Enniatin B 1-induced lysosomal membrane permeabilization in mouse embryonic fibroblasts

Mycotoxin Res. 2020 Feb;36(1):23-30. doi: 10.1007/s12550-019-00366-8. Epub 2019 Jul 1.

Abstract

The mycotoxin enniatin B1 (ENN B1) is widely present in grain-based feed and food products. In the present study, we have investigated how this lipophilic and ionophoric molecule can affect the lysosomal stability and chaperone-mediated autophagy (CMA) in wild-type (WT) and in lysosome-associated membrane proteins (LAMP)-1/2 double-deficient (DD) mouse embryonic fibroblasts (MEF). The cell viability and lysosomal pH were assessed using the Neutral Red (NR) cytotoxicity assay and the LysoSensor® Yellow/Blue DND-160, respectively. Changes in the expression of the CMA-related components LAMP-2 and the chaperones heat shock cognate (hsc) 70 and heat shock protein (hsp) 90 were determined in cytosolic extracts by immunoblotting. In the NR assay, LAMP-1/2 DD MEF cells were significantly less sensitive to ENN B1 than WT MEF cells after 24 h exposure to ENN B1 at levels of 2.5-10 μmol/L. Exposure to ENN B1 at concentrations below the half maximal effective concentration (EC50) (1.5-1.7 μmol/L) increased the lysosomal pH in WT MEF, but not in LAMP-1/2 DD cells, suggesting that lysosomal LAMP-2 is an early target of ENN B1-induced lysosomal alkalization and cytotoxicity in MEF cells. Additionally, cytosolic hsp90 and LAMP-2 levels slightly increased after exposure for 4 h, indicating lysosomal membrane permeabilization (LMP). In summary, it appeared that ENN B1 can destabilize the LAMP-2 complex in the lysosomal membrane at concentrations close to the EC50, resulting in the alkalinization of lysosomes, partial LMP, and thereby leakage of CMA-associated components into the cytosol.

Keywords: Chaperone-mediated autophagy (CMA); Cytotoxic activity; Enniatin B1 (ENN B1); Lysosomal pH; Mouse embryonic fibroblasts (MEF).

MeSH terms

  • Animals
  • Chaperone-Mediated Autophagy / drug effects
  • Depsipeptides / toxicity*
  • Fibroblasts
  • Gene Deletion
  • HSC70 Heat-Shock Proteins / drug effects
  • HSC70 Heat-Shock Proteins / metabolism
  • HSP90 Heat-Shock Proteins / drug effects
  • HSP90 Heat-Shock Proteins / metabolism
  • Hydrogen-Ion Concentration / drug effects
  • Intracellular Membranes / drug effects*
  • Lysosomal-Associated Membrane Protein 2 / drug effects
  • Lysosomal-Associated Membrane Protein 2 / genetics
  • Lysosomal-Associated Membrane Protein 2 / metabolism
  • Lysosomes / pathology*
  • Mice
  • Molecular Chaperones / drug effects
  • Molecular Chaperones / metabolism
  • Mycotoxins / toxicity*
  • Permeability / drug effects*

Substances

  • Depsipeptides
  • HSC70 Heat-Shock Proteins
  • HSP90 Heat-Shock Proteins
  • Lysosomal-Associated Membrane Protein 2
  • Molecular Chaperones
  • Mycotoxins
  • enniatins