Université de Pau et des Pays de l'Adour, E2S UPPA, INRAE, UMR1419 Nutrition Métabolisme et Aquaculture, Saint-Pée-sur-Nivelle, France.
INRAE, UR1037 Laboratory of Fish Physiology and Genomics, Campus de Beaulieu, Rennes, France.
Autophagy. 2024 Apr;20(4):752-768. doi: 10.1080/15548627.2023.2267415. Epub 2023 Oct 12.
Chaperone-mediated autophagy (CMA) is a major pathway of lysosomal proteolysis critical for cellular homeostasis and metabolism, and whose defects have been associated with several human pathologies. While CMA has been well described in mammals, functional evidence has only recently been documented in fish, opening up new perspectives to tackle this function under a novel angle. Now we propose to explore CMA functions in the rainbow trout (RT, ), a fish species recognized as a model organism of glucose intolerance and characterized by the presence of two paralogs of the CMA-limiting factor Lamp2A (lysosomal associated membrane protein 2A). To this end, we validated a fluorescent reporter (KFERQ-PA-mCherry1) previously used to track functional CMA in mammalian cells, in an RT hepatoma-derived cell line (RTH-149). We found that incubation of cells with high-glucose levels (HG, 25 mM) induced translocation of the CMA reporter to lysosomes and/or late endosomes in a KFERQ- and Lamp2A-dependent manner, as well as reduced its half-life compared to the control (5 mM), thus demonstrating increased CMA flux. Furthermore, we observed that activation of CMA upon HG exposure was mediated by generation of mitochondrial reactive oxygen species, and involving the antioxidant transcription factor Nfe2l2/Nrf2 (nfe2 like bZIP transcription factor 2). Finally, we demonstrated that CMA plays an important protective role against HG-induced stress, primarily mediated by one of the two RT Lamp2As. Together, our results provide unequivocal evidence for CMA activity existence in RT and highlight both the role and regulation of CMA during glucose-related metabolic disorders.: AREs: antioxidant response elements; CHC: α-cyano -4-hydroxycinnamic acid; Chr: chromosome; CMA: chaperone-mediated autophagy; CT: control; DMF: dimethyl fumarate; Emi: endosomal microautophagy; HG: high-glucose; HMOX1: heme oxygenase 1; HO: hydrogen peroxide; KFERQ: lysine-phenylalanine-glutamate-arginine-glutamine; LAMP1: lysosomal associated membrane protein 1; LAMP2A: lysosomal associated membrane protein 2A; MCC: Manders' correlation coefficient; Manders' correlation coefficient Mo: morpholino oligonucleotide; NAC: N-acetyl cysteine; NFE2L2/NRF2: NFE2 like bZIP transcription factor 2; PA-mCherry: photoactivable mCherry; PCC: Pearson's correlation coefficient; ROS: reactive oxygen species; RT: rainbow trout; siRNAs: small interfering RNAs; SOD: superoxide dismutase; Tsg101: tumor susceptibility 101; TTFA: 2-thenoyltrifluoroacetone; WGD: whole-genome duplication.
伴侣介导的自噬（CMA）是溶酶体蛋白水解的主要途径，对细胞内稳态和代谢至关重要，其缺陷与几种人类病理有关。虽然 CMA 在哺乳动物中得到了很好的描述，但最近才在鱼类中记录到其功能证据，为从新的角度解决这一功能开辟了新的前景。现在，我们提议研究虹鳟鱼（RT）中的 CMA 功能，虹鳟鱼是一种葡萄糖不耐受的模型生物，其特征是存在 Lamp2A（溶酶体相关膜蛋白 2A）的两个 paralogs。为此，我们在一种源自虹鳟鱼肝癌的细胞系（RTH-149）中验证了先前用于跟踪哺乳动物细胞中功能性 CMA 的荧光报告物（KFERQ-PA-mCherry1）。我们发现，用高葡萄糖水平（HG，25 mM）孵育细胞会以 KFERQ 和 Lamp2A 依赖的方式将 CMA 报告物易位到溶酶体和/或晚期内体中，并且与对照相比其半衰期降低（5 mM），从而表明 CMA 通量增加。此外，我们观察到，HG 暴露时 CMA 的激活是由线粒体活性氧的产生介导的，并涉及抗氧化转录因子 Nfe2l2/Nrf2（nfe2 样 bZIP 转录因子 2）。最后，我们证明 CMA 在 HG 诱导的应激中发挥重要的保护作用，主要由两个 RT Lamp2As 之一介导。总之，我们的研究结果为 RT 中 CMA 活性的存在提供了明确的证据，并强调了 CMA 在与葡萄糖相关的代谢紊乱中的作用和调节。
