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:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Huntington disease (HD) inclusions are stained with anti-ubiquitin and anti-proteasome antibodies. This, together with proteasome activity studies on transfected cell, suggested that alterations in the ubiquitin-proteasome system (UPS) might contribute to HD pathogenesis. In previous work we reported that in a conditional mouse model of Huntington's disease (HD94 mice), the chymiotrypsin- and trypsin-"like" activities of the proteasome are increased selectivity in the affected and aggregate-containing brain regions: striatum, and cortex. Moreover, in these areas a neuronal increase in the interferon-inducible subunits of the immunoproteasome LMP2 and LPM7 was observed. In order to test if the expression of N-terminal mutant huntingtin (htt) by itself is sufficient to induce the change in proteasome catalytic activities as well as in LMP2 subunit expression, we performed activities of the proteasome and western blot experiments in striatal cultured neurons from HD94 mice free of glial contamination. We found no changes in any of the activities in these cells. Furthermore, western blot analysis performed with specific antibody against LMP2 subunits, revealed no difference in levels of this subunit in striatal neurons from HD94 compared to control cultures were treated with interferon-gamma (IFN-gamma) during 72 hours, a clear increase in LMP2 levels was observed in control neuronal cultures. Interestingly, this increase was much more pronounced (95% higher) in HD94 striatal cultures. These results indicate that although expression of mutant htt is not sufficient to induce the changes in proteasome catalytic core observed in HD, it synergizes the changes induced by IFN-gamma. Furthermore, immunocytochemical studies revealed that HD94 striatal neuron expressing high levels of LMP2 subunit showed a pre-apoptotic appearance. These results suggest that the correlation between neuronal induction of the immunoproteasome and neurodegeneration found in HD brains is secondary to inflammatory processes.
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PMID:Enhanced induction of the immunoproteasome by interferon gamma in neurons expressing mutant Huntingtin. 1565 1

A proteomics method has been developed to purify and identify the specific proteins modified by ubiquitin (Ub) from human cells. In purified samples, Ub and 21 other proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) spectra using SEQUEST. These proteins included several of the expected carriers of Ub including Ub-conjugating enzymes and histone proteins. To perform these experiments, a cell line coexpressing epitope tagged His(6X)-Ub and green fluorescent protein (GFP) was generated by stably transfecting HEK293 cells. Ubiquitinated proteins were purified using nickel-affinity chromatography and digested in solution with trypsin. Complex mixtures of peptides were separated by reversed phase chromatography and analyzed by nano LC-MS/MS using the LCQ quadrupole ion-trap mass spectrometer. Proteins identified from His(6X)-Ub-GFP transfected cells were compared to a list of proteins from HEK293 cells, which associate with nickel-nitrilotriacetic acid (Ni-NTA)-agarose in the absence of His-tagged Ub. In a proof of principle experiment, His(6X)-Ub-GFP transfected cells were treated with As (III) (10 microM, 24 h) in an attempt to identify substrates increasingly modified by Ub. In this experiment, proliferating cell nuclear antigen, a DNA repair protein and known ubiquitin substrate, was confidently identified. This proteomics method, developed for the analysis of ubiquitinated proteins, is a step towards large-scale characterization of Ub-protein conjugates in numerous physiological and pathological states.
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PMID:Proteomic identification of ubiquitinated proteins from human cells expressing His-tagged ubiquitin. 1585 47

