Nanobody-assisted structural and functional studies of the human ADP-ribosyltransferase ARTD4

• Applicant: Dr. Christian Löw, since 9/2018
• Subject Area: Biochemistry ; Structural Biology ; Cell Biology
• Term: from 2016 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 311060003

ADP-ribosyltransferases play regulatory roles in DNA repair, inflammation and apoptosis by transferring ADP-ribose from nicotinamide adenine dinucleotide (NAD+) to acceptor proteins. ARTD4/ vault PARP is one of 17 human ARTD isoforms with a peculiar structure. The catalytic domain of ARTD4 is followed by >1.000 amino acids harbouring three distinct protein domains not found in other family members. In cells, ARTD4 is present in complex with large MDa ribonucleoprotein particles - vaults - that are thought to play a role in drug resistance. ARTD4 also occurs in a free, unbound form. Interestingly, ARTD4 is strongly overexpressed in ovarian cancer cells and mesenchymal glioblastoma stem cells. However, the subcellular dynamics and the structure and function of ARTD4 remain largely unknown.Nanobodies - single domain antibodies derived from llama heavy chain antibodies - are emerging as powerful tools for structure/function analyses and for tracking proteins in living cells. The goal of the proposed project is to generate ARTD4-specific nanobodies as tools for elucidating the structure and function of ARTD4. Specifically, we will use these nanobodies i) as crystallization chaperones for structural analyses ii) to immunoprecipitate ARTD4 and ARTD4-associated proteins for further analyses by ADP-ribosylation assays and mass spectrometry, iii) to monitor the expression and subcellular dynamics of ARTD4 in normal and in cancer cells by immunofluorescence microscopy and flow cytometry, and iv) to block the ADP-ribosylation of ARTD4 target proteins in vitro and in transduced cells. We expect that the results of our studies will help to elucidate the functions and structure of ARTD4 and to provide a rational basis for developing more effective strategies to overcome drug resistance and to target ARTD4 overexpressing tumor cells.

• DFG Programme: Research Grants
• Ehemalige Antragstellerin: Dr. Mirjam Löw, until 8/2018