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1
Asokananthan, N., and M. H. Cake. "Stimulation of surfactant lipid secretion from fetal type II pneumocytes by gastrin-releasing peptide." American Journal of Physiology-Lung Cellular and Molecular Physiology 270, no. 3 (March 1, 1996): L331—L337. http://dx.doi.org/10.1152/ajplung.1996.270.3.l331.
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Gastrin-releasing peptide (GRP) and bombesin apparently enhance the rate of secretion of surfactant lipids from cultured fetal rat type II pneumocytes. This effect, evident within 1h of addition of the peptide, is concentration-dependent, with a maximal response at 3.0 nM. When the effect of GRP was assessed in comparison with other known secretagogues, it was found that, whereas GRP and isoproterenol were additive in their effect, there was no response to GRP in the presence of saturating concentrations of A23187 or phorbol 12-myristate 13-acetate. This suggests that the secretory response to GRP is via activation of Ca2+/calmodulin-dependent protein kinase and/or protein kinase C and is independent of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase. This conclusion is supported by the observation that the GRP-induced secretion is inhibited by calphostin C, an inhibitor of protein kinase C, but not by H-89, an inhibitor of cAMP-dependent protein kinase. The fact that GRP regulates surfactant secretion from type II pneumocytes suggests that it and/or related peptides may play a significant role in the physiological maturation of the lung.
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McLaughlin, Stuart, Steven O. Smith, Michael J. Hayman, and Diana Murray. "An Electrostatic Engine Model for Autoinhibition and Activation of the Epidermal Growth Factor Receptor (EGFR/ErbB) Family." Journal of General Physiology 126, no. 1 (June 13, 2005): 41–53. http://dx.doi.org/10.1085/jgp.200509274.
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We propose a new mechanism to explain autoinhibition of the epidermal growth factor receptor (EGFR/ErbB) family of receptor tyrosine kinases based on a structural model that postulates both their juxtamembrane and protein tyrosine kinase domains bind electrostatically to acidic lipids in the plasma membrane, restricting access of the kinase domain to substrate tyrosines. Ligand-induced dimerization promotes partial trans autophosphorylation of ErbB1, leading to a rapid rise in intracellular [Ca2+] that can activate calmodulin. We postulate the Ca2+/calmodulin complex binds rapidly to residues 645–660 of the juxtamembrane domain, reversing its net charge from +8 to −8 and repelling it from the negatively charged inner leaflet of the membrane. The repulsion has two consequences: it releases electrostatically sequestered phosphatidylinositol 4,5-bisphosphate (PIP2), and it disengages the kinase domain from the membrane, allowing it to become fully active and phosphorylate an adjacent ErbB molecule or other substrate. We tested various aspects of the model by measuring ErbB juxtamembrane peptide binding to phospholipid vesicles using both a centrifugation assay and fluorescence correlation spectroscopy; analyzing the kinetics of interactions between ErbB peptides, membranes, and Ca2+/calmodulin using fluorescence stop flow; assessing ErbB1 activation in Cos1 cells; measuring fluorescence resonance energy transfer between ErbB peptides and PIP2; and making theoretical electrostatic calculations on atomic models of membranes and ErbB juxtamembrane and kinase domains.
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VINTON, B. Betsy, L. Stacey WERTZ, Jaison JACOB, Joanna STEERE, M. Charles GRISHAM, S. David CAFISO, and J. Julianne SANDO. "Influence of lipid on the structure and phosphorylation of protein kinase C α substrate peptides." Biochemical Journal 330, no. 3 (March 15, 1998): 1433–42. http://dx.doi.org/10.1042/bj3301433.
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The structure and phosphorylation of two protein kinase C (PKC) α substrate peptides were investigated in varying lipid systems using enzyme activity assays and circular dichroism (CD) spectroscopy. The α-peptide, which exhibits the typical PKC α substrate motif and is based on the pseudosubstrate region of PKC α, was phosphorylated to a similar extent in bovine brain phosphatidylserine vesicles or diheptanoylphosphatidylcholine (PC7) micelles (both with 5 mol % 1,2-dioleoyl-sn-glycerol), whereas neuromodulin (NM)-peptide, which does not exhibit this motif by virtue of its primary structure, was phosphorylated to a much lesser extent in the PC7 micellar system. CD spectra of the peptides indicated that NM-peptide underwent a dramatic structural change in the presence of dimyristoylphosphatidylserine (DMPS) vesicles, whereas spectra acquired in PC7 micelles were similar to those acquired in buffer alone. No significant structural change was observed in the α-peptide in the presence of either lipid. PKC activity assays conducted with a series of NM-peptides successively substituted with nitroxide spin labels at each residue position suggested that several residues distal to the phosphorylation site are necessary for substrate recognition. The effect of these substitutions is not consistent with the binding of the NM-peptide to PKC in an extended structure, but is consistent with the binding of this peptide in a helical conformation. Furthermore, the docking of a helical NM-peptide to the substrate binding site of PKC suggests that the interaction is energetically feasible. These results suggest that PKC may recognize some non-linear substrate motifs and that lipid binding may convert a protein into a better PKC substrate.
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Mosior, M., and S. McLaughlin. "Peptides that mimic the pseudosubstrate region of protein kinase C bind to acidic lipids in membranes." Biophysical Journal 60, no. 1 (July 1991): 149–59. http://dx.doi.org/10.1016/s0006-3495(91)82038-0.
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STOYANOVA, Stefka, Ginette BULGARELLI-LEVA, Cornelia KIRSCH, Theodor HANCK, Reinhard KLINGER, Reinhard WETZKER, and Matthias P. WYMANN. "Lipid kinase and protein kinase activities of G-protein-coupled phosphoinositide 3-kinase γ: structure–activity analysis and interactions with wortmannin." Biochemical Journal 324, no. 2 (June 1, 1997): 489–95. http://dx.doi.org/10.1042/bj3240489.
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Signalling via seven transmembrane helix receptors can lead to a massive increase in cellular PtdIns(3,4,5)P3, which is critical for the induction of various cell responses and is likely to be produced by a trimeric G-protein-sensitive phosphoinositide 3-kinase (PI3Kγ). We show here that PI3Kγ is a bifunctional lipid kinase and protein kinase, and that both activities are inhibited by wortmannin at concentrations equal to those affecting the p85/p110α heterodimeric PI3K (IC50 approx. 2 nM). The binding of wortmannin to PI3Kγ, as detected by anti-wortmannin antisera, closely followed the inhibition of the kinase activities. Truncation of more than the 98 N-terminal amino acid residues from PI3Kγ produced proteins that were inactive in wortmannin binding and kinase assays. This suggests that regions apart from the core catalytic domain are important in catalysis and inhibitor interaction. The covalent reaction of wortmannin with PI3Kγ was prevented by preincubation with phosphoinositides, ATP and its analogues adenine and 5′-(4-fluorosulphonylbenzoyl)adenine. Proteolytic analysis of wortmannin-prelabelled PI3Kγ revealed candidate wortmannin-binding peptides around Lys-799. Replacement of Lys-799 by Arg through site-directed mutagenesis aborted the covalent reaction with wortmannin and the lipid kinase and protein kinase activities completely. The above illustrates that Lys-799 is crucial to the phosphate transfer reaction and wortmannin reactivity. Parallel inhibition of the PI3Kγ-associated protein kinase and lipid kinase by wortmannin and by the Lys-799 → Arg mutation reveals that both activities are inherent in the PI3Kγ polypeptide.
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Hemmings, Hugh C., and Anna I. B. Adamo. "Activation of Endogenous Protein Kinase C by Halothane in Synaptosomes." Anesthesiology 84, no. 3 (March 1, 1996): 652–62. http://dx.doi.org/10.1097/00000542-199603000-00021.
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Background Protein kinase C is a signal transducing enzyme that is an important regulator of multiple physiologic processes and a potential molecular target for general anesthetic actions. However, the results of previous studies of the effects of general anesthetics on protein kinase C activation in vitro have been inconsistent. Methods The effects of halothane on endogenous brain protein kinase C activation were analyzed in isolated rat cerebrocortical nerve terminals (synaptosomes) and in synaptic membranes. Protein kinase C activation was monitored by the phosphorylation of MARCKS, a specific endogenous substrate. Results Halothane stimulated basal Ca2+ dependent phosphorylation of MARCKS (Mr = 83,000) in lysed synaptic membranes (2.1-fold; P< 0.01) and in intact synaptosomes (1.4-fold; P< 0.01). The EC50 for stimulation of MARCKS phosphorylation by halothene in synaptic membranes was 1.8 vol%. A selective peptide protein kinase C inhibitor, but not a protein phosphatase inhibitor (okadaic acid) or a peptide inhibitor of Ca2+/calmodulin-dependent protein kinase II, another Ca2+/-dependent signal transducing enzyme, blocked halothane-stimulated MARCKS phosphorylation in synaptic membranes. Halothane did not affect the phosphorylation of synapsin 1, a synaptic vesicle-associated protein substrate for Ca2+/calmodulin-dependent protein kinase II and AMP-dependent protein kinase, in synaptic membranes or intact synaptosomes subjected to KC1-evoked depolarization. However, halothane stimulated synapsin 1 phosphorylation evoked by ionomycin (a Ca2+ ionophore that permeabilizes membranes to Ca2+) in intact synaptosomes. Conclusions Halothane acutely stimulated basal protein kinase C activity in synaptosomes when assayed with endogenous nerve terminal substrates, lipids, and protein kinase C. This effect appeared to be selective for protein kinases C, because two other structurally similar second messenger-regulated protein kinases were not affected. Direct determinations of anesthetic effects on endogenous protein kinase C activation, translocation, and/or down-regulation are necessary to determine the ultimate effect of anesthetics on the protein kinase C signaling pathway in intact cells.
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Divecha, N., O. Truong, J. J. Hsuan, K. A. Hinchliffe, and R. F. Irvine. "The cloning and sequence of the C isoform of PtdIns4P 5-kinase." Biochemical Journal 309, no. 3 (August 1, 1995): 715–19. http://dx.doi.org/10.1042/bj3090715.
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In this study we describe the purification and sequencing of the C isoform of platelet PtdIns4P 5-kinase. Subsequently a cDNA was isolated from a human circulating-leucocyte library, which when sequenced was shown to contain all of the peptides identified in the purified protein. In addition, expression of this cDNA in bacteria led to the production of a protein which was recognized by specific monoclonal antibodies raised to the bovine brain enzyme [Brooksbank, Hutchings, Butcher, Irvine and Divecha (1993) Biochem. J. 291, 77-82] and also led to the appearance of PtdIns4P 5-kinase activity in the bacterial lysates. Interestingly, the cDNA showed no similarity to any of the previously cloned inositide kinases. A search of the DNA databases showed that two proteins from Saccharomyces cerevisiae shared close similarity to this enzyme, one of which, the mss4 gene product, has been implicated in the yeast inositol lipid pathway. These data suggest that the PtdIns4P 5-kinases are a new family of inositide kinases unrelated to the previously cloned phosphoinositide 3/4-kinases.
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Knopp, Michael, Arianne M. Babina, Jónína S. Gudmundsdóttir, Martin V. Douglass, M. Stephen Trent, and Dan I. Andersson. "A novel type of colistin resistance genes selected from random sequence space." PLOS Genetics 17, no. 1 (January 7, 2021): e1009227. http://dx.doi.org/10.1371/journal.pgen.1009227.
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Antibiotic resistance is a rapidly increasing medical problem that severely limits the success of antibiotic treatments, and the identification of resistance determinants is key for surveillance and control of resistance dissemination. Horizontal transfer is the dominant mechanism for spread of resistance genes between bacteria but little is known about the original emergence of resistance genes. Here, we examined experimentally if random sequences can generate novel antibiotic resistance determinants de novo. By utilizing highly diverse expression libraries encoding random sequences to select for open reading frames that confer resistance to the last-resort antibiotic colistin in Escherichia coli, six de novo colistin resistance conferring peptides (Dcr) were identified. The peptides act via direct interactions with the sensor kinase PmrB (also termed BasS in E. coli), causing an activation of the PmrAB two-component system (TCS), modification of the lipid A domain of lipopolysaccharide and subsequent colistin resistance. This kinase-activation was extended to other TCS by generation of chimeric sensor kinases. Our results demonstrate that peptides with novel activities mediated via specific peptide-protein interactions in the transmembrane domain of a sensory transducer can be selected de novo, suggesting that the origination of such peptides from non-coding regions is conceivable. In addition, we identified a novel class of resistance determinants for a key antibiotic that is used as a last resort treatment for several significant pathogens. The high-level resistance provided at low expression levels, absence of significant growth defects and the functionality of Dcr peptides across different genera suggest that this class of peptides could potentially evolve as bona fide resistance determinants in natura.
