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1.
Annexin I is a glucocorticoid-induced mediator with anti-inflammatory activity in animal models of arthritis. We studied the effects of a bioactive annexin I peptide, ac 2-26, dexamethasone (DEX), and interleukin-1beta (IL-1beta) on phospholipase A2 (PLA2) and cyclooxygenase (COX) activities and prostaglandin E2 (PGE2) release in cultured human fibroblast-like synoviocytes (FLS). Annexin I binding sites on human osteoarthritic (OA) FLS were detected by ligand binding flow cytometry. PLA2 activity was measured using 3H-arachidonic acid release, PGE2 release and COX activity by ELISA, and COX2 content by flow cytometry. Annexin I binding sites were present on human OA FLS. Annexin I peptide ac 2-26 exerted a significant concentration-dependent inhibition of FLS constitutive PLA2 activity, which was reversed by IL-1beta. In contrast, DEX inhibited IL-1beta-induced PLA2 activity but not constitutive activity. DEX but not annexin I peptide inhibited IL-1beta-induced PGE2 release. COX activity and COX2 expression were significantly increased by IL-1beta. Annexin I peptide demonstrated no inhibition of constitutive or IL-1beta-induced COX activity. DEX exerted a concentration-dependent inhibition of IL-1beta-induced but not constitutive COX activity. Uncoupling of inhibition of PLA2 and COX by annexin I and DEX support the hypothesis that COX is rate-limiting for PGE2 synthesis in FLS. The effect of annexin I but not DEX on constitutive PLA2 activity suggests a glucocorticoid-independent role for annexin I in autoregulation of arachidonic acid production. The lack of effect of annexin I on cytokine-induced PGE2 production suggests PGE2-independent mechanisms for the anti-inflammatory effects of annexin I in vivo. 相似文献
2.
Kleinau G Claus M Jaeschke H Mueller S Neumann S Paschke R Krause G 《The Journal of biological chemistry》2007,282(1):518-525
A number of alanine mutations in extracellular loop two (ECL2) of the thyroid-stimulating hormone receptor (TSHR) were found to increase or decrease basal activity when compared with the wild type receptor. K565A was identified as a mutant with decreased basal activity, and strongly impaired hormone induced signaling activity. To gain insights into how ECL2 mutants affect basal activity, we focused on constitutively activating pathogenic mutant I568V in ECL2, which exhibits elevated basal activity. Because our molecular model suggests that Ile-568 is embedded in an environment of hydrophobic residues provided by transmembrane helix bundle, we tested mutants in this region to identify potential interaction partner(s) for Ile-568. Indeed, the double mutant I568V/I640L (ECL2/TMH6) suppresses the increased basal activity exhibited by I568V alone. We suggest a spatial and functional relationship between ECL2 and TMH6 in which side chain interaction between Ile-568 and Ile-640 constrains the receptor in a conformation with low basal activity. Although the single mutant I640L exhibits basal activity lower than wild type, its differently branched and bulkier side chain complements the reduced side chain bulk in I568V, restoring wild type basal activity to the double mutant. This scenario is confirmed by the reciprocal double mutant I640V/I568L. The combination of basally increased activity of I640V and basally decreased activity of mutant I568L also restores basal activity of wild type TSHR. These and other mutant phenotypes reported here support a dynamic interface between TMH6 and ECL2. Disruption of this critical interface for signaling by introduction of mutations in TSHR can either increase or decrease basal activity. 相似文献
3.
