luciferase assay for the detection of autophagy protease atg4b

High-Throughput Screening Analysis for 4B Cysteine Peptidase Autophagy Inhibitor

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.

Author Spotlight: A Selective Luciferase-Based Assay for Monitoring ATG4B 27 Activity in Cells 

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

The main aim of research in my lab is to understand the regulation of autophagy and in particular of the ATG4 cysteine protean and use this knowledge to develop small molecule inhibitors that target this pathway in particular for diseases such as pancreatic cancer. We use high content, high throughput screening technologies to identify drug targets as well as drug candidates and we focus on the ATG4 cysteine protease family because we think it's a very good drug target for various forms of cancer. We have been studying the ATG4 family members for quite some time now.We made significant discoveries into dysfunction of this protein family and cells. We've also identified novel post-translational mechanisms of regulation of the ATG4 family members and currently we are developing small molecule inhibitors and also activators of the ATG4 families for diseases such as cancer and neuro degeneration. There are not many assays to monitor ATG4 protease activity in cells.So we were one of the first to develop a luciferase based assay to monitor this activity. And our assay is quite unusual because it uses secreted, let's say phrase, therefore we don't need to license the cells and we can adapt this protocol easily to high throughput screening methods and lab automation. We have focused on the ATG4 B protein protease for quite some time now.We have generated knockout cell lines of the ATG4 family members. We have identified novel post-translational regulation mechanisms of ATG4, and ultimately we have also identified several novel small molecule compounds that could inhibit and even activate the ATG4 B protease. In the future, we'd like to continue our work on the ATG4 family members.We think that's quite a lot that is currently unknown and still remains to be uncovered. For example, how these proteins regulate autophagy in neurons is currently completely not understood. We'd also like to focus our work on validation of these small molecule compounds that we've identified and potentially to use activators in neurodegenerative diseases.

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  

Begin by opening the program and initialize the injector pump by clicking on the Dispenser Control, and select Pump 2, and then clicking on Init. To rinse the tubing with the ionized water, click on Rinse, then press Next. Select Pump 2 and set the tip mount to the desired tip.Then click on Next again. Click Start to begin tube rinsing. Repeat rinsing with methanol.Meanwhile, prepare a working substrate solution by diluting the substrate stock by 1 to 100. Finally, rinse the tubing with the working dilution of the substrate stock in PBS. Load the plate into the plate reader.And then, on Measurement settings, choose the aperture to 384 plate US luminescence aperture, followed by the distance between plate and detector to 0.6 millimeters, and the measurement time as 0.5 seconds. Once the measurement settings have been settled, on the Protocol-General settings tab, select the plate type as 384 Nunc, and choose the By columns bi-directional option as the measurement mode. Input 1 into the number of assay repeats tab.Now, on the Dispense tab, select the desired dispense measurement. Then set the measurement time for each well as one second and select the desired used pump. Dispensing speed of 200 microliters per second.And the dispensing volume to 50 microliters. Choose Dispensing volume as the syringe filling volume for Pump 2. And then set the Start dispensing at measurement number to 1.On the Well selection tab, use the Add/remove samples dropdown menu and choose the desired sample type, and select the wells to be measured. After that, start the measurement by clicking Run. The basal luminescence signal from DMSO is seen in column 1, and the decrease of luminescence signal on the presence of ATG4B inhibitor, FMK-9a, is seen in column 24.

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

Research

JoVE Journal

Biology

75 visualizações.  University College London. Comece abrindo o programa e inicialize a bomba injetora clicando no Controle do Dispensador, selecione Bomba 2 e, em seguida, clique em Iniciar. Para enxaguar a tubulação com a água ionizada, clique em Enxaguar e pressione Avançar. Selecione Bomba 2 e ajuste o suporte da ponta para a ponta desejada.Em seguida, clique em Avançar novamente. Clique em Iniciar para iniciar o enxágue do tubo. Repita o enxágue com metanol.Enquanto isso, prepare uma solução de substrato de tra...