Gaub Perrine, de Léon Andrès, Gibon Julien, Soubannier Vincent, Dorval Geneviève, Séguéla Philippe, Barker Philip A
Montreal Neurological Institute, Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec, H3A 2B4, Canada.
PLoS One. 2016 Mar 7;11(3):e0150601. doi: 10.1371/journal.pone.0150601. eCollection 2016.
Neurotrophins activate intracellular signaling pathways necessary for neuronal survival, growth and apoptosis. The most abundant neurotrophin in the adult brain, brain-derived neurotrophic factor (BDNF), is first synthesized as a proBDNF precursor and recent studies have demonstrated that proBDNF can be secreted and that it functions as a ligand for a receptor complex containing p75NTR and sortilin. Activation of proBDNF receptors mediates growth cone collapse, reduces synaptic activity, and facilitates developmental apoptosis of motoneurons but the precise signaling cascades have been difficult to discern. To address this, we have engineered, expressed and purified HBpF-proBDNF, an expression construct containing a 6X-HIS tag, a biotin acceptor peptide (BAP) sequence, a PreScission™ Protease cleavage site and a FLAG-tag attached to the N-terminal part of murine proBDNF. Intact HBpF-proBDNF has activities indistinguishable from its wild-type counterpart and can be used to purify proBDNF signaling complexes or to monitor proBDNF endocytosis and retrograde transport. HBpF-proBDNF will be useful for characterizing proBDNF signaling complexes and for deciphering the role of proBDNF in neuronal development, synapse function and neurodegenerative disease.
神经营养因子激活神经元存活、生长和凋亡所必需的细胞内信号通路。成年大脑中最丰富的神经营养因子，即脑源性神经营养因子（BDNF），最初是以proBDNF前体的形式合成的，最近的研究表明proBDNF可以分泌，并且它作为一种配体作用于含有p75NTR和sortilin的受体复合物。proBDNF受体的激活介导生长锥塌陷，降低突触活性，并促进运动神经元的发育性凋亡，但精确的信号级联反应一直难以辨别。为了解决这个问题，我们构建、表达并纯化了HBpF-proBDNF，这是一种表达构建体，包含一个6X-HIS标签、一个生物素受体肽（BAP）序列、一个PreScission™蛋白酶切割位点以及一个连接到小鼠proBDNF N端的FLAG标签。完整的HBpF-proBDNF具有与其野生型对应物难以区分的活性，可用于纯化proBDNF信号复合物或监测proBDNF的内吞作用和逆向运输。HBpF-proBDNF将有助于表征proBDNF信号复合物，并有助于解读proBDNF在神经元发育、突触功能和神经退行性疾病中的作用。