Topoisomerase I (topo I) is required to unwind DNA during synthesis and provides the unique target for camptothecin-derived chemotherapeutic agents, including Irinotecan and Topotecan. While these agents are highly effective anticancer agents, some tumors do not respond due to intrinsic or acquired resistance, a process that remains poorly understood. Because of treatment toxicity, there is interest in identifying cellular factors that regulate tumor sensitivity and might serve as predictive biomarkers of therapy sensitivity. Here we identify the serine kinase, protein kinase CK2, as a central regulator of topo I hyperphosphorylation and activity and cellular sensitivity to camptothecin. In nine cancer cell lines and three normal tissue-derived cell lines we observe a consistent correlation between CK2 levels and camptothecin responsiveness. Two other topo I-targeted serine kinases, protein kinase C and cyclin-dependent kinase 1, do not show this correlation. Camptothecin-sensitive cancer cell lines display high CK2 activity, hyperphosphorylation of topo I, elevated topo I activity, and elevated phosphorylation-dependent complex formation between topo I and p14ARF, a topo I activator. Camptothecin-resistant cancer cell lines and normal cell lines display lower CK2 activity, lower topo I phosphorylation, lower topo I activity, and undetectable topo I/p14ARF complex formation. Experimental inhibition or activation of CK2 demonstrates that CK2 is necessary and sufficient for regulating these topo I properties and altering cellular responses to camptothecin. The results establish a cause and effect relationship between CK2 activity and camptothecin sensitivity and suggest that CK2, topo I phosphorylation, or topo I/p14ARF complex formation could provide biomarkers of therapy-responsive tumors. 相似文献
4.
Four phosphoprotein phosphatases, with the ability to act upon hydroxymethylglutaryl (HMG)-CoA reductase, phosphorylase, and glycogen synthase have been purified from rat liver cytosol through a process that involves DEAE-cellulose, aminohexyl-Sepharose-4B, and Bio-Gel A 1.5 m chromatographies. Protein phosphatase II (Mr 180,000) was the major enzyme (68%) with a very broad substrate specificity, showing similar activity toward the three substrates. Phosphatases I1 (Mr 180,000) and I3 (Mr 250,000) accounted for only 12 and 15% of the total activity, respectively, and they were also able to dephosphorylate the three substrates. In contrast, phosphatase I2 (Mr 200,000) showed only phosphorylase phosphatase activity with insignificant dephosphorylating capacity toward HMG-CoA reductase and glycogen synthase. Upon ethanol treatment at room temperature, the Mr of all phosphatases changed; protein phosphatases I2, I3, and II were brought to an Mr of 35,000, while phosphatase I1 was reduced to an Mr of 69,000. Glycogen synthase phosphatase activity was decreased in all four phosphatases. There was also a decrease in phosphatase I1 activity toward HMG-CoA reductase and phosphorylase as substrates. The HMG-CoA reductase phosphatase and phosphorylase phosphatase activities of phosphatases I2, I3, and II were increased after ethanol treatment. Each protein phosphatase showed a different optimum pH, which changed depending on the substrate. The four phosphatases increased their activity in the presence of Mn2+ and Mg2+. In general, Mn2+ was a better activator than Mg2+, and phosphatase I1 showed a stronger dependency on these cations than any other phosphatase. Phosphorylase was a competitive substrate in the HMG-CoA reductase phosphatase and glycogen synthase phosphatase reactions of protein phosphatases I1, I3, and II. HMG-CoA reductase was also able to compete with phosphorylase and glycogen synthase for phosphatase activity. Glycogen synthase phosphatase activity presented less inhibition in the low-Mr forms. A comparison has been made with other protein phosphatases previously reported in the literature. 相似文献
5.
Tobacco nectarin I. Purification and characterization as a germin-like, manganese superoxide dismutase implicated in the defensE of floral reproductive tissues 总被引:13,自引:0,他引:13
Nectarin I, a protein that accumulates in the nectar of Nicotiana sp. , was determined to contain superoxide dismutase activity by colorimetric and in-gel assays. This activity was found to be remarkably thermostable. Extended incubations at temperatures up to 90 degrees C did not diminish the superoxide dismutase activity of nectarin I. This attribute allowed nectarin I to be purified to homogeneity by heat denaturation of the other nectar proteins. By SDS-polyacrylamide gel electrophoresis, nectarin I appeared as a 29-kDa monomer. If the protein sample was not boiled prior to loading the gel, then nectarin I migrated as 165-kDa oligomeric protein. By matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, the protomer subunit was found to be a 22.5-kDa protein. Purified nectarin I contained 0.5 atoms of manganese/monomer, and the superoxide dismutase activity of nectarin I was not inhibited by either H(2)O(2) or NaCN. Following denaturation, the superoxide dismutase activity was restored after Mn(2+) addition. Addition of Fe(2+), Cu(2+), Zn(2+), and Cu(2+)/Zn(2+) did not restore superoxide dismutase activity. The quaternary structure of the reconstituted enzyme was examined, and only tetrameric and pentameric aggregates were enzymatically active. The reconstituted enzyme was also shown to generate H(2)O(2). Putative nectarin I homologues were found in the nectars of several other plant species. 相似文献
6.
