Journal
LIFE SCIENCE ALLIANCE
Volume 6, Issue 1, Pages -Publisher
LIFE SCIENCE ALLIANCE LLC
DOI: 10.26508/lsa.202201455
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Funding
- Confocal Microscopy Facility, a Core Facility of the Interdisciplinary Center for Clinical Research (IZKF) Aachen within the Faculty of Medicine at RWTH Aachen University
- START Program of the Medical Faculty of RWTH Aachen University
- German Research Foundation DFG
- [LU466/16-2]
- [FE1423/3-1]
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This study investigates the function of ADP-ribosylation in cells and optimizes sample preparation methods, defining the suitable reagents for specific methods and substrates.
The modification of substrates with ADP-ribose (ADPr) is im-portant in, for example, antiviral immunity and cancer. Recently, several reagents were developed to detect ADP-ribosylation; however, it is unknown whether they recognise ADPr, specific amino acid-ADPr linkages, or ADPr with the surrounding protein backbone. We first optimised methods to prepare extracts con-taining ADPr-proteins and observe that depending on the amino acid modified, the modification is heatlabile. We tested the re-activity of available reagents with diverse ADP-ribosylated pro-tein and RNA substrates and observed that not all reagents are equally suited for all substrates. Next, we determined cross -reactivity with adenylylated RNA, AMPylated proteins, and me-tabolites, including NADH, which are detected by some reagents. Lastly, we analysed ADP-ribosylation using confocal microscopy, where depending on the fixation method, either mitochondrion, nucleus, or nucleolus is stained. This study allows future work dissecting the function of ADP-ribosylation in cells, both on protein and on RNA substrates, as we optimised sample prepa-ration methods and have defined the reagents suitable for specific methods and substrates.
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