Systematic analysis of post-translational regulation of autophagy protease ATG4B

Lead Research Organisation: University College London
Department Name: MRC Cell Biology Unit

Abstract

Autophagy can be regarded as a cellular self-cleaning process through which damaged and potentially toxic components are removed through degradation. This process is involved in the clearance of protein aggregates (as for example in neurodegenerative disorders), damaged organelles (induced by cellular stresses or extracellular toxins) or pathogens (such as bacteria or viruses). In addition, autophagy counteracts the deleterious effects associated with ageing as it helps clear the accumulation of toxic protein products and damaged material that accumulates over time. Accordingly, it is of high importance that this process is tightly controlled. Dysregulation of autophagy is commonly associated with severe diseases that can lead to cancer, Parkinson's disease or Alzheimer's disease, just to name a few.
Autophagy can be induced through multiple mechanisms, including starvation, hypoxia, DNA damage or pathogen infection resulting in the formation of a double-membrane structure, the autophagosome. The autophagosome can remove defective organelles or complexes from cells and in most cases rescues the cell from potential harmful effects of the initial stressor. To understand these complex physiological conditions, it is of high importance to identify the molecular components involved and understand their interactions and regulation.
The autophagy machinery has been well characterized both in human cells and in yeast. There is one main question remaining in the field: that is to characterize the molecular mechanisms by which these proteins interact. Further, there is a huge interest in drug discovery to utilize this knowledge to develop therapeutic strategies for diseases associated with autophagy. Here, we aim to identify the molecular regulation of one of the key enzymes involved in autophagy, ATG4B. In order to identify the regulation of this protein, we will make use of recent technical advances in genomics and proteomics and use a combination of high-throughput screening technologies and proteomic profiling. There will be a huge benefit in identifying specific molecular targets within the autophagy pathway as this will enable more focused drug discovery efforts.
This project will shed light onto the signaling cascades that control an early step in autophagy and will provide to a general understanding of autophagy that will lead to the identification of potential novel therapeutic targets.

Technical Summary

Autophagy is a stress response that results in the activation of a lysosomal degradation pathway. Autophagy is a cellular process essential for homeostasis and has been implicated in the progression of ageing as well as various diseases including neurodegenerative diseases and cancer. Understanding this process at the molecular level is of high importance not only in the context of development of potential therapeutic strategies for disease, but also to understand this fundamental cellular process at the molecular level.
The signaling pathways that initiate autophagy are poorly understood and have centered on the serine/threonine kinase mammalian target of rapamycin (mTOR) as an inhibitor of autophagosome formation. In addition, several signaling molecules have been implicated in mTOR-dependent and mTOR-independent regulation of autophagy, such as phosphatidyl-inositol-3-kinase classes I and III, AKT1, PTEN, beclin-1 and Bcl2.
Mass spectrometry data have clearly indicated that autophagy proteins are controlled by post-translational modifications. However, none of the regulators of these modifications have yet been identified. The proposed project focuses on the identification of the molecular regulation of one specific autophagy gene, the autophagy protease ATG4B, which is the key regulator of LC3 proteolytic processing. We have previously described a cell-based assay system to monitor ATG4B proteolytic activity and used this system to identify kinases that control ATG4B activity in the basal state. Here, we plan to study the specific regulation of ATG4B activity under conditions that induce autophagy, such as oxidative stress, starvation and DNA damage induction using siRNA-based high-throughput screening and mass spetrometry. This study will provide important insights into the regulation of ATG4B and help elucidate signaling cascades that modulate autophagosome formation.

