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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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 4

RNF114 promotes CRYAA ubiquitination during hypothermia-rewarming in GS iLECs.

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RNF114 promotes CRYAA ubiquitination during hypothermia-rewarming in GS ...
(A) Intracellular localization of α-, β-, and γ-crystallins in HLECs after cold treatment and rewarming. Red arrows: protein aggregates. Scale bars: 20 μm. Intensity of intracellular fluorescence spots was also quantified (2-tailed Student’s t test, n = 3, 50 cells per experiment). (B) Colocalization of CRYAA and Ub in GS iLECs and HLECs after cold treatment. Scale bars: 40 μm. (C and D) Immunoprecipitation assay to detect interaction between CRYAA and ubiquitin. Exogenous Myc-ubiquitin with αA. Endogenous ubiquitin with αA. Controls with goat anti-mouse IgG–coated magnetic beads are included. (n ≥ 3 independent experiments.) (E) qPCR analysis to assess the level of RNF114 mRNA in HLECs and GS iLECs after low-temperature treatment (n = 3 independent experiments). (F–H) CRYAA-RNF114 interaction in GS iLECs after cold rewarming. (F) Endogenous CRYAA with RNF114. (G) Exogenous CRYAA with RNF114. “Vector” refers to the transfection of the empty vector pCEP4-tetR. (H) Exogenous RNF114 with CRYAA. (I) Construction of the C-terminal truncated recombinant protein RNF114ΔC (1–200 aa). UIM, ubiquitin interaction motif. (J) Immunoprecipitation assay to investigate the interaction between endogenous CRYAA and ubiquitin, as well as their regulation by RNF114. “RNF114 KD” refers to the transfection of RNF114 siRNA to knock down the expression of RNF114. “RNF114 KD + RNF114 WT/ΔC” refers to the transfection of RNF114 WT/ΔC plasmid into cells where RNF114 has been knocked down. (2-tailed Student’s t tests followed by Holm-Šidák correction, n = 3 independent experiments.) (K and L) Immunoblotting was used to assess expression levels of endogenous CRYAA (K), exogenous CRYAA WT, and the Y118D mutant (L) in GS iLECs under normal temperature conditions, subject to RNF114 regulation (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 < 0.01, ***P < 0.001.)