Affiliation
Institute of Chemical Epigenetics
LMU München
Faculty of Chemistry and Pharmacy
Contact
D-81375 München
Email:
pavel.kielkowski@cup.lmu.de
Website:
https://www.cup.lmu.de/oc/kielkowski/
Website:
https://www.ice-m.de/
Research Focus
1. Project title:
Discovering the connection between tyrosination and other microtubule modifications using chemoproteomics.
Project description:
Post-translational modifications (PTMs) of microtubules (MTs) equip them with specific properties that are essential for plethora of cellular functions, such as neuronal polarization and axonal transport. Alpha- and beta-tubulins are main components of microtubules. Their unstructured C-terminus is modified by multiple PTMs, which are collectively called a ‘tubulin code’ and include tyrosination, (poly)glutamylation, (poly)glycylation and (poly)amination. The malfunction of these modifications has been associated with variety of neurodegenerative diseases. We have developed a chemoproteomic approach to characterize tyrosination of tubulins in living cells. However, the functional consequences and connection between the tyrosination and other modifications is not yet understood. The aim of this project is to characterize regulatory mechanisms behind tyrosination and how it influences other modifications. This will be done by usage of our in-gel based fluorescence and the state-of-the-art mass spectrometry-based chemoproteomics approaches. The resulting data will be analyzed and integrated by set of bioinformatic tools. The project promises to yield first insights into regulatory mechanisms behind microtubules tyrosination.
Project supervisor: Andreas Wiest
Required lab skills:
- Cell culture of human cancer cell lines (must-have)
- Preparation and protein separation on SDS-PAGE (must-have)
- Theoretical background in mass spectrometry-based proteomics and analysis of proteomics data (nice-to-have)
- Bioinformatics (nice-to-have)
2. Project title:
Functional characterization of AMPylation on beta-hexosamidase subunit beta (HEXB).
Project description:
Protein AMPylation is transient protein modification regulating important cellular pathways including protein stress response, immune signaling and neuronal differentiation. Recently, we have developed efficient chemoproteomics strategy enabling to profile AMPylated proteins including HEXB AMPylation leveraging from synthetic adenosine monophosphate analogue probe. However, the identification of an exact AMPylated amino acid in proteins remains challenging due to low abundance, poor ionization of negatively charged phosphate group of adenosine monophosphate and low stability of phosphodiester linkage during mass spectrometric fragmentation. In our laboratory, the AMPylation site identification is now possible by advanced mass spectrometry techniques and application of the state-of-the-art bioinformatic search algorithms to analyze the proteomics data. The aim of this project is site identification and functional characterization of AMPylation on HEXB. Dysregulation of HEXB protein has been linked to sever neurodevelopmental issues that result in brain malformations and Sandhoff disease. The project will include cloning of HEXB gene into vector with the pCMV promotor and FLAG-tag for overexpression in human cancer cell lines. The ‘click chemistry’ and in-gel fluorescence analysis will be used to test the functionality of prepared plasmid. This will be followed by immunoprecipitation of the FLAG-HEXB protein, preparation of samples for proteomics analysis by high-resolution orbitrap-based mass spectrometer and bioinformatic analysis of the mass spectra for site identification. Finally, the correct assignment of the AMPylation sites on HEXB will be validate by point mutation of the HEXB gene and reanalysis to confirm the lack of AMPylation. The work will provide important insights into HEXB regulation and lay down the basis for follow up works in neurons.
Project supervisor: Laura Hoffmann
Required lab skills:
- Cell culture of human cancer cell lines (must-have)
- Plasmid cloning and amplification (must-have)
- Side directed mutagenesis (should-have)
- Transfection of human cancer cell lines with plasmids (should have)
- Experience with protein immunoprecipitation (IP) (nice-to-have)
- Theoretical background in mass spectrometry-based proteomics and analysis of proteomics data (nice-to-have)