State Key Laboratory of Chemical Oncogenomics, Key Laboratory of Chemical Genomics, Peking University Shenzhen Graduate School, Shenzhen University Town, China.
Ciechanover Institute of Precision and Regenerative Medicine, School of Life and Health Sciences, The Chinese University of Hong Kong, Shenzhen, China.
FEBS J. 2021 Dec;288(23):6783-6794. doi: 10.1111/febs.16104. Epub 2021 Jul 13.
SARM1, an executioner in axon degeneration, is an autoinhibitory NAD-consuming enzyme, composed of multiple domains. NMN and its analogs, CZ-48 and VMN, are the only known activators, which can release the inhibitory ARM domain from the enzymatic TIR domain. Here, we document that acid can also activate SARM1, even more efficiently than NMN, possibly via the protonation of the negative residues. Systematic mutagenesis revealed that a single mutation, E689Q in TIR, led to the constitutive activation of SARM1. It forms a salt bridge with R216 in the neighboring ARM, maintaining the autoinhibitory structure. Using this 'acid activation' protocol, mutation K597E was found to inhibit activation, while H685A eliminated SARM1 catalytic activity, revealing two distinct inhibitory mechanisms. The protocol has also been applied to differentiate two classes of chemical inhibitors. NAD, dHNN, disulfiram, CHAPS, and TRX-100 mainly inhibited the activation process, while nicotinamide and Tweens mainly inhibited SARM1 catalysis. Taken together, we demonstrate a new mechanism for SARM1 activation and decipher two distinct inhibitory mechanisms of SARM1.
SARM1 是轴突退化的执行者，它是一种自动抑制 NAD 消耗的酶，由多个结构域组成。NMN 及其类似物 CZ-48 和 VMN 是唯一已知的激活剂，它们可以将抑制性 ARM 结构域从酶的 TIR 结构域中释放出来。在这里，我们记录到酸也可以激活 SARM1，其效率甚至比 NMN 更高，可能是通过对负电荷残基的质子化作用。系统的突变分析显示，单个突变，即 TIR 中的 E689Q，导致 SARM1 的组成性激活。它与相邻 ARM 中的 R216 形成盐桥，维持自抑制结构。使用这种“酸激活”方案，发现突变 K597E 抑制了激活，而 H685A 消除了 SARM1 的催化活性，揭示了两种不同的抑制机制。该方案还被应用于区分两类化学抑制剂。NAD、dHNN、双硫仑、CHAPS 和 TRX-100 主要抑制激活过程，而烟酰胺和吐温主要抑制 SARM1 催化。总之，我们证明了 SARM1 激活的一种新机制，并解析了 SARM1 的两种不同抑制机制。
