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.

中文翻译：

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Enniatin B1诱导的小鼠胚胎成纤维细胞溶酶体膜通透性。

霉菌毒素肾上腺素B ₁（ENN B ₁）广泛存在于谷物饲料和食品中。在本研究中，我们研究了该亲脂性和离子载体性分子如何影响野生型（WT）和溶酶体相关膜蛋白（LAMP）-1/2 double-lys的溶酶体稳定性和分子伴侣介导的自噬（CMA）。缺陷（DD）小鼠胚胎成纤维细胞（MEF）。分别使用中性红（NR）细胞毒性试验和LysoSensor®Yellow / Blue DND-160评估细胞活力和溶酶体pH。通过免疫印迹测定胞质提取物中CMA相关成分LAMP-2和分子伴侣热休克同源蛋白（hsc）70和热休克蛋白（hsp）90表达的变化。在NR分析中，LAMP-1 / 2 DD MEF细胞对ENN B _1的敏感性明显降低在浓度为2.5-10μmol/ L的ENN B ₁中暴露24小时后，野生型MEF细胞比野生型MEF细胞多。浓度低于最大有效浓度一半（EC ₅₀）（1.5–1.7μmol/ L）的ENN B _1会增加WT MEF的溶酶体pH，但不会导致LAMP-1 / 2 DD细胞的溶酶体pH升高，表明溶酶体LAMP-2是ENN B ₁诱导MEF细胞溶酶体碱化和细胞毒性的早期靶标。另外，暴露4小时后胞质hsp90和LAMP-2水平略有增加，表明溶酶体膜通透性（LMP）。总之，似乎ENN B ₁可以在接近EC _50的浓度下使溶酶体膜中的LAMP-2复合物不稳定。导致溶酶体，部分LMP碱化，从而导致CMA相关成分渗入细胞质。