ABSTRACT

Dysfunction of macroautophagy/autophagy has been postulated as a major cellular toxicological response to nanomaterials. It has been reported that excessive autophagy activation, induced by silica nanoparticles (SiNPs), contributes to autophagy dysfunction, whereas little is known how SiNPs trigger autophagy activation. Here, we treated normal rat kidney (NRK) cells using 3 different sizes of SiNPs (16, 29, and 51 nm) and observed that 16-nm SiNPs, with a final concentration of 60 μg/mL, dramatically induce autophagy activation without reducing cell viability. We further conducted a transcriptomic, proteomic, and phosphoproteomic profiling, and detected 23 autophagy-related (Atg) genes and 35 autophagy regulators regulated on at least one omic layer. To identify key regulators from the multi-omics data, we developed a new algorithm of computational prediction of master autophagy-regulating kinases (cMAK) to detect 21 candidates and revealed the CDK7-CDK4 cascade to be functional. The silence or inhibition of Cdk7 or Cdk4 significantly attenuated autophagic activation but not influenced autophagic flux blockage induced by 16-nm SiNPs. Further computational modeling indicated that the CDK7-CDK4 signaling axis potentially triggers autophagy activation by phosphorylating RB1 (RB transcriptional corepressor 1), activating two critical transcription factors, E2F1 (E2F transcription factor 1) and FOXO3 (forkhead box O3), and enhancing the transcriptional levels of at least 8 Atg genes and autophagy regulators in response to SiNPs. Our studies not only established a powerful method for predicting regulatory kinases from the multi-omics data but also revealed a potential mechanism of SiNP-triggered autophagy activation through modulating the CDK7-CDK4 cascade.

Abbreviations: 3-MA: 3-methyladenine; Atg: autophagy-related; BECN1: beclin 1; CCK-8: cell counting kit-8; CDK4: cyclin dependent kinase 4; CDK7: cyclin dependent kinase 7; cMAK: computational prediction of master autophagy-regulating kinases; CQ: chloroquine; DMEM: Dulbecco’s modified Eagle’s medium; DMSO: dimethyl sulfoxide; E-ratio: enrichment ratio; E2F1: E2F transcription factor 1; EBSS: Earle’s balanced salt solution; ER: endoplasmic reticulum; FOXO3: forkhead box O3; FPKM: fragments per kilobase of exon per million fragments mapped; GO: gene ontology; H₂O₂: hydrogen peroxide; iGPS: in vivo GPS; KEGG: Kyoto Encyclopedia of Genes and Genomes; LC-MS/MS: liquid chromatography–tandem mass spectrometry; LDH: lactate dehydrogenase; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; NRK: normal rat kidney; p-site: phosphorylation site; PBS: phosphate-buffered saline; PDI: polydispersity index; PTM: post-translational modification; QKS: quantitative kinase state; RB1: RB transcriptional corepressor 1; RBHs: reciprocal best hits; RNA-Seq: RNA sequencing; ROS: reactive oxygen species; rSiNPs: SiNPs fluorescently labeled with rhodamine B; SEM: scanning electronic microscopy; SiNPs: silica nanoparticles; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; ssKSR: site-specific kinase-substrate relation; TEM: transmission electron microscopy; tfLC3: mRFP-GFP tandem fluorescent-tagged LC3.

摘要

巨自噬/自噬功能障碍被认为是对纳米材料的主要细胞毒理学反应。据报道，由二氧化硅纳米粒子 (SiNPs) 诱导的过度自噬激活会导致自噬功能障碍，而对 SiNPs 如何触发自噬激活知之甚少。在这里，我们使用 3 种不同大小的 SiNP（16、29 和 51 nm）处理正常大鼠肾脏 (NRK) 细胞，并观察到最终浓度为 60 μg/mL 的 16 nm SiNP 显着诱导自噬激活，而不会降低细胞活力。我们进一步进行了转录组学、蛋白质组学和磷酸蛋白质组学分析，并检测到 23 个与自噬相关的 ( Atg) 基因和 35 个自噬调节因子在至少一个组学层上进行调节。为了从多组学数据中识别关键调节因子，我们开发了一种新的计算预测主自噬调节激酶 (cMAK) 的算法来检测 21 个候选物，并揭示 CDK7-CDK4 级联是有功能的。Cdk7或Cdk4的沉默或抑制显着减弱自噬激活，但不影响由 16-nm SiNPs 诱导的自噬通量阻塞。进一步的计算模型表明，CDK7-CDK4 信号轴可能通过磷酸化 RB1（RB 转录辅抑制因子 1）、激活两个关键转录因子 E2F1（E2F 转录因子 1）和 FOXO3（叉头盒 O3）并增强转录来触发自噬激活。响应SiNP 的至少 8 个Atg基因和自噬调节剂的水平。我们的研究不仅建立了一种从多组学数据预测调节激酶的强大方法，而且还揭示了通过调节 CDK7-CDK4 级联反应激活 SiNP 触发自噬的潜在机制。

缩写： 3-MA：3-甲基腺嘌呤；Atg：自噬相关；BECN1: beclin 1; CCK-8：细胞计数试剂盒-8；CDK4：细胞周期蛋白依赖性激酶 4；CDK7：细胞周期蛋白依赖性激酶 7；cMAK：主自噬调节激酶的计算预测；CQ：氯喹；DMEM：Dulbecco 改良的 Eagle 培养基；DMSO：二甲亚砜；E-ratio：富集比；E2F1：E2F 转录因子 1；EBSS：厄尔平衡盐溶液；ER：内质网；FOXO3：叉头盒O3；FPKM：映射的每百万个外显子千碱基片段；GO：基因本体；H ₂ O ₂：过氧化氢；iGPS：体内全球定位系统; KEGG：京都基因和基因组百科全书；LC-MS/MS：液相色谱-串联质谱；LDH：乳酸脱氢酶；MAP1LC3B/LC3：微管相关蛋白 1 轻链 3 β；NRK：正常大鼠肾脏；p-site：磷酸化位点；PBS：磷酸盐缓冲盐水；PDI：多分散指数；PTM：翻译后修饰；QKS：定量激酶状态；RB1：RB 转录抑制因子 1；RBHs：相互最佳命中；RNA-Seq：RNA测序；ROS：活性氧；rSiNPs：用罗丹明 B 荧光标记的 SiNPs；SEM：扫描电子显微镜；SiNPs：二氧化硅纳米粒子；siRNA：小干扰RNA；SQSTM1/p62：sequestosome 1；ssKSR：位点特异性激酶-底物关系；TEM：透射电子显微镜；tfLC3：mRFP-GFP 串联荧光标记 LC3。