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Systematic analysis of post-translational regulation of autophagy protease ATG4B

Reference	BB/J015881/1
Principal Investigator / Supervisor	Professor Robin Ketteler
Co-Investigators / Co-Supervisors
Institution	University College London
Department	MRC Cell Biology Unit
Funding type	Research
Value (£)	255,375
Status	Completed
Type	Research Grant
Start date	01/01/2013
End date	31/01/2016
Duration	37 months

Abstract

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.

Summary

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.

Impact Summary

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.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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