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Mozaffari, Saghar, Emira Bousoik, Farideh Amirrad, Robert Lamboy, Melissa Coyle, Ryley Hall, Abdulaziz Alasmari, Parvin Mahdipoor, Keykavous Parang, and Hamidreza Montazeri Aliabadi. "Amphiphilic Peptides for Efficient siRNA Delivery." Polymers 11, no. 4 (April 17, 2019): 703. http://dx.doi.org/10.3390/polym11040703.
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A number of amphiphilic cyclic peptides—[FR]4, [WR]5, and [WK]5—containing hydrophobic and positively-charged amino acids were synthesized by Fmoc/tBu solid-phase peptide methods and evaluated for their efficiency in intracellular delivery of siRNA to triple-negative breast cancer cell lines, MDA-MB-231 and MDA-MB-468, in the presence and absence of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). Among the peptides, [WR]5, which contains alternate tryptophan (W) and arginine (R) residues, was found to be the most efficient in the delivery of siRNA by improving the delivery by more than 3-fold when compared to other synthesized cyclic peptides that were not efficient. The data also showed that co-formulation of [WR]5 with lipid DOPE significantly enhanced the efficiency of siRNA delivery by up to ~2-fold compared to peptide alone. Based on the data indicating the efficiency of [WR]5 in siRNA delivery, peptides containing arginine residues on the ring and tryptophan residues on the side chain, [R6K]W6 and [R5K]W5, were also evaluated, and demonstrated improved delivery of siRNA. The presence of DOPE again enhanced the siRNA delivery in most cases. [WR]5, [R5K]W5, and [R6K]W6 did not show any significant toxicity in MDA-MB-231, MDA-MB-468, and AU565 WT cells at N/P ratios of 20:1 or less, in the presence and absence of DOPE. Silencing of kinesin spindle protein (KSP) and Janus kinase 2 (JAK2) was evaluated in MDA-MB-231 cells in the presence of the peptides. The addition of DOPE significantly enhanced the silencing efficiency for all selected peptides. In conclusion, peptides containing tryptophan and arginine residues were found to enhance siRNA delivery and to generate silencing of targeted proteins in the presence of DOPE.
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Wei, Ying, Xiuwei Yang, Qiumei Liu, John A. Wilkins, and Harold A. Chapman. "A Role for Caveolin and the Urokinase Receptor in Integrin-mediated Adhesion and Signaling." Journal of Cell Biology 144, no. 6 (March 22, 1999): 1285–94. http://dx.doi.org/10.1083/jcb.144.6.1285.
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The assembly of signaling molecules surrounding the integrin family of adhesion receptors remains poorly understood. Recently, the membrane protein caveolin was found in complexes with β1 integrins. Caveolin binds cholesterol and several signaling molecules potentially linked to integrin function, e.g., Src family kinases, although caveolin has not been directly implicated in integrin-dependent adhesion. Here we report that depletion of caveolin by antisense methodology in kidney 293 cells disrupts the association of Src kinases with β1 integrins resulting in loss of focal adhesion sites, ligand-induced focal adhesion kinase (FAK) phosphorylation, and adhesion. The nonintegrin urokinase receptor (uPAR) associates with and stabilizes β1 integrin/caveolin complexes. Depletion of caveolin in uPAR-expressing 293 cells also disrupts uPAR/integrin complexes and uPAR-dependent adhesion. Further, β1 integrin/caveolin complexes could be disassociated by uPAR-binding peptides in both uPAR-transfected 293 cells and human vascular smooth muscle cells. Disruption of complexes by peptides in intact smooth muscle cells blocks the association of Src family kinases with β1 integrins and markedly impairs their migration on fibronectin. We conclude that ligand-induced signaling necessary for normal β1 integrin function requires caveolin and is regulated by uPAR. Caveolin and uPAR may operate within adhesion sites to organize kinase-rich lipid domains in proximity to integrins, promoting efficient signal transduction.
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Liu, Aizhuo, Dexin Sui, Dianqing Wu, and Jian Hu. "The activation loop of PIP5K functions as a membrane sensor essential for lipid substrate processing." Science Advances 2, no. 11 (November 2016): e1600925. http://dx.doi.org/10.1126/sciadv.1600925.
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Phosphatidylinositol 4-phosphate 5-kinase (PIP5K), a representative member of the phosphatidylinositol phosphate kinase (PIPK) family, is a major enzyme that biosynthesizes the signaling molecule PI(4,5)P2 (phosphatidylinositol 4,5-bisphosphate) in eukaryotic cells. The stringent specificity toward lipid substrates and the high sensitivity to the membrane environment strongly suggest a membrane-sensing mechanism, but the underlying structural basis is still largely unknown. We present a nuclear magnetic resonance (NMR) study on a peptide commensurate with a PIP5K’s activation loop, which has been reported to be a determinant of lipid substrate specificity and subcellular localization of PIP5K. Although the activation loop is severely disordered in the crystal structure of PIP5K, the NMR experiments showed that the largely unstructured peptide folded into an amphipathic helix upon its association with the 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) micellar surface. Systematic mutagenesis and functional assays further demonstrated the crucial roles of the amphipathic helix and its hydrophobic surface in kinase activity and membrane-sensing function, supporting a working model in which the activation loop is a critical structural module conferring a membrane-sensing mechanism on PIP5K. The activation loop, surprisingly functioning as a membrane sensor, represents a new paradigm of kinase regulation by the activation loop through protein-membrane interaction, which also lays a foundation on the regulation of PIP5K (and other PIPKs) by membrane lipids for future studies.
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Chapnik, Nava, Yoni Genzer, Avraham Ben-Shimon, Masha Y. Niv, and Oren Froy. "AMPK-derived peptides reduce blood glucose levels but lead to fat retention in the liver of obese mice." Journal of Endocrinology 221, no. 1 (January 29, 2014): 89–99. http://dx.doi.org/10.1530/joe-13-0625.
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AMP-activated protein kinase (AMPK) is a regulator of energy balance at both the cellular and the whole-body levels. Direct activation of AMPK has been highlighted as a potential novel, and possibly safer, alternative to treat type II diabetes and obesity. In this study, we aimed to design and characterize novel peptides that mimic the αG region of the α2 AMPK catalytic domain to modulate its activity by inhibiting interactions between AMPK domains or other interacting proteins. The derived peptides were tested in vivo and in tissue culture. The computationally predicted structure of the free peptide with the addition of the myristoyl (Myr) or acetyl (Ac) moiety closely resembled the protein structure that it was designed to mimic. Myr-peptide and Ac-peptide activated AMPK in muscle cells and led to reduced adipose tissue weight, body weight, blood glucose levels, insulin levels, and insulin resistance index, as expected from AMPK activation. In addition, triglyceride, cholesterol, leptin, and adiponectin levels were also lower, suggesting increased adipose tissue breakdown, a result of AMPK activation. On the other hand, liver weight and liver lipid content increased due to fat retention. We could not find an elevated pAMPK:AMPK ratio in the liver in vivo or in hepatocytes ex vivo, suggesting that the peptide does not lead to AMPK activation in hepatocytes. The finding that an AMPK-derived peptide leads to the activation of AMPK in muscle cells and in adipose tissue and leads to reduced glucose levels in obese mice, but to fat accumulation in the liver, demonstrates the differential effect of AMPK modulation in various tissues.
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SCHMITZ-PEIFFER, Carsten, Carol L. BROWNE, and Trevor J. BIDEN. "Characterization of two forms of protein kinase C α, with different substrate specificities, from skeletal muscle." Biochemical Journal 320, no. 1 (November 15, 1996): 207–14. http://dx.doi.org/10.1042/bj3200207.
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We have investigated protein kinase C (PKC) in skeletal muscle cytosol and demonstrated the presence of two major activities. These did not correspond to different PKC isoenzymes but seemed to represent two species of PKC α as deduced by: elution during hydroxyapatite chromatography at KH2PO4 concentrations expected of PKC α; detection of the two species by three specific but unrelated anti-(PKC α) antibodies; immunodepletion of both activities with anti-(PKC α) antibody; and demonstration of identical requirements of both Ca2+ ions and lipid for activation. These species, termed PKC α1 and PKC α2, phosphorylated the modified conventional PKC pseudosubstrate peptide (19–31, Ser-25) equally well. Importantly, however, the activities differed in that PKC α1 phosphorylated histone IIIS, and also peptides derived from the EGF receptor and glycogen synthase, to a much greater extent than did PKC α2. Similarly, incubation of crude muscle extracts with either PKC α1 or α2 gave rise to different protein phosphorylation patterns. The involvement of proteolysis, dephosphorylation or oxidative modification in the interconversion of PKC α1 and PKC α2 during preparation was ruled out. Although some PKC-binding proteins were detected in overlay assays, their presence did not explain the anomalous PKC α2 activity. The results suggest that a modification of PKC α in situ limits its substrate specificity, and indicate an additional level of control of the kinase that may be a site for modulation of PKC-mediated signal transduction.
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Su, Wen-Min, Gil-Soo Han, Prabuddha Dey, and George M. Carman. "Protein kinase A phosphorylates the Nem1–Spo7 protein phosphatase complex that regulates the phosphorylation state of the phosphatidate phosphatase Pah1 in yeast." Journal of Biological Chemistry 293, no. 41 (September 10, 2018): 15801–14. http://dx.doi.org/10.1074/jbc.ra118.005348.
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The Nem1–Spo7 protein phosphatase plays a role in lipid synthesis by controlling the membrane localization of Pah1, the diacylglycerol-producing phosphatidate (PA) phosphatase that is crucial for the synthesis of triacylglycerol in the yeast Saccharomyces cerevisiae. By dephosphorylating Pah1, Nem1–Spo7 facilitates its translocation to the nuclear/endoplasmic reticulum membrane for catalytic activity. Like its substrate Pah1, Nem1–Spo7 is phosphorylated in the cell, but the specific protein kinases involved remain to be identified. In this study, we demonstrate that the Nem1–Spo7 complex is phosphorylated by protein kinase A (PKA), which is associated with active cell growth, metabolic activity, and membrane phospholipid synthesis. In vitro phosphorylation of purified Nem1–Spo7 and of their synthetic peptides revealed that both subunits of the phosphatase complex are PKA substrates. Using phosphoamino acid and phosphopeptide-mapping analyses coupled with site-directed mutagenesis, we identified Ser-140 and Ser-210 of Nem1 and Ser-28 of Spo7 as PKA-targeted phosphorylation sites. Immunodetection of the phosphatase complex from the cell with anti-PKA substrate antibody confirmed the in vivo phosphorylations of Nem1 and Spo7 on the serine residues. Lipid-labeling analysis of cells bearing phosphorylation-deficient alleles of NEM1 and SPO7 indicated that the PKA phosphorylation of the phosphatase complex stimulates phospholipid synthesis and attenuates the synthesis of triacylglycerol. This work advances the understanding of how PKA-mediated posttranslational modifications of Nem1 and Spo7 regulate lipid synthesis in yeast.
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Frühbeck, Gema, Leire Méndez-Giménez, José-Antonio Fernández-Formoso, Secundino Fernández, and Amaia Rodríguez. "Regulation of adipocyte lipolysis." Nutrition Research Reviews 27, no. 1 (May 28, 2014): 63–93. http://dx.doi.org/10.1017/s095442241400002x.
