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)

(1) RNase Ms was inactivated by iodoacetate. The inactivation was most rapid at pH 6.0, and was inhibited in the presence of a denaturant such as 8 m urea or 6 m guanidine-HCL. (2) Competitive inhibitors protected RNase Ms from inactivation by iodoacetate; the effect was in the order 2',(3')-GTP greater than 2',(3')-AMP, 2',(3')-UMP greater than or equal to 2',(3')-CMP. The order is not consistent with that of the binding constants of the 4 nucleotides towards RNase Ms (A is greater than C greater than G greater than U). (3) RNase Ms was inactivated with the concomitant incorporation of one molar equivalent of carboxymethly group. The following evidence indicated that the carboxymethyl group was incorporated into the carboxyl group of an aspartic acid or glutamic acid residue. (i) The carboxymethyl group incorporated into RNase Ms was liberated by treatment with 0.1 n NaOH or 1 m hydroxylamine. (ii) The amino acid composition of carboxymethylated RNase Ms (CM RNase Ms) after acid hydrolysis is similar to that of RNase Ms. (4) 14C-Labeled CM RNase Ms was digested successively with alkaline protease and amino-peptidase M. The radioactive amino acid released was eluted just before aspartate on an amino acid analyzer. After hydrolysis with 6 n HCL, glutamic acid was produced exclusively from the radioactive amino acid. The specific radioactivity of this amino acid calculated from the radioactivity and glutamic acid formed was practctically the same as that of CM RNase Ms. Thus, it was concluded that a carboxymethyl group was incorporated at the carboxyl group of a glutamic acid residue of RNnase Ms. (5) CM RNase Ms bound with 2'-AMP to the same extent as native RNase Ms, but bound to a lesser extent with 2',(3')-GMP. (6) Although the conformation of CM RNase Ms as judged from the CD spectrum was practically the same as that of native RNase Ms, the reactivity of CM RNase Ms towards dinitrofluorobenzene was different from that of native RNase Ms, indicating some difference in the conformation. (7) These results indicate that one glutamic acid residue is involved in the active of RNase Ms.
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PMID:Carboxymethylation of a minor ribonuclease from Aspergillus saitoi. 47 29

Ethylenediamine-soluble glycoproteins were extracted from isolated Microsporum gypseum hyphal walls during sporulation and from spore coats before and after germination. This study was carried out to identify a sporulation-specific cell wall protein that possibly served as a substrate for the alkaline protease which initiated the macroconidial germination of this fungus. Analyses revealed that water-insoluble glycoprotein accounted for 10% of the ungerminated spore coat but only for 4 to 5% of the mycelial wall dry weight. This fraction was modified in its amino acid composition during sporulation, and it decreased in protein content during spore germination. Water-soluble glycoprotein, which accounted for approximately 3 to 3.5% of either the spore coat or mycelial wall dry weight, was of similar amino acid composition from both sources and did not decrease in protein content upon spore germination. The water-insoluble glycoprotein was found to be rich in leucine, aspartic acid, glycine, glutamic acid, and phenylalanine residues. The water-soluble glycoprotein was rich in proline, threonine, glycine, serine, glutamic acid, and alanine.
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PMID:Changes in Microsporum gypseum mycelial wall and spore coat glycoproteins during sporulation and spore germination. 440 13

