Decoding protein ADP-ribosylation networks in neurons using a chemical genetic ap

描述（由申请人提供）：拟议研究的目的是产生化学工具，将扩大我们对神经元生理学中ADP-核糖基化的理解。ADP-核糖基化最初被认为是由单一的酶，ARTD 1（ADP-核糖基转移酶1）催化，但现在在人类中发现了一个17种蛋白质家族，与ARTD 1催化结构域具有结构同源性。ARTD 1，也许还有其他的ARTD，在神经元的细胞通路中起着重要的作用，这些神经元介导了长期记忆（LTM），然而，它们在这些过程中的作用还没有得到很好的理解。此外，单个ARTD在神经元中的直接蛋白靶点尚不清楚，这阻碍了我们完全描绘神经元中ARTD的能力。 从ARTD激活到LTM的途径。 我们目前对ARTD 1和其他ARTD在神经元和其他细胞类型中的特定作用缺乏了解，这受到缺乏单个家族成员抑制剂以及无法在细胞环境中识别单个ARTD的直接靶点的严重限制。为了克服这些限制，本申请首次描述了ARTD 1突变体的（1）单选择性抑制剂和（2）正交NAD+底物类似物的设计和合成，所述ARTD 1突变体被工程化以含有野生型ARTD中不存在的独特口袋，但保留酶活性。这些正交NAD+类似物将用于鉴定神经元中ARTD 1的直接靶标。虽然最初的研究将集中在ARTD 1在神经元中的作用，但我们预计我们的策略可以推广到其他ARTD，从而可能对它们在生理学和病理生理学中的作用提供前所未有的见解。

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.