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)

The proteasome is a multisubunit enzyme complex that plays a central role in the regulation of proteins that control cell-cycle progression and apoptosis, and has therefore become an important target for anticancer therapy. Before a protein is degraded, it is first flagged for destruction by the ubiquitin conjugation system, which ultimately results in the attachment of a polyubiquitin chain on the target protein. The proteasome's 19S regulatory cap binds the polyubiquitin chain, denatures the protein, and feeds the protein into the proteasome's proteolytic core. The proteolytic core is composed of 2 inner beta rings and 2 outer alpha rings. The 2 beta rings each contain 3 proteolytic sites named for their trypsin-like, post-glutamyl peptide hydrolase-like (PGPH) (i.e., caspase-like), or chymotrypsin-like activity. Inhibition of the proteasome results in cell-cycle arrest and apoptosis. In in vitro and in vivo animal studies, inhibition of the proteasome via bortezomib (VELCADE; formerly, PS-341, LDP-341, and MLN341) had antitumor activity against numerous tumor types either alone or in combination with conventional chemotherapeutic agents; these results provided the rationale for a broad clinical trial program. Bortezomib is currently in phase III trials for myeloma and is in early clinical development for numerous other tumor types.
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PMID:The proteasome: structure, function, and role in the cell. 1273 38

There is a growing need for techniques that can identify and characterize protein modifications on a large or global scale. We report here a proteomics approach to enrich, recover, and identify ubiquitin conjugates from Saccharomyces cerevisiae lysate. Ubiquitin conjugates from a strain expressing 6xHis-tagged ubiquitin were isolated, proteolyzed with trypsin and analyzed by multidimensional liquid chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for amino acid sequence determination. We identified 1,075 proteins from the sample. In addition, we detected 110 precise ubiquitination sites present in 72 ubiquitin-protein conjugates. Finally, ubiquitin itself was found to be modified at seven lysine residues providing evidence for unexpected diversity in polyubiquitin chain topology in vivo. The methodology described here provides a general tool for the large-scale analysis and characterization of protein ubiquitination.
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PMID:A proteomics approach to understanding protein ubiquitination. 1287 31

To construct a high information content assay for examination of the function of the cellular ubiquitin system, we added his-tagged ubiquitin, ATP, and an ATP-regenerating system to endogenous human cellular ubiquitin system enzymes, and labeled cellular proteins with hexa-histidine tagged ubiquitin in vitro. Labeling depended on ATP, the ATP recycling system, the proteasome inhibitor MG132, and the ubiquitin protease inhibitor ubiquitin aldehyde, and was inhibited by iodoacetamide. Quadruplicate affinity extracted proteins were digested with trypsin, and the peptides were analyzed by 2D capillary LC-MS/MS, SEQUEST, MEDUSA, and support vector machine calculations. Identified proteins included 22 proteasome subunits or associated proteins, 18 E1, E2, or E3 ubiquitin system enzymes or related proteins, 4 ubiquitin domain proteins and 36 proteins in functional clusters associated with redox processes, endocytosis/vesicle trafficking, the cytoskeleton, DNA damage/repair, calcium binding, and mRNA splicing. This suggests a link between the ubiquitin system and these cellular processes. This map of cellular ubiquitin-associated proteins may be useful for further studies of ubiquitin system function.
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PMID:Multiple functional categories of proteins identified in an in vitro cellular ubiquitin affinity extract using shotgun peptide sequencing. 1293 29

