Chemical Proteomics Approach for Profiling the NAD Interactome

Nicotinamide adenine dinucleotide
(NAD+) is a multifunctional
molecule. Beyond redox metabolism, NAD+ has an equally
important function as a substrate for post-translational modification
enzymes, the largest family being the poly-ADP–ribose polymerases
(PARPs, 17 family members in humans). The recent surprising discoveries
of noncanonical NAD (NAD+/NADH)-binding proteins suggests
that the NAD interactome is likely larger than previously thought;
yet, broadly useful chemical tools for profiling and discovering NAD-binding
proteins do not exist. Here, we describe the design, synthesis, and
validation of clickable, photoaffinity labeling (PAL) probes, 2- and
6-ad-BAD, for interrogating the NAD interactome. We found that 2-ad-BAD
efficiently labels PARPs in a UV-dependent manner. Chemical proteomics
experiments with 2- and 6-ad-BAD identified known and unknown NAD+/NADH-binding proteins. Together, our study shows the utility
of 2- and 6-ad-BAD as clickable PAL NAD probes.

烟酰胺腺嘌呤二核苷酸（Nicotinamide adenine dinucleotide, NAD+）是一种多功能分子。除参与氧化还原代谢外，NAD+还具备另一项同等重要的功能：作为翻译后修饰酶的底物，其中家族规模最大的一类为多聚ADP核糖聚合酶（poly-ADP–ribose polymerases, PARPs），人类体内共含17个该家族成员。近期有关非经典NAD（NAD+/NADH）结合蛋白的意外发现提示，NAD相互作用组的规模可能远超此前认知；然而目前尚无广泛适用的、用于筛选与发现NAD结合蛋白的化学工具。本研究介绍了可点击化学光亲和标记（photoaffinity labeling, PAL）探针2-ad-BAD与6-ad-BAD的设计、合成与验证方案，用于解析NAD相互作用组。研究发现，2-ad-BAD能够以紫外线依赖的方式高效标记PARPs。通过使用2-ad-BAD与6-ad-BAD开展的化学蛋白质组学实验，本研究成功鉴定出已知与未知的NAD+/NADH结合蛋白。综上，本研究证实了2-ad-BAD与6-ad-BAD作为可点击化学PAL型NAD探针的应用价值。