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In adipocytes the hydrolysis of TAG to produce fatty acids and glycerol under fasting conditions or times of elevated energy demands is tightly regulated by neuroendocrine signals, resulting in the activation of lipolytic enzymes. Among the classic regulators of lipolysis, adrenergic stimulation and the insulin-mediated control of lipid mobilisation are the best known. Initially, hormone-sensitive lipase (HSL) was thought to be the rate-limiting enzyme of the first lipolytic step, while we now know that adipocyte TAG lipase is the key enzyme for lipolysis initiation. Pivotal, previously unsuspected components have also been identified at the protective interface of the lipid droplet surface and in the signalling pathways that control lipolysis. Perilipin, comparative gene identification-58 (CGI-58) and other proteins of the lipid droplet surface are currently known to be key regulators of the lipolytic machinery, protecting or exposing the TAG core of the droplet to lipases. The neuroendocrine control of lipolysis is prototypically exerted by catecholaminergic stimulation and insulin-induced suppression, both of which affect cyclic AMP levels and hence the protein kinase A-mediated phosphorylation of HSL and perilipin. Interestingly, in recent decades adipose tissue has been shown to secrete a large number of adipokines, which exert direct effects on lipolysis, while adipocytes reportedly express a wide range of receptors for signals involved in lipid mobilisation. Recently recognised mediators of lipolysis include some adipokines, structural membrane proteins, atrial natriuretic peptides, AMP-activated protein kinase and mitogen-activated protein kinase. Lipolysis needs to be reanalysed from the broader perspective of its specific physiological or pathological context since basal or stimulated lipolytic rates occur under diverse conditions and by different mechanisms.
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Taskén, K., and A. J. Stokka. "The molecular machinery for cAMP-dependent immunomodulation in T-cells." Biochemical Society Transactions 34, no. 4 (July 21, 2006): 476–79. http://dx.doi.org/10.1042/bst0340476.
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cAMP inhibits Src-family kinase signalling by PKA (protein kinase A)-mediated phosphorylation and activation of Csk (C-terminal Src kinase). The PKA type I–Csk pathway is assembled and localized in membrane microdomains (lipid rafts) and regulates immune responses activated through the TCR (T-cell receptor). PKA type I is targeted to the TCR–CD3 complex during T-cell activation via an AKAP (A-kinase-anchoring protein) that serves as a scaffold for the cAMP–PKA/Csk pathway in lipid rafts of the plasma membrane during T-cell activation. Displacement of PKA by anchoring disruption peptides prevents cAMP/PKA type I-mediated inhibition of T-cell activation. These findings provide functional evidence that PKA type I regulation of T-cell responses is dependent on AKAP anchoring. Furthermore, we show that upon TCR/CD28 co-ligation, β-arrestin in complex with PDE4 (phosphodiesterase 4) is recruited to lipid rafts. The CD28-mediated recruitment of PDE4 to lipid rafts potentiates T-cell immune responses and counteracts the local, TCR-induced production of cAMP that produces negative feedback in the absence of a co-receptor stimulus. The specific recruitment of PDE4 thus serves to abrogate the negative feedback by cAMP which is elicited in the absence of a co-receptor stimulus.
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Nuñez-Durán, Esther, Belén Chanclón, Silva Sütt, Joana Real, Hanns-Ulrich Marschall, Ingrid Wernstedt Asterholm, Emmelie Cansby, and Margit Mahlapuu. "Protein kinase STK25 aggravates the severity of non-alcoholic fatty pancreas disease in mice." Journal of Endocrinology 234, no. 1 (July 2017): 15–27. http://dx.doi.org/10.1530/joe-17-0018.
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Characterising the molecular networks that negatively regulate pancreatic β-cell function is essential for understanding the underlying pathogenesis and developing new treatment strategies for type 2 diabetes. We recently identified serine/threonine protein kinase 25 (STK25) as a critical regulator of ectopic fat storage, meta-inflammation, and fibrosis in liver and skeletal muscle. Here, we assessed the role of STK25 in control of progression of non-alcoholic fatty pancreas disease in the context of chronic exposure to dietary lipids in mice. We found that overexpression of STK25 in high-fat-fed transgenic mice aggravated diet-induced lipid storage in the pancreas compared with that of wild-type controls, which was accompanied by exacerbated pancreatic inflammatory cell infiltration, stellate cell activation, fibrosis and apoptosis. Pancreas of Stk25 transgenic mice also displayed a marked decrease in islet β/α-cell ratio and alteration in the islet architecture with an increased presence of α-cells within the islet core, whereas islet size remained similar between genotypes. After a continued challenge with a high-fat diet, lower levels of fasting plasma insulin and C-peptide, and higher levels of plasma leptin, were detected in Stk25 transgenic vs wild-type mice. Furthermore, the glucose-stimulated insulin secretion was impaired in high-fat-fed Stk25 transgenic mice during glucose tolerance test, in spite of higher net change in blood glucose concentrations compared with wild-type controls, suggesting islet β-cell dysfunction. In summary, this study unravels a role for STK25 in determining the susceptibility to diet-induced non-alcoholic fatty pancreas disease in mice in connection to obesity. Our findings highlight STK25 as a potential drug target for metabolic disease.
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Solinas, G., S. Summermatter, D. Mainieri, M. Gubler, J. P. Montani, J. Seydoux, S. R. Smith, and A. G. Dulloo. "Corticotropin-Releasing Hormone Directly Stimulates Thermogenesis in Skeletal Muscle Possibly through Substrate Cycling between de Novo Lipogenesis and Lipid Oxidation." Endocrinology 147, no. 1 (January 1, 2006): 31–38. http://dx.doi.org/10.1210/en.2005-1033.
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The mechanisms by which CRH and related peptides (i.e. the CRH/urocortin system) exert their control over thermogenesis and weight regulation have until now focused only upon their effects on brain centers controlling sympathetic outflow. Using a method that involves repeated oxygen uptake determinations in intact mouse skeletal muscle, we report here that CRH can act directly on skeletal muscle to stimulate thermogenesis, an effect that is more pronounced in oxidative than in glycolytic muscles and that can be inhibited by a selective CRH-R2 antagonist or blunted by a nonselective CRH receptor antagonist. This thermogenic effect of CRH can also be blocked by interference along pathways of de novo lipogenesis and lipid oxidation, as well as by inhibitors of phosphatidylinositol 3-kinase or AMP-activated protein kinase. Taken together, these studies demonstrate that CRH can directly stimulate thermogenesis in skeletal muscle, and in addition raise the possibility that this thermogenic effect, which requires both phosphatidylinositol 3-kinase and AMP-activated protein kinase signaling, might occur via substrate cycling between de novo lipogenesis and lipid oxidation. The effect of CRH in directly stimulating thermogenesis in skeletal muscle underscores a potentially important peripheral role for the CRH/urocortin system in the control of thermogenesis in this tissue, in its protection against excessive intramyocellular lipid storage, and hence against skeletal muscle lipotoxicity and insulin resistance.
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Park, Junsoo, Nam-Hyuk Cho, Joong-Kook Choi, Pinghui Feng, Joonho Choe, and Jae U. Jung. "Distinct Roles of Cellular Lck and p80 Proteins in Herpesvirus Saimiri Tip Function on Lipid Rafts." Journal of Virology 77, no. 16 (August 15, 2003): 9041–51. http://dx.doi.org/10.1128/jvi.77.16.9041-9051.2003.
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ABSTRACT Lipid rafts are proposed to function as platforms for both receptor signaling and trafficking. Following interaction with antigenic peptides, the T-cell receptor (TCR) rapidly translocates to lipid rafts, where it transmits signals and subsequently undergoes endocytosis. The Tip protein of herpesvirus saimiri (HVS), which is a T-lymphotropic tumor virus, interacts with cellular Lck tyrosine kinase and p80, a WD domain-containing endosomal protein. Interaction of Tip with p80 induces enlarged vesicles and recruits Lck and TCR complex into these vesicles for trafficking. We report here that Tip is constitutively present in lipid rafts and that Tip interaction with p80 but not with Lck is necessary for its efficient localization in lipid rafts. The Tip-Lck interaction was required for recruitment of the TCR complex to lipid rafts, and the Tip-p80 interaction was critical for the aggregation and internalization of lipid rafts. These results suggest the potential mechanism for Tip-mediated TCR downregulation: Tip interacts with Lck to recruit TCR complex to lipid rafts, and it subsequently interacts with p80 to initiate the aggregation and internalization of the lipid raft domain and thereby downregulate the TCR complex. Thus, the signaling and targeting functions of HVS Tip rely on two functionally and genetically separable mechanisms that independently target cellular Lck tyrosine kinase and p80 endosomal protein.
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Cybulski, Larisa Estefanía, Joost Ballering, Anastassiia Moussatova, Maria Eugenia Inda, Daniela B. Vazquez, Tsjerk A. Wassenaar, Diego de Mendoza, D. Peter Tieleman, and J. Antoinette Killian. "Activation of the bacterial thermosensor DesK involves a serine zipper dimerization motif that is modulated by bilayer thickness." Proceedings of the National Academy of Sciences 112, no. 20 (May 4, 2015): 6353–58. http://dx.doi.org/10.1073/pnas.1422446112.
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DesK is a bacterial thermosensor protein involved in maintaining membrane fluidity in response to changes in environmental temperature. Most likely, the protein is activated by changes in membrane thickness, but the molecular mechanism of sensing and signaling is still poorly understood. Here we aimed to elucidate the mode of action of DesK by studying the so-called “minimal sensor DesK” (MS-DesK), in which sensing and signaling are captured in a single transmembrane segment. This simplified version of the sensor allows investigation of membrane thickness-dependent protein–lipid interactions simply by using synthetic peptides, corresponding to the membrane-spanning parts of functional and nonfunctional mutants of MS-DesK incorporated in lipid bilayers with varying thicknesses. The lipid-dependent behavior of the peptides was investigated by circular dichroism, tryptophan fluorescence, and molecular modeling. These experiments were complemented with in vivo functional studies on MS-DesK mutants. Based on the results, we constructed a model that suggests a new mechanism for sensing in which the protein is present as a dimer and responds to an increase in bilayer thickness by membrane incorporation of a C-terminal hydrophilic motif. This results in exposure of three serines on the same side of the transmembrane helices of MS-DesK, triggering a switching of the dimerization interface to allow the formation of a serine zipper. The final result is activation of the kinase state of MS-DesK.
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Vázquez, María-Isabel, and Mariano Esteban. "Identification of Functional Domains in the 14-Kilodalton Envelope Protein (A27L) of Vaccinia Virus." Journal of Virology 73, no. 11 (November 1, 1999): 9098–109. http://dx.doi.org/10.1128/jvi.73.11.9098-9109.1999.
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ABSTRACT The mechanism of entry of vaccinia virus (VV) into cells is still a poorly understood process. A 14-kDa protein (encoded by the A27L gene) in the envelope of intracellular mature virus (IMV) has been implicated in virus-cell attachment, virus-cell fusion, and virus release from cells. We have previously described the structural organization of the VV 14-kDa protein, consisting of a triple-stranded coiled-coil region responsible for oligomer formation and a predicted Leu zipper-like third alpha helix with an important role in the interaction with a 21-kDa membrane protein (encoded by the A17L gene) thought to anchor the 14-kDa protein to the envelope of IMV (M.-I. Vázquez, G. Rivas, D. Cregut, L. Serrano, and M. Esteban, J. Virol. 72:10126–10137, 1998). To identify the functional domains important for virus entry and release, we have generated VV recombinants containing a copy of the A27L gene regulated by the lacIoperator-repressor system of Escherichia coli (VVIndA27L) in the thymidine kinase locus and a mutant form of the A27L gene in the hemagglutinin locus but expressed constitutively under the control of an early-late VV promoter. Cells infected with a VV recombinant that expresses a mutant 14-kDa form lacking the first 29 amino acids at the N terminus failed to form extracellular enveloped virus (EEV). Fusion-from-without assays with purified virus confirmed that the fusion process was mediated by the 14-kDa protein and the fusion domain to be contained within amino acids 29 to 43 of the N-terminal region. Competitive inhibition of the infection process with soluble heparin and synthetic peptides and in vitro experiments with purified mutant proteins identified the heparin binding domain within amino acids 21 to 33, suggesting that this domain is involved in virus-cell binding via heparan sulfate. Thus, the N terminus of the 14-kDa protein contains a heparin binding domain, a fusion domain, and a domain responsible for interacting with proteins or lipids in the Golgi stacks for EEV formation and virus spread.
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Valicherla, Guru Raghavendra, Zakir Hossain, Sushil K. Mahata, and Jiaur R. Gayen. "Pancreastatin is an endogenous peptide that regulates glucose homeostasis." Physiological Genomics 45, no. 22 (November 15, 2013): 1060–71. http://dx.doi.org/10.1152/physiolgenomics.00131.2013.
