Abstract:
Excitotoxicity, a neuronal death process in neurological disorders such as stroke, is initiated by the overstimulation of ionotropic glutamate receptors. Although dysregulation of proteolytic signaling networks is critical for excitotoxicity, the identity of affected proteins and mechanisms by which they induce neuronal cell death remain unclear. To address this, we used quantitative N-terminomics to identify proteins modified by proteolysis in neurons undergoing excitotoxic cell death. We found that most proteolytically processed proteins in excitotoxic neurons are likely substrates of calpains, including key synaptic regulatory proteins such as CRMP2, doublecortin-like kinase I, Src tyrosine kinase and calmodulin-dependent protein kinase IIβ (CaMKIIβ). Critically, calpain-catalyzed proteolytic processing of these proteins generates stable truncated fragments with altered activities that potentially contribute to neuronal death by perturbing synaptic organization and function. Blocking calpain-mediated proteolysis of one of these proteins, Src, protected against neuronal loss in a rat model of neurotoxicity. Extrapolation of our N-terminomic results led to the discovery that CaMKIIα, an isoform of CaMKIIβ, undergoes differential processing in mouse brains under physiological conditions and during ischemic stroke. In summary, by identifying the neuronal proteins undergoing proteolysis during excitotoxicity, our findings offer new insights into excitotoxic neuronal death mechanisms and reveal potential neuroprotective targets for neurological disorders.
摘要:
兴奋毒性,神经疾病如中风中的神经元死亡过程,是由离子型谷氨酸受体的过度刺激引发的。尽管蛋白水解信号网络的失调对于兴奋性毒性至关重要,受影响的蛋白质的身份和它们诱导神经元细胞死亡的机制仍不清楚。为了解决这个问题,我们使用定量N-末端组学在经历兴奋毒性细胞死亡的神经元中鉴定通过蛋白水解修饰的蛋白质.我们发现兴奋性毒性神经元中大多数蛋白水解加工的蛋白质可能是钙蛋白酶的底物,包括关键的突触调节蛋白,如CRMP2,双曲肽样激酶I,Src酪氨酸激酶和钙调蛋白依赖性蛋白激酶IIβ(CaMKIIβ)。严重的,钙蛋白酶催化的这些蛋白质的蛋白水解加工产生稳定的截短片段,其活性改变,可能通过扰乱突触组织和功能而导致神经元死亡。阻断钙蛋白酶介导的这些蛋白质之一的蛋白水解,Src,在大鼠神经毒性模型中防止神经元丢失。对我们的N-末端结果的推断导致发现CaMKIIα,CaMKIIβ的同种型,在生理条件下和缺血性中风期间在小鼠大脑中经历差异处理。总之,通过鉴定在兴奋性毒性过程中经历蛋白水解的神经元蛋白,我们的发现为兴奋性毒性神经元死亡机制提供了新的见解,并揭示了神经系统疾病的潜在神经保护靶点。