RESUMO
ATP2B1 is a known regulator of calcium (Ca2+) cellular export and homeostasis. Diminished levels of intracellular Ca2+ content have been suggested to impair SARS-CoV-2 replication. Here, we demonstrate that a nontoxic caloxin-derivative compound (PI-7) reduces intracellular Ca2+ levels and impairs SARS-CoV-2 infection. Furthermore, a rare homozygous intronic variant of ATP2B1 is shown to be associated with the severity of COVID-19. The mechanism of action during SARS-CoV-2 infection involves the PI3K/Akt signaling pathway activation, inactivation of FOXO3 transcription factor function, and subsequent transcriptional inhibition of the membrane and reticulum Ca2+ pumps ATP2B1 and ATP2A1, respectively. The pharmacological action of compound PI-7 on sustaining both ATP2B1 and ATP2A1 expression reduces the intracellular cytoplasmic Ca2+ pool and thus negatively influences SARS-CoV-2 replication and propagation. As compound PI-7 lacks toxicity in vitro, its prophylactic use as a therapeutic agent against COVID-19 is envisioned here.
Assuntos
COVID-19 , Cálcio , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , SARS-CoV-2 , Transdução de Sinais , Replicação Viral , Humanos , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Replicação Viral/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , COVID-19/virologia , COVID-19/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Cálcio/metabolismo , Animais , Proteína Forkhead Box O3/metabolismo , Proteína Forkhead Box O3/genética , Chlorocebus aethiops , Tratamento Farmacológico da COVID-19 , Células Vero , Feminino , ATPases Transportadoras de Cálcio/metabolismo , ATPases Transportadoras de Cálcio/genética , MasculinoRESUMO
Centrosome amplification results into genetic instability and predisposes cells to neoplastic transformation. Supernumerary centrosomes trigger p53 stabilization dependent on the PIDDosome (a multiprotein complex composed by PIDD1, RAIDD and Caspase-2), whose activation results in cleavage of p53's key inhibitor, MDM2. Here, we demonstrate that PIDD1 is recruited to mature centrosomes by the centriolar distal appendage protein ANKRD26. PIDDosome-dependent Caspase-2 activation requires not only PIDD1 centrosomal localization, but also its autoproteolysis. Following cytokinesis failure, supernumerary centrosomes form clusters, which appear to be necessary for PIDDosome activation. In addition, in the context of DNA damage, activation of the complex results from a p53-dependent elevation of PIDD1 levels independently of centrosome amplification. We propose that PIDDosome activation can in both cases be promoted by an ANKRD26-dependent local increase in PIDD1 concentration close to the centrosome. Collectively, these findings provide a paradigm for how centrosomes can contribute to cell fate determination by igniting a signalling cascade.
Assuntos
Proteína Adaptadora de Sinalização CRADD/metabolismo , Caspase 2/metabolismo , Centrossomo/metabolismo , Cisteína Endopeptidases/metabolismo , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Células A549 , Proteína Adaptadora de Sinalização CRADD/genética , Caspase 2/genética , Diferenciação Celular , Cisteína Endopeptidases/genética , Dano ao DNA , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Transdução de Sinais , Proteína Supressora de Tumor p53/genéticaRESUMO
53BP1 acts at the crossroads between DNA repair and p53-mediated stress response. With its interactors p53 and USP28, it is part of the mitotic surveillance (or mitotic stopwatch) pathway (MSP), a sensor that monitors the duration of cell division, promoting p53-dependent cell cycle arrest when a critical time threshold is surpassed. Here, we show that Polo-like kinase 1 (PLK1) activity is essential for the time-dependent release of 53BP1 from kinetochores. PLK1 inhibition, which leads to 53BP1 persistence at kinetochores, prevents cytosolic 53BP1 association with p53 and results in a blunted MSP. Strikingly, the identification of CENP-F as the kinetochore docking partner of 53BP1 enabled us to show that measurement of mitotic timing by the MSP does not take place at kinetochores, as perturbing CENP-F-53BP1 binding had no measurable impact on the MSP. Taken together, we propose that PLK1 supports the MSP by generating a cytosolic pool of 53BP1 and that an unknown cytosolic mechanism enables the measurement of mitotic duration.
Assuntos
Proteínas de Ciclo Celular , Proteínas Serina-Treonina Quinases , Humanos , Proteínas de Ciclo Celular/metabolismo , Células HeLa , Cinetocoros/metabolismo , Mitose , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Supressora de Tumor p53/genética , Ubiquitina Tiolesterase/genéticaRESUMO
hTERT-RPE1 cells are genetically stable near diploid cells widely used to model cell division, DNA repair, or ciliogenesis in a non-transformed context. However, poor transfectability and limited homology-directed repair capacity hamper their amenability to gene editing. Here, we describe a protocol for rapid and efficient generation of diverse homozygous knockins. In contrast to other approaches, this strategy bypasses the need for molecular cloning. Our approach can also be applied to a variety of cell types including cancer and induced pluripotent stem cells (iPSCs).