Name: Image Acquisition and Analysis for the Detection of Autophagy Protease ATG4B Using Luciferase Assay
Uploaded: 2023-06-30T12:10:00
Description: Watch this Scientific Journal Video about Image Acquisition and Analysis for the Detection of Autophagy Protease ATG4B Using Luciferase Assay at JoVE.com

image acquisition and analysis for the detection of autophagy protease atg4b using luciferase assay

4B सिस्टीन पेप्टिडेज़ ऑटोफैगी अवरोधक के लिए उच्च-थ्रूपुट स्क्रीनिंग विश्लेषण

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.

लेखक स्पॉटलाइट: कोशिकाओं में ATG4B 27 गतिविधि की निगरानी के लिए एक चयनात्मक लूसिफ़ेरेज़-आधारित परख 

ऑटोफैगी से संबंधित 4 बी सिस्टीन पेप्टिडेस के अवरोधकों के लिए सेल-आधारित दवा स्क्रीनिंग

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 Image Acquisition and Analysis for the Detection of Autophagy Protease ATG4B Using Luciferase Assay at JoVE.com

Image Acquisition and Analysis for the Detection of Autophagy Protease ATG4B Using Luciferase Assay  

ल्यूसिफेरस परख का उपयोग करके ऑटोफैगी प्रोटीज एटीजी 4 बी का पता लगाने के लिए छवि अधिग्रहण और विश्लेषण

माइक्रोस्कोप ऑपरेटिंग सॉफ्टवेयर लॉन्च करके शुरू करें। सेटअप टैब के अंतर्गत, सही पूर्वनिर्धारित प्लेट प्रकार चुनें. प्लेट को स्थिति में रखने के लिए प्लेट चरण तक पहुंचने के लिए इजेक्ट दबाएं और फिर प्लेट को माइक्रोस्कोप में लोड करने के लिए लोड दबाएं।इसके बाद, 0.4 के संख्यात्मक अपर्चर के साथ 20 गुना एयर ऑब्जेक्टिव चुनें। चुने हुए प्लेट प्रकार की प्लेट मोटाई के लिए 20x वायु उद्देश्य के सुधार कॉलर को समायोजित करें। Hoechst 33342 पर चैनल चयन सेट करें, और Hoechst चैनल के लिए इमेजिंग पैरामीटर समायोजित करें।प्लेट के भीतर एक प्रतिनिधि का चयन करके चुनी गई सेटिंग्स का परीक्षण करें। निर्धारित लेआउट पर, सभी प्लेट कुओं और फ़ील्ड्स का चयन करें. फिर ऑनलाइन विश्लेषण सॉफ्टवेयर में डेटा स्थानांतरित करने के लिए ऑनलाइन नौकरियों के तहत संबंधित फ़ोल्डर चुनें।प्रयोग सेटिंग्स सहेजी जाती हैं. रन प्रयोग टैब के तहत, जिस प्लेट को चित्रित किया जाने वाला है, उसका नाम रखा गया है, और इमेजिंग प्रक्रिया शुरू की गई है। छवि विश्लेषण शुरू करने के लिए, पहले सॉफ्टवेयर लॉन्च करें, विश्लेषण के लिए छवियों का चयन करें, और फिर छवि विभाजन शुरू करने के लिए छवि विश्लेषण टैब पर क्लिक करें।इसके बाद, एक नया बिल्डिंग ब्लॉक जोड़ने के लिए इनपुट छवि टैब में प्लस साइन पर क्लिक करें। सूची से, नाभिक खोजें पर क्लिक करें। छवि पर खंडित वस्तुओं का निरीक्षण करने के बाद सबसे उपयुक्त विभाजन विधि चुनें।विभाजन विधि के भीतर पहचान पैरामीटर समायोजित करें। परिणाम परिभाषित करने पर क्लिक करें और विधि के रूप में मानक आउटपुट चुनें। विश्लेषण पाइपलाइन को सहेजने के लिए डेटाबेस में विश्लेषण सहेजें पर क्लिक करें।बैच विश्लेषण टैब के तहत, सहेजी गई पाइपलाइन पर क्लिक करें जो विधि विकल्प के तहत दिखाई देगा। फिर विश्लेषण किए जाने वाले डेटा का चयन करें। सेल नंबरों के लिए डेटासेट उत्पन्न करने के लिए प्रारंभ विश्लेषण पर क्लिक करके विश्लेषण शुरू करें।वर्णित सेल-आधारित परख एक रिपोर्टर रीडआउट पर निर्भर करती है जो अप्रत्यक्ष रूप से एटीजी 4 बी गतिविधि में परिवर्तन को दर्शाती है, जिससे एटीजी 4 बी गतिविधि के अवरोधकों और सक्रियकर्ताओं दोनों का पता लगाने में मदद मिलती है। प्रत्येक यौगिक की एटीजी 4 बी गतिविधि का अनुपात इसकी सेल व्यवहार्यता के लिए एटीजी 4 बी अवरोधकों की पहचान के लिए कटऑफ मूल्य के रूप में उपयोग किया जाता है। एक से अधिक अनुपात संभावित एटीजी 4 बी सक्रियकर्ताओं को इंगित करते हैं और एक से कम अनुपात संभावित एटीजी 4 बी अवरोधकों को इंगित करते हैं।

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JoVE Journal

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Image Acquisition and Analysis for the Detection of Autophagy Protease ATG4B Using Luciferase Assay

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 ...