Planned Impact

Autophagy is a fundamental process in cell biology that is tightly connected to multiple cellular pathways such as cellular homeostasis, the metabolic state, energy production, mitogenic signaling, apoptosis and protein trafficking. The immediate goal of this project is to increase human knowledge and understanding about the regulation of this process. The importance of autophagy for cell survival and growth, as well as its involvement in multiple diseases, underscores its relevance for general health and well-being. The main benefectors from this study will be the research community that spans from areas of cell biology and biomedicine to drug discovery.
In the long-term, we aim to establish a relevance of the investigated pathway for biomedical research. Specifically, regulation of autophagy is involved in the development of disease. As such, the identification of the underlying pathways will help to determine potential disease marker and drug targets. Further, this information will help to identify strategies for the development of disease therapeutics. Accordingly, there is a high chance that intellectual property rights will be developed as a consequence of this study. To this end, we maintain links with MRC Technology and Transfer to discuss the potential generation of patentable ideas on a regular basis.
In a previous project, we have identified small molecules that modulate autophagy with the aim to develop potential therapeutic lead compounds. The proposed study is a direct follow-up of that work in order to identify the molecular targets for some of these compounds. As many pharmaceutical companies are desperately searching for new therapeutic targets at this moment, there will be a huge benefit in identifying kinases that modulate autophagy. In combination with our previous successful small molecule screening, there is a high chance for translational benefits of this project. We have already initiated discussions with MRCT about potential commercialization of this part of the project.
Training of skilled researchers is another major goal. The lab offers a perfect environment for providing a very broad and technology-driven training that includes expertise in cell biology, biotechnology engineering and bioinformatics. Researchers in my lab have access to a unique set of robotic automation resources that is not often found in academic settings. The postdoctoral research fellow will gain access to these instruments and learn how to operate them and how to handle large datasets. These are skills that are very useful and are only accessible in laboratories similar to the Translational Research Resource Centre at MRC LMCB.
In addition, this study will foster collaboration within different research groups across the world. This can be mainly achieved by exchange of knowledge in the form of publication and presentation at conferences, and in addition could take the form of national and international collaborations. For instance, there is already a close collaboration with Dr. Joern Dengjel at FRIAS in Germany in place. Dr. Dengjel is one of the world leading experts in proteomic analysis of autophagy proteins. As part of this project, visits to the mass spectrometry facility in Freiburg will take place and funds to facilitate these visits are requested.
Breakthrough findings will be communicated to the UCL press office. In addition, we will make all of our data publicly available, as well as any research tools including novel assay systems or bioinformatic databases.
As a consequence of this work, the prestige of our institute will increase in the scientific community, which will attract further financial support from funding agencies. The underlying mechanisms have implications for the development of biotechnology tools, thus attracting interest from the commercial sectors as well.

Publications

10 25 50
 
Description This grant has helped us to understand the regulation of autophagy, the cellular stress response, in more detail. We have been able to identify genes involved in the control of this process and discovered a novel mechanism of regulation that was previously not anticipated.
Exploitation Route The novel mechanisms of autophagy regulation that we have uncovered will generate multiple novel lines of research. For instance, a new concept of localized control of autophagy by kinases emerges that will be important in other areas of cell biology as well. Furthermore, there is the potential that some of the discovered genes are novel drug targets for pharmaceutical approaches. Also, this work has initiated discussions with pharmaceutical industry partners (AstraZeneca, J&J, Apollo Therapeutics, Samsara Therapeutics) and the identification of small molecule compounds for treatments of various forms of cancer.
Sectors Chemicals,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Our findings are of high interest to a cell biology audience and will have impact for several areas: 1. the data help generate novel lines of research; 2. the data help understand animal health; 3. we have identified chemical compounds as novel research tools;
First Year Of Impact 2013
Sector Chemicals,Education,Healthcare,Pharmaceuticals and Medical Biotechnology
 
Description Membership of Advisory Panel UCL-Eisai Collaboration
Geographic Reach Multiple continents/international 
Policy Influence Type Participation in a advisory committee
 
Description Michael J Fox Foundation Parkin Consortium
Geographic Reach Multiple continents/international 
Policy Influence Type Membership of a guideline committee
 
Description Scientific Advisor to AstraZeneca High-Content Biology
Geographic Reach Europe 
Policy Influence Type Participation in a advisory committee
 
