自噬调节 p16INK4a 的定位和降解。,Aging Cell

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自噬调节 p16INK4a 的定位和降解。
Aging Cell ( IF 8.0 ) Pub Date : 2020-07-13 , DOI: 10.1111/acel.13171
Philip R Coryell ₁ , Supreet K Goraya ₁ , Katherine A Griffin ₁ , Margaret A Redick ₁ , Samuel R Sisk ₁ , Jeremy E Purvis _{1,

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Affiliation

肿瘤抑制蛋白 p16 ^INK4a(p16) 是公认的衰老标志，可诱导细胞衰老以应对压力。以前的研究主要集中在转录水平的 p16 调节上。关于蛋白质的细胞内定位和降解知之甚少。自噬-溶酶体途径与各种应激反应蛋白的亚细胞运输和周转有关，也被证明可以减轻与年龄相关的病理，但尚不清楚 p16 是否参与该途径。在这里，我们研究了自噬、囊泡运输和溶酶体降解对人类上皮细胞中 p16 表达和定位的作用。使用内源性 p16-mCherry 报告基因的延时荧光显微镜显示血清饥饿、依托泊苷、和过氧化氢刺激自噬并在 4 小时内驱动 p16 募集到酸性细胞质囊泡。用 leupeptin 和氯化铵阻断溶酶体蛋白酶导致 p16 在溶酶体内积累并增加总 p16 水平，表明 p16 被该途径降解。此外，自噬阻滞剂氯喹和巴弗洛霉素 A1 在含有自噬体标志物 LC3 的停滞囊泡内引起 p16 聚集。这些囊泡内 p16 的增加与 LC3-II 的积累一致。自噬体伴侣 p62 的敲除减弱了溶酶体中 p16 聚集体的形成，表明 p16 被 p62 靶向这些囊泡。总之，这些结果表明自噬途径是 p16 降解和定位的新调节剂，

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Autophagy regulates the localization and degradation of p16INK4a.

The tumor suppressor protein p16^INK4a (p16) is a well‐established hallmark of aging that induces cellular senescence in response to stress. Previous studies have focused primarily on p16 regulation at the transcriptional level; comparatively little is known about the protein's intracellular localization and degradation. The autophagy–lysosomal pathway has been implicated in the subcellular trafficking and turnover of various stress‐response proteins and has also been shown to attenuate age‐related pathologies, but it is unclear whether p16 is involved in this pathway. Here, we investigate the role of autophagy, vesicular trafficking, and lysosomal degradation on p16 expression and localization in human epithelial cells. Time‐lapse fluorescence microscopy using an endogenous p16‐mCherry reporter revealed that serum starvation, etoposide, and hydrogen peroxide stimulate autophagy and drive p16 recruitment to acidic cytoplasmic vesicles within 4 hr. Blocking lysosomal proteases with leupeptin and ammonium chloride resulted in the accumulation of p16 within lysosomes and increased total p16 levels suggesting that p16 is degraded by this pathway. Furthermore, autophagy blockers chloroquine and bafilomycin A1 caused p16 aggregation within stalled vesicles containing autophagosome marker LC3. Increase of p16 within these vesicles coincided with the accumulation of LC3‐II. Knockdown of autophagosome chaperone p62 attenuated the formation of p16 aggregates in lysosomes, suggesting that p16 is targeted to these vesicles by p62. Taken together, these results implicate the autophagy pathway as a novel regulator of p16 degradation and localization, which could play a role in the etiology of cancer and age‐related diseases.

更新日期：2020-07-13

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