2'-5'-Oligoadenylate synthetase (OAS), an interferon (IFN) induced enzyme, synthesizes 2'-5'-oligoadenylate (2-5A) from ATP when activated by dsRNA. Chicken OAS (ChOAS) has a ubiquitin-like (UbL) domain of two consecutive sequences (UbL1 and UbL2) at its carboxyl-terminus. The OAS gene has at least two alleles, OAS*A and OAS*B. OAS-A is the wild-type (wt) and OAS-B is a mutant deleted of a highly hydrophobic region of UbL1. To study the function of the UbL domain, enzymatic and physiologic properties were compared between OAS-A and OAS-B. OAS-B was more susceptible to trypsin than OAS-A and was converted very quickly into p38, deleting a greater part of the UbL domain. The p38 has the enzymatic activity to synthesize 2-5A. Thermal inactivation of OAS-B occurred at a lower temperature than that of OAS-A and p38, with loss of the ability to bind dsRNA. In contrast to OAS-A, the content of OAS-B in erythrocytes decreased during growth to a very low level. However, red blood cells (RBC) from anemic B/B chickens synthesized OAS-B at a high level comparable to A/A, although OAS-B levels decreased sharply again during maturation to erythrocytes. Thus, OAS-B carrying the mutated UbL domain is unstable compared with OAS-A in vitro and in vivo, and the wt UbL domain may contribute to the stability of the protein structure of ChOAS.
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PMID:Function of ubiquitin-like domain of chicken 2'-5'-oligoadenylate synthetase in conformational stability. 1465 81

Huntington's disease (HD) inclusions are stained with anti-ubiquitin and anti-proteasome antibodies. This, together with proteasome activity studies on transfected cells, suggest that an impairment of the ubiquitin-proteasome system (UPS) may be key in HD pathogenesis. To test whether proteasome activity is impaired in vivo, we performed enzymatic assays for the three peptidase activities of the proteasome in brain extracts from the HD94 conditional mouse model of HD. We found no inhibition of any of the activities, suggesting that if UPS impairment happens in vivo, it is not at the level of the proteasome catalytic core. Intriguingly, the chymotrypsin- and trypsin-like activities increased selectively in the affected and aggregate-containing regions: cortex and striatum. Western blot analysis revealed no difference in total proteasome content whereas an increase in the interferon-inducible subunits of the immunoproteasome, LMP2 and LMP7, was observed. These subunits confer to the proteasome catalytic properties that are optimal for MHC-I peptide presentation. Immunohistochemistry in control mouse brain revealed LMP2 and LMP7 mainly in neurons. Accordingly, their increase in HD94 mice predominantly took place in neurons, and 5% of the ubiquitin-positive cortical aggregates were also LMP2-positive. Ultrastructural analysis of neurons with high level of immunoproteasome subunits revealed signs of neurodegeneration like nuclear indentation or fragmentation and dark cell appearance. The neuronal induction of LMP2 and LMP7 and the associated signs of neurodegeneration were also found in HD postmortem brains. Our results indicate that LMP2 and LMP7 participate in normal neuronal physiology and suggest a role in HD neurodegeneration.
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PMID:Neuronal induction of the immunoproteasome in Huntington's disease. 1468 67

Green tea has been shown to lower plasma cholesterol, associated with up-regulation of the low-density lipoprotein receptor (LDLR) although the responsible molecular mechanism is unknown. Previously, we reported that ester bond-containing green tea polyphenols (GTPs), such as (-)-epigallocatechin-3-gallate [(-)-EGCG], potently inhibit the tumor cellular proteasome activity, which may contribute to the cancer-preventative effect of green tea. In the current study, we hypothesize that the proteasome is a heart disease-associated molecular target of GTPs. We have shown that ester bond-containing GTPs, including (-)-EGCG, potently inhibit the proteasomal activity in intact hepatocellular carcinoma HepG2 and cervical carcinoma HeLa cells, as evident by accumulation of ubiquitinated proteins and three natural proteasome targets (p27, IkappaB-alpha and Bax). (-)-EGCG selectively inhibits the chymotrypsin-like, but not trypsin-like, activity of the proteasome. Associated with proteasome inhibition by ester bond-containing GTPs, there was a significant, time- and concentration-dependent increase in levels of the cleaved, activated, but not the precursor, form of sterol regulatory element-binding protein 2 (SREBP-2), an essential factor for LDLR transcription. Subsequently, LDL receptor expression was increased dramatically in HepG2 and HeLa cells treated with (-)-EGCG. Our results suggest that ester bond-containing GTPs inhibit ubiquitin/proteasome-mediated degradation of the active SREBP-2, resulting in up-regulation of LDLR. This identified molecular mechanism may be related to the previously reported cholesterol-lowering and heart disease-preventative effects of green tea.
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PMID:Direct inhibition of the ubiquitin-proteasome pathway by ester bond-containing green tea polyphenols is associated with increased expression of sterol regulatory element-binding protein 2 and LDL receptor. 1515 50