1. RNase St was inactivated by iodoacetate. The inactivation was most rapid at pH 5.0-7.0. Competitive inhibitors protected RNase St from inactivation by iodoacetate. The protective effect of 2'-GMP was most effective among nucleotides tested. 2. RNase St was inactivated with the concomitant incorporation of one molar equivalent of carboxymethyl group. The carboxymethyl group incorporated into RNase St was liberated by treatment with 0.2 N NaOH or 1 M hydroxylamine. Thus the incorporation of a carboxymethyl group into a carboxyl group was demonstrated. 3. 14C-labeled CM-RNase St was digested successively with alkaline protease and aminopeptidase M. The 14C-labeled amino acid was identified as the carboxymethyl ester of glutamic acid by means of column chromatography. 4. By digestion of reduced carboxymethylated CM-RNase St with trypsin, a peptide containing a 14C-carboxymethyl group was isolated by Dowex AG-50W colum chromatography. alpha-Chymotryptic digestion of the radioactive tryptic peptide, Glu48-Lys65, produced a tetrapeptide containing a 14C-carboxymethyl group, that is, Tyr59-His60-Glu61-Tyr62. Therefore, it was concluded that Glu61 in RNase St was the site of carboxymethylation. 5. When RNase St was inactivated by iodoacetamide at pH 8.0, about 2 histidine residues were modified. The molar ratio of the products of carboxyamidomethylation were 52.3%, 21.7%, 21.0%, and 4.8% for 3-CAM-His, 1,3-di-CAM-His, 1-CAM-His, and di-CM-Lys, respectively. 6. CD spectra of CM-RNase St and CAM-RNase St were practically the same as that of the native RNase St indicating the maintenance of the native conformation during modification. 7. The binding constants of CM-RNase St and CAM-RNase St with 2'-GMP were about 1/150 and 1/38 of that of the native enzyme, respectively.
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PMID:Alkylation of a ribonuclease from Streptomyces erythreus with iodoacetate and iodoacetamide. 728 72

The rapid turnover of spermidine/spermine N1-acetyltransferase (SSAT), a key enzyme in the regulation of polyamine levels, was found to be mediated via ubiquitination and the proteasomal system. SSAT degradation was blocked by the binding of polyamines or of the polyamine analog, N1,N12-bis(ethyl)spermine (BE-3-4-3), to the protein, providing a mechanism for the increase of SSAT activity in response to these agents. Site-directed mutagenesis indicated that a number of residues including arginine 19, cysteine 122, histidine 126, glutamic acid 152, arginine 155, and methionine 167 were needed for protection of SSAT by BE-3-4-3. These residues have previously been shown to reduce the affinity for the binding of polyamines to the SSAT protein, and these results indicate that the change in protein configuration brought about by this binding renders the protein resistant to proteasomal degradation. Mutations to alanines of residues arginine 7, cysteine 14, and lysine 141 also prevented the protection by BE-3-4-3, and these residues may be required for the formation of the protected conformation. The rapid degradation of SSAT required the carboxyl-terminal region of the protein, and the two terminal glutamic acid residues at positions 170 and 171 were found to be of critical importance. Truncation of the protein to remove these residues or the mutation of either of these acidic residues to glutamine completely abolished the rapid degradation of SSAT. The addition of two extra lysine residues at the carboxyl terminus or the conversion of the glutamic acids at positions 170 and 171 to lysines also prevented SSAT degradation by the proteasome. These results show the key role of the acidic residues at the carboxyl terminus of the protein in reacting with the proteasome. In contrast, mutation of lysine 166 to alanine, which extends the length of the acidic region in the carboxyl-terminal fragment of SSAT, actually increased the rate of degradation of SSAT without affecting its stabilization by BE-3-4-3. The binding of BE-3-4-3 or polyamines is therefore likely to change the configuration of the SSAT protein in a way that prevents the exposure of the carboxyl-terminal region of the ubiquitinated protein to the proteasome.
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PMID:Proteasomal degradation of spermidine/spermine N1-acetyltransferase requires the carboxyl-terminal glutamic acid residues. 911 88

Two proteasome activators PA28alpha and beta, which have been implicated in antigen processing for loading class I MHC molecules, are synthesized in response to Ifn-gamma. The human genes encoding these activators (PSME1 and PSME2, respectively) were analyzed by sequencing. Each gene comprised 11 exons, consistent with gene duplication during vertebrate evolution. The intron/exon organization of both genes was highly conserved, the major difference being the absence of the exon encoding the lysine and glutamic acid-rich 'KEKE' motif in PA28beta. Two other genes of relevance to the immune system were located close to those for PA28 at 14q11.2 including ISGF3G, a protein involved in transcription after IFNalpha signalling. These sequences were also characterized.
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PMID:Organization of the genes encoding the human proteasome activators PA28alpha and beta. 1019 20

