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)

Membrane cofactor protein (MCP; CD46), a widely distributed regulatory protein of the complement system, was analyzed for expression in polarized epithelial cells. Both a human and a simian (Vero C1008) cell line were found to contain endogenous MCP mainly on the basolateral surface. Transfected Madin-Darby canine kidney cells stably expressing human MCP delivered this protein also predominantly to the basolateral surface. A deletion mutant lacking the cytoplasmic tail was transported in a nonpolarized fashion, indicating that the targeting signal for the basolateral transport is located in the cytoplasmic domain. A characteristic feature of MCP is the presence of various isoforms that contain either of two different cytoplasmic tails as a consequence of alternative splicing. Two isoforms differing only in the cytoplasmic tail (tail 1 or 2) were analyzed for polarized expression in Madin-Darby canine kidney cells. Surface biotinylation, as well as confocal immunofluorescence microscopy, indicated that both proteins were transported to the basolateral surface. Because no sequence similarity has been observed, the two tails contain different basolateral targeting signals. A deletion mutant lacking the only tyrosine residue in tail 1 retained the polarized expression indicating that, in contrast to most basolateral sorting signals, the transport signal of the tail 1 isoform is not dependent on tyrosine. The maintenance of a targeting motif in two distinct cytoplasmic tails suggests that the basolateral expression of MCP in polarized epithelial cells is of physiological importance.
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PMID:Two different cytoplasmic tails direct isoforms of the membrane cofactor protein (CD46) to the basolateral surface of Madin-Darby canine kidney cells. 870 45

Succinyl-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide (Suc-LLVY-MCA) hydrolyzing activities of the 20S and 26S proteasomes in the gerbil cortex following transient forebrain ischemia were examined. Using extraction solutions without ATP, only 20S proteasome activity was noted after separation with glycerol gradient centrifugation. When these extracts were incubated with ATP and an ATP-regenerating system prior to glycerol gradient separation, both 20S and 26S proteasome activities were detected. Following 10 min of ischemia, the activity of the 26S proteasomes decreased, whereas the 20S proteasome activity increased after 30 min of reperfusion. These changes returned to the control level after 1 h. The active 26S proteasomes were formed with ATP-dependent association with the 20S proteasomes and several subunits and the 26S proteasomes degraded ubiquitin-protein conjugates. These results indicate that proteasome activity might not be irreversibly impaired after transient ischemia. However, transient inhibition of ATP-dependent conversion of 20S to 26S proteasomes in vitro must be one of the causes of the accumulation of the ubiquitin-protein conjugates in the early reperfusion period.
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PMID:Changes in proteasome activity following transient ischemia. 871 10

PA28, also referred to as 11S regulator, is a potent activator of the peptidase activities of the proteasome (multicatalytic proteinase complex). Although the role(s) of PA28-20S proteasome complexes in cellular proteolytic processes remain to be defined, these particles have been implicated in antigen processing of major histocompatibility complex (MHC) class I molecules. Our results demonstrate that PA28 is phosphorylated as evidenced by 32P incorporation into a single PA28 species in rabbit reticulocytes. In reticulocytes as well as human erythrocytes, PA28 is normally found in a phosphorylated state as detected by phosphoserine antibody. In human erythrocytes, this antibody recognizes three polypeptides which are also detected by antibody to PA28 on Western blot analysis. Dephosphorylation with alkaline phosphatase treatment completely abolishes the ability of PA28 to activate hydrolysis of Suc-Leu-Leu-Val-Tyr by proteasomes. After exposure to phosphatase, the three polypeptides are no longer recognized by phosphoserine antibody, although binding to PA28 antibody is unaffected. These results suggest that phosphorylation may function in transduction of cytokine and growth factor signals that, in turn, modulate antigen presentation and other processes which involve PA28-20S proteasome complexes.
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PMID:Phosphorylation of the proteasome activator PA28 is required for proteasome activation. 878 Jul 2

