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 that exposure of aconitase to moderate concentrations of peroxynitrite, 3-morpholinosydnonimine (SIN-1; a superoxide- and nitric oxide-liberating substance), or hydrogen peroxide, inhibits the enzyme and enhances susceptibility to proteolytic digestion by the isolated 20 S proteasome. Exposure to more severe levels of oxidative stress, from these same agents, causes further inhibition of the enzymatic activity of aconitase but actually decreases its proteolytic breakdown by proteasome. It should be noted that the superoxide and nitric oxide liberated by SIN-1 decomposition react to form a steady flux of peroxynitrite. S-Nitroso-N-acetylpenicillamine, a compound that liberates nitric oxide alone, causes only a small loss of aconitase activity (25% or less) and has no effect on the proteolytic susceptibility of the enzyme. Proteasome also seems to be the main protease in cell lysates that can degrade aconitase after it has been oxidatively modified by exposure to peroxynitrite, SIN-1, or hydrogen peroxide. Using cell lysates isolated from K562 cells treated for several days with an antisense oligodeoxynucleotide to the initiation codon region of the C2 subunit of proteasome (a treatment which diminishes proteasome activity by 50-60%), the enhanced degradation of moderately damaged aconitase was essentially abolished. Other model proteins as well as complex mixtures of proteins, such as cell lysates, also exhibit enhanced proteolytic susceptibility after moderate SIN-1 treatment. Therefore we conclude that peroxynitrite reacts readily with proteins and that mild modification by peroxynitrite results in selective recognition and degradation by proteasome.
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PMID:Peroxynitrite increases the degradation of aconitase and other cellular proteins by proteasome. 955 59

The capacity of readily exchanging electrons makes iron not only essential for fundamental cell functions, but also a potential catalyst for chemical reactions involving free-radical formation and subsequent oxidative stress and cell damage. Cellular iron levels are therefore carefully regulated in order to maintain an adequate substrate while also minimizing the pool of potentially toxic 'free iron'. Iron homoeostasis is controlled through several genes, an increasing number of which have been found to contain non-coding sequences [i.e. the iron-responsive elements (IREs)] which are recognized at the mRNA level by two cytoplasmic iron-regulatory proteins (IRP-1 and IRP-2). The IRPs belong to the aconitase superfamily. By means of an Fe-S-cluster-dependent switch, IRP-1 can function as an mRNA-binding protein or as an enzyme that converts citrate into isocitrate. Although structurally and functionally similar to IRP-1, IRP-2 does not seem to assemble a cluster nor to possess aconitase activity; moreover, it has a distinct pattern of tissue expression and is modulated by means of proteasome-mediated degradation. In response to fluctuations in the level of the 'labile iron pool', IRPs act as key regulators of cellular iron homoeostasis as a result of the translational control of the expression of a number of iron metabolism-related genes. Conversely, various agents and conditions may affect IRP activity, thereby modulating iron and oxygen radical levels in different pathobiological settings. As the number of mRNAs regulated through IRE-IRP interactions keeps growing, the definition of IRPs as iron-regulatory proteins may in the near future become limiting as their role expands to other essential metabolic pathways.
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PMID:Iron regulatory proteins in pathobiology. 1108 15

