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The Ketteler lab is interested in signal transduction pathways and the regulation of autophagy. We employ high-throughput screening platforms using cDNA, siRNA and CRISPR libraries to investigate growth factor signaling cascades and identify therapeutic targets for cancer and neuro-degeneration.
1) Identification of proteins involved in EGFR signaling
Classical EGFR signaling involves the binding of adaptor molecules such as Grb2 to the receptor and converts an extracellular signal into an intracellular response. This signal conversion involves protein-protein interactions and activation of intracellular signaling complexes through post-translational modifications. We and others have recently developed novel assays that enable the characterization of these signaling cascades in a systematic manner. We utilize a mammalian two-hybrid system, originally developed by Julia Petschnigg and Igor Stagljar, in order to identify novel EGFR interaction partners (Petschnigg et al. 2014). Julia recently joined my lab to introduce this technique at the LMCB. In combination with a previously developed assay from our lab to identify localized signaling via the adaptor Grb2 (Freeman et al. 2012), we were able to identify novel genes involved in trafficking and signaling of the EGFR.
The main findings so far are:
1. A large number of genes was identified that bind to Grb2 or to the EGFR that were previously not implicated in signalling via this pathway.
2. Some genes were shown to preferentially interact with oncogenic versions of the EGFR when compared to wild-type EGFR.
3. Grb2 complexes were identified in unexpected subcellular locations such as the nucleus, the centrosome, autophagosomes and sites of protein aggregation.
These studies indicate that very little is known about the spatial and temporal regulation of classical signalling pathways that we plan to address in the future.
2) Systematic analysis of post-translational modification of autophagy proteins
Autophagy is a cellular stress response to diverse stimuli such as starvation, infection and DNA damage. Despite recent advances in our understanding of the autophagy machinery, the molecular regulation of autophagy is not very well understood. We are interested in understanding how autophagy core proteins are controlled by post-translational modifications. In particular, we are interested in the regulation of autophagy by kinase signaling. Using a luciferase-based assay and siRNA screening (Ketteler et al. 2008; Ketteler and Seed, 2008), we have identified genes that regulate the cellular activity of ATG4B. We have identified multiple kinases and phosphatases that regulate ATG4B activity and autophagosome formation. The main findings so far are:
1. Several kinases and phosphatases were identified that control the proteolytic activity of ATG4B and the formation of autophagosomes.
2. ATG4B activity is controlled locally by phosphorylation/de-phosphorylation events with an inhibitory phosphorylation at the autophagosome initiation site and an activating de-phosphorylation at the autophagosome-lysosome fusion site.
The local control of autophagy signalling is very intriguing and forms the basis for future studies where we aim to understand this in more mechanistic detail.
3) Identification of small molecule compounds that modulate autophagy
Autophagy plays important roles in the progression of various diseases including cancer and Parkinson’s disease. In particular, it has been proposed that the up-regulation of autophagy in neuro-degenerative diseases can be benefitial for neuron survival. We have completed a small molecule screen of ~50,000 compounds in order to identify chemical compounds that target either ATG4B directly or autophagosome formation in general. Through various collaborations with chemists and bioinformaticians, we have identified four main classes of compounds:
- Activators of ATG4B protease activity
- Inhibitors of ATG4B protease activity
- Activators of autophagic flux
- Inhibitors of autophagic flux
These compounds can find applications in various disease models such as cancer and neuro-degeneration.
Our work has been supported by the Medical Research Council, BBSRC, Wellcome Trust, Michael J Fox Foundation and University College London CiC/TIF funding.
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