Description The Royal Society Newton Fellowship panel member
Geographic Reach Multiple continents/international 
Policy Influence Type Membership of a guideline committee
 
Description Action Medical Research PhD Fellowship
Amount £230,000 (GBP)
Organisation Action Medical Research 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2016 
End 08/2018
 
Description Michael J Fox Foundation
Amount $78,000 (USD)
Organisation Michael J Fox Foundation 
Sector Charity/Non Profit
Country United States
Start 01/2014 
End 12/2014
 
Description UCL Confidence in Concept Award
Amount £99,208 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 06/2014 
End 05/2015
 
Description UCL Eisai collaboration
Amount £5,000,000 (GBP)
Organisation Eisai Ltd 
Sector Private
Country Japan
Start 11/2017 
End 04/2019
 
Description UCL-Eisai Collaboration
Amount £12,500 (GBP)
Organisation Eisai Ltd 
Sector Private
Country Japan
Start 10/2016 
End 03/2017
 
Description WT Institutional Strategic Support Fund
Amount £65,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 11/2015 
End 12/2017
 
Title ATG4ABC CRISPR knockout cell line 
Description HeLa cells with knockout of three proteins (ATG4A, B and C) generated by CRISPR/Cas9 genome editing 
Type Of Material Cell line 
Year Produced 2018 
Provided To Others? Yes  
Impact This cell line will be useful for further research into the function of ATG4 proteases and can be used in early stage drug discovery for target validation. 
 
Title Antibody for phosphorylated ATG4B. 
Description We have raised a polyclonal antibody for phosphorylated autophagy protease ATG4B. 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? Yes  
Impact This is the only available antibody to detect phosphorylated ATG4B at Serine 316 and will be a useful research tool to study the function of this protein. We expect that researchers will request this once our publication is accepted. We will happily provide this antibody to anyone interested. 
 
Title Inhibitor and activator compounds of ATG4B protease. 
Description We have identified inhibitors, as well as activators of the autophagy protease ATG4B. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? No  
Impact These compounds have therapeutic potential and we are further developing them into lead compounds for the treatment of cancer. 
 
Title Modified luciferase release assay 
Description This new version of a luciferase-based assay to monitor autophagy protease ATG4 activity can be used in academic research and drug target identification. The new version includes assays to monitor other protein targets of ATG4, such as LC3A and mutants thereof. 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? Yes  
Impact We have given the plasmid encoding this reporter to several other research groups. We are actively sharing this reagents with multiple labs and hope that this will advance research in this field. 
 
Description ATG4B compounds BC Cancer Agency 
Organisation British Columbia Cancer Agency (BCCA)
Country Canada 
Sector Public 
PI Contribution We are assaying compounds that inhibit autophagy in our cell-based assays.
Collaborator Contribution Provision of chemical compounds for testing.
Impact No outcomes yet.
Start Year 2014
 
Description Arrayed CRISPR Library Screening 
Organisation Thermo Fisher Scientific
Country United States 
Sector Private 
PI Contribution We are developing protocols for arrayed CRISPR library screening in partnership with ThermoFisher.
Collaborator Contribution ThermoFisher has provided us with early access to a lentivirus library that is not yet commercially available. Also, Thermofisher is providing consumables and reagents for this research project.
Impact We are currently preparing experiments for publication. Also, a Postdoc in the lab has presented preliminary results at the Wellcome Trust-AstraZeneca CRISPR conference in January 2016. Further, I have been invited to present these data at the CRISPR conference in Oxford in April 2016 and the GE/Dharmacon Functional Genomics meeting in London in April 2016.
Start Year 2015
 
Description Arrayed CRISPR library screening 
Organisation Thermo Fisher Scientific
Country United States 
Sector Private 
PI Contribution "We are developing protocols for arrayed CRISPR library screening in partnership with ThermoFisher."
Collaborator Contribution "ThermoFisher has provided us with early access to a lentivirus library that is not yet commercially available. Also, Thermofisher is providing consumables and reagents for this research project."
Impact "We are currently preparing experiments for publication. Also, a Postdoc in the lab has presented preliminary results at the Wellcome Trust-AstraZeneca CRISPR conference in January 2016. Further, I have been invited to present these data at the CRISPR conference in Oxford in April 2016 and the GE/Dharmacon Functional Genomics meeting in London in April 2016."
Start Year 2015
 
