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)

The crystal structure of Serratia protease from Serratia sp. E-15 was solved by the single isomorphous replacement method supplemented with anomalous scattering effects from both the Zn atom in the native crystal and the Sm atom in the derivative crystal, and refined at 2.0 A resolution to a crystallographic R-factor of 0.194. The enzyme consists of N-terminal catalytic and C-terminal beta-sandwich domains, as observed in alkaline protease from Pseudomonas aeruginosa IFO3080. The catalytic domain with a five-stranded antiparallel beta-sheet and five alpha-helices shares a basically common folding topology with those of other zinc metalloendoproteases. The catalytic zinc ion at the bottom of the active site cleft is ligated by His176, His180, His186, Tyr216, and a water molecule in a distorted trigonalbipyramidal manner. The C-terminal domain is a beta-strand-rich domain containing eighteen beta-strands and a short alpha-helix, and has seven Ca2+ ions bound to calcium binding loops. An unusual beta-sheet coil motif is observed in this domain, where the beta-strands and calcium binding loops are alternately incorporated into an elliptical right-handed spiral so as to form a two-layer untwisted beta-sandwich structure. The Ca2+ ions in the C-terminal domain seem to be very important for the folding and stability of the beta-sheet coil structure.
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PMID:Crystal structure of Serratia protease, a zinc-dependent proteinase from Serratia sp. E-15, containing a beta-sheet coil motif at 2.0 A resolution. 879 82

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

In normal subjects and diabetic patients, insulin suppresses whole body proteolysis suggesting that the loss of lean body mass and muscle wasting in insulinopenia is related to increased muscle protein degradation. To document how insulinopenia affects organ weights and to identify the pathway for accelerated proteolysis in muscle, streptozotocin-treated and vehicle-injected, pair-fed control rats were studied. The weights of liver, adipose tissue, and muscle were decreased while muscle protein degradation was increased 75% by insulinopenia. This proteolytic response was not eliminated by blocking lysosomal function and calcium-dependent proteases at 7 or 3 d after streptozotocin. When ATP synthesis in muscle was inhibited, the rates of proteolysis were reduced to the same level in insulinopenic and control rats suggesting that the ATP-dependent, ubiquitin-proteasome pathway is activated. Additional evidence for activation of this pathway in muscle includes: (a) an inhibitor of proteasome activity eliminated the increased protein degradation; (b) mRNAs encoding ubiquitin and proteasome subunits were increased two- to threefold; and (c) there was increased transcription of the ubiquitin gene. We conclude that the mechanism for muscle protein wasting in insulinopenia includes activation of the ubiquitin-proteasome pathway with increased expression of the ubiquitin gene.
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PMID:Muscle wasting in insulinopenic rats results from activation of the ATP-dependent, ubiquitin-proteasome proteolytic pathway by a mechanism including gene transcription. 887 19

Monocyte chemoattractant protein-1 (MCP-1) is a member of the chemokine family of chemotactic cytokines and signals via activation of a G protein-coupled seven-transmembrane domain receptor to mediate chemotaxis. Monocyte activation is limited by desensitization and internalization of the MCP-1R, but these mechanisms are not well understood. In this study, we show that the type B MCP-1R (MCP-1RB/CCR2B) is rapidly phosphorylated and internalized in response to nanomolar concentrations of MCP-1. Co-expression of CCR2B in Xenopus oocytes with beta-adrenergic receptor kinase 2 (beta ark2), but not beta ark1 or rhodopsin kinase, specifically blocked receptor activation by MCP-1. Mutation of serine (Ser) and threonine (Thr) residues in the terminal carboxyl-tail of the receptor, which are potential targets of beta ark-mediated phosphorylation, prevented inhibition of receptor activation by beta ark2 in microinjected oocytes. Finally, a construct in which multiple Ser and Thr residues in the carboxyl-tail were changed to alanine significantly prolonged the agonist-dependent intracellular calcium flux and inhibited receptor internalization in transfected human embryonic kidney (HEK)-293 cells. These studies demonstrate that phosphorylation of Ser and Thr residues in the carboxyl-tail of CCR2B mediates receptor desensitization and internalization and may serve to limit the chemotactic response of leukocytes to MCP-1 and related chemokines.
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PMID:Phosphorylation by a G protein-coupled kinase inhibits signaling and promotes internalization of the monocyte chemoattractant protein-1 receptor. Critical role of carboxyl-tail serines/threonines in receptor function. 895 13

