Transmembrane TACE Activity: Development Of An In-situ Assay And The Effect Of Membrane Lipid Microenvironment

Part 1 Establishment of Peptidesubstrate-HPLC Method for the Determination of Activity of TACE in Situ[Background]Tumor necrosis factor-alpha converting enzyme(TACE) is the main secretase by which membrane-type tumor necrosis factor-α(mTNF-α) can be converted into soluble tumor necrosis factorα(sTNF-α).Moreover,it can also mediate the processing and the shedding of many other cytokine receptors and thus plays an important role in many physiological and pathological processes such as inflammatory response,immune response,the regulation of proliferation and differentiation,as well as the occurrence and the development of tumor.However, the mechanisms researchers have been interested in such as how TACE to identify specifically molecular substrates and realize activity regulation and control etc are still unknown.The most important prerequisite for making clear these questions is to find a fast,accurate and high-repetitive detection method which can change the activity of enzyme in the process of observational study.At present,there are two kinds of common methods to determine the activity of enzyme:one is based on enzyme-substrate reaction products such as Peptidesubstrate-HPLC method, substrate gel electrophoresis,MTT cytotoxicity test etc and another auxiliary method is based on the determination of the expression of enzyme such as enzyme-linked immunosorbent assay(ELISA),Western Blot,flow cytometry(FACS), indirect immunofluorescence assay etc.The former method is mainly used in the study on the regulation of enzyme activity such as the regulatory mechanism of activity of enzyme and enzyme Inhibitors for drugs etc,and the latter one is regarded as an auxiliary method and it is mainly used to research the regulatory mechanism on the change of enzyme activity.Peptidesubstrate-HPLC method is a fast,accurate method for the determination of enzyme activity,which integrates artificial peptide substrate and high-performance liquid chromatography(HPLC) and can be used for high-throughput screening.The peptide substrate is a synthetic oligopeptide with the function of substrate can be identified and hydrolyzed specifically by the active site of enzyme but not be degraded easily by protease.Establish the hydrolysis reaction system of enzyme and peptide substrate,quantitatively analyze the changes of reaction products with HPLC,and then evaluate the specificity and the catalytic activity of enzyme for specific substrates.If ultroviolet or fluorescent group is adopted to mark peptidesubstrate molecule,then the detection limit can be decreased to nanogram-level and thus increase its detection sensitivity finally.By means of the method above,the catalytic efficiency of enzyme to specific substrates can be monitored directly and thus it is the most direct and effective evaluation index for the enzyme activity.At present,Peptidesubstrate-HPLC method has been used widely in the studies on enzyme activity for its advantages such as high-speed, perfect repeatability,high-sensitivity and be suitable for high-throughput screening drug studies etc.[Objectives]1.Establish TNFα-mimic Peptidesubstrate-HPLC method to determine the activity of TACE and evaluate the method with Substrate Gel Electrophoresis (Zymography),MTT cytotoxicity method and FACS etc.2.Study the catalytic kinetics of recombinant human TACE(rhTACE) to peptide substrate with the established Peptidesubstrate-HPLC method and evaluate the substrate specificity and the hydrolysis activity of rhTACE by determining kinetic constants such as K_m,V_max,k_cat/K_m etc.3.Carry out cell membrane TACE activity study with the established method above.[Methods]1.Simulate the amino acid sequence around the hydrolytic site of TACE-specific substrate---TNFαso as to synthesize high-purity(95%) peptide substrate with UV markers(Dnp-SPLAQAVRSSSR-NH₂). 