Description Chemistry of ATG4B Inhibitors 
Organisation University College London
Country United Kingdom 
Sector Academic/University 
PI Contribution We have developed cell-based and in vitro assays to monitor autophagy protease activity. We work with researchers at UCL for provision of chemical libraries and chemistry to enable drug discovery projects.
Collaborator Contribution Provision of chemical compounds and expertise in chemistry.
Impact This partnership is multi-disciplinary and involves chemistry, biology and bioinformatics.
Start Year 2014
 
Description Eisai Pharmaceuticals 
Organisation Eisai Ltd
Country Japan 
Sector Private 
PI Contribution In collaboration with researchers at Eisai and funded through the Michael J Fox Foundation, we are exploring the role of De-ubiquitinating enzymes in Parkin-mediated mitophagy. We are providing the underlying cell biology for this study.
Collaborator Contribution Provision of a cDNA library and expertise in the field of neuro-degeneration. This has led to funding from the Michael J Fox Foundation for a pilot project. Since 2017, the partner has provided substantial funding for an early stage drug discovery project in my lab. This project has been initiated in 2017 and is ongoing.
Impact This collaboration has led to two successful funding applications. One is with the Michael J Fox Foundation. The other is with the industry partner itself, which is funding an early stage drug discovery project and a Postdoctoral scientist in the lab.
Start Year 2013
 
Description ICR Kirkin 
Organisation Institute of Cancer Research UK
Country United Kingdom 
Sector Academic/University 
PI Contribution This is a research collaboration, which is lead by my team at UCL.
Collaborator Contribution The collaborator has given us access to important research tools and reagents and will contribute by performing in vivo studies in the future.
Impact None so far
Start Year 2018
 
Description Johnson & Johnson 
Organisation Johnson & Johnson
Country United States 
Sector Private 
PI Contribution We are screening a small molecule library from J&J with the aim to identify tool compounds that modulate breast cancer cell growth and autophagy.
Collaborator Contribution We have received a small molecule library targeting cysteine proteases for screening.
Impact Exchange of know-how and materials.
Start Year 2017
 
Description Knockout Cell Panel Screening 
Organisation Horizon Genomics
Country United Kingdom 
Sector Private 
PI Contribution "We are developing protocols for the use of CRISPR-engineered knockout cell panels in arrayed screening."
Collaborator Contribution "Provision of knockout cell lines and knockout cell panels free of charge."
Impact "We are preparing experiments for publication."
Start Year 2015
 
Description Knockout Cell Panel Screening 
Organisation Horizon Genomics
Country United Kingdom 
Sector Private 
PI Contribution We are developing protocols for the use of CRISPR-engineered knockout cell panels in arrayed screening.
Collaborator Contribution Provision of knockout cell lines and knockout cell panels free of charge.
Impact We are preparing experiments for publication.
Start Year 2015
 
Description MRCT Index Compound Set 
Organisation MRC-Technology
Country United Kingdom 
Sector Academic/University 
PI Contribution High-throughput screening of small molecule compounds"
Collaborator Contribution "Provision of small molecule library"
Impact None to date
Start Year 2015
 
Description MRCT Index Compound Set 
Organisation MRC-Technology
Country United Kingdom 
Sector Academic/University 
PI Contribution High-throughput screening of small molecule compounds
Collaborator Contribution Provision of small molecule library
Impact None to date
Start Year 2015
 
Description MaMTH EGFR Interactions 
Organisation University of Toronto
Country Canada 
Sector Academic/University 
PI Contribution We are studying novel interaction partners of the EGFR that were identified using a technology developed in Igor Stagljar's lab at University of Toronto.
Collaborator Contribution Access to MaMTH, a novel method to identify protein-protein interactions.
Impact A publication has been published: Petschnigg et al. J Mol Biol 2017.
Start Year 2014
 
