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:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A missense mutation in the gene of tissue-nonspecific alkaline phosphatase, which replaces aspartic acid at position 289 with valine [TNSALP (D289V)], was reported in a lethal hypophosphatasia patient [Taillandier, A. et al. (1999) Hum. Mut. 13, 171-172]. To define the molecular defects of TNSALP (D289V), this mutant protein in transiently transfected COS-1 cells was analyzed biochemically and morphologically. TNSALP (D289V) exhibited no alkaline phosphatase activity and mainly formed a disulfide-linked high molecular mass aggregate. Cell-surface biotinylation, digestion with phosphatidylinositol-specific phospholipase C and an immunofluorescence study showed that the mutant protein failed to appear on the cell surface and was accumulated intracellularly. In agreement with this, pulse/chase experiments demonstrated that TNSALP (D289V) remained endo-beta-N-acetyl- glucosaminidase H-sensitive throughout the chase and was eventually degraded, indicating that the mutant protein is unable to reach the medial-Golgi. Proteasome inhibitors strongly blocked the degradation of TNSALP (D289V), and furthermore the mutant protein was found to be ubiquitinated. Besides, another naturally occurring TNSALP with a Glu(218)-->Gly mutation was also found to be polyubiquitinated and degraded in the proteasome. Since the acidic amino acids at positions 218 and 289 of TNSALP are thought to be directly involved in the Ca(2+) coordination, these results suggest the critical importance of calcium binding in post-translational folding and assembly of the TNSALP molecule.
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PMID:Tissue-nonspecific alkaline phosphatase with an Asp(289)-->Val mutation fails to reach the cell surface and undergoes proteasome-mediated degradation. 1294 72

The 5-hydroxytryptamine 2A receptor (5-HT2AR) is a member of the class I family of rhodopsin-related G protein-coupled receptors. The receptor is known to activate phospholipase C via the heterotrimeric G proteins Gq/11, but we showed previously that it can also signal through the phospholipase D (PLD) pathway in an ADP-ribosylation factor (ARF)-dependent manner that seems to be independent of Gq/11 (Mitchell et al., 1998). Both coimmunoprecipitation experiments and the effects of negative mutant ARF constructs on 5-HT2AR-induced PLD activation here suggested that ARF1 may play a greater role than ARF6 in the function of this receptor. Furthermore, we demonstrated using glutathione S-transferase (GST)-fusion proteins of receptor domains that ARF1 and ARF6 bind to the third intracellular loop (i3) and the carboxy terminal tail (ct) of the 5-HT2AR. The association of ARF1 with the ct domain of the receptor was stronger than its interaction with i3, or the interactions of ARF6 with either construct. Experiments using ARF mutants that are deficient in GTP loading, and the in vitro addition of GTPgammaS suggested that GTP loading enhances ARF1 binding to the receptor. The N376PxxY motif in the transmembrane 7 domain of the receptor (rather than a N376DPxxY mutant form) was shown to be essential for ARF-dependent PLD signaling and ARF1 coimmunoprecipitation. In GST-fusion proteins of the 5-HT2AR ct domain, mutation of Asn376 to Asp also markedly reduced ARF1-HA binding, although additional motifs in the Asn376-Asn384 sequence and to a lesser extent elsewhere, seem also to contribute to the interaction.
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PMID:Selective interaction of ARF1 with the carboxy-terminal tail domain of the 5-HT2A receptor. 1457 74

Two mutations, R69D and K115E, converted a bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) to a phosphatase with much higher specific activity toward glucose-6-phosphate than inositol-1-phosphate. PI-PLC single mutations R69D and K115E can cleave PI but lack any demonstrable phosphatase activity. The bacterial PI-PLC has no sequence homology with known glucose-6-phosphatase enzymes, which need His, Arg, and negatively charged residues (Asp or Glu) at the active site. The change in chemical reaction and substrate specificity can be rationalized by energy minimization of the mutant with I-1-P or G-6-P bound.
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PMID:Mutation of two active-site residues converts a phosphatidylinositol-specific phospholipase C to a glucose phosphatase. 1474 54

