Molecular mechanisms underlying the interaction of motuporin and microcystins with type-1 and type-2A protein phosphatases

1996 ◽  
Vol 74 (4) ◽  
pp. 569-578 ◽  
Author(s):  
Marcia Craig ◽  
Hue Anh Luu ◽  
Tara L. McCready ◽  
Charles F. B. Holmes ◽  
David Williams ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Molecular Mechanisms ◽  
Protein Phosphatases ◽  
Covalent Bond ◽  
Covalent Modification ◽  
Striking Difference ◽  
Functional Difference ◽  
Subsequent Formation ◽  
Covalent Adduct

Heptapeptide microcystin and pentapeptide motuporin (nodularin-V) are equipotent inhibitors of type-1 and type-2A protein phosphatase catalytic subunits (PP-1c and PP-2Ac). Herein we describe elucidation of the molecular mechanisms involved in the interaction of these structurally similar hepatotoxins with PP-1c/PP-2Ac and identification of an important functional difference between their mode of interaction with these enzymes. Microcystin-LR, microcystin-LA, and microcystin-LL were found to interact with PP-2Ac and PP-1c by a two-step mechanism involving rapid binding and inactivation of the protein phosphatase (PPase) catalytic subunit, followed by a slower covalent interaction (within hours). Covalent adducts comprising PPase–toxin complexes were separated from free PPase by C-18 reverse-phase liquid chromatography, thus allowing the time course of covalent adduct formation to be quantitated. In contrast to microcystins, motuporin (nodularin-V) and noduIarin-R were unable to form covalent complexes with either PP-1c or PP-2Ac even after 96 h incubation. Specific reduction of microcystin-LA to dihydromicrocystin-LA abolished the ability of the toxin to form a covalent adduct with PP-2Ac. Specific methyl esterification of the single Glu residue in microcystin-LR rendered this toxin inactive as a PPase inhibitor and abolished subsequent formation of a covalent adduct. Our data indicate that inactivation of PP-2Ac/PP-1c by microcystins precedes covalent modification of the PPases via a Michael addition reaction between a nucleophilic phosphatase residue and Mdha in the heptapeptide toxin. In contrast, following rapid inactivation of PP-2Ac/PP-1c by motuporin, the equivalent N-methyldehydrobutyrine residue in this toxin is unreactive and does not form a covalent bond with the PPases. These results are consistent with structural data for (i) the NMR solution structures of microcystin-LR and motuporin, which indicate a striking difference in the relative positions of their corresponding dehydroamino acids in the toxin peptide backbone, and (ii) X-ray crystallographic data on an inactive complex between PP-1c and microcystin-LR, which show a covalent bond between Cys-273 and the bound toxin.Key words: microcystins, nodularin, motuporin, protein phosphatases, protein phosphorylation.

scholarly journals Inhibitory effect of okadaic acid on the p-nitrophenyl phosphate phosphatase activity of protein phosphatases

1991 ◽  
Vol 275 (1) ◽  
pp. 233-239 ◽  
Author(s):  
A Takai ◽  
G Mieskes
Keyword(s):  
Phosphatase Activity ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Protein Phosphatases ◽  
Inhibitory Effect ◽  
Enzyme Concentration ◽  
Alkaline Phosphatases ◽  
Nitrophenyl Phosphate ◽  
Type 1 Phosphatase

The phosphatase activities of type 2A, type 1 and type 2C protein phosphatase preparations were measured against p-nitrophenyl phosphate (pNPP), a commonly used substrate for alkaline phosphatases. Of the three types of phosphatase examined, the type 2A phosphatase exhibited an especially high pNPP phosphatase activity (119 +/- 8 mumol/min per mg of protein; n = 4). This activity was strongly inhibited by pico- to nano-molar concentrations of okadaic acid, a potent inhibitor of type 2A and type 1 protein phosphatases that has been shown to have no effect on alkaline phosphatases. The dose-inhibition relationship was markedly shifted to the right and became steeper by increasing the concentration of the enzyme, as predicted by the kinetic theory for tightly binding inhibitors. The enzyme concentration estimated by titration with okadaic acid agreed well with that calculated from the protein content and the molecular mass for type 2A phosphatase. These results strongly support the idea that the pNPP phosphatase activity is intrinsic to type 2A protein phosphatase and is not due to contamination by alkaline phosphatases. pNPP was also dephosphorylated, but at much lower rates, by type 1 phosphatase (6.4 +/- 8 nmol/min per mg of protein; n = 4) and type 2C phosphatase (1.2 +/- 3 nmol/min per mg of protein; n = 4). The pNPP phosphatase activity of the type 1 phosphatase preparation shows a susceptibility to okadaic acid similar to that of its protein phosphatase activity, whereas it was interestingly very resistant to inhibitor 2, an endogenous inhibitory factor of type 1 protein phosphatase. The pNPP phosphatase activity of type 2C phosphatase preparation was not affected by up to 10 microM-okadaic acid.

