RESUMO
Cellular adaptation to stress is essential to ensure organismal survival. NRF2/NFE2L2 is a key determinant of xenobiotic stress responses, and loss of negative regulation by the KEAP1-CUL3 proteasome system is implicated in several chemo- and radiation-resistant cancers. Advantageously using C. elegans alongside human cell culture models, we establish a new WDR23-DDB1-CUL4 regulatory axis for NRF2 activity that operates independently of the canonical KEAP1-CUL3 system. WDR23 binds the DIDLID sequence within the Neh2 domain of NRF2 to regulate its stability; this regulation is not dependent on the KEAP1-binding DLG or ETGE motifs. The C-terminal domain of WDR23 is highly conserved and involved in regulation of NRF2 by the DDB1-CUL4 complex. The addition of WDR23 increases cellular sensitivity to cytotoxic chemotherapeutic drugs and suppresses NRF2 in KEAP1-negative cancer cell lines. Together, our results identify WDR23 as an alternative regulator of NRF2 proteostasis and uncover a cellular pathway that regulates NRF2 activity and capacity for cytoprotection independently of KEAP1.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Fator 2 Relacionado a NF-E2/genética , Neoplasias/genética , Proteínas Repressoras/genética , Motivos de Aminoácidos/genética , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Linhagem Celular Tumoral , Proteínas Culina/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Neoplasias/patologia , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica/genética , Proteínas Repressoras/metabolismo , Transdução de SinaisRESUMO
Maintaining a healthy cellular environment requires the constant control of proteostasis. E3 ubiquitin ligase complexes facilitate the post-translational addition of ubiquitin, which based on the quantity and specific lysine linkages, results in different outcomes. Our studies reveal the CUL4-DDB1 substrate receptor, WDR23, as both a positive and a negative regulator in cellular stress responses. These opposing roles are mediated by two distinct isoforms: WDR-23A in the cytoplasm and WDR-23B in the nucleus. C. elegans expressing only WDR-23A display activation of SKN-1 and enhanced survival to oxidative stress, whereas animals with restricted WDR-23B expression do not. Additionally, we identify GEN-1, a Holliday junction resolvase, as an evolutionarily conserved WDR-23 substrate and find that the nuclear and cytoplasmic isoforms of WDR-23 differentially affect double-strand break repair. Our results suggest that through differential ubiquitination, nuclear WDR-23B inhibits the activity of substrates, most likely by promoting protein turnover, while cytoplasmic WDR-23A performs a proteasome-independent role. Together, our results establish a cooperative role between two spatially distinct isoforms of WDR-23 in ensuring proper regulation of WDR-23 substrates.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Núcleo Celular/genética , Proteínas de Ligação a DNA/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genética , Animais , Caenorhabditis elegans/genética , Citoplasma/genética , Regulação da Expressão Gênica/genética , Estresse Oxidativo/genética , Proteólise , Proteostase/genética , Especificidade por Substrato , Ubiquitinação/genéticaRESUMO
Mechanisms that coordinate different metabolic pathways, such as glucose and lipid, have been recognized. However, a potential interaction between amino acid and lipid metabolism remains largely elusive. Here we show that during starvation of Caenorhabditis elegans, proline catabolism is coupled with lipid metabolism by SKN-1. Mutation of alh-6, a conserved proline catabolic enzyme, accelerates fat mobilization, enhances the expression of genes involved in fatty acid oxidation and reduces survival in response to fasting. This metabolic coordination is mediated by the activation of the transcription factor SKN-1/Nrf2, possibly due to the accumulation of the alh-6 substrate P5C, and also requires the transcriptional co-regulator MDT-15. Constitutive activation of SKN-1 induces a similar transcriptional response, which protects animals from fat accumulation when fed a high carbohydrate diet. In human cells, an orthologous alh-6 enzyme, ALDH4A1, is also linked to the activity of Nrf2, the human orthologue of SKN-1, and regulates the expression of lipid metabolic genes. Our findings identify a link between proline catabolism and lipid metabolism, and uncover a physiological role for SKN-1 in metabolism.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ligação a DNA/metabolismo , Metabolismo dos Lipídeos , Fator 2 Relacionado a NF-E2/metabolismo , Fatores de Transcrição/metabolismo , 1-Pirrolina-5-Carboxilato Desidrogenase/metabolismo , Animais , Caenorhabditis elegans , Ácidos Graxos/química , Alimentos , Células HEK293 , Humanos , Metabolismo , Estresse Oxidativo , Oxigênio/química , Prolina/genética , Prolina/metabolismo , Interferência de RNARESUMO
SKN-1/Nrf plays multiple essential roles in development and cellular homeostasis. We demonstrate that SKN-1 executes a specific and appropriate transcriptional response to changes in available nutrients, leading to metabolic adaptation. We isolated gain-of-function (gf) alleles of skn-1, affecting a domain of SKN-1 that binds the transcription factor MXL-3 and the mitochondrial outer membrane protein PGAM-5. These skn-1(gf) mutants perceive a state of starvation even in the presence of plentiful food. The aberrant monitoring of cellular nutritional status leads to an altered survival response in which skn-1(gf) mutants transcriptionally activate genes associated with metabolism, adaptation to starvation, aging, and survival. The triggered starvation response is conserved in mice with constitutively activated Nrf and may contribute to the tumorgenicity associated with activating Nrf mutations in mammalian somatic cells. Our findings delineate an evolutionarily conserved metabolic axis of SKN-1/Nrf, further establishing the complexity of this pathway.