimulation. However, mIkappaB beta completely blocks NF-kappaB activity after IkappaB alpha degradation. In addition, as a consequence of this inhibition, ikba expression is down regulated, along with that of other NF-kappaB-regulated genes. These transgenic mice have a significant reduction in the peripheral T-cell population, especially CD8+ cells. The remaining T cells have impaired proliferation in response to phorbol 12-myristate 13-acetate plus phytohemagglutinin or calcium ionophore but not to anti-CD3/anti-CD28 costimulation. As a result of these alterations, transgenic animals present defects in immune responses such as delayed-type hypersensitivity and the generation of specific antibodies against T-cell-dependent antigens. These results show that in nonstimulated T cells, IkappaB beta cannot efficiently displace IkappaB alpha bound to RelA-containing complexes and that persistent NF-kappaB activity is required for proper T-cell responses in vivo.
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PMID:Expression of constitutively active IkappaB beta in T cells of transgenic mice: persistent NF-kappaB activity is required for T-cell immune responses. 941 95

The well-known Rel/NF-kappaB family of vertebrate transcription factors comprises a number of structurally related, interacting proteins that bind DNA as dimers and whose activity is regulated by subcellular location. This family includes many members (p50, p52, RelA, RelB, c-Rel, ...), most of which can form DNA-binding homo- or hetero-dimers. All Rel proteins contain a highly conserved domain of approximately 300 amino-acids, called the Rel homology domain (RH), which contains sequences necessary for the formation of dimers, nuclear localization, DNA binding and IkappaB binding. Nuclear expression and consequent biological action of the eukaryotic NF-kappaB transcription factor complex are tightly regulated through its cytoplasmic retention by ankyrin-rich inhibitory proteins known as IkappaB. The IkappaB proteins include a group of related proteins that interact with Rel dimers and regulate their activities. The interaction of a given IkappaB protein with a Rel complex can affect the Rel complex in distinct ways. In the best characterized example, IkappaB-alpha interacts with a p50/RelA (NF-kappaB) heterodimer to retain the complex in the cytoplasm and inhibit its DNA-binding activity. The NF-kappaB/IkappaB-alpha complex is located in the cytoplasm of most resting cells, but can be rapidly induced to enter the cell nucleus. Upon receiving a variety of signals, many of which are probably mediated by the generation of reactive oxygen species (ROS), IkappaB-alpha undergoes phosphorylation at serine residues by a ubiquitin-dependent protein kinase, is then ubiquitinated at nearby lysine residues and finally degraded by the proteasome, probably while still complexed with NF-kappaB. Removal of IkappaB-alpha uncovers the nuclear localization signals on subunits of NF-kappaB, allowing the complex to enter the nucleus, bind to DNA and affect gene expression. Like proinflammatory cytokines (e.g. IL-1, TNF), various ROS (peroxides, singlet oxygen, ...) as well as UV (C to A) light are capable of mediating NF-kappaB nuclear translocation, while the sensor molecules which are sensitive to these agents and trigger IkappaB-alpha proteolysis are still unidentified. We also show that a ROS-independent mechanism is activated by IL-1beta in epithelial cells and seems to involve the acidic sphingomyelinase/ceramide transduction pathway.
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PMID:Multiple redox regulation in NF-kappaB transcription factor activation. 942 83

Human membrane cofactor protein (MCP, CD46) has been suggested, although no convincing evidence has been proposed, to be a fertilization-associated protein, in addition to its primary functions as a complement regulator and a measles virus receptor. We have cloned a cDNA encoding the murine homologue of MCP. This cDNA showed 45% identity in deduced protein sequence and 62% identity in nucleotide sequence with human MCP. Its ectodomains were four short consensus repeats and a serine/threonine-rich domain, and it appeared to be a type 1 membrane protein with a 23-amino acid transmembrane domain and a short cytoplasmic tail. The protein expressed on Chinese hamster ovary cell transfectants was 47 kDa on SDS/PAGE immunoblotting, approximately 6 kDa larger than the murine testis MCP. It served as a cofactor for factor I-mediated inactivation of the complement protein C3b in a homologous system and, to a lesser extent, in a human system. Strikingly, the major message of murine MCP was 1.5 kb and was expressed predominantly in the testis. It was not detected in mice defective in spermatogenesis or with immature germ cells (until 23 days old). Thus, murine MCP may be a sperm-dominant protein the message of which is expressed selectively in spermatids during germ-cell differentiation.
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PMID:Molecular cloning of a murine homologue of membrane cofactor protein (CD46): preferential expression in testicular germ cells. 946 5

