Study On Mechanism Of Slingshot N-terminal Domain Auto-inhibition And Its Activation Mechanism By F-actin

Slingshot belongs to the DUSPs family which were in the type I tyrosine phosphatase family(PTPs).DUSPs differ from classical PTPs were that they can both dephosphorylate tyrosine(pY)and dephosphorylate Ser/Thr(pS/T).Currently the Slingshot-specific substrate is known as phosphorylated Cofilin protein(p-Cofilin).Slingshot can affect the polymerization and dissociation of F-actin by regulating the dephosphorylation of the substrate Cofilin and thus participate in cytoskeletal dynamics.On the other hand,F-actin can bind Slingshot certain specific amino acids or domains,which can greatly increase the activity of Slingshot phosphatase.Recent studies have found abnormal increase in Slingshot phosphatase activity in some diseases,for example,cancers,Alzheimer's disease and certain vascular diseases.Although the Slingshot2(SSH2)catalytic domain crystal structure was resolved in 2006,there is no detailed description of the more comprehensive substrate catalytic mechanism of SSH2 phosphatase and how it is activated by F-actin.Objectives1.Basic enzymatic studies of Slingshot phosphatase;the basic catalytic mechanism of Slingshot;2.Slingshot substrate selectivity mechanism study;3.How does F-actin activate Slingshot,and the activation mechanism;4.How to efficiently screen Slingshot small molecule inhibitors or agonists by our own established methods;Methods1.A series of Slingshot truncated plasmids were constructed by PCR and expressed in vitro by E.coli BL21 competent cells for later enzyme activity detection;2.Select small molecular substrates with slightly different structures and physiological substrate to detect the selection mechanism of the Slingshot truncated protein on the substrate;3.pH profile assay to detect the acid-base catalytic mechanism of Slingshot phosphatase;4.The leaving group dependence assay detects the dependence of Slingshot on the small molecule dissociation group of the product;5.Preparation of F-actin in vitro using muscle acetone powder was used to detect Slingshot enzyme activity using pNPP and p-Cofilin as substrates.6.Using the leaving group dependence assay to detect the mechanism by which F-actin activates Slingshot enzyme activity;7.Trypsin digestion assay in vitro to idetify that F-actin activates Slingshot which will cause a conformational change in Slingshot;8.Introduce the Cys mutation at a specific site of the Slingshot protein,label the single Cys with a small-molecule fluorescent substrate mBBr,and detect the activation of Slingshot with continuous fluorescence spectral scanning while causing Slingshot structure changes when Slingshot activated by F-actin;9.FlAsH BRET assay verified that when MCF-7 cells were stimulated with NRG agonists,the activation mechanism of Slingshot was consistent with the results of in vitro experiments;10.Development of Slingshot enzyme activity detection probe using FlAsH BRET,which can be used to screen Slingshot small molecule inhibitors or agonists;Results1.SSH2 N-terminal domain acted as auto-inhibitionThrough the small-scale expression of the constructed SSH2 different lengths or different truncated proteins,purification in vitro,and according to the different domains of SSH2,A,B,P,long C-terminal domain protein expression screening,the final detection of enzymes was found that with the continuous truncation of the N-terminal(1-305)of SSH2,the SSH2 enzyme activity gradually increased.On the contrary,the presence of the N-terminal domain inhibited the SSH2 enzyme activity.For the detection of pNPP(monocyclic),DiFMUP(bicyclic),OMFP(multi-ring),the structure of small molecule substrates showed that SSH2 has better selectivity for polycyclic substrates relative to monocyclic substrates;The detection of p-Cofilin protein activity also found that the N-terminal of SSH2 also has auto-inhibition effect on physiological substrates,and unlike the small molecule substrate,the N-terminal 233-305 domain of SSH2 plays a key role in the recognition of physiological substrates.Intracellular detection of SSH2 full length and truncation of SSH2 233-full length enzyme activities,also found that the N-terminal of SSH2 has an automatic inhibition.2.N-terminal region auto-inhibition was through non-competitive modeThe SSH2 N-terminal 1-227 