Publication Highlights: January 2017-April 2017 CFATG
Publication Highlights: January 2017-April 2017
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Publication Highlights: Articles on autophagy research published by french laboratories and selected by CFATG.

Previous work in the field of autophagy has demonstrated that membrane damage induced by invasive bacteria elicits selective autophagy to eliminate the pathogen via lysosomal degradation. In our work published in PlosPathogens (Montespan et al. 2017) we show that adenoviruses (AdV) upon entry during which they provoke membrane damage, also activate autophagy. We further demonstrate how the virus uses a small PPxY peptide motif in the endosomolytic capsid protein VI to seize control of the autophagic machinery.
AdV are non-enveloped, ds-DNA containing viruses that enter cells by receptor-mediated endocytosis. Inside the endosomal compartment the virus releases the membrane lytic internal capsid protein VI (PVI) to rupture the endosomal membrane and access cytosolic transport means towards the nucleus. In our work we show by using mutant viruses that AdV membrane damage is necessary to trigger autophagy and that the damaged membrane is subsequently recognized via galectin-8 and autophagy receptors such as NDP52 and p62. Upon endosome lysis wild type AdV recruits a cellular ubiquitin ligase Nedd4.2 via the PPxY motif in PVI, rapidly escapes from the endosome and prevents autophagic degradation by halting autophagosome maturation. Our work further showed that AdV subsequently uses the PPxY motif to subvert the autophagic machinery and to recruit the autophagosomal marker LC3 for accelerated transport to the nucleus suggesting that the autophagic machinery is hijacked by the virus to promote AdV infection. Employing an AdV mutant with a mutated PPxY in PVI we show that these viruses remain trapped in ruptured endosomes, are unable to limit autophagosome maturation and are subject to galectin-8 dependent autophagic elimination.
Thus, our work has identified the PPxY motif as the first virus encoded molecular determinant enabling incoming membrane lytic viruses to actively take control of antiviral cellular defenses such as autophagy.

PLoS Pathog. 2017 Feb 13;13(2):e1006217.

The canonical autophagy pathway represents an attractive therapeutic target for a range of diseases. Many autophagy modulating drugs that have been identified target lysosomes and inhibit autophagic flux. However, identification of these autophagy modulating drugs commonly relies on measuring LC3 lipidation as a read out. Our lab now clearly shows that LC3 lipidation is not a specific marker for the canonical autophagy pathway.
Non-canonical autophagy is a pathway that utilises some autophagy proteins to modify membranes of the endolysosomal system. During non-canonical autophagy, the lipidation of LC3 onto single-membrane endolysosomal compartments requires components of the lipidation machinery but is independent of mTOR signalling, the ULK complex and autophagosome formation. An example of non-canonical autophagy is LC3-associated phagocytosis (LAP), during which the lipidation of LC3 to phagosomes housing pathogens or dead cells plays an important role in immune responses and homeostasis.
Using cell lines that are deficient for canonical autophagy and human tissue samples, we show that a range of clinically relevant drugs thought to inhibit autophagosome flux, including chloroquine and lidocaine, are able to promote LC3 lipidation to endolysosomal compartments. Thus, these drugs can activate non-canonical autophagy in parallel to their known effects on canonical autophagy. Importantly, the autophagy activators CCCP and amiodarone can also activate non-canonical autophagy independently of autophagosome formation.
In this paper, we highlight unexpected functions of autophagy modulators and demonstrate that drug-induced non-canonical autophagy is common feature of drugs with lysosomotropic or ionophore properties. This finding emphasises the need for a better understanding of the functional consequences of endolysosomal LC3 lipidation and also raises important questions regarding the design and analysis of high-throughput screens for autophagy modulators.

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MCF10 cells stably expressing GFP-LC3 treated for 2h with 5 mM Procainamide. Confocal imaging of GFP-LC3 (green), endogenous LAMP1 immunostaining (red) and DAPI staining (blue). Treatment with Procainamide induces a dramatic vacuolation of MCF10A cells and a relocalisation of GFP-LC3 onto LAMP1 positive compartments.

Autophagy. 2017 May 4;13(5):854-867.

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