Reconstitution of calyculin-inhibited K-Cl cotransport in dog erythrocyte ghosts by exogenous PP-1

1996 ◽  
Vol 270 (3) ◽  
pp. C898-C902 ◽  
Author(s):  
T. Krarup ◽  
P. B. Dunham
Keyword(s):  
Protein Phosphatase ◽  
Direct Evidence ◽  
Potent Inhibitor ◽  
Protein Phosphatases ◽  
Calyculin A ◽  
Osmotic Swelling ◽  
Maximal Activation ◽  
Resealed Ghosts ◽  
K Transport

Osmotic swelling of dog and other mammalian erythrocytes activates Cl-dependent K transport, K-Cl cotransport. This activation can be abolished by nanomolar concentrations of calyculin, a potent inhibitor of serine-threonine protein phosphatases. Therefore, K-Cl cotransport is probably activated by dephosphorylation by a type 1 and/or type 2A protein phosphatase (PP-1 and PP-2A, respectively). This was tested directly by incorporating exogenous protein phosphatases into resealed ghosts made from dog erythrocytes previously exposed to calyculin. K-Cl cotransport was nearly completely inhibited in the ghosts. Incorporation of PP-1 reconstituted K-Cl cotransport. Maximal reconstitution was up to 90% of the control flux in the ghosts and 0.1 U PP-1/ml lysate gave half-maximal reconstitution of cotransport. In contrast, PP-2A had no effect. This result with PP-1 provides direct evidence that K-Cl cotransport is activated by PP-1 in dog erythrocytes. Half-maximal activation of K-Cl cotransport required approximately 180 molecules of PP-1 per ghost.

scholarly journals Molecular determinants of SR-B1-dependent Plasmodium sporozoite entry into hepatocytic cells

2020 ◽  
Author(s):  
Anne-Claire Langlois ◽  
Giulia Manzoni ◽  
Laetitia Vincensini ◽  
Romain Coppée ◽  
Carine Marinach ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Scavenger Receptor ◽  
Host Cells ◽  
Functional Difference ◽  
Structural Determinants ◽  
Functional Region ◽  
Scavenger Receptor Class ◽  
Class B ◽  
Liver Infection

ABSTRACTSporozoite forms of the malaria parasite Plasmodium are transmitted by mosquitoes and first infect the liver for an initial round of replication before parasite proliferation in the blood. The molecular mechanisms involved during sporozoite invasion of hepatocytes remain poorly understood. Two receptors of the Hepatitis C virus (HCV), the tetraspanin CD81 and the scavenger receptor class B type 1 (SR-B1), play an important role during the entry of Plasmodium sporozoites into hepatocytic cells. In contrast to HCV entry, which requires both CD81 and SR-B1 together with additional host factors, CD81 and SR-B1 operate independently during malaria liver infection. Sporozoites from human-infecting P. falciparum and P. vivax rely respectively on CD81 or SR-B1. Rodent-infecting P. berghei can use SR-B1 to infect host cells as an alternative pathway to CD81, providing a tractable model to investigate the role of SR-B1 during Plasmodium liver infection. Here we show that mouse SR-B1 is less functional as compared to human SR-B1 during P. berghei infection. We took advantage of this functional difference to investigate the structural determinants of SR-B1 required for infection. Using a structure-guided strategy and chimeric mouse/human SR-B1 constructs, we could map the functional region of human SR-B1 within apical loops, suggesting that this region of the protein may play a crucial role for interaction of sporozoite ligands with host cells and thus the very first step of Plasmodium infection.IMPORTANCEMalaria is caused by Plasmodium parasites and remains one of the deadliest parasitic diseases worldwide. The parasite is transmitted by a blood feeding mosquito and first invades the liver for an initial, obligatory and silent round of replication. The liver infection is an attractive target for antimalarial vaccine strategies, however the molecular mechanisms of parasite invasion of hepatocytes remain to be fully elucidated. Two hepatocyte surface proteins are known to be important for parasite entry into hepatocytes, the tetraspanin CD81 and the scavenger receptor class B type 1 (SR-B1). These receptors constitute independent gateways depending on the Plasmodium species. Here, we identified the structural determinants of SR-B1, an important hepatocyte entry factor for human-infecting P. vivax. This study paves the way toward a better characterization of the molecular interactions underlying the crucial early stages of infection, a pre-requisite for the development of novel malaria vaccine strategies.

