Dissection of the role of p62/Sqstm1 in activation of Nrf2 during xenophagy

Several mechanisms are involved in controlling intracellular survival of pathogenic mycobacteria in host macrophages, but how these mechanisms are regulated remains poorly understood. We report a role for Kelch-like ECH-associated protein 1 (Keap1), an oxidative stress sensor, in regulating inflammation induced by infection with Mycobacterium avium in human primary macrophages. By using confocal microscopy, we found that Keap1 associated with mycobacterial phagosomes in a time-dependent manner, whereas siRNA-mediated knockdown of Keap1 increased M. aviuminduced expression of inflammatory cytokines and type I interferons (IFNs). We show evidence of a mechanism whereby Keap1, as part of an E3 ubiquitin ligase complex with Cul3 and Rbx1, facilitates ubiquitination and degradation of IκB kinase (IKK)-β thus terminating IKK activity. Keap1 knockdown led to increased nuclear translocation of transcription factors NF-κB, IFN regulatory factor (IRF) 1, and IRF5 driving the expression of inflammatory cytokines and IFN-β. Furthermore, knockdown of other members of the Cul3 ubiquitin ligase complex also led to increased cytokine expression, further implicating this ligase complex in the regulation of the IKK family. Finally, increased inflammatory responses in Keap1-silenced cells contributed to decreased intracellular growth of M. avium in primary human macrophages that was reconstituted with inhibitors of IKKβ or TANK-binding kinase 1 (TBK1). Taken together, we propose that Keap1 acts as a negative regulator for the control of inflammatory signaling in M. avium-infected human primary macrophages. Although this might be important to avoid sustained or overwhelming inflammation, our data suggest that a negative consequence could be facilitated growth of pathogens like M. avium inside macrophages.Keap1 | human primary macrophages | infection | Mycobacterium avium | inflammation

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“…p62-dependent fashion (34). However, a physiological significance of this association remains to be determined.…”

Section: Discussionmentioning

confidence: 96%

Keap1 regulates inflammatory signaling in Mycobacterium avium -infected human macrophages

Awuh

Haug

Mildenberger

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Several groups, including ours, have reported that p62 competes with NRF2 for binding to KEAP1, resulting in stabilization of NRF2, whereas KEAP1 is sequestered into p62 bodies and subsequently degraded by autophagy (31)(32)(33)(34). It was also recently shown that phosphorylation of the KEAP1-interacting region (KIR) motif of p62 enhanced binding to KEAP1 (35,36). We reported earlier that NRF2 bound to an ARE site in the p62 promoter and induced p62 expression upon oxidative stress (31).…”

mentioning

confidence: 79%

p62/Sequestosome-1, Autophagy-related Gene 8, and Autophagy in Drosophila Are Regulated by Nuclear Factor Erythroid 2-related Factor 2 (NRF2), Independent of Transcription Factor TFEB

Jain

Rusten

Katheder

et al. 2015

Journal of Biological Chemistry

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“…Particularly, phosphorylation of Ser-403, located in its UBA domain, mediates its interaction with Lys-63-polyubiquitinated proteins, inducing their degradation (19,75,76). Furthermore, SQSTM1 phosphorylation on Ser-351 has a crucial role in SQSTM1 interaction with Keap1 (9,17). We determined the level of phosphorylation of both serines in TIVE cells and LTC (Fig.…”

Section: Fig 4 Analysis Of Nrf2 Influence On Host Gene Expression (Amentioning

confidence: 99%

“…SQSTM1 is a key molecule involved in targeting specific proteins for degradation through selective autophagy (14,15). The interaction of SQSTM1 with its targets is highly dependent on the posttranslational modifications of both proteins, notably the phosphorylation of SQSTM1 and the Lys-63 polyubiquitination of the targeted protein (9,(16)(17)(18)(19). To activate Nrf2 signaling, phosphorylated SQSTM1 binds to the Nrf2-binding site of Lys-63-polyubiquitinated Keap1, competitively inhibiting Keap1-Nrf2 interaction and inducing Keap1 degradation (9)(10)(11)(12)(13)20).…”

mentioning

confidence: 99%

Kaposi's Sarcoma-Associated Herpesvirus Induces Nrf2 Activation in Latently Infected Endothelial Cells through SQSTM1 Phosphorylation and Interaction with Polyubiquitinated Keap1

