Departments of Cellular & Molecular Pharmacology, University of California San Francisco, San Francisco, California, USA.
Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California, USA.
Mol Cell Proteomics. 2020 Dec;19(12):1968-1986. doi: 10.1074/mcp.RA120.002316. Epub 2020 Sep 10.
Mallory-Denk-bodies (MDBs) are hepatic protein aggregates associated with inflammation both clinically and in MDB-inducing models. Similar protein aggregation in neurodegenerative diseases also triggers inflammation and NF-κB activation. However, the precise mechanism that links protein aggregation to NF-κB-activation and inflammatory response remains unclear. Herein we find that treating primary hepatocytes with MDB-inducing agents (N-methylprotoporphyrin (NMPP), protoporphyrin IX (PPIX), or Zinc-protoporphyrin IX (ZnPP)) elicited an IκBα-loss with consequent NF-κB activation. Four known mechanisms of IκBα-loss the canonical ubiquitin-dependent proteasomal degradation (UPD), autophagic-lysosomal degradation, calpain degradation and translational inhibition, were all probed and excluded. Immunofluorescence analyses of ZnPP-treated cells coupled with 8 M urea/CHAPS-extraction revealed that this IκBα-loss was due to its sequestration along with IκBβ into insoluble aggregates, thereby releasing NF-κB. Through affinity pulldown, proximity biotinylation by antibody recognition, and other proteomic analyses, we verified that NF-κB subunit p65, which stably interacts with IκBα under normal conditions, no longer binds to it upon ZnPP-treatment. Additionally, we identified 10 proteins that interact with IκBα under baseline conditions, aggregate upon ZnPP-treatment, and maintain the interaction with IκBα after ZnPP-treatment, either by cosequestering into insoluble aggregates or through a different mechanism. Of these 10 proteins, the nucleoporins Nup153 and Nup358/RanBP2 were identified through RNA-interference, as mediators of IκBα-nuclear import. The concurrent aggregation of IκBα, NUP153, and RanBP2 upon ZnPP-treatment, synergistically precluded the nuclear entry of IκBα and its consequent binding and termination of NF-κB activation. This novel mechanism may account for the protein aggregate-induced inflammation observed in liver diseases, thus identifying novel targets for therapeutic intervention. Because of inherent commonalities this MDB cell model is a protoporphyric model, making these findings equally relevant to the liver inflammation associated with clinical protoporphyria.
Mallory-Denk 小体 (MDBs) 是与炎症相关的肝蛋白聚集体，无论是在临床上还是在诱导 MDB 的模型中都是如此。神经退行性疾病中的类似蛋白聚集也会引发炎症和 NF-κB 激活。然而，将蛋白聚集与 NF-κB 激活和炎症反应联系起来的确切机制仍不清楚。在此，我们发现用诱导 MDB 的试剂（N-甲基原卟啉 (NMPP)、原卟啉 IX (PPIX) 或锌原卟啉 IX (ZnPP)）处理原代肝细胞会引起 IκBα 丧失，进而导致 NF-κB 激活。我们探查并排除了 IκBα 丧失的四种已知机制——经典的泛素依赖性蛋白酶体降解 (UPD)、自噬溶酶体降解、钙蛋白酶降解和翻译抑制。用 8 M 尿素/CHAPS 提取对 ZnPP 处理的细胞进行免疫荧光分析表明，这种 IκBα 丧失是由于其与 IκBβ 一起被隔离到不溶性聚集体中，从而释放 NF-κB。通过亲和下拉、抗体识别的邻近生物素化以及其他蛋白质组学分析，我们证实了 NF-κB 亚基 p65 在正常条件下与 IκBα 稳定相互作用，而在 ZnPP 处理后不再与之结合。此外，我们鉴定出 10 种在基线条件下与 IκBα 相互作用、在 ZnPP 处理后聚集、并在 ZnPP 处理后通过共隔离到不溶性聚集体或通过不同机制与 IκBα 保持相互作用的蛋白质。在这 10 种蛋白质中，核孔蛋白 Nup153 和 Nup358/RanBP2 通过 RNA 干扰被鉴定为 IκBα 核输入的介体。ZnPP 处理后 IκBα、NUP153 和 RanBP2 的同时聚集，协同阻止了 IκBα 的核内进入及其随后与 NF-κB 激活的结合和终止。这种新的机制可能解释了在肝脏疾病中观察到的蛋白聚集体诱导的炎症，从而为治疗干预确定了新的靶点。由于固有的共性，这种 MDB 细胞模型是一种原卟啉模型，因此这些发现与临床原卟啉症相关的肝脏炎症同样相关。
