PINK1, Keap1, and Rtnl1 regulate selective clearance of endoplasmic reticulum during development.

Selective clearance of organelles, including endoplasmic reticulum (ER) and mitochondria, by autophagy plays an important role in cell health. Here, we describe a developmentally programmed selective ER clearance by autophagy. We show that Parkinson's disease-associated PINK1, as well as Atl, Rtnl1, and Trp1 receptors, regulate ER clearance by autophagy. The E3 ubiquitin ligase Parkin functions downstream of PINK1 and is required for mitochondrial clearance while having the opposite function in ER clearance. By contrast, Keap1 and the E3 ubiquitin ligase Cullin3 function downstream of PINK1 to regulate ER clearance by influencing Rtnl1 and Atl. PINK1 regulates a change in Keap1 localization and Keap1-dependent ubiquitylation of the ER-phagy receptor Rtnl1 to facilitate ER clearance. Thus, PINK1 regulates the selective clearance of ER and mitochondria by influencing the balance of Keap1- and Parkin-dependent ubiquitylation of substrates that determine which organelle is removed by autophagy.

EMSY inhibits homologous recombination repair and the interferon response, promoting lung cancer immune evasion.

Non-small cell lung cancers (NSCLCs) harboring KEAP1 mutations are often resistant to immunotherapy. Here, we show that KEAP1 targets EMSY for ubiquitin-mediated degradation to regulate homologous recombination repair (HRR) and anti-tumor immunity. Loss of KEAP1 in NSCLC induces stabilization of EMSY, producing a BRCAness phenotype, i.e., HRR defects and sensitivity to PARP inhibitors. Defective HRR contributes to a high tumor mutational burden that, in turn, is expected to prompt an innate immune response. Notably, EMSY accumulation suppresses the type I interferon response and impairs innate immune signaling, fostering cancer immune evasion. Activation of the type I interferon response in the tumor microenvironment using a STING agonist results in the engagement of innate and adaptive immune signaling and impairs the growth of KEAP1-mutant tumors. Our results suggest that targeting PARP and STING pathways, individually or in combination, represents a therapeutic strategy in NSCLC patients harboring alterations in KEAP1.

A Cellular Mechanism to Detect and Alleviate Reductive Stress.

Metazoan organisms rely on conserved stress response pathways to alleviate adverse conditions and preserve cellular integrity. Stress responses are particularly important in stem cells that provide lifetime support for tissue formation and repair, but how these protective systems are integrated into developmental programs is poorly understood. Here we used myoblast differentiation to identify the E3 ligase CUL2FEM1B and its substrate FNIP1 as core components of the reductive stress response. Reductive stress, as caused by prolonged antioxidant signaling or mitochondrial inactivity, reverts the oxidation of invariant Cys residues in FNIP1 and allows CUL2FEM1B to recognize its target. The ensuing proteasomal degradation of FNIP1 restores mitochondrial activity to preserve redox homeostasis and stem cell integrity. The reductive stress response is therefore built around a ubiquitin-dependent rheostat that tunes mitochondrial activity to redox needs and implicates metabolic control in coordination of stress and developmental signaling.

The Oncogenic Action of NRF2 Depends on De-glycation by Fructosamine-3-Kinase.

The NRF2 transcription factor controls a cell stress program that is implicated in cancer and there is great interest in targeting NRF2 for therapy. We show that NRF2 activity depends on Fructosamine-3-kinase (FN3K)-a kinase that triggers protein de-glycation. In its absence, NRF2 is extensively glycated, unstable, and defective at binding to small MAF proteins and transcriptional activation. Moreover, the development of hepatocellular carcinoma triggered by MYC and Keap1 inactivation depends on FN3K in vivo. N-acetyl cysteine treatment partially rescues the effects of FN3K loss on NRF2 driven tumor phenotypes indicating a key role for NRF2-mediated redox balance. Mass spectrometry reveals that other proteins undergo FN3K-sensitive glycation, including translation factors, heat shock proteins, and histones. How glycation affects their functions remains to be defined. In summary, our study reveals a surprising role for the glycation of cellular proteins and implicates FN3K as targetable modulator of NRF2 activity in cancer.

Nrf2 Activation Promotes Lung Cancer Metastasis by Inhibiting the Degradation of Bach1.