Recent work suggests that the proteolytic degradation of the nuclear lamins is a common event in apoptosis, although the nature of the proteases involved is still not clear. Our previous work showed that the degradation of lamin B1 in glucocorticoid-treated thymocytes occurs via a Ca2+-sensitive mechanism and that exogenous Ca2+ promotes lamin degradation in isolated thymocyte nuclei from untreated cells. Here we demonstrate that peptide-based inhibitors of the interleukin 1beta-converting enzyme family of cysteine proteases (Tyr-Val-Ala-Asp fluoromethyl ketone) and of the nuclear scaffold multicatalytic proteinase (Ala-Pro-Phe chloromethyl ketone) block the degradation of lamin B1 to a 21-kDa fragment in thymocytes treated with glucocorticoid, the Ca2+-mobilizing agent thapsigargin, or antibodies to the T cell receptor. However, among a panel of inhibitors specific for several different proteases implicated in apoptosis, only tosylphenylalanyl chloromethyl ketone and the nuclear scaffold protease inhibitor block lamin degradation, histone H1 cleavage, and DNA fragmentation in isolated thymocyte nuclei incubated with Ca2+. Overexpression of human BCL-2 in nuclei by stable transfection resulted in an inhibition of Ca2+-stimulated lamin degradation and DNA fragmentation, suggesting that endogenous nuclear BCL-2 regulates activation of the nuclear scaffold protease. The results demonstrate the existence of an alternative pathway of lamin degradation and DNA fragmentation mediated by a resident Ca2+-stimulated nuclear protease that is not directly dependent upon activation of the interleukin 1beta-converting enzyme family of cell death regulators.
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PMID:Calcium-dependent, interleukin 1-converting enzyme inhibitor-insensitive degradation of lamin B1 and DNA fragmentation in isolated thymocyte nuclei. 879 2

PA28 is a protein activator of the 20S proteasome. It has a native molecular weight of approximately 200,000 and is composed of six 28,000-dalton subunits arranged in a ring-shaped complex. Purified preparations of PA28 contain two polypeptides, alpha and beta, which are about 50% identical in primary structure. It has been unclear whether native PA28 consists of two distinct homohexameric proteins or of a single protein containing both alpha and beta subunits. To distinguish between these possibilities, we prepared antibodies that reacted specifically with either the alpha or beta subunit and used these subunit-specific antibodies in two types of experiments designed to elucidate PA28 quaternary structure. In the first experiment, the alpha and beta subunits were completely co-immunoprecipitated by each subunit-specific antibody, indicating that both subunits were part of a single protein complex. In the second experiment, PA28 was chemically cross-linked using bis(sulfosuccinimidyl)suberate. When the cross-linked products were immunoblotted after SDS-polyacrylamide gel electrophoresis, indistinguishable patterns were obtained with each subunit-specific antibody. These results confirm that the alpha and beta subunits were part of the same protein complex. The pattern of cross-linked products also provided insight as to the relative abundance and arrangement of the subunits within the PA28 complex and indicated that the ring-shaped PA28 hexamer may be composed of alternating alpha and beta subunits with a stoichiometry of (alphabeta)3. PA28 was inactivated by treatment with carboxypeptidase Y, which cleaved Tyr and Ile residues from the carboxyl terminus of the alpha subunit but had very little effect on the beta subunit. This selective and limited proteolysis prevented binding of both alpha and beta subunits to the proteasome and therefore provides additional evidence of the heterodimeric nature of PA28. These results indicate that a short carboxyl-terminal sequence of the alpha subunit is critical for binding of native PA28 to the proteasome. To learn about the relative functions of the alpha and beta subunits, PA28alpha was expressed in Escherichia coli and purified to homogeneity. Purified PA28alpha stimulated proteasome activity but required 5-10-fold greater concentrations than the heterodimeric PA28 to achieve a given level of activity. These results suggest that the heterodimeric structure of PA28 is required for maximal proteasome activation.
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PMID:A model for the quaternary structure of the proteasome activator PA28. 882 98

