Abstract
The dysregulation of autophagy, an evolutionarily conserved lysosomal degradation process, has been implicated in a wide variety of human diseases, and thus, small chemicals that modulate autophagy have therapeutic potential. Here, we assessed the ability of active components isolated from Bupleurum falcatum, a popular Chinese herb, to modulate autophagy. We found that saikosaponin D (SsD) and A (SsA) but not C (SsC) potently and reversibly inhibited the fusion of autophagosomes and lysosomes, resulting in the accumulation of autophagosomes, an increased lysosomal pH, and TFEB nuclear translocation. RAB5A knockdown or the expression of a dominant-negative RAB5 mutant significantly reduced the ability of SsD or SsA to block autophagy. Enterovirus A71 (EV-A71), the cause of hand-foot-mouth disease, has been shown to induce autophagy. We found that SsD potently inhibited EV-A71 RNA replication and subsequent viral protein synthesis, thereby preventing EV-A71-induced cell death. ATG5 knockdown inhibited EV-A71 viral protein synthesis, whereas autophagy induction by rapamycin promoted synthesis. Taken together, our data indicate that SsD and SsA are potent late-stage autophagy inhibitors that can be used to prevent EV-A71 infection.
Highlights
Autophagy is an evolutionarily conserved lysosomal degradation process that is essential for maintaining the homeostasis of eukaryotic cells.[1,2,3] Among the three types of autophagy— macroautophagy, microautophagy, and chaperone-mediated autophagy—the most common and best-studied type is macroautophagy, which is hereafter referred to as “autophagy”
We found that the accumulated LC3-II and p62 (SQSTM1) in saikosaponin D (SsD)-treated cells was reduced after washing out SsD, indicating that the effects of SsD on autophagy are reversible (Fig. 1g)
We found that SsD or saikosaponin A (SsA) treatment increased lysosomal pH and induced the nuclear localization of TFEB (Fig. 3) but did not affect ER or lysosome Ca2+ pools (Fig. 4)
Summary
Autophagy is an evolutionarily conserved lysosomal degradation process that is essential for maintaining the homeostasis of eukaryotic cells.[1,2,3] Among the three types of autophagy— macroautophagy, microautophagy, and chaperone-mediated autophagy—the most common and best-studied type is macroautophagy, which is hereafter referred to as “autophagy”. Autophagy is initiated when cytoplasmic components, e.g. damaged organelles or misfolded proteins, are sequestered by an isolated membrane called a phagophore. After cargo is sequestered by elongating this membrane, a double-membrane vesicle called an autophagosome forms. A fusion process occurs between the autophagosome and a lysosome, thereby forming an autolysosome in which the sequestered components are degraded by acidic lysosomal hydrolases. The digested products, such as amino acids or fatty acids, are recycled back to the cytosol to maintain cell homeostasis.[4]
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