p97-dependent retrotranslocation and proteolytic processing govern formation of active Nrf1 upon proteasome inhibition.
p97-dependent retrotranslocation and proteolytic processing govern formation of active Nrf1 upon proteasome inhibition.
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作者：
Radhakrishnan SK;den Besten W;Deshaies RJ
蛋白酶体抑制引起进化上保守的应答，其中蛋白酶体亚基mRNA被上调，导致恢复（即，“反弹”）。我们以前证明，转录因子Nrf 1/NFE 2L 1介导的哺乳动物细胞中的这种稳态反应。我们在这里表明，Nrf 1最初易位到内腔的ER，但迅速和有效的retrotranslocated的方式，依赖于p97/VCP的细胞质侧的膜。正常情况下，逆转位的Nrf 1迅速降解的蛋白酶体和活性物质不积累。然而，在具有受损蛋白酶体的细胞中，逆易位的Nrf 1逃避降解，并被切割成Leu-104的N-末端，以产生不再束缚于ER膜的片段。重要的是，这种切割事件是必不可少的蛋白酶体抑制后的蛋白酶体基因表达的Nrf 1依赖性激活。我们的数据揭示了一个意想不到的作用p97在激活转录因子的重新定位它从内质网腔的胞质溶胶。http://dx.doi.org/10.7554/eLife.01856.001细胞暴露在高温、感染和其他形式的压力下，经常会产生氧离子和过氧化物分子，这些分子会对蛋白质和DNA造成损害。因此，细胞依靠称为蛋白酶体的分子机器来消除受损的蛋白质，以免它们造成太大的伤害。两种相关的转录因子--与DNA相互作用以“打开”基因表达的蛋白质--参与细胞对压力的反应，但方式不同。Nrf 2开启限制氧离子和过氧化物分子造成的损伤的基因，而Nrf 1开启编码蛋白酶体组分的基因。因此，Nrf 1有助于重新启动蛋白酶体活动，如果它已经关闭-一种被称为“反弹”的现象。在细胞内，已知Nrf 1开始嵌入称为内质网的结构的膜内。然而，目前尚不清楚激活的Nrf 1如何离开这层膜并进入细胞核与细胞的DNA相互作用。现在，Radhakrishnan等人表明，当Nrf 1产生时，它的大部分长度都在内质网内，只有一小部分锚定在周围的膜上。这与先前描述的与内质网相关的转录因子不同，这些转录因子粘附在内质网结构的外部。Radhakrishnan等人还发现Nrf 1的激活依赖于一种称为p97或VCP的酶。这种酶有助于将Nrf 1从内质网内部翻转到其外表面。在大多数细胞中，蛋白酶体然后分解Nrf 1的这一部分。然而，如果蛋白酶体受到抑制，一种未知的酶会将Nrf 1从内质网中切割出来，使其迁移到细胞核，并促进更多蛋白酶体组分的产生，以抵消抑制作用。有趣的是，抑制蛋白酶体的药物被用来对抗癌症，因为受损蛋白质的积累对癌细胞是有毒的。通过显示p97促进蛋白酶体的“反弹”，Radhakrishnan等人的工作表明，将现有的蛋白酶体抑制剂与抑制p97的药物相结合，最终可能导致癌症或其他疾病的新的，更有效的治疗方法。DOI：http://dx.doi.org/10.7554/eLife.01856.002网站
Proteasome inhibition elicits an evolutionarily conserved response wherein proteasome subunit mRNAs are upregulated, resulting in recovery (i.e., ‘bounce-back’) of proteasome activity. We previously demonstrated that the transcription factor Nrf1/NFE2L1 mediates this homeostatic response in mammalian cells. We show here that Nrf1 is initially translocated into the lumen of the ER, but is rapidly and efficiently retrotranslocated to the cytosolic side of the membrane in a manner that depends on p97/VCP. Normally, retrotranslocated Nrf1 is degraded promptly by the proteasome and active species do not accumulate. However, in cells with compromised proteasomes, retrotranslocated Nrf1 escapes degradation and is cleaved N-terminal to Leu-104 to yield a fragment that is no longer tethered to the ER membrane. Importantly, this cleavage event is essential for Nrf1-dependent activation of proteasome gene expression upon proteasome inhibition. Our data uncover an unexpected role for p97 in activation of a transcription factor by relocalizing it from the ER lumen to the cytosol. DOI: http://dx.doi.org/10.7554/eLife.01856.001 Cells exposed to high temperatures, infections and other forms of stress often produce oxygen ions and peroxide molecules that can cause damage to proteins and DNA. Cells therefore rely on molecular machines called proteasomes to eliminate damaged proteins, before they cause too much harm. Two related transcription factors—proteins that interact with DNA to ‘switch on’ the expression of genes—are involved in a cell’s responses to stress, but in different ways. Nrf2 switches on genes that limit the damage caused by oxygen ions and peroxide molecules, while Nrf1 switches on the genes that encode the components of the proteasome. As such, Nrf1 helps to restart proteasome activity if it has been shut off—a phenomenon known as ‘bounce-back’. Within a cell, Nrf1 is known to start off embedded within the membranes of a structure called the endoplasmic reticulum. However, it is not clear how activated Nrf1 leaves this membrane and enters the nucleus to interact with the cell’s DNA. Now, Radhakrishnan et al. show that when Nrf1 is produced, most of its length is found inside the endoplasmic reticulum, with only a small piece being anchored in the surrounding membrane. This is unlike previously described transcription factors that associate with the endoplasmic reticulum, which are stuck to the outside of this structure. Radhakrishnan et al. also discovered that the activation of Nrf1 depends on an enzyme called p97 or VCP. This enzyme helps to flip Nrf1 from the inside of the endoplasmic reticulum to its outside surface. In most cells, the proteasome then breaks down this part of Nrf1. However, if the proteasome is inhibited, an unknown enzyme cuts Nrf1 free from the endoplasmic reticulum, allowing it to migrate to the nucleus and promote the production of more proteasome components to counteract the inhibition. Interestingly, drugs that inhibit the proteasome are used to combat cancer because the build-up of damaged proteins is toxic to the cancer cells. By showing that p97 promotes the ‘bounce-back’ of the proteasome, the work of Radhakrishnan et al. suggests that combining existing proteasome inhibitors with drugs that inhibit p97 could eventually lead to new, more effective, therapies for cancer or other diseases. DOI: http://dx.doi.org/10.7554/eLife.01856.002