| Background and objective Autophagy plays an important role in maintaining cell homeostasis.It is one of the main proteins degradation pathways and involved in a variety of metabolic processes.Functional defects of autophagy can induce neurodegenerative diseases which is closely related to aging.Autophagy is regulated by many kinases,including m TOR and AMPK.However,autophagy is a complex process that requires multiple protein synthesis.For the coordination of multiple steps and protein synthesis associated with autophagy,a master regulator is needed.TFEB is the key regulatory transcription factor for autophagy.TFEB belongs to the basic helix-loop-helix-leucine-zipper transcription factor.TFEB can regulate the transcription of a variety of genes involved in autophagy and lysosomal biogenesis.TFEB has been shown to bind to the promoter regions of numerous autophagy genes and to induce autophagosome biogenesis and autophagosome–lysosome fusion.In resting cells,under nutrient-rich conditions,TFEB is phosphorylated,inactive and in cytosolic.Upon starvation or under conditions of lysosomal dysfunction,the kinase activity is inhibited and TFEB are dephosphorylated.TFEB is transferred to the nucleus and plays transcriptional function.However,the specific mechanism(s)by which TFEB is transferred to the nucleus are unclear.Hsp90 is a highly conserved chaperone protein and widely exists in eukaryotic cells.Under the ATP-rich condition,Hsp90 can bind with client proteins,including a variety of autophagy related proteins,to stabilize their structure.In addition,Hsp90 can promote many transcription factors transferred to the nucleus and playing the transcription function.Previous studies have shown that Hsp90 plays an important role in the process of autophagy,but the specific mechanism regulating autophagy especially about transcription remains unclear.Cdk5 is an important kinase protein in the central nervous system.Stress stimulation can affect the function of neurons by regulating the activity of Cdk5,including starvation stimulation which can cause autophagy.However,there are few studies on the changes of Cdk5 activity induced by starvation.The specific mechanism of starvation-cdk5-autophagy regulation pathway remains to be explored.Cdk5 is a proline-directed kinase that phosphorylates serine and threonine directly upstream of proline residues.Sequence analysis revealed the potential phosphorylation site of Cdk5 in the sequence of Hsp90.This suggests that Hsp90 may play a key role in the Cdk5 regulation of autophagy.The purpose of this research is to explore the function of Hsp90 to regulate TFEB and affect autophagy activity.Studying the phosphorylation of Hsp90 by Cdk5 and the biological effects.Finding Cdk5,Hsp90 and TFEB relationship between each molecule in the process of autophagy.Finally finding out the specific functions of these protains through regulation of autophagy.Methods In this study,we found that Hsp90 played a key regulatory role in starvation-induced autophagy by western blot and the expression of RFP-GFP-LC3 observed by fluorescence microscope.Meanwhile,after Hsp90 was inhibited,TFEB expression was detected in cytosolic or nuclear by western blot.The effect of Hsp90 on TFEB activity was tested by luciferase reporter gene assay.The transcription level of TFEB downstream target genes was tested by q PCR.The interaction between Hsp90 and TFEB was detected by immunoprecipitation and immunofluorescence.Secondly,the expression p35 and p25 which belong to Cdk5 activator were detected by western blot under starvation stimulation.The phosphorylation activity of Cdk5 was detected by in vitro kinase assay.The effect of Cdk5 on autophagy was explored by western blot and RFP-GFP-LC3 observed by fluorescence microscope.Third,the phosphorylation of Hsp90 by Cdk5 was tested by in vitro kinase assay and immunoprecipitation.The phosphorylation sites of Hsp90 were identified by transfection of mutant plasmids of Hsp90.The effect of Cdk5 on TFEB's localization and transcriptional activity was also detected in cytosolic or nuclear by western blot,by reporter gene assay,and by q PCR.Finally,various strains of C.elegans were used to detect the effect of Hsp90 on TFEB localization and transcriptional activity,as well as on autophagy by fluorescence assay and q PCR.The effect of Hsp90 mutation on starvation-induced longevity was tested by lifespan assay.Through the hybridization of different nematodes' s strains,TFEB was overexpressed in the Hsp90 mutant strain and in this new strain,starvation could induce TFEB's activity and the longevity was tested under starvation.In addition,by interfering the expression of Cdk5 and Hsp90 with RNAi,the lifespan was detected.Result Experiment 1: Hsp90 mediated TFEB transferred into the nucleus for transcriptional activity(1)The effect of Hsp90 on autophagy was explored by detecting the expression of LC3 II,the marker of autophagy,and autophagy flux.The results showed that inhibition of Hsp90 expression and function could significantly inhibit autophagy activity.