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Pancreastatin (PST) is a regulatory peptide containing 49 amino acids, first isolated from porcine pancreas. Intracellular and extracellular processing of the prohormone Chromogranin A (Chga) results various bioactive peptides of which PST has dysglycemic activity. PST regulates glucose, lipid, and protein metabolism in liver and adipose tissues. It also regulates the secretion of leptin and expression of leptin and uncoupling protein 2 in adipose tissue. In Chga knockout mice, PST induces gluconeogenesis in the liver. PST reduces glucose uptake in mice hepatocytes and adipocytes. In rat hepatocytes, PST induces glycogenolysis and glycolysis and inhibits glycogen synthesis. In rat adipocytes, PST inhibits lactate production and lipogenesis. These metabolic effects are confirmed in humans. In the dual signaling mechanism of PST receptor, mostly PST activates Gαq/11 protein leads to the activation of phospholipase C β3-isoform, therefore increasing cytoplasmic free calcium and stimulating protein kinase C. PST inhibits the cell growth in rat HTC hepatoma cells, mediated by nitric oxide and cyclic GMP production. Elevated levels of PST correlating with catecholamines have been found in gestational diabetes and essential hypertension. Rise in the blood PST level in Type 2 diabetes suggests that PST is a negative regulator of insulin sensitivity and glucose homeostasis.
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Jazvinšćak Jembrek, Maja, Patrick R. Hof, and Goran Šimić. "Ceramides in Alzheimer’s Disease: Key Mediators of Neuronal Apoptosis Induced by Oxidative Stress and AβAccumulation." Oxidative Medicine and Cellular Longevity 2015 (2015): 1–17. http://dx.doi.org/10.1155/2015/346783.
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Alzheimer’s disease (AD), the most common chronic and progressive neurodegenerative disorder, is characterized by extracellular deposits of amyloidβ-peptides (Aβ) and intracellular deposits of hyperphosphorylated tau (phospho-tau) protein. Ceramides, the major molecules of sphingolipid metabolism and lipid second messengers, have been associated with AD progression and pathology via Aβgeneration. Enhanced levels of ceramides directly increase Aβthrough stabilization ofβ-secretase, the key enzyme in the amyloidogenic processing of Aβprecursor protein (APP). As a positive feedback loop, the generated oligomeric and fibrillar Aβinduces a further increase in ceramide levels by activating sphingomyelinases that catalyze the catabolic breakdown of sphingomyelin to ceramide. Evidence also supports important role of ceramides in neuronal apoptosis. Ceramides may initiate a cascade of biochemical alterations, which ultimately leads to neuronal death by diverse mechanisms, including depolarization and permeabilization of mitochondria, increased production of reactive oxygen species (ROS), cytochrome c release, Bcl-2 depletion, and caspase-3 activation, mainly by modulating intracellular signalling, particularly along the pathways related to Akt/PKB kinase and mitogen-activated protein kinases (MAPKs). This review summarizes recent findings related to the role of ceramides in oxidative stress-driven neuronal apoptosis and interplay with Aβin the cascade of events ending in neuronal degeneration.
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Robia, Seth L., Misuk Kang, and Jeffery W. Walker. "Novel determinant of PKC-ε anchoring at cardiac Z-lines." American Journal of Physiology-Heart and Circulatory Physiology 289, no. 5 (November 2005): H1941—H1950. http://dx.doi.org/10.1152/ajpheart.01111.2004.
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The Z-line represents a critical link between the transverse tubule network and cytoskeleton of cardiac cells with a role in anchoring structural proteins, ion channels, and signaling molecules. Protein kinase C-ε (PKC-ε) regulates cardiac excitability, cardioprotection, and growth, possibly as a consequence of translocation to the Z-line/T tubule region. To investigate the mechanism of PKC-ε translocation, fragments of its NH2-terminal 144-amino acid variable domain, εV1, were fused with green fluorescent protein and evaluated by quantitative Fourier image analysis of decorated myocytes. Deletion of 23 amino acids from the NH2-terminus of εV1, including an EAVSLKPT motif important for binding to a receptor for activated C kinase (RACK2), reduced but did not abolish Z-line binding. Further deletions of up to 84 amino acids from the NH2-terminus of εV1 also did not prevent Z-line decoration. However, deletions of residues 85–144 from the COOH-terminus strongly reduced Z-line binding. COOH-terminal deletions caused 2.5-fold greater loss of binding energy (ΔΔG) than did NH2-terminal deletions. Synthetic peptides derived from these regions modulated εV1 binding and cardiac myocyte function, but also revealed considerable heterogeneity within populations of adult cardiac myocytes. The COOH-terminal subdomain important for Z-line anchoring maps to a surface in the εV1 crystal structure that complements the eight-amino acid RACK2 binding site and two previously identified membrane docking motifs. PKC-ε anchoring at the cardiac Z-line/T tubule appears to rely on multiple points of contact probably involving protein-lipid and protein-protein interactions.
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Turovsky, Egor A., Maria V. Turovskaya, and Vladimir V. Dynnik. "Deregulation of Ca2+-Signaling Systems in White Adipocytes, Manifested as the Loss of Rhythmic Activity, Underlies the Development of Multiple Hormonal Resistance at Obesity and Type 2 Diabetes." International Journal of Molecular Sciences 22, no. 10 (May 12, 2021): 5109. http://dx.doi.org/10.3390/ijms22105109.
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Various types of cells demonstrate ubiquitous rhythmicity registered as simple and complex Ca2+-oscillations, spikes, waves, and triggering phenomena mediated by G-protein and tyrosine kinase coupled receptors. Phospholipase C/IP3-receptors (PLC/IP3R) and endothelial NO-synthase/Ryanodine receptors (NOS/RyR)–dependent Ca2+ signaling systems, organized as multivariate positive feedback generators (PLC-G and NOS-G), underlie this rhythmicity. Loss of rhythmicity at obesity may indicate deregulation of these signaling systems. To issue the impact of cell size, receptors’ interplay, and obesity on the regulation of PLC-G and NOS-G, we applied fluorescent microscopy, immunochemical staining, and inhibitory analysis using cultured adipocytes of epididumal white adipose tissue of mice. Acetylcholine, norepinephrine, atrial natriuretic peptide, bradykinin, cholecystokinin, angiotensin II, and insulin evoked complex [Ca2+]i responses in adipocytes, implicating NOS-G or PLC-G. At low sub-threshold concentrations, acetylcholine and norepinephrine or acetylcholine and peptide hormones (in paired combinations) recruited NOS-G, based on G proteins subunits interplay and signaling amplification. Rhythmicity was cell size- dependent and disappeared in hypertrophied cells filled with lipids. Contrary to control cells, adipocytes of obese hyperglycemic and hypertensive mice, growing on glucose, did not accumulate lipids and demonstrated hormonal resistance being non responsive to any hormone applied. Preincubation of preadipocytes with palmitoyl-L-carnitine (100 nM) provided accumulation of lipids, increased expression and clustering of IP3R and RyR proteins, and partially restored hormonal sensitivity and rhythmicity (5–15% vs. 30–80% in control cells), while adipocytes of diabetic mice were not responsive at all. Here, we presented a detailed kinetic model of NOS-G and discussed its control. Collectively, we may suggest that universal mechanisms underlie loss of rhythmicity, Ca2+-signaling systems deregulation, and development of general hormonal resistance to obesity.
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LESLIE, Nick R., Alex GRAY, Ian PASS, Elaine A. ORCHISTON, and C. Peter DOWNES. "Analysis of the cellular functions of PTEN using catalytic domain and C-terminal mutations: differential effects of C-terminal deletion on signalling pathways downstream of phosphoinositide 3-kinase." Biochemical Journal 346, no. 3 (March 7, 2000): 827–33. http://dx.doi.org/10.1042/bj3460827.
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The tumour suppressor protein, PTEN (phosphatase and tensin homolog deleted on chromosome 10), is a phosphatase that can dephosphorylate tyrosine-containing peptides, Shc, focal adhesion kinase and phosphoinositide substrates. In cellular assays, PTEN has been shown to antagonize the PI-3K-dependent activation of protein kinase B (PKB) and to inhibit cell spreading and motility. It is currently unclear, however, whether PTEN accomplishes these effects through its lipid- or protein-phosphatase activity, although strong evidence has demonstrated the importance of the latter for tumour suppression by PTEN. By using a PTEN G129E (Gly129 → Glu) mutant that has lost its lipid phosphatase activity, while retaining protein phosphatase activity, we demonstrated a requirement for the lipid phosphatase activity of PTEN in the regulation of PKB activity, cell viability and membrane ruffling. We also made a small C-terminal deletion of PTEN, removing a putative PDZ (PSD95, Dlg and ZO1)-binding motif, with no detectable effect on the phosphatase activity of the protein expressed in HEK293 cells (human embryonic kidney 293 cells) assayed in vitro. Surprisingly, expression of this mutant revealed differential requirements for the C-terminus in the different functional assays. Wild-type and C-terminally deleted PTEN appeared to be equally active in down-regulating PKB activity, but this mutant enzyme had no effect on platelet-derived growth factor (PDGF)-induced membrane ruffling and was only partially active in a cell viability assay. These results stress the importance of the lipid phosphatase activity of PTEN in the regulation of several signalling pathways. They also identify a mutation, similar to mutations that occur in some human tumours, which removes the effect of PTEN on membrane ruffling but not that on PKB.
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Somogyi, L., Z. Lasić, S. Vukičević, and H. Banfić. "Collagen type IV stimulates an increase in intracellular Ca2+ in pancreatic acinar cells via activation of phospholipase C." Biochemical Journal 299, no. 3 (May 1, 1994): 603–11. http://dx.doi.org/10.1042/bj2990603.
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Intracellular Ca2+ responses to extracellular matrix molecules were studied in suspensions of pancreatic acinar cells loaded with Fura-2. Collagen type I, laminin, fibrinogen and fibronectin were unable to raise cytosolic free Ca2+ concentration ([Ca2+]i), whereas collagen type IV, at concentrations from 5 to 50 micrograms/ml, significantly increased it. The effect of collagen type IV was not due to possible contamination with type-I transforming growth factor beta or plasminogen, as neither of these agents was able to increase [Ca2+]i. Using highly specific mass assays, concentrations of inositol lipids, 1,2-diacylglycerol (DAG) and Ins(1,4,5) P3 were measured in pancreatic acinar cells stimulated with collagen type IV. A decrease in the concentrations of PtdIns(4,5) P2 and PtdIns4 P with a concomitant increase in the concentrations of DAG and InsP3 mass were observed, showing that collagen type IV increases [Ca2+]i by activation of phospholipase C. The observed [Ca2+]i signals had two components, the first resulting from Ca2+ release from the intracellular stores, and the second resulting from Ca2+ flux from the extracellular medium through the verapamil-insensitive channels. A tyrosine kinase inhibitor (tyrphostine) was able to block inositol lipid signalling caused by collagen type IV, which together with the insensitivity of this pathway to cholera toxin and pertussis toxin or to preactivation of protein kinase C, the longer duration of the increase in [Ca2+]i and a longer lag period needed for observation of increases in DAG and InsP3 concentration with collagen type IV than with carbachol (50 mM) suggest that activation of phospholipase C by collagen type IV is caused by tyrosine kinase activation. Inositol lipid signalling and increases in [Ca2+]i were also observed with Arg-Gly-Asp (RGD)-containing peptide but not with Arg-Asp-Gly (RDG)-containing peptide. Collagen type IV and RGD-containing peptide, but not carbachol, competed in increasing [Ca2+]i and DAG concentration, suggesting that the binding site of collagen type IV responsible for phospholipase C activation contains the RGD sequence. Together the present results suggest that, in pancreatic acinar cells, RGD sequence(s) within collagen type IV molecules cause activation of tyrosine kinase, probably through one of the integrin receptors, which then stimulates phospholipase C and increases [Ca2+]i.
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Myeong, Jongyun, Cheon-Gyu Park, Byung-Chang Suh, and Bertil Hille. "Compartmentalization of phosphatidylinositol 4,5-bisphosphate metabolism into plasma membrane liquid-ordered/raft domains." Proceedings of the National Academy of Sciences 118, no. 9 (February 22, 2021): e2025343118. http://dx.doi.org/10.1073/pnas.2025343118.
