Reversible cold-induced lens opacity in a hibernator reveals a molecular target for treating cataracts
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
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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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 3

The UPS promotes the degradation of aggregated crystallin.

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The UPS promotes the degradation of aggregated crystallin. (A) GO pathwa...
(A) GO pathway analysis of upregulated proteins in GS and rat lens capsules after cold treatment. Red boxes: ubiquitin-related pathways. (B) Interactions among upregulated ubiquitin enzymes in GS lens capsules post-cold treatment. Gray: E1, blue: E2, red: E3. (C) Immunoblotting to detect expression levels of total ubiquitin protein in GS iLECs and HLECs after 30 minutes of rewarming following 24 and 48 hours of cold treatment. (D) Corresponding statistical graph of C (1-way ANOVA, n = 3 independent experiments). (E) Localization of GFP-tagged mutant crystalline proteins in HLECs and GS iLECs after cold treatment and rewarming. Scale bars: 8 μm. (F) Statistical analysis of fluorescence spot intensity in the live-cell imaging experiment described in E (2-tailed Student’s t tests followed by Holm-Šidák correction, n = 3, 10 cells per experiment). (G and H) Live-cell imaging of GS iLECs showing intracellular localization and relative fluorescence intensity of exogenous αA (Y118D) under conditions 1–5 (1-way ANOVA, n = 3, 5 fields of view per experiment). Scale bar: 20 μm. (I–L) Immunoblotting to detect expression levels of relevant proteins (2-tailed Student’s t test, n = 3 independent experiments). (I) Protein levels of exogenous CRYAA(Y118D) in GS iLECs after 24 hours of low-temperature rewarming. (J) Effect of 3MA+NH4Cl on p62 protein levels. PBS treatment used as control. (K) Effect of MG132 on ERK2 protein levels; DMSO treatment used as control. (L) Protein levels of exogenous CRYAA(Y118D) in GS iLECs after 24 hours of low-temperature rewarming. (M and N) CRYAA (Y118D) localization in GS iLECs after cold treatment and rewarming. Scale bars: 8 μm. Red arrows indicate prominent protein aggregates. Intensity of intracellular fluorescence spots was also quantified (2-tailed Student’s t tests followed by Holm-Šidák correction, n = 3, 10 cells per experiment). (All values are represented as mean ± SD, with *P < 0.05, **P < 0.01, and ***P < 0.001 indicating statistical significance.)