Mammalian Ubc9 (mUbc9) is required for rapid degradation of the E2A proteins E12 and E47 by the ubiquitin-proteasome system. We have shown elsewhere that mUbc9 interacts with amino acids 477-530 of E12/E47. Here we test the hypothesis that this region, rich in proline, glutamic acid, serine, and threonine (PEST) residues, serves as the E2A protein degradation domain (DD). An E2A protein lacking this region, E47Delta(478-531), was significantly more stable than wild-type E47(half-life of more than 6 h versus 55 min). Deletion of the E2A DD had no effect on the E-box-binding and transcriptional activity of E47. We mapped two discreet mUbc9-interacting regions within the E2A DD: amino acids 476-494 and 505-513. E2A(505-513) interacted with mUbc9 but not with human Ubc5, MyoD, Id3, or the polymyositis-scleroderma autoantigen. Substitution of the E2A(505-513) central hydrophobic residues with basic residues abolished interaction with mUbc9. Also, full-length E47 lacking the second mUbc9-interacting region was significantly more stable than wild-type E47. Reintroduction of the E2A DD into the long-lived, naturally occurring chimeric oncoprotein E2A-HLF (hepatic leukemic factor) destabilized it, suggesting that this domain can transfer a degradation signal to a heterologous protein. E2A-HLF-DD chimeric protein was stabilized by the proteasome inhibitor LLNL, indicating the role of the ubiquitin-proteasome system mediating degradation through the E2A degradation domain. Our experiments indicate that the E2A DD mediates E2A protein interactions with the ubiquitin-proteasome system and that the E2A DD is required for metabolism of these widely expressed proteins.
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PMID:Characterization of the mUBC9-binding sites required for E2A protein degradation. 1049 39

The basic helix-loop-helix/leucine zipper (bHLH/ZIP) microphthalmia-associated transcription factor (MITF) regulates transcription of genes encoding enzymes essential for melanin biosynthesis in melanocytes and retinal pigmented epithelial cells. To determine how MITF activity is regulated, we used the yeast two-hybrid system to identify proteins expressed by human melanoma cells that interact with MITF. The majority of clones that showed positive interaction with a 158-amino-acid region of MITF containing the bHLH/ZIP domain (aa 168-325) encoded the ubiquitin conjugating enzyme hUBC9. The association of MITF with hUBC9 was further confirmed by an in vitro GST pull-down assay. Although hUBC9 is known to interact preferentially with SENTRIN/SUMO1, in vitro transcription/translation analysis demonstrated greater association of MITF with ubiquitin than with SENTRIN. Importantly, cotransfection of MITF and hUBC9 expression vectors resulted in MITF protein degradation. MITF protein was stabilized by the proteasome inhibitor MG132, indicating the role of the ubiquitin-proteasome system in MITF degradation. Serine 73, which is located in a region rich in proline, glutamic acid, serine, and threonine (PEST), regulates MITF protein stability, since a serine to alanine mutation prevented hUBC9-mediated MITF (S73A) degradation. Furthermore, we identified lysine 201 as a potential ubiquitination site. A lysine to arginine mutation abolished MITF (K201R) degradation by hUBC9 in vivo. Our experiments indicate that by targeting MITF for proteasome degradation, hUBC9 is a critical regulator of melanocyte differentiation.
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PMID:Regulation of microphthalmia-associated transcription factor MITF protein levels by association with the ubiquitin-conjugating enzyme hUBC9. 1069 30