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Possible segregation of plasma membrane (PM) phosphoinositide metabolism in membrane lipid domains is not fully understood. We exploited two differently lipidated peptide sequences, L10 and S15, to mark liquid-ordered, cholesterol-rich (Lo) and liquid-disordered, cholesterol-poor (Ld) domains of the PM, often called raft and nonraft domains, respectively. Imaging of the fluorescent labels verified that L10 segregated into cholesterol-rich Lo phases of cooled giant plasma-membrane vesicles (GPMVs), whereas S15 and the dye FAST DiI cosegregated into cholesterol-poor Ld phases. The fluorescent protein markers were used as Förster resonance energy transfer (FRET) pairs in intact cells. An increase of homologous FRET between L10 probes showed that depleting membrane cholesterol shrank Lo domains and enlarged Ld domains, whereas a decrease of L10 FRET showed that adding more cholesterol enlarged Lo and shrank Ld. Heterologous FRET signals between the lipid domain probes and phosphoinositide marker proteins suggested that phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and phosphatidylinositol 4-phosphate (PtdIns4P) are present in both Lo and Ld domains. In kinetic analysis, muscarinic-receptor-activated phospholipase C (PLC) depleted PtdIns(4,5)P2 and PtdIns4P more rapidly and produced diacylglycerol (DAG) more rapidly in Lo than in Ld. Further, PtdIns(4,5)P2 was restored more rapidly in Lo than in Ld. Thus destruction and restoration of PtdIns(4,5)P2 are faster in Lo than in Ld. This suggests that Lo is enriched with both the receptor G protein/PLC pathway and the PtdIns/PI4-kinase/PtdIns4P pathway. The significant kinetic differences of lipid depletion and restoration also mean that exchange of lipids between these domains is much slower than free diffusion predicts.
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Lin, Feng, Mohammed Akhter Hossain, Stephanie Post, Galina Karashchuk, Marc Tatar, Pierre De Meyts, and John D. Wade. "Total Solid-Phase Synthesis of Biologically Active Drosophila Insulin-Like Peptide 2 (DILP2)." Australian Journal of Chemistry 70, no. 2 (2017): 208. http://dx.doi.org/10.1071/ch16626.
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In the fruit fly Drosophila melanogaster, there are eight insulin-like peptides (DILPs) with DILPs 1–7 interacting with a sole insulin-like receptor tyrosine kinase (DInR) while DILP8 interacts with a single G protein-coupled receptor (GPCR), Lgr3. Loss-of-function dilp mutation studies show that the neuropeptide DILP2 has a key role in carbohydrate and lipid metabolism as well as longevity and reproduction. A better understanding of the processes whereby DILP2 mediates its specific actions is required. Consequently we undertook to prepare DILP2 as part of a larger, detailed structure–function relationship study. Use of our well established insulin-like peptide synthesis protocol that entails separate solid-phase assembly of each of the A- and B-chains with selective cysteine S-protection followed by sequential S-deprotection and simultaneous disulfide bond formation produced DILP2 in good overall yield and high purity. The synthetic DILP2 was shown to induce significant DInR phosphorylation and downstream signalling, with it being more potent than human insulin. This peptide will be a valuable tool to provide further insights into its binding to the insulin receptor, the subsequent cell signalling, and role in insect metabolism.
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Skibola, Christine F. "Serum Proteomic Profiling in Diffuse Large B-Cell Lymphoma." Blood 126, no. 23 (December 3, 2015): 3887. http://dx.doi.org/10.1182/blood.v126.23.3887.3887.
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Abstract Background: The aim of this study was to conduct an exploratory serum proteomic profiling study of diffuse large B-cell lymphoma (DLBCL) using mass spectrometry (MS) analysis to identify potential biomarkers that may provide further clues to disease mechanisms. Methods: Serum samples from 57 chemotherapy-naive male DLBCL cases and 30 healthy controls matched by age and BMI from a San Francisco Bay Area case-control study were divided into single-use aliquots and stored at -80°C until proteomic analysis. Serum samples were depleted of the 12 most abundant proteins by filtration on immunoaffinity spin columns prior to MS analysis. Proteomic analysis was performed using a GeLC approach. Specifically, equal amounts of protein from serologic specimens were separated by one-dimensional denaturing gel electrophoresis. Each lane was cut into six equal MW fractions followed by in-gel digestion with trypsin. The resultant peptides were analyzed using nano-liquid chromatography MS. Because MS is more qualitative than quantitative, the most significant and biologically relevant proteins of interest were chosen for further confirmation by ELISA in the "sandwich" configuration to specifically and quantitatively measure protein concentrations. ELISA assays were performed on unprocessed serum samples without depletion of the major serum proteins, providing a direct, one-step measurement, thereby avoiding any artifacts due to uncontrolled parameters common in preprocessing. Statistically significant differentially expressed serum peptides between the cases and controls as detected by MS were further analyzed using Systems biology/Pathway analysis. Results: MS data computed as normalized spectral counts of peptides revealed 1,207 protein IDs with >99% confidence, and 44 statistically significant candidates that were differentially expressed between the DLBCL cases and controls. We confirmed 4 [adiponectin (AdipoQ), extracellular matrix protein 1 (ECM1), CD14, and SerpinA3 (ACT)] of the 6 top candidates chosen for further confirmation by ELISA, which were elevated by 68.8, 62.9, 33.6 and 28.8 %, respectively, in DLBCL sera compared to controls. Adiponectin is an adipocyte-derived cytokine that regulates the metabolism of lipids and glucose. There is accumulating evidence that adiponectin also exhibits pro- and anti-tumorigenic activity, depending on the tumor type. Our study confirms previous reports of significantly elevated adiponectin levels in patients with adult non-Hodgkin lymphoma (NHL) and DLBCL, childhood NHL, and Hodgkin lymphoma compared to controls (PMID: 22547160). CD14 is a myeloid differentiation marker found primarily on monocytes and macrophages. Soluble CD14 levels have been positively associated with AIDS-related NHL risk (PMID: 23169327). ECM1 regulates cell migration, invasion and stem-like properties via induction of EMT progression and cancer stem cell formation. High ECM1 levels have been detected in various epithelial cancers, including invasive breast ductal carcinoma, esophageal squamous carcinoma, and gastric and colorectal cancer. ECM1 also has been associated with invasiveness and poor prognosis in thyroid and breast cancer. ACT is a metastasis-associated protein. Elevated ACT levels in serum have been associated with poor overall survival in acute leukemia (PMID: 24179540). The proteins identified in this study were critically analyzed using pathway analysis tools, and were found to be strongly associated with FcR signaling, and response to kinase activation including MAPK, membrane PTK, and neurotrophin TRK. These activities, and the proteins identified appear to involve IL-6 (Jak1/Stat3), ESR1/ CREB1, RXR1/ PPAR1, NF-kB, and HIF-1 signaling pathways at different stages of disease vs. controls. Based on these data, the predicted downstream effectors include regulation of immune processes that involve acute-phase and inflammatory responses, and regulation of lipid metabolism and transport. Conclusions: Through serum proteomic profiling studies, we have identified 3 novel (ECM1, CD14, and ACT) and 1 previously reported biomarker (adiponectin) of DLBCL that may have potential biological relevance in the pathogenesis or progression of the disease. Prospective epidemiology and clinical studies will be needed to determine whether these markers are involved in disease etiology and their possible prognostic value. Disclosures No relevant conflicts of interest to declare.
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Boyd, Robert S., Rebekah Jukes-Jones, Renata Walewska, David Brown, Martin J. S. Dyer, and Kelvin Cain. "Protein Profiling of Plasma Membranes and Lipid Rafts Defines Aberrant Signaling Pathways in Mantle Cell Lymphoma." Blood 112, no. 11 (November 16, 2008): 374. http://dx.doi.org/10.1182/blood.v112.11.374.374.
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Abstract Mantle cell lymphoma (MCL) remains incurable with current therapeutic approaches. Proteins expressed aberrantly at the MCL plasma membrane are candidate molecules for the development of targeted therapy. In the current study, we characterized globally the expression of proteins in the MCL plasma membrane and in purified lipid raft fractions in comparison with normal B cells using proteomic and bioinformatic analyses. Plasma membrane fractions were prepared from MCL patients in leukemic phase of disease and were then separated on 1D SDS-PAGE gels; sequential gel slices were digested with trypsin and the extracted peptides identified by LC/MS-MS. This approach overcomes the limitations of 2D gels for membrane proteins and also provides information on protein localization and post-translational modifications. Bioinformatics identified 111 intrinsic transmembrane proteins, from which we profiled selected proteins across primary MCL cases, MCL derived cell lines and normal B cells by reverse-transcription polymerase chain reaction (RT-PCR) and Western blotting. Several transmembrane proteins, including CD27/CD70 and CD31 (PECAM) were abnormally expressed when compared to normal B cells. CD70 was significantly up-regulated (> 10 fold) in MCL patients along with its cognate receptor, CD27 which was also up-strongly up-regulated (4–9 fold), suggesting that MCL cells may undergo autocrine stimulation via this signaling pathway. Activated calpain I and PKC-βII were also detected in plasma membranes, suggesting constitutive activation of these proteins in MCL. Since activated PKC-βII has been reported to be recruited to lipid rafts during BCR signaling and to control IêB kinase lipid raft recruitment and activation, we undertook shotgun proteomics and protein profiling of MCL lipid rafts, purified using sucrose gradient centrifugation. This analysis revealed an abnormal composition of lipid rafts in MCL. Raftlin a myristoylated lipid raft B-cell specific protein, required for the integrity of lipid rafts and BCR signal transduction, was markedly down-regulated in MCL, as was the lymphoid transmembrane adaptor protein, Cbp/PAG (PAG1), which controls lymphocyte activation. These data were confirmed by RT-PCR which showed significant down-regulation of both genes in MCL. In comparison, other constitutive lipid raft proteins, such as Csk, Blk, Fyn and Lyn kinases and flotillin 1 were expressed within lipid rafts at levels similar to normal B lymphocytes. However, PKC-bII was not localized within lipid rafts indicating aberrant signaling via this molecule in MCL. Conversely, 5-lipoxygenase (5-LO/ALOX5) a key enzyme in leukotriene biosynthesis, which is normally expressed in either the nucleus or cytoplasm, was unexpectedly associated with lipid rafts isolated from MCL cells and was up-regulated ~7-fold in MCL as compared to normal B cells. Comparable results were obtained in chronic lymphocytic leukemia (CLL). Aberrant expression of 5-lipoxygenase has been associated with increased proliferation and suppression of apoptosis in other malignancies. To assess the possible functional activity of this pathway in malignant B-cells, the effects of inhibitors of 5-LO activity (AA861) and FLAP (MK886) its activating enzyme, were assessed on MCL cell lines and primary CLL cells. Both inhibitors induced high levels of apoptosis in a time-dependent and concentration-dependent manner in MCL cell lines and CLL cells, indicating an important role for this enzyme and the leukotriene biosynthetic pathway in MCL and other B-cell malignancies. Thus, using shotgun proteomics and protein expression profiling we have identified a subset of transmembrane proteins with aberrant expression and aberrant subcellular localization in MCL plasma membranes that may contribute to the pathology of the disease and are potential therapeutic targets in treating MCL.
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Barnes, Stephen, Erin M. Shonsey, Shannon M. Eliuk, David Stella, Kerri Barrett, Om P. Srivastava, Helen Kim, and Matthew B. Renfrow. "High-resolution mass spectrometry analysis of protein oxidations and resultant loss of function." Biochemical Society Transactions 36, no. 5 (September 19, 2008): 1037–44. http://dx.doi.org/10.1042/bst0361037.
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MS, with or without pre-analysis peptide fractionation, can be used to decipher the residues on proteins where oxidative modifications caused by peroxynitrite, singlet oxygen or electrophilic lipids have occurred. Peroxynitrite nitrates tyrosine and tryptophan residues on the surface of actin. Singlet oxygen, formed by the interaction of UVA light with tryptophan, can oxidize neighbouring cysteine, histidine, methionine, tyrosine and tryptophan residues. Dose–response inactivation by 4HNE (4-hydroxynonenal) of hBAT (human bile acid CoA:amino acid N-acyltransferase) and CKBB (cytosolic brain isoform of creatine kinase) is associated with site-specific modifications. FT-ICR (Fourier-transform ion cyclotron resonance)–MS using nanoLC (nano-liquid chromatography)–ESI (electrospray ionization)–MS or direct-infusion ESI–MS with gas-phase fractionation identified 14 4HNE adducts on hBAT and 17 on CKBB respectively. At 4HNE concentrations in the physiological range, one member of the catalytic triad of hBAT (His362) was modified; for CKBB, although all four residues in the active site that were modifiable by 4HNE were ultimately modified, only one, Cys283, occurred at physiological concentrations of 4HNE. These results suggest that future in vivo studies should carefully assess the critical sites that are modified rather than using antibodies that do not distinguish between different modified sites.
