Identification and characterisation of enzymes that control mono(ADP-ribosyl)ation in mitochondria

• Applicant: Karla Feijs, Ph.D.
• Subject Area: Cell Biology ; Biochemistry
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 494549563

ADP-ribosylation is well known as a posttranslational modification of proteins. The role of poly(ADP-ribosyl)ation by PARP1 as part of the DNA damage response has been well studied and has resulted in the usage of PARP1/2 inhibitors as cancer treatment. In contrast to PARP1, the majority of PARP enzymes transfer only one ADP-ribose moiety from co-factor NAD+ onto their targets, referred to as MARylation. Exciting recent break-throughs hint that MARylation is present on proteins in mitochondria and interestingly, can also be added onto RNA and DNA in in vitro assays. Our own data hint that loss of a mitochondrial ADP-ribosylhydrolase, MACROD1, leads to changes in mitochondrial respiration as well as slight morphological alterations, implying cellular stress upon loss of MACROD1. These findings give rise to many questions: what is the function of MARylation in mitochondria, which transferase is adding this modification, how does it influence the modified proteins and what is the role of MACROD1 in mitochondria? We are pursuing two research aims to address these questions: first, we will use two different approaches to identify the mitochondrial transferase(s), including a targeted study of the known ADP-ribosyltransferases, as well as an unbiased study of all mitochondrial proteins using BioID. We will further characterise the identified transferases as well as selected substrates to study the function of MARylation in mitochondria. Second, we will investigate the function of MACROD1 by combining different datasets: we will determine which proteins and RNAs are affected by loss of MACROD1, perform gene ontology (GO) analysis and determine overlap with the GO analysis of our previous, published BioID which identified proteins in close proximity to MACROD1. The most promising candidates identified in multiple approaches will be taken for further investigations of the role of MACROD1 in mitochondria.This work thus aims to identify mitochondrial ADP-ribosyltransferase(s), study the consequence of the modification for modified substrates and investigate the function of MACROD1 in mitochondria in more detail.

• DFG Programme: Research Grants