[논문]Determination of HIF-1α degradation pathways via modulation of the propionyl mark

Kwanyoung Jeong; Jinmi Choi; Ahrum Choi; Joohee Shim; Young Ah Kim; Changseok Oh; Hong-Duk Youn; Eun-Jung Cho

doi:10.5483/bmbrep.2022-0191

Determination of HIF-1α degradation pathways via modulation of the propionyl mark 원문보기

BMB reports, v.56 no.4, 2023년, pp.252 - 257

Kwanyoung Jeong (School of Pharmacy, Sungkyunkwan University) , Jinmi Choi (School of Pharmacy, Sungkyunkwan University) , Ahrum Choi (School of Pharmacy, Sungkyunkwan University) , Joohee Shim (School of Pharmacy, Sungkyunkwan University) , Young Ah Kim (Department of Molecular Medicine & Biopharmaceutical Sciences, Graduate School of Convergence Science, Seoul National University College of Medicine) , Changseok Oh (School of Pharmacy, Sungkyunkwan University) , Hong-Duk Youn (Department of Biomedical Sciences, Stochastic Stemness Research Center, Seoul National University College of Medicine) , Eun-Jung Cho (School of Pharmacy, Sungkyunkwan University)

Abstract ▼ AI-Helper

The hypoxia-inducible factor-1α (HIF-1α) is a key regulator of hypoxic stress under physiological and pathological conditions. HIF-1α protein stability is tightly regulated by the ubiquitin-proteasome system (UPS) and autophagy in normoxia, hypoxia, and the tumor environment to mediate the hypoxic response. However, the mechanisms of how the UPS and autophagy interplay for HIF-1α proteostasis remain unclear. Here, we found a HIF-1α species propionylated at lysine (K) 709 by p300/CREB binding protein (CBP). HIF-1α stability and the choice of degradation pathway were affected by HIF-1α propionylation. K709-propionylation prevented HIF-1α from degradation through the UPS, while activated chaperon-mediated autophagy (CMA) induced the degradation of propionylated and nonpropionylated HIF-1α. CMA contributed to HIF-1α degradation in both normoxia and hypoxia. Furthermore, the pan-cancer analysis showed that CMA had a significant positive correlation with the hypoxic signatures, whereas SIRT1, responsible for K709-depropionylation correlated negatively with them. Altogether, our results revealed a novel mechanism of HIF-1α distribution into two different degradation pathways.

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표/그림 (4)

그림 Fig. 1. HIF-1α is propionylated at K709 by p300. (A) IP with anti-HIF-1α antibody from HEK293T cells expressing HA-HIF-1α and HA-p300. WB with anti-pan-K-acyl antibodies. (B) IP with an anti-HIF-1α antibody from HEK293T cells expressing HA-HIF-1α and either HA-p300 or Myc-GCN5. WB with anti-K-propionyl antibody. (C, D) HEK293T cells were transfected with HA-HIF-1α WT, K674A, or K709A together with Myc-p300 or empty vector. IP was performed with anti-HA antibody. (E) IP with anti K709-propionyl antibody. Lamin B1 was used as a loading control.
그림 Fig. 2. K709-propionylated HIF-1α is transcriptionally active and preserved from degradation. (A, C) WB for the K709-propionyl levels under hypoxia (Hx, 1% O₂ for 4 h) or normoxia (Nx) with DMOG (1 mM), Baf A1 (400 nM), or MG132 (25 μM) for 4 h. Lamin B1 and α-Tubulin are loading controls. (B, D) Chromatin was IPed with rabbit IgG, HIF-1α, or K709-propionyl antibodies and precipitates were analyzed by RT-qPCR. (E, F) HeLa cells were treated with control or EP300-specific siRNAs for 48 h and then exposed to 1% O₂ for 4 h (Hx). All data in the graphs are presented as the means ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001, and ns; not significant.
그림 Fig. 3. CMA is efficient for propionylated HIF-1α degradation and important for HIF-1α proteostasis under hypoxia. (A) Pearson correlation coefficients and the corresponding P-values were calculated between hypoxia signatures and VHL or CMA-genes. (B) Correlation of the hypoxia signature levels with CMA or macroautophagy (MA) genes. n = 2773 for each group. ***P < 0.001, and ns; not significant. (C, D) HeLa cells were treated with Baf A1 (400 nM), MG132 (25 μM) for 4 h or 6-AN (50 μM) for 24 h with hypoxia (1% O₂ for last 4 h). p62 and LC3B are autophagy markers, while p53 is proteasomal inhibition marker. (E) HEK293T cells were cotransfected with HA-HIF-1α WT or K709A/Q/R and Flag-CHIP for 48 h.
그림 Fig. 4. SIRT1 mediates HIF-1α depropionylation and correlates negatively with the hypoxia signatures. (A) Kaplan-Meier survival curve of patients with cancers of high- or low-hypoxia signatures according to SIRT1 levels. (B) Pearson correlation coefficients and the corresponding P-values were calculated between the hypoxia signatures and SIRT1 or CMA-genes. (C) HeLa cells were transfected with Flag- SIRT1 or empty vector for 48 h and then exposed to 1% O₂ for 4 h with or without MG132 (25 μM). (D) HeLa cells were treated with a control or SIRT1-specific siRNAs for 48 h and then exposed to 1% O₂ or DMOG (1 mM) for 4 h. (E) Comparison of hypoxia signature levels in the SIRT1-high vs. SIRT1-low groups. n = 2773 for each. ***P < 0.001.

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