Age-related increase in protein oxidation in brain coupled to an impairment of proteasomal activity may underline neuronal loss but differences in susceptibility between species and brain regions remain unexplained. We now investigate differences in proteasomal activity, measured as chymotrypsin-, trypsin- and peptidylglutamyl-like hydrolysing activities between brain regions in rats, mice and common marmosets. In aged rats and mice, proteasomal activity was decreased in the cortex, striatum, cerebellum, globus pallidus and substantia nigra overall when compared to young animals. However, in the aged brain only chymotrypsin-like activity was decreased in the cortex and the globus pallidus while only trypsin-like activity was reduced in the cerebellum. In contrast, in the striatum, both chymotrypsin-like and trypsin-like activities were reduced and in the substantia nigra, all the three catalytic activities of proteasome were significantly impaired. Chymotrypsin-like and trypsin-like activities were significantly higher in all the brain regions of marmosets compared to those of mice and rats. Peptidylglutamyl-like activity was only significantly higher in the cerebellum and striatum of marmoset compared to rodents. The data suggest that there is higher proteasome activity in common marmoset brain compared to rat and mouse and that the basal ganglia are more prone to an age-related decrease in proteasomal activity. This may explain the involvement of altered ubiquitin-proteasome system activity in Parkinson's disease and the relationship to ageing.
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PMID:Proteasomal activity in brain differs between species and brain regions and changes with age. 1588 31

We studied the role of the ubiquitin-proteasome system in rat skeletal muscle during sepsis and subsequent recovery. Sepsis was induced with intraperitoneal zymosan injections. This model allows one to study a sustained and reversible catabolic phase and mimics the events that prevail in septic and subsequently recovering patients. In addition, the role of the ubiquitin-proteasome system during muscle recovery is poorly documented. There was a trend for increased ubiquitin-conjugate formation in the muscle wasting phase, which was abolished during the recovery phase. The trypsin- and chymotrypsin-like peptidase activities of the 20S proteasome peaked at day 6 following zymosan injection (i.e. when both muscle mass and muscle fiber cross-sectional area were reduced the most), but remained elevated when muscle mass and muscle fiber cross-sectional area were recovering (11 days). This clearly suggests a role for the ubiquitin-proteasome pathway in the muscle remodeling and/or recovery process. Protein levels of 19S complex and 20S proteasome subunits did not increase throughout the study, pointing to alternative mechanisms regulating proteasome activities. Overall these data support a role for ubiquitin-proteasome dependent proteolysis in the zymosan septic model, in both the catabolic and muscle recovery phases.
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PMID:Ubiquitin-proteasome-dependent proteolytic activity remains elevated after zymosan-induced sepsis in rats while muscle mass recovers. 1595 21

TGF-beta1 is a well-known immunosuppressive cytokine that inhibits inducible nitric oxide synthase (iNOS) gene expression in various cells including macrophages. In this study, we investigated the suppressive mechanisms of TGF-beta1 on IFN-gamma-induced iNOS gene expression using the murine macrophage-like cell line RAW 264.7. TGF-beta1 decreased iNOS protein amount through enhanced degradation, although TGF-beta1 did not affect IFN-gamma-induced iNOS mRNA level or stability. In addition, the enhancement of iNOS protein degradation by TGF-beta1 treatment was almost completely blocked by MG132, a proteasome inhibitor. Furthermore, TGF-beta1 enhanced the trypsin-like activity of proteasomes in the presence of IFN-gamma, although did not enhance the peptidylglutamyl-peptide hydrolyzing and chymotrypsin-like activities of proteasomes. The level of ubiquitinated iNOS protein was not significantly altered by IFN-gamma or IFN-gamma plus TGF-beta1 treatment. Because MG132 inhibited iNOS protein degradation and IFN-gamma plus TGF-beta1 treatment increased the trypsin-like activity of proteasomes, we hypothesized that TGF-beta1 might enhance iNOS protein degradation via the ubiquitin-proteasome pathway in the presence of IFN-gamma. We propose that these mechanisms of TGF-beta1 in the posttranslational regulation of iNOS gene expression may contribute to suppression of excess nitric oxide during inflammatory processes.
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PMID:TGF-beta1 enhances degradation of IFN-gamma-induced iNOS protein via proteasomes in RAW 264.7 cells. 1596 25