2.Optimize chromatographic operating conditions which are suitable for the separation of peptide substrate and hydrolyzed peptide products of TACE.It includes the optimization and the selection of chromatographic parameter such as UV absorption wavelength,the conditions of gradient elution,mobile phase flow rate and sample amount etc.3.Optimize TACE-Peptidesubstrate reaction system,which includes the selection of reaction buffer system,optimum pH value,the effects of temperature and reaction terminating conditions etc.4.Carry out the studies on enzymatic kinetics,which includes making certain the amount of enzyme needed at initial reaction phase,the relation between the initial reaction rate and different substrate concentrations under the influence of a certain amount of enzyme,and working out kinetic constants(Km,V_max and k_cat/K_m) with Lineweaver-Burk double reciprocal plot according to Michaelis-Menten equation.5.Establish microlitre-level Cell-Peptidesubstrate reaction systems with U937 and HEK293 cells respectively,make use of HPLC method to observe the influences which are brought about by different number of cells(and thus the different concentration of membrane TACE) on the hydrolysis reaction with a certain concentration of peptide substrate and observe the impacts caused by cellular enzyme activator or inhibitor on enzyme activity.6.Multi-method comparative evaluation:determine the hydrolysis activity of Metalloproteinases(MMP) and rhTACE on glutin by means of substrate gel electrophoresis,make use of MMT method to observe the cell toxicity on L929 brought about by dissolvable TNFα(sTNF-αin the supernatant of PMNLPS stimulated cell;determine the expression of TACE on the cell membrane after LPS stimulation with FACS method,and evaluate the sensitivities and the application ranges of all the methods above.[Results]1.The analysis conditions for the RP-HPLC of TACE-Peptidesubstrate reaction system have been made certain(Method:MS): [6]C8 Reversed-phase column(4.6mm×50mm);[7]Detection wavelength:350nm;[8]Mobile phase:A:20%acetonitrile-t-80%water,B:60%acetonitrile+ 40%water(containing 1%TFA);[9]Conditions for gradient elution:100%A and 5min balance,then convert 100%A into 100%B linearly within 20min;[10]Mobile phase flow rate:0.8mL/min;sample size:10-15μl.2.The composition of TACE-Peptidesubstrate reaction buffer system has been made certain:25 mM Tris-HCl(pH 8.5),2.5μM ZnCl₂,5%Glycerol;add 1%TFA to stop enzyme hydrolysis.3.Based on the analysis conditions for the RP-HPLC(M5),pure peptide standard working curve has been made certain and it can be used to calibrate the relationship between the peak area and actual concentration of peptide measured by HPLC.4.The amount of enzyme needed at the initial velocity phase of enzymatic reaction has been made certain:when substrate concentration is 20μM,then TACE should be no more than 1.5ng/μl,therefore,we always adopted TACE 1ng/μl in the 10-80μM substrate peptide reaction systems.5.The results of the determination of enzyme kinetic constants(PH 8.5):Michaelis constant(K_m):62.84 pM(37℃) and 37.02 pM(25℃);catalytic constant(k_cat): 1.5407 s^-1(37℃) and 0.5161 s^-1(25℃);specificity constant(k_cat/K_m):2.4×10⁴ M^-1·s^-1(37℃) and 1.4×10⁴ M^-1·s^-1(25℃).6.Owing to HEK293 cell line can highly express TACE(ADAM17) without MMP companion on the surface of cell membrane;therefore,it can be used as a perfect cell model of the Peptidesubstrate-HPLC for its application in the cell system.Although the product peak of TACE can almost not be determined in the 100μl natural cell reaction system within 20min,however,after TACE was processed with metalloproteinase-activator PMA and a drug(berberine) and reacted with the peptide substrate again,it could be detected accurately within a relatively short time(≤20min);at the same time,a reduced product peak area of it could be observed and it showed a linear relationship with processing time and concentration respectively.The product peaks of pure enzyme system and cell enzyme system have been made sure by means of HPLC-MASS joint analysis.7.Although the results of substrate gel electrophoresis showed that the expression of MMP of LPS stimulated-U937 increased obviously;however,positive result has not been obtained even if a high dose of rhTACE(50ng) was adopted;the results of cytotoxicity experiment showed that the changes in the expressions of sTNF-αof HL-60 and U937 could be observed after they were stimulated by LPS and PMA respectively;comparing with the control group,the kill rate of the cell supernatant of stimulation group on L929 increased obviously;The results of FACS showed the distribution of TACE has not been impacted distinctly by LPS stimulation.