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.

中文翻译：

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热激因子4通过调节眼晶状体中的αB-晶状体蛋白-ATP6V1A-mTOR复合物来调节溶酶体的活性。

背景技术热休克因子4（HSF4）的种系突变导致先天性白内障。以前，我们已经证明HSF4参与体外调节小鼠晶状体上皮细胞中的溶酶体pH值。但是，其潜在机制仍不清楚。方法采用HSF4缺陷型小鼠晶状体上皮细胞系和斑马鱼。免疫印迹和定量RT-PCR用于表达分析。蛋白质-蛋白质相互作用用GST-pull downs测试。通过超速离心分离溶酶体。结果HSF4缺乏或αB-晶状体蛋白的敲低会提高溶酶体的pH值，并增加蛋白酶体的泛素化和ATP6V1A的降解。αB-晶状蛋白部分位于溶酶体中，并且仅与V-ATPase V1复合物的ATP6V1A蛋白相互作用。此外，在GST下拉试验中，αB-晶状蛋白可以与mTORC1和ATP6V1A共沉淀。雷帕霉素或siRNA对mTORC1的抑制作用可导致αB-晶状体蛋白从ATP6V1A和mTORC1复合物中解离，从而缩短ATP6V1A的半衰期并增加溶酶体pH。ATP6V1A / S441A的突变（预测的mTOR磷酸化位点）降低了其与αB-晶状体蛋白的缔合。在斑马鱼模型中，HSF4缺乏会降低晶状体组织中的αB-晶状体蛋白表达并提高溶酶体pH值。结论HSF4通过控制αB-晶状体蛋白与ATP6V1A和mTOR的缔合并调节ATP6V1A蛋白质的稳定来调节溶酶体酸化。一般意义该研究揭示了αB-晶状体蛋白的新功能，证明αB-晶状体蛋白可以通过与ATP6V1A和mTOR结合来调节溶酶体ATP6V1A蛋白质的稳定性。这突显了一种新的机制，HSF4通过该机制调节晶状体发育过程中细胞器的蛋白水解过程。