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Chemical biology of autophagy

Final Report Summary - CHEMBIO_ATG (Chemical biology of autophagy)

Autophagy is a cellular pathway that regulates the degradation and recycling of proteins and organelles. A normal function of this process is crucial to maintain cell survival under starving conditions, to prevent pathogen infection or to eliminate protein aggregates. However, there are growing evidences that a malfunctioning of autophagy is also related with several pathologies and associated with aging.
The Atg8 family of ubiquitin-like proteins are the best characterized markers of autophagy. Theses protein exists in two forms, a soluble and a lipidated, bearing a phosphatidylethanolamine (PE) unit at the C-terminus, that associates with the membrane of the autophagosome and regulates its elongation and closure. In mammalian cells, there are six Atg8 orthologues that are divided into the LC3 (LC3A, LCB, LC3C) and GABARAP (GABARAP, GABARAPL1, and GABARAPL2) subfamilies.
The main goal of this project was the development of a chemical biology approach that enables the characterization of the molecular mechanism controlling autophagy with special emphasis on the role of the lipid posttranslational modification of LC3-II on the regulation of protein function and localization. With this aim, we have generated a semisynthetic lipidated LC3 protein and it has been employed to investigate the function of LC3-II in membrane tethering and fusion processes. Another part of the project was directed to characterize the PEs species bound to LC3-II. Hence, PEs can contain two different fatty acids, one at the position sn-1 that tends to be saturated or monounsaturated and another one at the sn-2 position, often mono- unsaturated or polyunsaturated. This diversity in lipid composition may have a strong impact on protein function. Hence, saturated fatty acids make membranes thicker whereas unsaturated lipids increase their fluidity. Moreover, whereas DOPE is considered a cone-shaped lipid, saturated lipids such as DPPE are considered cylindrical. This shape together with the spontaneous curvature caused by PEs may strongly influence membrane fission and fusion processes. As a result, our aim is to investigate the structural diversity in PEs residues on LC3-II and their consequences on protein localization and function. To achieve this, we will set-up a lipidomics approach based on mass-spectroscopy techniques and employed it to analyse the chemical diversity of the lipids present in LC3-II.
Knowing in more detail the molecular mechanisms regulating autophagy remain critical in fighting age-related disorders and improving healthspan in the aging population. Despite the recent advances seen in the field of autophagy in the last decade, there are many questions that remain unanswered. We expect that the advances seen during this project may contribute to our better understanding of the physiology and pathology of autophagy.