scholarly journals Reconstruction of spatial structure of plant protein phosphatase type 1, 2a and 4 in complexes with microcystin-LR

1970 ◽  
pp. 339-344
Author(s):  
D. A. Samofalova ◽  
P. A. Karpov ◽  
O. V. Raievskyi ◽  
Ya. B. Blume
Keyword(s):  
Molecular Docking ◽  
Spatial Structure ◽  
Protein Phosphatase ◽  
Protein Phosphatases ◽  
Specific Interaction ◽  
Specific Protein ◽  
Plant Protein ◽  
Scoring Functions ◽  
High Level

Aim. The major toxicity of Microcystin-LR (MCLR) has been ascribed to its potent ability to inhibit serine/threonine-specific protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A). Although MCLR is widely used in animal models its selectivity for these enzymes of plant origin is not still investigated in details for phylogenetically diversified sources. Methods. The spatial structure of plant PP1, PP2A, PP4 protein phosphatases was reconstructed with homology modeling method. Flexible docking of MCLR was performed using CCDC GOLD Suite 5.3. For docking evaluations, GOLD scoring functions were used. Results. Information about amino acids, involved in ligand binding, was obtained from 8 experimentally proved human MCLR-PP1 and PP2A complexes. The sites of microcystin-LR binding with plant protein phosphatases (type-1, 2A and 4) were proved by comparative analysis and molecular docking. A high level of sequence and structure identity of plant and animal phosphatases allow us to conclude similarity of MCLR binding in PP1, PP2A and PP4. Keywords: microcystin-LR, protein phosphatase, specific interaction, molecular docking.

Serine/threonine protein phosphatases and regulation of K-Cl cotransport in human erythrocytes

1999 ◽  
Vol 277 (5) ◽  
pp. C926-C936 ◽  
Author(s):  
Isabel Bize ◽  
Birol Güvenç ◽  
Aeisha Robb ◽  
Guido Buchbinder ◽  
Carlo Brugnara
Keyword(s):  
Ionic Strength ◽  
Protein Phosphatase ◽  
Protein Phosphatases ◽  
Human Erythrocytes ◽  
Phosphatase Inhibitors ◽  
Pp2a Activity ◽  
Highly Sensitive ◽  
Protein Phosphatase Inhibitors ◽  
Membrane Bound

Activation of K-Cl cotransport is associated with activation of membrane-bound serine/threonine protein phosphatases (S/T-PPases). We characterize red blood cell S/T-PPases and K-Cl cotransport activity regarding protein phosphatase inhibitors and response to changes in ionic strength and cell size. Protein phosphatase type 1 (PP1) activity is highly sensitive to calyculin A (CalA) but not to okadaic acid (OA). PP2A activity is highly sensitive to CalA and OA. CalA completely inhibits K-Cl cotransport activity, whereas OA partially inhibits K-Cl cotransport. Membrane PP1 and membrane PP2A activities are elevated in cells suspended in hypotonic solutions, where K-Cl cotransport is elevated. Increases in membrane PP1 activity (62 ± 10% per 100 meq/l) result from decreases in intracellular ionic strength and correlate with increases in K-Cl cotransport activity (54 ± 10% per 100 meq/l). Increases in membrane PP2A activity (270 ± 77% per 100 mosM) result from volume increases and also correlate with increases in K-Cl cotransport activity (420 ± 47% per 100 mosM). The characteristics of membrane-associated PP1 and PP2A are consistent with a role for both phosphatases in K-Cl cotransport activation in human erythrocytes.

scholarly journals Role for protein phosphatases in the cell-cycle-regulated phosphorylation of stathmin

1998 ◽  
Vol 334 (1) ◽  
pp. 23-29 ◽  
Author(s):  
Sucharita J. MISTRY ◽  
Heng-Chun LI ◽  
George F. ATWEH
Keyword(s):  
Cell Cycle ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Protein Phosphatases ◽  
Specific Protein ◽  
G1 Phase ◽  
Major Increase ◽  
Protein Phosphatase Type