Proteolytic cleavage of a limited number of cellular proteins is a central biochemical feature of apoptosis. Aspartate-specific cysteine proteases, the so-called 'caspases', are the main enzymes involved in this process. At least ten homologues of interleukin-1 beta converting enzyme (ICE), the first described human caspase, have been identified so far. The purified active proteins are heterodimers with a long and a short subunit derived from a common inactive precursor. Crystallized ICE has an original tetrameric structure. The various caspases tend to show high degrees of homology around the active site Cys. Proteolysis by caspases minimally requires a tetrapeptide substrate in which Asp is an absolute requirement in P1 position, the P4 substrate residue is unique to each homologue, and much more widespread amino acid substitution is observed in P2 and P3. Caspase activation might involve a proteolytic cascade similar to that of the coagulation cascade but the molecular ordering of these proteases in vivo remains to be established clearly. Calpains, serine proteases, granzymes and the proteasome-ubiquitin pathway of protein degradation are other proteolytic pathways that have been suggested to play a role in apoptosis. Substrate proteins can be either activated or degraded during cell death and the consequences of their cleavage remains mostly ill-understood. Nevertheless, the recent demonstration that protease inhibitors can rescue mice undergoing acute liver destruction indicates the accuracy of therapeutic strategies aiming to inhibit cell death-associated proteolysis.
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PMID:Proteases, proteolysis, and apoptosis. 955 23

The interferon regulatory factors (IRF) consist of a growing family of related transcription proteins first identified as regulators of the alpha beta interferon (IFN-alpha/beta) gene promoters, as well as the interferon-stimulated response element (ISRE) of some IFN-stimulated genes. IRF-3 was originally identified as a member of the IRF family based on homology with other IRF family members and on binding to the ISRE of the ISG15 promoter. IRF-3 is expressed constitutively in a variety of tissues, and the relative levels of IRF-3 mRNA do not change in virus-infected or IFN-treated cells. In the present study, we demonstrate that following Sendai virus infection, IRF-3 is posttranslationally modified by protein phosphorylation at multiple serine and threonine residues, which are located in the carboxy terminus of IRF-3. A combination of IRF-3 deletion and point mutations localized the inducible phosphorylation sites to the region -ISNSHPLSLTSDQ- between amino acids 395 and 407; point mutation of residues Ser-396 and Ser-398 eliminated virus-induced phosphorylation of IRF-3 protein, although residues Ser-402, Thr-404, and Ser-405 were also targets. Phosphorylation results in the cytoplasm-to-nucleus translocation of IRF-3, DNA binding, and increased transcriptional activation. Substitution of the Ser-Thr sites with the phosphomimetic Asp generated a constitutively active form of IRF-3 that functioned as a very strong activator of promoters containing PRDI-PRDIII or ISRE regulatory elements. Phosphorylation also appears to represent a signal for virus-mediated degradation, since the virus-induced turnover of IRF-3 was prevented by mutation of the IRF-3 Ser-Thr cluster or by proteasome inhibitors. Interestingly, virus infection resulted in the association of IRF-3 with the CREB binding protein (CBP) coactivator, as detected by coimmunoprecipitation with anti-CBP antibody, an interaction mediated by the C-terminal domains of both proteins. Mutation of residues Ser-396 and Ser-398 in IRF-3 abrogated its binding to CBP. These results are discussed in terms of a model in which virus-inducible, C-terminal phosphorylation of IRF-3 alters protein conformation to permit nuclear translocation, association with transcriptional partners, and primary activation of IFN- and IFN-responsive genes.
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PMID:Virus-dependent phosphorylation of the IRF-3 transcription factor regulates nuclear translocation, transactivation potential, and proteasome-mediated degradation. 956 18