Zhao LX Moon YS Basnet A Kim EK Jahng Y Park JG Jeong TC Cho WJ Choi SU Lee CO Lee SY Lee CS Lee ES 《Bioorganic & medicinal chemistry letters》2004,14(5):1333-1337
For the development of new anticancer agents, phenyl, 2-pyridyl, 2-furyl, 2-thienyl, 2-furylvinyl and 2-thienylvinyl substituted derivatives on 2,4,6-position in pyridine moiety were prepared and evaluated for their topoisomerase I inhibitory activity. Among the thirteen prepared compounds, four compounds exhibited strong topoisomerase I inhibitory activity. A structure-activity relationship study indicated that the 2-thienyl-4-furylpyridine skeleton was important for topoisomerase I inhibitory activity. 相似文献
7.
Human wild-type cardiac troponin T, I, C and five troponin T mutants (I79N, R92Q, F110I, E244D, and R278C) causing familial hypertrophic cardiomyopathy were expressed in Escherichia coli, and then were purified and incorporated into rabbit cardiac myofibrils using a troponin exchange technique. The Ca2+-sensitive ATPase activity of these myofibrillar preparations was measured in order to examine the functional consequences of these troponin mutations. An I79N troponin T mutation was found to cause a definite increase in Ca2+ sensitivity of the myofibrillar ATPase activity without inducing any significant change in the maximum level of ATPase activity. A detailed analysis indicated the inhibitory action of troponin I to be impaired by the I79N troponin T mutation. Two more troponin T mutations (R92Q and R278C) were also found to have a Ca2+-sensitizing effect without inducing any change in maximum ATPase activity. Two other troponin T mutations (F110I and E244D) had no Ca2+-sensitizing effects on the ATPase activity, but remarkably potentiated the maximum level of ATPase activity. These findings indicate that hypertrophic cardiomyopathy-linked troponin T mutations have at least two different effects on the Ca2+-sensitive ATPase activity, Ca2+-sensitization and potentiation of the maximum level of the ATPase activity. 相似文献
8.
Topoisomerase I is the target for a potent class of chemotherapeutic drugs derived from the plant alkaloid camptothecin that includes irinotecan and topotecan. In this study we have identified a novel site of CK2-mediated topoisomerase I (topo I) phosphorylation at serine 506 (PS506) that is relevant to topo I function and to cellular responses to these topo I-targeted drugs. CK2 treatment induced hyperphosphorylation of recombinant topo I and expression of the PS506 epitope, and resulted in increased binding of topo I to supercoiled plasmid DNA. Hyperphosphorylated topo I was approximately three times more effective than the basal phosphorylated enzyme at relaxing plasmid supercoils but had similar DNA cleavage activity once bound to DNA. The PS506 epitope was expressed in cancer cell lines with elevated CK2 activity, hyperphosphorylated topo I, and increased sensitivity to camptothecin. In contrast, PS506 was not detected in normal cells or cancer cell lines with lower levels of CK2 activity. By experimentally manipulating CK2 activity in cancer cell lines, we demonstrate a cause and effect relationship between CK2 activity, PS506 expression, camptothecin-induced cellular DNA damage, and cellular camptothecin sensitivity. Our results show that the PS506 epitope is an indicator of dysregulated, hyperphosphorylated topo I in cancer cells, and may thus serve as a diagnostic or prognostic biomarker and predict tumor responsiveness to widely used topo I-targeted therapies. 相似文献
9.
