AS_ETHZ_IEF_2012 · The Role of Atg8 Posttranslational Modifications in Autophagy
FP7 — People (Marie Curie Actions)
- Duration
- 2013-03-01 → 2015-02-28
- EU contribution
- €184,709
- Participants
- 1
- Scheme
- MC-IEF
Lines connect the coordinator with its partners.
Results in brief
The Role of Atg8 Posttranslational Modifications in Autophagy
Autophagy is a cellular degradation pathway required for eukaryotic protein homeostasis, development, differentiation and cellular defence. Deregulation of autophagy has been shown to cause the development of various human pathologies, while an increased basal level of autophagy has been shown to correlate with improved health. Autophagy involves the formation of double-membrane vesicles, called autophagosomes, which sequester portions of the cytosol (bulk autophagy) or whole organelles, pathogens or aggregates (selective autophagy). Autophagosomes then fuse with the lysosome or vacuole (in yeast and plants), where their cargo is degraded. Recent proteomics studies have reported various posttranslational modifications of autophagy related (Atg) proteins. However, their functional implications are currently unknown. In order to bridge the knowledge gap between large-scale proteomics data and essential functional and mechanistic understanding we proposed to study the functional significance of posttranslational modifications of Atg proteins with a particular focus on the ubiquitin-like protein Atg8. To this end I developed an approach, which uses the in vitro phosphorylation sites of recombinantly expressed and purified Atg proteins as determined by mass spectrometry as leads for the functional screen in vivo. By this means I showed that Atg8 is phosphorylated by Atg1 and that this is required for autophagy progression. In a major expansion of the scope of my project, I have discovered many more novel Atg1 substrates amongst all the known Atg proteins involved in macroautophagy, which I am currently investigating further. For example the Vps34 complex is a direct target of Atg1 dependent phosphorylation. Having expressed and purified this and other large multisubunit complexes for the Atg1 target discovery study opened up a second major scientific direction - to use these complexes for structural determination by electron microscopy (EM) and single particle analysis. The Vps34 complex turned out to be a promising sample for structural studies allowing us to determine the negative stain EM structure for its autophagy and endosome specific variants. In summary, our highly sensitive and largely unbiased in vitro approach has already yielded novel insights into the currently unknown signaling transduction pathways involved in the onset and progression of autophagy and provided a starting point for structural studies of the major multisubunit complexes involved in autophagy. Moreover, the data generated during the course of this study have paved the way for a significant conceptual advancement of our mechanistic understanding of autophagy in the imminent future.
Data: CORDIS, © European Union
Project objective
Autophagy is an important cellular degradation pathway required for eukaryotic protein homeostasis. Deregulation of autophagy has been shown to cause the development of various human pathologies, including aggregation diseases and cancer. Autophagy involves the formation of double-membrane vesicles, called autophagosomes, which sequester portions of the cytosol (general autophagy) or whole protein aggregates, organelles or pathogens (selective autophagy). Autophagosomes then fuse with the lysosome or vacuole, where the cargo is degraded. Atg8 is a key regulator of autophagy facilitating autophagosome formation, lysosomal fusion and cargo recruitment. Atg8 is conjugated to the lipid phosphatidylethanolamine and is thereby inserted into the arising autophagsomal membrane, in which it promotes autophagosomal membrane expansion and cargo recruitment through direct binding of LIR-motif containing cargo adaptors.Interestingly, large-scale proteomics data suggest that Atg8 is regulated by several posttranslational modifications (PTMs). Here we propose to study the functional implications of these Atg8 modifications for general and selective autophagy. Intriguingly, our preliminary data suggest that Atg8 undergoes Atg1- and Atg7-dependent polyubiquitination. We propose that Atg8 polyubiquitination may cause a switch from selective to general autophagy to promote cell survival or, alternatively, that it may play a role in autophagosome-vacuole fusion. Moreover, we will investigate the enzymatic machinery and regulatory mechanisms underlying Atg8 PTMs. We anticipate bridging the knowledge gap between large-scale proteomics data and essential functional and mechanistic understanding of Atg8 PTMs. Based on our promising preliminary data, we are confident that this study will provide important insights into the regulation of autophagy while also contributing to the identification of novel drug targets for the treatment of autophagy-related diseases.""
Original text from CORDIS.
Participants
- EIDGENOESSISCHE TECHNISCHE HOCHSCHULE ZUERICH · ZuerichCoordinatorSwitzerland
Links
Data: CORDIS, © European Union