Poly-GR And Poly-PR Inhibit Autophagosomal Formation

Objective:To study the effects of dipeptide repeat proteins Poly-GR and Poly-PR on autophagy pathway and explore the mechanism of these effects.Methods:Cell lines such as HEK 293,HEK 293T,ATG5-WT MEF and ATG5-KO MEF were cultured in vitro as cell models;Cells were transfected with siRNA targeting Beclin 1 gene using RNAiMax to construct a cell model of Beclinl gene deficiency;The plasmids were transfected with Lipofectamine 2000,and overexpressed proteins in cells to construct a cell experimental model;Cell starvation model was constructed by starving cells with Earle’s Balanced Salt Solution(EBSS);Western blot was used to detect the level of LC3,GFP-fusing protein,GAPDH,p70S6K,phosphorylated p70S6K,Beclin 1,total and phosphorylated Bcl-2;The cell nucleus was stained with DAPI,and the proteins in the cell were detected by immunofluorescence assay;The expression,aggregation,localization and colocalization of related proteins in cells were observed by Zeiss laser confocal microscope or Nikon fluorescence microscope;ImageJ was used to analyze the fluorescence intensity.Results:(1)Arginine-containing Poly-GR and Poly-PR can significantly reduce the level of LC3-II in cells and inhibit the formation of autophagosomes,The effect was in a dose dependent manner(0.1μg,0.5μg,3.5μg).(2)According to autophagy flux detected by mCherry-EGFP-LC3,Poly-GR can significantly inhibit the production of autophagosomes,but has no effect on the fusion of autophagosomes with lysosomes;In the case of starvation,Poly-GR and Poly-PR can significantly inhibit autophagy degradation of Htt-60Q and Htt-150Q,and have similar inhibition in ATG5-WT MEF,but not in ATG5-KO MEF.(3)Poly-GR and Poly-PR can change the lysosomal localization of mTOR,inhibit the activity of mTOR,and promote the TFEB nuclear translocation.(4)Poly-PR affected the autophagy initial proteins,ATG5,ATG9,ATG16 and DFCP1,under starvation condition.(6)Poly-PR reduced the phosphorylation level of Bcl-2 at S87 and S70 sites,promoted the binding of Bcl-2 to Beclin1,and the expression of autophagosome was significantly reduced in si-Beclinl cells.(7)Overexpression of GFP-LC3-RFP-LC3 AG in cells,analysis of fluorescence intensity showed that Poly-GR protein significantly reduced the autophagy degradation under starvation condition.Conclusion:The arginine-containing Poly-GR and Poly-PR reduced the phosphorylation level of Bcl-2,promoted the binding of Beclinl and Bcl-2,and affected the autophagy initiation which is associated with ATG5,ATG9,ATG16 and DFCP1.Thereby,Poly-GR and Poly-PR reduced the level of LC3-Ⅱ and inhibited the production of autophagosomes.