Clostridium pasteurianum's hydrogenase I, an important constitutive metabolic enzyme, has been shown to function as a 'novel selenite reductase'. Selenite reductase activity was found to co-purify with hydrogenase I activity; the fold purification and specific activities for these two activities paralleled each other throughout the purification steps. The highly purified hydrogenase I apparent K(m) for the selenite substrate was 0.2 mM. The stoichiometry for the enzymatic reduction of SeO3(2-) to Se(0) via H2 oxidation, was determined to be 2.3:1 (H2:Se(0)), very close to the theoretical ratio of 2:1 for this reduction reaction. Known electron carriers required for hydrogenase I activity were also found to couple its selenite reductase activity, the most efficient one being ferredoxin. The purified hydrogenase I not only reduced selenite but also tellurite, and its selenite activity was completely inhibited by O2 and CuSO4, potent inhibitors of hydrogenase I activity. 相似文献
10.
Brassica juncea glyoxalase I (S-lactoylglutathione-lyase, EC 4.4.1. 5) is a 56 kDa, heterodimeric protein. It requires magnesium (Mg2+) for its optimal activity. In this report we provide biochemical evidence for modulation of glyoxalase I activity by calcium/calmodulin (Ca2+/CaM). In the presence of Ca2+ glyoxalase I showed a significant (2.6-fold) increase in its activity. It also showed a Ca2+ dependent mobility shift on denaturing gels. Its Ca2+ binding was confirmed by Chelex-100 assay and gel overlays using 45CaCl2. Glyoxalase I was activated by over 7-fold in the presence of Ca2+ (25 microM) and CaM (145 nM) and this stimulation was blocked by the CaM antibodies and a CaM inhibitor, trifluroperazine (150 microM). Glyoxalase I binds to a CaM-Sepharose column and was eluted by EGTA. The eluted protein fractions also showed stimulation by CaM. The stimulation of glyoxalase I activity by CaM was maximum in the presence of Mg2+ and Ca2+; however, magnesium alone also showed glyoxalase I activation by CaM. 相似文献
11.
12.
Deoxyribonuclease I (DNase I) was purified about 850,000-fold from human kidney using a rabbit anti-human urine DNase I antibody and sensitive DNase I activity assay. On SDS-PAGE, the purified kidney DNase I gave a single major band, and its molecular mass was estimated to be 38,000 Da. The activity of purified kidney DNase I was dependent on the presence of Mg2+ and Ca2+. G-Actin inhibited the activity, as did the anti-urine DNase I antibody. The properties of the kidney DNase I were the same as those of urine DNase I. 相似文献
13.
14.
In order to determine the functional consequences of the Arg145Gly mutation in troponin I found in familial hypertrophic cardiomyopathy, human cardiac troponin I and its mutant were expressed in Escherichia coli and purified, and then their effects on the ATPase activity of porcine cardiac myofibrillar preparations from which both troponins C and I had been depleted were examined. Both the wild-type and mutant troponin Is suppressed the ATPase activity of the troponin C.I-depleted myofibrils, but the maximum inhibition caused by mutant troponin I was weaker than that by wild-type troponin I. In the Ca(2)(+)-activation profile of the myofibrillar ATPase activity after reconstitution with both troponins I and C, the Ca(2)(+)-sensitivity with mutant troponin I was higher than that with wild-type troponin I, whereas the maximum level of the ATPase activity with mutant troponin I was lower than that with wild-type troponin I. These findings strongly suggest that the Arg145Gly mutation in human cardiac troponin I modulates the Ca(2)(+)-regulation of contraction by impairing the interaction of troponin I with both actin-tropomyosin and troponin C. 相似文献
15.
