Neuroscience and Molecular Pharmacology, Wolfson and Davidson Buildings, Faculty of Biomedical & Life Sciences, University of Glasgow, Glasgow, G12 8QQ, Scotland, United Kingdom.
Cell Signal. 2010 Oct;22(10):1576-96. doi: 10.1016/j.cellsig.2010.06.003. Epub 2010 Jun 19.
Chronic challenge of cyclic AMP phosphodiesterase-4A4 (PDE4A4) with certain PDE4 selective inhibitors causes it to reversibly form intracellular aggregates that are not membrane-encapsulated. These aggregates are neither stress granules (SGs) nor processing bodies (PBs) as they contain neither PABP-1 nor Dcp1a, respectively. However, the PDE4 inhibitor rolipram decreases arsenite-induced SGs and increases the amount of PBs, while arsenite challenge ablates rolipram-induced PDE4A4 aggregates. PDE4A4 aggregates are neither autophagic vesicles (autophagosomes) nor aggresomes, although microtubule disruptors ablate PDE4A4 aggregate formation. PDE4A4 constitutively co-immunoprecipitates with p62 protein (sequestosome1, SQSTM1), which locates to both PDE4A4 aggregates and autophagosomes in cells constitutively challenged with rolipram. The mTor inhibitor, rapamycin, activates autophagy, prevents PDE4A4 from forming intracellular aggregates and triggers the loss of bound p62 from PDE4A4. siRNA-mediated knockdown of p62 attenuates PDE4A4 aggregate formation. The p62-binding protein, light chain 3 (LC3), is not found in PDE4A4 aggregates. Blockade of proteasome activity and activation of autophagy with MG132 both increases the level of ubiquitinated proteins found associated with PDE4A4 and inhibits PDE4A4 aggregate formation. Activation of autophagy with either thapsigargin or ionomycin inhibits PDE4A4 aggregate formation. Inhibition of autophagy with either wortmannin or LY294002 activates PDE4A4 aggregate formation. The protein kinase C inhibitors, RO 320432 and GO 6983, and the ERK inhibitors UO 126 and PD 98059 all activated PDE4A4 aggregate formation, whilst roscovitine, thalidomide and the tyrosine kinase inhibitors, genistein and AG17, all inhibited this process. We suggest that the fate of p62-containing protein aggregates need not necessarily be terminal, through delivery to autophagic vesicles and aggresomes. Instead, we propose a novel regulatory mechanism where a sub-population of p62-containing protein aggregates would form in a rapid, reversible manner so as to sequester specific cargo away from their normal, functionally important site(s) within the cell. Thus an appropriate conformational change in the target protein would confer reversible recruitment into a sub-population of p62-containing protein aggregates and so provide a regulatory function by removing these cargo proteins from their functionally important site(s) in a cell.
慢性挑战环磷酸腺苷磷酸二酯酶 4A4（PDE4A4）与某些 PDE4 选择性抑制剂可逆地形成细胞内聚集体，这些聚集体不被膜包裹。这些聚集体既不是应激颗粒（SGs）也不是加工体（PBs），因为它们分别不含有 PABP-1 或 Dcp1a。然而，PDE4 抑制剂罗利普兰减少亚砷酸盐诱导的 SGs 并增加 PB 的数量，而亚砷酸盐挑战则消除罗利普兰诱导的 PDE4A4 聚集体。PDE4A4 聚集体既不是自噬小体（自噬体）也不是聚集体，尽管微管破坏剂消除 PDE4A4 聚集体的形成。PDE4A4 聚集体与 p62 蛋白（自噬体 1，SQSTM1）组成性地共免疫沉淀，该蛋白在细胞中持续受到罗利普兰的挑战时定位于 PDE4A4 聚集体和自噬体。mTOR 抑制剂雷帕霉素激活自噬，阻止 PDE4A4 形成细胞内聚集体，并触发与 PDE4A4 结合的 p62 从 PDE4A4 上脱落。siRNA 介导的 p62 敲低可减轻 PDE4A4 聚集体的形成。LC3 轻链 3（LC3）未在 PDE4A4 聚集体中发现。蛋白酶体活性的阻断和用 MG132 激活自噬均增加与 PDE4A4 相关的泛素化蛋白的水平，并抑制 PDE4A4 聚集体的形成。用他普西卡丁或离子霉素激活自噬抑制 PDE4A4 聚集体的形成。用渥曼青霉素或 LY294002 抑制自噬激活 PDE4A4 聚集体的形成。蛋白激酶 C 抑制剂 RO 320432 和 GO 6983 以及 ERK 抑制剂 UO 126 和 PD 98059 均激活 PDE4A4 聚集体的形成，而罗司维丁、沙利度胺和酪氨酸激酶抑制剂，染料木黄酮和 AG17，均抑制该过程。我们建议，p62 包含的蛋白质聚集体的命运不一定是终末的，而是通过递送到自噬小体和聚集体中。相反，我们提出了一种新的调节机制，其中亚群 p62 包含的蛋白质聚集体将以快速、可逆的方式形成，从而将特定的货物从细胞内其正常的、功能重要的位置隔离出来。因此，靶蛋白的适当构象变化会将其可逆地募集到亚群 p62 包含的蛋白质聚集体中，从而通过将这些货物蛋白从细胞内其功能重要的位置去除来提供调节功能。