A crude fraction that contains ubiquitin-protein ligases contains also a proteolytic activity of approximately 100 kDa that cleaves p53 to several fragments. The protease does not require ATP and is inhibited in the crude extract by an endogenous approximately 250 kDa inhibitor. The proteinase can be inhibited by chelating the Ca2+ ions, by specific cysteine proteinase inhibitors and by peptide aldehyde derivatives that inhibit calpains. Purified calpain demonstrates an identical activity that can be inhibited by calpastatin, the specific protein inhibitor of the enzyme. Thus, it appears that the activity we have identified in the extract is catalyzed by calpain. The calpain in the extract degrades also N-myc, c-Fos and c-Jun, but not lysozyme. In crude extract, the calpain activity can be demonstrated only when the molar ratio of the calpain exceeds that of its native inhibitor. Recent experimental evidence implicates both the ubiquitin proteasome pathway and calpain in the degradation of the tumor suppressor, and it was proposed that the two pathways may play a role in targeting the protein under various conditions. The potential role of the two systems in this important metabolic process is discussed.
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PMID:On the involvement of calpains in the degradation of the tumor suppressor protein p53. 910 77

p53 is a short-lived transcription factor that is frequently mutated in tumor cells. Work by several laboratories has already shown that the ubiquitin-proteasome pathway can largely account for p53 destruction, at least under specific experimental conditions. We report here that, in vitro, wild-type p53 is a sensitive substrate for milli- and microcalpain, which are abundant and ubiquitous cytoplasmic proteases. Degradation was dependent on p53 protein conformation. Mutants of p53 with altered tertiary structure displayed a wide range of susceptibility to calpains, some of them being largely resistant to degradation and others being more sensitive. This result suggests that the different mutants tested here adopt slightly different conformations to which calpains are sensitive but that cannot be discriminated by using monoclonal antibodies such as PAb1620 and PAb240. Inhibition of calpains by using the physiological inhibitor calpastatin leads to an elevation of p53 steady-state levels in cells expressing wild-type p53. Conversely, activation of calpains by calcium ionophore led to a reduction of p53 in mammalian cells, and the effect was blocked by cell-permeant calpain inhibitors. Cotransfection of p53-null cell lines with p53 and calpastatin expression vectors resulted in an increase in p53-dependent transcription activity. Taken together, these data support the idea that calpains may also contribute to the regulation of wild-type p53 protein levels in vivo.
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PMID:Proteolysis by calpains: a possible contribution to degradation of p53. 911 52

Chronic stimulation of WB rat liver epithelial cells by angiotensin II (Ang II) resulted in the down-regulation of both type I and type III myo-inositol 1,4,5-trisphosphate receptors (IP3Rs). Stimulation with vasopressin, bradykinin, epidermal growth factor, or 12-O-tetradecanoylphorbol-13-acetate was without effect. Ang II-induced down-regulation of IP3Rs could be detected within 2 h and resulted in an inhibition of IP3-induced Ca2+ release from permeabilized cells. IP3R down-regulation was reversible, and both homo- and heterooligomers of IP3Rs were equally susceptible to Ang II-induced degradation. Chloroquine and NH4Cl increased the basal levels of IP3Rs by 2-fold, suggesting that the basal turnover of IP3Rs occurs via a lysosomal pathway. However, Ang II-induced degradation of IP3R was not affected by these inhibitors, suggesting that stimulated degradation of IP3Rs occurs via a non-lysosomal pathway. The cysteine protease and proteasomal inhibitor N-acetyl-Leu-Leu-norleucinal completely prevented Ang II-mediated down-regulation of IP3Rs, whereas the structural analog N-acetyl-Leu-Leu-methioninal was without effect. Lactacystin, a highly specific proteasome inhibitor, also blocked Ang II-mediated IP3R degradation. Stimulation with Ang II increased the amount of IP3R immunoprecipitated by anti-ubiquitin antibodies. We conclude that Ang II-stimulated IP3R degradation involves enhanced ubiquitination of the protein and degradation by the proteasome pathway.
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PMID:Angiotensin II-induced down-regulation of inositol trisphosphate receptors in WB rat liver epithelial cells. Evidence for involvement of the proteasome pathway. 913 93