To explore membrane-permeable synthetic inhibitors that discriminate between endogenous calpain and proteasome in cells, we examined the inhibition of profiles against calpain and proteasome in vitro and in vivo of peptidyl aldehydes possessing di-leucine and tri-leucine. The tripeptide aldehyde benzyloxycarbonyl-leucyl-leucinal (ZLLLal) strongly inhibited calpain and proteasome activities in vitro. The concentration required for 50% inhibition (IC50) of the casein-degrading activity of calpain was 1.25 microM, and the IC50s for the succinyl-leucyl-leucyl-valyl-tyrosine-4-methylcoumaryl-7-amide (Suc-LLVY-MCA)- and benzyloxycarbonyl-leucyl-leucyl-leucine-4-methylcoumaryl -7-amide (ZLLL-MCA)-degrading activities of proteasome were 850 and 100 nM, respectively. On the other hand, the synthetic dipeptide aldehyde benzyloxycarbonyl-leucyl-leucinal (ZLLal) strongly inhibited the casein degrading activity of calpain (IC50 1.20 microM), but the inhibition of proteasome was weak (IC50S for SucLLVY-MCA- and ZLLL-MCA-degrading activities were 120 and 110 microM, respectively). Thus, while calpain was inhibited by similar concentrations of ZLLal and ZLLLal, the inhibitory potencies of ZLLLal against the ZLLL-MCA- and Suc-LLVY-MCA-degrading activities in proteasome were 1,100 and 140 times stronger than those of ZLLal, respectively. To evaluate the effectiveness of these inhibitors on intracellular proteasome, the induction of neurite outgrowth in PC12 cells caused by proteasome inhibition was examined. ZLLLal and ZLLal initiated neurite outgrowth with optimal concentrations of 20 nM and 10 microM, respectively, again showing a big difference in the effective concentrations for the proteasome inhibition as in vitro. As for the effect on intracellular calpain, the concentration of ZLLLal and ZLLal required for the inhibition of the autolytic activation of calpain in rabbit erythrocytes were 100 and 100 microM or more, respectively. The almost equal inhibitory potencies of ZLLLal and ZLLal were in agreement with the inhibition of calpain in vitro. These differential effects of inhibitors against calpain and proteasome are potentially useful for identifying the functions of calpain and proteasome in cell physiology and pathology.
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PMID:Differential inhibition of calpain and proteasome activities by peptidyl aldehydes of di-leucine and tri-leucine. 883 56

The transcription factor NF-kappaB is retained in the cytoplasm by its interaction with the inhibitory subunit known as IkappaB. Signal-induced serine phosphorylation and subsequent ubiquitination of IkappaBalpha target it for degradation by the 26 S proteasome. Recently, pervanadate, a protein-tyrosine phosphatase inhibitor, was shown to block the degradation of IkappaBalpha, thus inhibiting NF-kappaB activation. We investigated the mechanism by which pervanadate inhibits the degradation of IkappaBalpha. Western blot analysis of IkappaBalpha from tumor necrosis factor-treated cells revealed a slower migrating IkappaBalpha species that was subsequently degraded. However, pervanadate-treated cells also revealed a slower migrating species of IkappaBalpha that appeared in a time- and dose-dependent manner and was not degraded by tumor necrosis factor. The slower migrating species of IkappaBalpha from pervanadate-treated cells was tyrosine-phosphorylated as revealed by cross-reactivity with anti-phosphotyrosine antibodies, by the ability of the specific tyrosine phosphatase PTP1B to dephosphorylate it, and by phosphoamino acid analysis of IkappaBalpha immunoprecipitated from 32P-labeled cells. By site-specific mutagenesis and deletion analysis, we identified Tyr-42 on IkappaBalpha as the phosphoacceptor site. Furthermore, in an in vitro reconstitution system, tyrosine-phosphorylated IkappaBalpha was protected from degradation. Our results demonstrate that inducible phosphorylation and degradation of IkappaBalpha are negatively regulated by phosphorylation at Tyr-42, thus preventing NF-kappaB activation.
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PMID:Site-specific tyrosine phosphorylation of IkappaBalpha negatively regulates its inducible phosphorylation and degradation. 894 99

