n2STAR SEMINAR
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Speaker : Prof. Volker Dötsch, Goethe University, Frankfurt, Germany
Title :Designed Ankyrin Repeat Proteins as research tools and potential therapeutics
Date :Thursday, March 30, 2023
Time :3:00 PM, ISTANBUL
Place : Zoom Webinar
Abstract:
Inactivation of the tumor suppressor p53, also known as the guardian of the genome, is one of the most common hallmarks of human cancer. In more than 50 % of all tumors, this inactivation is caused by mutations that are mainly located at several hotspots within the DNA binding domain. Some of the hotspot mutations do not only lead to the inactivation of p53 but also its destabilization causing conformational changes of the DBD or even unfolding and the exposure of aggregation-prone regions. Those mutants are described as temperature-sensitive mutants as they adopt a wild-type conformation at lower temperatures but get destabilized at elevated temperatures. Unfolding can lead to co-aggregation with other proteins including p53 family members, thus repressing their transcriptional function and hence promoting cancer cell survival and metastasis. In order to target and stabilize those cancer hotspot mutants we selected Designed Ankyrin Repeat Proteins (DARPins) against the wild type and mutant p53 DBD. Characterization of the selected DARPins revealed that DARPin C10, a DARPin binding to both, mutant and wild-type p53, stabilizes a variety of common cancer mutants of p53. Furthermore, we were able to show that DARPin C10 restores the transcriptional activity of p53 in transactivation assays. Additionally, we found that DARPin C10 can stabilize and restore the transcriptional activity of p53 in HeLa cells by blocking the HPV‑E6-mediated degradation of p53. The described DARPins could present a new approach to stabilize p53 mutants and target HPV-induced tumorigenesis.
Proteins containing iron-sulfur (Fe-S) clusters arose early in evolution and are essential to life. Organisms have evolved machinery consisting of specialized proteins that operate together to assemble Fe-S clusters efficiently so as to minimize cellular exposure to their toxic constituents: iron and sulfide ions. To date, the best studied system is the iron sulfur cluster (ISC) operon of Escherichia coli, and the eight ISC proteins it encodes. We employed nuclear magnetic resonance (NMR) spectroscopy, a powerful technique of elucidating structural and dynamic features of proteins and protein-protein interactions, and have characterized (1) two functional conformational states of the Fe-S cluster assembly protein (IscU), (2) the complex interaction network among the ISC proteins, and (3) the intriguing preferences of some ISC proteins that interact more with one conformational state of IscU over the other. In the upcoming seminar, along with these experimental observations, I will show our up-to-date model for Fe-S cluster assembly and delivery, of which physiological implication and relevance to the higher organisms, such as humans, are to be discussed.
Short Bio:
Volker Dötsch is a Professor of Biophysical Chemistry at Goethe University and a member of the Magnetic Resonance Center Frankfurt. He studied chemistry at the University of Göttingen and obtained a Ph.D. from the ETH in Zürich. As a postdoctoral fellow, he used NMR to determine the structure of protein-DNA complexes at Harvard Medical School. In 1998 he moved as an assistant professor at the University of California San Francisco (UCSF). In 2003 he was appointed professor at the Institute of Biophysical Chemistry of Goethe University in Frankfurt. His research interests focus on the structural and functional characterization of members of the p53 protein family, in particular p63, and its involvement in genetic quality control in germ cells. In addition, his laboratory uses a combination of NMR spectroscopy and cell-free protein expression to investigate the structure and function of membrane proteins and studies molecular interactions regulating autophagy. His lab uses a wide variety of biophysical methods including NMR spectroscopy and combines these studies with investigations in cell culture experiments and mouse models. Volker Dötsch is an elected EMBO member.