[Conclusions]This study has successfully established the Peptidesubstrate-HPLC method for the determination of the activity of TACE,made certain the optimum reaction conditions of TACE-Peptidesubstrate reaction system and the optimum separation conditions of HPLC for peptide substrate and TACE cleavage products;in addition, we determined the kinetics constants of TACE-Peptidesubstrate reaction such as K_m,V_max and k_cat/K_m etc and the results showed that all the constants accorded with enzyme kinetics.In the references they determined the metalloproteinase activity with similar methods,k_cat/K_m was usually within 10⁴～10⁵ M^-1·s^-1 which are accordant with the results of our study[2.4×10⁴ M^-1·s^-1(37℃) and 1.4×10⁴ M^-1·s^-1(25℃)];all these data above fully showed that the Peptidesubstrate-HPLC method for the determination of the activity of TACE established by us is reasonable and practicable.The Peptidesubstrate-HPLC method for the determination of the activity of TACE has been applied tentatively in the viable cell system firstly by us and relatively ideal results have been obtained finally.The key factor for simplifying the spectral peak of HPLC of cleavage products is to take HEK293 as cell model.The regulatory roles of enzyme activators and inhibitors on the enzyme activity can be evaluated directly and effectively by means of the peak area of enzyme cleavage products on the chromatogram of HPLC.The existing methods for the determination of TACE activity have many technical disadvantages such as substrate gel electrophoresis will consume a lot of substrates and it can not be used for the detection of living cell;MMT method is indirect,complicated and unrepeatable;FACS method only plays a supporting role in providing the activity information of enzyme etc,all these shortcomings above will not be resolved by themselves.However,with the application of the Peptidesubstrate-HPLC method in the cell system,the activity of enzyme can be determined directly and accurately and it is possible for us to realize the determination in the microlitre system,which will provide important technical supports for the further study on the mechanism of regulatory on TACE activity and the initial screening of inhibitors.Part 2 Regulation of membrane TACE activity by Its Lipid Microenvironment[Background]Lipid raft is a sub domain in the bimolecular lipid membrane.It contains relatively high cholesterol and its fluidity is lower than adjacent plasma membrane. The main function of lipid raft is to provide an assembly platform for membrane protein so as to facilitate their aggregation and interaction;moreover,it is helpful for membrane protein to form effective conformation.Appropriate cholesterol is necessary for lipid raft to maintain its stability and the decrease of cholesterol will increase membrane fluidity and thus lead to the disintegration of lipid raft and thus some enzymes or its substrates molecules fall off and conformational changes occur;as a result,they contact with each other,hydrolyze and consequently trigger a variety of physiological and pathological responses.It has been confirmed that the changes in cholesterol content will influence the extracellular shedding of many transmembrane substrates of TACE such as APP (amyloid precursor protein),CD30,L-selectin and adhesion molecule L1 etc,which suggests that there may be a certain link between cholesterol content and TACE activity.The existing common method to reduce cholesterol content in the plasma membrane is making use of methyl-β-cyclodextrin(M^βCD) to deplete the cholesterol in the cells,which will not influence other physiological and biochemical processes of cell.Determining membrane fluidity with fluorescent probes combined with lipid molecules in plasma membrane can evaluate the changes of cholesterol content.Berberine(BBR) is an active traditional Chinese medicine,it is confirmed that berberine can obviously reduce cell cholesterol content.Originally,BBR has been clinically used as a heat-clearing and detoxifying drug and a antibacterial drug, subsequently,it was found that BBR can provide many various pharmacological effects such as blood sugar-reducing effect,anti-platelet effect,anti-senile plaque effect and anti-tumor growth effects etc.The occurrence and the development of the diseases above are close contact with TACE-related processing and the shedding of substrate molecules.However,the issues whether these pharmacological effects are related with membrane fluidity and the interaction between TACE and substrate molecules both on the lipid raft or not are still unknown and that's exactly what we are interested in now.[Objectives]1.Fstablish the detection method of membrane fluidity which is based on FACS technology.2.Study the changes in membrane fluidity and TACF activity brought about by M^βCD-related decrease of cholesterol content in the cell membrane,and thus provide basis for the study on confirming the causation between membrane fluidity and TACE activity.3.Observe the impacts of BBR on membrane fluidity and TACF activity. 4.Discuss the mechanism BBR influence membrane TACE activity from the point of view of membrane fluidity.