luciferase assay for the detection of autophagy protease atg4b

Análisis de cribado de alto rendimiento para el inhibidor de la autofagia de la peptidasa de cisteína 4B

Autophagy is a conserved metabolic process that allows cells to keep intracellular homeostasis and respond to stress by degrading aged, defective, or unnecessary cellular contents via the lysosomes1,2,3. Under some pathophysiologic conditions, this process acts as a crucial cellular response to nutrient and oxygen deprivation, resulting in recycled nutrients and lipids, allowing the cells to adapt to their metabolic needs2,3,4. Autophagy has also been identified as a cellular stress response related to several diseases, such as neurodegenerative disorders, pathogen infection, and various types of cancer. The function of autophagy in cancer is complex and dependent on the type, stage, and status of the tumor. It can suppress tumorigenesis through autophagic degradation of damaged cells, but can also promote the survival of advanced tumors by improving cell survival during stressful conditions, such as hypoxia, nutrient deprivation, and cytotoxic damage2,4,5,6.
Several studies have shown that autophagy inhibition provides a benefit as an anticancer strategy. Thus, the inhibition of critical steps, such as autophagosome formation or its fusion with the lysosome, could be an effective method for cancer control2,4,5,6. Growing evidence has shown that ATG4B is involved in certain pathological conditions, and it has gained attention as a potential anticancer target2,3,4. For instance, it was observed that colorectal cancer cells and human epidermal growth factor receptor 2 (HER2)-positive breast cancer cells had significantly higher ATG4B expression levels than adjacent normal cells2,4. In prostate cancer cells, inhibition of ATG4B resulted in a cell line-specific susceptibility to chemotherapy and radiotherapy7. Recently, strong evidence has emerged that pancreatic ductal adenocarcinoma (PDAC) is particularly vulnerable to ATG4B inhibition. For instance, in a genetically engineered mouse model, it was shown that intermittent loss of ATG4B function reduces PDAC tumor growth and increases survival3,4. Overall, ATG4B is highly overexpressed in some cancer types, is related to the progression of tumor, and is linked to cancer therapy resistance2,4,8.
The ATG4 cysteine proteases in mammals have four family members, ATG4A-ATG4D. These proteins exhibit some target selectivity toward the LC3/GABARAP (ATG8) family of proteins9,10,11 and may have additional functions not linked to their protease activity12,13. Furthermore, ATG4 functions in regulating a novel type of post-translational modification, the ATG8-ylation of proteins11,12. While ATG4B and its main substrate LC3B are the most widely studied, a picture is emerging that suggests a complex role for each subfamily member in the regulation of autophagic and non-autophagic processes. This is further corroborated by a complex network of post-translational modifications that regulate ATG4B activity via phosphorylation, acetylation, glycosylation, and nitrosylation9,10,11,12,13.
Several known ATG4B inhibitors have been published2,4,14,15. While these are suitable as research tools, their pharmacodynamic profile, selectivity, or potency have yet precluded them from development as preclinical candidates4,16. Overall, there is an urgent need to identify more potent and selective compounds. Often, the compounds are good biochemical inhibitors of protein function, yet their efficacy in cell-based assays is poor. There are multiple assays to monitor ATG4B activity, including biochemical methods and cell-based assays4. We have previously developed a simple, luminescence-based, high-throughput assay for monitoring ATG4B activity in cells8,17. This assay utilizes a luciferase protein from Gaussia princeps (GLUC) that is stable and active in the extracellular milieu and can be inducibly released from cells in response to ATG4B proteolytic activity18,19.
In this reporter construct, dNGLUC is linked to the actin cytoskeleton of cells. A protease-specific linker can be introduced between the &#946;-actin anchor and dNGLUC, turning the secretion dependent on cleavage of the linker. We used the full-length open reading frame of LC3B between &#946;-actin and dNGLUC, to be able to monitor LC3B cleavage17,18,19. Although the secretion mechanism of dNGLUC is poorly understood, it is specific for monitoring ATG4B activity, does not depend on overall autophagy as it occurs in ATG5 knockout cells, and is mediated by non-conventional mechanisms that do not require a classical signal peptide4,18,19. We have successfully used this reporter to screen small molecules and siRNA libraries, and have identified novel regulators of ATG4B activity, such as the Akt protein kinases8. This paper describes a detailed protocol for the use of this luciferase reporter in a semi-automated, high-throughput screening format.