The localization of ubiquitin (UB) signals in the acrosomes of rat spermiogenic cells was investigated by immunoelectron microscopy using two anti-UB antibodies: UB1, reacting with ubiquitinated proteins and free UB; and FK1, recognizing polyubiquitinated proteins but not monoubiquitinated proteins or free UB. Labeling of UB by UB1 (UB1 signal) was detected in the acrosomes at any stage of differentiation. In step 1 spermatids, UB1 signals were detected on the cytoplasmic surface and in the matrix of transport vesicles located between the trans-Golgi network and the acrosome. Weak signals were detected in acrosomal granules within acrosome vesicles that had not yet attached to the nucleus. In step 4-5 spermatids, the acrosome vesicles had enlarged and attached to the nucleus. Strong gold labeling was noted in a narrow space between the outer acrosomal membrane and the developing acrosomal granule, where a dense fibrous material was observed on routine electron microscopy, whereas the acrosomal granule was weakly stained by UB1 antibody. In step 6-8 spermatids, UB1 signals were detected in the fibrous material that expanded laterally to form a narrow electronless dense zone between the acrosomal granule and the outer acrosomal membrane. Labeling in the acrosomal granule increased. In step 9-11 spermatids, UB1 signals were confined to the narrow zone from the tip of the head to the periphery of the ventral fin. The matrix of the acrosome was weakly stained. In epididymal sperm, UB1 labeling in the acrosome decreased without any pretreatment, whereas staining was noted in a spot in the neck region and in the dorsal fin after trypsin digestion. On the other hand, the staining pattern with FK1 was quite different from that with UB1. The trans-Golgi network was weakly stained but the cis-Golgi network was strongly stained. The dense fibrous material just beneath the outer membrane was never stained with FK1. The results suggest that UB on the surface of transport vesicles is involved in anterograde transport from the Golgi apparatus to the acrosome. The physiological role of UB in acrosomes is not clear. Two candidates for monoubiquitinated proteins in the acrosome, which have a UB-interacting motif, were found by cyber screening.
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PMID:Ubiquitin signals in the developing acrosome during spermatogenesis of rat testis: an immunoelectron microscopic study. 1550 34

Prolonged mechanical ventilation (MV) results in diaphragmatic atrophy due, in part, to an increase in proteolysis. These experiments tested the hypothesis that MV-induced diaphragmatic proteolysis is accompanied by increased expression of key components of the ubiquitin-proteasome pathway (UPP). To test this postulate, we investigated the effect of prolonged MV on UPP components and determined the trypsin-like and peptidylglutamyl peptide hydrolyzing activities of the 20S proteasome. Adult Sprague-Dawley rats were assigned to either control or 12-h MV groups (n=7/group). MV animals were anesthetized, tracheostomized, and ventilated with room air for 12 h. Animals in the control group were acutely anesthetized but not exposed to MV. Compared with controls, MV animals demonstrated increased diaphragmatic mRNA levels of two ubiquitin ligases, muscle atrophy F-box (+8.3-fold) and muscle ring finger 1 (+19.0-fold). However, MV did not alter mRNA levels of 14-kDa ubiquitin-conjugating enzyme, polyubiquitin, proteasome-activating complex PA28, or 20S alpha-subunit 7. Protein levels of 14-kDa ubiquitin-conjugating enzyme and proteasome-activating complex PA28 were not altered following MV, but 20S alpha-subunit 7 levels declined (-17.7%). MV increased diaphragmatic trypsin-like activity (+31%) but did not alter peptidylglutamyl peptide hydrolyzing activity. Finally, compared with controls, MV increased ubiquitin-protein conjugates in both the myofibrillar (+24.9%) and cytosolic (+54.7%) fractions of the diaphragm. These results are consistent with the hypothesis that prolonged MV increases diaphragmatic levels of key components within the UPP and that increases in 20S proteasome activity contribute to MV-induced diaphragmatic proteolysis and atrophy.
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PMID:Mechanical ventilation induces alterations of the ubiquitin-proteasome pathway in the diaphragm. 1555 10

