Selective autophagy and cell signalling: Regulation of p62/SQSTM1 and functional studies of novel LIR-containing proteins

Autophagy literally means eating oneself and describes a degradation process in our cells where a double membrane encapsulates part of the cells cytoplasm forming a closed autophagosome. The autophagosome eventually fuses with the lysosome, the content is degraded and building blocks recirculated. Autophagy was long regarded as a non-selective, bulk degradation route. However, recent research, including data from our group, has shown that autophagy can be very selective. This way, the cell can degrade larger structures in a targeted manner. This includes protein aggregates, damaged or superfluous organelles like mitochondria and peroxisomes, and invading intracellular bacteria and viruses. We were the first to describe the first selective autophagy receptor in mammalian cells, p62/SQSTM1. We showed that p62 acts as a renovator that can recognize material destined for destruction. Such material is tagged with ubiquitin which p62 binds to, and subsequently couples the tagged material to the autophagy machinery. In this selective autophagy process p62 is itself degraded. We identified a so-called LIR (LC3 Interacting Region) motif, which p62 uses to bind to a family of proteins bound to the autophagosomal membrane. This family of proteins consists of LC3A, LC3B, LC3C, GABARAP, GABARAPL1 and GABARAPL2, that can be bound via LIR motifs. The current project aims to provide novel insight into roles played in cell signaling and selective autophagy by p62/SQSTM1 and novel proteins identified to harbor the LIR motif. In collaboration with Peter Kim?s group in Toronto we have shown that a close relative of p62, NBR1, can act as a selective autophagy receptor for degradation of peroxisomes. During this work we found that NBR1 binds to membranes and that it sees both the membrane and the ubiquitin tag on the peroxisomes and therefore binds to them and leads to selective autophagy of peroxisomes (J. Cell Sci. 126, 939-952, 2013). In another important study we collaborated with the group of Vojo Deretic in New Mexico describing a novel selective autophagy receptor, TRIM5alpha. TRIM5alpha binds to the HIV virus capsid and can remove it after the virus has entered the cell. The viral capsid is degraded by selective autophagy following recognition by TRIM5alpha. Rhesus monkey TRIM5alpha efficiently recognizes the HIV capsid and mediates its destruction after entry into the cell. Human TRIM5alpha is unfortunately not very effective in recognizing HIV that has entered our cells. TRIM5alpha also binds to p62, and p62 is also required for effective degradation of HIV capsids (Dev. Cell, 30, 394-409, 2014). Related to the work on TRIM5 alpha we also collaborated with the group of Martine Biard-Piechaczyk on the role of the small HIV protein Vif (Viral infectivity factor). Interestingly, Vif bound to LC3B and inhibited autophagy during HIV infection of T cells(AIDS 29, 275-286, 2015). Our collaboration with Deretic´s group continues and has lead to the novel concept precision autophagy. Several TRIM proteins can bind specific proteins directly and target them for autophagic degradation. They also recruit components of the autophagy machinery. The TRIM20 and TRIM21 proteins are important in regulating the defense against infections and inflammation. Remarkably, the autophagic function of TRIM20 is affected by mutations associated with familial Mediterranean fever (J. Cell. Biol. 210, 973-989, 2015). Studying gene regulation of p62 we found that the cofactor SPBP is induced by sulforaphane and functions as a coactivator of NRF2. NRF2 regulates the expression of the p62 gene, and a number of genes that are induced as a response to oxidative stress. Sulforaphane is a substance that is found in several green vegetables that leads to activation of NRF2 (PLOS One, 9,e85262. doi: 10.1371, 2014). We explored the relation between NRF2 and p62 in the fruit fly in collaboration with Tor Erik Rusten, Norwegian Radium Hospital. Strikingly, in the fruit fly NRF2 induced increased autophagy in fat body and larval gut tissues. The results extend the intimate relationship between oxidative stress-sensing NRF2 transcription factors and autophagy and suggest that NRF2 may regulate autophagic activity in other organisms too (J. Biol. Chem. 290, 14945-14962, 2015). We initiated a collaboration with Carsten Sachse, an expert on cryo-electron microscopy. The first cryo-EM structural analyses of p62 explained how p62 assemblies provide a large molecular scaffold for the nascent autophagosome and reveal how they can bind ubiquitinated cargo (Cell Reports 11, 748-758, 2015). Degradation of nuclear components by autophagy is emerging. We have contributed to a study led by the lab of Shelley Berger in Philadelphia demonstrating that lamin B1 is degraded by autophagy during oncogene-induced senescence. The study suggests that this new function of autophagy acts as a guarding mechanism protecting cells from tumorigenesis (Nature 527, 105-109, 2015).

(Macro)autophagy is a lysosomal degradation pathway for cytosolic macromolecules and organelles that plays a crucial role eliminating damaged organelles and toxic protein aggregates which cannot be degraded by proteasomes. There is great interest in deter mining the significance of autophagy in cancer and neurodegenerative diseases, inflammation and innate immunity. Autophagy has been considered a non-specific, bulk degradation pathway. However, we identified p62 and NBR1 as the first selective autophagy s ubstrates and receptors for autophagic degradation of ubiquitinated targets. p62 also plays a pivotal role in several important cell signalling pathways. We will now determine how the protein level and activity of p62 is regulated in response to different stressors. The level of p62 is upregulated significantly in cells exposed to oxidative stress, ER stress, bacteria, viruses, various drugs and signalling molecules. Regulation of the p62 level is of vital importance both in cell signalling and selective autophagy. We will study all major stress response pathways for their effects on p62 gene transcription. We will study the regulation of p62 mRNA levels by microRNA, and how post translational modifications may regulate p62 activities. Another focus of r esearch in our group is based on our discovery of the LIR motif mediating interaction with ATG8 family proteins. An increasing number of LIR-containing ATG8-interaction partners have been identified. Here, we will focus on basal autophagy proteins and the protein kinases binding to ATG8. Functional roles of interactions of LIR-containing proteins in the ULK1/2 and class III PtdIns 3-kinase autophagy complexes and atypical PKCs, STK3 and STK4 will be studied in Drosophila and cell culture models. The proje ct will involve confocal, live cell, fluorescence- and electron microscopy imaging, proteomics, structural biology, cell- and animal models.