Reversible cold-induced lens opacity in a hibernator reveals a molecular target for treating cataracts
Hao Yang, … , Wei Li, Xingchao Shentu
Hao Yang, … , Wei Li, Xingchao Shentu
Published September 17, 2024
Citation Information: J Clin Invest. 2024;134(18):e169666. https://doi.org/10.1172/JCI169666.
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Research Article Ophthalmology

Reversible cold-induced lens opacity in a hibernator reveals a molecular target for treating cataracts

Abstract

Maintaining protein homeostasis (proteostasis) requires precise control of protein folding and degradation. Failure to properly respond to stresses disrupts proteostasis, which is a hallmark of many diseases, including cataracts. Hibernators are natural cold-stress adaptors; however, little is known about how they keep a balanced proteome under conditions of drastic temperature shift. Intriguingly, we identified a reversible lens opacity phenotype in ground squirrels (GSs) associated with their hibernation-rewarming process. To understand this “cataract-reversing” phenomenon, we first established induced lens epithelial cells differentiated from GS-derived induced pluripotent stem cells, which helped us explore the molecular mechanism preventing the accumulation of protein aggregates in GS lenses. We discovered that the ubiquitin-proteasome system (UPS) played a vital role in minimizing the aggregation of the lens protein αA-crystallin (CRYAA) during rewarming. Such function was, for the first time to our knowledge, associated with an E3 ubiquitin ligase, RNF114, which appears to be one of the key mechanisms mediating the turnover and homeostasis of lens proteins. Leveraging this knowledge gained from hibernators, we engineered a deliverable RNF114 complex and successfully reduced lens opacity in rats with cold-induced cataracts and zebrafish with oxidative stress–related cataracts. These data provide new insights into the critical role of the UPS in maintaining proteostasis in cold and possibly other forms of stresses. The newly identified E3 ubiquitin ligase RNF114, related to CRYAA, offers a promising avenue for treating cataracts with protein aggregates.

Authors

Hao Yang, Xiyuan Ping, Jiayue Zhou, Hailaiti Ailifeire, Jing Wu, Francisco M. Nadal-Nicolás, Kiyoharu J. Miyagishima, Jing Bao, Yuxin Huang, Yilei Cui, Xin Xing, Shiqiang Wang, Ke Yao, Wei Li, Xingchao Shentu

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Figure 5

RNF114 promotes CRYAA proteasomal degradation in HLECs.

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RNF114 promotes CRYAA proteasomal degradation in HLECs. (A) Immunofluore...
(A) Immunofluorescence images depict the intracellular localization of endogenous CRYAA in GS iLECs, HLECs, and HLECs transfected with RNF114 WT or RNF114 ΔC after 24 or 48 hours of low-temperature treatment followed by 30 minutes of rewarming. Red arrows indicate prominent protein aggregates. Scale bars: 20 μm. (B) Statistical analysis of fluorescence spot intensity from A (2-tailed Student’s t test, n = 3 independent experiments, 50 cells per experiment). (C) Immunoblotting to detect protein levels of endogenous CRYAA in cells subjected to the aforementioned treatments. (D) Statistical analysis of protein expression levels from C (2-tailed Student’s t tests followed by Holm-Šidák correction, n = 3 independent experiments). (E) qPCR analysis to measure mRNA expression levels of CRYAA in GS iLECs after 24 hours of low-temperature rewarming and subsequent statistical analysis (n = 3 independent experiments). (F) Live-cell imaging was used to assess the impact of ubiquitin system inhibition on the function of exogenous RNF114 WT in relation to WT CRYAA and Y118D. The effects of overexpressing RNF114 WT and RNF114 ΔC on the intracellular localization of Y118D in HLECs were also observed. Scale bars: 8 μm. (G) Statistical analysis of fluorescence spot intensity from F (n = 3 independent experiments, 10 cells per experiment). (H) Immunoblotting to detect expression levels of knocked-in CRYAA(Y118D) in treated HLECs to validate the RNF114-mediated reduction mechanism via ubiquitin (2-tailed Student’s t tests followed by Holm-Šidák correction, n = 3 independent experiments). (All values are presented as mean ± SD, *P < 0.05, ***P < 0.001.)