A reduction in proteasome activity and accumulation of oxidized proteins may play a role in alcoholic liver disease. The current study assessed proteasome peptidase activities and oxidative modifications of proteasomes during oxidative stress generated by CYP2E1. The model of toxicity by arachidonic acid (AA) and iron [ferric-nitrilotriacetate (Fe-NTA)] in HepG2 cells overexpressing CYP2E1 (E47 cells) and control C34 cells was used. AA/Fe-NTA treatment decreased trypsin-like (T-L) activity of the proteasome in E47 cells but not in C34 cells. This inhibition was abolished by antioxidants. Chymotrypsin-like activity of the proteasome was increased in E47 cells, and activity was not altered by AA/Fe-NTA treatment. There were no changes in content of subunits of 20S proteasomes or 19S regulator ATPase subunits S4 and p42 by AA/Fe-NTA treatment. An increased content of the PA28alpha subunit of the 11S regulator of proteasomes was detected in E47 cells. In proteasome pellets, the decline of T-L activity was accompanied by increased content of carbonyl adducts, suggesting oxidative modification of proteasomes. Higher levels of ubiquitinated, 3-nitrotyrosine- and 4-hydroxynonenal-modified proteins and lower levels of free ubiquitin were detected in untreated E47 cells in comparison with C34 cells. Accumulation of protein cross-linked, detergent-insoluble aggregates was increased with AA/Fe-NTA treatment in E47 cells. Thus, reactive oxygen species generated upon CYP2E1-dependent oxidative stress mediated a decline in T-L proteasome function, increased carbonyl adducts in proteasomes, and promoted protein aggregate formation; this may alter the balance among protein oxidation, ubiquitination, and degradation.
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PMID:The effect of CYP2E1-dependent oxidant stress on activity of proteasomes in HepG2 cells. 1600 58

The proteome of a Medicago truncatula cell suspension culture was analyzed using two-dimensional electrophoresis and nanoscale HPLC coupled to a tandem Q-TOF mass spectrometer (QSTAR Pulsar i) to yield an extensive protein reference map. Coomassie Brilliant Blue R-250 was used to visualize more than 1661 proteins, which were excised, subjected to in-gel trypsin digestion, and analyzed using nanoscale HPLC/MS/MS. The resulting spectral data were queried against a custom legume protein database using the MASCOT search engine. A total of 1367 of the 1661 proteins were identified with high rigor, yielding an identification success rate of 83% and 907 unique protein accession numbers. Functional annotation of the M. truncatula suspension cell proteins revealed a complete tricarboxylic acid cycle, a nearly complete glycolytic pathway, a significant portion of the ubiquitin pathway with the associated proteolytic and regulatory complexes, and many enzymes involved in secondary metabolism such as flavonoid/isoflavonoid, chalcone, and lignin biosynthesis. Proteins were also identified from most other functional classes including primary metabolism, energy production, disease/defense, protein destination/storage, protein synthesis, transcription, cell growth/division, and signal transduction. This work represents the most extensive proteomic description of M. truncatula suspension cells to date and provides a reference map for future comparative proteomic and functional genomic studies of the response of these cells to biotic and abiotic stress.
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PMID:A two-dimensional electrophoresis proteomic reference map and systematic identification of 1367 proteins from a cell suspension culture of the model legume Medicago truncatula. 1604 9

We investigated the proteome of the anterior pituitary gland (AP) in a species in which the genome has been sequenced. Subcellular fractions of APs from 2-month-old male mice were prepared for protein denaturation, treatment with trypsin and analyses utilizing micro liquid chromatography tandem mass spectrometry and the database search software SEQUEST. In the nuclear, non-nuclear 100,000 g and cytosolic fractions, we identified 49, 36 and 68 different proteins, respectively. A total of 115 distinct proteins were detected. We identified growth hormone, prolactin, pro-opiomelanocortin, the alpha-subunit for the glycoprotein hormones, and luteinizing hormone-beta. Groups of other identified proteins included hormone-processing, secretion granule-associated, non-hormonal endoplasmic reticulum-associated, calcium-binding, protein kinase C-associated, histones, non-histone chromosomal, other RNA-binding, heterogeneous nuclear ribonucleoproteins, splicing factors, helicases, lamins, ribosomal, microtubule-associated, microfilament-associated, adenosine triphosphate- and guanosine triphosphate-associated, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation, enzymes in glycolysis and the tricarboxylic and urea cycles and the pentose phosphate path, heat-shock, glutathione-associated, peroxidases, ubiquitin-associated, catabolic, protease inhibitors, other, and blood proteins. The 115 proteins reported in this study and the 145 proteins reported in a previous study on the AP of the adult male Golden Syrian hamster are compared and form a foundation for defining the proteome in normal adult male AP.
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PMID:Mouse anterior pituitary gland: analysis by ion trap mass spectrometry. 1610 33