The rat cytomegalovirus (RCMV) R33 gene encodes a G protein-coupled receptor (GPCR), pR33, which possesses agonist-independent, constitutive signalling activity. To characterize this activity further, we generated a series of point and deletion mutants of pR33. Both expression of and signalling by the mutants was evaluated. Several point mutants were generated that contained modifications in the NRY motif. This motif, at aa 130-132 of pR33, is the counterpart of the common DRY motif of GPCRs, which is known to be involved in G protein coupling. We found that mutation of the asparagine residue within the NRY motif of pR33 (N(130)) to aspartic acid resulted in a mutant (N(130)D) with similar signalling characteristics to the wild-type (WT) protein, indicating that N(130) is not the determinant of constitutive activity of pR33. Interestingly, a mutant carrying an alanine at aa 130 (N(130)A) was severely impaired in G(q/11)-mediated, constitutive activation of phospholipase C, whereas it displayed similar levels of activity to pR33 in G(i/0)-mediated signalling. Another protein that contained a modified NRY motif, R(131)A, did not show constitutive activity, whereas mutants Y(132)F and Y(132)A displayed similar activities to the WT receptor. This indicated that residue R(131) is critical for pR33 function in vitro, whereas Y(132) is not. Finally, we identified two consecutive arginines within the C-terminal tails of both pR33 and its homologue from human CMV, pUL33, which are important for correct cell-surface expression of these receptors.
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PMID:Mutational analysis of the R33-encoded G protein-coupled receptor of rat cytomegalovirus: identification of amino acid residues critical for cellular localization and ligand-independent signalling. 1503 32

Phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis catalyzes the cleavage of the phosphorus-oxygen bond in phosphatidylinositol. The focus of this work is to dissect the roles of the carboxylate side chain of Asp(274) in the Asp(274)-His(32) dyad, where a short strong hydrogen bond (SSHB) was shown to exist based on NMR criteria. A regular hydrogen bond (HB) was observed in D274N, and no low field proton resonance was detected for D274E and D274A. Comparison of the activity of wild type, D274N, and D274A suggested that the regular HB contributes significantly (approximately 4 kcal/mol) to catalysis, whereas the SSHB contributes only an additional 2 kcal/mol. The mutant D274E displays high activity similar to wild type, suggesting that the negative charge is sufficient for the catalytic role of Asp(274). To further support this interpretation and rule out possible contribution of regular HB or SSHB in D274E, we showed that the activity of D274G can be rescued by exogenous chloride ions to a level comparable with that of D274E. Comparison between different anions suggested that the ability of an anion to rescue the activity is due to the size and the charge of the anion not the property as a HB acceptor. In conclusion, a major fraction of the functional role of Asp(274) in the Asp(274)-His(32) dyad can be attributed to a negative charge (as in D274E and D274G-Cl(-)), and the SSHB in the wild type enzyme provides minimal contribution to catalysis. These results represent novel insight for an Asp-His catalytic dyad and for the mechanism of phosphatidylinositol-specific phospholipase C.
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PMID:The catalytic role of aspartate in a short strong hydrogen bond of the Asp274-His32 catalytic dyad in phosphatidylinositol-specific phospholipase C can be substituted by a chloride ion. 1515 21

Aspergillus fumigatus causes a wide range of diseases that include mycotoxicosis, allergic reactions and systemic diseases (invasive aspergillosis) with high mortality rates. Pathogenicity depends on immune status of patients and fungal strain. There is no unique essential virulence factor for development of this fungus in the patient and its virulence appears to be under polygenetic control. The group of molecules and genes associated with the virulence of this fungus includes many cell wall components, such as beta-(1-3)-glucan, galactomannan, galactomannanproteins (Afmp1 and Afmp2), and the chitin synthetases (Chs; chsE and chsG), as well as others. Some genes and molecules have been implicated in evasion from the immune response, such as the rodlets layer (rodA/hyp1 gene) and the conidial melanin-DHN (pksP/alb1 gene). The detoxifying systems for Reactive Oxygen Species (ROS) by catalases (Cat1p and Cat2p) and superoxide dismutases (MnSOD and Cu, ZnSOD), had also been pointed out as essential for virulence. In addition, this fungus produces toxins (14 kDa diffusible substance from conidia, fumigaclavin C, aurasperon C, gliotoxin, helvolic acid, fumagilin, Asp-hemolysin, and ribotoxin Asp fI/mitogilin F/restrictocin), allergens (Asp f1 to Asp f23), and enzymatic proteins as alkaline serin proteases (Alp and Alp2), metalloproteases (Mep), aspartic proteases (Pep and Pep2), dipeptidyl-peptidases (DppIV and DppV), phospholipase C and phospholipase B (Plb1 and Plb2). These toxic substances and enzymes seems to be additive and/or synergistic, decreasing the survival rates of the infected animals due to their direct action on cells or supporting microbial invasion during infection. Adaptation ability to different trophic situations is an essential attribute of most pathogens. To maintain its virulence attributes A. fumigatus requires iron obtaining by hydroxamate type siderophores (ornitin monooxigenase/SidA), phosphorous obtaining (fos1, fos2, and fos3), signal transductional falls that regulate morphogenesis and/or usage of nutrients as nitrogen (rasA, rasB, rhbA), mitogen activated kinases (sakA codified MAP-kinase), AMPc-Pka signal transductional route, as well as others. In addition, they seem to be essential in this field the amino acid biosynthesis (cpcA and homoaconitase/lysF), the activation and expression of some genes at 37 degrees C (Hsp1/Asp f12, cgrA), some molecules and genes that maintain cellular viability (smcA, Prp8, anexins), etc. Conversely, knowledge about relationship between pathogen and immune response of the host has been improved, opening new research possibilities. The involvement of non-professional cells (endothelial, and tracheal and alveolar epithelial cells) and professional cells (natural killer or NK, and dendritic cells) in infection has been also observed. Pathogen Associated Molecular Patterns (PAMP) and Patterns Recognizing Receptors (PRR; as Toll like receptors TLR-2 and TLR-4) could influence inflammatory response and dominant cytokine profile, and consequently Th response to infec tion. Superficial components of fungus and host cell surface receptors driving these phenomena are still unknown, although some molecules already associated with its virulence could also be involved. Sequencing of A. fumigatus genome and study of gene expression during their infective process by using DNA microarray and biochips, promises to improve the knowledge of virulence of this fungus.
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PMID:Genes and molecules involved in Aspergillus fumigatus virulence. 1581 78

