Autophagy-regulated Signalosomes in Cellular Stress and Disease Pathways

Our project concerns a cellular process known as autophagy (greek for "self-eating"). Autophagy is a renovation or cleaning process in our cells that helps keeping us healthy and makes it possible for us to enjoy long lives. We are elucidating how autophagy can be targeted, or selective, and how selective autophagy is able to regulate signaling systems in our cells. Certain signaling pathways are not regulated properly in diseases such as cancer, diabetes, neurodegeneration, and inflammatory disorders. Proteins can be envisioned as workers and consultants that carry out important functions that are needed to keep our cells alive and healthy. Proteins also make up part of the physical infrastructure in the cells such as the "highways" and various molecular machines. Signaling is often organized into protein complexes containing a number of different proteins acting together in a "signalosome". This organization facilitates sensitive and efficient signaling. Autophagy is a cellular renovation process that degrades damaged proteins and non-functional or aged organelles. Autophagy is called upon under stress conditions, such as starvation. The cell degrades part of itself to recycle building blocks and release energy to ensure its own survival. Recent discoveries implicate autophagy in important physiological and pathological processes such as development, immunity, cell death and cancer. Work on the scaffold and signaling protein p62 led us to the discovery of selective autophagy at the molecular level. Hence, we found that p62 was as factor that could "sort the garbage". When cells are stressed in various ways we saw that p62 formed dynamic, round bodies in the cells. We asked whether these bodies could represent hubs where signaling involving p62 may occur, so-called signalosomes. p62 is, via these signalosomes, involved in regulating signaling. To test our hypothesis we considered stress signaling. The most studied stress increasing autophagy is starvation. We found that cells elicit a very rapid response upon starvation where a subset of proteins are degraded. Among these is p62 and several other selective autophagy receptors. They are degraded via two routes. The most rapid one is via organelles called late endosomes. Together with researchers from the Radium Hospital and from Germany we found that p62 exists as filaments inside membraneless round bodies in cells. These filaments are vital for p62 to get cellular garbage degraded by autophagy. Earlier we identified a LIR (LC3 interacting region) motif which p62 uses to bind to ATG8 proteins attached to the membrane of the forming autophagosome. Autophagosomes fuse with lysosomes, and the content is degraded and recycled. We found that several proteins needed to form autophagosomes bind to ATG8 proteins via LIR motifs. This places them in position to facilitate formation of autophagosomes. We showed this for ATG4B and three members of class III phosphatidylinositol 3-kinase complex I (ATG14L, BECLIN1 and VPS34). All are needed to form autophagosomes. Together with Sharon Toozes group in London we obtained detailed structural information on how these proteins bind to ATG8 proteins. We also found that several protein kinases have LIRs, bind to ATG8 proteins and can phosphorylate the binding site on the ATG8 protein LC3B. This way the kinase NEK9 inhibits the degradation of p62 by autophagy. We have in this project proudly discovered four new receptors for degradation of mitochondria by autophagy: FKBP8, SAMM50, NIPSNAP1 and -2. We also discovered CALCOCO1 as a novel autophagy receptor for degradation of endoplasmic reticulum (ER) and parts of the Golgi apparatus upon starvation. FKBP8 helps remove damaged mitochondria, but escapes itself destruction. Mitochondria are the power stations of the cells and produces energy needed. Mitochondria acquire damage to their machinery leading to release of toxic oxygen radicals which damage DNA and other cellular components. The damaged mitochondria needs to be recognized and eliminated. Collaborating with Anne Simonsens group in Oslo we found that the proteins NIPSNAP1 and -2 act as eat me signals for damaged mitochondria so that they are seen and degraded by autophagy. In healthy mitochondria NIPSNAP1 and -2 are inside, while in damaged mitochondria they sit outside and attract autophagy receptors. We found that SAMM50 helps remove worn out parts making up a structure required for efficient energy production in mitochondria by a piecemeal autophagy process. When mitochondria work maximally SAMM50 cooperates with SQSTM1 to mediate efficient piecemeal mitophagy to keep the cells healthy. Our cells respond to stress by making more of ER and Golgi, membrane structures important for folding and secretion of proteins. To return to normal the surplus must be degraded. We found that CALCOCO1 is a new autophagy receptor removing surplus ER and parts of Golgi. This is the first discovery of a receptor for autophagy of the Golgi.

Målgruppen for våre resultater er først og fremst andre forskere som studerer autofagi og forskere som ser på rollene til autofagi i alvorlige sykdommer som kreft, nevrodegenererende sykdommer, betennelsestilstander, virusinfeksjoner og innate immunitet. De langsiktige effektene tror vi kan bli betydelige. Vi bygger en kunnskapsbase som kan utnyttes i framtidig forskning på de store folkesykdommene og utvikling av nye medikamenter. Autofagiforskning vil bli veldig viktig i sunn aldring og i kampen mot demens og nevrodegenerende sykdommer i en befolkning som stadig blir eldre. Ser vi på hva våre tidligere funn har ført til så kan det nevnes kliniske studier av kreft og nevrodegenererende sykdommer der p62 er en viktig biomarkør. Det er også utviklet en DNA vaksine mot kreft med p62 plasmid som testes i forsøksdyr. Prosjektdeltakerne har økt sin tekniske forskningskompetanse, lært nye metoder og fått betydelig innsikt i rollene til autofagi og særlig selektiv autofagi i cellene våre.

An important challenge for our cells is to properly regulate the potent signalling pathways affecting differentiation, proliferation, inflammation, and cell death. Dysregulation is implicated in development of diseases like cancer, diabetes, neurodegeneration, and inflammatory disorders. Signalosomes are multimolecular protein complexes that facilitate sensitive and efficient signaling. They integrate upstream signals and control downstream effectors, and play important roles in cellular behavior and adaptation during various stress conditions, differentiation, inflammation and proliferation. Autophagy is an evolutionary conserved cellular process that serves as a mechanism for degradation and recycling of dysfunctional proteins, damaged or aged organelles, and intracellular pathogens. Recent discoveries implicate important roles of autophagy in specific physiological and pathological processes such as development, immunity, energy homeostasis, cell death and tumorigenesis. Our work on the scaffold and signaling protein p62 led to the discovery of selective autophagy at the molecular level, mediated by specific proteins termed autophagy receptor proteins. Triggering cellular stress conditions by starvation, ROS or protein aggregation, results in rapid formation of dynamic p62 bodies. Our hypothesis is that these bodies represent p62-mediated signalosomes, and that p62 and p62 like autophagy receptors have a pivotal role in regulating formation, signal integration, transmission, and termination of the associated signalosomes. To evaluate this hypothesis we will characterise the signaling events, the key signaling components, the structural requirements, and the role of selective autophagy in p62 signalosomes. This new knowledge on selectivity and specificity associated with autophagy-regulated signaling will be applied to identify new therapeutic targets for neurodegenerative diseases, tumorigenesis and inflammation.