Biological Function Studies Of RhoB Using CRISPR/Cas9 Technology

Autophagy provides amimo acids,fatty acids and nucleosides for the synthesis process by targeting proteins and organelles for degradation,especially under certain stresses.The signals that may active the initiation of autophagy usually come from various stress conditions,such as starvation,hypoxia,oxidative stress,DNA damage and so on.It is reported that in mammalian cells AMPK is activated under the condition of energy deficiency,reactive oxygen species(ROS)or activated nitrogen treat,which inhibits mTORC1 activity,leading to activation of the ULK1 complex and initiation of autophagic process.RhoB,as one of Rho family proteins,has various important biological functions.Our previous work found that RhoB can be sumoylated under DNA damage condition,which changes its location and is critical for recruiting TSC complex to lysosomest inhibit mTORC1 activity.In addition,RhoB has important roles in regulating protein transportation,cellular inflammatory response,ansiogenesis vascular function and the development of tumors.Here,we study the biological function of RhoB under various stress conditions that can trigger autophagy.In this paper,we knocked out RhoB in HeLa cells using CRISPR/Cas9 system.RhoB knockout cells and control cells are treated with different conditions including no glucose,no FBS,no amino acid,no glutamine and hydrogen peroxide.We found that inactivation of mTORC1 was blocked by knockout of RhoB specifically under treatment of hydrogen peroxide.Morever,we found that hydrogen peroxide treatment enhanced the sumolation of RhoB.However,whether sumolation of RhoB is related to hydrosen peroxide-induced autophagy remains to be further elucidated.