RESUMO
BACKGROUND INFORMATION: Comprehensive libraries of plasmids for SARS-CoV-2 proteins with various tags (e.g., Strep, HA, Turbo) are now available. They enable the identification of numerous potential protein-protein interactions between the SARS-CoV-2 virus and host proteins. RESULTS: We present here a large library of SARS CoV-2 protein constructs fused with green and red fluorescent proteins and their initial characterisation in various human cell lines including lung epithelial cell models (A549, BEAS-2B), as well as in budding yeast. The localisation of a few SARS-CoV-2 proteins matches their proposed interactions with host proteins. These include the localisation of Nsp13 to the centrosome, Orf3a to late endosomes and Orf9b to mitochondria. CONCLUSIONS AND SIGNIFICANCE: This library should facilitate further cellular investigations, notably by imaging techniques.
Assuntos
COVID-19/virologia , Biblioteca de Peptídeos , SARS-CoV-2/metabolismo , Proteínas Virais/metabolismo , Células A549 , Linhagem Celular , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , SARS-CoV-2/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Imagem com Lapso de Tempo , Proteínas Virais/genética , Proteína Vermelha FluorescenteRESUMO
Two-pore (2P)-domain K+ channels have been shown recently to play a critical role in both cell apoptosis and tumorigenesis. The activity of two-pore, (TWIK)-related acid-sensitive-3 (TASK-3) K+ channels, is responsible for K+-dependent apoptosis of cultured cerebellar granule neurons. Neuron death can be prevented by conditions that specifically reduce K+ efflux through the TASK-3 channels. Moreover, genetic transfer of TASK subunits into hippocampal neurons that lack TASK-3, induces apoptosis. These results indicate a direct link between TASK K+ channel activity and the physiological process of programmed cell death. The TASK-3 K+ channel gene has also been shown to be amplified genomically and over-expressed in a significant number of breast tumours. TASK-3 has a potent oncogenic potential that appears to be related directly to its K+ channel function. In the present review, we will examine the pro-apoptotic and oncogenic properties of TASK-3. We will discuss: (1) the molecular and functional properties of the novel family of mammalian 2P domain K+ channels; (2) the role of TASK-3 in cerebellar granule neuron apoptosis and (3) the role of TASK-3 in breast tumorigenesis.
Assuntos
Apoptose/fisiologia , Canais de Potássio de Domínios Poros em Tandem/química , Canais de Potássio de Domínios Poros em Tandem/fisiologia , Animais , Neoplasias da Mama/fisiopatologia , Cerebelo/fisiologia , Humanos , Neurônios/metabolismo , Neurônios/patologiaAssuntos
Anestésicos Gerais/farmacologia , Canais de Potássio de Domínios Poros em Tandem , Canais de Potássio/efeitos dos fármacos , Animais , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Humanos , Técnicas In Vitro , Proteínas do Tecido Nervoso/efeitos dos fármacos , Canais de Potássio/química , Canais de Potássio/metabolismo , Estrutura Terciária de Proteína , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismoRESUMO
Rat mature cerebellar granule, unlike hippocampal neurons, die by apoptosis when cultured in a medium containing a physiological concentration of K+ but survive under high external K+ concentrations. Cell death in physiological K+ parallels the developmental expression of the TASK-1 and TASK-3 subunits that encode the pH-sensitive standing outward K+ current IKso. Genetic transfer of the TASK subunits in hippocampal neurons, lacking IKso, induces cell death, while their genetic inactivation protects cerebellar granule neurons. Neuronal death of cultured rat granule neurons is also prevented by conditions that specifically reduce K+ efflux through the TASK-3 channels such as extracellular acidosis and ruthenium red. TASK leak K+ channels thus play an important role in K+-dependent apoptosis of cerebellar granule neurons in culture.