domain was expressed and purified in vitro,and incubated with SSH2 233-490 and SSH2 305-490 to detect the Ki inhibition constant of N-terminal domain against SSH2.And found that with the concentration of 1-227 protein increasing,SSH2 233-490 and SSH2 305-490 have no change in the affity Km value of pNPP,but Vmax.decreased;since 233-305 is necessary for the recognition of physiological substrate cofilin,So we detected 1-227 with SSH2 233-490 the Ki inhibition constant.Results showed that the inhibition of N-terminal region of SSH2 is a non-competitive way,that is,the N-terminal region of SSH2 does not compete with the substrate for the SSH2 active center,and may act on some key amino acids or domains outside the active center.3.The catalytic mechanism of SSH2 is acid-base catalyzed,and the auto-inhibition destroys the stability of the general acid to the products dissociation groupThe basic catalytic mechanism of SSH2 1-490,SSH2 233-490 and SSH2 305-490 was detected by pH profile.The catalytic mechanism of SSH2 is similar to that of most classical phosphatases.Slingshot is also acid-base catalyzed,which are regulated by general acid and general base.From the pH profile,the order of SSH2 enzyme activity is:SSH2 305-490>SSH2 233-490>SSH2 1-490,showing N terminal region inhibition trend;meanwhile,based on the lim value in the pH profile,the smaller the lim value,the weaker the role of the general acid in the catalytic process.So the 1-49 lim value is the smallest,probably because the N terminal region destroys the stability of the general acid on the substrate dissociation group.In addition,in the pH profile,when pH>6,the pH curve of 1-490 is significantly lighter than the pH curves of 233-490 and 305-490,indicating that the general acid has a significantly weaker effect in the catalytic process of 1-490 compared with in 233-490 and 305-490.Leaving group dependence experiments show that the absolute value of ?1 of 1-490 is significantly larger than that of the other two proteins,and the slope of the straight line is significantly larger than that of the other two proteins,indicating that the role of the general acid is restricted in the presence of N terminal region.The stability of the product dissociation group is reduced.4.F-actin activates SSH2 enzyme activity by acting on the general acid to relieving N-terminal auto-inhibition,increasing the stability of substrate dissociation groupsF-actin was incubated with SSH2 1-490,SSH2 233*490,and SSH2 305-490.respectively.The enzyme activity of SSH2 with different truncation on physiological substrate was detected.The results showed that the enzyme activity of 1-490 was significantly increased after adding F-actin.However,there was no change in the enzyme activities of 233-490 and 305-490.Switching to pNPP small molecule substrate detection,also found that after adding F-actin the enzyme activities of 1-490 and 1-455 were significantly increased,while the activities of other truncated proteins did not change significantly.In order to verify the activation mechanism of F-actin,we examined the leaving group dependence of SSH2 on small molecule substrates with different pKa values after adding F-actin.The results showed that the slope of the linear line of 1-490 became smaller after adding F-actin.,close to the p1 value of 233-490,indicating that after the addition of F-actin,the general acid begins tobe function,the stability of the substrate dissociation group increased,the N-terminal auto-inhibition is released,and the SSH2 enzyme activity increased.5.F-actin association leads to SSH2 conformational rearrangementsIn vitro Trypsin digestion assay confirmed that the size of the SSH2 1-490-cleaved protein fragment was different from that when F-actin was added.The size of the fragment was 40kDa and 28 kDa when digested with 1-490 alone.After addition of F-actin,the size of the fragment was changed to a single protein fragment of only 44 kDa.According Adman N-terminal degradation sequencing method and the SSH21-490 primary structure amino acid sequence analysis,when 1-490 was alone,the restriction sites were at R66,K291 and K450;after adding F-actin,K291 was protected by F-actin instead of originally exposed on the surface of the structure;R332 located on the loop between the 2nd alpha helix and the 3rd beta fold is hidden in the absence of F-actin,exposed