FRIAS Project: Spatio-temporal protein dynamics during autophagy
Cells possess two major degradation pathways: the ubiquitin/proteasome system and the autophagosomal/lysosomal system. Autophagy is an evolutionary conserved process wherein catabolism of cytoplasm generates energy which allows cell survival under condition of reduced nutrient availability. Autophagy is initiated by a flat membrane cistern enwrapping parts of the cytoplasm, thus, forming an autophagosome with a characteristic double membraned organization. The autophagosome matures in a stepwise process which may involve fusion with endosomal vesicles, before it finally fuses with the lysosome leading to degradation of the autophagosomal material.
Autophagy is thought to be important for the turn-over of whole organelles and long-lived proteins. However, prolonged autophagy can lead to type II programmed cell death. Manny aspects of autophagy regulation are still not fully understood. The best-characterized inhibitory pathway includes a class I PI3K and mTOR.
|On the other hand, a class III PI3K is needed for autophagy activation. Autophagy has been linked to several diseases amongst others cancer and neurodegenerative diseases. ||Schematic depiction of autophagosomal maturation and important underlying signaling pathways. We focus on four research areas aiming to decipher basic cell biological processes involved in autophagy. We characterize signaling networks by global phosphoproteomics approaches (1). In a targeted approach we investigate the influence of autophagy on the plasma membrane (2). To understand cargo selection we study inter-organelle crosstalk and autophagosomal dynamics inter alia after virus infection (3). Using cell culture and animal models we investigate the influence of autophagy on protein turnover, synthesis, degradation, and the global constitution of the proteome (4).|
|Furthermore, autophagy is regarded as an unspecific bulk degradation pathway. However, in a recent study we analyzed protein dynamics during amino acid starvation and found that the subcellular localization of proteins had an influence on their degradation dynamics. Proteins were degraded in an ordered fashion, where cytosolic proteins and proteins involved in translation were degraded initially, followed by multiprotein complexes and proteins situated in organelles. |
Looking at proteasomal and lysosomal degradation ample cross-talk between the two degradation pathways became evident. Our data implies that protein degradation during starvation-induced autophagy is far from being unspecific, and is rather tightly regulated.
We are interested in cellular pathways which are deregulated in cancer. One of our focuses is autophagy which is thought to be primarily a pro-survival response helping cancer cells to survive if only limited nutrition supplies are available. We characterize autophagy using a combination of techniques including quantitative mass spectrometry (MS)-based proteomics, confocal-imaging, RNA interference (RNAi), and modeling. Currently, we study signal transduction kinetics during autophagy by quantitative phosphoproteomics approaches to compare signaling events involved in autophagy and in type I programmed cell death pathways (apoptosis). Although the two processes are morphologically distinct, they are both characterized by lack of tissue inflammatory responses and may share signaling pathways. A special focus is the interplay between autophagosomes and the plasma membrane. By organellar proteomics we investigate the autophagosome, the double-membrane bound vacuole containing cytoplasmic material destined for degradation, with the aim to identify human proteins related to autophagy. We are also interested in global protein dynamics during long-term starvation to characterize the influence of different types of autophagy, macroautophagy and chaperone-mediated autophagy (CMA), on the cellular proteome.
To reveal new components in the analyzed organelle and signaling networks we are using MS-based proteomics in combination with stable isotope labelling by amino acids in cell culture (SILAC). SILAC is a quantitative proteomic strategy that metabolically labels the entire proteome, thus, making it distinguishable by MS analysis. Different populations of cells can be grown in medium containing distinct forms of arginine (Arg) and lysine (Lys). Subsequently, cell populations can be mixed and analyzed in one MS experiment. This allows the quantitation of proteins from different cellular states. Depending on the setup we are able to describe an organellar proteome or to follow site-specific phosphorylation changes in signaling pathways over a certain timeframe. The newest mass spectrometers allow specific screening for phosphopeptides on a routine basis. As sensitivity is down to the subfemtomolar range it is now possible to perform systemic analyses on as few as 10^5 cells.