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)

The degradation of misfolded, aged, or no longer needed cytosolic proteins depends largely on the ubiquitin-proteasome system. Proteasomes degrade their substrates into fragments of 3-20 amino acids. Human 20S proteasomes can be purified from human erythrocytes by batch adsorption to DEAE-cellulose, ammonium sulfate precipitation, anion-exchange fast protein liquid chromatography (FPLC), and glycerol density gradient ultracentrifugation. 20S proteasomes purified by this method are suitable for the in vitro digestion of synthetic peptides as well as full-length proteins. The degradation products produced by proteasomes are separated by reversed-phase HPLC using an acetonitrile gradient. The obtained fractions are further analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and Edman degradation, which allows a quantitative analysis of the digestion products.
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PMID:Assays of proteasome-dependent cleavage products. 1591 29

Activation of dendritic cells (DC) by Th-dependent (CD40) or -independent (LPS, CpG, or immune complexes) agonistic stimuli strongly enhances the expression of the proteasome activator PA28alphabeta complex. Upon activation of DC, increased MHC class I presentation occurred of the melanocyte-associated epitope tyrosinase-related protein 2(180-188) in a PA28alphabeta-dependent manner. In contrast to other cell types, regulation of PA28alphabeta expression in DC after maturation was found to be IFN-gamma independent. In the present study, we show that expression of PA28alpha and beta subunits was differentially regulated. Firstly, PA28alpha expression is high in both immature and mature DC. In contrast, PA28beta expression is low in immature DC and strongly increased in mature DC. Secondly, we show the presence of a functional NF-kappaB site in the PA28beta promoter, which is absent in the PA28alpha promoter, indicating regulation of PA28beta expression by transcription factors of the NF-kappaB family. In addition, glycerol gradient analysis of DC lysates revealed elevated PA28alphabeta complex formation upon maturation. Thus, induction of PA28beta expression allows proper PA28alphabeta complex formation, thereby enhancing proteasome activity in activated DC. Therefore, maturation of DC not only improves costimulation but also MHC class I processing. This mechanism enhances the CD8(+) CTL (cross)-priming capacity of mature DC.
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PMID:Differential expression regulation of the alpha and beta subunits of the PA28 proteasome activator in mature dendritic cells. 1594 86

The COP9 signalosome (CSN) is a multimeric protein complex that occurs in all eukaryotic cells. Originally described in plants as a regulator of photomorphogenesis, its purification and characterization from mammalian cells revealed significant sequence homologies to subunits of the 26S proteasome lid complex, as well as of the eukaryotic translation initiation factor 3. Recent studies disclosed its participation in processes such as DNA repair, cell cycle regulation, development, and angiogenesis. At the moment, the pleiotropic effects of the CSN point to a regulatory role in the ubiquitin/26S proteasome system, but its exact function still remains to be clarified. This chapter describes the method to purify human CSN from red blood cells. Two outdated erythrocyte concentrates are sufficient to prepare approximately 0.5 mg of CSN. Washed cells are first lysed and then proteins are separated by a DEAE anion-exchange column. The CSN-containing fractions are pooled and subjected to an ammonium sulfate precipitation followed by dialysis. The concentrated proteins are then loaded onto a glycerol density gradient and ultracentrifugation is performed. The purification procedure is continued using two succeeding anion-exchange columns, resulting in a sufficiently pure CSN complex. Optionally, an additional density gradient centrifugation can be attached. The purified CSN complex possesses kinase, deneddylase, and deubiquitinase activities and can be stored for at least 2 months on ice at 4 degrees .
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PMID:Purification method of the COP9 signalosome from human erythrocytes. 1627 52

The production of (R)-3-chloro-1,2-propanediol [(R)-MCP] from prochiral 1,3-dichloro-2-propanol (DCP) was examined with a bacterial strain identified as a Corynebacterium strain. The addition of glycerol as a carbon source or some chlorinated alcohols to a medium was effective for the induction of activity catalyzing the transformation of DCP into MCP. The optimum pH for (R)-MCP production by the resting cell reaction was around 8.0. The optical purity of (R)-MCP formed was improved by keeping the level of DCP in the reaction mixture at a low concentration. (R)-MCP was obtained from 77.5 mM DCP with a 97.3% molar conversion yield and an 83.8% enantiomeric excess of its optical purity by periodic feeding of the substrate.
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PMID:Production of (R)-3-Chloro-1,2-Propanediol from Prochiral 1,3-Dichloro-2-Propanol by Corynebacterium sp. Strain N-1074. 1634 46

Chaperones, particularly the heat-shock proteins, are considered as key players in the maintenance of protein homeostasis and are associated with longevity and cellular immortalization. In this study, we investigated the geroprotective activity of the chemical chaperone glycerol. Glycerol showed significant chaperoning activity in refolding heat-denatured luciferase in vivo and in protecting cells from heat stress-induced cytotoxicity. This was accompanied by decrease in p53, an upregulation of a stress chaperone mortalin/mtHsp70, and an increase in proteasome activity in the presence of oxidative stress.
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PMID:Geroprotection by glycerol: insights to its mechanisms and clinical potentials. 1680 31

