aDepartment of Pharmacology, College of Medicine, University of Arizona, Tucson, AZ, USA. Dr Dustrude is now with the Department of Psychiatry, Institute of Psychiatric Research, Indiana University School of Medicine, Indianapolis, IN, USA and Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, IN, USA bDepartment of Chemistry and Biochemistry, College of Science, University of Arizona, Tucson, AZ, USA cDepartment of Anesthesiology, College of Medicine, University of Arizona, Tucson, AZ, USA dNeuroscience Graduate Interdisciplinary Program, College of Medicine, University of Arizona, Tucson, AZ, USA.
Pain. 2017 Nov;158(11):2203-2221. doi: 10.1097/j.pain.0000000000001026.
Neurofibromatosis type 1 (NF1), a genetic disorder linked to inactivating mutations or a homozygous deletion of the Nf1 gene, is characterized by tumorigenesis, cognitive dysfunction, seizures, migraine, and pain. Omic studies on human NF1 tissues identified an increase in the expression of collapsin response mediator protein 2 (CRMP2), a cytosolic protein reported to regulate the trafficking and activity of presynaptic N-type voltage-gated calcium (Cav2.2) channels. Because neurofibromin, the protein product of the Nf1 gene, binds to and inhibits CRMP2, the neurofibromin-CRMP2 signaling cascade will likely affect Ca channel activity and regulate nociceptive neurotransmission and in vivo responses to noxious stimulation. Here, we investigated the function of neurofibromin-CRMP2 interaction on Cav2.2. Mapping of >275 peptides between neurofibromin and CRMP2 identified a 15-amino acid CRMP2-derived peptide that, when fused to the tat transduction domain of HIV-1, inhibited Ca influx in dorsal root ganglion neurons. This peptide mimics the negative regulation of CRMP2 activity by neurofibromin. Neurons treated with tat-CRMP2/neurofibromin regulating peptide 1 (t-CNRP1) exhibited a decreased Cav2.2 membrane localization, and uncoupling of neurofibromin-CRMP2 and CRMP2-Cav2.2 interactions. Proteomic analysis of a nanodisc-solubilized membrane protein library identified syntaxin 1A as a novel CRMP2-binding protein whose interaction with CRMP2 was strengthened in neurofibromin-depleted cells and reduced by t-CNRP1. Stimulus-evoked release of calcitonin gene-related peptide from lumbar spinal cord slices was inhibited by t-CNRP1. Intrathecal administration of t-CNRP1 was antinociceptive in experimental models of inflammatory, postsurgical, and neuropathic pain. Our results demonstrate the utility of t-CNRP1 to inhibit CRMP2 protein-protein interactions for the potential treatment of pain.
神经纤维瘤病 1 型（NF1）是一种遗传疾病，与 Nf1 基因的失活突变或纯合缺失有关，其特征是肿瘤发生、认知功能障碍、癫痫发作、偏头痛和疼痛。对人类 NF1 组织的组学研究表明， collapsin 反应介质蛋白 2（CRMP2）的表达增加，CRMP2 是一种细胞质蛋白，据报道可调节突触前 N 型电压门控钙（Cav2.2）通道的运输和活性。由于神经纤维瘤蛋白，即 Nf1 基因的蛋白产物，与 CRMP2 结合并抑制其活性，因此神经纤维瘤蛋白-CRMP2 信号级联可能会影响钙通道活性并调节伤害性神经传递和体内对有害刺激的反应。在这里，我们研究了神经纤维瘤蛋白-CRMP2 相互作用对 Cav2.2 的功能。在神经纤维瘤蛋白和 CRMP2 之间的>275 个肽段进行映射，确定了一个 15 个氨基酸的 CRMP2 衍生肽，当与 HIV-1 的 tat 转导结构域融合时，抑制背根神经节神经元中的钙内流。该肽模拟了神经纤维瘤蛋白对 CRMP2 活性的负调节。用 tat-CRMP2/神经纤维瘤蛋白调节肽 1（t-CNRP1）处理的神经元表现出 Cav2.2 膜定位减少，以及神经纤维瘤蛋白-CRMP2 和 CRMP2-Cav2.2 相互作用的解偶联。纳米盘溶解膜蛋白文库的蛋白质组学分析鉴定出 syntaxin 1A 为一种新型的 CRMP2 结合蛋白，其与 CRMP2 的相互作用在神经纤维瘤蛋白耗尽的细胞中增强，并被 t-CNRP1 减弱。刺激诱发的腰椎脊髓切片中降钙素基因相关肽的释放被 t-CNRP1 抑制。鞘内给予 t-CNRP1 在炎症、手术后和神经性疼痛的实验模型中具有抗伤害作用。我们的结果证明了 t-CNRP1 抑制 CRMP2 蛋白-蛋白相互作用的有效性，可用于治疗疼痛。
