The Function Of SUMOylation On SMN Protein And Its Role In SMA Disease

Spinal muscular atrophy(SMA),a degenerative motor neuron disease,is caused by loss of functional survival motor neuron(SMN)protein due to SMN1 gene mutation.It is one of the most common lethal genetic diseases with a morbidity of 1/6000-10000.Recently SMA treatment has some advances,but it remains to be further investigated.SUMO(Small ubiquitin-like modifier)modification is a dynamic and reversible posttranslational modification,which covalently modifies a large number of cellular proteins,and affects the localization and function of target proteins to participate in regulating many cellular life processes.In this study,SENP2(Sentrin/SUMO-specific protease 2)knockout cells and mice were used to study the SUMOylation of SMN and its roles in SMA.Previous studies found that SENP2 deficient mice develop spontaneous seizures and sudden death.Further observation found they lost motor ability before death.Elevated plus-maze experiments also revealed a loss of motor ability in SENP2 deficient mice.Righting reflex experiments also showed the neuromuscular function was defective in SENP2 deficient mice.Histological analysis showed a significant decrease in the number of muscle fibers in distal hind limb and motor neurons in spinal cord of SENP2 deficient mice.Further experiments showed that the transcription level of SMN was not significantly changed in mouse embryonic fibroblasts(MEF),brain and spinal cord,whereas the protein level was significantly decreased in SENP2 deficient mice.These results suggested SENP2 deficiency causes a decrease of SMN protein,and in turn leads to development of SMA.The main role of SENP2 is to remove SUMOylation,hence SENP2 could remove SMN SUMOylation.A series of biochemical experiments proved SMN protein was mainly modified by SUMO2,and SENP2 could specifically remove SUMO conjugation of SMN,and PIAS2a was the major E3 ligase of SMN.K55 and K119 were the major SUMO sites of SMN.The results indicated SENP2 deficiency leads to a decrease of SMN protein by increasing SUMOylation of SMN,which leads to SMA,so SENP2 might affect the stability of SMN protein.Overexpressing SUMO2 in HEK293T cells,the degradation of endogenous SMN was significantly accelerated.And overexpressing SENP2,its degradation was slowed down.Further,the degradation of SUMO site-mutated SMN protein was accelerated compared to wild-type.These results show that SUMOylation accelerates SMN degradation to reduce the stability of SMN protein,while SENP2 can remove its SUMOylation to enhance its stability.The pathway by which SUMOylation regulates SMN degradation was further investigated.It was found that SUMOylation enhances SMN ubiquitination to promote degradation of SMN by increasing the protein expression of UBE1 and the interaction of SMN and ubiquitin E3 ligase ITCH.SMN participates in CBs(Cajal Bodies)formation in the nucleus.It was found that mutated SMN caused a significant increase of CBs.It was reported acetylation site of SMN is K119,and its acetylation inhibits CBs formation,thus there might be a relationship between SUMOylation and acetylation of SMN.The experimental results suggested SUMOylation enhanced SMN acetylation.Therefore,SUMOylation inhibits CBs formation by enhancing SMN acetylation.In summary,using mouse and cell models to behavioral and histological studies,it was found SENP2 deficient mice developed a SMA phenotype.In molecular level,it revealed SENP2 deficiency didn't affect transcription of SMN,but downregulated its protein level.SMN is mainly modified by SUM02,and the major SUMOylation E3 ligase is PIAS2a.SENP2 specially mediates deSUMOylation of SMN.SUMOylation accelerates degradation of SMN by ubiquitin-proteasome,and increases the acetylation of SMN to inhibit CBs formation.In this paper,it was mainly found that SENP2 deficiency causes hyper-SUMOylation of SMN protein,which further affects the stability and functions of SMN protein,and eventually leads to SMA.These results provide a new strategy for the development of targeted therapeutic drugs for SMA disease.