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Mau, S. E., M. R. Witt, and H. Vilhardt. "Mastoparan, a wasp venom peptide, stimulates release of prolactin from cultured rat anterior pituitary cells." Journal of Endocrinology 142, no. 1 (July 1994): 9–18. http://dx.doi.org/10.1677/joe.0.1420009.
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Abstract Studies have shown that mastoparan and other amphiphilic peptides induce exocytosis of hormones from anterior pituitary cells. We have studied the effect of mastoparan on the secretion of prolactin from cultured rat anterior pituitary cells and on the concomitant functional status of signal-transducing pathways in lactotroph-enriched cell cultures. Mastoparan stimulation of prolactin secretion was dose-dependent, time-dependent, reversible and required the presence of calcium. Pretreatment of pituitary cell cultures with cholera and pertussis toxin had no effect on the secretory response, whereas encapsulation of guanosine 5-[β-thio]diphosphate (GDP-β-S) by reversible electropermeabilization inhibited mastoparan-stimulated secretion. Incubation of mastoparan with myo[3H]inositol-labelled lactotroph-enriched anterior pituitary cell cultures resulted in increased formation of inositol phosphates compared with control cells, and encapsulation of GDP-β-S blocked mastoparan-induced inositol lipid hydrolysis. Mastoparan caused translocation of protein kinase C activity from a soluble to a membrane-attached form. Mastoparan was able to increase the intracellular Ca2+ concentration in Fura-2-loaded individual lactotrophs. Omission of Ca2+ from the extracellular medium did not change the Ca2+ response in lactotrophs when stimulated with mastoparan. On the basis of these results it is concluded that mastoparan-induced release of prolactin is preceded by activation of the inositol(1,4,5)trisphosphate/diacylglycerol pathway with resulting translocation of protein kinase activity and increment in intracellular Ca2+. However, other signal-transducing pathways may be involved in the secretory process. Journal of Endocrinology (1994) 142, 9–18
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Kawasaki, Kiyoshi, Robert K. Ernst, and Samuel I. Miller. "Inhibition of Salmonella enterica Serovar Typhimurium Lipopolysaccharide Deacylation by Aminoarabinose Membrane Modification." Journal of Bacteriology 187, no. 7 (April 1, 2005): 2448–57. http://dx.doi.org/10.1128/jb.187.7.2448-2457.2005.
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ABSTRACT Salmonella enterica serovar Typhimurium remodels the lipid A component of lipopolysaccharide, a major component of the outer membrane, to survive within animals. The activation of the sensor kinase PhoQ in host environments increases the synthesis of enzymes that deacylate, palmitoylate, hydroxylate, and attach aminoarabinose to lipid A, also known as endotoxin. These modifications promote bacterial resistance to antimicrobial peptides and reduce the host recognition of lipid A by Toll-like receptor 4. The Salmonella lipid A 3-O-deacylase, PagL, is an outer membrane protein whose expression is regulated by PhoQ. In S. enterica serovar Typhimurium strains that had the ability to add aminoarabinose to lipid A, 3-O-deacylated lipid A species were not detected, despite the PhoQ induction of PagL protein expression. In contrast, strains defective for the aminoarabinose modification of lipid A demonstrated in vivo PagL activity, indicating that this membrane modification inhibited PagL's enzymatic activity. Since not all lipid A molecules are modified with aminoarabinose upon PhoQ activation, these results cannot be ascribed to the substrate specificity of PagL. PagL-dependent deacylation was detected in sonically disrupted membranes and membranes treated with the nonionic detergent n-octyl-β-d-glucopyranoside, suggesting that perturbation of the intact outer membrane releases PagL from posttranslational inhibition by aminoarabinose-containing membranes. Taken together, these results suggest that PagL enzymatic deacylation is posttranslationally inhibited by membrane environments, which either sequester PagL from its substrate or alter its conformation.
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Erbeznik, Milutin, Herbert J. Strobel, Karl A. Dawson, and Chris R. Jones. "The d-Xylose-Binding Protein, XylF, from Thermoanaerobacter ethanolicus 39E: Cloning, Molecular Analysis, and Expression of the Structural Gene." Journal of Bacteriology 180, no. 14 (July 15, 1998): 3570–77. http://dx.doi.org/10.1128/jb.180.14.3570-3577.1998.
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ABSTRACT Immediately downstream from the Thermoanaerobacter ethanolicus xylAB operon, comprising genes that encoded-xylose isomerase and d-xylulose kinase, lies a 1,101-bp open reading frame that exhibits 61% amino acid sequence identity to the Escherichia coli d-xylose binding periplasmic receptor, XylF, a component of the high-affinity binding-protein-dependent d-xylose transport. The 25-residue N-terminal fragment of the deduced T. ethanolicus XylF has typical features of bacterial leader peptides. The C-terminal portion of this leader sequence matches the cleavage consensus for lipoproteins and is followed by a 22-residue putative linker sequence rich in serine, threonine, and asparagine. The putative mature 341-amino-acid-residue XylF (calculated molecular mass of 37,069 Da) appears to be a lipoprotein attached to the cell membrane via a lipid anchor covalently linked to the N-terminal cysteine, as demonstrated by metabolic labelling of the recombinant XylF with [14C]palmitate. The induced E. coli avidly bound d-[14C]xylose, yielding additional evidence that T. ethanolicus XylF is thed-xylose-binding protein. On the basis of sequence comparison of XylFs to other monosaccharide-binding proteins, we propose that the sequence signature of binding proteins specific for hexoses and pentoses be refined as (KDQ)(LIVFAG)3IX3(DN)(SGP)X3(GS)X(LIVA)2X2A. Transcription of the monocistronic 1.3-kb xylF mRNA is inducible by xylose and unaffected by glucose. Primer extension analysis indicated that xylF transcription initiates from two +1 sites, both situated within the xylAB operon. Unlike in similar transport systems in other bacteria, the genes specifying the membrane components (e.g., ATP-binding protein and permease) of the high-affinity d-xylose uptake system are not located in the vicinity of xylF in T. ethanolicus. This is the first report of a gene encoding a xylose-binding protein in a gram-positive or thermophilic bacterium.
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BEETON, Carolyn A., Edwin M. CHANCE, Lazaros C. FOUKAS, and Peter R. SHEPHERD. "Comparison of the kinetic properties of the lipid- and protein-kinase activities of the p110α and p110β catalytic subunits of class-Ia phosphoinositide 3-kinases." Biochemical Journal 350, no. 2 (August 23, 2000): 353–59. http://dx.doi.org/10.1042/bj3500353.
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Growth factors regulate a wide range of cellular processes via activation of the class-Ia phosphoinositide 3-kinases (PI 3-kinases). We directly compared kinetic properties of lipid- and protein-kinase activities of the widely expressed p110α and p110β isoforms. The lipid-kinase activity did not display Michaelis–Menten kinetics but modelling the kinetic data demonstrated that p110α has a higher Vmax and a 25-fold higher Km for PtdIns than p110β. A similar situation occurs with PtdIns(4,5)P2, because at low concentration of PtdIns(4,5)P2 p110β is a better PtdIns(4,5)P2 kinase than p110α, although this is reversed at high concentrations. These differences suggest different functional roles and we hypothesize that p110β functions better in areas of membranes containing low levels of substrate whereas p110α would work best in areas of high substrate density such as membrane lipid rafts. We also compared protein-kinase activities. We found that p110β phosphorylated p85 to a lower degree than did p110α. We used a novel peptide-based assay to compare the kinetics of the protein-kinase activities of p110α and p110β. These studies revealed that, like the lipid-kinase activity, the protein-kinase activity of p110α has a higher Km (550µM) than p110β (Km 8µM). Similarly, the relative Vmax towards peptide substrate of p110α was three times higher than that of p110β. This implies differences in the rates of regulatory autophosphorylation in vivo, which are likely to mean differential regulation of the lipid-kinase activities of p110α and p110β in vivo.
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Borsatto, Alberto, Valerio Marino, Gianfranco Abrusci, Gianluca Lattanzi, and Daniele Dell’Orco. "Effects of Membrane and Biological Target on the Structural and Allosteric Properties of Recoverin: A Computational Approach." International Journal of Molecular Sciences 20, no. 20 (October 10, 2019): 5009. http://dx.doi.org/10.3390/ijms20205009.
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Recoverin (Rec) is a prototypical calcium sensor protein primarily expressed in the vertebrate retina. The binding of two Ca2+ ions to the functional EF-hand motifs induces the extrusion of a myristoyl group that increases the affinity of Rec for the membrane and leads to the formation of a complex with rhodopsin kinase (GRK1). Here, unbiased all-atom molecular dynamics simulations were performed to monitor the spontaneous insertion of the myristoyl group into a model multicomponent biological membrane for both isolated Rec and for its complex with a peptide from the GRK1 target. It was found that the functional membrane anchoring of the myristoyl group is triggered by persistent electrostatic protein-membrane interactions. In particular, salt bridges between Arg43, Arg46 and polar heads of phosphatidylserine lipids are necessary to enhance the myristoyl hydrophobic packing in the Rec-GRK1 assembly. The long-distance communication between Ca2+-binding EF-hands and residues at the interface with GRK1 is significantly influenced by the presence of the membrane, which leads to dramatic changes in the connectivity of amino acids mediating the highest number of persistent interactions (hubs). In conclusion, specific membrane composition and allosteric interactions are both necessary for the correct assembly and dynamics of functional Rec-GRK1 complex.
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Portal-Núñez, S., J. A. Ardura, D. Lozano, I. Martínez de Toda, M. De la Fuente, G. Herrero-Beaumont, R. Largo, and P. Esbrit. "Parathyroid hormone-related protein exhibits antioxidant features in osteoblastic cells through its N-terminal and osteostatin domains." Bone & Joint Research 7, no. 1 (January 2018): 58–68. http://dx.doi.org/10.1302/2046-3758.71.bjr-2016-0242.r2.
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Objectives Oxidative stress plays a major role in the onset and progression of involutional osteoporosis. However, classical antioxidants fail to restore osteoblast function. Interestingly, the bone anabolism of parathyroid hormone (PTH) has been shown to be associated with its ability to counteract oxidative stress in osteoblasts. The PTH counterpart in bone, which is the PTH-related protein (PTHrP), displays osteogenic actions through both its N-terminal PTH-like region and the C-terminal domain. Methods We examined and compared the antioxidant capacity of PTHrP (1-37) with the C-terminal PTHrP domain comprising the 107-111 epitope (osteostatin) in both murine osteoblastic MC3T3-E1 cells and primary human osteoblastic cells. Results We showed that both N- and C-terminal PTHrP peptides at 100 nM decreased reactive oxygen species production and forkhead box protein O activation following hydrogen peroxide (H2O2)-induced oxidation, which was related to decreased lipid oxidative damage and caspase-3 activation in these cells. This was associated with their ability to restore the deleterious effects of H2O2 on cell growth and alkaline phosphatase activity, as well as on the expression of various osteoblast differentiation genes. The addition of Rp-cyclic 3′,5′-hydrogen phosphorothioate adenosine triethylammonium salt (a cyclic 3',5'-adenosine monophosphate antagonist) and calphostin C (a protein kinase C inhibitor), or a PTH type 1 receptor antagonist, abrogated the effects of N-terminal PTHrP, whereas protein phosphatase 1 (an Src kinase activity inhibitor), SU1498 (a vascular endothelial growth factor receptor 2 inhibitor), or an anti osteostatin antiserum, inhibited the effects of C-terminal PTHrP. Conclusion These findings indicate that the antioxidant properties of PTHrP act through its N- and C-terminal domains and provide novel insights into the osteogenic action of PTHrP. Cite this article: S. Portal-Núñez, J. A. Ardura, D. Lozano, I. Martínez de Toda, M. De la Fuente, G. Herrero-Beaumont, R. Largo, P. Esbrit. Parathyroid hormone-related protein exhibits antioxidant features in osteoblastic cells through its N-terminal and osteostatin domains. Bone Joint Res 2018;7:58–68. DOI: 10.1302/2046-3758.71.BJR-2016-0242.R2.
