14-3-3 proteins regulate cullin 7-mediated Eag1 degradation

Chang-Heng Hsieh; Chia-Cheng Chou; Ya-Ching Fang; Po-Hao Hsu; Yi-Hung Chiu; Chi-Sheng Yang; Guey-Mei Jow; Chih-Yung Tang; Chung-Jiuan Jeng

doi:10.1186/s13578-023-00969-w

14-3-3 proteins regulate cullin 7-mediated Eag1 degradation

Cell Biosci. 2023 Jan 30;13(1):18. doi: 10.1186/s13578-023-00969-w.

Authors

Chang-Heng Hsieh¹, Chia-Cheng Chou², Ya-Ching Fang^{1

3}, Po-Hao Hsu^{1

3}, Yi-Hung Chiu¹, Chi-Sheng Yang¹, Guey-Mei Jow⁴, Chih-Yung Tang⁵, Chung-Jiuan Jeng^{6

7}

Affiliations

¹ Institute of Anatomy and Cell Biology, College of Medicine, National Yang Ming Chiao Tung University, Taipei, 112, Taiwan.
² National Laboratory Animal Center, National Applied Research Laboratories, Taipei, Taiwan.
³ Department of Physiology, College of Medicine, National Taiwan University, Taipei, 100, Taiwan.
⁴ School of Medicine, Fu-Jen Catholic University, New Taipei City, Taiwan.
⁵ Department of Physiology, College of Medicine, National Taiwan University, Taipei, 100, Taiwan. tang@ntu.edu.tw.
⁶ Institute of Anatomy and Cell Biology, College of Medicine, National Yang Ming Chiao Tung University, Taipei, 112, Taiwan. cjjeng@nycu.edu.tw.
⁷ Brain Research Center, National Yang Ming Chiao Tung University, Taipei, Taiwan. cjjeng@nycu.edu.tw.

Abstract

Background: Mutations in the human gene encoding the neuron-specific Eag1 (K_V10.1; KCNH1) potassium channel are linked to congenital neurodevelopmental diseases. Disease-causing mutant Eag1 channels manifest aberrant gating function and defective protein homeostasis. Both the E3 ubiquitin ligase cullin 7 (Cul7) and the small acid protein 14-3-3 serve as binding partners of Eag1. Cul7 mediates proteasomal and lysosomal degradation of Eag1 protein, whereas over-expression of 14-3-3 notably reduces Eag1 channel activity. It remains unclear whether 14-3-3 may also contribute to Eag1 protein homeostasis.

Results: In human cell line and native rat neurons, disruptions of endogenous 14-3-3 function with the peptide inhibitor difopein or specific RNA interference up-regulated Eag1 protein level in a transcription-independent manner. Difopein hindered Eag1 protein ubiquitination at the endoplasmic reticulum and the plasma membrane, effectively promoting the stability of both immature and mature Eag1 proteins. Suppression of endogenous 14-3-3 function also reduced excitotoxicity-associated Eag1 degradation in neurons. Difopein diminished Cul7-mediated Eag1 degradation, and Cul7 knock-down abolished the effect of difopein on Eag1. Inhibition of endogenous 14-3-3 function substantially perturbed the interaction of Eag1 with Cul7. Further structural analyses suggested that the intracellular Per-Arnt-Sim (PAS) domain and cyclic nucleotide-binding homology domain (CNBHD) of Eag1 are essential for the regulatory effect of 14-3-3 proteins. Significantly, suppression of endogenous 14-3-3 function reduced Cul7-mediated degradation of disease-associated Eag1 mutant proteins.

Conclusion: Overall these results highlight a chaperone-like role of endogenous 14-3-3 proteins in regulating Eag1 protein homeostasis, as well as a therapeutic potential of 14-3-3 modulators in correcting defective protein expression of disease-causing Eag1 mutants.

Keywords: Lysosomal degradation; Potassium channel; Proteasomal degradation; Protein interaction; Protein stability; Ubiquitin ligase.

Abstract

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