Prof. Dr. Marcus Krüger

Research Area: Quantitative proteomics

Website: http://krueger.cecad-labs.de

1. Research Background:

The increasing number of people with type 2 diabetes is mainly due to insulin resistance as a dominant symptom. In addition, this resistance is not only the driving force behind type 2 diabetes, but is also associated with many other diseases such as high blood pressure, obesity, skeletal muscle remodeling, and neurodegeneration.

Enhanced insulin secretion induces the activation of the insulin signalling pathway, translocate GLUT4 vesicles to the plasma membrane, allowing them to transport glucose into the cell. The cascade starts with the activation of the insulin receptor and induces the phosphorylation of signalling molecules, including IRS1, PI-3K, and the AKT kinase as the key regulator for glucose transport. So far much effort has been invested in identifying proteins that impair intracellular insulin signalling, which can lead to the development of insulin resistance. Using state-of-the-art mass spectrometry in combination with transgenic diabetes mouse models, the Krüger laboratory is interested to decipher insulin-dependent cellular networks under regular and disease-related conditions. In particular, we ask which posttranslational modifications (PTMs) are associated with the insulin signaling and control glucose uptake, protein expression and metabolic activity. In addition, we are interested how insulin regulates the cross-talk to other signaling cascades.

2. Research questions addresses by the group:

  1. Decipher the insulin-dependent network and its posttranslational modifications by quantitative mass spectrometry.
  2. How do kinases and insulin-dependent E3 ubiquitin ligases control glucose uptake and cellular homeostasis?
  3. How does insulin stimulation control protein-protein interactions?

3. Possible projects:

  1. How does the inactivation of insulin-dependent E3-ligases affect glucose uptake in living animals?
    F-box proteins are important components of the Skp1-Cullin1-F-box (SCF) E3 ubiquitin ligase complex and they bind substrates for ubiquitin-mediated proteolysis. We previously found that the ablation of an F-box E3-ligase influences the insulin signaling and glucose uptake in mice. To explore the physiological role of the E3-ligase in more detail, we will generate a knock out mouse to examine whether the inactivation will also influence insulin sensitivity and glucose uptake in living animals. The enrichment of PTMs such as phosphopeptides and di-glycine remnants in combination with mass spectrometric analysis will help to identify dysregulated signaling molecules and assess the function of insulin-dependent E3-ligases.   
  2. High-throughput protein-protein interaction study in adipocytes
    To identify novel components and modulators of the insulin signaling pathway, we will perform a global protein-protein interaction study based on affinity enrichment (AE-MS) and cross mass-linker mass spectrometry (XL-MS) in white and brown adipocytes. We have generated more than 120 expression constructs for insulin associated signaling molecules and a high-throughput interactome screen based on SILAC quantification and high resolution mass spectrometry will allow us to obtain a reliable set of protein-protein interactions. These dataset will decipher the core network underlying the insulin signaling pathway and will endeavor novel signaling molecules belonging to the insulin signaling pathway.

4. Applied Methods and model organisms:

Both projects focusing on high resolution mass spectrometry using quadrupole Orbitrap (QExactive HF-X) mass spectrometer. Sample preparation and PTM enrichment will be achieved with chromatographic separation techniques, including size exclusion and high pH reversed phase chromatography. In addition, kinase motif antibodies will be used to enrich specific phosphorylation sites to gain a more comprehensive view on activated signaling pathways. Bioinformatics analysis using different software tools will be an important part of the project to analyse the complex datasets. Protein quantification will be achieved by the stable isotope labeling of amino acids in cell culture (SILAC) approach, with in vivo SILAC, and with label free protein quantification.

Transgenic mouse models are established in the laboratory and further models based on the Crisper/Cas9 approach will be generated upon demand. Standard immunohistochemistry (IHC) methods and RNA hybridization will be used to visualize protein and gene expression changes. In collaboration with the laboratory of Jens Brüning from the MPI for Metabolomics, we will perform hyperinsulinemic-euglycemic clamp experiments to assess insulin sensitivity in vivo.

In the protein-protein interaction project we will also use an Orbitrap Fusion Lumos mass spectrometer which allows different peptide fragmentation modes such as Collision-induced Dissociation (CID) and Electron-transfer/higher-Energy Collision Dissociation (EThcD) to identify cross-linked proteins. In collaboration with the laboratory of Fan Liu (Potsdam), we have established the XL-MS approach, which used different fragmentation techniques for optimized identification of direct protein-protein interactions.

5. Desirable skills and qualifications:

Basic knowledge in cell and molecular biology, quantitative proteomics and interest in bioinformatics analysis of large-scale datasets.

6. References:

  • Wiederstein, J.L., Nolte, H., Günther, S., Piller, T., Baraldo, M., Kostin, S., Bloch, W., Schindler, N., Sandri, M., Blaauw, B., Braun, T., Hölper, S., Krüger, M. (2018). Skeletal Muscle-Specific Methyltransferase METTL21C Trimethylates p97 and Regulates Autophagy-Associated Protein Breakdown. Cell Reports 23(5), 1342-56.
  • Lang, F., Aravamudhan, S., Nolte, H., Türk, C., Hölper, S., Müller, S., Günther, S., Blaauw, B., Braun, T., Krüger, M. (2017). Dynamic changes in the mouse skeletal muscle proteome during denervation-induced atrophy. Dis Model Mech. 10(7), 881-96.
  • Islam, S., Nolte, H., Jacob, W., Ziegler, A.B., Pütz, S., Grosjean, Y., Szczepanowska, K., Trifunovic, A., Braun, T., Heumann, H., Heumann, R., Hovemann, B., Moore, D.J., Krüger, M. (2016). Human R1441C LRRK2 regulates the synaptic vesicle proteome and phosphoproteome in a Drosophila model of Parkinson's disease. Hum Mol Genet. 25(24), 5365-82.
  • Hölper, S., Nolte, H., Bober, E., Braun, T., Krüger, M. (2015). Dissection of metabolic pathways in the Db/Db mouse model by integrative proteome and acetylome analysis Molecular BioSystems 3, 908-22.