Gjyshi

Flaherty

Veettil

et al. 2015

J Virol

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Nuclear factor erythroid 2-related factor 2 (Nrf2), the cellular master regulator of the antioxidant response, dissociates from its inhibitor Keap1 when activated by stress signals and participates in the pathogenesis of viral infections and tumorigenesis. Early during de novo infection of endothelial cells, KSHV induces Nrf2 through an intricate mechanism involving reactive oxygen species (ROS) and prostaglandin E2 (PGE2). When we investigated the Nrf2 activity during latent KSHV infection, we observed increased nuclear serine-40-phosphorylated Nrf2 in human KS lesions compared to that in healthy tissues. Using KSHV long-term-infected endothelial cells (LTC) as a cellular model for KS, we demonstrated that KSHV infection induces Nrf2 constitutively by extending its half-life, increasing its phosphorylation by protein kinase C (PKC) via the infection-induced cyclooxygenase-2 (COX-2)/PGE2 axis and inducing its nuclear localization. Nrf2 knockdown in LTC decreased expression of antioxidant genes and genes involved in KS pathogenesis such as the NAD(P)H quinone oxidase 1 (NQO1), gamma glutamylcysteine synthase heavy unit (␥GCSH), the cysteine transporter (xCT), interleukin 6 (IL-6), and vascular endothelial growth factor A (VEGF-A) genes. Nrf2 activation was independent of oxidative stress but dependent on the autophagic protein sequestosome-1 (SQSTM1; p62). SQSTM1 levels were elevated in LTC, a consequence of protein accumulation due to decreased autophagy and Nrf2-mediated transcriptional activation. SQSTM1 was phosphorylated on serine-351 and -403, while Keap1 was polyubiquitinated with lysine-63-ubiquitin chains, modifications known to increase their mutual affinity and interaction, leading to Keap1 degradation and Nrf2 activation. The latent KSHV protein Fas-associated death domain-like interleukin-1␤-converting enzyme-inhibitory protein (vFLIP) increased SQSTM1 expression and activated Nrf2. Collectively, these results demonstrate that KSHV induces SQSTM1 to constitutively activate Nrf2, which is involved in the regulation of genes participating in KSHV oncogenesis. IMPORTANCEThe transcription factor Nrf2 is activated by stress signals, including viral infection, and responds by activating the transcription of cytoprotective genes. Recently, Nrf2 has been implicated in oncogenesis and was shown to be activated during de novo KSHV infection of endothelial cells through ROS-dependent pathways. The present study was undertaken to determine the mechanism of Nrf2 activation during prolonged latent infection of endothelial cells, using an endothelial cell line latently infected with KSHV. We show that Nrf2 activation was elevated in KSHV latently infected endothelial cells independently of oxidative stress but dependent on the autophagic protein sequestosome-1 (SQSTM1), which was involved in the degradation of the Nrf2 inhibitor Keap1. Furthermore, our results indicated that the KSHV latent protein vFLIP participates in Nrf2 activation. This study suggests that KSHV hijacks the host's autophagic protein SQS...

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Dissection of the role of p62/Sqstm1 in activation of Nrf2 during xenophagy

Cited by 62 publications

References 23 publications

Keap1 regulates inflammatory signaling in Mycobacterium avium -infected human macrophages

Keap1 regulates inflammatory signaling in Mycobacterium avium -infected human macrophages

p62/Sequestosome-1, Autophagy-related Gene 8, and Autophagy in Drosophila Are Regulated by Nuclear Factor Erythroid 2-related Factor 2 (NRF2), Independent of Transcription Factor TFEB

Kaposi's Sarcoma-Associated Herpesvirus Induces Nrf2 Activation in Latently Infected Endothelial Cells through SQSTM1 Phosphorylation and Interaction with Polyubiquitinated Keap1

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