Approximately 30% of human lung cancers acquire mutations in either Keap1 or Nfe2l2, resulting in the stabilization of Nrf2, the Nfe2l2 gene product, which controls oxidative homeostasis. Here, we show that heme triggers the degradation of Bach1, a pro-metastatic transcription factor, by promoting its interaction with the ubiquitin ligase Fbxo22. Nrf2 accumulation in lung cancers causes the stabilization of Bach1 by inducing Ho1, the enzyme catabolizing heme. In mouse models of lung cancers, loss of Keap1 or Fbxo22 induces metastasis in a Bach1-dependent manner. Pharmacological inhibition of Ho1 suppresses metastasis in a Fbxo22-dependent manner. Human metastatic lung cancer display high levels of Ho1 and Bach1. Bach1 transcriptional signature is associated with poor survival and metastasis in lung cancer patients. We propose that Nrf2 activates a metastatic program by inhibiting the heme- and Fbxo22-mediated degradation of Bach1, and that Ho1 inhibitors represent an effective therapeutic strategy to prevent lung cancer metastasis.

Proteasomal and Autophagic Degradation Systems.

Autophagy and the ubiquitin-proteasome system are the two major quality control pathways responsible for cellular homeostasis. As such, they provide protection against age-associated changes and a plethora of human diseases. Ubiquitination is utilized as a degradation signal by both systems, albeit in different ways, to mark cargoes for proteasomal and lysosomal degradation. Both systems intersect and communicate at multiple points to coordinate their actions in proteostasis and organelle homeostasis. This review summarizes molecular details of how proteasome and autophagy pathways are functionally interconnected in cells and indicates common principles and nodes of communication that can be therapeutically exploited.

Chemical Proteomics Identifies Druggable Vulnerabilities in a Genetically Defined Cancer.

The transcription factor NRF2 is a master regulator of the cellular antioxidant response, and it is often genetically activated in non-small-cell lung cancers (NSCLCs) by, for instance, mutations in the negative regulator KEAP1. While direct pharmacological inhibition of NRF2 has proven challenging, its aberrant activation rewires biochemical networks in cancer cells that may create special vulnerabilities. Here, we use chemical proteomics to map druggable proteins that are selectively expressed in KEAP1-mutant NSCLC cells. Principal among these is NR0B1, an atypical orphan nuclear receptor that we show engages in a multimeric protein complex to regulate the transcriptional output of KEAP1-mutant NSCLC cells. We further identify small molecules that covalently target a conserved cysteine within the NR0B1 protein interaction domain, and we demonstrate that these compounds disrupt NR0B1 complexes and impair the anchorage-independent growth of KEAP1-mutant cancer cells. Our findings designate NR0B1 as a druggable transcriptional regulator that supports NRF2-dependent lung cancers.

Phosphorylation of phase-separated p62 bodies by ULK1 activates a redox-independent stress response.

NRF2 is a transcription factor responsible for antioxidant stress responses that is usually regulated in a redox-dependent manner. p62 bodies formed by liquid-liquid phase separation contain Ser349-phosphorylated p62, which participates in the redox-independent activation of NRF2. However, the regulatory mechanism and physiological significance of p62 phosphorylation remain unclear. Here, we identify ULK1 as a kinase responsible for the phosphorylation of p62. ULK1 colocalizes with p62 bodies, directly interacting with p62. ULK1-dependent phosphorylation of p62 allows KEAP1 to be retained within p62 bodies, thus activating NRF2. p62S351E/+ mice are phosphomimetic knock-in mice in which Ser351, corresponding to human Ser349, is replaced by Glu. These mice, but not their phosphodefective p62S351A/S351A counterparts, exhibit NRF2 hyperactivation and growth retardation. This retardation is caused by malnutrition and dehydration due to obstruction of the esophagus and forestomach secondary to hyperkeratosis, a phenotype also observed in systemic Keap1-knockout mice. Our results expand our understanding of the physiological importance of the redox-independent NRF2 activation pathway and provide new insights into the role of phase separation in this process.

UBE2M Is a Stress-Inducible Dual E2 for Neddylation and Ubiquitylation that Promotes Targeted Degradation of UBE2F.