Iron regulatory protein 1 (IRP1) binds to mRNA iron-responsive elements (IREs) and thereby controls the expression of IRE-containing mRNAs. In iron-replete cells, assembly of a cubane [4Fe-4S] cluster inhibits IRE-binding activity and converts IRP1 to a cytosolic aconitase. Earlier experiments with Saccharomyces cerevisiae suggested that phosphomimetic mutations of Ser-138 negatively affect the stability of the cluster (N. M. Brown, S. A. Anderson, D. W. Steffen, T. B. Carpenter, M. C. Kennedy, W. E. Walden, and R. S. Eisenstein, Proc. Natl. Acad. Sci. USA 95:15235-15240, 1998). Along these lines, we show here that a highly purified preparation of recombinant human IRP1 bearing a phosphomimetic S138E substitution (IRP1(S138E)) lacks aconitase activity, which is a hallmark of [4Fe-4S] cluster integrity. Similarly, IRP1(S138E) expressed in mammalian cells fails to function as aconitase. Furthermore, we demonstrate that the impairment of [4Fe-4S] cluster assembly in mammalian cells sensitizes IRP1(S138E) to iron-dependent degradation. This effect can be completely blocked by the iron chelator desferrioxamine or by the proteasome inhibitors MG132 and lactacystin. As expected, the stability of wild-type or phosphorylation-deficient IRP1(S138A) is not affected by iron manipulations. Ser-138 and flanking sequences appear to be highly conserved in the IRP1s of vertebrates, whereas insect IRP1 orthologues and nonvertebrate IRP1-like molecules contain an S138A substitution. Our data suggest that phosphorylation of Ser-138 may provide a basis for an additional mechanism for the control of vertebrate IRP1 activity at the level of protein stability.
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PMID:A phosphomimetic mutation at Ser-138 renders iron regulatory protein 1 sensitive to iron-dependent degradation. 1297 14

Nitric oxide (NO) has been demonstrated to be the principal effector molecule mediating intracellular killing of Leishmania, both in vitro and in vivo. We investigated the type of cell death process induced by NO for the intracellular amastigote stage of the protozoa Leishmania. Specific detection methods revealed a rapid and extensive cell death with morphological features of apoptosis in axenic amastigotes exposed to NO donors, in intracellular amastigotes inside in vitro - activated mouse macrophages and also in activated macrophages of regressive lesions in a leishmaniasis-resistant mouse model. We extended our investigations to the dog, a natural host-reservoir of Leishmania parasites, by demonstrating that co-incubation of infected macrophages with autologous lymphocytes derived from dogs immunised with purified excreted-secreted antigens of Leishmania resulted in a significant NO-mediated apoptotic cell death of intracellular amastigotes. From the biochemical point of view, NO-mediated Leishmania amastigotes apoptosis did not seem to be controlled by caspase activity as indicated by the lack of effect of cell permeable inhibitors of caspases and cysteine proteases, in contrast to specific proteasome inhibitors, such as lactacystin or calpain inhibitor I. Moreover, addition of the products of two NO molecular targets, cis-aconitase and glyceraldehyde-3-phosphate dehydrogenase, also had an inhibitory effect on the cell death induced by NO. Interestingly, activities of these two enzymes plus 6-phosphogluconate dehydrogenase, parasitic enzymes involved in both glycolysis and respiration processes, are overexpressed in amastigotes selected for their NO resistance. This review focuses on cell death of the intracellular stage of the pathogen Leishmania induced by nitrogen oxides and gives particular attention to the biochemical pathways and the molecular targets potentially involved. Questions about the role of Leishmania amastigotes NO-mediated apoptosis in the overall infection process are raised and discussed.
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PMID:Phenotypical characteristics, biochemical pathways, molecular targets and putative role of nitric oxide-mediated programmed cell death in Leishmania. 1701 62

In recent years, a growing number of proteins have been shown to be localized in more than one subcellular location, although encoded from a single gene. Fundamental aspects in the research of such dual-distributed proteins involve determination of their subcellular localization and their location-specific functions. The lack of sensitive and suitable tools to address these issues has led us to develop a novel tool for functional detection of cytosolic/nuclear isoproteins in the cell, which we term location-specific depletion or subcellular knockout. The depletion of the protein occurs post-translationally via degradation by the ubiquitin-proteasome system, which operates only in the cytosol and the nucleus. As an example, we fused the yeast tricarboxylic acid (TCA) cycle enzyme aconitase to a degron sequence (SL17) recognizable by the ubiquitin-proteasome system. This fusion resulted in the degradation of the cytosolic enzyme, specifically eliminating its activity within the cytosolic glyoxylate shunt without disrupting the protein's activity within the mitochondrial TCA cycle. We show that the degradation of the fusion protein can be attributed specifically to the ubiquitin-proteasome system and that inhibition of this degradation restores its cytosolic activity. This novel tool can be used to detect small subpopulations of dual-targeted proteins, thereby revealing isoproteins that were considered to be confined to a single compartment. The particular advantage of this specific subcellular depletion is that it can reveal the functions of the cytosolic/nuclear isoproteins.
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PMID:Location-specific depletion of a dual-localized protein. 1724 46

