Regulation Of Heat Shock Factor 4 By UBA52

BackgroundCataract is the mainly cause of blindness worldwide. 60-80% of aged people suffers cataracts, while the congenital cataract morbidity is 0.015-0.03%. Genetic mutation is the predominant cause of congenital cataract. Genetic mutation of HSF4 can lead to dominant inheritary cataracts in human. Knocking out HSF4 gene cause mouse postnatal cataract characterized with lens epithelial cell hyperporliferation and abnormal differentiation. Those data demonstrate that HSF4 is the important regulator of postnatal lens. HSF4 is a family member of heat shock transcription factors. Compared with the typical heat shock transcription factor 1(HSF1), HSF4 lacks an HR-C hydrophobic domain, which is used for inhibition of homotrimerization. Therefore, the signal pathways underlying HSF4 transcription activity is quite different from HSF1, which is still under investigation. HSF4 mainly expresses in lens, and its transcription activity is regulated during lens development. However, the signal pathways that regulate HSF4 transcription activity remain unclear.To study the regulator of HSF4, we screened HSF4 interacting proteins by using mouse embryo cDNA by using yeast two hybrid, and found that HSF4 is interacted with UBA52, a fusion protein of ubiquitin and L40. Ubiquitin is a regulatory factor that can modify protein by forming isopeptide bonds between glycin of Ub and lysine of target proteins. Ubiquitination is important for protein quality control by regulating protein-protein interaction and protein degradation through ubiquitin-proteasome or lysosome pathways. It is reported that ubiquitin is indispensible for lens development. Mutation of Ub/K6 W causes cataract with abnormal fiber cell differentiation. According to these, we hypothesize that UBA52 couples the protein quality control stress to the activation of heat shock response by interacting with HSF4. ObjectTo explore the regulatory mechanism of UBA52 on HSF4’s transcription activity. Methodes1. The K48 R, K63 R and G75/76 A point mutations of UBA52 were generated using PCR overlap extension technology.2. The UBA52 overexpressed stably cell lines were generated using retroviral.3. The interaction among UBA52, HSF4 b, Rpb1 was detected by immunoprecipitation, GST-pull down and ubiquitin-enrichment assays. Immunofluorescence was used to observe the colocalization of these proteins.4. We detected the proteins expression in the lens epithelial cells or the tissues of mice lens by Immunoblotting or Real-time PCR.5. We detected HSF4b’s DNA-binding capacity in the UBA52 overexpressed cells by Chromatin Immunoprecipitation or Dual luciferase report experiment. Results1. The epithelial and fiber tissues of lens can both express UBA52 in different development stages. The Ub hybrid protein UBA52 in the lens epithelial cells can mature to a single Ub and a carboxyl extension protein-L40, which consists of 52 basis amino acids. The ubiquitin can be interaction with HSF4 b, but HSF4 b can not be covalently modified by the ubiquitin of UBA52 directly.2. UBA52 enhances HSF4 binding to the promoters of hsp25 and alpha B-crystallin, which results in upregulation of HSF4’s transcription activity.3. IF some sites of UBA52 is mutanted, the maturity and location of UBA52 will be changed. UBA52 mutants can downregulate the transcription activity of HSF4 b by affecting the interaction of UBA52 and HSF4 b.4. UBA52 is associated with the complex of HSF4-Rpb1 and regulates HSF4’s transcription activity. ConclusionUBA52 is constitutively expressed in lens tissue and cleaved into ubiquitin and L40 in lens epithelial cells. UBA52 is associated with the complex of HSF4-Rpb1 and enhances HSF4 binding to the promoters of hsp25 and alpha B-crystallin, which results in upregulation of HSF4’s transcription activity. These data reveal a novel signal pathway underlying HSF4 transcription activity by which the HSF4 survey the protein quality control stress during lens development.