Interactin And Regulatory Effect Between UBA52and Hsf4b

BackgroundThe study found that the mutation of Hsf4can cause neonatal congenital cataract,when hsf4geneof newborn mice was knocked out, it could lead to lens developmental disorders in animal experiments, sohsf4genes is crucial to the development of the lens of newborns.The family of heat shock transcription factor has four members, Hfs4is one of them, and it hastwo subtypes, they are Hsf4a and Hsf4b respectively, In the eye lens,it only expresses Hsf4b. Studies haveshown that the regulation of Hsf4b transcription is essential to the early development of the lens, Hsf4b ismainly expressed in lens epithelial cells and secondary fiber cells,and it can activate the expression of smallheat shock protein molecules such as Hsp25, γ protein and β protein and regulate the proliferation of lensepithelial cells and the differentiation of fiber cells. The study found that the ubiquitin-proteasome isactivated in the process of lens epithelial cells and fibers cells differentiation and it involves in theregulation of protein degradation and removing membranous organelles within the cells. Using yeast twohybrid technology, We found that a new protein which is UBA52can interact with hsf4b inside cells.UBA52is called Ubiquitin A-52residue ribosomal protein and it consists of ubiquitin atthe N-terminus and ribosomal protein L40at the C-terminus. The72th locus amino acids of UBA52can becut by protease and transform into an active ubiquitin molecule and a L40ribosome molecule. UBA52is anexistence form of ubiquitin molecules in cells and it can transport the protein to the proteasome todegradation or mediate the transportation and interaction of protein within cells. This experiment verifiesthat UBA52can be able to interact with Hsf4b and UBA52has no effect on the transcription activity ofHsf4b, as to the acting mechanism between them and the biological effect, it remains to be further explored.ObjectiveUsing overexpression of UBA52and Hsf4b protein in-vitro assays, we research whether Hsf4bcan interact with UBA52, we observe their subcellular localization through immunofluorescence and thenpreliminary explore the effection on ubiquitination by Hsf4b. In the end we verify whether UBA52has an effect on the transcription activity of Hsf4b.MethodsThrough Pull Down and Co-Immunoprecipitation,we verify the interaction between UBA52andHsf4b and the part of functional domains which they interact. we use immunofluorescence to observetheir subcellular localization and explore the effection on ubiquitination by Hsf4b through prokaryoticexpression technology and Western Blot. At last we try to find whether UBA52has an effect on thetranscription activity of Hsf4b.Results(1) Co-Immunoprecipitation suggests that Hsf4b can combine with ubiquitin molecules, and thecombining functional domains of Hsf4b with UBA52is Hsf4b(196-493). Immunofluorescence showes thatboth of them are located in the nucleus.(2) An UBA52molecule can be cut into two molecules in the body, Western Blot showes Hsf4bhas no effect on the shearing of UBA52.(3) It shows that although UBA52may interact with Hsf4b through Co-Immunoprecipitation butUBA52may modify Hsf4b through ubiqutilation indirectly.(4) UBA52protein has not been cut through the prokaryotic expression, When the protein ofUBA52is incubated with cell lysis solution of wild type mlec and cell lysis solution of mlec which canstable express Hsf4b respectively under a certain temperature, we find that UBA52in vitro can also besheared, Western Blot shows ubiquitination increase significantly When the protein of UBA52is incubatedwith cell lysis solution of mlec which can stable express Hsf4b, this can preliminary descripte that Hsf4bcan promote cell ubiquitination.(5) Western Blot shows UBA52can increase the expression of αB protein.ConclusionsUBA52can interact with Hsf4b. Although UBA52may interact with Hsf4b but UBA52maymodify Hsf4b through ubiqutilation indirectly. Hsf4b has no effect on the shearing of UBA52. Hsf4b can promote cellular ubiquitination and UBA52can increase gene expression of αB.