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Hua, Pengpeng, Zhiying Yu, Yu Xiong, Bin Liu, and Lina Zhao. "Regulatory Efficacy of Spirulina platensis Protease Hydrolyzate on Lipid Metabolism and Gut Microbiota in High-Fat Diet-Fed Rats." International Journal of Molecular Sciences 19, no. 12 (December 13, 2018): 4023. http://dx.doi.org/10.3390/ijms19124023.
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Lipid metabolism disorder (LMD) is a public health issue. Spirulina platensis is a widely used natural weight-reducing agent and Spirulina platensis is a kind of protein source. In the present study, we aimed to evaluate the effect of Spirulina platensis protease hydrolyzate (SPPH) on the lipid metabolism and gut microbiota in high-fat diet (HFD)-fed rats. Our study showed that SPPH decreased the levels of triglyceride (TG), total cholesterol (TC), low-density-lipoprotein cholesterol (LDL-c), alanine transaminase (ALT), and aspartate transaminase (AST), but increased the level of high-density-lipoprotein cholesterol (HDL-c) in serum and liver. Moreover, SPPH had a hypolipidemic effect as indicated by the down-regulation of sterol regulatory element-binding transcription factor-1c (SREBP-1c), acetyl CoA carboxylase (ACC), SREBP-1c, and peroxisome proliferator-activated receptor-γ (PPARγ) and the up-regulation of adenosine 5’-monophosphate (AMP)-activated protein kinase (AMPK) and peroxisome proliferator-activated receptorα (PPARα) at the mRNA level in liver. SPPH treatment enriched the abundance of beneficial bacteria. In conclusion, our study showed that SPPH might be produce glucose metabolic benefits in rats with diet-induced LMD. The mechanisms underlying the beneficial effects of SPPH on the metabolism remain to be further investigated. Collectively, the above-mentioned findings illustrate that Spirulina platensis peptides have the potential to ameliorate lipid metabolic disorders, and our data provides evidence that SPPH might be used as an adjuvant therapy and functional food in obese and diabetic individuals.
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Yang, Moua, Calvin Harberg, Wenjing Chen, Sarah L. Wynia-Smith, Kate S. Carroll, Jacek Zielonka, Roy L. Silverstein, and Brian C. Smith. "Protein Cysteine Sulfenylation By CD36-Dependent Reactive Oxygen Species Signaling Promotes Platelet Activation." Blood 134, Supplement_1 (November 13, 2019): 2338. http://dx.doi.org/10.1182/blood-2019-122465.
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Arterial thrombosis in the setting of dyslipidemia produces clinically significant events, including myocardial infarction and stroke. Oxidized lipids in circulating lipoproteins (oxLDL) are a risk factor for atherothrombosis in dyslipidemia and are recognized by platelet scavenger receptor CD36. OxLDL binding to CD36 promotes platelet activation and thrombosis by generating intracellular reactive oxygen species (ROS), such as hydrogen peroxide (H2O2). Downstream signaling events initiated by ROS in this setting are largely unknown. We hypothesize that H2O2 generated by CD36 signaling promotes oxidative cysteine modification of cellular regulators of arterial thrombosis. Platelets isolated from healthy human donors were stimulated with oxLDL and H2O2 levels were measured by HPLC fluorescence quantification of 7-hydroxycoumarin generated by H2O2-specific oxidation of the coumarin boronic acid probe. OxLDL induced time- and concentration-dependent H2O2 formation (up to 80 ±13% greater than unstimulated platelets), while control "native" LDL showed negligible H2O2 formation. Pre-treatment of platelets with a CD36-blocking antibody or with PEG-catalase, an enzyme that degrades H2O2, normalized H2O2 formation by oxLDL to levels observed in unstimulated platelets. To mimic pathophysiologic conditions platelets were sensitized with oxLDL before stimulating with classic activators: adenosine diphosphate (ADP) and collagen-related peptide (CRP). Sensitization synergistically increased H2O2 formation through collagen receptors, but not ADP receptors. Since H2O2 can induce transient protein cysteine sulfenylation as a posttranslational oxidative modification, we assayed platelet cysteine sulfenylation using an alkyne-containing benzothiazine-based probe, BTD. BTD was loaded into platelets prior to exposure to oxLDL and detected by click-chemistry with biotin-PEG-azide followed by detection by western blot with streptavidin. OxLDL, but not LDL, induced two-fold increase in sulfenylation in the platelet proteome within 15 minutes. Src family kinases (SFK) are known to be recruited to and activated by CD36 in a ligand-dependent manner and SFK were also previously shown to be sulfenylated by H2O2 at Cys185 and Cys277, maintaining the kinase in an activated state. To determine if SFK cysteines are oxidatively modified by CD36 signaling, lysates from BTD-loaded, oxLDL-stimulated platelets were biotinylated as above, immunoprecipitated with anti-Src antibody, and then analyzed by immunoblot to detect BTD incorporation. SFK were sulfenylated in a time-dependent manner and this was blocked by a CD36 blocking monoclonal antibody or by treatment with PEG-catalase. We then showed by immunoblot that SFK cysteine sulfenylation by CD36/H2O2 was associated with phosphorylation at Y416, a signature activation motif in the kinase domain. OxLDL induced Src Y416 phosphorylation was prevented by PEG-catalase. Platelet aggregometry was used to determine the functional impact of protein sulfenylation and revealed that oxLDL-induced platelet aggregation was inhibited in a concentration-dependent manner (IC50 2.02 mM) by modifying sites of sulfenylation with BTD. BTD had no impact on aggregation induced by low- or high-concentration of ADP or CRP, suggesting these physiologic activators alone do not generate sufficient H2O2 to promote cysteine sulfenylation. OxLDL/CD36 induced procoagulant phosphatidylserine externalization, assessed by flow cytometry using fluorophore-tagged annexin V, was also prevented by BTD. In conclusion, our studies show that platelet CD36 signaling in response to oxLDL induces intracellular H2O2 generation which in turn induces cysteine sulfenylation of Src family kinases to promote platelet activation. Cysteine sulfenylation by CD36 could potentially be targeted to reduce the risk for clinically significant thrombotic events while maintaining hemostasis. Disclosures No relevant conflicts of interest to declare.
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Abdel-Reheim, Eman Salah. "Ameliorative effects of gallic acid on cardio-renal complexity induced by isoproterenol." International Journal of Bioassays 5, no. 10 (October 1, 2016): 4963. http://dx.doi.org/10.21746/ijbio.2016.10.0010.
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Myocardial infarction (MI) arises out many risk factors, which work in concert and give rise to a lot of unfavorable outcome especially on the kidney. In the present study, we investigate the effect of gallic acid (GA), a natural antioxidant on cardio-renal complexity induced by isoproterenol (ISO). The myocardial infarcted rats showed deterioration in the heart function measured by lactate dehydrogenase (LDH) and creatin kinase (CK) and kidney function measured by urea, uric acid and creatinine. There were relative overweight in both the organs. Abnormal oxidation in lipids of their membrane indicated by the increased malomdialdhyde (MDA) content and nitrite level indicating the increase in their nitric oxide (NO). However, the result indicated a decrease in glutathione (GSH) content and superoxide dismutase (SOD) and peroxidase (POX) activities. These results were assured by other measurements as brain naturetic peptide (BNP) and myoglobin (Mgb) which indicated a damage happened in the myocytes, C-reactive protein (CRP) indicated the inflammatory response and homocystain, angiotensin and aldosterone levels which indicating the kidney hormones secretion. In conclusion, GA ascertained its efficacy in ameliorating the heart function biomarkers, kidney function testes and hormones and oxidation state.
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HEID, Hans W., Martina SCHNÖLZER, and Thomas W. KEENAN. "Adipocyte differentiation-related protein is secreted into milk as a constituent of milk lipid globule membrane." Biochemical Journal 320, no. 3 (December 15, 1996): 1025–30. http://dx.doi.org/10.1042/bj3201025.
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Milk lipid globules from humans, cows and rats contained a protein identified as adipocyte differentiation-related protein (ADRP) associated with the globule surface membrane material. This protein, previously believed to be specific to adipocytes, was a major constituent of the globule surface and was present in a detergent-insoluble complex that contained stoichiometric amounts of butyrophilin and xanthine oxidase. Identification of ADRP was by sequence similarity of tryptic peptides from cow and human proteins with the sequence inferred from the cDNA for mouse ADRP. The putative ADRP of lipid globules from cow, human and rat milk was recognized specifically by antisera raised against a peptide synthesized to duplicate the N-terminal 26 residues of the mouse protein. In homogenates of lactating mammary gland, ADRP was found only in endoplasmic reticulum and in lipid droplet fractions. ADRP was modified, apparently post-translationally, and one modification apparently was acylation, primarily with C14, C16 and C18 fatty acids. Two isoelectric variants of ADRP were present in cow globule membrane material. In vitro, ADRP served as a substrate for protein kinases associated with milk lipid globule membrane, but this protein did not seem to become phosphorylated intracellularly.
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Wang, Xiaoyu, Susan V. McLennan, Terri J. Allen, Tatiana Tsoutsman, Christopher Semsarian, and Stephen M. Twigg. "Adverse effects of high glucose and free fatty acid on cardiomyocytes are mediated by connective tissue growth factor." American Journal of Physiology-Cell Physiology 297, no. 6 (December 2009): C1490—C1500. http://dx.doi.org/10.1152/ajpcell.00049.2009.
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Diabetic cardiomyopathy is characterized by interstitial fibrosis and cardiomyocyte hypertrophy and apoptosis. Also known as CCN2, connective tissue growth factor (CTGF) is implicated in the fibrosis; however, whether it contributes to cardiomyocytes changes and adverse effects of high glucose and lipids on these cells remains unknown. Hearts from streptozotocin-induced diabetic rats had elevated CTGF and changes of pathological myocardial hypertrophy, fibrosis, and cardiomyocyte apoptosis. Rat H9c2 cardiomyocytes were then treated with recombinant human (rh)CTGF, high glucose, or the saturated free fatty acid palmitate. Each reagent induced cell hypertrophy, as indicated by the ratio of total protein to cell number, cell size, and gene expression of cardiac hypertrophy marker genes atrial natriuretic peptide (ANP), and α-skeletal actin. Each treatment also caused apoptosis measured by increased caspase3/7 activity, apoptotic cells by transferase-mediated dUTP nick end labeling (TUNEL) assay, and lower viable cell number. Further studies showed CTGF mRNA was rapidly induced by high glucose and palmitate in H9c2 cells and in mouse neonatal cardiomyocyte primary cultures. small interfering RNA against CTGF blocked the high glucose and palmitate induction of hypertrophy and apoptosis. In addition, these CTGF effects were through the tyrosine kinase A (TrkA) receptor with tyrosine kinase activity, which has previously been implicated in CTGF signaling: TrkA was phosphorylated by CTGF, and a specific TrkA blocker abrogated CTGF-induced effects on hypertrophy and apoptosis. For the first time in any system, fatty acid is newly identified as a regulator of CTGF, and this work implicates autocrine CTGF as a mediator of adverse effects of high glucose and fatty acids in cardiomyocytes.
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Scheving, Lawrence A., Xiuqi Zhang, Oscar A. Garcia, Rebecca F. Wang, Mary C. Stevenson, David W. Threadgill, and William E. Russell. "Epidermal growth factor receptor plays a role in the regulation of liver and plasma lipid levels in adult male mice." American Journal of Physiology-Gastrointestinal and Liver Physiology 306, no. 5 (March 1, 2014): G370—G381. http://dx.doi.org/10.1152/ajpgi.00116.2013.
