McMillan Joseph D, Wang Shuai, Wohlfahrt Jessica, Guergues Jennifer, Stevens Stanley M, Thinakaran Gopal
Byrd Alzheimer's Center and Research Institute, University of South Florida, Tampa, FL 33613; Department of Molecular Medicine, University of South Florida, Tampa, FL 33612.
Department of Molecular Biosciences, University of South Florida, Tampa, FL 33620.
Mol Cell Proteomics. 2025 Aug 18:101055. doi: 10.1016/j.mcpro.2025.101055.
The gene BIN1 is the second-largest genetic risk factor for late-onset Alzheimer's disease (LOAD). It is expressed in neurons and glia in the brain as cell-type-specific and ubiquitous isoforms. BIN1 is an adaptor protein that regulates membrane dynamics in many cell types. Previously, we reported that BIN1 predominantly localizes to presynaptic terminals in neurons and regulates presynaptic vesicular release. However, the function of neuronal BIN1 in relation to LOAD is not yet fully understood. A significant gap in the field is the unbiased characterization of neuronal BIN1-interacting proteins and proximal neighbors. To address this gap and help define the functions of neuronal BIN1 in the brain, we employed TurboID-based proximity labeling to identify proteins biotinylated by the neuronal BIN1 isoform 1-TurboID fusion protein (BIN1iso1-TID) in cultured mouse neuroblastoma (N2a) cells in vitro and in adult mouse brain neurons in vivo. Label-free quantification-based proteomic analysis of the BIN1iso1-TID biotinylated proteins led to the discovery of 360 proteins in N2a cells and 897 proteins in mouse brain neurons, identified as BIN1iso1-associated (proximal) or interacting proteins. A total of 92 proteins were common in both datasets, indicating that these are high-confidence BIN1-interacting or proximity proteins. SynapticGO analysis of the mouse brain dataset revealed that BIN1iso1-TurboID labeled 159 synaptic proteins, with 60 corresponding to the synaptic vesicle cycle. Based on phosphorylation site analysis of the neuronal BIN1iso1-TID interactome and related kinase prediction, we selected and validated AAK1, CDK16, SYNJ1, PP2BA, and RANG through immunostaining and proximity ligation assays as members of the BIN1 interactome in the mouse brain. This study establishes a foundation for further investigations into the function of neuronal BIN1 by identifying several previously unknown proximal and potential interacting proteins of BIN1.
基因BIN1是晚发性阿尔茨海默病（LOAD）的第二大遗传风险因素。它以细胞类型特异性和普遍存在的异构体形式在大脑中的神经元和胶质细胞中表达。BIN1是一种衔接蛋白，可调节多种细胞类型中的膜动力学。此前，我们报道BIN1主要定位于神经元的突触前末端，并调节突触前囊泡释放。然而，神经元BIN1与LOAD相关的功能尚未完全了解。该领域的一个重大空白是对神经元BIN1相互作用蛋白和近端邻居的无偏表征。为了填补这一空白并帮助定义神经元BIN1在大脑中的功能，我们采用基于TurboID的邻近标记来鉴定在体外培养的小鼠神经母细胞瘤（N2a）细胞和体内成年小鼠脑神经元中被神经元BIN1异构体1-TurboID融合蛋白（BIN1iso1-TID）生物素化的蛋白。对BIN1iso1-TID生物素化蛋白进行基于无标记定量的蛋白质组分析，在N2a细胞中发现了360种蛋白，在小鼠脑神经元中发现了897种蛋白，这些蛋白被鉴定为与BIN1iso1相关（近端）或相互作用的蛋白。两个数据集中共有92种蛋白，表明这些是高可信度的BIN1相互作用或邻近蛋白。对小鼠脑数据集的SynapticGO分析表明，BIN1iso1-TurboID标记了159种突触蛋白，其中60种与突触囊泡循环相关。基于对神经元BIN1iso1-TID相互作用组的磷酸化位点分析和相关激酶预测，我们通过免疫染色和邻近连接试验选择并验证了AAK1、CDK16、SYNJ1、PP2BA和RANG作为小鼠脑中BIN1相互作用组的成员。本研究通过鉴定几种先前未知的BIN1近端和潜在相互作用蛋白，为进一步研究神经元BIN1的功能奠定了基础。
