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)

Previous studies provided evidence that sepsis-induced muscle proteolysis in experimental animals is caused by increased ubiquitin-proteasome-dependent protein breakdown. It is not known if a similar mechanism accounts for muscle proteolysis in patients with sepsis. We determined mRNA levels for ubiquitin and the 20 S proteasome subunit HC3 by Northern blot analysis in muscle tissue from septic (n = 7) and non-septic (n = 11) patients. Plasma and muscle amino acid concentrations and concentrations in urine of 3-methylhistidine (3-MH), creatinine, and cortisol were measured at the time of surgery to assess the catabolic state of the patients. A three- to fourfold increase in mRNA levels for ubiquitin and HC3 was noted in muscle tissue from the septic patients concomitant with increased muscle levels of phenylalanine and 3-MH and reduced levels of glutamine. Total plasma amino acids were decreased by approximately 30% in the septic patients. The 3-MH/creatinine ratio in urine was almost doubled in septic patients. The cortisol levels in urine were higher in septic than in control patients but this difference did not reach statistical significance. The results suggest that sepsis is associated with increased mRNAs of the ubiquitin-proteasome pathway in human skeletal muscle.
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PMID:Sepsis is associated with increased mRNAs of the ubiquitin-proteasome proteolytic pathway in human skeletal muscle. 900 83

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

Three-week-old maize (Zea mays L.) plants were submitted to light/dark cycles and to prolonged darkness to investigate the occurrence of sugar-limitation effects in different parts of the whole plant. Soluble sugars fluctuated with light/dark cycles and dropped sharply during extended darkness. Significant decreases in protein level were observed after prolonged darkness in mature roots, root tips, and young leaves. Glutamine and asparagine (Asn) changed in opposite ways, with Asn increasing in the dark. After prolonged darkness the increase in Asn accounted for most of the nitrogen released by protein breakdown. Using polyclonal antibodies against a vacuolar root protease previously described (F. James, R. Brouquisse, C. Suire, A. Pradet, P. Raymond [1996] Biochem J 320: 283-292) or the 20S proteasome, we showed that the increase in proteolytic activities was related to an enrichment of roots in the vacuolar protease, with no change in the amount of 20S proteasome in either roots or leaves. Our results show that no significant net proteolysis is induced in any part of the plant during normal light/dark cycles, although changes in metabolism and growth appear soon after the beginning of the dark period, and starvation-related proteolysis probably appears in prolonged darkness earlier in sink than in mature tissues.
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PMID:Induction of a carbon-starvation-related proteolysis in whole maize plants submitted to Light/Dark cycles and to extended darkness 970 83

The c-myb oncogene has been a target of retroviral insertional mutagenesis in murine monocytic leukemias. One mechanism by which c-myb can be activated is through the integration of a retroviral provirus into the central portion of the locus, causing premature termination of c-myb transcription and translation. We had previously shown that a leukemia-specific c-Myb protein, truncated at the site of proviral integration by 248 amino acids, had approximately a fourfold-increased half-life compared to the normal c-Myb protein, due to its ability to escape rapid degradation by the ubiquitin-26S proteasome pathway. Here we provide evidence for the existence of more than one instability determinant in the carboxy-terminal region of the wild-type protein, which appear to act independently of each other. The data were derived from examination of premature termination mutants and deletion mutants of the normal protein, as well as analysis of another carboxy-terminally truncated protein expressed in leukemia. Evidence is provided that one instability determinant is located in the terminal 87 amino acids of the protein and another is located in the vicinity of the internal region that has leucine zipper homology. In leukemias, different degrees of protein stability are attained following proviral integration depending upon how many determinants are removed. Interestingly, although PEST sequences (rich in proline, glutamine, serine, and threonine), often associated with degradation, are found in c-Myb, deletion of PEST-containing regions had no effect on protein turnover. This study provides further insight into how inappropriate expression of c-Myb may contribute to leukemogenesis. In addition, it will facilitate further studies aimed at characterizing the specific role of individual regions of the normal protein in targeting to the 26S proteasome.
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PMID:Identification of protein instability determinants in the carboxy-terminal region of c-Myb removed as a result of retroviral integration in murine monocytic leukemias. 997 84

Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited disorder characterized by progressive loss of coordination, motor impairment and the degeneration of cerebellar Purkinje cells, spinocerebellar tracts and brainstem nuclei. Many dominantly inherited neurodegenerative diseases share the mutational basis of SCA1: the expansion of a translated CAG repeat coding for glutamine. Mice lacking ataxin-1 display learning deficits and altered hippocampal synaptic plasticity but none of the abnormalities seen in human SCA1; mice expressing ataxin-1 with an expanded CAG tract (82 glutamine residues), however, develop Purkinje cell pathology and ataxia. These results suggest that mutant ataxin-1 gains a novel function that leads to neuronal degeneration. This novel function might involve aberrant interaction(s) with cell-specific protein(s), which in turn might explain the selective neuronal pathology. Mutant ataxin-1 interacts preferentially with a leucine-rich acidic nuclear protein that is abundantly expressed in cerebellar Purkinje cells and other brain regions affected in SCA1. Immunolocalization studies in affected neurons of patients and SCA1 transgenic mice showed that mutant ataxin-1 localizes to a single, ubiquitin-positive nuclear inclusion (NI) that alters the distribution of the proteasome and certain chaperones. Further analysis of NIs in transfected HeLa cells established that the proteasome and chaperone proteins co-localize with ataxin-1 aggregates. Moreover, overexpression of the chaperone HDJ-2/HSDJ in HeLa cells decreased ataxin-1 aggregation, suggesting that protein misfolding might underlie NI formation. To assess the importance of the nuclear localization of ataxin-1 and its role in SCA1 pathogenesis, two lines of transgenic mice were generated. In the first line, the nuclear localization signal was mutated so that full-length mutant ataxin-1 would remain in the cytoplasm; mice from this line did not develop any ataxia or pathology. This suggests that mutant ataxin-1 is pathogenic only in the nucleus. To assess the role of the aggregates, transgenic mice were generated with mutant ataxin-1 without the self-association domain (SAD) essential for aggregate formation. These mice developed ataxia and Purkinje cell abnormalities similar to those seen in SCA1 transgenic mice carrying full-length mutant ataxin-1, but lacked NIs. The nuclear milieu is thus a critical factor in SCA1 pathogenesis, but large NIs are not needed to initiate pathogenesis. They might instead be downstream of the primary pathogenic steps. Given the accumulated evidence, we propose the following model for SCA1 pathogenesis: expansion of the polyglutamine tract alters the conformation of ataxin-1, causing it to misfold. This in turn leads to aberrant protein interactions. Cell specificity is determined by the cell-specific proteins interacting with ataxin-1. Submicroscopic protein aggregation might occur because of protein misfolding, and those aggregates become detectable as NIs as the disease advances. Proteasome redistribution to the NI might contribute to disease progression by disturbing proteolysis and subsequent vital cellular functions.
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PMID:Progress in pathogenesis studies of spinocerebellar ataxia type 1. 1043 9

Because parenteral feeding is associated with negative N balance and reduced rates of protein synthesis in intestinal mucosa, we hypothesized that luminal exposure to specific amino acids or energy fuels would stimulate intestinal protein synthesis. We studied the acute effects of luminal nutrients on mucosal protein synthesis in the absence of systemic influences. Multiple jejunal segments constructed in piglets deprived of food overnight (n = 6) were randomly assigned to luminal perfusion with saline, 30 mmol/L amino acid mixture with or without 50 mmol/L glucose, or 30 mmol/L glutamine for 90 min. Protein synthesis was then measured by luminal perfusion with L-[2,6-(3)H]-phenylalanine. Energy substrates (glucose, short-chain fatty acids or beta-hydroxybutyrate) had no effect on mucosal protein synthesis. Relative to saline, a 30 mmol/L amino acid mixture or 30 mmol/L glutamine suppressed mucosal protein synthesis by 20-25% (P < 0.05). On the basis of these surprising results, we speculated that a coordinate reduction of proteolytic processes would be required to maintain positive intestinal N balance. Although intestinal protein catabolism cannot be assessed directly, the 30 mmol/L amino acid mixture acutely suppressed mucosal levels of mRNA encoding ubiquitin, 14-kDa ubiquitin conjugating enzyme and the C9 subunit of the proteasome by 20-30% (P < 0.05), demonstrating the sensitivity of components of the ATP-ubiquitin proteolytic pathway to acute regulation by nutrients. The suppression of protein synthesis by luminal amino acids in the absorptive state might lower intestinal utilization of amino acids to ensure efficient allocation of absorbed nutrients to nonintestinal tissues.
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PMID:Luminal amino acids acutely decrease intestinal mucosal protein synthesis and protease mRNA in piglets. 1049 61

P-glycoprotein (P-gp), the product of human MDR1 gene, which functions as an ATP-dependent drug efflux pump, is N-linked glycosylated at asparagine residues 91, 94, and 99 located within the first extracellular loop. We report here the biochemical characterization of glycosylation-deficient (Gly(-)) P-gp using a vaccinia virus based transient expression system. The staining of HeLa cells expressing Gly(-) P-gp (91, 94, and 99N-->Q), with P-gp specific monoclonal antibodies, MRK-16, UIC2 and 4E3 revealed a 40 to 50% lower cell-surface expression of mutant P-gp compared to the wild-type protein. The transport function of Gly(-) P-gp, assessed using a variety of fluorescent compounds indicated that the substrate specificity of the pump was not affected by the lack of glycosylation. Additional mutants, Gly(-) D (91, 94, 99N-->D) and Gly(-) Delta (91, 94, 99 N deleted) were generated to verify that the reduced cell surface expression, as well as total expression, were not a result of the glutamine substitutions. Gly(-) D and Gly(-) Delta Pgps were also expressed to the same level as the Gly(-) mutant protein. (35)S-Methionine/cysteine pulse-chase studies revealed a reduced incorporation of (35)S-methionine/cysteine in full length Gly(-) P-gp compared to wild-type protein, but the half-life ( approximately 3 hr) of mutant P-gp was essentially unaltered. Since treatment with proteasome inhibitors (MG-132, lactacystin) increased only the intracellular level of nascent, mutant P-gp, the decreased incorporation of (35)S-methionine/cysteine in Gly(-) P-gp appears to be due to degradation of improperly folded mutant protein by the proteasome and endoplasmic reticulum-associated proteases. These results demonstrate that the unglycosylated protein, although expressed at lower levels at the cell surface, is functional and suitable for structural studies.
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PMID:Functional characterization of glycosylation-deficient human P-glycoprotein using a vaccinia virus expression system. 1066 16