Several observations have suggested that the enhanced proteolysis and atrophy of skeletal muscle in various pathological states is due primarily to activation of the ubiquitin-proteasome pathway. To test this idea, we investigated whether peptide aldehyde inhibitors of the proteasome, N-acetyl-leucyl-leucyl-norleucinal (LLN), or the more potent CBZ-leucyl-leucyl-leucinal (MG132) suppressed proteolysis in incubated rat skeletal muscles. These agents (e.g., MG132 at 10 microM) inhibited nonlysosomal protein breakdown by up to 50% (P < 0.01), and this effect was rapidly reversed upon removal of the inhibitor. The peptide aldehydes did not alter protein synthesis or amino acid pools, but improved overall protein balance in the muscle. Upon treatment with MG132, ubiquitin-conjugated proteins accumulated in the muscle. The inhibition of muscle proteolysis correlated with efficacy against the proteasome, although these agents could also inhibit calpain-dependent proteolysis induced with Ca2+. These inhibitors had much larger effects on proteolysis in atrophying muscles than in controls. In the denervated soleus undergoing atrophy, the increase in ATP-dependent proteolysis was reduced 70% by MG132 (P < 0.001). Similarly, the rise in muscle proteolysis induced by administering thyroid hormones was reduced 40-70% by the inhibitors. Finally, in rats made septic by cecal puncture, the increase in muscle proteolysis was completely blocked by MG132. Thus, the enhanced proteolysis in many catabolic states (including denervation, hyperthyroidism, and sepsis) is due to a proteasome-dependent pathway, and inhibition of proteasome function may be a useful approach to reduce muscle wasting.
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PMID:Inhibitors of the proteasome reduce the accelerated proteolysis in atrophying rat skeletal muscles. 920 72

A novel intracellular calcium-binding protein from Echinococcus granulosus is described in this work. A cDNA was isolated from a lambdagt11 protoscolex expression library and the deduced amino acid sequence has at least fifteen sequentially repeated twelve-residue repeats that resemble the calcium-binding loop of EF-hands; however, the dodecamer motif has no flanking helices. The cDNA was expressed in Escherichia coli using the pGEX vector, and a recombinant fusion protein (EgCaBP1-GST) was obtained. The recombinant fusion protein binds calcium when assayed with 45Ca. It is possible that the calcium-binding motifs present a secondary structure similar to the parallel beta roll structure described for an alkaline protease from Pseudomonas aeruginosa. A native protein of more than 300 kDa was recognized by an anti-EgCaBP1 monoclonal antibody by Western-blot analysis. Immunohistochemistry using a pool of anti-EgCaBP1-GST mouse sera demonstrated a strong association of the protein with calcareous corpuscles. The possible role of this protein and that of the calcareous corpuscles in the protoscolex are discussed.
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PMID:A protein with a novel calcium-binding domain associated with calcareous corpuscles in Echinococcus granulosus. 926 32

Cyclin D1, a critical positive regulator of G1 progression, has been implicated in the pathogenesis of certain cancers. Regulation of cyclin D1 occurs at the transcriptional and posttranscriptional level. Here we present evidence that cyclin D1 levels are regulated at the posttranscriptional level by the Ca2+-activated protease calpain. Serum starvation of NIH 3T3 cells resulted in rapid loss of cyclin D1 protein that was completely reversible by calpain inhibitors. Actinomycin D and lovastatin induced rapid loss of cyclin D1 in prostate and breast cancer cells that was reversible by calpain inhibitors and not by phenylmethylsulfonyl fluoride, caspase inhibitors, or lactacystin, a specific inhibitor of the 26 S proteasome. Treatment of intact NIH 3T3, prostate, and breast cancer cells with a calpain inhibitor dramatically increased the half-life of cyclin D1 protein. Addition of purified calpain to PC-3-M lysates resulted in Ca2+-dependent cyclin D1 degradation. Transient expression of the calpain inhibitor calpastatin increased cyclin D1 protein in serum-starved NIH 3T3 cells. Cyclins A, E, and B1 have been reported to be regulated by proteasome-associated proteolysis. The data presented here implicate calpain in cyclin D1 posttranslational regulation.
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PMID:Regulation of cyclin D1 by calpain protease. 935 8


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