Research and publications

• Research projects

  Phosphorylation-based regulation of autophagy
  Status: Ongoing

  Start	01.04.2023
  End	31.03.2027
  Funding	SNSF  Open project sheet
  The term autophagy summarizes constitutive and stress-induced lysosomal degradation pathways, which target cytoplasmic material and commonly function cytoprotective. A proper regulation of autophagy is vital for human health, as its dysregulation has been linked to numerous diseases, amongst others to metabolic diseases, cancer and protein aggregation-linked neurodegeneration. Autophagy induction is mainly organized by posttranslational mechanisms and the function of protein as well as of lipid kinases critical for autophagy initiation is rather well understood. We know much less about the function of protein phosphatases in autophagy induction. In the current project, we would like to study the function and interplay between autophagy and protein phosphatase (PP) 1 (PP1) and PP6 signaling with the help of mass spectrometry (MS)-based (phospho)proteomics. By studying the functions and interactions of the ULK1/2 kinase complex, the most proximal kinase complex critical for initiation of canonical autophagy, we recently identified PP1, PP2A and PP6 complex members as interacting partners and/or bona fide targets of ULK1. Whereas we could show that the PP2A complex member Striatin is involved in a positive feedback being activated by ULK1 and in turn removing inhibitory phosphorylation sites on ULK1 itself, we do not know how ULK1 modulates PP1 and PP6 complexes and activities. Using a combination of biochemical in vitro assays and in vivo signal transduction studies all relying in part on MS-based (phospho)proteomics as readout, we would like to study the role of ULK1 target sites on PP6 complex members and the regulation of PP1 complex interactions under autophagy. As regulatory subunits of protein phosphatases are being recognized as promising drug targets, our results will hold the potential to design phosphatase-based autophagy modulating therapeutic approaches.

  Replacement of xenograft mouse models by molecularly-defined 3D in vitro systems
  Status: Ongoing

  Start	01.10.2022
  End	30.09.2026
  Funding	SNSF  Open project sheet
  Xenograft mouse models, i.e. immunodeficient mice into which fresh biopsies of human tumours or tumour cell lines are transferred, are frequently used in drug development and personalized cancer therapy. In the current project, we will utilise cells isolated from human tumour biopsies to establish in vivo-like, molecularly and biophysically defined 3D in vitro cell culture models to reduce and ideally replace xenograft mouse models. As model disease we will use squamous cell carcinoma (SCC) of the skin, which belongs to the group of non-melanoma skin cancers that are by far the most frequent types of cancer. In an iterative fashion, we will use state-of-the-art ´omics´ approaches to comprehensively characterize the molecular composition of human tumour biopsies, compare them to xenograft specimens, characterize their biophysical properties and reconstruct 3D in vitro models that perfectly mimic the in vivo setup. In a proof-of-concept, these models will be used to study the effects of genetic and pharmacological perturbation on cancer cell survival, proliferation, and migration. By establishing robust in vivo-like 3D cell culture models we will increase throughput and ensure high experimental reproducibility.

  Metabolic reprogramming by selective autophagy
  Status: Completed

  Start	01.02.2020
  End	31.12.2024
  Funding	SNSF  Open project sheet
  Background: Autophagy is a highly conserved eukaryotic process for protein and organelles degradation. This catabolic pathway plays a crucial role in quality control and repair mechanisms necessary to maintain cellular homeostasis. Another key function of autophagy is to mediate metabolic adaptation and survival under stress and limited nutrient conditions, by degrading and recycling cytoplasmic components, and generating an internal pool of metabolic resources. While autophagic degradation was often considered to be non-selective, recent evidence has shown that autophagy can be highly selective. It remains largely unexplored, however, whether and under which conditions selective autophagy is necessary and/or sufficient to mediate the adaptation to environmental changes and scarce nutrients. Crucially, autophagy dysregulation has been implicated in various human diseases, including cancer, and hence a more systematic and quantitative understanding of how selective autophagy participates to cellular metabolic reprogramming can reveal conserved metabolic regulatory mechanisms and open new opportunities for therapeutic treatments. Main goal and research approach: The main and breakthrough objective of this project is to dissect the role of selective autophagy in metabolic adaptation to nutrient shifts. We will start by systematically investigating autophagy-mediated metabolic regulation in yeast Saccharomyces cerevisiae. In particular, we will monitor cargo material, i.e., proteins and organelles, selectively delivered to vacuoles by autophagosomes during dynamic transitions of different carbon sources and from rich to poor nutrient conditions, by using a combination of genetic techniques and quantitative mass spectrometry-based proteomics. Simultaneously, we will use high-throughput metabolomics to measure dynamic changes in extra- and intra-cellular metabolites, and resolve the metabolic rewiring during the studied nutrient transitions. Next, we will integrate proteomics and metabolomics datasets by using constraint-based and kinetic models to identify metabolic reactions regulated by selective degradation of enzymes or enzyme regulators (e.g., transcription factors) by autophagy. Model-based predictions and relevance for cell survival will then be tested by point mutating these enzymes or regulators to specifically inhibit their down-regulation by selective autophagy. Translational relevance: As autophagy and metabolic pathways are highly conserved among eukaryotes, we expect that discoveries obtained with yeast will be applicable to other systems. Thus, we will focus on those enzymes and regulators that are targeted by selective autophagy and have a central role in metabolic adaptations in yeast, and study them in human cancer cells. Cancer cells are often subjected to metabolic reprogramming and/or nutrient starvation in vivo, and as a result selective autophagy could play an important regulatory role. We have opted for colorectal cancer models because of their high medical relevance and our expertise. We will first confirm which of the yeast key enzymes and regulators are also mammalian autophagy substrates. Subsequently, we will deplete and point mutate those proven to be autophagosomal cargoes using knock-out and knock-in technologies, and study their relevance in metabolic reprogramming and cell survival during transformation or when cancer cells are subjected to nutritional shifts. Scientific and biomedical impact: The realization of this project will reveal how selective autophagy contributes to metabolic reprogramming, not only to adapt to nutrient changes, but also in cancer pathophysiology and eventually other processes in which selective autophagy has been shown to be important, such as cell differentiation and development.