Friedreich ataxia has frequently been associated with an increased susceptibility to oxidative stress. We used the yeast (Saccharomyces cerevisiae) model of Friedreich ataxia to study the physiological consequences of a shift from anaerobiosis to aerobiosis. Cells lacking frataxin (Deltayfh1) showed no growth defect when cultured anaerobically. Under these conditions, a significant amount of aconitase was functional, with an intact 4 Fe/4 S cluster. When shifted to aerobic conditions, aconitase was rapidly degraded, and oxidatively modified proteins (carbonylated and HNE-modified proteins) accumulated in both the cytosol and the mitochondria. The ATP-dependent mitochondrial protease Pim1 (Lon) was strongly activated, although its expression level remained unchanged, and the cytosolic activity of the 20S proteasome was greatly decreased, compared to that in wild-type cells. Analysis of the purified proteasome revealed that the decrease in proteasome activity was likely due to both direct inactivation of the enzyme and inhibition by cytosolic oxidized proteins. These features indicate that the cells were subjected to major oxidative stress triggered by oxygen. Accumulation of oxidatively modified proteins, activation of Pim1, and proteasome inhibition did not directly depend on the amount of mitochondrial iron, because these phenotypes remained unchanged when the cells were grown under iron-limiting conditions, and these phenotypes were not observed in another mutant (Deltaggc1) which overaccumulates iron in its mitochondrial compartment. We conclude that oxygen is primarily involved in generating the deleterious phenotypes that are observed in frataxin-deficient yeast cells.
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PMID:Oxidative stress and protease dysfunction in the yeast model of Friedreich ataxia. 1744 3

Mitochondria are a major intracellular source of free radicals and related oxidants. It is generally agreed that the mitochondrial production of such reactive oxygen and nitrogen species increases with age. Antioxidant systems in the mitochondria play an important role in limiting the amount of oxidative damage to tolerable levels. The Lon protease degrades oxidatively modified proteins in the mitochondrial matrix, a function similar to that of the 20S proteasome in the cytoplasm. Recently it was shown that inactive aconitase, a preferential substrate for the Lon protease, might be involved in the maintenance of the mitochondrial genome. Lon protease expression and activity declines with age, which may contribute to the accumulation of the oxidatively modified protein aggregates typically observed in aging and diseased cells. In addition, Lon has multiple functions, such as DNA binding and chaperone activity, for the assembly of respiratory complexes in the Electron Transport Chain. Taken together, Lon and aconitase may be key players in the maintenance of mitochondrial homeostasis under conditions of stress, and (partial) compromise of their function may contribute to both aging and degenerative diseases.
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PMID:Importance of the lon protease in mitochondrial maintenance and the significance of declining lon in aging. 1805 57

Iron regulatory protein 1 (IRP1) controls the translation or stability of several mRNAs by binding to iron responsive elements (IREs) within their untranslated regions. Its activity is regulated by an unusual iron-sulfur cluster (ICS) switch. Thus, in iron-replete cells, IRP1 assembles a cubane [4Fe-4S] cluster that prevents RNA-binding activity and renders the protein to cytosolic aconitase. We show that wild type or mutant forms of IRP1 that fail to assemble a [4Fe-4S] cluster are sensitized for iron-dependent degradation by the ubiquitin-proteasome pathway. The regulation of IRP1 abundance poses an alternative mechanism to prevent accumulation of inappropriately high IRE-binding activity when the ICS assembly pathway is impaired. To study functional aspects of IRP1, we overexpressed wild type or mutant forms of the protein in human H1299 lung cancer cells in a tetracycline-inducible fashion, and analyzed how this affects cell growth. While the induction of IRP1 did not affect cell proliferation in culture, it dramatically reduced the capacity of the cells to form solid tumor xenografts in nude mice. These data provide a first link between IRP1 and cancer.
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PMID:Insights on regulation and function of the iron regulatory protein 1 (IRP1). 1827 88