The Met tyrosine kinase receptor is a widely expressed molecule which mediates pleiotropic cellular responses following activation by its ligand, hepatocyte growth factor/scatter factor (HGF/SF). In this communication we demonstrate that significant Met degradation is induced by HGF/SF and that this degradation can be blocked by lactacystin, an inhibitor of proteasome activity. We also show that Met is rapidly polyubiquitinated in response to ligand and that polyubiquitinated Met molecules, which are normally unstable, are stabilized by lactacystin. Both HGF/SF-induced degradation and polyubiquitination of Met were shown to be dependent on the receptor possessing intact tyrosine kinase activity. Finally, we found that a normally highly labile 55-kDa fragment of the Met receptor is stabilized by lactacystin and demonstrate that it represents a cell-associated remnant that is generated following the ligand-independent proteolytic cleavage of the Met receptor in its extracellular domain. This truncated Met molecule encompasses the kinase domain of the receptor and is itself tyrosine phosphorylated. We conclude that the ubiquitin-proteasome pathway plays a significant role in the degradation of the Met tyrosine kinase receptor as directed by ligand-dependent and -independent signals. We propose that this proteolytic pathway may be important for averting cellular transformation by desensitizing Met signaling following ligand stimulation and by eliminating potentially oncogenic fragments generated via extracellular cleavage of the Met receptor.
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PMID:Degradation of the Met tyrosine kinase receptor by the ubiquitin-proteasome pathway. 900 Dec 34

The present study unequivocally demonstrated the expression of CD28 on murine bone marrow-derived cultured mast cells and a mast cell line, MCP-5. Stimulation of surface CD28 molecules on mast cells with anti-CD28 mAbs induced tyrosine phosphorylation of cellular proteins, including several protein tyrosine kinases and their substrates, such as Itk/Emt (Emt), Btk, Syk, c-Cbl, Shc, and Vav. CD28-stimulated tyrosine phosphorylation was followed by a rebound hypophosphorylation. Interestingly, CD28 stimulation alone elicited a low level secretion of TNF-alpha. On the other hand, cross-linking of the high affinity IgE receptor (Fc epsilon RI) on mast cells induces a set of activation events, i.e., degranulation, secretion of eicosanoids, secretion of cytokines, and DNA synthesis. Concurrent stimulation of mast cells through CD28 enhanced Fc epsilon RI-induced TNF-alpha secretion in a dose-dependent manner. Together, the present data suggest a role for CD28-mediated costimulation of mast cells in the initiation and progression of allergic responses and other diseases.
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PMID:Increased secretion of TNF-alpha by costimulation of mast cells via CD28 and Fc epsilon RI. 903 88

Interleukin-2 (IL-2) activates the receptor-associated Janus family tyrosine kinases, Jak1 and Jak3, which in turn phosphorylate and activate specific STAT proteins (signal transducers and activators of transcription), such as STAT5. Activation of Jak and STAT proteins by IL-2 is transient and the mechanism for the subsequent down-regulation of their activity is largely unknown. We report here that IL-2-induced DNA-binding activity and tyrosine phosphorylation of STAT5 are stabilized by a proteasome inhibitor MG132; however, no detectable ubiquitination of the STAT proteins is observed. This sustained STAT5 activation can be blocked by protein kinase inhibitors, which is consistent with the ability of the proteasome inhibitor to stabilize IL-2-induced tyrosine phosphorylation of Jak1 and Jak3. These results suggest that proteasome-mediated protein degradation modulates protein-tyrosine phosphatase activity that negatively regulates the Jak-STAT signaling pathways.
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PMID:Involvement of proteasomes in regulating Jak-STAT pathways upon interleukin-2 stimulation. 916 19


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