The binary Clostridium botulinum C2 toxin consists of the binding/translocation component C2IIa and the separate enzyme component C2I, which mono-ADP-ribosylates actin in eukaryotic cells. Pore formation of C2IIa in early endosomal membranes facilitates translocation of unfolded C2I into the cytosol. We discovered earlier that translocation of C2I depends on the activity of the host cell chaperone heat shock protein Hsp90. Here, we demonstrate that cyclosporin A, which inhibits the peptidyl-prolyl cis / trans isomerase activity of cyclophilins, inhibited intoxication of cells with C2 toxin and prevented uptake of C2I into the cytosol. Cyclosporin A blocked the pH-dependent translocation of C2I activity across membranes of intact cells and of partially purified early endosomes. In vitro , the addition of cytosol to C2 toxin-loaded endosomes induced translocation of C2I activity into the cytosol, which was prevented by pretreatment of the cytosol with an antibody against cyclophilin A. Pull-down experiments with lysates from C2 toxin-treated cells revealed specific binding of cyclophilin A to the N-terminal domain of C2I. In conclusion, our results suggest an essential role of cyclophilin A for translocation of C2I across endosomal membranes during the uptake of C2 toxin into mammalian cells. 相似文献
16.
Jun-Ping Liu Qun-Xing Zhang †Graham Baldwin Phillip J. Robinson 《Journal of neurochemistry》1996,66(5):2074-2081
Abstract: Synaptic vesicle recycling is a neuronal specialization of endocytosis that requires the GTPase activity of dynamin I and is triggered by membrane depolarization and Ca2+ entry. To establish the relationship between dynamin I GTPase activity and Ca2+ , we used purified dynamin I and analyzed its interaction with Ca2+ in vitro. We report that Ca2+ bound to dynamin I and this was abolished by deletion of dynamin's C-terminal tail. Phosphorylation of dynamin I by protein kinase C promoted formation of a dynamin I tetramer and increased Ca2+ binding to the protein. Moreover, Ca2+ inhibited dynamin I GTPase activity after stimulation by phosphorylation or by phospholipids but not after stimulation with a GST-SH3 fusion protein containing the SH3 domain of phosphoinositide 3-kinase. These results suggest that in resting nerve terminals, phosphorylation of dynamin I by protein kinase C converts it to a tetramer that functions as a Ca2+ -sensing protein. By binding to Ca2+ , dynamin I GTPase activity is specifically decreased, possibly to regulate synaptic vesicle recycling. 相似文献
17.
Acanthamoeba cofactor protein is a heavy chain kinase required for actin activation of the Mg2+-ATPase activity of Acanthamoeba myosin I. 总被引:5,自引:0,他引:5
We have purified a cofactor protein previously shown (Pollard, T. D., and Korn, E. D. (1973) J. Biol. Chem. 248, 4691-4697) to be required for actin activation of the Mg2+-ATPase activity of Acanthamoeba myosin I. The purified cofactor protein is a novel myosin kinase that phosphorylates the single heavy chain, but neither of the two light chains, of Acanthamoeba myosin I. Phosphorylation of Acanthamoeba myosin I by the purified cofactor protein requires ATP and Mg2+ but is Ca2+-independent. The Mg2+-ATPase activity of phosphorylated Acanthamoeba myosin I is highly activated by F-actin in the absence of cofactor protein. Actin-activated Mg2+-ATPase activity is lost when phosphorylated Acanthamoeba myosin I is dephosphorylated by platelet phosphatase. Phosphorylation and dephosphorylation have no effect on the (K+,EDTA)-ATPase and Ca2+-ATPase activities of Acanthamoeba myosin I. These results show that cofactor protein is an Acanthamoeba myosin I heavy chain kinase and that phosphorylation of the heavy chain of this myosin is required for actin activation of its Mg2+-ATPase activity. 相似文献
18.