  Functional characterisation of dynamic BRAF signalling complexes and their modulation by tumour specific mutations and clinically relevant kinase inhibitors
  Status: Completed

  Start	01.06.2019
  End	31.12.2022
  Funding	SNSF  Open project sheet
  BRAF plays a central role in the activation of RAS/ERK signaling. The activation cycle of this kinase is driven by RAS induced homo- or hetero-dimerization and tightly controlled by protein-protein interaction events and post-translational modifications (PTMs). BRAF is often dysregulated in cancer. The most common mutation, V600E, cuts the incompletely understood BRAF activation cycle short. Thereby, this mutation generates an oncoprotein in which its kinase domain maintains an active conformation that is only transiently assumed by wildtype BRAF (BRAFWT) following RAS induced activation. This allowed the development of BRAFV600E selective inhibitors that yield impressive initial response rates in various entities. Unfortunately, therapeutic responses are short-lived due to the emergence of drug resistance. BRAF inhibitor induced paradoxical ERK pathway activation represents a common resistance mechanism. This phenomenon is caused by the unforeseen property of clinically applied BRAF selective inhibitors to promote heterodimers between drug-bound BRAF and other RAF isoforms in the presence of RAS activity. Drug-bound BRAF acts as a potent allosteric activator of the drug-free RAF protomer, thereby causing ERK re-activation and tumor growth. It is likely that the paradoxical action of BRAF inhibitors exploits processes occurring during physiological RAS/ERK pathway activation. In order to develop more effective and safer inhibitors, it will be critical to understand the spatio-temporal dynamics of quaternary BRAF signaling complexes in both physiological and pharmacological settings. Using Blue Native PAGE and SEC-PCP-SILAC based mass spectrometry (MS), we demonstrated that BRAFWT and BRAFV600E organize multi-protein complexes of distinct size and composition. We also showed that RAS induces BRAFWT containing complexes of similar size as those formed by BRAFV600E. Moreover, clinically relevant drugs affect the stability of these complexes, e.g. in settings with desired or paradoxical effects of BRAF inhibitors. Based on this and other data, we posit that the activity status of the kinase domain dictates the assembly of BRAF signaling complexes. In the proposed project, we aim to confirm this hypothesis by conducting an in-depth characterization of the composition and PTM pattern of BRAF complexes formed under physiological conditions and in the presence of kinase inhibitors of (pre)clinical relevance. We will combine our MS protocols with novel biochemical approaches to identify short-lived dynamic interactions. We will extend our studies to complexes formed by selected non-V600E BRAF oncoproteins, which are increasingly detected by personalized medicine programs.

  Stimulus- and time-dependent protein dynamics in autophagy analyzed by quantitative mass spectrometry
  Status: Completed

  Start	01.04.2019
  End	31.03.2023
  Funding	SNSF  Open project sheet
  Macroautophagy, hereafter referred to as autophagy, is a cellular recycling pathway. We investigate molecular mechanisms regulating, or regulated by autophagy with the help of quantitative mass spectrometry (MS)-based proteomics. Next to the ubiquitin/proteasome system, the autophagosomal/lysosomal system is responsible for the majority of cellular proteolysis. Autophagy is a basic cellular degradation pathway but also a cytoprotective stress-response. Its dysregulation is involved in numerous human diseases, such as neurodegenerative disorders and cancer. We are interested in regulatory protein-protein interactions, especially in underlying posttranslational protein modifications in stress-induced autophagy. For this, we follow a two-tiered strategy: (i) we investigate proteins known to be involved in autophagy regulation, and (ii) perform MS-based proteomics screens for the identification of new proteins being critical for stress-induced autophagy. Our main model system are human cell lines and primary cells. We are specifically interested in kinases regulating autophagy, e.g. Unc-51-like kinase 1 (ULK1) and MTORC1. MTORC1 is a master regulator of cellular homeostasis and negatively regulated autophagy by phosphorylating ULK1. Next to phosphorylation-based signaling, we study ubiquitination-based signaling and its role in autophagosomal cargo selection. In parallel, we perform unbiased approaches studying protein-protein interactions in autophagy inducing conditions to identify new autophagy regulatory proteins. By combining unbiased screening approaches with targeted MS analyses and development of new MS-based methods, we aim to characterize new molecular mechanisms regulating autophagy and being important for human health.

