Novel biochemical and functional targets of PARP

项目总结

Project Summary The T helper 17 subset plays important roles in defense against microbes but also is pathogenic in inflammatory diseases such as experimental autoimmune encephalomyelitis (EAE), a mouse model used to generate leads for analysis of multiple sclerosis (MS). Targeting specific cytokine products (e.g., IL-17, IL-22) of the various forms of Th17 cells, or cytokine-receptor interactions vital for the efficiency of differentiation or expansion of a Th17 pool (IL-23), may prove to help MS but these interventions may prove either to lack sufficient efficacy or to impair host defenses too effectively. As such, there is great intrinsic value in discovering new mechanisms by which the Th17 subset is regulated and functions. Signaling mechanisms offer opportunities for new therapeutic approaches to treatments for immune- mediated disorders. We have discovered that a mammalian intracellular ADP-ribosyl transferase (ART), PARP14, promotes the differentiation of several T helper subsets. Importantly, its capacity to impact Th17 differentiation is dependent on the intrinsic ART activity. These findings are particularly notable for several reasons. First, the ways in which this protein affects T helper differentiation are quite different from PARP1, the most-studied mammalian ART. Indeed, whereas PARP1 and several of its relatives can catalyze branching polymers of ADP-ribose (ADPr) after placement of an initial adduct on target proteins, PARP14 appears unable to function as a polymerase and instead is an ADP-ribosyl mono-transferase (mART). As such, the findings open entirely new vistas for understanding how physiological regulation is effected by a post-translational modification that until now has been little studied in normal mammalian biology. Second, microbial pathogens often exploit or subvert signaling mechanisms in host cells. Indeed, a number of bacterial exotoxins function by intoxicating the mammalian cells through addition of ADP-ribose to intracellular proteins after being introduced inside, and in fact these toxins elicit Th17 responses. The proposed work may, therefore, shed light on endogenous pathways exploited by, for instance, pertussis toxin. With these points as backdrop, we will determine if disease severity in EAE is influenced by the ART activity of PARP14 by using bone marrow transfers and transduction of active or inactive PARP14 into cells (Aim 1), and identify lymphocyte types critical for the dependence of EAE on PARP14 (Aim 2). Further, we will use adduct tagging and proteomics to identify PARP14-dependent molecular targets for ADP-ribosylation in CD4 T cells and determine the overlap of these targets with those of pertussis toxin (PT) (Aim 3). The combined results from this work would finalize an exciting new insight into molecular regulation and lay the foundations for a more comprehensive and sustained elucidation of these processes.