Thromboxane A(2) (TXA(2)) is an important lipid mediator generated during oxidative stress and implicated in ischemic neural injury. This autacoid was recently shown to partake in this injury process by directly inducing endothelial cytotoxicity. We explored the mechanisms for this TXA(2)-evoked neural microvascular endothelial cell death. Stable TXA(2) mimetics 5-heptenoic acid, 7-[6-(3-hydroxy-1-octenyl)-2-oxabicyclo[2.2.1]hept-5-yl]-[1R-[1alpha,4alpha,5beta(Z),6alpha,(1E,3S)]]-9,11-dedioxy-9alpha,11alpha-methanolpoxy (U-46619) [as well as [1S-[1alpha,2alpha(Z),3beta(1E,3S(*)),4alpha]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.1.1]-hept-2-yl]-5-heptenoic acid; I-BOP] induced a retinal microvascular degeneration in rat pups in vivo and in porcine retinal explants ex vivo and death of porcine brain endothelial cells (in culture). TXA(2) dependence of these effects was corroborated by antagonism using the selective TXA(2) receptor blocker (-)-6,8-difluoro-9-p-methyl-sulfonyl-benzyl-1,2,3,4-tetrahydrocarbazol-1-yl-acetic acid (L670596). In all cases, neurovascular endothelial cell death was prevented by pan-calpain and specific m-calpain inhibitors but not by caspase-3 or pan-caspase inhibitors. Correspondingly, TXA(2) (mimetics) augmented generation of known active m-calpain (but not mu-calpain) form and increased the activity of m-calpain (cleavage of fluorogenic substrate N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin; and of alpha-spectrin into specific fragments) but not of pan-caspase or specific caspase-3 (respectively, using sulforhodamine-Val-Arg-Asp-fluoromethyl ketone and detecting its active 17- and 12-kDa fragments). Interestingly, these effects were phospholipase C (PLC)-dependent [associated with increase in inositol triphosphate and inhibited by PLC blocker 1-[6-[[17beta-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122)] and required calcium but were not associated with increased intracellular calcium. U-46619-induced calpain activation resulted in translocation of Bax to the mitochondria, loss of polarization of the latter (using potentiometric probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide; JC-1) and in turn release of cytochrome c into the cytosol and depletion of cellular ATP; these effects were all blocked by calpain inhibitors. Overall, this work identifies (specifically) m-calpain as a dominant protease in TXA(2)-induced neurovascular endothelial cell death.
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PMID:Dominant role for calpain in thromboxane-induced neuromicrovascular endothelial cytotoxicity. 1621 79