John F. Cannon 《Proteins》2020,88(2):366-384
This work explores how phosphorylation of an unstructured protein region in inhibitor-2 (I2) regulates protein phosphatase-1 (PP1) enzyme activity using molecular dynamics (MD). Free I2 is largely unstructured; however, when bound to PP1, three segments adopt a stable structure. In particular, an I2 helix (i-helix) blocks the PP1 active site and inhibits phosphatase activity. I2 phosphorylation in the PP1-I2 complex activates phosphatase activity without I2 dissociation. The I2 Thr74 regulatory phosphorylation site is in an unstructured domain in PP1-I2. PP1-I2 MD demonstrated that I2 phosphorylation promotes early steps of PP1-I2 activation in explicit solvent models. Moreover, phosphorylation-dependent activation occurred in PP1-I2 complexes derived from I2 orthologs with diverse sequences from human, yeast, worm, and protozoa. This system allowed exploration of features of the 73-residue unstructured human I2 domain critical for phosphorylation-dependent activation. These studies revealed that components of I2 unstructured domain are strategically positioned for phosphorylation responsiveness including a transient α-helix. There was no evidence that electrostatic interactions of I2 phosphothreonine74 influenced PP1-I2 activation. Instead, phosphorylation altered the conformation of residues around Thr74. Phosphorylation uncurled the distance between I2 residues Glu71 to Tyr76 to promote PP1-I2 activation, whereas reduced distances reduced activation. This I2 residue Glu71 to Tyr76 distance distribution, independently from Thr74 phosphorylation, controls I2 i-helix displacement from the PP1 active site leading to PP1-I2 activation. 相似文献
19.
The actin-activated Mg(2+)-ATPase activity of Acanthamoeba myosins I depends on phosphorylation of their single heavy chains by myosin I heavy chain kinase. Kinase activity is enhanced > 50-fold by autophosphorylation at multiple sites. The rate of kinase autophosphorylation is increased approximately 20-fold by acidic phospholipids independent of the presence of Ca2+ and diglycerides. We show in this paper that Ca(2+)-calmodulin inhibits phospholipid-stimulated autophosphorylation of myosin I heavy chain kinase and hence also inhibits the catalytic activity of unphosphorylated kinase in the presence of phospholipid. Ca(2+)-calmodulin does not inhibit kinase activity in the absence of phospholipid. Micromolar Ca(2+)-calmodulin also inhibits binding of myosin I heavy chain kinase to phospholipid vesicles and purified plasma membranes. Proteolytic removal of a 7-kDa NH2-terminal segment from the 97-kDa kinase prevents binding of both calmodulin and phospholipid; therefore, we propose that they bind to the same or overlapping sites. These data provide a mechanism by which Ca2+ could inhibit the actin-activated Mg(2+)-ATPase activity of the myosin I isozymes in vivo and thus regulate myosin I-dependent motile activities. 相似文献
20.
Sebastian Grünberg Baptiste Coxam Tien-Hao Chen Nan Dai Lana Saleh Ivan R Corrêa Jr. Nicole M Nichols Erbay Yigit 《Nucleic acids research》2021,49(9):5265
Since its initial characterization, Escherichia coli RNase I has been described as a single-strand specific RNA endonuclease that cleaves its substrate in a largely sequence independent manner. Here, we describe a strong calcium (Ca2+)-dependent activity of RNase I on double-stranded RNA (dsRNA), and a Ca2+-dependent novel hybridase activity, digesting the RNA strand in a DNA:RNA hybrid. Surprisingly, Ca2+ does not affect the activity of RNase I on single stranded RNA (ssRNA), suggesting a specific role for Ca2+ in the modulation of RNase I activity. Mutation of a previously overlooked Ca2+ binding site on RNase I resulted in a gain-of-function enzyme that is highly active on dsRNA and could no longer be stimulated by the metal. In summary, our data imply that native RNase I contains a bound Ca2+, allowing it to target both single- and double-stranded RNAs, thus having a broader substrate specificity than originally proposed for this traditional enzyme. In addition, the finding that the dsRNase activity, and not the ssRNase activity, is associated with the Ca2+-dependency of RNase I may be useful as a tool in applied molecular biology. 相似文献