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Dsk5 mice have a gain of function in the epidermal growth factor receptor (EGFR), caused by a point mutation in the kinase domain. We analyzed the effect of this mutation on liver size, histology, and composition. We found that the livers of 12-wk-old male Dsk5 heterozygotes (+/ Dsk5) were 62% heavier compared with those of wild-type controls (+/+). The livers of the +/ Dsk5 mice compared with +/+ mice had larger hepatocytes with prominent, polyploid nuclei and showed modestly increased cell proliferation indices in both hepatocytes and nonparenchymal cells. An analysis of total protein, DNA, and RNA (expressed relative to liver weight) revealed no differences between the mutant and wild-type mice. However, the livers of the +/ Dsk5 mice had more cholesterol but less phospholipid and fatty acid. Circulating cholesterol levels were twice as high in adult male +/ Dsk5 mice but not in postweaned young male or female mice. The elevated total plasma cholesterol resulted mainly from an increase in low-density lipoprotein (LDL). The +/ Dsk5 adult mouse liver expressed markedly reduced protein levels of LDL receptor, no change in proprotein convertase subtilisin/kexin type 9, and a markedly increased fatty acid synthase and 3-hydroxy-3-methyl-glutaryl-CoA reductase. Increased expression of transcription factors associated with enhanced cholesterol synthesis was also observed. Together, these findings suggest that the EGFR may play a regulatory role in hepatocyte proliferation and lipid metabolism in adult male mice, explaining why elevated levels of EGF or EGF-like peptides have been positively correlated to increased cholesterol levels in human studies.
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Tolias, Kimberley F., Anthony D. Couvillon, Lewis C. Cantley, and Christopher L. Carpenter. "Characterization of a Rac1- and RhoGDI-Associated Lipid Kinase Signaling Complex." Molecular and Cellular Biology 18, no. 2 (February 1, 1998): 762–70. http://dx.doi.org/10.1128/mcb.18.2.762.
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ABSTRACT Rho family GTPases regulate a number of cellular processes, including actin cytoskeletal organization, cellular proliferation, and NADPH oxidase activation. The mechanisms by which these G proteins mediate their effects are unclear, although a number of downstream targets have been identified. The interaction of most of these target proteins with Rho GTPases is GTP dependent and requires the effector domain. The activation of the NADPH oxidase also depends on the C terminus of Rac, but no effector molecules that bind to this region have yet been identified. We previously showed that Rac interacts with a type I phosphatidylinositol-4-phosphate (PtdInsP) 5-kinase, independent of GTP. Here we report the identification of a diacylglycerol kinase (DGK) which also associates with both GTP- and GDP-bound Rac1. In vitro binding analysis using chimeric proteins, peptides, and a truncation mutant demonstrated that the C terminus of Rac is necessary and sufficient for binding to both lipid kinases. The Rac-associated PtdInsP 5-kinase and DGK copurify by liquid chromatography, suggesting that they bind as a complex to Rac. RhoGDI also associates with this lipid kinase complex both in vivo and in vitro, primarily via its interaction with Rac. The interaction between Rac and the lipid kinases was enhanced by specific phospholipids, indicating a possible mechanism of regulation in vivo. Given that the products of the PtdInsP 5-kinase and the DGK have been implicated in several Rac-regulated processes, and they bind to the Rac C terminus, these lipid kinases may play important roles in Rac activation of the NADPH oxidase, actin polymerization, and other signaling pathways.
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Huang, Sheng-He, Chu-Hua Wu, Shibo Jiang, Ingrid Bahner, Albert S. Lossinsky, and Ambrose Y. Jong. "HIV-1 gp41 ectodomain enhances Cryptococcus neoformans binding to human brain microvascular endothelial cells via gp41 core-induced membrane activities." Biochemical Journal 438, no. 3 (August 26, 2011): 457–66. http://dx.doi.org/10.1042/bj20110218.
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Cryptococcus neoformans causes life-threatening meningoencephalitis, particularly prevalent in AIDS patients. The interrelationship between C. neoformans and HIV-1 is intriguing, as both pathogens elicit severe neuropathological complications. We have previously demonstrated that the HIV-1 gp41 ectodomain fragments gp41-I33 (amino acids 579–611) and gp41-I90 (amino acids 550–639) can enhance C. neoformans binding to HBMECs (human brain microvascular endothelial cells). Both peptides contain the loop region of gp41. In the present study, we used immunofluorescence microscopy and transmission and scanning electron microscopy to explore the underlying mechanisms. Our findings indicated that both C. neoformans and gp41-I90 up-regulated ICAM-1 (intercellular adhesion molecule 1) on the HBMECs and elicited membrane ruffling on the surface of HBMECs. The HIV-1 gp41 ectodomain could also induce CD44 and β-actin redistribution to the membrane lipid rafts, but it could not enhance PKCα (protein kinase Cα) phosphorylation like C. neoformans. Instead, gp41-I90 was able to induce syncytium formation on HBMECs. The results of the present study suggest HIV-1 gp41-enhanced C. neoformans binding to HBMECs via gp41 core domain-induced membrane activities, revealing a potential mechanism of invasion for this pathogenic fungus into the brain tissues of HIV-1-infected patients.
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Kompaneets, Ivan Yu, Evgeny A. Ermakov, Sergey E. Sedykh, Valentina N. Buneva, and Georgy A. Nevinsky. "IgGs from Human Milk Hydrolyze microRNAs." Molecules 25, no. 10 (May 20, 2020): 2366. http://dx.doi.org/10.3390/molecules25102366.
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Mother’s milk provides breast-fed infants with various nutrients, including peptides, proteins, DNA, RNA, antibodies, and other bioactive components promoting neonatal growth and protecting infants from viral and bacterial infection. The functions of many human milk components regarding the nutrition and protection of newborns may be very different compared to those of various biological fluids of healthy adults. For example, human milk contains catalytic antibodies (abzymes) with protein, lipid, and oligosaccharide kinase activities, which are absent in the biological fluids of healthy people and autoimmune patients. Obviously, the nutrition of infants with fresh breast milk is a special phenomenon having a very specific and important role. Here, we have shown that mother’s milk IgGs effectively split homo-(pN)23, and four miRNAs: miR-137, miR-219a-5p, miR-219-2-3p, and miR-9-5p. It was shown that ribonuclease activity is a unique property of milk IgGs. On average, individual IgGs hydrolyze (pA)23, (pU)23, and (pC)23 nonspecifically and with comparable efficiency, whereas the hydrolysis of four miRNAs is predominately site-specific. The specific sites of the hydrolysis of four miRNAs by IgGs from the blood of schizophrenic (SCZ) patients and secretory immunoglobulins A (sIgAs) from human milk were found earlier. The sites of the hydrolysis of four miRNAs by milk IgGs and sIgA-abzymes are almost the same, but are significantly different in comparison with those for SCZ IgGs. In addition, in contrast to the SCZ IgGs, milk IgGs and sIgAs efficiently hydrolyzed miRNAs in the duplex regions formed by their terminal sequences.
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Thomas, Daniel, Joanna Woodcock, Jason A. Powell, Emma F. Barry, Angel F. Lopez, and Mark A. Guthridge. "Lipid and Protein Substrates of PI3K in Cytokine Receptor Survival Signalling: Deregulation in Leukemia." Blood 112, no. 11 (November 16, 2008): 3864. http://dx.doi.org/10.1182/blood.v112.11.3864.3864.
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Abstract New therapeutic approaches to acute myeloid leukemia (AML) must ultimately target cell survival pathways in leukemic cells in order to be effective. We have identified a serine residue (Ser585) in the cytoplasmic domain of the common GM-CSF and IL-3 receptor beta subunit which is phosphorylated in response to sub-picomolar concentrations of growth factor and is involved in signalling cytokine-mediated survival via 14-3-3 zeta phosphoserine adaptor. While Serine 585 is tightly controlled in non-transformed haematopoietic cells from normal donors, Serine 585 is constitutively phosphorylated in AML blasts suggesting a role in AML cell survival and a novel target for anti-leukaemic therapy. We attempted to isolate Ser585 kinase activity from leukemic blasts and characterise this activity in response to serine/threonine kinase inhibitors in biochemical and biological assays. Results: Cell extracts from primary AML blasts (>99% blasts by flow/morphology) obtained from adult patients were fractionated and assayed for intrinsic serine 585 peptide (13-mer) kinase activity via 32P gamma-ATP in vitro kinase assay. A single peak of Ser585 kinase activity was isolated and tested against a panel of serine/threonine kinase inhibitors. Kinase activity was selectively sensitive to LY294002, wortmannin and quercelin suggesting a role for the PI3K family of kinases in activating this residue. Ser585 kinase activity was also directly present in both p85 and p110 alpha PI3K immunoprecipitates from AML blasts and leukemic cell lines tested on both Ser585 peptide and recombinant beta cytoplasmic domain protein substrates. Serine 585 phosphorylation induced by sub-picomolar concentrations of GM-CSF in TF1.8 cells was inhibited by three different isoform selective p110 alpha inhibitors used at low nanomolar ranges consistent with reported IC50s. These results suggest a novel role for protein kinase rather than lipid kinase activity of PI3K alpha subunit in low dose cytokine signalling. We also show induction of serine phosphorylation of p85 PI3K regulatory subunit on Ser608 by GM-CSF, a previously reported protein substrate of PI3K. Furthermore, p110 alpha and delta inhibitors abrogate GM-CSF dependent survival of murine lineage negative bone marrow progenitor cells and also exert apoptotic activity on flow-sorted CD34+CD38−CD123+ sub-populations of primary AML blasts. Conclusions: Inhibition of Ser585 phosphorylation by targetting PI3K protein kinase activity by isoform selective inhibitors represents a novel approach toward the eradication of residual leukemic stem cells.
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FAUX, Maree C., Emily N. ROLLINS, Amelia S. EDWARDS, Lorene K. LANGEBERG, Alexandra C. NEWTON, and John D. SCOTT. "Mechanism of A-kinase-anchoring protein 79 (AKAP79) and protein kinase C interaction." Biochemical Journal 343, no. 2 (October 8, 1999): 443–52. http://dx.doi.org/10.1042/bj3430443.
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The A-kinase-anchoring protein AKAP79 co-ordinates the location of cAMP-dependent protein kinase, phosphatase 2B (PP2B/calcineurin) and protein kinase C (PKC) at postsynaptic sites in neurons. In this report we focus on the mechanism of interaction between AKAP79 and PKC. We show that neither lipid activators nor kinase activation are required for association with AKAP79. The anchoring protein binds and inhibits the conserved catalytic core of PKCβII. AKAP79 also associates with conventional, novel and atypical isoforms of PKC in vitro andin vivo, and immunofluorescence staining of rat hippocampal neurons demonstrates that the murine anchoring-protein homologue AKAP150 is co-distributed with PKCα/β, PKCε or PKCℓ. Binding of the AKAP79(31-52) peptide, which inhibits kinase activity, exposes the pseudosubstrate domain of PKCβII, allowing endoproteinase Arg-C proteolysis in the absence of kinase activators. Reciprocal experiments have identified two arginine residues at positions 39 and 40 that are essential for AKAP79(31-52) peptide inhibition of PKCβII. Likewise, the same mutations in the full-length anchoring protein reduced inhibition of PKCβII. Thus AKAP79 associates with multiple PKC isoforms through a mechanism involving protein-protein interactions at the catalytic core where binding of the anchoring protein inhibits kinase activity through displacement of the pseudosubstrate.
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Villalba, M., M. A. Pajares, M. F. Renart, and J. M. Mato. "Protein kinase C catalyses the phosphorylation and activation of rat liver phospholipid methyltransferase." Biochemical Journal 241, no. 3 (February 1, 1987): 911–16. http://dx.doi.org/10.1042/bj2410911.
Full textAbstract:
When a partially purified rat liver phospholipid methyltransferase is incubated with [gamma-32P]ATP and rat brain protein kinase C, phospholipid methyltransferase (Mr 50,000, pI 4.75) becomes phosphorylated. Phosphorylation of the enzyme showed Ca2+/lipid-dependency. Protein kinase C-dependent phosphorylation of phospholipid methyltransferase was accompanied by an approx. 2-fold activation of the enzyme activity. Activity changes and enzyme phosphorylation showed the same time course. Activation of the enzyme also showed Ca2+/lipid-dependency. Protein kinase C mediates phosphorylation of predominantly serine residues of the methyltransferase. One major peak of phosphorylation was identified by analysis of tryptic phosphopeptides by isoelectrofocusing. This peak (pI 5.2) differs from that phosphorylated by the cyclic AMP-dependent protein kinase (pI 7.2), demonstrating the specificity of phosphorylation of protein kinase C. Tryptic-peptide mapping by h.p.l.c. of the methyltransferase phosphorylated by protein kinase C revealed one major peak of radioactivity, which could be resolved into two labelled phosphopeptides by t.l.c. The significance of protein kinase C-mediated phosphorylation of phospholipid methyltransferase is discussed.