UBE2M and UBE2F are two family members of neddylation E2 conjugating enzyme that, together with E3s, activate CRLs (Cullin-RING Ligases) by catalyzing cullin neddylation. However, whether and how two E2s cross-talk with each other are largely unknown. Here, we report that UBE2M is a stress-inducible gene subjected to cis-transactivation by HIF-1 and AP1, and MLN4924, a small molecule inhibitor of E1 NEDD8-activating enzyme (NAE), upregulates UBE2M via blocking degradation of HIF-1α and c-JUN. UBE2M is a dual E2 for targeted ubiquitylation and degradation of UBE2F, acting as a neddylation E2 to activate CUL3-Keap1 E3 under physiological conditions but as a ubiquitylation E2 for Parkin-DJ-1 E3 under stressed conditions. UBE2M-induced UBE2F degradation leads to CRL5 inactivation and subsequent NOXA accumulation to suppress the growth of lung cancer cells. Collectively, our study establishes a negative regulatory axis between two neddylation E2s with UBE2M ubiquitylating UBE2F, and two CRLs with CRL3 inactivating CRL5.

Combinatorial CRISPR-Cas9 Metabolic Screens Reveal Critical Redox Control Points Dependent on the KEAP1-NRF2 Regulatory Axis.

The metabolic pathways fueling tumor growth have been well characterized, but the specific impact of transforming events on network topology and enzyme essentiality remains poorly understood. To this end, we performed combinatorial CRISPR-Cas9 screens on a set of 51 carbohydrate metabolism genes that represent glycolysis and the pentose phosphate pathway (PPP). This high-throughput methodology enabled systems-level interrogation of metabolic gene dispensability, interactions, and compensation across multiple cell types. The metabolic impact of specific combinatorial knockouts was validated using 13C and 2H isotope tracing, and these assays together revealed key nodes controlling redox homeostasis along the KEAP-NRF2 signaling axis. Specifically, targeting KEAP1 in combination with oxidative PPP genes mitigated the deleterious effects of these knockouts on growth rates. These results demonstrate how our integrated framework, combining genetic, transcriptomic, and flux measurements, can improve elucidation of metabolic network alterations and guide precision targeting of metabolic vulnerabilities based on tumor genetics.

CRISPR metabolic screen identifies ATM and KEAP1 as targetable genetic vulnerabilities in solid tumors.

Cancer treatments targeting DNA repair deficiencies often encounter drug resistance, possibly due to alternative metabolic pathways that counteract the most damaging effects. To identify such alternative pathways, we screened for metabolic pathways exhibiting synthetic lethality with inhibition of the DNA damage response kinase Ataxia-telangiectasia-mutated (ATM) using a metabolism-centered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 library. Our data revealed Kelch-like ECH-associated protein 1 (KEAP1) as a key factor involved in desensitizing cancer cells to ATM inhibition both in vitro and in vivo. Cells depleted of KEAP1 exhibited an aberrant overexpression of the cystine transporter SLC7A11, robustly accumulated cystine inducing disulfide stress, and became hypersensitive to ATM inhibition. These hallmarks were reversed in a reducing cellular environment indicating that disulfide stress was a crucial factor. In The Cancer Genome Atlas (TCGA) pan-cancer datasets, we found that ATM levels negatively correlated with KEAP1 levels across multiple solid malignancies. Together, our results unveil ATM and KEAP1 as new targetable vulnerabilities in solid tumors.

The KEAP1-NRF2 pathway regulates TFEB/TFE3-dependent lysosomal biogenesis.

The maintenance of redox and metabolic homeostasis is integral to embryonic development. Nuclear factor erythroid 2-related factor 2 (NRF2) is a stress-induced transcription factor that plays a central role in the regulation of redox balance and cellular metabolism. Under homeostatic conditions, NRF2 is repressed by Kelch-like ECH-associated protein 1 (KEAP1). Here, we demonstrate that Keap1 deficiency induces Nrf2 activation and postdevelopmental lethality. Loss of viability is preceded by severe liver abnormalities characterized by an accumulation of lysosomes. Mechanistically, we demonstrate that loss of Keap1 promotes aberrant activation of transcription factor EB (TFEB)/transcription factor binding to IGHM Enhancer 3 (TFE3)-dependent lysosomal biogenesis. Importantly, we find that NRF2-dependent regulation of lysosomal biogenesis is cell autonomous and evolutionarily conserved. These studies identify a role for the KEAP1-NRF2 pathway in the regulation of lysosomal biogenesis and suggest that maintenance of lysosomal homeostasis is required during embryonic development.

