SUMOlyzation Of Katanin P60 To Promote Hippocampal Neuron Neurite Outgrowth

Objective: To explore wether Katanin p60 can be modified with SUMOylation(Small ubiquitin-like modifiers)and how SUMOylation of Katanin p60 affects neuronal growth and development.Methods: First,cellular immunofluorescence,Pulldown,and Co-IP were used to detect whether Katanin p60 interacted with SUMO protein in vivo and in vitro.Potential SUMOization sites of Katanin p60 were predicted,and corresponding plasmids were constructed and their SUMO sites were verified.Changes of acetylated microtubules after Tubacin treatment and their effects on the cutting ability of Katanin p60;Katanin p60 and SUMO2 were co-transfected with COS7 to verify the effect of SUMO-modified Katanin p60 on microtubules.Cellular immunofluorescence was used to observe acetylated hippocampal neurons Microtubules were transfected,and wild-type and mutant plasmids were transfected into hippocampal neurons to observe the growth and development of hippocampal neuronal processes.Results:(1)Bioinformatics software predicts the SUMOylation site of Katanin p60(2)GST and GST-SUMO1-3 proteins were successfully purified,and GST Pulldown experiments were performed.The results showed that SUMO2 protein could chromatograph Katanin p60 protein.(3)Incubation of antibodies to Katanin and SUMO2 in 3 DIV hippocampal neurons.Cellular immunochemical experiments showed that Katanin p60 and SUMO2 protein co-localized in hippocampal neurons.(4)Western Blot and immunofluorescence staining showed that the acetylation level of cells increased after Tubacin treatment.(5)Katanin p60 was transfected in COS7 cells,and the microtubules were cut into small fragments by the Tubacin treatment group,while the DMSO group was not able to cut microtubules.(6)Construction of Katanin p60 has 3 potential mutation sites and total mutation plasmids for SUMOylation site,respectively transfected wild type and mutant into COS7 cells,and found that the ability of K330 R and PAN to cut microtubules was lost;(7)In the 293 T cell line,Co-IP experiments show that wild-type Katanin p60 can bind to SUMO2 protein,but K330 R cannot;(8)Katanin p60 and SUMO2 co-transformed COS7 cells,and the fluorescence intensity of cell microtubules was significantly weakened compared with the control group.(9)In neurons of 2-4 DIV,cellular immunofluorescence chemistry experiments show that hippocampal neurons are rich in acetylated microtubules;(10)Transfection of wild-type and mutant plasmids into 2 DIV hippocampal neurons,it was found that Katanin p60 wild-type K77 R and K157 R can promote the increase of the number of branches and length of hippocampal neuronal processes,while neither K330 R nor PAN can promote the development of processes;Conclusions: 1.Katanin p60 can be SUMO-modified and the modified site is at K330;2.Katanin p60 cutting acetylation of microtubules;3.SUMOylation can enhance the ability of Katanin p60 to cut microtubules.Mutation of the K330 at the SUMOylation site of Katanin p60 loses the function of cutting microtubules after mutation.4.The mutation of K330 in the SUMOylation site of Katanin p60 cannot promote the growth of neurons.