Restoration of Clostridium difficile toxin-B-inhibited phospholipase D by phosphatidylinositol 4,5-bisphosphate.

6533b870fe1ef96bd12d06cb

RESEARCH PRODUCT

Restoration of Clostridium difficile toxin-B-inhibited phospholipase D by phosphatidylinositol 4,5-bisphosphate.

Christoph Von Eichel-streiber Ulrich Rümenapp Karl Jakobs Martina Schmidt Sabine Ott Christoph Nehls Jutta Keller

subject

Phosphatidylinositol 4 5-Diphosphate GTP'Bacterial Toxins Clostridium difficile toxin B Biology Biochemistry Cell Line chemistry.chemical_compound Bacterial Proteins GTP-Binding Proteins Phosphatidylcholine RhoB GTP-Binding Protein Phospholipase D Humans Phosphatidylinositol Enzyme Inhibitors rhoB GTP-Binding Protein Phospholipase D Clostridioides difficile HEK 293 cells Cell Membrane Membrane Proteins Receptors Muscarinic Cell biology Enzyme Activation enzymes and coenzymes (carbohydrates)chemistry Phosphatidylinositol 4 5-bisphosphate Guanosine 5'-O-(3-Thiotriphosphate)lipids (amino acids peptides and proteins)

description

Receptor signalling to phospholipase D (PLD) in human embryonic kidney (HEK) cells stably expressing the m3 muscarinic acetylcholine receptor apparently involves Rho proteins. Since phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] has been recognized as an essential cofactor for PLD activity and since activated Rho proteins have been reported to stimulate the synthesis of PtdIns(4,5)P2, we studied whether in HEK cells PLD activity is regulated by PtdIns(4,5)P2 and, in particular, whether PtdIns(4,5)P2 can restore PLD activity inhibited by Clostridium difficile toxin B, which inactivates Rho proteins. Addition of MgATP to permeabilized HEK cells increased basal PLD activity and potentiated PLD stimulation by the stable GTP analogue, guanosine 5'-[gamma-thio]triphosphate (GTP[S]), concomitant with a large increase in PtdIns(4,5)P2. On the other hand, neomycin, which binds to PtdIns(4,5)P2, inhibited basal and GTP[S]-stimulated PLD activities. Addition of PtdIns(4,5)P2 increased PLD activity in HEK cell membranes by 2-3-fold, whereas various other phospholipids were ineffective. Prior treatment of HEK cells with toxin B reduced the level of PtdIns(4,5)P2, measured either in intact cells or in membrane preparations, by about 40%. In membranes of toxin-B-treated cells, basal and GTP[S]-stimulated PLD activities were reduced, when measured with exogenous phosphatidylcholine as enzyme substrate. Inclusion of PtdIns(4,5)P2 with phosphatidylcholine in the substrate vesicles or addition of PtdIns(4,5)P2 fully restored basal and GTP[S]-stimulated PLD activities in membranes of toxin-B-treated cells. In conclusion, the data indicate that PtdIns(4,5)P2 is an essential cofactor for PLD activity in HEK cells and that inhibition of PLD activity by the Rho-inactivating toxin B is apparently caused by depletion of the PLD cofactor, PtdIns(4,5)P2.

year	journal	country	edition	language
1996-09-15	European journal of biochemistry

10.1111/j.1432-1033.1996.0707h.x https://pubmed.ncbi.nlm.nih.gov/8856074