Targeting NRF2 and Its Downstream Processes: Opportunities and Challenges.

The transcription factor NRF2 coordinates the expression of a vast array of cytoprotective and metabolic genes in response to various stress inputs to restore cellular homeostasis. Transient activation of NRF2 in healthy tissues has been long recognized as a cellular defense mechanism and is critical to prevent cancer initiation by carcinogens. However, cancer cells frequently hijack the protective capability of NRF2 to sustain the redox balance and meet their metabolic requirements for proliferation. Further, aberrant activation of NRF2 in cancer cells confers resistance to commonly used chemotherapeutic agents and radiotherapy. During the last decade, many research groups have attempted to block NRF2 activity in tumors to counteract the survival and proliferative advantage of cancer cells and reverse resistance to treatment. In this review, we highlight the role of NRF2 in cancer progression and discuss the past and current approaches to disable NRF2 signaling in tumors.

AMPK activates the Nrf2-Keap1 pathway to govern dendrite pruning via the insulin pathway in Drosophila.

During Drosophila metamorphosis, the ddaC dendritic arborisation sensory neurons selectively prune their larval dendrites in response to steroid hormone ecdysone signalling. The Nrf2-Keap1 pathway acts downstream of ecdysone signalling to promote proteasomal degradation and thereby dendrite pruning. However, how the Nrf2-Keap1 pathway is activated remains largely unclear. Here, we demonstrate that the metabolic regulator AMP-activated protein kinase (AMPK) plays a cell-autonomous role in dendrite pruning. Importantly, AMPK is required for Mical and Headcase expression and for activation of the Nrf2-Keap1 pathway. We reveal that AMPK promotes the Nrf2-Keap1 pathway and dendrite pruning partly via inhibition of the insulin pathway. Moreover, the AMPK-insulin pathway is required for ecdysone signalling to activate the Nrf2-Keap1 pathway during dendrite pruning. Overall, this study reveals an important mechanism whereby ecdysone signalling activates the Nrf2-Keap1 pathway via the AMPK-insulin pathway to promote dendrite pruning, and further suggests that during the nonfeeding prepupal stage metabolic alterations lead to activation of the Nrf2-Keap1 pathway and dendrite pruning.

Hyperoside inhibits EHV-8 infection via alleviating oxidative stress and IFN production through activating JNK/Keap1/Nrf2/HO-1 signaling pathways.

Equine herpesvirus type 8 (EHV-8) causes abortion and respiratory disease in horses and donkeys, leading to serious economic losses in the global equine industry. Currently, there is no effective vaccine or drug against EHV-8 infection, underscoring the need for a novel antiviral drug to prevent EHV-8-induced latent infection and decrease the pathogenicity of this virus. The present study demonstrated that hyperoside can exert antiviral effects against EHV-8 infection in RK-13 (rabbit kidney cells), MDBK (Madin-Darby bovine kidney), and NBL-6 cells (E. Derm cells). Mechanistic investigations revealed that hyperoside induces heme oxygenase-1 expression by activating the c-Jun N-terminal kinase/nuclear factor erythroid-2-related factor 2/Kelch-like ECH-associated protein 1 axis, alleviating oxidative stress and triggering a downstream antiviral interferon response. Accordingly, hyperoside inhibits EHV-8 infection. Meanwhile, hyperoside can also mitigate EHV-8-induced injury in the lungs of infected mice. These results indicate that hyperoside may serve as a novel antiviral agent against EHV-8 infection.IMPORTANCEHyperoside has been reported to suppress viral infections, including herpesvirus, hepatitis B virus, infectious bronchitis virus, and severe acute respiratory syndrome coronavirus 2 infection. However, its mechanism of action against equine herpesvirus type 8 (EHV-8) is currently unknown. Here, we demonstrated that hyperoside significantly inhibits EHV-8 adsorption and internalization in susceptible cells. This process induces HO-1 expression via c-Jun N-terminal kinase/nuclear factor erythroid-2-related factor 2/Kelch-like ECH-associated protein 1 axis activation, alleviating oxidative stress and triggering an antiviral interferon response. These findings indicate that hyperoside could be very effective as a drug against EHV-8.

