Lee Hyuna, Kim Mi-Nam, Ryu Kwon-Yul
Department of Life Science, University of Seoul, Seoul 02504, Republic of Korea.
Department of Life Science, University of Seoul, Seoul 02504, Republic of Korea.
Biochem Biophys Res Commun. 2017 May 6;486(3):839-844. doi: 10.1016/j.bbrc.2017.03.146. Epub 2017 Mar 28.
Oxidative stress induced by arsenite [As(III)] affects protein folding and results in increased levels of misfolded proteins or protein aggregates. Accumulation of misfolded protein aggregates may act as a cue signal for the oligomerization of the autophagic adaptor protein p62, which facilitates recognition of misfolded protein aggregates that are polyubiquitinated with K63 linkages. However, as the autophagic flux is impaired under exposure to As(III), p62 oligomers cannot be cleared by autophagy and accumulate as aggregates with Keap1. This results in the sequestration of Keap1 and the stabilization of Nrf2, which activates the non-canonical Nrf2-Keap1 pathway as an antioxidant response. In this study, we found that polyubiquitination of p62 itself increased upon exposure to As(III) to prevent further oligomerization of p62 and to increase the availability of functional free monomeric p62. We also found that monomeric p62 could also interact with ubiquitinated proteins and that the forced dimerization of p62 was sufficient to increase the interactions with ubiquitinated proteins, probably polyubiquitinated with K63 linkages. Upon exposure to As(III), (1) inability to form oligomeric p62 because of a mutation in the PB1 dimerization domain, or (2) reduced capability to generate monomeric p62 owing to diminished polyubiquitination of p62 itself, resulted in reduced viability of cells. Therefore, upon exposure to As(III), p62 initially needs to form oligomers to activate an antioxidant response pathway. Subsequently, p62 is polyubiquitinated to prevent further oligomerization and ensure the availability of free p62 monomers. We propose that the polyubiquitination of p62 under exposure to As(III) plays an important role in overcoming the impaired autophagic flux by regulating the oligomerization status of p62.
亚砷酸盐[As(III)]诱导的氧化应激会影响蛋白质折叠，并导致错误折叠蛋白或蛋白质聚集体水平升高。错误折叠蛋白聚集体的积累可能作为自噬衔接蛋白p62寡聚化的信号提示，促进对通过K63连接多聚泛素化的错误折叠蛋白聚集体的识别。然而，由于在As(III)暴露下自噬通量受损，p62寡聚物无法通过自噬清除，并与Keap1一起聚集成聚集体。这导致Keap1被隔离以及Nrf2稳定，从而激活非经典Nrf2-Keap1途径作为抗氧化反应。在本研究中，我们发现暴露于As(III)后，p62自身的多聚泛素化增加，以防止p62进一步寡聚化，并增加功能性游离单体p62的可用性。我们还发现单体p62也可以与泛素化蛋白相互作用，并且p62的强制二聚化足以增加与泛素化蛋白的相互作用，这些泛素化蛋白可能是通过K63连接多聚泛素化的。暴露于As(III)后，(1)由于PB1二聚化结构域突变而无法形成p62寡聚物，或(2)由于p62自身多聚泛素化减少而产生单体p62的能力降低，均导致细胞活力下降。因此，暴露于As(III)后，p62最初需要形成寡聚物以激活抗氧化反应途径。随后，p62被多聚泛素化以防止进一步寡聚化，并确保游离p62单体的可用性。我们提出，暴露于As(III)时p62的多聚泛素化通过调节p62的寡聚化状态，在克服受损的自噬通量中起重要作用。
