Chaperone-mediated autophagy protects against hyperglycemic stress

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, <i>Oncorhynchus mykiss</i>), 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. <b>Abbreviations</b>: 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; H₂O₂: 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.

分子伴侣介导的自噬（Chaperone-mediated autophagy, CMA）是溶酶体蛋白水解的核心途径之一，对细胞稳态与代谢至关重要，其功能缺陷与多种人类疾病密切相关。尽管哺乳动物体内的CMA已有充分研究，但鱼类中CMA的功能证据直到近年才被报道，为从全新视角解析该通路的功能提供了新方向。本研究拟探索虹鳟（rainbow trout, RT，学名Oncorhynchus mykiss）体内的CMA功能：虹鳟是公认的葡萄糖不耐受研究模式生物，其体内存在CMA限速因子溶酶体相关膜蛋白2A（lysosomal associated membrane protein 2A, LAMP2A）的两个旁系同源基因。为此，我们在虹鳟肝癌来源的细胞系（RTH-149）中验证了此前用于示踪哺乳动物细胞中功能性CMA的荧光报告基因（KFERQ-PA-mCherry1）。研究发现，将细胞置于高糖环境（HG，25 mM）中培养，可使CMA报告基因以KFERQ和LAMP2A依赖的方式向溶酶体和/或晚期内体易位，且与对照组（5 mM）相比，报告基因的半衰期缩短，证明CMA通量有所提升。此外，我们观察到高糖暴露下CMA的激活由线粒体活性氧（reactive oxygen species, ROS）的产生所介导，且涉及抗氧化转录因子Nfe2l2/Nrf2（nfe2 like bZIP transcription factor 2）。最后，我们证实CMA在对抗高糖诱导的应激中发挥重要保护作用，且该作用主要由虹鳟两个LAMP2A同源基因中的一个所介导。综上，本研究为虹鳟体内存在CMA活性提供了确凿证据，并阐明了CMA在葡萄糖相关代谢紊乱中的作用与调控机制。<b>缩略词表</b>：AREs：抗氧化反应元件（antioxidant response elements）；CHC：α-氰基-4-羟基肉桂酸（α-cyano -4-hydroxycinnamic acid）；Chr：染色体（chromosome）；CMA：分子伴侣介导的自噬（Chaperone-mediated autophagy）；CT：对照组（control）；DMF：富马酸二甲酯（dimethyl fumarate）；Emi：内体微自噬（endosomal microautophagy）；HG：高糖（high-glucose）；HMOX1：血红素氧合酶1（heme oxygenase 1）；H₂O₂：过氧化氢（hydrogen peroxide）；KFERQ：赖氨酸-苯丙氨酸-谷氨酸-精氨酸-谷氨酰胺（lysine-phenylalanine-glutamate-arginine-glutamine）；LAMP1：溶酶体相关膜蛋白1（lysosomal associated membrane protein 1）；LAMP2A：溶酶体相关膜蛋白2A（lysosomal associated membrane protein 2A）；MCC：曼德斯相关系数（Manders’ correlation coefficient）；Mo：吗啉寡聚核苷酸（morpholino oligonucleotide）；NAC：N-乙酰半胱氨酸（N-acetyl cysteine）；NFE2L2/NRF2：NFE2样bZIP转录因子2（NFE2 like bZIP transcription factor 2）；PA-mCherry：光活化mCherry（photoactivable mCherry）；PCC：皮尔逊相关系数（Pearson’s correlation coefficient）；ROS：活性氧（reactive oxygen species）；RT：虹鳟（rainbow trout）；siRNAs：小干扰RNA（small interfering RNAs）；SOD：超氧化物歧化酶（superoxide dismutase）；Tsg101：肿瘤易感基因101（tumor susceptibility 101）；TTFA：2-噻吩甲酰三氟丙酮（2-thenoyltrifluoroacetone）；WGD：全基因组复制（whole-genome duplication）。