Activation of the p62-Keap1-Nrf2 pathway improves pulmonary arterial hypertension in MCT-induced rats by inhibiting autophagy.

Autophagy is implicated in the pathogenesis of pulmonary arterial hypertension (PAH). We aimed to investigate whether the p62-Keap1-Nrf2 pathway affects the development of PAH by mediating autophagy. A PAH rat model was established using monocrotaline (MCT). Pulmonary artery smooth muscle cells (PASMCs) were extracted, and the changes in proliferation, migration, autophagy, and oxidative stress were analyzed following overexpression or knockdown of p62. The impact of p62 on the symptoms of PAH rats was assessed by the injection of an adenovirus overexpressing p62. We found that the knockdown of p62 increased the proliferation and migration of PASMCs, elevating the oxidative stress of PASMCs and upregulating gene expression of NADPH oxidases. Co-IP assay results demonstrated that p62 interacted with Keap1. p62 knockdown enhanced Keap1 protein stability and Nrf2 ubiquitination. LC3II/I and ATG5 were expressed more often when p62 was knocked down. Treating with an inhibitor of autophagy reversed the impact of p62 knockdown on PASMCs. Nrf2 inhibitor treatment reduced the expression of Nrf2 and p62, while increasing the expression of Keap1, LC3II/I, and ATG5 in PASMCs. However, overexpressing p62 diminished mRVP, SPAP, and Fulton index in PAH rats and attenuated pulmonary vascular wall thickening. Overexpression of p62 also decreased the expression of Keap1, LC3II/I, and ATG5 and increased the nuclear expression of Nrf2 in PAH rats. Importantly, overexpression of p62 reduced oxidative stress and the NADPH oxidase expression in PAH rats. Overall, activation of the p62-Keap1-Nrf2 positive feedback signaling axis reduces the proliferation and migration of PASMCs and alleviates PAH by inhibiting autophagy and oxidative stress.

Plumbagin ameliorates LPS-induced acute lung injury by regulating PI3K/AKT/mTOR and Keap1-Nrf2/HO-1 signalling pathways.

Acute lung injury (ALI) is a major pathophysiological problem characterized by severe inflammation, resulting in high morbidity and mortality. Plumbagin (PL), a major bioactive constituent extracted from the traditional Chinese herb Plumbago zeylanica, has been shown to possess anti-inflammatory and antioxidant pharmacological activities. However, its protective effect on ALI has not been extensively studied. The objective of this study was to investigate the protective effect of PL against ALI induced by LPS and to elucidate its possible mechanisms both in vivo and in vitro. PL treatment significantly inhibited pathological injury, MPO activity, and the wet/dry ratio in lung tissues, and decreased the levels of inflammatory cells and inflammatory cytokines TNF-α, IL-1ß, IL-6 in BALF induced by LPS. In addition, PL inhibited the activation of the PI3K/AKT/mTOR signalling pathway, increased the activity of antioxidant enzymes CAT, SOD, GSH and activated the Keap1/Nrf2/HO-1 signalling pathway during ALI induced by LPS. To further assess the association between the inhibitory effects of PL on ALI and the PI3K/AKT/mTOR and Keap1/Nrf2/HO-1 signalling, we pretreated RAW264.7 cells with 740Y-P and ML385. The results showed that the activation of PI3K/AKT/mTOR signalling reversed the protective effect of PL on inflammatory response induced by LPS. Moreover, the inhibitory effects of PL on the production of inflammatory cytokines induced by LPS also inhibited by downregulating Keap1/Nrf2/HO-1 signalling. In conclusion, the results indicate that the PL ameliorate LPS-induced ALI by regulating the PI3K/AKT/mTOR and Keap1-Nrf2/HO-1 signalling, which may provide a novel therapeutic perspective for PL in inhibiting ALI.

SUMOylation of the ubiquitin ligase component KEAP1 at K39 upregulates NRF2 and its target function in lung cancer cell proliferation.

