Andrea C. Becker

Identification of Autophagosome-associated Proteins and Regulators by Quantitative Proteomic Analysis and Genetic Screens

Autophagy is one of the major intracellular catabolic pathways, but little is known about the composition of autophagosomes. To study the associated proteins, we isolated autophagosomes from human breast cancer cells using two different biochemical methods and three stimulus types: amino acid deprivation or rapamycin or concanamycin A treatment. The autophagosome-associated proteins were dependent on stimulus, but a core set of proteins was stimulus-independent. Remarkably, proteasomal proteins were abundant among the stimulus-independent common autophagosome-associated proteins, and the activation of autophagy significantly decreased the cellular proteasome level and activity supporting interplay between the two degradation pathways. A screen of yeast strains defective in the orthologs of the human genes encoding for a common set of autophagosome-associated proteins revealed several regulators of autophagy, including subunits of the retromer complex. The combined spatiotemporal proteomic and genetic data sets presented here provide a basis for further characterization of autophagosome biogenesis and cargo selection.

ErbB2-associated changes in the lysosomal proteome

Late endosomes and lysosomes (hereafter referred to as lysosomes) play an essential role in the turnover of cellular macromolecules and organelles. Their biochemical characterization has so far depended on purification methods based on either density gradient centrifugations or magnetic purification of iron‐loaded organelles. Owing to dramatic changes in lysosomal density and stability associated with lysosomal diseases and cancer, these methods are not optimal for the comparison of normal and pathological lysosomes. Here, we introduce an efficient method for the purification of intact lysosomes by magnetic immunoprecipitation with antibodies against the vacuolar‐type H+‐ATPase. Quantitative MS‐based proteomics analysis of the obtained lysosomal membranes identified 60 proteins, most of which have previously been associated with the lysosomal compartment. Interestingly, the lysosomal membrane proteome was significantly altered by the ectopic expression of an active form of the ErbB2 oncogene, which renders the cells highly metastatic. The furthermost ErbB2‐associated changes included increased levels of CD63, S100A11 and ferritin heavy chain. Overall, our data introduce the antibody‐based purification of lysosomes as a suitable method for the characterization of lysosomes from a variety of pathological conditions with altered lysosomal density and stability.

Characterization of early autophagy signaling by quantitative phosphoproteomics

Under conditions of nutrient shortage autophagy is the primary cellular mechanism ensuring availability of substrates for continuous biosynthesis. Subjecting cells to starvation or rapamycin efficiently induces autophagy by inhibiting the MTOR signaling pathway triggering increased autophagic flux. To elucidate the regulation of early signaling events upon autophagy induction, we applied quantitative phosphoproteomics characterizing the temporal phosphorylation dynamics after starvation and rapamycin treatment. We obtained a comprehensive atlas of phosphorylation kinetics within the first 30 min upon induction of autophagy with both treatments affecting widely different cellular processes. The identification of dynamic phosphorylation already after 2 min demonstrates that the earliest events in autophagy signaling occur rapidly after induction. The data was subjected to extensive bioinformatics analysis revealing regulated phosphorylation sites on proteins involved in a wide range of cellular processes and an impact of the treatments on the kinome. To approach the potential function of the identified phosphorylation sites we performed a screen for MAP1LC3-interacting proteins and identified a group of binding partners exhibiting dynamic phosphorylation patterns. The data presented here provide a valuable resource on phosphorylation events underlying early autophagy induction.

