Yan Longjun, Li Jing, Hu Jialin, Qu Junwei, Li Kejia, Wang Mingli, An Shuang-Shuang, Ke Cun-Cun, Li Hui, Yuan Fengling, Guo Weikai, Hu Mengyue, Zhang Jing, Yang Zhengyan, Mu Hongmei, Zhang Fengyan, Zhang Jun, Cui Xiukun, Hu Yanzhong
National-Joint Laboratory for Antibody Drug Engineering, The First Affiliated Hospital of Henan University, Henan International Union Lab of Antibody Medicine, Department of Cell Biology and Genetics, Henan University School of Basic Medical Sciences, Kaifeng, China.
Kaifeng Key Lab for Cataract and Myopia, Institute of Eye Disease, Kaifeng Central Hospital, Kaifeng, China.
Biochem Biophys Rep. 2022 Feb 5;30:101227. doi: 10.1016/j.bbrep.2022.101227. eCollection 2022 Jul.
Genetic mutations in HSF4 cause congenital cataracts. HSF4 exhibits both positive and negative regulation on the transcription of heat shock and non-heat shock proteins during lens development, and its activity is regulated by posttranslational modifications. Biotin is an essential vitamin that regulates gene expression through protein biotinylation. In this paper, we report that HSF4b is negatively regulated by biotinylation. Administration of biotin or ectopic bacterial biotin ligase BirA increases HSF4b biotinylation at its C-terminal amino acids from 196 to 493. This attenuates the HSF4b-controlled expression of αB-crystallin in both lens epithelial cells and tested HEK293T cells. HSF4b interacts with holocarboxylase synthetase (HCS), a ubiquitous enzyme for catalyzing protein biotinylation in mammal. Ectopic HA-HCS expression downregulates HSF4b-controlled αB-crystallin expression. Lysine-mutation analyses indicate that HSF4b/K444 is a potential biotinylation site. Mutation K444R reduces the co-precipitation of HSF4b by streptavidin beads and biotin-induced reduction of αB-crystallin expression. Mutations of other lysine residues such as K207R/K209R, K225R, K288R, K294R and K355R in HSF4's C-terminal region do not affect HSF4's expression level and the interaction with streptavidin, but they exhibit distinct regulation on αB-crystallin expression through different mechanisms. HSF4/K294R leads to upregulation of αB-crystallin expression, while mutations K207R/K209R, K225R, K288R, K255R and K435R attenuate HSF4's regulation on αB-crystallin expression. K207R/K209R blocks HSF4 nuclear translocation, and K345R causes HSF4 destabilization. Taken together, the data reveal that biotin maybe a novel factor in modulating HSF4 activity through biotinylation.
热休克因子4（HSF4）的基因突变会导致先天性白内障。在晶状体发育过程中，HSF4对热休克蛋白和非热休克蛋白的转录具有正负调控作用，其活性受翻译后修饰的调节。生物素是一种必需维生素，通过蛋白质生物素化调节基因表达。在本文中，我们报道HSF4b受生物素化负调控。给予生物素或异位表达细菌生物素连接酶BirA会增加HSF4b在其C末端196至493位氨基酸的生物素化。这减弱了HSF4b在晶状体上皮细胞和受试HEK293T细胞中对αB-晶状体蛋白表达的调控。HSF4b与全羧化酶合成酶（HCS）相互作用，HCS是哺乳动物中催化蛋白质生物素化的一种普遍存在的酶。异位表达HA-HCS会下调HSF4b调控的αB-晶状体蛋白表达。赖氨酸突变分析表明HSF4b/K444是一个潜在的生物素化位点。K444R突变减少了链霉亲和素磁珠对HSF4b的共沉淀作用以及生物素诱导的αB-晶状体蛋白表达的降低。HSF4 C末端区域的其他赖氨酸残基如K207R/K209R、K225R、K288R、K294R和K355R的突变不影响HSF4的表达水平和与链霉亲和素的相互作用，但它们通过不同机制对αB-晶状体蛋白表达表现出不同的调控作用。HSF4/K294R导致αB-晶状体蛋白表达上调，而K207R/K209R、K225R、K288R、K255R和K435R突变减弱了HSF4对αB-晶状体蛋白表达的调控。K207R/K209R阻止HSF4核转位，K345R导致HSF4不稳定。综上所述，数据表明生物素可能是通过生物素化调节HSF4活性的一个新因子。
