Studies On The In Vivo Interaction Between Md-2 And Tlr4 Based On Fret

Sepsis is still associated with a high mortality rate in severe clinical patients who have a serious infection,trauma,shock,major operation,and so on. Lipopolysaccharide(LPS,endotoxin),an integral component of the gram-negative bacterial outer membrane,plays a critical role in the pathophysiology of sepsis.LPS in blood plasma binds to LPS binding protein(LBP) first,and then LBP transfers LPS to cluster of differentiation-14(CD14).CD14 binds and accumulates LPS as a signal, but it lacks transmembrane structure and intracytoplasm domain,and can't transfer signal into cytoplasm,so it is suggested that there must have one or several transmembrane receptor for LPS signal transduction.Toll-like receptor 4(TLR4) belongs to pattern recognition receptor(PRR), comprising of extracellular leucine-rich repeats,transmembrane domain and intracellular domain and Toll/IL-1 receptor homologous region(TIR).Human TLR4 which relative molecular weight is about 96 kD contains 839 amino residues,and mainly distributes in spleen,liver,lungs and placenta etc.,especially in macrophages and lymphocytes.Poltorak A and Hoshino K found that TLR4 was the main receptor for LPS transmembrane signaling tranduction by researching the TLR4 gene mutant mice C3H/HeJ and C57BL/10ScCr.It has been known that TLR4 can regulate signal transduction through MyD88-dependent pathway and MyD88-independent pathway. The former pathway mediates NF-κB activation,cytokines generation,while the latter pathway induces the expression of IFN-inducible protein 10,glucocorticoid attenuated respinse gene16 and IFN-regulated genel under LPS stimulate,and promotes dendritic cells' maturation.TLR4 is the main receptor of LPS transmembrane signaling.However,in vitro transfer TLR4 gene into human embryonic kidney-derived 293 cell line(HEK293) or mouse IL-3-dependent pro-B cell line(Ba/F3) could only constitutive activate NF-κB without LPS signal transduction.The following investigation found that TLR4 mediated LPS signal transduction associated with a 25-30 kD protein named myeloid differentation-2(MD-2).Consisted of 160 amino acid residues with a signal peptide of 16 amino acid residues at the end of N terminal,MD-2 is a secretory protein and widely distributed in the hematopoietic system,nervous system and the reproductive system,and can be expressed in B cells,dendritic cells,macrophages,endothelial cells,and so on.LPS signal recognition and transduction is the key for host defense reaction to gram-negative bacteria,and TLR4/MD-2 complex play an important role in the course as LPS recoginiton receptors.The early research considered that the binding domain of MD-2 and TLR4 mainly on Cys⁹⁵ and Cys¹⁰⁵,but recent research showed that Cys³⁷-Cys⁵¹ loop,Cys⁹⁵-Cys¹⁰ loop and nearby amino acid sides also participated in binding to TLR4.Although resent sepsis research is focus on the interaction domain between MD-2 and TLR4,the interaction mechanism,the exact domain,TLR4 polymer forming stimulated by LPS,role of MD-2 and too many factors are still uncertain.In our study,the fluorescence resonance energy transfer technology(FRET) was used to study the interaction mechanism between TLR4 and MD-2.FRET is considered as one of the best methods used for intracellular protein-protein interaction study.Compared to traditional research methods of protein interaction,for example Co-Immunoprecipitation and affinity chromatography,FRET needn't smudge cells,which can retaining the proteins under normal physiological conditions in cells,and provide the spatial distribution information of interaction proteins.Based on these characteristics,FRET becomes one of the most reliable methods in current protein interaction studies in living cells.FRET was most early proposed by F(o|")rster as a physical phenomenon in 1948, and its basic principle is the energy can be transferred to another molecular neighbor to it after a fluorophore absorbs energy.This is a kind of non-radiation dipole-dipole coupling process,and electron excitation can be transferred from donor fluorescent molecular to very short distance acceptor fluorescent molecular in this process.It could be manifested by acceptor luminescence after excited donor molecular during the transient energy transfer process.Energy transfer efficiency E=R₀6/(R₀⁶+r⁶), which R₀ represents the distance that Energy transfer efficiency is 50%,r represents the distance between two fluorescent molecular.Because FRET efficiency is proportional to 6th power of fluorescent molecular distance,in order to reach 5% detection limit,FRET detection distance approx 1.6×R₀,usually no more than 10nm. In our research,cyan flurescent protein(CFP) was used as donor while yellow flurescent protein(YFP) was used as acceptor,then we can detect FRET signal in 7.8nm with 4.9nm R₀.FRET assayed by microscope can be generally divided into two types:one is