  Characterization of Biomolecules by Mass Spectrometry
  Status: Completed

  Start	01.01.2018
  End	31.12.2018
  Funding	SNSF  Open project sheet
  January 2017 the joined Metabolomics/Proteomics Platform (MAPP) of the Department of Biology, University of Fribourg was officially launched (https://www.unifr.ch/go/mapp). The aim of MAPP is to provide all research groups of the University of Fribourg and the associated NCCR Bio-Inspired Materials with mass spectrometry (MS) expertise. So far, the offered services include quantitative expression proteomics analyses, analyses of protein-protein interactions by affinity-purification (AP)-MS, characterization of posttranslational modifications (PTMs) including phosphorylation and ubiquitination, and the analysis of lipids and lipophilic metabolites. Currently MAPP is equipped with two liquid chromatography (LC)-MS/MS systems, of which one, a 12 year old LTQ-Orbitap Classic, is outdated and only used for method development. Thus, almost the entire workload is handled by one Q-Exactive Plus system, which creates a serious bottleneck in sample throughput and causes increasingly long waiting times. We apply for a new generation Thermo Scientific Q-Exactive HF system offering enhanced resolution and sequencing speed. We exemplify with eight diverse research projects that this state-of-the-art system is essential for the future functioning of MAPP and for the entire natural science community in Fribourg: Project 1 characterizes ubiquitination-dependent signaling events in stress-induced autophagy employing human cell culture systems. Autophagy is an essential cellular turnover path-way often deregulated in human diseases such as cancer and neurodegeneration. Project 2 studies phosphorylation-dependent signaling events regulating cell growth. Using yeast Saccharomyces cerevisiae as model the TORC1 signaling network is analyzed mechanistically. In Project 3 we employ crosslinking MS to study the topology of membrane proteins and how it changes upon movement from the lipid bilayer of the endoplasmic reticulum onto the putative monolayer membrane of lipid droplets. Project 4 deals with the role of the transcription factor Creb (cAMP-response-element binding protein) in long-term memory formation using Drosophila melanogaster as model system. In Project 5 top-down MS is applied to study the regulation of small molecular neurotransmitters, neuropeptides and proteins in animal models of major depression disorder. Project 6 characterizes organelles and protein-protein interactions important in arbuscular mycorrhizal symbiosis in the model plant Petunia hybrid. In Project 7 we investigate the antimicrobial effects of silver ions and silver nanoparticles and how the gram-negative bacterium Geobacter sulfurreducens copes with it. Project 8 aims at characterizing the cellular uptake of newly developed nanoparticles and their interaction with serum and cellular proteins. Thus, with these projects we cover the major research areas in Fribourg having need for a state-of-the-art LC-MS/MS system and highlight the importance of MAPP for the research location Fribourg.

  Stimulus and time-dependent protein dynamics in autophagy analyzed by quantitative mass spectrometry
  Status: Completed

  Start	01.04.2016
  End	31.03.2019
  Funding	SNSF  Open project sheet
  We investigate stimulus- and time-dependent protein dynamics in active macroautophagy, hereafter referred to as autophagy, by quantitative mass spectrometry (MS)-based proteomics and delineate molecular, autophagy-relevant processes under physiological conditions. The autophagosomal/lysosomal system is next to the ubiquitin/proteasome system responsible for the majority of cellular proteolysis. It is a basic cellular degradation pathway whose deregulation is involved in numerous diseases, inter alia in cancer and neurodegenerative disorders. The aim is to mechanistically characterize protein-protein interactions and posttranslational protein modifications in stress-induced autophagy in order to foster the design of therapeutic strategies addressing autophagy in diverse disease settings. We (a) target proteins identified to be important for autophagy regulation under physiological conditions, and (b) perform proteomics screens to identify new proteins important in stress-induced autophagy. Using human cells, we study the crosstalk between protein translation and autophagy by targeted approaches. In addition, the roles of kinases, e.g. Unc-51-like kinase 1 (ULK1), in autophagy regulation are analyzed. ULK1 is a central kinase linking growth factor, nutrient and energy sensing. Complementary to these targeted approaches, we globally characterize the dynamics of macromolecular multiprotein complexes during active autophagy to identify so far unknown autophagy-relevant, regulatory proteins and underlying molecular mechanisms. The combination of targeted and unbiased approaches and development of new analytical methods using MS-based proteomics will allow us to identify new mechanisms of autophagy regulation, having potential implications on human pathophysiology.

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