UC San Diego

The post-translational modification ADP-ribosylation, whereby ADP-ribose subunits are attached to proteins to alter their function, has been previously implicated in processes including DNA damage repair and certain stress responses. However, the mediators of this modification are poorly understood; in particular, little is known about a diverse class of ADP-ribose binding domains called macrodomains, beyond a general affinity to bind to ADP- ribose. Experiments were thus undertaken to determine if different macrodomains show differences in their ADP-ribose binding activity. In addition, as several members of the macrodomain family are suspected to be involved in some host-pathogen conflict, potential alterations of macrodomains’ ADP-ribose binding in response to immunological signals were also investigated. Initial experiments utilized an Ni-NTA column to purify proteins based on their ability to bind to a macrodomain; although initial experiments proved that such a system could purify ADP-ribosylated proteins, further refinement to this system is needed. Further experiments to address this question of differential binding of ADP-ribose by macrodomains utilized fluorescent proteins fused to macrodomains, which allow for the localization of the macrodomain inside a cell to be observed. These experiments were successful in demonstrating that different macrodomains localize to different cellular compartments, suggesting that they each bind to different sets of ADP-ribosylated proteins. Experiments to identify alterations in ADP-ribosylation in cells following exposure to immunological signals, however, were unsuccessful. Refinement of a pulldown assay to purify ADP-ribosylated proteins would be advantageous for future exploration of macrodomain binding activity.