Friedreich's ataxia (FRDA) is a devastating orphan disease, with no specific treatment. The disease is caused by reduced expression of the protein frataxin, which results in mitochondrial defects and oxidative damage. Levels of residual frataxin critically affect onset and progression of the disease. Understanding the molecular mechanisms that regulate frataxin stability and degradation may, therefore, be exploited for the design of effective therapeutics. Here we show that frataxin is degraded by the ubiquitin-proteasome system and that K(147) is the critical residue responsible for frataxin ubiquitination and degradation. Accordingly, a K(147)R substitution generates a more stable frataxin. We then disclose a set of lead compounds, computationally selected to target the molecular cleft harboring K(147), that can prevent frataxin ubiquitination and degradation, and increase frataxin levels in cells derived from FRDA patients. Moreover, treatment with these compounds induces substantial recovery of aconitase activity and adenosine-5'-triphosphate levels in FRDA cells. Thus, we provide evidence for the therapeutic potential of directly interfering with the frataxin degradation pathway.
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PMID:Preventing the ubiquitin-proteasome-dependent degradation of frataxin, the protein defective in Friedreich's ataxia. 2121 78

Trichosporon asahii is a yeast pathogen implicated in opportunistic infections. Cultures of an isolate collected from industrial wastewater were exposed for 2 days to 100 mg/L sodium arsenite (NaAsO2) and cadmium (CdCl2). Both metals reduced glutathione transferase (GST) activity but had no effect on superoxide dismutase or catalase. NaAsO2 exposure increased glutathione reductase activity while CdCl2 had no effect. Protein thiols were labeled with 5-iodoacetamido fluorescein followed by one dimensional electrophoresis which revealed extensive protein thiol oxidation in response to CdCl2 treatment but thiol reduction in response to NaAsO2. Two dimensional electrophoresis analyses showed that the intensity of some protein spots was enhanced on treatment as judged by SameSpots image analysis software. In addition, some spots showed decreased IAF fluorescence suggesting thiol oxidation. Selected spots were excised and tryptic digested for identification by MALDI-TOF/TOF MS. Twenty unique T. asahii proteins were identified of which the following proteins were up-regulated in response to NaAsO2: 3-isopropylmalate dehydrogenase, phospholipase B, alanine-glyoxylate aminotransferase, ATP synthase alpha chain, 20S proteasome beta-type subunit Pre3p and the hypothetical proteins A1Q1_08001, A1Q2_03020, A1Q1_06950, A1Q1_06913. In addition, the following showed decreased thiol-associated fluorescence consistent with thiol oxidation; aconitase; aldehyde reductase I; phosphoglycerate kinase; translation elongation factor 2; heat shock protein 70 and hypothetical protein A1Q2_04745. Some proteins showed both increase in abundance coupled with decrease in IAF fluorescence; 3-hydroxyisobutyryl-CoA hydrolase; homoserine dehydrogenase Hom6 and hypothetical proteins A1Q2_03020 and A1Q1_00754. Targets implicated in redox response included 10 unique metabolic enzymes, heat shock proteins, a component of the 20S proteasome and translation elongation factor 2. These data suggest extensive proteomic alterations in response to metal-induced oxidative stress in T. asahii. Amino acid metabolism, protein folding and degradation are principally affected.
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PMID:Redox proteomics changes in the fungal pathogen Trichosporon asahii on arsenic exposure: identification of protein responses to metal-induced oxidative stress in an environmentally-sampled isolate. 2506 82


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