Description Mass Spectrometry of Autophagy 
Organisation Albert Ludwigs University of Freiburg
Country Germany 
Sector Hospitals 
PI Contribution In collaboration with Dr. Joern Dengjel at the University of Freiburg, we are using Mass Spectrometry to study signal transduction in autophagy.
Collaborator Contribution Access to mass spectrometer.
Impact Preliminary work has resulted in data for a BBSRC-funded project grant.
Start Year 2010
 
Description Medicinal Chemistry of Autophagy modulators 
Organisation University College London
Department School of Pharmacy
Country United Kingdom 
Sector Academic/University 
PI Contribution "Testing small molecule compounds generated by the School of Pharmacy in autophagy assays."
Collaborator Contribution "Generation and provision of 26 compounds for drug discovery."
Impact "Invention Declaration Form has been filed with UCL-B."
Start Year 2015
 
Description Medicinal Chemistry of Autophagy modulators 
Organisation University College London
Department School of Pharmacy
Country United Kingdom 
Sector Academic/University 
PI Contribution Testing small molecule compounds generated by the School of Pharmacy in autophagy assays.
Collaborator Contribution Generation and provision of 60 compounds for drug discovery.
Impact Invention Declaration Form has been filed with UCL-B. Discussions about licensing of compounds with industry partners started.
Start Year 2015
 
Description Academic RNAi Screening meeting 
Form Of Engagement Activity Scientific meeting (conference/symposium etc.)
Part Of Official Scheme? No
Type Of Presentation workshop facilitator
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact 120 Researchers attended a conference co-organized by Mike Howell, Steve Brown, Emma Shanks and myself. There were talks and panel discussions on this one-day meeting.

The majority of attendants felt that the conference attracted important scientists in the field and many expressed the notion that this will impact the way they plan to do their research. Industrial partners have expressed satisfaction with the level of engagement with researchers, which will give them leads for commercialisation of their products.
Year(s) Of Engagement Activity 2011,2013,2014
 
Description Autophagy UK Network meeting 2017 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact 150 researchers, students, representatives from companies and journals came together to discuss the latest advances in the field of autophagy research in Longon. I was one of two organisers for the entire event.
Year(s) Of Engagement Activity 2017
URL http://autophagy.uk
 
Description Royal Reception 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Supporters
Results and Impact The charitable organization Action Medical Research invited our research team to present at a Royal Reception at Buckingham Palace. HRH Prince Philip was hosting the event and there were approx 50-60 potential charitable donors present who engaged with our research presentation. The donors were very impressed and as a consequence willing to donate funding towards research into childhood diseases. A lot of the people reported that they learned something new and everyone was engaging in discussions around health, scientific research and patient benefit.
Year(s) Of Engagement Activity 2017
 
Description School Visit (UCL Syddanmark, Denmark) 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact 14 Students attended a workshop presentation and a tour of the high-throughput screening facility, which led to very fruitful discussion afterwards.

The students were looking to explore career track options at that time. Several students indicated that they would now consider a career in science rather than as medical assistant.
Year(s) Of Engagement Activity 2014
 
Description School visit 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact School visit with a tour of the MRC funded screening facility and demonstration of experiments
Year(s) Of Engagement Activity 2012,2013,2014,2015,2016
 
Description Scientific Advisory Board Expert Panel for the Dementia Discovery Fund 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact The Scientific Advisory Board of the Dementia Discovery Fund in collaboration with Alzheimers Research UK, invited me as an expert panel member to discuss the role of Autophagy in Alzheimer's disease. The SAB (composed of leading experts in the pharmaceutical industry and charities) explored the possbility to invest in the development of autophagy modulating drugs for treatment of Alzheimer's disease. This discussion had an immediate impact on funding decisions and the policy of the Dementia Discovery Fund going forward in this area.
Year(s) Of Engagement Activity 2019