RESUMO
Although oxidative phosphorylation is best known for producing ATP, it also yields reactive oxygen species (ROS) as invariant byproducts. Depletion of ROS below their physiological levels, a phenomenon known as reductive stress, impedes cellular signaling and has been linked to cancer, diabetes, and cardiomyopathy. Cells alleviate reductive stress by ubiquitylating and degrading the mitochondrial gatekeeper FNIP1, yet it is unknown how the responsible E3 ligase CUL2FEM1B can bind its target based on redox state and how this is adjusted to changing cellular environments. Here, we show that CUL2FEM1B relies on zinc as a molecular glue to selectively recruit reduced FNIP1 during reductive stress. FNIP1 ubiquitylation is gated by pseudosubstrate inhibitors of the BEX family, which prevent premature FNIP1 degradation to protect cells from unwarranted ROS accumulation. FEM1B gain-of-function mutation and BEX deletion elicit similar developmental syndromes, showing that the zinc-dependent reductive stress response must be tightly regulated to maintain cellular and organismal homeostasis.
Assuntos
Estresse Fisiológico , Aminoácidos/química , Animais , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Feminino , Humanos , Íons , Camundongos , Proteínas Mutantes/metabolismo , Mutação/genética , Ligação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Relação Estrutura-Atividade , Especificidade por Substrato/efeitos dos fármacos , Complexos Ubiquitina-Proteína Ligase/química , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitinação/efeitos dos fármacos , Zinco/farmacologiaRESUMO
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.
Assuntos
Proteínas de Transporte/metabolismo , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Antioxidantes/metabolismo , Proteínas de Transporte/genética , Diferenciação Celular , Células HEK293 , Homeostase , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Mitocôndrias , Desenvolvimento Muscular/fisiologia , Mioblastos/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Synthetic lethality provides an attractive strategy for developing targeted cancer therapies. For example, cancer cells with high levels of microsatellite instability (MSI-H) are dependent on the Werner (WRN) helicase for survival. However, the mechanisms that regulate WRN spatiotemporal dynamics remain poorly understood. Here, we used single-molecule tracking (SMT) in combination with a WRN inhibitor to examine WRN dynamics within the nuclei of living cancer cells. WRN inhibition traps the helicase on chromatin, requiring p97/VCP for extraction and proteasomal degradation in a MSI-H dependent manner. Using a phenotypic screen, we identify the PIAS4-RNF4 axis as the pathway responsible for WRN degradation. Finally, we show that co-inhibition of WRN and SUMOylation has an additive toxic effect in MSI-H cells and confirm the in vivo activity of WRN inhibition using an MSI-H mouse xenograft model. This work elucidates a regulatory mechanism for WRN that may facilitate identification of new therapeutic modalities, and highlights the use of SMT as a tool for drug discovery and mechanism-of-action studies.
Assuntos
Cromatina , Proteínas Inibidoras de STAT Ativados , Proteína com Valosina , Helicase da Síndrome de Werner , Helicase da Síndrome de Werner/metabolismo , Helicase da Síndrome de Werner/genética , Humanos , Animais , Cromatina/metabolismo , Proteína com Valosina/metabolismo , Proteína com Valosina/genética , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteínas Inibidoras de STAT Ativados/genética , Camundongos , Linhagem Celular Tumoral , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Instabilidade de Microssatélites , Proteólise/efeitos dos fármacos , Sumoilação/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Ensaios Antitumorais Modelo de Xenoenxerto , FemininoRESUMO
Cell-cell fusion is a frequent and essential event during development, and its dysregulation causes diseases ranging from infertility to muscle weakness. Fusing cells need to repeatedly remodel their plasma membrane through orchestrated formation and disassembly of actin filaments, but how the dynamic reorganization of the cortical actin cytoskeleton is controlled is still poorly understood. Here, we identified a ubiquitin-dependent toggle switch that establishes reversible actin bundling during mammalian cell fusion. We found that EPS8-IRSp53 complexes stabilize cortical actin bundles at sites of cell contact to promote close membrane alignment. EPS8 monoubiquitylation by CUL3KCTD10 displaces EPS8-IRSp53 from membranes and counteracts actin bundling, a dual activity that restricts actin bundling to allow paired cells to progress with fusion. We conclude that cytoskeletal rearrangements during development are precisely controlled by ubiquitylation, raising the possibility of modulating the efficiency of cell-cell fusion for therapeutic benefit.
Assuntos
Citoesqueleto de Actina/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fusão Celular , Proteínas Culina/metabolismo , Mioblastos/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Ubiquitina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Comunicação Celular , Membrana Celular/metabolismo , Proteínas Culina/genética , Células HEK293 , Humanos , Camundongos , Mioblastos/citologia , Proteínas do Tecido Nervoso/genéticaRESUMO
The lipophilic electron-transport cofactor rhodoquinone (RQ) facilitates anaerobic metabolism in a variety of bacteria and selected eukaryotic organisms in hypoxic environments. We have shown that an intact rquA gene in Rhodospirillum rubrum is required for RQ production and efficient growth of the bacterium under anoxic conditions. While the explicit details of RQ biosynthesis have yet to be fully delineated, ubiquinone (Q) is a required precursor to RQ in R. rubrum, and the RquA gene product is homologous to a class I methyltransferase. In order to identify any additional requirements for RQ biosynthesis or factors influencing RQ production in R. rubrum, we performed transcriptome analysis to identify differentially expressed genes in anoxic, illuminated R. rubrum cultures, compared with those aerobically grown in the dark. To further select target genes, we employed a bioinformatics approach to assess the likelihood that a given differentially expressed gene under anoxic conditions may also have a direct role in RQ production or regulation of its levels in vivo. Having thus compiled a list of candidate genes, nine were chosen for further study by generation of knockout strains. RQ and Q levels were quantified using liquid chromatography-mass spectrometry, and rquA gene expression was measured using the real-time quantitative polymerase chain reaction. In one case, Q and RQ levels were decreased relative to wild type; in another case, the opposite effect was observed. These results comport with the crucial roles of rquA and Q in RQ biosynthesis, and reveal the existence of potential modulators of RQ levels in R. rubrum.
Assuntos
Genes Bacterianos , Rhodospirillum rubrum/genética , Rhodospirillum rubrum/metabolismo , Ubiquinona/análogos & derivados , Sequência de Bases , Vias Biossintéticas/genética , Cromatografia Líquida , DNA Bacteriano/genética , Expressão Gênica , Técnicas de Inativação de Genes , Espectrometria de Massas por Ionização por Electrospray , Ubiquinona/biossíntese , Ubiquinona/genéticaRESUMO
A transcription factor called SALL4 could be the missing link between thalidomide and the limb defects caused by the drug.