Activation of transcription factor NF-kappa B involves the signal-dependent degradation of basally phosphorylated inhibitors such as I kappa B alpha. In response to proinflammatory cytokines or mitogens, the transduction machinery has recently been characterized, but the activation mechanism upon oxidative stress remains unknown. In the present work, we provide several lines of evidence that NF-kappa B activation in a T lymphocytic cell line (EL4) by hydrogen peroxide (H2O2) did not involve phosphorylation of the serine residues 32 and 36 in the amino-terminal part of I kappa B alpha. Indeed, mutation of Ser32 and Ser36 blocked IL-1 beta- or PMA-induced NF-kappa B activation, but had no effect on its activation by H2O2. Although I kappa B alpha was phosphorylated upon exposure to H2O2, tyrosine residue 42 and the C-terminal PEST (proline-glutamic acid-serine-threonine) domain played an important role. Indeed, mutation of tyrosine 42 or serine/threonine residues of the PEST domain abolished NF-kappa B activation by H2O2, while it had no effect on activation by IL-1 beta or PMA-ionomycin. This H2O2-inducible phosphorylation was not dependent on I kappa B kinase activation, but could involve casein kinase II, because an inhibitor of this enzyme (5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole) blocks NF-kappa B activation. H2O2-induced I kappa B alpha phosphorylation was followed by its degradation by calpain proteases or through the proteasome. Taken together, our findings suggest that NF-kappa B activation by H2O2 involves a new mechanism that is totally distinct from those triggered by proinflammatory cytokines or mitogens.
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PMID:Crucial role of the amino-terminal tyrosine residue 42 and the carboxyl-terminal PEST domain of I kappa B alpha in NF-kappa B activation by an oxidative stress. 1075 28

All RNA viruses known to date encode an RNA-dependent RNA polymerase (RdRp) that is required for replication of the viral genome. We have expressed and purified the turnip yellow mosaic virus (TYMV) RdRp in insect cells using a recombinant baculovirus, either in its native form, or fused to an hexa-histidine tag. Phosphorylation of the protein was demonstrated by labelling experiments in vivo, as well as phosphatase treatment of the purified protein in vitro. Phospho amino acid analysis and immunoblotting experiments identified serine and threonine residues as being the subject of phosphorylation. Peptide mass mapping using MS analysis of a protein digest revealed that phosphorylation sites are localized within a putative PEST sequence [a sequence rich in proline (P), glutamic acid (E), serine (S) and threonine (T) residues] in the N-terminal region of the protein. Using monoclonal antibodies specific for ubiquitin conjugates, we were able to demonstrate that the TYMV RdRp is conjugated to ubiquitin molecules when expressed in insect cells. These observations suggest that the TYMV RdRp may be processed selectively by the ubiquitin/proteasome degradation system upon phosphorylation of the PEST sequence.
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PMID:Evidence for phosphorylation and ubiquitinylation of the turnip yellow mosaic virus RNA-dependent RNA polymerase domain expressed in a baculovirus-insect cell system. 1088 Mar 40

Targeted protein degradation plays an important regulatory role in the cell, but only a few protein degradation signals have been characterized in plants. Here we describe three instability determinants in the termini of the cauliflower mosaic virus (CaMV) capsid protein precursor, of which one is still present in the mature capsid protein p44. A modified ubiquitin protein reference technique was used to show that these motifs are still active when fused to a heterologous reporter gene. The N-terminus of p44 contains a degradation motif characterized by proline, glutamate, aspartate, serine and threonine residues (PEST), which can be inactivated by mutation of three glutamic acid residues to alanines. The signals from the precursor do not correspond to known degradation motifs, although they confer high instability on proteins expressed in plant protoplasts. All three instability determinants were also active in mammalian cells. The PEST signal had a significantly higher degradation activity in HeLa cells, whereas the precursor signals were less active. Inhibition studies suggest that only the signal within the N-terminus of the precursor is targeting the proteasome in plants. This implies that the other two signals may target a novel degradation pathway.
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PMID:Degradation signals within both terminal domains of the cauliflower mosaic virus capsid protein precursor. 1153 79


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