A Noncanonical Mechanism of Nrf2 Activation by Autophagy Deficiency: Direct Interaction between Keap1 and p62

Abstract

In response to stress, cells can utilize several cellular processes, such as autophagy, which is a bulklysosomal degradation pathway, to mitigate damages and increase the chances of cell survival. Deregulation of autophagy causes upregulation of p62 and the formation of p62-containing aggregates, which are associated with neurodegenerative diseases and cancer. The Nrf2-Keap1 pathway functions as a critical regulator of the cell's defense mechanism against oxidative stress by controlling the expression of many cellular protective proteins. Under basal conditions, Nrf2 is ubiquitinated by the Keap1-Cul3-E3 ubiquitin ligase complex and targeted to the 26S proteasome for degradation. Upon induction, the activity of the E3 ubiquitin ligase is inhibited through the modification of cysteine residues in Keap1, resulting in the stabilization and activation of Nrf2. In this current study, we identified the direct interaction between p62 and Keap1 and the residues required for the interaction have been mapped to 349-DPSTGE-354 in p62 and three arginines in the Kelch domain of Keap1. Accumulation of endogenous p62 or ectopic expression of p62 sequesters Keap1 into aggregates, resulting in the inhibition of Keap1-mediated Nrf2 ubiquitination and its subsequent degradation by the proteasome. In contrast, overexpression of mutated p62, which loses its ability to interact with Keap1, had no effect on Nrf2 stability, demonstrating that p62-mediated Nrf2 upregulation is Keap1 dependent. These findings demonstrate that autophagy deficiency activates the Nrf2 pathway in a noncanonical cysteine-independent mechanism.

The Keap1–Nrf2 pathway: promising therapeutic target to counteract ROS-mediated damage in cancers and neurodegenerative diseases

“…Another protein, p62 (sequestosome 1 protein), which modulates the activity of Nrf2, is a scaffold protein that binds to the polyubiquitinated proteins and targets aggregated proteins and damaged organelles for degradation. p62 directly interacts with the kelch domain of Keap1 via its STGE motif that is similar to the Nrf2 ETGE motif, thereby disrupting the Keap1-Nrf2 complex (Komatsu et al 2010;Lau et al 2010). It causes a decrease in the ubiquitination of Nrf2, an increase in Nrf2 stability and, ultimately, leads to the enhanced expression of ARE-bearing genes.…”

p62/SQSTM1 at the interface of aging, autophagy, and disease

“…Finally, p62/SQSTM1 binds to Kelch-like ECHassociated protein 1 (Keap1) through a Keap1 interacting region (KIR) (aa 346-355) (Jain et al 2010;Komatsu et al 2010;Lau et al 2010). The KIR binds to Keap1 on a site essential for Keap1 to interact with and repress nuclear factor erythroid 2-like 2 (NFE2L2)'s activation, albeit with much lower affinity (Komatsu et al 2010).…”

“…NFE2L2 can also be activated by p62/SQSTM1 in conditions where oxidative stress is not present: p62/ SQSTM1 binds to Keap1 with the KIR on the same sequence that binds to the ETGE domain of NFE2L2 (Jain et al 2010;Komatsu et al 2010;Lau et al 2010;Copple et al 2010). The KIR has a much weaker affinity for Keap1 than NFE2L2 (Komatsu et al 2010), hence it has been proposed that p62/SQSTM1 can activate the NFE2L2 pathway only in pathological conditions, for example, when autophagy is impaired.…”

“…Alternatively, posttranslational modifications of either Keap1 or p62/SQSTM1 itself may affect binding between the two proteins and provide a physiological mechanism for this alternative NFE2L2 activation. p62/SQSTM1 brings Keap1 into sequestosomes, effectively impairing its ability to bind NFE2L2 (Lau et al 2010), and Keap1 presence into sequestosomes seems to facilitate their clearance through autophagy (Fan et al 2010), which would further reduce Keap1 levels and its ability to bind NFE2L2. p62/SQSTM1 relevance in aging and age-related pathologies p62/SQSTM1 is involved in a variety of cellular processes that are relevant for aging, including protein degradation (Pankiv et al 2007;Seibenhener et al 2004), mitophagy (Narendra et al 2010a;Geisler et al 2010a;Tang et al 2011), oxidative stress response (Jain et al 2010;Komatsu et al 2010;Lau et al 2010), metabolism regulation (Moscat and Diaz-Meco 2011), and inflammation (Lee et al 2012).…”

“…p62/SQSTM1 brings Keap1 into sequestosomes, effectively impairing its ability to bind NFE2L2 (Lau et al 2010), and Keap1 presence into sequestosomes seems to facilitate their clearance through autophagy (Fan et al 2010), which would further reduce Keap1 levels and its ability to bind NFE2L2. p62/SQSTM1 relevance in aging and age-related pathologies p62/SQSTM1 is involved in a variety of cellular processes that are relevant for aging, including protein degradation (Pankiv et al 2007;Seibenhener et al 2004), mitophagy (Narendra et al 2010a;Geisler et al 2010a;Tang et al 2011), oxidative stress response (Jain et al 2010;Komatsu et al 2010;Lau et al 2010), metabolism regulation (Moscat and Diaz-Meco 2011), and inflammation (Lee et al 2012). The role of p62/ SQSTM1 in aging and age-related diseases is not fully understood yet, although experimental evidence suggests it is likely a critical factor: expression of p62/ SQSTM1 declines with age in mice (Kwon et al 2012), loss of p62/SQSTM1 reduces lifespan and shows several age-related effects in animal models, and reduced p62/SQSTM1 correlates with age-related pathologies in human tissues.…”