Laboratory of Protein Dynamics and Signaling, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA.
Proc Natl Acad Sci U S A. 2010 Sep 21;107(38):16554-9. doi: 10.1073/pnas.1008302107. Epub 2010 Sep 7.
Botulinum neurotoxins (BoNTs) are the most potent natural toxins known. The effects of BoNT serotype A (BoNT/A) can last several months, whereas the effects of BoNT serotype E (BoNT/E), which shares the same synaptic target, synaptosomal-associated protein 25 (SNAP25), last only several weeks. The long-lasting effects or persistence of BoNT/A, although desirable for therapeutic applications, presents a challenge for medical treatment of BoNT intoxication. Although the mechanisms for BoNT toxicity are well known, little is known about the mechanisms that govern the persistence of the toxins. We show that the recombinant catalytic light chain (LC) of BoNT/E is ubiquitylated and rapidly degraded in cells. In contrast, BoNT/A LC is considerably more stable. Differential susceptibility of the catalytic LCs to ubiquitin-dependent proteolysis therefore might explain the differential persistence of BoNT serotypes. In this regard we show that TRAF2, a RING finger protein implicated in ubiquitylation, selectively associates with BoNT/E LC and promotes its proteasomal degradation. Given these data, we asked whether BoNT/A LC could be targeted for rapid proteasomal degradation by redirecting it to characterized ubiquitin ligase domains. We describe chimeric SNAP25-based ubiquitin ligases that target BoNT/A LC for degradation, reducing its duration in a cellular model for toxin persistence.
肉毒神经毒素（BoNTs）是已知最有效的天然毒素。BoNT 血清型 A（BoNT/A）的作用可以持续数月，而具有相同突触靶标突触小体相关蛋白 25（SNAP25）的 BoNT 血清型 E（BoNT/E）的作用仅持续数周。BoNT/A 的长效作用或持久性对于治疗应用是理想的，但这对 BoNT 中毒的医疗治疗提出了挑战。尽管 BoNT 毒性的机制众所周知，但对于控制毒素持久性的机制知之甚少。我们表明，BoNT/E 的重组催化轻链（LC）在细胞中被泛素化并迅速降解。相比之下，BoNT/A LC 则稳定得多。因此，催化 LC 对泛素依赖性蛋白水解的不同敏感性可能解释了 BoNT 血清型的不同持久性。在这方面，我们表明 TRAF2，一种涉及泛素化的 RING 指蛋白，与 BoNT/E LC 选择性结合并促进其蛋白酶体降解。考虑到这些数据，我们询问是否可以通过将 BoNT/A LC 重定向到已鉴定的泛素连接酶结构域来靶向其进行快速蛋白酶体降解。我们描述了基于 SNAP25 的嵌合泛素连接酶，这些连接酶可靶向 BoNT/A LC 进行降解，从而减少其在毒素持久性的细胞模型中的持续时间。