Mammals survive starvation by activating proteolysis and lipolysis in many different tissues. These responses are triggered, at least in part, by changing hormonal and neural statuses during starvation. Pathways of proteolysis that are activated during starvation are surprisingly diverse, depending on tissue type and duration of starvation. The ubiquitin-proteasome system is primarily responsible for increased skeletal muscle protein breakdown during starvation. However, in most other tissues, lysosomal pathways of proteolysis are stimulated during fasting. Short-term starvation activates macroautophagy, whereas long-term starvation activates chaperone-mediated autophagy. Lipolysis also increases in response to starvation, and the breakdown of triacylglycerols provides free fatty acids to be used as an energy source by skeletal muscle and other tissues. In addition, glycerol released from triacylglycerols can be converted to glucose by hepatic gluconeogenesis. During long-term starvation, oxidation of free fatty acids by the liver leads to the production of ketone bodies that can be used for energy by skeletal muscle and brain. Tissues that cannot use ketone bodies for energy respond to these small molecules by activating chaperone-mediated autophagy. This is one form of interaction between proteolytic and lipolytic responses to starvation.
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PMID:Proteolytic and lipolytic responses to starvation. 1681 97

Two-dimensional gel electrophoresis (2DE) and MALDI-TOF MS were used to obtain a global view of the cytoplasmic proteins expressed by Thermoplasma acidophilum. In addition, glycerol gradient ultracentrifugation coupled to 2DE-MALDI-TOF MS analysis was used to identify subunits of macromolecular complexes. With the 2DE proteomics approach, over 900 spots were resolved of which 271 proteins were identified. A significant number of these form macromolecular complexes, among them the ribosome, proteasome, and thermosome, which are expressed at high levels. In the glycerol gradient heavy fractions, 10 as yet uncharacterized proteins (besides the well known ribosomal subunits, translation initiation factor eIF-6-related protein, elongation factor 1, and DNA-dependent RNA polymerase) were identified that are putative building blocks of protein complexes. These proteins belong to the categories of hypothetical or conserved hypothetical proteins, and they are present in the cytosol at low concentrations. Although these proteins exhibit homology to known sequences, their structures, subunit compositions, and biological functions are not yet known.
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PMID:Proteomics analysis of Thermoplasma acidophilum with a focus on protein complexes. 1715 Oct 18

Apparent mineralocorticoid excess (AME) is a severe form of hypertension that is caused by impaired activity of 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), which converts biologically active cortisol into inactive cortisone. Mutations in HSD11B2 result in cortisol-induced activation of mineralocorticoid receptors and cause hypertension with hypokalemia, metabolic alkalosis, and suppressed circulating renin and aldosterone concentrations. This study uncovered the first patient with AME who was described in the literature, identified the genetic defect in HSD11B2, and provided evidence for a novel mechanism of reduced 11beta-HSD2 activity. This study identified a cluster of amino acids (335 to 339) in the C-terminus of 11beta-HSD2 that are essential for protein stability. The cluster includes Tyr(338), which is mutated in the index patient, and Arg(335) and Arg(337), previously reported to be mutated in hypertensive patients. It was found that wild-type 11beta-HSD2 is a relatively stable enzyme with a half-life of 21 h, whereas that of Tyr(338)His and Arg(337)His was 3 and 4 h, respectively. Enzymatic activity of Tyr(338)His was partially retained at 26 degrees C or in the presence of the chemical chaperones glycerol and dexamethasone, indicating thermodynamic instability and misfolding. The results provide evidence that the degradation of both misfolded mutant Tyr(338)His and wild-type 11beta-HSD2 occurs through the proteasome pathway. Therefore, impaired 11beta-HSD2 protein stability rather than reduced gene expression or loss of catalytic activity seems to be responsible for the development of hypertension in some individuals with AME.
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PMID:Impaired protein stability of 11beta-hydroxysteroid dehydrogenase type 2: a novel mechanism of apparent mineralocorticoid excess. 1731 22

Only few enzymes from haloalkaliphiles are biochemically characterized for their kinetic behaviour and stability. In view of this realization, an alkaline protease from Bacillus sp. AH-6, displaying salt-dependent resistance against chemical denaturation by Urea and Guanidium hydrochloride was investigated for denaturation and in vitro protein folding. The crude enzyme was highly resistant against urea (8 M) denaturation up to 72 h; however, on purification, it turned sensitive and got denatured within 2 h. Interestingly, the purified enzyme regained the resistance in the presence of NaCl. Effective refolding of the purified enzyme was achieved with glycerol; however, other approaches such as lower protein concentrations, rapid dilution and slow removal of the denaturant did not further add to refolding. The results are important from the viewpoint that only few enzymes from haloalkaliphilic bacteria are characterized. Since the resistance against chemical denaturation is a rare phenomenon, the findings would enrich the knowledge on protein stability and denaturation. Besides, such biocatalysts would definitely have novel applications under harsh chemical environments.
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PMID:Salt dependent resistance against chemical denaturation of alkaline protease from a newly isolated haloalkaliphilic Bacillus sp. 1821 18

Aging is associated with accumulation of toxic intracellular and extracellular protein aggregates. Cells manage "aged" proteins by mobilizing their molecular chaperones or heat shock proteins that are also considered as determinants of lifespan in diverse species. In this study, we tested whether an exogenous addition of the non-toxic chemical chaperone 'glycerol' could elicit stress and geronto-protective activities. We found that glycerol enhanced chaperoning of heat-denatured proteins. In addition to stimulating proteasome activity, glycerol led to an increased expression of the stress chaperone 'mortalin' and decreased p53 function in human cells. Glycerol-fed worms exhibited thermo-tolerance and lower level of age-associated auto-fluorescence. Through the combined stimulation of the proteasome and chaperoning activities of mortalin, in particular, glycerol treatment resulted in increased survival and fitness against oxidative- and heat-stress. These results may have significant implications in the use of glycerol as a candidate geronto-modulator in development of practical interventions for "healthy aging".
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PMID:Glycerol stimulates innate chaperoning, proteasomal and stress-resistance functions: implications for geronto-manipulation. 1834 10


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