(2)The effect of Hsp90 on the localization of TFEB which is the key transcription factor of autophagy was investigated.The results showed that inhibiting Hsp90 expression and function significantly decreased TFEB's expression in nucleus.(3)Exploring the effect of Hsp90 on TFEB transcriptional activity.The reporter gene and q RCP results showed that inhibition of Hsp90 expression and function can decrease TFEB transcriptional activity as well as the transcriptional levels of TFEB downstream target gene.(4)Immunoprecipitation showed that TFEB could bind directly with Hsp90,and starvation promoted the combination of TFEB and Hsp90.Also we found that the middle domain of Hsp90 plays a key role in the combination.Experiment 2: kinase Cdk5 regulated autophagy activity(1)Western blotting experiments showed that the expression levels of p35 and p25 which are Cdk5's activators were significantly decreased under starvation.The phosphorylation activity of Cdk5 was significantly reduced under starvation by in vitro kinase assay.(2)Overexpression of Cdk5 and p35 could significantly inhibit the expression of LC3 II,autophagy marker,induced by starvation and inhibit the autophagy flux.Experiment 3: Cdk5 regulated TFEB activity by phosphorylating Hsp90(1)In vitro kinase assay and immunoprecipitation showed that Cdk5 could directly phosphorylate Hsp90.After transfection with Hsp90S595 A mutant plasmid,the results showed that Cdk5 could not phosphorylate Hsp90,suggesting that the phosphorylation site was Ser 595.(2)Hsp90S595D mutation can lead to the phosphorylation of Ser 595.The results of Hsp90 dimer experiment and immunoprecipitation showed that the phosphorylation at Ser 595 could significantly reduce the expression level of Hsp90 dimer,as well as the binding ability of Hsp90 to its common client Hsp70.(3)TFEB localization and transcriptional activity were detected after Cdk5 and p35 overexpression.The results showed that overexpression of Cdk5 and p35 could significantly reduce starvation-induced TFEB nuclear localization,TFEB transcriptional activity and m RNA levels of downstream TFEB target genes.Experiment 4: hsp90-TFEB-autophagy regulation pathway influence on lifespan and neurodegenerative disease(1)Hsp90 mutant and wild type nematodes were used to detect the effect of Hsp90 on TFEB and autophagy.The results showed that inhibition of Hsp90 inhibited both the starvation-induced TFEB nuclear localization and the transcriptional levels of downstream TFEB target genes,as well as the starvation-induced autophagy.(2)Inhibiting the function of Hsp90 can inhibit the starvation-induced lifespan extension in nematodes.After overexpression of TFEB is in the Hsp90 mutant nematodes,the result showed that the effect of Hsp90 on lifespan is involved with TFEB.Interference with the expression of Cdk5 and Hsp90 in C.elegans showed that interference with the expression of Hsp90 alone had no significant effect on the lifespan of C.elegans.Interference with the expression of Cdk5 alone could significantly increase the lifespan of C.elegans,meanwhile interference with the expression of Hsp90 could restore the lifespan of C.elegans to a normal state.(3)CR could protect the dopaminergic neurons in the substantia nigra and the striatum in the MPTP mouse model.After inhibiting the function of Hsp90,CR could not have the protective effect on the neurons.Conclusion In this study,we firstly found that TFEB was transferred into the nucleus for transcriptional activity with the coordination of Hsp90,which regulated the autophagy.We also found that Cdk5 phosphorylates Hsp90 at Ser 595.Cdk5 could regulate TFEB's nuclear location and transcriptional activity by inhibiting the function of Hsp90,and then affect the autophagy.Cdk5-Hsp90-TFEB-autophagy regulation pathway was established,and its important role in extending lifespan and improving Parkinson's disease was preliminarily studied,which provided a new target and treatment strategy for antiaging and treatment of Parkinson's disease. |
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