Nuclear Localization of the Autism Candidate Gene Neurobeachin and Functional Interaction with the NOTCH1 Intracellular Domain Indicate a Role in Regulating Transcription

Background
Neurobeachin (NBEA) is an autism spectrum disorders (ASD) candidate gene. NBEA deficiency affects regulated secretion, receptor trafficking, synaptic architecture and protein kinase A (PKA)-mediated phosphorylation. NBEA is a large multidomain scaffolding protein. From N- to C-terminus, NBEA has a concanavalin A-like lectin domain flanked by armadillo repeats (ACA), an A-kinase anchoring protein domain that can bind to PKA, a domain of unknown function (DUF1088) and a BEACH domain, preceded by a pleckstrin homology-like domain and followed by WD40 repeats (PBW). Although most of these domains mediate protein-protein interactions, no interaction screen has yet been performed.
Methods
Yeast two-hybrid screens with the ACA and PBW domain modules of NBEA gave a list of interaction partners, which were analyzed for Gene Ontology (GO) enrichment. Neuro-2a cells were used for confocal microscopy and nuclear extraction analysis. NOTCH-mediated transcription was studied with luciferase reporter assays and qRT-PCR, combined with NBEA knockdown or overexpression.
Results
Both domain modules showed a GO enrichment for the nucleus. PBW almost exclusively interacted with transcription regulators, while ACA interacted with a number of PKA substrates. NBEA was partially localized in the nucleus of Neuro-2a cells, albeit much less than in the cytoplasm. A nuclear localization signal was found in the DUF1088 domain, which was shown to contribute to the nuclear localization of an EGFP-DPBW fusion protein. Yeast two-hybrid identified the Notch1 intracellular domain as a physical interactor of the PBW domain and a role for NBEA as a negative regulator in Notch-mediated transcription was demonstrated.
Conclusion
Defining novel interaction partners of conserved NBEA domain modules identified a role for NBEA as transcriptional regulator in the nucleus. The physical interaction of NBEA with NOTCH1 is most relevant for ASD pathogenesis because NOTCH signaling is essential for neural development.

研究背景
神经海滩蛋白（Neurobeachin, NBEA）是自闭症谱系障碍（Autism Spectrum Disorders, ASD）的候选基因。NBEA缺失会影响调控型分泌、受体运输、突触结构以及蛋白激酶A（Protein Kinase A, PKA）介导的磷酸化过程。NBEA属于大型多结构域支架蛋白。从N端到C端，NBEA依次包含一个由臂重复序列（armadillo repeats）侧翼结合的伴刀豆球蛋白A样凝集素结构域（concanavalin A-like lectin domain, ACA）、一个可结合PKA的A激酶锚定蛋白结构域（A-kinase anchoring protein domain）、一个功能未知结构域（Domain of Unknown Function 1088, DUF1088）以及一个BEACH结构域（BEACH domain）；该蛋白序列前接一个类似pleckstrin同源结构域（pleckstrin homology-like domain），后接WD40重复序列（WD40 repeats, PBW）。尽管上述多数结构域均介导蛋白质-蛋白质相互作用，但目前尚未开展相关相互作用筛选实验。

实验方法
本研究采用针对NBEA的ACA与PBW结构域模块的酵母双杂交筛选（yeast two-hybrid screens），获得了一系列相互作用靶点，并对其进行基因本体（Gene Ontology, GO）富集分析。使用Neuro-2a细胞开展共聚焦显微镜成像（confocal microscopy）与核提取物分析（nuclear extraction analysis）。通过荧光素酶报告基因实验（luciferase reporter assays）、定量实时聚合酶链反应（quantitative real-time polymerase chain reaction, qRT-PCR）结合NBEA敲低或过表达处理，探究NOTCH介导的转录调控过程。

实验结果
两类结构域模块均在细胞核相关功能上呈现GO富集。PBW结构域几乎仅与转录调控因子发生相互作用，而ACA结构域则可结合多种PKA底物。NBEA在Neuro-2a细胞核中存在部分定位，尽管其核内表达量远低于胞质。研究人员在DUF1088结构域中发现了一段核定位信号（nuclear localization signal），该信号可促进EGFP-DPBW融合蛋白（EGFP-DPBW fusion protein）的核定位。酵母双杂交实验证实，Notch1胞内结构域（Notch1 intracellular domain）为PBW结构域的物理相互作用靶点，并验证了NBEA作为NOTCH介导转录过程的负调控因子的功能。

研究结论
通过对保守型NBEA结构域模块的新型相互作用靶点进行鉴定，本研究明确了NBEA作为细胞核内转录调控因子的功能。NBEA与NOTCH1的物理相互作用与自闭症谱系障碍的发病机制密切相关，因为NOTCH信号通路对神经发育至关重要。