The Degradative Inventory of the Cell: Proteomic Insights

SIGNIFICANCE Protein degradation has been identified as being deregulated in numerous human diseases. Hence, proteins involved in proteasomal as well as lysosomal degradation are regarded as interesting potential drug targets and are thoroughly investigated in clinical studies. RECENT ADVANCES Technical advances in the field of quantitative mass spectrometry (MS)-based proteomics allow for detailed investigations of protein degradation dynamics and identifications of responsible protein-protein interaction networks enabling a systematic analysis of the degradative inventory of the cell and its underlying molecular mechanisms. CRITICAL ISSUES In the current review we outline recent technical advances and their limitations in MS-based proteomics and discuss their use for the analysis of protein dynamics involved in degradation processes. FUTURE DIRECTIONS In the next years the analysis of crosstalk between different posttranslational modifications (PTMs) will be a major focus of MS-based proteomics studies. Increasing evidence highlights the complexity of PTMs with positive and negative feedbacks being discovered. In this regard, the generation of absolute quantitative proteomic data will be essential for theoretical scientists to construct predictive network models that constitute a valuable tool for fast hypothesis testing and for explaining underlying molecular mechanisms.

Degradation of protein translation machinery by amino acid starvation-induced macroautophagy

ABSTRACT Macroautophagy is regarded as a nonspecific bulk degradation process of cytoplasmic material within the lysosome. However, the process has mainly been studied by nonspecific bulk degradation assays using radiolabeling. In the present study we monitor protein turnover and degradation by global, unbiased approaches relying on quantitative mass spectrometry-based proteomics. Macroautophagy is induced by rapamycin treatment, and by amino acid and glucose starvation in differentially, metabolically labeled cells. Protein dynamics are linked to image-based models of autophagosome turnover. Depending on the inducing stimulus, protein as well as organelle turnover differ. Amino acid starvation-induced macroautophagy leads to selective degradation of proteins important for protein translation. Thus, protein dynamics reflect cellular conditions in the respective treatment indicating stimulus-specific pathways in stress-induced macroautophagy.

The Atypical Kinase RIOK1 Promotes Tumor Growth and Invasive Behavior

Despite being overexpressed in different tumor entities, RIO kinases are hardly characterized in mammalian cells. We investigated the role of these atypical kinases in different cancer cells. Using isogenic colon-, breast- and lung cancer cell lines, we demonstrate that knockdown of RIOK1, but not of RIOK2 or RIOK3, strongly impairs proliferation and invasiveness in conventional and 3D culture systems. Interestingly, these effects were mainly observed in RAS mutant cancer cells. In contrast, growth of RAS wildtype Caco-2 and Bcr-Abl-driven K562 cells is not affected by RIOK1 knockdown, suggesting a specific requirement for RIOK1 in the context of oncogenic RAS signaling. Furthermore, we show that RIOK1 activates NF-κB signaling and promotes cell cycle progression. Using proteomics, we identified the pro-invasive proteins Metadherin and Stathmin1 to be regulated by RIOK1. Additionally, we demonstrate that RIOK1 promotes lung colonization in vivo and that RIOK1 is overexpressed in different subtypes of human lung- and breast cancer. Altogether, our data suggest RIOK1 as a potential therapeutic target, especially in RAS-driven cancers.

Autophagosomal Proteome Analysis by Protein Correlation Profiling-SILAC

Autophagy is one of the two major degradation pathways within eukaryotic cells. Nevertheless, little is known about the protein composition of autophagosomes, the vesicles shuttling proteins to lysosomes for degradation. Protein correlation profiling in combination with stable isotope labeling by amino acids in cell culture is a stringent method to investigate the dynamics of the autophagosomal proteome. It enables the discrimination between autophagosomal and co-purifying proteins identifying organellar candidate proteins for further investigation.

Friend or food: different cues to the autophagosomal proteome.

A hallmark of macroautophagy is the formation of autophagosomes, double-membrane vesicles that enwrap cellular components destined for lysosomal degradation. We examined autophagosomal protein dynamics under various inducing stimuli using a comprehensive mass spectrometry-based proteomics approach in combination with functional studies in yeast and human cell cultures. Time frame and stimuli type influenced the autophagosome proteome, underlining the dynamic constitution of the organelle. We identified both a core set of proteins always localizing to autophagosomes and stimulus-dependent components that will serve as a resource for further characterization of the autophagosomal machinery and cargo selection. Among the core proteins were newly discovered autophagy regulators found to be conserved from yeast to humans, as well as the proteasome.