Identification of critical residues within the conserved and specificity patches of nerve growth factor leading to survival or differentiation.
Identification of critical residues within the conserved and specificity patches of nerve growth factor leading to survival or differentiation.
复制标题
鉴定神经生长因子的保守和特异性斑块内的关键残基，从而导致存活或分化。
DOI：
10.1074/jbc.m109.058420
复制
发表时间：
2009
期刊：
影响因子：
--
通讯作者：
Neet,KennethE
中科院分区：
文献类型：
--
作者：
Mahapatra,Sidharth;Mehta,Hrishikesh;Woo,SangB;Neet,KennethE
阿尔茨海默病中受影响的神经元已经显示出在细胞表面处TrkA和p75 NTR表达的不平衡，并且已经考虑并尝试施用神经生长因子（NGF）用于治疗。然而，野生型神经生长因子引起神经突的广泛加工，同时提供生存支持。本研究旨在开发重组NGF突变蛋白，不支持轴突发生，同时维持生存反应。关键残基被确定在配体-受体界面点突变，在neuritogenesversussurvival发挥了更大的重要性。通过组合点突变，两种存活选择性重组NGF突变蛋白，即/ 7-84-103和KKE/7-84-103。两种突变蛋白均使PC 12（TrkA+/p75 NTR+）细胞中的轴突发生减少> 90%，同时在MG 139（仅TrkA+）和PCNA成纤维细胞（仅p75 NTR+）细胞中保持接近野生型的存活活性。此外，在幼稚和终末分化的PC 12细胞中的存活率显示为介于NGF和阴性对照之间。7-84-103的剂量反应曲线显示，分化曲线移动了约100倍，而存活的EC 50仅增加了3.3倍。表面等离子体共振分析揭示7-84-103与TrkA的结合降低200倍。细胞存活的保持归因于通过Akt存活途径的信号传导的维持，其中用于分化的MAPK信号传导减少。关键突变沿着NGF受体界面的作用在细胞内传递，从而能够产生存活选择性重组NGF突变蛋白，其可能代表用于改善阿尔茨海默病的新型药理学先导剂。
Afflicted neurons in Alzheimer disease have been shown to display an imbalance in the expression of TrkA and p75NTRat the cell surface, and administration of nerve growth factor (NGF) has been considered and attempted for treatment. However, wild-type NGF causes extensive elaboration of neurites while providing survival support. This study was aimed at developing recombinant NGF muteins that did not support neuritogenesis while maintaining the survival response. Critical residues were identified at the ligand-receptor interface by point mutagenesis that played a greater importance in neuritogenesisversussurvival. By combining point mutations, two survival-selective recombinant NGF muteins,i.e./7-84-103 and KKE/7-84-103, were generated. Both muteins reduced neuritogenesis in PC12 (TrkA+/p75NTR+) cells by >90%, while concurrently retaining near wild-type survival activity in MG139 (TrkA+only) and PCNA fibroblast (p75NTR+-only) cells. Additionally, survival in both naive and terminally differentiated PC12 cells was shown to be intermediate between NGF and negative controls. Dose-response curves with 7-84-103 showed that the differentiation curve was shifted by about 100-fold, whereas the EC50for survival was only increased by 3.3-fold. Surface plasmon resonance analysis revealed a 200-fold decrease in binding of 7-84-103 to TrkA. The retention of cell survival was attributed to maintenance of signaling through the Akt survival pathway with reduced MAPK signaling for differentiation. The effect of key mutations along the NGF receptor interface are transmitted inside the cell to enable the generation of survival-selective recombinant NGF muteins that may represent novel pharmacologic lead agents for the amelioration of Alzheimer disease.