Michael S. Cohen, Ph.D. - US grants

DESCRIPTION (provided by applicant): The objective of the proposed research is to generate chemical tools that will expand our understanding of ADP-ribosylation in neuronal physiology. ADP-ribosylation was originally thought to be catalyzed by a single enzyme, ARTD1 (ADP-ribosyltransferases 1), but a family of 17 proteins is now recognized in humans that shares structural homology to the ARTD1 catalytic domain. ARTD1, and perhaps other ARTDs, play essential roles in cellular pathways in neurons that mediate long-term memory (LTM)~ however, their roles in these processes are not well understood. Moreover, the direct protein targets of individual ARTDs in neurons are not known, hindering our ability to fully delineate the pathway from ARTD activation to LTM. Our current lack of understanding of the specific role of ARTD1, and other ARTDs, in neurons and in other cell types has been severely limited by the lack of inhibitors of individual family members and the inability to identify the direct targets fr individual ARTDs in a cellular context. To overcome these limitations, this application describes, for the first time, the design and synthesis of (1) mono-selective inhibitors and (2) orthogonal NAD+ substrate analogs of ARTD1 mutants that are engineered to contain a unique pocket absent from wild-type ARTDs, but retain enzymatic activity. These orthogonal NAD+ analogs will be used for the identification of direct targets of ARTD1 in neurons. While initial studies wll focus on the role of ARTD1 in neurons, we anticipate that our strategy can be generalized to other ARTDs, thereby potentially providing unprecedented insights into their roles in physiology and pathophysiology.

Project Summary Astrocytes are critical regulators of innate immunity in the central nervous system (CNS). Stimulation of CNS innate immunity by neuroinflammatory activators such as pathogens and brain injury, as well as in response to neurodegeneration, cause astrocytes to undergo a transition to a reactive phenotype called astrogliosis. While it is well accepted that astrogliosis can act as a protective mechanism to minimize CNS damage, the mechanisms that regulate astrogliosis are not well understood. Our preliminary results and data from the literature support our general hypothesis that PARP7 controlled MARylation critically shapes the innate immune responses in the CNS. Our long-term goal is to understand the role of PARP7 in astrogliosis and whether PARP7 represents an actionable target for CNS pathologies that arise as a consequence of activation of CNS innate immunity. The objective of the proposed work is elucidate the mechanisms by which PARP7 regulates innate immunity in astrocytes. PARP7 has emerged as a critically important member of a large enzyme family known as PARPs, especially in the innate immune response. Similar to other PARP family members, PARP7 catalyzes the post- translational modification known as mono-ADP-ribosylation (MARylation), which involves the transfer of ADP-ribose from NAD+ to amino acids on target proteins. The MARylation targets of PARP7 in astrocytes are unknown. To decode the mechanisms by which PARP7 regulates innate immunity in astrocyte, we need to identify the direct targets of PARP7 in astrocytes. Identifying the direct targets of PARP7 has been challenging, however, due to the fact that PARPs share the same substrate NAD+. To overcome this limitation, we describe the development of engineered PARP7?orthogonal NAD+ analogue pairs for identifying the direct targets of PARP7 in astrocytes lysates (Aim I). We also describe the generation of membrane-permeant variants of our orthogonal NAD+ analogues, which are critical for identifying PARP7 targets in intact astrocytes using stimuli that activate the innate immune response in astrocytes (Aim II). Lastly, we describe a strategy for improving the selectivity of PARP7 inhibitors (Aim III). Selective inhibitors of PARP7 are essential chemical probes for evaluating the function of PARP7-mediated MARylation in the innate immune response in astrocytes. We anticipate that these studies will not only clarify our understanding of the function of PARP7-mediated MARylation in innate immunity in astrocytes, but could also lead to new therapeutic strategies for CNS pathologies, particularly neuroinflammatory (e.g. multiple sclerosis) and neurodegenerative diseases (e.g. Alzheimer's disease). More generally, the results obtained from these studies will have far-reaching impact on our understanding of MARylation in cell signaling.