Autor destacado: Un ensayo selectivo basado en luciferasa para monitorear la actividad de ATG4B 27 en células 

Detección de fármacos basados en células para inhibidores de la 4B cisteína peptidasa relacionada con la autofagia

Watch this Scientific Journal Video about Luciferase Assay for the Detection of Autophagy Protease ATG4B at JoVE.com

Luciferase Assay for the Detection of Autophagy Protease ATG4B  

Ensayo de luciferasa para la detección de la proteasa de autofagia ATG4B

Comience abriendo el programa e inicialice la bomba inyectora haciendo clic en el Control del dispensador, seleccione Bomba 2 y luego haga clic en Iniciar. Para enjuagar el tubo con agua ionizada, haga clic en Enjuagar y, a continuación, pulse Siguiente. Seleccione la bomba 2 y ajuste el soporte de la punta a la punta deseada.A continuación, vuelva a hacer clic en Siguiente. Haga clic en Iniciar para comenzar el enjuague de la tubería. Repita el enjuague con metanol.Mientras tanto, prepare una solución de sustrato de trabajo diluyendo el material de sustrato de 1 a 100. Finalmente, enjuague el tubo con la dilución de trabajo del sustrato en PBS. Cargue la placa en el lector de placas.Y luego, en Configuración de medición, elija la apertura a la apertura de luminiscencia de 384 placas de EE. UU., seguida de la distancia entre la placa y el detector a 0,6 milímetros y el tiempo de medición a 0,5 segundos. Una vez que se hayan establecido los ajustes de medición, en la pestaña Configuración general de protocolo, seleccione el tipo de placa como 384 Nuc y elija la opción Por columnas bidireccionales como modo de medición. Introduzca 1 en la pestaña número de repeticiones de ensayo.Ahora, en la pestaña Dispensar, seleccione la medida de dispensación deseada. A continuación, ajuste el tiempo de medición para cada pozo a un segundo y seleccione la bomba usada deseada. Velocidad de dispensación de 200 microlitros por segundo.Y el volumen de dispensación a 50 microlitros. Elija Volumen de dispensación como el volumen de llenado de la jeringa para la bomba 2. Y luego establezca el Iniciar dispensación en el número de medición en 1.En la pestaña Selección de pocillos, utilice el menú desplegable Agregar o quitar muestras, elija el tipo de muestra deseado y seleccione los pocillos que se van a medir. Después de eso, inicie la medición haciendo clic en Ejecutar. La señal de luminiscencia basal del DMSO se observa en la columna 1, y la disminución de la señal de luminiscencia en presencia del inhibidor de ATG4B, FMK-9a, se observa en la columna 24.

luciferase-assay-for-the-detection-of-autophagy-protease-atg4b

Research

JoVE Journal

Biology

Luciferase Assay for the Detection of Autophagy Protease ATG4B

76 vistas. Comience abriendo el programa e inicialice la bomba inyectora haciendo clic en el Control del dispensador, seleccione Bomba 2 y luego haga clic en Iniciar. Para enjuagar el tubo con agua ionizada, haga clic en Enjuagar y, a continuación, pulse Siguiente. Seleccione la bomba 2 y ajuste el soporte de la punta a la punta deseada.A continuación, vuelva a hacer clic en Siguiente. Haga clic en Iniciar para comenzar el enjuague de la tubería. Repita el enjuague con metanol.Mientras ...

Video: Luciferase Assay for the Detection of Autophagy Protease ATG4B  

Cell-Based Drug Screening for Inhibitors of Autophagy Related 4B Cysteine Peptidase

Growing evidence has shown that high autophagic flux is related to tumor progression and cancer therapy resistance. Assaying individual autophagy proteins is a prerequisite for therapeutic strategies targeting this pathway. Inhibition of the autophagy protease ATG4B has been shown to increase overall survival, suggesting that ATG4B could be a potential drug target for cancer therapy. Our laboratory has developed a selective luciferase-based assay for monitoring ATG4B activity in cells. For this assay, the substrate of ATG4B, LC3B, is tagged at the C-terminus with a secretable luciferase from the marine copepod Gaussia princeps (GLUC). This reporter is linked to the actin cytoskeleton, thus keeping it in the cytoplasm of cells when uncleaved. ATG4B-mediated cleavage results in the release of GLUC by non-conventional secretion, which then can be monitored by harvesting supernatants from cell culture as a correlate of cellular ATG4B activity. This paper presents the adaptation of this luciferase-based assay to automated high-throughput screening. We describe the workflow and optimization for exemplary high-throughput analysis of cellular ATG4B activity.

Here, we describe a detailed protocol for the use of a luciferase-based reporter assay in a semi-automated, high-throughput screening format.

Growing evidence has shown that high autophagic flux is related to tumor progression and cancer therapy resistance. Assaying individual autophagy proteins ...