Fahim Ammad, Rehman Zaira, Bhatti Muhammad Faraz, Ali Amjad, Virk Nasar, Rashid Amir, Paracha Rehan Zafar
Atta-ur-Rahman School of Applied Biosciences (ASAB), National University of Sciences and Technology (NUST), Islamabad, Pakistan.
Army Medical College, National University of Medical Sciences, Rawalpindi, Pakistan.
PeerJ. 2018 Jun 14;6:e4978. doi: 10.7717/peerj.4978. eCollection 2018.
Npas4 is an activity dependent transcription factor which is responsible for gearing the expression of target genes involved in neuro-transmission. Despite the importance of Npas4 in many neuronal diseases, the tertiary structure of Npas4 protein along with its physico-chemical properties is limited. In the current study, first we perfomed the phylogenetic analysis of Npas4 and determined the content of hydrophobic, flexible and order-disorder promoting amino acids. The protein binding regions, post-translational modifications and crystallization propensity of Npas4 were predicted through different in-silico methods. The three dimensional model of Npas4 was predicted through LOMET, SPARSKS-X, I-Tasser, RaptorX, MUSTER and Pyhre and the best model was selected on the basis of Ramachandran plot, PROSA, and Qmean scores. The best model was then subjected to further refinement though MODREFINER. Finally the interacting partners of Npas4 were identified through STRING database. The phylogenetic analysis showed the human Npas4 gene to be closely related to other primates such as chimpanzees, monkey, gibbon. The physiochemical properties of Npas4 showed that it is an intrinsically disordered protein with N-terminal ordered region. The post-translational modification analyses indicated absence of acetylation and mannosylation sites. Three potential phosphorylation sites (S108, T130 and T136) were found in PAS A domain whilst a single phosphorylation site (S273) was present in PAS B domain. The predicted tertiary structure of Npas4 showed that bHLH domain and PAS domain possess tertiary structures while the rest of the protein exhibited disorder property. Protein-protein interaction analysis revealed NPas4 interaction with various proteins which are mainly involved in nuclear trafficking of proteins to cytoplasm, activity regulated gene transcription and neurodevelopmental disorders. Moreover the analysis also highlighted the direct relation to proteins involved in promoting neuronal survival, plasticity and cAMP responsive element binding protein proteins. The current study helps in understanding the physicochemical properties and reveals the neuro-modulatory role of Npas4 in crucial pathways involved in neuronal survival and neural signalling hemostasis.
Npas4是一种活性依赖的转录因子，负责调控参与神经传递的靶基因的表达。尽管Npas4在许多神经元疾病中具有重要作用，但其蛋白质的三级结构及其物理化学性质仍知之甚少。在本研究中，我们首先对Npas4进行了系统发育分析，并确定了疏水、柔性和促进有序-无序的氨基酸含量。通过不同的计算机模拟方法预测了Npas4的蛋白质结合区域、翻译后修饰和结晶倾向。通过LOMET、SPARSKS-X、I-Tasser、RaptorX、MUSTER和Pyhre预测了Npas4的三维模型，并根据拉氏图、PROSA和Qmean评分选择了最佳模型。然后通过MODREFINER对最佳模型进行进一步优化。最后通过STRING数据库鉴定了Npas4的相互作用伙伴。系统发育分析表明，人类Npas4基因与黑猩猩、猴子、长臂猿等其他灵长类动物密切相关。Npas4的物理化学性质表明它是一种具有N端有序区域的内在无序蛋白。翻译后修饰分析表明不存在乙酰化和甘露糖基化位点。在PAS A结构域中发现了三个潜在的磷酸化位点（S108、T130和T136），而在PAS B结构域中存在一个磷酸化位点（S273）。预测的Npas4三级结构表明，bHLH结构域和PAS结构域具有三级结构，而蛋白质的其余部分表现出无序特性。蛋白质-蛋白质相互作用分析揭示了NPas4与各种蛋白质的相互作用，这些蛋白质主要参与蛋白质从细胞核到细胞质的运输、活性调节基因转录和神经发育障碍。此外，分析还突出了与促进神经元存活、可塑性和cAMP反应元件结合蛋白的蛋白质的直接关系。本研究有助于理解Npas4的物理化学性质，并揭示其在神经元存活和神经信号止血的关键途径中的神经调节作用。