Interaction of many infectious agents with eukaryotic host cells is known to cause activation of the ubiquitous transcription factor nuclear factor kappaB (NF-kappaB) (U. Siebenlist, G. Franzoso, and K. Brown, Annu. Rev. Cell Biol. 10:405-455, 1994). Recently, we reported a biphasic pattern of NF-kappaB activation in cultured human umbilical vein endothelial cells consequent to infection with Rickettsia rickettsii, an obligate intracellular gram-negative bacterium and the etiologic agent of Rocky Mountain spotted fever (L. A. Sporn, S. K. Sahni, N. B. Lerner, V. J. Marder, D. J. Silverman, L. C. Turpin, and A. L. Schwab, Infect. Immun. 65:2786-2791, 1997). In the present study, we describe activation of NF-kappaB in a cell-free system, accomplished by addition of partially purified R. rickettsii to endothelial cell cytoplasmic extracts. This activation was rapid, reaching maximal levels at 60 min, and was dependent on the number of R. rickettsii organisms added. Antibody supershift assays using monospecific antisera against NF-kappaB subunits (p50 and p65) confirmed the authenticity of the gel-shifted complexes and identified both p50-p50 homodimers and p50-p65 heterodimers as constituents of the activated NF-kappaB pool. Activation occurred independently of the presence of endothelial cell membranes and was not inhibited by removal of the endothelial cell proteasome. Lack of involvement of the proteasome was further confirmed in assays using the peptide-aldehyde proteasome inhibitor MG 132. Activation was not ATP dependent since no change in activation resulted from addition of an excess of the unhydrolyzable ATP analog ATPgammaS, supplementation with exogenous ATP, or hydrolysis of endogenous ATP with ATPase. Furthermore, Western blot analysis before and after in vitro activation failed to demonstrate phosphorylation of serine 32 or degradation of the cytoplasmic pool of IkappaB alpha. This lack of IkappaB alpha involvement was supported by the finding that R. rickettsii can induce NF-kappaB activation in cytoplasmic extracts prepared from T24 bladder carcinoma cells and human embryo fibroblasts stably transfected with a superrepressor phosphorylation mutant of IkappaB alpha, rendering NF-kappaB inactivatable by many known signals. Thus, evidence is provided for a potentially novel NF-kappaB activation pathway wherein R. rickettsii may interact with and activate host cell transcriptional machinery independently of the involvement of the proteasome or known signal transduction pathways.
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PMID:Proteasome-independent activation of nuclear factor kappaB in cytoplasmic extracts from human endothelial cells by Rickettsia rickettsii. 957 57

Improperly processed secretory proteins are degraded by a hydrolytic system that is associated with the endoplasmic reticulum (ER) and appears to involve re-export of lumenal proteins into the cytoplasm for ultimate degradation by the proteasome. The chimaeric protein hGHDAF28, which contains a crippled glycosylphosphatidylinositol (GPI) C-terminal signal peptide, is degraded by a pathway highly similar to that for other ER-retained proteins and is characterized by formation of disulphide-linked aggregates, failure to reach the Golgi complex and intracellular degradation with a half life of approximately 2 h. Here we show that N-acetyl-leucinal-leucinal-norleucinal, MG-132 and lactacystin, all inhibitors of the proteasome, protect hGHDAF28; hGHDAF28 is still proteolytically cleaved in the presence of lactacystin or MG-132, by the removal of approximately 2 kDa, but the truncated fragment is not processed further. We demonstrate that the ubiquitination system accelerates ER-degradation of hGHDAF28, but is not essential to the process. Overall, these findings indicate that GPI quality control is mediated by the cytoplasmic proteasome. We also show that the presence of a cysteine residue in the GPI signal of hGHDAF28 is required for retention and degradation, as mutation of this residue to serine results in secretion of the fusion protein, implicating thiol-mediated retention as a mechanism for quality control of some GPI signals. Removal of the cysteine also prevents inclusion of hGHDAF28 in disulphide-linked aggregates, indicating that aggregate formation is an additional retention mechanism for this class of protein. Therefore our data suggest that an unpaired terminal cysteine is the retention motif of the hGHDAF28 GPI-processing signal and that additional information may be required for efficient engagement of ER quality control systems by the majority of GPI signals which lack cysteine residues.
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PMID:Proteasome and thiol involvement in quality control of glycosylphosphatidylinositol anchor addition. 957 58

The reverse transcription polymerase chain reaction (RT-PCR) with primers specific for each of the 14 exons of the human complement regulatory protein membrane cofactor protein (MCP;CD46) has been utilized to determine MCP mRNA transcript expression in peripheral blood mononuclear cells (PBMC). An additional transcript of a larger size than predicted was consistently detected in reactions with a sense primer for exon 7, that encodes the first alternatively spliced serine-threonine-rich region (ST-A), together with an antisense exon 12 primer, RT-PCR with primers for other exons both 5' and 3' of exon 7 further showed that these MCP transcripts contain additional sequences immediately both 5' and 3' to the exon 7-encoded sequence. Comparison of genomic DNA with cDNA by PCR, in combination with sequence analysis, demonstrated the presence of the complete invariant sequences of both introns adjacent to exon 7, i.e. intron 6 (411 bp) and intron 7 (127 bp). RT-PCR using primers specific for the intron 6 sequence, together with Southern and Northern blotting using an intron 6-specific probe, confirmed retention of this intron within a novel 4.8-kb mRNA transcript in human PBMC. Due to the presence of a stop codon within intron 6, translation would result in a novel truncated MCP isoform (MCPi) containing the four invariant short consensus repeat (SCR) regions and a unique C-terminal 39 amino acid transmembrane and cytoplasmic tail region that may promote endoplasmic reticulum retention.
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PMID:A novel isoform of human membrane cofactor protein (CD46) mRNA generated by intron retention. 966 62


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