Nuclear Factor Erythroid 2-Related Factor 2 (NRF2) is important for the expression of genes associated with oxidative stress. The levels of NRF2 are controlled by Kelch-like ECH-associated protein 1 (KEAP1)-dependent degradation. Although oxidative stress is known to suppress KEAP1 activity to stabilize the levels of NRF2, the mechanism for this control is unclear. Here, we identify that KEAP1 is modified by SUMO1 at the lysine residue position 39 (K39). Arginine replacement of this lysine (K39R) in KEAP1 did not affect its stability, subcellular localization, or dimerization but promoted the formation of the Cullin 3 ubiquitin ligase and increased NRF2 ubiquitination. This was accompanied by decreased NRF2 expression. Gene reporter assays showed that the transcription of antioxidant response elements was heightened in KEAP1-WT cells compared to cells expressing the KEAP1-K39R SUMO1 substrate mutant. Consistent with this, chromatin immunoprecipitation assays revealed higher NRF2 binding to the promoter regions of antioxidant genes in cells expressing the KEAP1-WT compared to the KEAP1-K39R mutant protein in H1299 lung cancer cell. The significance of this suppression of KEAP1 activity by its SUMOylation was tested in a subcutaneous tumor model of H1299 lung cancer cell lines that differentially expressed the WT and K39R KEAP1 constructs. This model showed that mutating the SUMOylation site on KEAP1 altered the production of reactive oxygen species and suppressed tumor growth. Taken together, our study recognizes that NRF2-dependent redox control is regulated by the SUMOylation of KEAP1. These findings identify a potential new therapeutic option to counteract oxidative stress.

Exosomes From Muscle-Derived Stem Cells Repair Peripheral Nerve Injury by Inhibiting Ferroptosis via the Keap1-Nrf2-Ho-1 Axis.

Currently, the clinical outcomes of peripheral nerve injuries are suboptimal, highlighting the urgent need to understand the mechanisms of nerve injury to enhance treatment strategies. Muscle-derived stem cells (MDSCs) are a diverse group of multipotent cells that hold promise for peripheral nerve regeneration due to their strong antioxidant and regenerative properties. Our research has revealed that severe ferroptosis occurs in the sciatic nerve and ipsilateral dorsal root ganglion following sciatic nerve injury. Interestingly, we have observed that MDSC-derived exosomes effectively suppress cell ferroptosis and enhance cell viability in Schwann cells and dorsal root ganglion cells. Treatment with exosomes led to increased expression of BDNF and P62 in Schwann cells, decreased expression of Keap1, Nrf2, and HO-1 in Schwann cells, and upregulated dorsal root ganglion cells. Rats treated with exosomes exhibited improvements in sciatic nerve function, sensitivity to stimuli, and reduced muscle atrophy, indicating a positive impact on post-injury recovery. In conclusion, our findings demonstrate the occurrence of ferroptosis in the sciatic nerve and dorsal root ganglion post-injury, with MDSC exosomes offering a potential therapeutic strategy by inhibiting ferroptosis, activating the Keap1-Nrf2-HO-1 pathway, and optimizing the post-injury repair environment.

Homocysteine-potentiated Kelch-like ECH-associated protein 1 promotes senescence of neuroblastoma 2a cells via inhibiting ubiquitination of ß-catenin.

Elevated serum homocysteine (Hcy) level is a risk factor for Alzheimer's disease (AD) and accelerates cell aging. However, the mechanism by which Hcy induces neuronal senescence remains largely unknown. In this study, we observed that Hcy significantly promoted senescence in neuroblastoma 2a (N2a) cells with elevated ß-catenin and Kelch-like ECH-associated protein 1 (KEAP1) levels. Intriguingly, Hcy promoted the interaction between KEAP1 and the Wilms tumor gene on the X chromosome (WTX) while hampering the ß-catenin-WTX interaction. Mechanistically, Hcy attenuated the methylation level of the KEAP1 promoter CpG island and activated KEAP1 transcription. However, a slow degradation rate rather than transcriptional activation contributed to the high level of ß-catenin. Hcy-upregulated KEAP1 competed with ß-catenin to bind to WTX. Knockdown of both ß-catenin and KEAP1 attenuated Hcy-induced senescence in N2a cells. Our data highlight a crucial role of the KEAP1-ß-catenin pathway in Hcy-induced neuronal-like senescence and uncover a promising target for AD treatment.