ARK5 is a tumor progression-associated factor that is directly phosphorylated by AKT at serine 600 in the regulatory domain, but phosphorylation at the conserved threonine residue on the active T loop has been found to be required for its full activation. In this study, we identified serine/threonine protein kinase NDR2 as a protein kinase that phosphorylates and activates ARK5 during insulin-like growth factor (IGF)-1 signaling. Upon stimulation with IGF-1, NDR2 was found to directly phosphorylate the conserved threonine 211 on the active T loop of ARK5 and to promote cell survival and invasion of colorectal cancer cell lines through ARK5. During IGF-1 signaling, phosphorylation at three residues (threonine 75, serine 282, and threonine 442) was also found to be required for NDR2 activation. Among these three residues, phosphorylation of serine 282 seemed to be the most important for NDR2 activation (the same as for the mouse homologue) because its aspartic acid-converted mutant (NDR2/S282D) induced ARK5-mediated cell survival and invasion activities even in the absence of IGF-1. As in the mouse homologue, threonine 75 in NDR2 was required for interaction with S100B, and binding was in a calcium ion- and phospholipase C-gamma-dependent manner. We also found that PDK-1 plays an important role in NDR2 activation especially in the phosphorylation of threonine 442. Based on the results of this study, we report here that NDR2 is an upstream kinase of ARK5 that plays an essential role in tumor progression through ARK5.
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PMID:NDR2 acts as the upstream kinase of ARK5 during insulin-like growth factor-1 signaling. 1648 89

Thromboxane A(2) (TXA(2)), an unstable arachidonic acid metabolite, elicits diverse physiological/pathophysiological actions, including platelet aggregation and smooth muscle contraction. TXA(2) has been shown to be involved in allergies, modulation of acquired immunity, atherogenesis, neovascularization, and metastasis of cancer cells. The TXA(2) receptor (TP) communicates mainly with G(q) and G(13), resulting in phospholipase C activation and RhoGEF activation, respectively. In addition, TP couples with G(11), G(12), G(13), G(14), G(15), G(16), G(i), G(s) and G(h). TP is widely distributed in the body, and is expressed at high levels in thymus and spleen. The second extracellular loop of TP is an important ligand-binding site, and Asp(193) is a key amino acid. There are two alternatively spliced isoforms of TP, TPalpha and TPbeta, which differ only in their C-terminals. TPalpha and TPbeta communicate with different G proteins, and undergo hetero-dimerization, resulting in changes in intracellular traffic and receptor protein conformations. TP cross-talks with receptor tyrosine kinases, such as EGF receptor, to induce cell proliferation and differentiation. TP is glycosylated in the N-terminal region for recruitment to plasma membranes. Furthermore, TP conformation is changed by coupling to G proteins, showing several states of agonist binding. Finally, several drugs modify TP-mediated events; these include cyclooxygenase inhibitors, TXA(2) synthase inhibitors and TP antagonists. Some flavonoids of natural origin also have TP receptor antagonistic activity. Recent advances in TP research have clarified TXA(2)-mediated events in detail, and further study will supply more beneficial information about TXA(2) pathophysiology.
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PMID:Thromboxane A2: physiology/pathophysiology, cellular signal transduction and pharmacology. 1837 20

Class A (rhodopsin-like) G protein-coupled receptors possess conserved residues and motifs that are important for their specific activity. In the present study, we examined the role of residue Asp97(2.50) as well as residues Glu147(3.49), Arg148(3.50), and Tyr149(3.51) of the ERY motif on the functionality of the urotensin II receptor (UT). Mutations D97(2.50)A, R148(3.50)A, and R148(3.50)H abolished the ability of UT to activate phospholipase C, whereas mutations E147(3.49)A and Y149(3.51)A reduced the ability to activate PLC by 50%. None of the mutants exhibited constitutive activity. However, R148(3.50)A and R148(3.50)H promoted ERK1/2 activation, which was abolished by 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478), an inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase activity. Both these mutants were capable of directly activating EGFR, which confirmed that they activated the mitogen-activated protein kinase (MAPK) pathway by a Galpha(q/11)-independent transactivation of EGFR. The D97(2.50)A, R148(3.50)A, and R148(3.50)H mutants did not readily internalize and did not promote translocation or colocalize with beta-arrestin2-GFP. Finally, the agonist-induced internalization of the E147(3.49)A mutant receptor was significantly increased compared with wild-type receptor. This study highlights the major contribution of the conserved Asp(2.50) residue to the functionality of the UT receptor. The Arg residue in the ERY motif of UT is an important structural element in signaling crossroads that determine whether Galpha(q/11)-dependent and -independent events can occur.
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PMID:Mutational analysis of the conserved Asp2.50 and ERY motif reveals signaling bias of the urotensin II receptor. 1850 66


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