Mutations in familial Parkinson's disease (PD) have been associated with the failure of protein degradation through the ubiquitin-proteasome system (UPS). Impairment of proteasome function has also been suggested to play a role in the pathogenesis of sporadic PD. We examined the proteasome activity in PC12 cells treated with 6-hydroxydopamine (6-OHDA), the dopamine synthetic derivate used in models of PD. We found that 6-OHDA treatment increased protein oxidation, as indicated by carbonyl group accumulation, and increased caspase-3 activity. In addition, there was an increase in trypsin-, chymotrypsin-, and postacidic-like proteasome activities in cells treated with 10-100 microM 6-OHDA, whereas higher doses caused a marked decline. 6-OHDA exposure also increased mRNA expression of the 19S regulatory subunit in a dose-dependent manner, whereas the expression of 20S- and 11S-subunit mRNAs did not change. Administration of the antioxidant N-acetylcysteine to 6-OHDA-treated cells prevented the alteration in proteasome functions. Moreover, reduction in cell viability owing to administration of proteasome inhibitor MG132 or lactacystin was partially prevented by the endogenous antioxidant-reduced glutathione. In conclusion, our data indicate that mild oxidative stress elevates proteasome activity in response to increase in protein damage. Severe oxidative insult might cause UPS failure, which leads to protein aggregation and cell death. Moreover, in the case of UPS inhibition or failure, the blockade of physiological reactive oxygen species production during normal aerobic metabolism is enough to ameliorate cell viability. Control of protein clearance by potent, brain-penetrating antioxidants might act to slow down the progression of PD.
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PMID:Oxidative stress, induced by 6-hydroxydopamine, reduces proteasome activities in PC12 cells: implications for the pathogenesis of Parkinson's disease. 1565 61

In this work, synthetic peptides were used to determine the fragmentation behavior of ubiquitinated peptides and to find ions diagnostic for peptide ubiquitination. The ubiquitin-calmodulin peptide1 was chosen as the model peptide for naturally occurring ubiquitinated proteins cleaved with endoproteinase gluC. In addition, the fragmentation behavior of model ubiquitinated peptides produced by tryptic digestion was also of great interest since the standard protocols for proteomics-based protein identification use trypsin as the protease. Attachment of ubiquitin to a target protein results in a branched structure, but only ions from the ubiquitin side chain (and the lysine to which it is attached) can be used as diagnostic ions, since fragment ions that contain other amino acids from the parent protein will vary in mass. Characteristic b-type fragment ions from the gluC cleavage of the ubiquitin side chain (designated as b ions) were found which involve only the ubiquitin tail (b2, b3, b4, b5 and b6 ions at m/z 189.06, 302.12, 439.18, 552.30 and 651.30, respectively). Maximum production of these ions occurred at a collision energy of 45 eV in a Q-TOF instrument. Although a non-ubiquitinated peptide may produce isobaric fragment ions, it is unlikely that it can produce these ions in combination. With liquid chromatography/tandem mass spectrometry (LC/MS/MS) experiments, ubiquitinated peptides can readily be determined by surveying the reconstructed or extracted ion chromatograms of the diagnostic fragment ions for common peaks. Characteristic ions resulting from tryptic cleavage of the side chain were found in cleavage products with a missed cleavage, resulting in a LRGG- tag instead of a GG- tag. For the LRGG-tagged peptide, diagnostic MS/MS fragment ions (at m/z 270.17 and 384.21) from the ubiquitin tail (b2 and b4, respectively) were found, along with an internal fragment ion (LRGGK-28) at m/z 484.30. These ions should prove useful in precursor-ion scanning experiments for identifying peptides modified by attachment of ubiquitin, and for locating the site of ubiquitin attachment.
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PMID:Electrospray ionization tandem mass spectrometry of model peptides reveals diagnostic fragment ions for protein ubiquitination. 1565


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