Stathmin is a major cytosolic phosphoprotein that regulates microtubule dynamics during the assembly of the mitotic spindle. The activity of stathmin itself is regulated by changes in its state of phosphorylation during the transition from interphase to metaphase. For a better understanding of the regulation of stathmin activity during the cell cycle, we explored the mechanism(s) responsible for the decrease in the level of phosphorylation of stathmin as cells complete mitosis and enter a new G1 phase. We show that stathmin mRNA and protein are expressed constitutively throughout the different phases of the cell cycle. This suggests that the non-phosphorylated stathmin that predominates during G1 is not generated by degradation of phosphorylated stathmin in mitosis and synthesis of new unphosphorylated stathmin as cells enter a new G1 phase. This suggested that protein phosphatases might be responsible for dephosphorylating stathmin as cells enter a new cell cycle. Okadaic acid-mediated inhibition of protein phosphatases in vivoshowed a major increase in the level of phosphorylation of stathmin. Dephosphorylation studies in vitro showed differential patterns of site-specific dephosphorylaton of stathmin to protein phosphatase type 1, protein phosphatase type 2A and protein phosphatase type 2B. Thus stathmin might be a target for okadaic acid-sensitive protein phosphatase(s), and its activity in eukaryotic cells might be modulated by the sequential activity of specific protein kinases and phosphatases.

Sequence of the human glycogen-associated regulatory subunit of type 1 protein phosphatase and analysis of its coding region and mRNA level in muscle from patients with NIDDM

Diabetes ◽  
1994 ◽  
Vol 43 (10) ◽  
pp. 1234-1241 ◽  
Author(s):  
Y. H. Chen ◽  
L. Hansen ◽  
M. X. Chen ◽  
C. Bjorbaek ◽  
H. Vestergaard ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Mrna Level ◽  
Regulatory Subunit ◽  
Coding Region

scholarly journals The Landscape of microRNAs in βCell: Between Phenotype Maintenance and Protection

2021 ◽  
Vol 22 (2) ◽  
pp. 803
Author(s):  
Giuseppina Emanuela Grieco ◽  
Noemi Brusco ◽  
Giada Licata ◽  
Daniela Fignani ◽  
Caterina Formichi ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Metabolic Disorders ◽  
Molecular Mechanisms ◽  
Β Cell ◽  
Physiological Mechanisms ◽  
Effective Strategies ◽  
Chronic Hyperglycaemia ◽  
Shed Light

Diabetes mellitus is a group of heterogeneous metabolic disorders characterized by chronic hyperglycaemia mainly due to pancreatic β cell death and/or dysfunction, caused by several types of stress such as glucotoxicity, lipotoxicity and inflammation. Different patho-physiological mechanisms driving β cell response to these stresses are tightly regulated by microRNAs (miRNAs), a class of negative regulators of gene expression, involved in pathogenic mechanisms occurring in diabetes and in its complications. In this review, we aim to shed light on the most important miRNAs regulating the maintenance and the robustness of β cell identity, as well as on those miRNAs involved in the pathogenesis of the two main forms of diabetes mellitus, i.e., type 1 and type 2 diabetes. Additionally, we acknowledge that the understanding of miRNAs-regulated molecular mechanisms is fundamental in order to develop specific and effective strategies based on miRNAs as therapeutic targets, employing innovative molecules.

scholarly journals Unfolded protein response triggers differential apoptotic mechanisms in ovaries and early embryos exposed to maternal type 1 diabetes

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Aslı Okan ◽  
Necdet Demir ◽  
Berna Sozen
Keyword(s):  
Embryonic Development ◽  
Er Stress ◽  
Unfolded Protein Response ◽  
Molecular Mechanisms ◽  
Early Embryonic Development ◽  
Unfolded Protein ◽  
Caspase 12 ◽  
Early Embryos ◽  
Protein Response

AbstractDiabetes mellitus (DM) has profound effects on the female mammalian reproductive system, and early embryonic development, reducing female reproductive outcomes and inducing developmental programming in utero. However, the underlying cellular and molecular mechanisms remain poorly defined. Accumulating evidence implicates endoplasmic reticulum (ER)-stress with maternal DM associated pathophysiology. Yet the direct pathologies and causal events leading to ovarian dysfunction and altered early embryonic development have not been determined. Here, using an in vivo mouse model of Type 1 DM and in vitro hyperglycaemia-exposure, we demonstrate the activation of ER-stress within adult ovarian tissue and pre-implantation embryos. In diabetic ovaries, we show that the unfolded protein response (UPR) triggers an apoptotic cascade by the co-activation of Caspase 12 and Cleaved Caspase 3 transducers. Whereas DM-exposed early embryos display differential ER-associated responses; by activating Chop in within embryonic precursors and Caspase 12 within placental precursors. Our results offer new insights for understanding the pathological effects of DM on mammalian ovarian function and early embryo development, providing new evidence of its mechanistic link with ER-stress in mice.

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