Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The REG homologs, alpha, beta and gamma, activate mammalian proteasomes in distinct ways. REGalpha and REGbeta activate the trypsin-like, chymotrypsin-like and peptidylglutamyl-preferring active sites, whereas REGgamma only activates the proteasome's trypsin-like subunit. The three REG homologs differ in carboxyl-terminal sequences that are located next to activation loops on their proteasome binding surface. To assess the importance of these carboxyl-terminal sequences in the activation of specific proteasome beta catalytic subunits, we characterized chimeras in which 8 or 12 residues were exchanged among the three proteins. Like the wild-type molecule, REGalpha chimeras activated all three proteasome catalytic subunits regardless of the carboxyl-terminal sequence. However, REGalpha-beta chimeras activated the proteasome at lower concentrations than wild-type REGalpha and higher levels of REGalpha-gamma chimeras were needed for maximal activation because exchanged carboxyl-terminal sequences can stabilize (REGalpha-beta) or destabilize (REGalpha-gamma) the REGalpha heptamer. REGgamma chimeras were equivalent to REGgamma in their activation properties, but they bound the proteasome less tightly than the wild-type molecule. REGbeta chimeras also bound the proteasome more weakly than wild-type REGbeta and were virtually unable to activate it. Our findings demonstrate that the carboxyl-terminal sequences of REG subunits can affect heptamer stability and proteasome affinity, but they do not determine which proteasome beta subunits become activated.
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PMID:The proteasome activator 11 S REG or PA28: chimeras implicate carboxyl-terminal sequences in oligomerization and proteasome binding but not in the activation of specific proteasome catalytic subunits. 1083 74

The activation kinetics of constitutive and IFNgamma-stimulated 20S proteasomes obtained with homomeric (recPA28alpha, recPA28beta) and heteromeric (recPA28alphabeta) forms of recombinant 11S regulator PA28 was analysed by means of kinetic modelling. The activation curves obtained with increasing concentrations of the individual PA28 subunits (RecP28alpha/RecP28beta/RecP28alpha + RecP28beta) exhibit biphasic characteristics which can be attributed to a low-level activation by PA28 monomers and full proteasome activation by assembled activator complexes. The dissociation constants do not reveal significant differences between the constitutive and the immunoproteasome. Intriguingly, the affinity of the proteasome towards the recPA28alphabeta complex is about two orders of magnitude higher than towards the homomeric PA28alpha and PA28beta complexes. Striking similarities can been revealed in the way how PA28 mediates the kinetics of latent proteasomes with respect to three different fluorogenic peptides probing the chymotrypsin-like, trypsin-like and peptidylglutamyl-peptide hydrolyzing like activity: (a) positive cooperativity disappears as indicated by a lack of sigmoid initial parts of the kinetic curves, (b) substrate affinity is increased, whereby (c), the maximal activity remains virtually constant. As these kinetic features are independent of the peptide substrates, we conclude that PA28 exerts its activating influence on the proteasome by enhancing the uptake (and release) of shorter peptides.
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PMID:Kinetic evidences for facilitation of peptide channelling by the proteasome activator PA28. 1101 76

We investigated the expression of interferon gamma (IFN-gamma)-regulated subunits and the enzymatic activity of proteasomes purified from tumor-derived and normal B lymphocytes representing different stages of B-cell activation/differentiation. The catalytic beta subunits (Lmp2 and Lmp7) and the regulatory subunits (PA28alpha and PA28beta) were expressed at equally high levels in Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs), freshly isolated B-chronic lymphocytic leukemia (B-CLL) cells and normal CD23(-) B lymphocytes. Lmp2 and Lmp7 were selectively down-regulated in germinal center cell-derived Burkitt's lymphoma (BL) and Hodgkin's lymphoma (HD) cell lines. There was a direct correlation between the expression of Lmp2/7 and the chymotrypsin and trypsin-like activities in proteasomes purified from LCLs, BLs and CLL cells, whereas 5 HD cell lines expressing B or T-cell markers exhibited a variable pattern of subunit expression and enzymatic activity. Poor hydrolysis of the fluorogenic substrates by proteasomes from BL cells correlated with a distinct pattern of cleavage of a reference 50mer peptide, production of different sets of degradation products and significantly reduced recovery of a known cytotoxic T-lymphocyte (CTL) target epitope. The enzymatic activity of proteasomes from normal CD23(-) "resting" B lymphocytes resembled that of BL cells in spite of high Lmp2/7 expression. This pattern was not reversed by treatment with the B-cell mitogen, lipopolysaccharide (LPS). The results suggest that different stages of B-cell activation/differentiation are associated with distinct profiles of IFN-gamma-regulated subunit composition and enzymatic activity of the proteasome. This may have important implications for the analysis and manipulation of tumor-specific immune responses.
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PMID:Variations in proteasome subunit composition and enzymatic activity in B-lymphoma lines and normal B cells. 1109 9

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

Following cellular stress or tissue injury, the proteasome plays a critical role in protein degradation and signal transduction. The present study examined the beta-subunit expression of constitutive proteasomes (beta1, beta2, and beta5), immunoproteasomes (beta1i, beta2i, and beta5i) and the 11S proteasome activator, PA28alpha, in the rat CNS after traumatic brain injury (TBI). Concomitant measures assessed changes in proteasome activities. Quantitative real time PCR results indicated that beta1 and beta2 mRNA levels were not changed, while beta5 mRNA levels were significantly decreased in injured CNS following TBI. However, beta1i, beta2i, beta5i, and PA28alpha mRNA levels were significantly increased in the injured CNS. Western blotting studies found that beta1, beta2, beta5, beta2i, and beta5i subunit protein levels remained unchanged in the injured CNS, but beta1i and PA28alpha protein levels were significantly elevated in ipsilateral cerebral cortex and hippocampus. Proteasome activity assays found that peptidyl glutamyl peptide hydrolase-like and chymotrypsin-like activity were significantly reduced in the CNS after TBI, and that trypsin-like proteasome activity was increased in the injured cerebral cortex. Our results demonstrated that both proteasome composition and function in the CNS were affected by trauma. Treatments that preserve proteasome function following CNS injury may be beneficial as an approach to cerebral neuroprotection.
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PMID:Alterations of cerebral cortex and hippocampal proteasome subunit expression and function in a traumatic brain injury rat model. 1794 70