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O-GlcNAcylation stabilizes the autophagy-initiating kinase ULK1 by inhibiting chaperone-mediated autophagy upon HPV infection
Journal of Biological Chemistry ( IF 5.5 ) Pub Date : 2022-08-03 , DOI: 10.1016/j.jbc.2022.102341
Yingxin Shi 1 , Sheng Yan 1 , Guang-Can Shao 2 , Jinglong Wang 3 , Yong-Ping Jian 4 , Bo Liu 1 , Yanqiu Yuan 5 , Ke Qin 6 , Shanshan Nai 1 , Xiahe Huang 7 , Yingchun Wang 7 , Zhenghui Chen 8 , Xing Chen 6 , Meng-Qiu Dong 2 , Yiqun Geng 8 , Zhi-Xiang Xu 4 , Jing Li 9
Affiliation  

Human papillomaviruses (HPVs) cause a subset of head and neck squamous cell carcinomas (HNSCCs). Previously, we demonstrated that HPV16 oncogene E6 or E6/E7 transduction increases the abundance of O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT), but OGT substrates affected by this increase are unclear. Here, we focus on the effects of O-GlcNAcylation on HPV-positive HNSCCs. We found that upon HPV infection, Unc-51-like kinase 1 (ULK1), an autophagy-initiating kinase, is hyper-O-GlcNAcylated, stabilized, and linked with autophagy elevation. Through mass spectrometry, we identified that ULK1 is O-GlcNAcylated at Ser409, which is distinct from the previously reported Thr635/Thr754 sites. It has been demonstrated that PKCα mediates phosphorylation of ULK1 at Ser423, which attenuates its stability by shunting ULK1 to the chaperone-mediated autophagy (CMA) pathway. Using biochemical assays, we demonstrate that ULK1 Ser409Ser410 O-GlcNAcylation antagonizes its phosphorylation at Ser423. Moreover, mutations of Ser409A and its neighboring site Ser410A (2A) render ULK1 less stable by promoting interaction with the CMA chaperone HSC70 (heat shock cognate 70 kDa protein). Furthermore, ULK1-2A mutants attenuate the association of ULK1 with STX17, which is vital for the fusion between autophagosomes and lysosomes. Analysis of The Cancer Genome Atlas (TCGA) database reveals that ULK1 is upregulated in HPV-positive HNSCCs, and its level positively correlates with HNSCC patient survival. Overall, our work demonstrates that O-GlcNAcylation of ULK1 is altered in response to environmental changes. O-GlcNAcylation of ULK1 at Ser409 and perhaps Ser410 stabilizes ULK1, which might underlie the molecular mechanism of HPV-positive HNSCC patient survival.



中文翻译:

O-GlcNAcylation 通过抑制 HPV 感染后伴侣介导的自噬来稳定自噬起始激酶 ULK1

人乳头瘤病毒 (HPV) 会导致一部分头颈部鳞状细胞癌 (HNSCC)。以前,我们证明了 HPV16 癌基因E6E6/E7转导增加了 O-linked β- N的丰度-乙酰氨基葡萄糖(O-GlcNAc)转移酶(OGT),但受这种增加影响的OGT底物尚不清楚。在这里,我们关注 O-GlcNAcylation 对 HPV 阳性 HNSCC 的影响。我们发现,在 HPV 感染后,Unc-51 样激酶 1 (ULK1),一种自噬起始激酶,被过度 O-GlcNAcylated、稳定并与自噬升高相关。通过质谱分析,我们发现 ULK1 在 Ser409 位点是 O-GlcNAcylated,这与之前报道的 Thr635/Thr754 位点不同。已经证明 PKCα 介导 ULK1 在 Ser423 位点的磷酸化,这通过将 ULK1 分流到伴侣介导的自噬 (CMA) 途径来减弱其稳定性。使用生化测定,我们证明 ULK1 Ser409Ser410 O-GlcNAcylation 拮抗其在 Ser423 处的磷酸化。而且,Ser409A 及其邻近位点 Ser410A (2A) 的突变通过促进与 CMA 伴侣 HSC70(热休克同源 70 kDa 蛋白)的相互作用而降低了 ULK1 的稳定性。此外,ULK1-2A 突变体减弱了 ULK1 与 STX17 的结合,这对于自噬体和溶酶体之间的融合至关重要。对癌症基因组图谱 (TCGA) 数据库的分析表明,ULK1 在 HPV 阳性 HNSCC 中上调,其水平与 HNSCC 患者存活率正相关。总体而言,我们的工作表明,ULK1 的 O-GlcNAcylation 会随着环境变化而改变。ULK1 在 Ser409 和 Ser410 的 O-GlcNAcylation 可能稳定 ULK1,这可能是 HPV 阳性 HNSCC 患者存活的分子机制的基础。

更新日期:2022-08-03
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