P5-ATPases are conserved in all eukaryotes and malfunctions in human are associated with severe neurological diseases, such as familial early-onset parkinsonism and autism/language disorders, and with phenotypical traits in yeasts. They belong to the P-type ATPase superfamily, which encompass a range of essential membrane transporters for ions and lipids. Ion pumps such as Na,K-ATPase and Ca2+-ATPase have been studied in great detail during the last decades. However, astonishingly little is known about the P5-ATPases and their actual function, despite their physiological importance in all eukaryotes. The current proposal focuses on substrate identification and structural characterization of P5-ATPases, as well as investigations of their cellular interaction network. Human P5-ATPases (ATP13A1 through 5, ATP13A2 also known as PARK9) and the yeast orthologues Spf1p and Ypk9p will be subjects of this study. Target proteins will be expressed in their native host (yeast or HEK cells) and subsequently purified and used for activity assays, structural studies, and identification of interaction partners. Native mass spectrometry will identify bound substrates and cofactors, and activity studies will elucidate structure-function relationships. 3D-structures obtained by single-particle cryo-electron microscopy (cryo-EM) and/or X-ray crystallography will reveal catalytic mechanisms and mutational effects. Structural and functional characterization of P5-ATPases can therefore serve as a basis for understanding molecular mechanisms of e.g. neurodegenerative and cognitive disorders and guide novel strategies in disease treatments and drug discovery. Using my profound experience from my PhD with crystallography of biotechnologically relevant proteins, I wish to pursue a postdoc focused on membrane proteins with a strong potential in molecular medicine and to expand my knowledge of methods in structural biology and molecular cell biology, in particular cryo-EM.

Regulation and fidelity of gene expression is fundamental to the differentiation and maintenance of living organisms. Moreover, understanding how genes are regulated is an essential research question of importance for biomedical application. Although our knowledge about key factors influencing gene expression has increased substantially over the past years, the complexity of gene expression regulation remains elusive. In this project, I intend to discover novel gene expression regulatory circuits operating in mammalian genomes facilitated by pervasive transcription. Although some scattered examples of how pervasive transcription may regulate gene promoter activity exist, no systematic study has been conducted. Here, I will address this question by using a new protein depletion strategy, developed in the host laboratory, to inactivate the nuclear exosome, a major 3’-to-5’ ribonuclease complex, and its co-factors in HeLa and mouse embryonic stem (mES) cells, thus, to create an ideal situation to observe ‘hidden’ cellular transcription events, which would not normally be visible. I intend to assess whether and how this ‘hidden’ transcription contributes to the regulation of promoter activity in HeLa and mES cells, further expanding our knowledge of the regulation of protein-coding genes and ultimately revealing the extent of regulation instigated by pervasive transcription. This study will lay a foundation for future research in the field and at the same time provide novel conceptual information, which might be exploited in prevention or treatment of human diseases.

Dramatically increasing cancer cases around the world call for extra research efforts to improve cancer therapies. Radiation therapy or radiotherapy is one of the most common treatment methods. A way to enhance radiotherapy is inserting ‘radiosensitizers (RSs)’ and ‘radioprotectors (RPs)’ into the patient’s body. RSs in tumor cells make them more sensitive to radiation damage, allowing one to use reduced radiation doses, thus minimizing side effects. In contrast, RPs inhibit the damage of healthy cells from radiation. RSs and RPs are actively studied mostly in clinical trials. However, the fundamental mechanisms causing damage or death of cancer cells are not fully understood. Therefore, this project aims at elucidating the elementary steps of radiation damage, their enhancement by RSs, and their inhibition by RPs. The technique combines beams of mixed molecular clusters and doped helium nanodroplets uniquely with synchrotron spectroscopy, electron spectroscopy, and ion mass spectrometry. The main goals are to unravel the photochemistry of selected organic RS compounds (nimorazole, NIMO, bromoadenine, WR-1065 dihydrochloride), metal ions (Mg2+, Ca2+, K+), and gold (RS) and silver (RP) nanoparticles in the state of controlled microhydration and contact with DNA components (thymine, cytosine, tetrahydrofuran). Emission of slow electrons, water fragmentation, and anions formation are observables for radiation damage enhanced by RSs. A time-resolved experiment on the tetrahydrofuran-water complex will elucidate the ultrafast dynamics of intermolecular energy transfer causing dissociation, a mechanism recently identified to play an important role in radiation damage. A better understanding of the radiochemistry of RPs and RSs obtained with this project may help develop new schemes for efficient cancer treatment and identify new types of molecules or nanoparticles with improved RS or RP properties.