[Methods]1.The establishment of detection method of membrane fluidity:Make use of flow cytometry(FACS) to determine the two-dimensional scatter and the frequency distribution of membrane lipid after it is combined with fluorescent probe MC540;determine the degree of accumulation of lipid molecules and thus reflect the membrane lipid fluidity finally.2.Make clear the changes in membrane fluidity after reducing membrane cholesterol content physically from two different aspects below:(1) Observe morphological changes of HEK293 and U937 under a microscope after M^βCD treatment;(2) Use FACS to determine the changes in accumulation degree of lipid molecules of HEK293 and U937 after M^βCD treatment;3.Evaluate the influence of M^βCD on TACE activity from two different aspects:(1) Use MTT cytotoxicity method to study the changes of sTNFαin the supernatants of HEK293 and U937 after treated with M^βCD;(2) Use Peptidesubstrate-HPLC method to study the yield change of Cell-Peptidesubstrate reaction after the cells above were treated with M^βCD.4.Observe the changes of membrane fluidity of HEK293 and U937 at the different time points after it was processed by M^βCD with different concentrations,and thus make sure the relationship between the membrane fluidity and the BBR treatment.5.Make use of the methods above mentioned to observe if the activity changes of TACE were time and dose dependent on BBR treatment.That is to say,use Peptidesubstrate-HPLC method to study the changes of Cell-Peptidesubstrate reaction yield after the cells were treateded with BBR;use MTT cytotoxicity method to study the changes of sTNFαin the supernatants of HEK293 and U937 after it was treated with M^βCD;at last,compare the M^βCD group and the PMA group to make sure the relationship between the activity of TACE and BBR treatment.6.Make use of RT-PCR method to study the influence of BBR on the expression of TACE mRNA of HEK293 and U937 to exclude other possible ways that BBR impacts TACE activity.[Results]1.The establishment of detection method of membrane fluidity:FACS results of the two-dimensional scatter and the frequency distribution spectrum showed that,after membrane cholesterol was reduced by M^βCD,both suspended cell U937 and adherent cell HEK293 diffused obviously to high fluorescence intensity zone and showed a relatively loose distribution,which showed the volumes of membrane lipid molecules increased thus indicated an increased membrane fluidity.The results above accorded with the previous literatures,confirm that the detection method is reasonable and practicable.2.The results of membrane fluidity test:(1) Adherent spindle cell HEK293 converted into irregular oval-shaped cell after it was treated with M^βCD;moreover,this morphological change is time and dose-dependent on M^βCD treatment.The edges of HEK293 cell membrane became fuzzy and the cell did not adhere firmly after 1 and 2 hours of M^βCD treatment at 8mM and 16mM respectively,and thus showed a tendency of apoptosis.(2) The fluorescence intensities on the surfaces of U937 and HEK293 increased obviously and the cells dispersed further into higher fluorescence intensity zone upon M^βCD treatment;moreover,the fluorescence intensity increased along with the increasing concentration of M^βCD(HEK293:149,470,904;U937:177,892, 2242).Especially,the increase of fluorescence intensity of U937 was more obvious than that of HEK293.The results showed M^βCD treatment can improve the cell membrane fluidity.(3) U937 and HEK293 cells dispersed and distributed distinctly in the high fluorescence intensity zone upon 10～40μg/mL BBR treatment;moreover,the degree of dispersion increased(HEK293:BBR 0,5,10,20,40,60 and 80μg/mL, fluorescence intensity:606,687,718,1045,1165,832,730;U937:BBR 0,5,10, 20,30 and 40μg/mL,fluorescence intensity:175,180,218,264,262 and 208). With the increase of BBR concentration,the high fluorescence intensity zone enlarged gradually,which indicated cell membrane fluidity increased accordingly. The results showed that treating the cells with BBR and M^βCD had same effects and both of two processes can increase the cell membrane fluidity.3.The results of cytotoxicity experiment:(1) 2 hours after we treated cell U937 with different doses of M^βCD(4mM and 8mM) respectively,the corresponding kill rates on cell A549 were 11.2%and 16.8%respectively,which showed that the secretion of sTNF-αincreased accordingly along with the increase of concentration of M^βCD.