Hydrogen peroxide depletes phosphatidylinositol-3-phosphate from endosomes in a p38 MAPK-dependent manner and perturbs endocytosis

Abstract

Phosphatidylinositol-3-phosphate (PI3P) is a lipid that is enriched specifically in early endosomes. Given that early endosomes containing PI3P act as a microdomain to recruit proteins that contain a PI3P-binding domain (FYVE domain), the equilibrium between the production and degradation of PI3P influences a variety of processes, including endocytosis and signal transduction via endosomes. In the study reported herein, we have developed a novel analytical method to quantify the amount of PI3P in endosomes by introducing a GST-2xFYVE protein probe into semi-intact cells. The GST-2xFYVE probe was targeted specifically to intracellular PI3P-containing endosomes, which retained their small punctate structure, and allowed the semi-quantitative measurement of intracellular PI3P. Using the method, we found that treatment of HeLa cells with H(2)O(2) decreased the amount of PI3P in endosomes in a p38 MAPK-dependent manner. In addition, H(2)O(2) treatment delayed transport through various endocytic pathways, especially post-early endosome transport; the retrograde transport of cholera toxin was especially dependent on the amount of PI3P in endosomes. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

A Resealed-Cell System for Analyzing Pathogenic Intracellular Events: Perturbation of Endocytic Pathways under Diabetic Conditions

“…The results of the morphological and biochemical analysis of the WT and Db cells indicated that p38 MAPK was activated in Db cells, which resulted in a decrease in phosphatidylinositol-3-phosphate (PI3P) in endosomes. This finding was consistent with our previous report that hydrogen peroxide (H 2 O 2 ) treatment of HeLa cells decreased intracellular PI3P in a p38 MAPK-dependent manner [22]. We could reconstitute successfully the typical endocytic pathways in WT and Db cells, and identified several perturbations of endocytic pathways that occurred specifically under diabetic conditions.…”

“…We found that > 0.2 µg/ml SLO was sufficient to permeabilize ∼90% of HeLa cells. Previously, we showed that the concentration of SLO required to give this level of permeabilization depended on the cell type [8], [13]–[16], [22], probably due to the differences in cholesterol content in the plasma membranes of different cells. Subsequent experiments were performed with 0.13 µg/ml SLO.…”

“…Therefore, we examined the level of intracellular PI3P in WT and Db cells by the FYVE-targeting assay. We established this assay previously to measure semi-quantitatively by immunofluorescence the amount of intracellular PI3P using probes that contain the PI3P binding (FYVE) domain [22]. Semi-intact HeLa cells were incubated with WT or Db liver cytosol for 30 min, then further with GST-2xFYVE proteins for 15 min to label the intracellular PI3P.…”

Cytotoxic Effects of Transthyretin Aggregates in an Epidermoid Cell Line

“…Is a keratinocyte TTR receptor involved in agTTR uptake and in mediating agTTR-dependent oxidative stress? How does such oxidative stress influence endocytic transport pathways of skin cells [40,41] ?…”

What can lipidomics tell us about the pathogenesis of Alzheimer disease?

“…Phosphatidylinositol 3-phosphate (PI3P) is found predominantly on early endosomal membranes, as well as in the intraluminal vesicles of multivescular endosomes (Simonsen et al, 2001). In Hela cells treated with hydrogen peroxide, PI3P was found to be obliterated from endosomes resulting in the perturbations in endocytosis pathway (Kano et al, 2011). PI3P has been shown to regulate receptor sorting but not transport in endolysosomal pathway (Petiot et al, 2003).…”