RESUMO
Metabolic adaptation is essential for cell survival during nutrient deprivation. We report that eukaryotic elongation factor 2 kinase (eEF2K), which is activated by AMP-kinase (AMPK), confers cell survival under acute nutrient depletion by blocking translation elongation. Tumor cells exploit this pathway to adapt to nutrient deprivation by reactivating the AMPK-eEF2K axis. Adaptation of transformed cells to nutrient withdrawal is severely compromised in cells lacking eEF2K. Moreover, eEF2K knockdown restored sensitivity to acute nutrient deprivation in highly resistant human tumor cell lines. In vivo, overexpression of eEF2K rendered murine tumors remarkably resistant to caloric restriction. Expression of eEF2K strongly correlated with overall survival in human medulloblastoma and glioblastoma multiforme. Finally, C. elegans strains deficient in efk-1, the eEF2K ortholog, were severely compromised in their response to nutrient depletion. Our data highlight a conserved role for eEF2K in protecting cells from nutrient deprivation and in conferring tumor cell adaptation to metabolic stress. PAPERCLIP:
Assuntos
Caenorhabditis elegans/metabolismo , Quinase do Fator 2 de Elongação/metabolismo , Neoplasias/fisiopatologia , Elongação Traducional da Cadeia Peptídica , Transdução de Sinais , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Neoplasias Encefálicas/fisiopatologia , Caenorhabditis elegans/genética , Sobrevivência Celular , Transformação Celular Neoplásica , Quinase do Fator 2 de Elongação/genética , Privação de Alimentos , Glioblastoma/fisiopatologia , Células HeLa , Humanos , Camundongos , Camundongos Nus , Células NIH 3T3 , Transplante de Neoplasias , Fator 2 de Elongação de Peptídeos/metabolismo , Transplante HeterólogoRESUMO
Ubiquitylation entails the concerted action of E1, E2, and E3 enzymes. We recently reported that OTUB1, a deubiquitylase, inhibits the DNA damage response independently of its isopeptidase activity. OTUB1 does so by blocking ubiquitin transfer by UBC13, the cognate E2 enzyme for RNF168. OTUB1 also inhibits E2s of the UBE2D and UBE2E families. Here we elucidate the structural mechanism by which OTUB1 binds E2s to inhibit ubiquitin transfer. OTUB1 recognizes ubiquitin-charged E2s through contacts with both donor ubiquitin and the E2 enzyme. Surprisingly, free ubiquitin associates with the canonical distal ubiquitin-binding site on OTUB1 to promote formation of the inhibited E2 complex. Lys48 of donor ubiquitin lies near the OTUB1 catalytic site and the C terminus of free ubiquitin, a configuration that mimics the products of Lys48-linked ubiquitin chain cleavage. OTUB1 therefore co-opts Lys48-linked ubiquitin chain recognition to suppress ubiquitin conjugation and the DNA damage response.
Assuntos
Cisteína Endopeptidases/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Proteínas Ubiquitinadas/metabolismo , Substituição de Aminoácidos , Linhagem Celular , Cristalografia por Raios X , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Enzimas Desubiquitinantes , Humanos , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Organismos Geneticamente Modificados , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitinação , Leveduras/genética , Leveduras/crescimento & desenvolvimentoRESUMO
The passage of genetic information during meiosis requires exceptionally high fidelity to prevent birth defects and infertility. Accurate chromosome segregation during the first meiotic division relies on the formation of crossovers between homologous chromosomes and a series of precisely controlled steps to exchange genetic information. Many studies have hinted at a role for p53 in meiosis, but how it functions in this process is poorly understood. Here, we have identified a cooperative role for the p53-like protein CEP-1 and the meiotic protein HIM-5 in maintaining genome stability in the C. elegans germline. Loss of cep-1 and him-5 results in synthetic lethality that is dependent on the upstream DNA damage checkpoint but independent of the downstream core apoptotic pathway. We show that this synthetic lethality is the result of defective crossover formation due to reduced SPO-11-dependent double-strand breaks. Using cep-1 separation-of-function alleles, we show that cep-1 and him-5 also suppress inappropriate activation of the nonhomologous end joining (NHEJ) pathway. This work reveals an ancestral function for the p53 family in ensuring the fidelity of meiosis and establishes CEP-1 as a critical determinant of repair pathway choice.