(2) After we treated cell U937 with different doses of BBR(15μg/mL and 30μg/mL) respectively,the corresponding kill rates on cell A549 were 0.5%and 0.7%respectively.For PMA treated group(100ng/mL),the kill rate reached 19.1%;however,it decreased to 7.5%after U937 was treated with PMA (100ng/mL) and BBR(30μg/mL) simultaneously.Besides,when the concentration of BBR was more than 40μg/mL,we found it will induce the apoptosis of A549.The results above showed that the secretion of sTNF-αcan not be induced by BBR and the stimulating effect of PMA on U937 can be inhibited by BBR too.sTNF-αcytotoxicity experiment can evaluate indirectly the activity of TACE which is the main secretase of sTNF-α.The results showed the effects of BBR treatment were not accorded with that of M^βCD and PMA treatment,which was probably caused by the inhibitory effect of BBR on endotoxin.In addition,we also found that the apoptosis of A549 will occur if the concentration of BBR was more than 40μg/mL or incubation time exceeded 24 hours.4.The results of TACE activity determination by Peptidesubstrate-HPLC method:(1) 30min after we treated HEK293 with different doses of M^βCD(8mM and 16mM) respectively,we observed the hydrolysis activity of proteinases on cell membranes on the peptidesubstrate Dnp-SPLAQAVRSSSR-NH₂ which is mimic for TNF-α.The results showed that the ratio of peak area of cleavage peptide products of TACE increased obviously and it increased accordingly along with the increases of the concentration(8～16mM) of M^βCD and the catalytic reaction time(5-15min).(2) 6h and 24h after we treated HEK293 with different doses of BBR(10μg/mL and 20μg/mL) respectively,we co-cultured the cells and peptide substrate and found that,comparing with untreated group,the ratio of peak area of specific cleavage peptide products of TACE increased at each time point during initial reaction(5-15min);moreover,it showed a time and dose dependence with BBR treatment.The increased peak area of specific cleavage peptide products of TACE indicated an increase of efficiency of enzymatic reaction and an enhanced hydrolysis activity of TACE.Treating the cells with BBR and M^βCD had similar effects,which showed that both of them can enhance TACE activity.5.RT-PCR results:We took housekeeping geneβ-actin as reference of RT-PCR and the results showed that there was no significant difference between the expressions of TACE mRNA of treatment group and the control group(p＜0.05), which indicated that BBR can not impact the activity of TACE by means of enhancing the gene transcription or the stability of TACE mRNA.[Conclusions]This study successfully established the detection method of membrane fluidity which is based on FACS technology.The method is applicable for the detections of both suspension cells and adherent cells.It has many advantages such as simple operation,high accuracy and repeatable quantitative analysis etc.in our study,we made use of the method to investigate if cholesterol depletion enhanced the cell membrane fluidity and the results are fully accordant with that of related literatures.Previous study has been confirmed that berberine(BBR),a kind of non-toxic traditional Chinese medicine,can reduce the cholesterol content in the cell membrane and thus be applicable for the treatment of hyperlipidemia;in addition,it has many pharmacological effects such as reducing the proliferation of vascular smooth muscle,anti-platelet effect,anti-inflammatory effect,anti-senile plaque effect and anti-tumor effect etc.Therefore,the promising method will be widely used in the treatments of cardiovascular and nervous system diseases in the near future. With the hope of establishing a link between the pharmacological activities above and the increase of membrane fluidity,the study discussed the possible mechanism that berberine influences the activity of TACE by means of observing the berberine influences on the hydrolysis activity of TACE instead of the expression of TACE mRNA.We first made sure that physically deplete membrane cholesterol can enhance membrane fluidity and the hydrolysis activity of membrane enzyme;then we confirmed BBR also has the effects above but not increase the expression of TACE mRNA.The results of the study indicated that the possible mechanism for BBR increases TACE activity is by the changes of microenvironment of cell membrane through membrane fluidity changes,which will cause the conformational variations of enzyme molecules on the lipid rafts;it is not supported that BBR enhanced TACE activity by increasing the expression of TACE mRNA.This study provided a new interpretation and research idea for the pharmacological effects such as lipid regulating effect,anti-senile plaque effect and anti-tumor effect etc.of BBR.