Autophagy Induced By Natural Small Compound X15-2

Autophagy is a dynamic process in which cellular components of misfolded or damaded organelles are sequestered within double-membrane vesicle termed autophagosomes. The autophagosome cargo is delivered to lysosome for degradation and the resulting macromolecules, such as amino acids can be recycled back to the cytosol for reuse after release back into the cytosol. Beclin1has a central role in autophagy, recently a report indicates that Beclin2also functions in autophagy and interacts with several known Beclin1partners. Autophagy is not only involved in many Physiological processes, but also plays an important role in resistance outside pathogen invading and pathological process.It’s reported that some drugs that induce autophagy can relieve the Parkinson symptoms. First we take advantage of GFP-LC3stable HeLa cell line and high-throughput screening assays. We find X15-2,a small compound derived from fungi,can efficiently induce autophagy. Next we futher confirm X15-2indeed induce autophagy by immunofluorescence and western blotting assay. We also find X15-2can induce mitochondria fragmentation, the loss of membrane potential and the significant increase of reactive oxygen sepsis(ROS). ROS is sufficient for X15-2inducing autophagy and NAC, a ROS scavenger, can inhibit autophagy induce by X15-2. Knockdowning Atg5, the key autophagy gene, can reduce X15-2-induced autophagy. Importantly, knowdowning Beclin1or Beclin2has no influence on X15-2-induced autophagy, but autophagy is interrupted when Beclin1and Beclin2knockdown simultaneously. We conclude that X15-2-induced autophagy is Atg5-dependent and both Beclin1-and Beclin2-dependent.The formation of Lewy bodies (LBs) has been considered to be a marker for neuronal degeneration. It’s reported that some drugs that can induce autophagy can relieve the Parkinson symptoms. Hence, we want to know if X15-2can degradate a-synuclein by autophagy. We construct GFP tagged a-synuclein stable HeLa cell line and tetracycline (Tet)-inducible expression of Myc-a-synuclein in HeLa cell line. We find X15-2can induce a-synuclein degradation by autophagy-lysosome pathway, and it’s Atg5-dependent.Taken together, we have find a small compound X15-2can induce autophagy, and ROS is essential for X15-2-inducing autophagy. X15-2-induced autophagy is Atg5-dependent, and both Beclin1-and Beclin2-dependent. X15-2can induce a-synuclein degradation via autophagy-lysosome pathway. The mechanism of X15-2-inducing autophagy is still unclear, we will futher study how Beclin1and Beclin2coordinate regulate autophagy.