Phosphoylation Modification On The Regulation Of HSF4b Transcription Activity

BackgroundCataract is due to eye lens part or all of the opacification in vision loss. It is divided into early style(congenital) cataract and hereditary cataract. Congenital cataract is pointed out that within one year afterborn in lens turbidity, who was one of the main causes of vision loss. Nature cataract genetic means hasautosomal dominant, autosomal recessive, and x-linked recessive inheritance, the former is given priorityto.So far, through the use of genetic linkage analysis in the big family of congenital cataract, we havemore detailed understanding on genetic background. About forty genetic loci are related to the cataract, Inaccordance with its encoding protein function, human autosomal dominant congenital cataract (ADCC)related gene divided into four class: Crystalline genes, transcription factor genes, cytoskeleton protein geneand membrane transport protein gene. The regulation of transcription factor HSF4gene plays an importantrole, also has the progress in the research, plays an important role in lens fiber cell growth and maturesprocess, but the mechanism of its regulation of transcription is not too clear. In recent years the number ofmutations associated with cataract has more than one hundrend.In the human body; HSF4gene missensemutation can lead to cataract. In mice, the lack of HSF4gene can lead to mice appeared lens fiber celldifferentiation flaws and early symptoms of cataract.HSF4belongs to the heat shock transcription factor family (HSF), binding heat shock element (HSE)in the outside world under the condition of different stimulation, and transcript activation of the heat shockprotein (HSP27, HSP70, and HSP90). HSF4has two splice variants: HSF4a transcription inhibition activity,while HSF4b has a role in transcriptional activation, HSF4b also inhibited transcription. HSF4b is the mainsubtype of crystalline expression in vivo. In HSF4b deficient mice reduced expression of Hsp25,r-crystallin and CP49protein.Protein phosphorylation is one of the most important post-translational modifications, plays a veryimportant role in the transfer process of cell signal, HSF4is phosphorylated state under physiologicalconditions, can also be regulated by MAP kinase phosphorylated modification. HSF4b is phosphorylation regulation of P38, JNK and ERKl\/2, activation expression of its downstream heat shock protein,phosphorylation of HSF4b induced ubiquitination, and SUM0inhibited HSF4b transcription activity invitro. The HSF4contains a PDSM (SUMO dependent phosphorylation modification motif) motif, PDSMexists in the conservative two different functional member of the heat shock factor family, HSF1andHSF4b, similar to SUMO, PDSM relies on the SUMO by phosphotion, to inhibit the transcription activityof substrate.Combination of14-3-3and target protein mainly by the phosphorylation of the serine/threonineprotein in target recognition sequence. According to the sequence motif can speculate whether some proteininteracts with14-3-3protein, S299motif containing serine, we guess whether it interacts with14-3-3.Three mutants were cotransfected with the14-3-3to observation the effect of their binding, thoughexpression of downstream proteins to analysis the site of phosphorylation after transcription regulation ofHSF4b.In order to further understand the molecular mechanisms that control the transcriptional activity ofHSF4b, We discussed mechanism of regulation of HSF4b by phosphorylation, predicted protein sequencepossible phosphorylation sites, the site-directed mutagenesis study research and mechanism; find thephosphorylation of kinases and the pathway.In this experiment, through the construction of HSF4b mutant, determine transcriptional activity ofHSF4b, determine the regulatory phosphorylation site effectively and the regulation on the downstreamgenes.ObjectivesIn this study, through the construction of HSF4b mutant, determine transcriptional activity of HSF4b;determine the regulation of downstream phosphorylation sites and HSF4b gene effectively.Methods1Application of PCR technology, the mutation primer was amplified by PWZL-blast-HSF4b (hereinafterreferred to as HSF4b), product with Dpn I digestion, extraction of plasmid was transformed to Dh5,ECOR â… , digestion of enzyme, sequencing.2After Sequencing, the correct mutant plasmids were transfected into293T cells for eukaryoticexpression. Using the method of SDS-PAGE and Western blot experiments for the identification the expression of HSF4b and Hsp27protein; determine initially identified phosphorylation siteseffectively.3Using MLEC HSF4b-/-cells, transfected with mutant plasmid and establishment of stable strains,MLEC HSF4b, MLEC HSF4b/S299A, MLEC HSF4b/S299D and MLEC HSF4b/S299E. Westernblot experimental identification expression of HSF4b, Hsp27, alpha A-crystallin, alpha B-crystallin andHsp70protein in vivo. Trimer experimental identification HSF4b and mutant protein structure has nochange.4Through the luciferase assay experiment, to verify the HSF4b/S299A, HSF4b/S299D, HSF4b/S299Eon the downstream gene B-crystal protein (alpha B-crystallin) effect.Results11The mutation plasmid of HSF4b/S42, A, HSF4b/S49A, HSF4b/S66A, HSF4b/T173A, HSF4b/S85A,HSF4b/T221A, HSF4b/S243A, HSF4b/S269A, HSF4b/S278A, HSF4b/S299A, HSF4b/S340A,HSF4b/S372A, HSF4b/T446A, HSF4b/T471A, HSF4b/S489A, HSF4b/S401A, HSF4b/S299D andHSF4b/S299E which expression of HSF4b were successful constructed, and sequencing correct.2The mutant plasmids were transfected into293T cells,Western blot experimental display identificationthe expression of HSF4b and Hsp27proteins in vivo: All mutant plasmids were normal expression ofHSF4b, the expression of Hsp27protein are different. The Hsp27expression of HSF4b/S66A,HSF4b/S269A, HSF4b/T446A, HSF4b/T471A are Decreased, while HSF4b/S299A is increased.3The establishment of stable strains MLEC/HSF4b, MLEC/HSF4b/S299A, MLEC/HSF4b/S299D andMLEC/HSF4b/S299E, the Western blot experiments show that S299is phosphorylated siteseffectively, the inhibition of expression.4Luciferase assay experiment shows the HSF4b/S299A have activity effect on the downstream gene B-crystal protein (alpha B-crystallin), HSF4b/S299D and HSF4b/S299E inhibit the expression of thedownstream gene alpha B-crystal protein (alpha B-crystallin).Conclusions1successfully established stable strain2Initially identified6effective phosphorylation sites, including HSF4b/S66A, HSF4b/S269A,HSF4b/T446A, HSF4b/T471A performance activation on the transcription of downstream gene, HSF4b/S299A showed a transcriptional repression..