Sumoylation Affects Tau Phosphorylation And Its Degradation

Part Ⅰ SUMOylation promotes tau hyperphosphorylationBackground:Alzheimer's disease (AD) is a neurodegenerative disease, the clinical symptoms of which are extracellular senile plaques (SP) and intracellular neurofibrillary tangles (NTFs). Tau is an important microtubule associated protein, which modulates the stability of axonal microtubules and maintain the function of microtubules. Hyperphophorylated Tau protein induces disaggregation of microtubules and aggregation of neurofibrillary tangles (NFTs). which induces neurodegenerative disease. SUMO (small ubiquitin-like modifer) is a ubiquitin-like modifer molecule, including SUMO-1, SUMO-2, SUMO-3and SUMO-4. SUMO regulates transcription, function and location of substrate proteins via modifying the substrate proteins. SUMOylation of substrate proteins regulates the binding of substrate proteins with other proteins and exposes the association-sites of other proteins, which changes the conformation of proteins. The target sites of SUMOylation may be the same one of ubiquitylation, so that SUMOylation compete againsts ubiquitylation. Some research found that Tau was modified by SUMO-1; SUMO-1immunoreaction could be colocalized with Tau in AD transigenic model. Thus, we suppose that phosphotylation of tau may be related with SUMOylation. Objective:To explore the relation between SUMOylation with phophorylation of Tau.Methods:SUMO-1plasmids are transfected into293/Tau cells. Tau and SUMO-1, mutant Tau K(340)R and SUMO-1plasmids are transiently transfected into HEK293WT cells for48h, and then phosphorylation of Tau is tested by Western blotting; Phosphorylation of Tau are tested when SUMO-1plasmids are transfected for32hours to continue to treatment with OA for16hours, an inhibitor of PP2Ac in SUMO-1-overexpression cells. Phosphorylation of Tau is tested after treatment with Ginkgolice acid in cells. The effect of SUMO-1on phosphorylation of Tau is observed by immunofluorescence stain in cells. The level of SUMO-1and SUMOlation of Tau are tested after upregulating activation of GSK-3β in animals. Distribution and merge of phosphorylated Tau and SUMO-1is observed in AD brain slices.Results:We found:(1) overexpression of SUMO-1induces Tau SUMOylation at Thr-205, Ser-214, Thr-231, Ser-262, Ser-396and Ser-404in HEK293/Tau cells.(2) site-specific mutation of Tau at Lys340(HEK293/Tau340R) abolishes the SUMO-induced tau hyperphosphorylation.(3) down-regulation of PP2A or up-regulation of GSK-3β promotes Tau SUMOylation with a simultaneous Tau hyperphosphorylation in HEK293/Tau cells. SUMO-1immunoreactivity is co-localized with p-tau in AD brain.Conclusion:Tau SUMOylation stimulates its phosphorylation, and Tau hyperphosphorylation promotes its SUMOylation. Part ⅡSUMOylation increases Tau accumulation via inhibiting its ubiquitination Background:Hyperphosphorylated Tau is the major component of neurofibrillary tangles (NFTs) in AD. The normal physiological function of this microtubule-associated protein is to promote the accumulation of microtubulin to form microtubules and maintain microtubule function stability. When Tau protein is abnormally hyperphosphorylated, it will lose the function of stabilizing microtubules, lead to the formation of NFTs and thus participate in the pathogenesis and development of a variety of neurodegenerative diseases. The degradation of abnormal proteins is mainly achieved through three major degradation systems-ubiquitin-proteasome degradation system (UPS), lysosomes-autophagic system and endoplasmic reticulum degradation pathway. UPS mainly contributes to the degradation of most intracellular abnormal proteins or long-lived proteins. Hyperphosphorylated Tau protein can also be degraded through UPS. However, it is still not well clarified that why in AD patients, NFTs can be modified by ubiquitin but still continuously deposited in AD brains. SUMO-1is the small ubiquitin-like modifier molecule, its function is to change the stabilities, functions, localizations in subcelluar organelles and the transcriptional regulations of substrate proteins through SUMO modification. SUMO-1and ubiquitin may have the common target protein binding sites-lysine residues, the target protein SUMOylation and ubiquitination may counteract to each other, meanwhile, SUMOylation of target proteins can lead to the changes of target proteins spatial structures thus affects their degradations through UPS pathway. It has been demonstrated that Tau protein can be modified both by ubiquitin and SUMO-1, and the inhibition of proteasome leads to decreased Tau protein SUMOylation; Tau phosphorylation also promotes Tau SUMOylation. These results indicate that Tau SUMOylation and its ubiquitination correlate to each other, but it is still unknown that whether Tau SUMOylation exerts certain effects on its aggregation.Objective:To investigate the effect of SUMOylation on Tau degradation.Methods:At the cellular level, SUMO-1plasmid was transfected into the stably transfected HEK293/Tau cell lines, and then we treated cells with cycloheximide (GFX) to conduct the protein degradation experiments, and used immunoblotting technique to detect Tau protein degradation and explore its underlying mechanism. Similarly, Tau plasmid and SUMO-1plasmid were transiently transfected into HEK293/wt cell lines simultaneously and then we treated cells with cycloheximide (GFX) to detect Tau protein degradation. At the same time, we extracted soluble and insoluble of Tau from the above cells which were overexpressed Tau, mutated Tau K340R and SUMO-1to detect aggregation of Tau protein.Results:(1) overexpression of SUMO-1inhibits Tau degradation in HEK293/Tau cells but not in wt HEK293/TauK340R cells.(2) overexpression of SUMO-1inhibits ubiquitin expression in both WT HEK293and HEK293/Tau cell lines and decreases ubiquitylation of Tau.(3) SUMOylation decreases the solubity of Tau proteins.Conclusion:SUMOylation increases Tau aggregation through inhibiting its ubiquitination and degradation.