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. N-terminal asparagine and glutamine are tertiary destabilizing residues, in that they are enzymatically deamidated to yield secondary destabilizing residues aspartate and glutamate, which are conjugated to arginine, a primary destabilizing residue. N-terminal arginine of a substrate protein is bound by the Ubr1-encoded E3alpha, the E3 component of the ubiquitin-proteasome-dependent N-end rule pathway. We describe the construction and analysis of mouse strains lacking the asparagine-specific N-terminal amidase (Nt(N)-amidase), encoded by the Ntan1 gene. In wild-type embryos, Ntan1 was strongly expressed in the branchial arches and in the tail and limb buds. The Ntan1(-/-) mouse strains lacked the Nt(N)-amidase activity but retained glutamine-specific Nt(Q)-amidase, indicating that the two enzymes are encoded by different genes. Among the normally short-lived N-end rule substrates, only those bearing N-terminal asparagine became long-lived in Ntan1(-/-) fibroblasts. The Ntan1(-/-) mice were fertile and outwardly normal but differed from their congenic wild-type counterparts in spontaneous activity, spatial memory, and a socially conditioned exploratory phenotype that has not been previously described with other mouse strains.
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PMID:Altered activity, social behavior, and spatial memory in mice lacking the NTAN1p amidase and the asparagine branch of the N-end rule pathway. 1080 55

Polyglutamine expansions in proteins are implicated in at least eight inherited neurodegenerative disorders, including Huntington's disease. These mutant proteins can form aggregates within the nucleus and processes of neurons possibly due to misfolding of the proteins. Polyglutamine aggregates are ubiquitinated and sequester molecular chaperone proteins and proteasome components. To investigate other protein components of polyglutamine aggregates, cerebral cortex and striata from patients with Huntington's disease and full-length cDNA transgenic mouse models for this disease were examined immunohistochemically for alpha-synuclein reactivity. Our findings demonstrate that alpha-synuclein can be used as a marker for huntingtin polyglutamine aggregates in both human and mice. Moreover in the HD transgenic mice, the intensity of immunoreactivity increases with age. The significance of recruitment of alpha-synuclein into huntingtin aggregates and its translocation away from the synapses remains to be determined. We propose that aberrant interaction of mutant huntingtin with other proteins, including alpha-synuclein, may influence disease progression.
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PMID:Alpha-synuclein immunoreactivity of huntingtin polyglutamine aggregates in striatum and cortex of Huntington's disease patients and transgenic mouse models. 1089 1

Expansion of CAG repeats within the coding region of target genes is the cause of several autosomal dominant neurodegenerative diseases including Huntington's disease (HD). A hallmark of HD is the proteolytic production of N-terminal fragments of huntingtin containing polyglutamine repeats that form ubiquitinated aggregates in the nucleus and cytoplasm of the affected neurons. In this study, we used an ecdysone-inducible stable mouse neuro2a cell line that expresses truncated N-terminal huntingtin (tNhtt) with different polyglutamine length, along with mice transgenic for HD exon 1, to demonstrate that the ubiquitin-proteasome pathway is involved in the pathogenesis of HD. Proteasomal 20S core catalytic component was redistributed to the polyglutamine aggregates in both the cellular and transgenic mouse models. Proteasome inhibitor dramatically increased the rate of aggregate formation caused by tNhtt protein with 60 glutamine (60Q) repeats, but had very little influence on aggregate formation by tNhtt protein with 150Q repeats. Both normal and polyglutamine-expanded tNhtt proteins were degraded by proteasome, but the rate of degradation was inversely proportional to the repeat length. The shift of the proteasomal components from the total cellular environment to the aggregates, as well as the comparatively slower degradation of tNhtt with longer polyglutamine, decreased the proteasome's availability for degrading other key target proteins, such as p53. This altered proteasomal function was associated with disrupted mitochondrial membrane potential, released cytochrome c from mitochondria into the cytosol and activated caspase-9- and caspase-3-like proteases. These results suggest that the impaired proteasomal function plays an important role in polyglutamine protein-induced cell death.
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PMID:Altered proteasomal function due to the expression of polyglutamine-expanded truncated N-terminal huntingtin induces apoptosis by caspase activation through mitochondrial cytochrome c release. 1133 15


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