This study describes a new protein digestion protocol in which a variety of detergents can be used to solubilize membrane proteins and facilitate trypsin digestion with higher efficiency. In this protocol, proteins are dissolved in solutions containing various detergents and directly incorporated into a polyacrylamide gel matrix without electrophoresis. Detergents are subsequently eliminated from the gel matrix while proteins are still immobilized in the gel matrix. After in-gel digestion of proteins, LC-MS/MS is used to analyze the extracted peptides for protein identification. The uniqueness of the protocol is that it allows usage of a variety of detergents in the starting solution without interfering with LC-MS/MS analysis. We hereby demonstrate that different detergents, including ionic SDS, non-ionic Triton X-100 and n-octyl beta-d-glucopyranoside, and zwitterionic CHAPS, can be used to achieve maximum solubilization of membrane proteins with minimal interference with LC-MS/MS analysis. Enhanced digestions, i.e. improved number and intensity of detected peptides, are also demonstrated for digestion-resistant proteins such as myoglobin, ubiquitin, and bacteriorhodopsin. An additional advantage of the Tube-Gel digestion protocol is that, even without electrophoresis separation, it allows high throughput analysis of complex protein mixtures when coupled with LC-MS/MS. The protocol was used to analyze a complex membrane protein mixture prepared from prostate cancer cells. The protocol involves only a single digestion and 2.5 h of LC-MS/MS analysis and identified 178 membrane proteins. In comparison, the same membrane fraction was resolved by SDS-PAGE, and 20 gel slices were excised and individually digested and analyzed by LC-MS/MS. The more elaborate effort demanded more than 50 h of LC-MS/MS analysis and identified 268 proteins. The new Tube-Gel digestion protocol is an alternative method for high throughput analysis of membrane proteins.
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PMID:Tube-gel digestion: a novel proteomic approach for high throughput analysis of membrane proteins. 1615 Aug 70

Heat shock proteins (Hsps) with chaperoning function work together with the ubiquitin-proteasome pathway to prevent the accumulation of misfolded, potentially toxic proteins, as well as to control catabolism of the bulk of cytoplasmic, cellular protein. There is evidence for the involvement of both systems in neurodegenerative disease, and a therapeutic target is the heat shock transcription factor, Hsf1, which mediates upregulation of Hsps in response to cellular stress. The mechanisms regulating expression of proteasomal proteins in mammalian cells are less well defined. To assess any direct effect of Hsf1 on expression of proteasomal subunits and activity in mammalian cells, a plasmid encoding a constitutively active form of Hsf1 (Hsf1act) was expressed in mouse embryonic fibroblasts lacking Hsf1 and in cultured human myoblasts. Plasmid encoding an inactivatible form of Hsf1 (Hsf1inact) served as control. In cultures transfected with plasmid hsf1act, robust expression of the major stress-inducible Hsp, Hsp70, occurred but not in cultures transfected with hsf1inact. No significant changes in the level of expression of representative proteasomal proteins (structural [20Salpha], a nonpeptidase beta subunit [20Sbeta3], or 2 regulatory subunits [19S subunit 6b, 11 Salpha]) or in chymotrypsin-, trypsin-, and caspaselike activities of the proteasome were measured. Thus, stress-induced or pharmacological activation of Hsf1 in mammalian cells would upregulate Hsps but not directly affect expression or activity of proteasomes.
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PMID:Proteasome activity or expression is not altered by activation of the heat shock transcription factor Hsf1 in cultured fibroblasts or myoblasts. 1618 68


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