after F-actin is added.6.The molecular mechanism of SSH2 by F-actin activatesA cysteine mutation was introduced at the specific site(19,63,78,98,146,161)at the N-terminus of SSH2,and the above-mentioned mutant cysteine was labeled with a small molecule fluorescent substrate mBBr.Then the Y in the VYD loop was mutated to W,the results shown the amino acids at the N-termimus were involved in the conformational change of SSH2 by the continuous scanning of the fluorescence spectrum.Fluorescence quenching experiments confirmed that C19 and C146 are further away from VYD loop when SSH2 is in a resting state,while C63,C78,C98,and C161 are closer to VYD loop.When F-actin activates SSH2,C63 Starting away from the VYD loop,C19 starts to approach the VYD loop.7.The molecular mechanism of SSH2 activation by F-actin under physiological conditionsThe FlAsH-BRET experiment was used to verify that the mechanism of F-actin activation of SSH2 proposed in vitro was also applicable to the activation mechanism under physiological conditions.The eukaryotic expression vector SSH2 full-length plasmid was inserted into Rluc(luciferase)at the N-position of the position 23,and a specific amino acid sequence of CCPGCC was inserted into the VYD loop.The BRET signal intensity was weakened before and after NRG stimulation.It was judged that 23 approached the VYD loop after cell stimulation,and the BRET signal intensity increased after the cell stimulation.The BRET signal intensity decreased indicating that the L63 amino acids were far from the VYD loop after the cells were stimulated.8.Extend SSH2-63-FIAsH to a molecular probe that can detect SSH2 activityIn the FlAsH-BRET experiment,we verified that there is a positive correlation between the signal of ABRET and the degree of dephosphorylation of p-Cofilin,that is,when the BRET value increases,the SSH2 phosphatase activity increases.According to previous research reported,when the serine 21,25,32,and 36 at the N terminal positions of SSH2 were phosphorylated by GSK kinase,the phosphatase activity of SSH2 was decreased;based on the SSH2-63-FlAsH plasmid,the N-terminus 21,25,32,36 were mutated to acidic amino acids D and E respectively,and a new SSH2-DDEE-63-FIAsH plasmid was constructed.After the new plasmid was over expressed in the cell,the ABRET signal was detected,and the ABRET signal intensity was weakened.The phosphatase activity of SSH2 was decreased after the mutation,maybe because of the N-terminal domain could not be dissociated from the VYD loop after the N-terminal amino acid mutation.Conclusions1.The N-terminal domain of SSH2 has an enzyme auto-inhibition effect,which is acted as non-competitive inhibition.2.The catalytic mechanism of SSH2 belongs to acid-base catalysis.The N-terminal domain acts with the general acid D361 in SSH2 VYD loop,which restr:icts its activity and reduces the stability of the product dissociation group.3.F-actin can activate SSH2 enzyme activity,and its activation mechanism is to remove the restriction of the N-terminal domain on the general acid D361 and improve the stability of the product dissociation group.4.When F-actin activates SSH2,the SSH2 conformational changes and belongs to the allosteric activation mechanism.5.Fluorescence quenching experiment,continuous fluorescence spectroscopy detection found that the N-terminal key amino acid molecule of SSH2 is involved in the enzyme activity regulation mechanism.63C is close to the VYD loop when SSH2 is resting,and away from the VYD loop when activated;19C is away from the VYD loop in the resting state of SSH2,close to the VYD loop when activated.6.The confirmed F-actin activation of the SSH2 mechanism in vitro is also applicable to the SSH2 activation mechanism under physiological conditions.7.Expanded the new FlAsH-BRET probe for screening changes in phosphatase activity under different conditions of SSH2 and screening for SSH2 small molecule inhibitors/agonists.SignificanceOur study found the autoinhibition of the N-terminal domain of SSH2.Elucidate the autoinhibitory mechanism and the allosteric regulation mechanism of F-actin activation of SSH2.It provides a theoretical basis for understanding various clinical diseases which caused by abnormal Slingshot functions and that has the great significance for the prevention and treatment of related diseases in the future.