Xiukun Cui1, Ruiping Feng1, Jungai Wang1, Chunxiao Du1, Xiahui Pi1, Danling Chen1, Jing Li1, Hui Li1, Jun Zhang1, Jing Zhang1, Hongmei Mu2, Fengyan Zhang3, Mugen Liu4, Yanzhong Hu5. 1. Joint National Laboratory for Antibody Drug Engineering, Henan International Union Lab of Antibody Medicine, Henan University School of Medicine, Kaifeng, China. 2. Kaifeng Key Lab of Myopia and Cataract, Institute of Eye Disease, Kaifeng Central Hospital, Kaifeng, China. 3. Department of Ophthalmology, the First Affiliate Hospital of Zhengzhou University, China. 4. College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei 430074, China. 5. Joint National Laboratory for Antibody Drug Engineering, Henan International Union Lab of Antibody Medicine, Henan University School of Medicine, Kaifeng, China; Department of Ophthalmology, the First Affiliate Hospital of Zhengzhou University, China; Kaifeng Key Lab of Myopia and Cataract, Institute of Eye Disease, Kaifeng Central Hospital, Kaifeng, China. Electronic address: hyz@henu.edu.cn.
Abstract
BACKGROUND: Germline mutations in heat shock factor 4 (HSF4) cause congenital cataracts. Previously, we have shown that HSF4 is involved in regulating lysosomal pH in mouse lens epithelial cell in vitro. However, the underlying mechanism remains unclear. METHODS: HSF4-deficient mouse lens epithelial cell lines and zebrafish were used in this study. Immunoblotting and quantitative RT-PCR were used for expression analysis. The protein-protein interactions were tested with GST-pull downs. The lysosomes were fractioned by ultracentrifugation. RESULTS: HSF4 deficiency or knock down of αB-crystallin elevates lysosomal pH and increases the ubiquitination and degradation of ATP6V1A by the proteasome. αB-crystallin localizes partially in the lysosome and interacts solely with the ATP6V1A protein of the V1 complex of V-ATPase. Furthermore, αB-crystallin can co-precipitate with mTORC1 and ATP6V1A in GST pull down assays. Inhibition of mTORC1 by rapamycin or siRNA can lead to dissociation of αB-crystallin from the ATP6V1A and mTORC1complex, shortening the half-life of ATP6V1A and increasing the lysosomal pH. Mutation of ATP6V1A/S441A (the predicted mTOR phosphorylation site) reduces its association with αB-crystallin. In the zebrafish model, HSF4 deficiency reduces αB-crystallin expression and elevates the lysosomal pH in lens tissues. CONCLUSION: HSF4 regulates lysosomal acidification by controlling the association of αB-crystallin with ATP6V1A and mTOR and regulating ATP6V1A protein stabilization. GENERAL SIGNIFICANCE: This study uncovers a novel function of αB-crystallin, demonstrating that αB-crystallin can regulate lysosomal ATP6V1A protein stabilization by complexing to ATP6V1A and mTOR. This highlights a novel mechanism by which HSF4 regulates the proteolytic process of organelles during lens development.
BACKGROUND: Germline mutations in heat shock factor 4 (HSF4) cause congenital cataracts. Previously, we have shown that HSF4 is involved in regulating lysosomal pH in mouse lens epithelial cell in vitro. However, the underlying mechanism remains unclear. METHODS:HSF4-deficient mouse lens epithelial cell lines and zebrafish were used in this study. Immunoblotting and quantitative RT-PCR were used for expression analysis. The protein-protein interactions were tested with GST-pull downs. The lysosomes were fractioned by ultracentrifugation. RESULTS:HSF4 deficiency or knock down of αB-crystallin elevates lysosomal pH and increases the ubiquitination and degradation of ATP6V1A by the proteasome. αB-crystallin localizes partially in the lysosome and interacts solely with the ATP6V1A protein of the V1 complex of V-ATPase. Furthermore, αB-crystallin can co-precipitate with mTORC1 and ATP6V1A in GST pull down assays. Inhibition of mTORC1 by rapamycin or siRNA can lead to dissociation of αB-crystallin from the ATP6V1A and mTORC1complex, shortening the half-life of ATP6V1A and increasing the lysosomal pH. Mutation of ATP6V1A/S441A (the predicted mTOR phosphorylation site) reduces its association with αB-crystallin. In the zebrafish model, HSF4 deficiency reduces αB-crystallin expression and elevates the lysosomal pH in lens tissues. CONCLUSION:HSF4 regulates lysosomal acidification by controlling the association of αB-crystallin with ATP6V1A and mTOR and regulating ATP6V1A protein stabilization. GENERAL SIGNIFICANCE: This study uncovers a novel function of αB-crystallin, demonstrating that αB-crystallin can regulate lysosomal ATP6V1A protein stabilization by complexing to ATP6V1A and mTOR. This highlights a novel mechanism by which HSF4 regulates the proteolytic process of organelles during lens development.