based on fluorescence intensity and another one is based on fluorescence dynamic attenuation.In our research,the calcultion,which is based on the calculation of three channels' fluorescence intensity and suitable for living cell FEET research,was adopted.Theoretically,accurate quantitative measurement of three channels' FRET signal needs calibration before calculation,which includes three aspects:cell background signal,fluorescence overlap signal and the influence of donor and acceptor concentration.To solve above three aspects,following ways were adopted to calibrate the signal.Produced by German Carl Zeiss Company,filter groups which can curtail fluorescence overlap signal significantly and improve signal noise ratio were choosed, including:CFP filter,YFP filter and CFP-YFP-FRET filter.As fluorescence overlap signal is proportional to protein expression level and best exposure time is inversely proportional to protein expression level,CFP and YFP were expressed respectively and applying Youvan algorithm to calculate donor/acceptor correction factor to solve the contradiction.100 different "best exposure time" and cells' "Donor Correction Factor" were collected and their curve estimation was analyzed by SPSS 13.0.The results showed that Donor Correction Factor=19.6766+0.025×Exposure Time-0.00003×Exposure Time²+0.000000012×Exposure time,the correlation coefficient was 0.822,and tens of Acceptor Correction Factor under best exposure time was 0.The results mightbe related with that YFP and FRET channel's signal overlap could be effectively eliminate by the filter.In order to compare FRET values between different cells and eliminate the influence of donor's expression level,Xia algorithm was applied to standardize the FRET value.In Xia algorithm,FRET gray value calculated by Youvan algorithm was divided by square root of donor and acceptor signal value,and the obtained FRET value had few correlation with donor and acceptor's expression level.Based on above,CY-15P which was fused by CFP and YFP through 15 neutral amino acids was used as positive control,while co-expressed CFP and YFP proteins were used as negative control.20 cells were selected for signal collect in each control, and AxioVision FRET 4.6 and Xia algorithm were used to calculate FRET value,then SPSS 13.0 statistical analysis software was used to analyze the data.By independend-samples T test,its P value was less than 0.01 which indicated that positive control and negative control had significant difference.The result proved our FRET system had been built successfully.To explore the interaction between MD-2 and TLR4,five possible sites Arg⁹⁰. Lys⁹¹,Asp⁹⁹,Asp¹⁰⁰ and Tyr¹⁰² near MD-2 Cys⁹⁵-Cys¹⁰⁵ loop which may interact with TLR4 were mutated into Ala.These five pECFP-SP-MD-2 mutant genes were transformed individually and cotransformed with pEYFP-SP-TLR4 respectively to HEK293,and the result showed that no MD-2 and TLR4 expression level was changed.We also studied the interaction between MD-2 R90A mutant and TLR4. Found site mutation in Arg⁹⁰ didn't affect the binding of MD-2 and TLR4.Depending on the FRET platform we built,TLR4 and MD-2 interaction domain was researched. A TLR4 mutant which lack of 18 amino acids at N terminal(Glu²⁴-Met⁴¹) was built, and FRET value were compared between MD-2+wild type TLR4 and MD-2+ mutant type TLR4.By SPSS13.0 statistical analysis software,the results showed that TLR4 mutant which lack N terminal Glu²⁴-Met⁴¹ decreased its binding ability to MD2 significantly,which implied that Glu²⁴-Met⁴¹ mightbe TLR4's domain binding to MD-2. In order to further research the effect of deletion N terminal Glu²⁴-Met⁴¹ on TLR4 binding ability,CFP or YFP to wild type TLR4 and mutant type TLR4 were fused respectively in Hela cell line.And then their FRET values were assayed after LPS stimulation.The result showed that wild type TLR4 FRET value transient increased in 1 min to 2 min,and then rapid decreased and disappeared in 5 min. Meanwhile,no FRET value was observed in the mutant TLR4 which lack of N terminal Glu²⁴-Met⁴¹.These suggested N terminal Glu²⁴-Met⁴¹ of TLR4 could affect TLR4 polymers by binding to MD-2 or participating TLR4 polymer forming directly.In the study,a FRET system platform was built successfully through Carl Zeiss living cell imaging system,and we found that N terminal Glu²⁴-Met⁴¹ of TLR4 might be the domain binding to MD-2 or participated TLR4 polymer forming by measuring FRET value changes of wild type and mutant type TLR4 after LPS stimulates.We also found that any MD-2 site mutation in Arg⁹⁰,Lys⁹¹,Asp⁹⁹,Asp¹⁰⁰ and Tyr¹⁰² didn't affect the MD-2 and TLR4's expression level and MD-2 site mutation in Arg⁹⁰ didn't affect the binding of MD-2 and TLR4.Furthermore,we successfully selected YFP-TLR4 steady expression cell line in HEK293.It is expected to reduce the inflammation cascade by making an in-depth study and effective intervention in MD-2-TLR4 interaction.