Targeting the autophagy pathway

Autophagy is catabolic process that involves the formation of double-membrane vesicles, autophagosomes, which sequester cytosolic components and deliver them to the lysosome for degradation. Resulting nutrients are recycled and participate in de novo synthesis or energy production.

Basal autophagy plays a critical role in cellular homeostasis by continuously removing damaged organelles, protein aggregates and long-lived proteins. Additionally, autophagy can be dramatically induced in response to various environmental stresses, such as starvation, hypoxia, oxidative stress, radiation and pathogen infection.

As an intracellular quality control pathway, autophagy is able, for instances, to selectively degrade damaged or surplus organelles and protein aggregates. Therefore, the autophagy machinery needs to distinguish between “normal” and “anomalous” cellular components and specifically target the latter for degradation. During the last years, our group has been interested in the mechanisms of selective autophagy in mammalian cells. In particular, we contributed to the discovery of novel autophagy receptors, as NBR1, NIX and OPTINEURIN.

In the field of autophagy, we are focusing on two main projects in our laboratory:

Project I:  We plan to develop selective modulators of general and selective autophagy by using a phage display method that has already been successfully applied for targeting the Ub system (Ernst et al Science 2013). This project is funded by the HFSP and is done in collaboration with Dev Sidhu’s and Andreas Ernst’s laboratories. We aim at targeting molecular core machinery of autophagy (e.g. ATG3, ATG7) and autophagy receptors (e.g. p62, NBR1, OPTINEURIN) by applying engineered variants of LC3 and GABARAP modifiers. We also plan to selectively target the LIR-binding interfaces motives (LIR: LC3 interaction region) of all six human ATG8 homologues by screening various phage-displayed peptide libraries.


Highlighted are randomized amino acid residues in LC3B mutants in the autophagy receptor binders screen (a) and in the core autophagic machinery binders screen (b).

This procedure will yield protein variants and peptides which bind with high affinity and excellent selectivity to specific ATG proteins, receptors or LC3 isoforms. Obtained variants will be tested for binding specificity in vitro and the successful clones will be further characterized by biochemical and biophysical methods (pull-down, IP, mass spectrometry, SPR, ITC, crystallography, NMR). The functional in vivo assays will involve live imaging as well as construction of transgenic mice which will enable inducible, tissue specific expression of the engineered protein variants and peptides.


Schematic depiction of phage display selections of LC3B/GATE-16 mutants for targeting autophagy pathways.

Our approach will yield highly specific autophagy inhibitors and will allow us to precisely address the functional relevance of various receptors in diverse pathways of selective autophagy.


Project II: The autophagy machinery is highly conserved from yeast to mammals. Autophagy-core proteins were mainly identified in yeast. Many of these proteins have orthologs in higher eukaryotes - C. elegans, Drosophila and mammals. Our group, in collaboration with Dr. Christian Pohl’s group, is now interested in investigating the molecular mechanisms of autophagy in vivo, in C. elegans. This approach will allow us to investigate the role autophagy and autophagy receptors in disease and development.


Autophagy can be monitored in C. elegans expressing GFP::LGG-1. Upon induction of autophagy by fasting, GFP::LGG-1 is recruited to the autophagosome membrane, appearing as fluorescent puncta. When autophagy is inhibited by Bec-1 depletion, no GFP::LGG-1 re-localization to puncta is observed after fasting.

 

Functional characterization of linear ubiquitination

Linear Ubiquitin chain Assembly Complex (LUBAC) is an E3 ligase complex, specifically generating Met1-linked (linear) ubiquitin chains. It consists of a catalytic protein HOIP/ Rnf31and two other critical subunits, Sharpin and HOIL-1L/ Rbck1. LUBAC is required for activation of the nuclear factor kappa-light-chain-enhancer of activated B cell (NF-kB) signaling cascade induced by inflammatory cytokines, such as tumor necrosis factor-a (TNF-a), Interleukin-1b (IL1-b), and bacterial component, lipopolysaccharide (LPS). Recently, the deubiquitinase that specifically disassembles linear ubiquitin chains, OTULIN has been shown to inhibit LUBAC-induced the NF-κB pathway. Our group was interested in the regulation of these opposing activities. We recently showed that HOIP and OTULIN interact and act as a bimolecular editing pair for linear ubiquitin signals in vivo. The HOIP PUB domain binds to the PIM (PUB interacting motif) of OTULIN and the chaperone VCP/p97. Our structural studies revealed the basis of high affinity interaction with the OTULIN PIM. The conserved Tyr56 of OTULIN makes critical contacts with the HOIP PUB domain and its phosphorylation negatively regulates this interaction. Moreover, we showed that HOIP binding to OTULIN is required for the recruitment of OTULIN to the TNF receptor complex and to counteract HOIP-dependent activation of the NF-κB pathway.
Proteomic and bioinformatic analyses have identified new proteins that bind to the HOIP PUB domain. We are now interested in investigating whether these proteins play a function on ubiquitin signaling. Our approach comprises biochemistry, cell biology, structural analysis and functional studies.




HOIP and OTULIN interact and act as a bimolecular editing pair for linear ubiquitination, controlling NF-κB signalling.