Heller Abigail T, Bhattacharya Aniket, Li Haorong, Turkalj Luka, Thiyagarajan Shruthi, Suzuki Emma, Mossa Adele, Zheng Haiyan, Hao Ling, Manzini M Chiara
bioRxiv. 2025 Jun 28:2025.06.26.661826. doi: 10.1101/2025.06.26.661826.
Loss of the protein scaffold Coiled-coil and C2 domain containing 1A (CC2D1A) leads to intellectual disability (ID), autism spectrum disorder (ASD), and other neurodevelopmental presentations in humans. CC2D1A interactions have been studied in different cell lines proposing diverse roles in endolysosomal maturation and intracellular signaling, but the composition and functional mechanisms of the CC2D1A interactome remain poorly understood, especially in the brain. We performed comprehensive proteomic analyses to characterize CC2D1A binding partners, first comparing immunoprecipitations with three different anti-CC2D1A antibodies in HEK293 cells and then probing the mouse hippocampus. In HEK cells, Gene Ontology (GO) analysis revealed broad interaction networks in the nucleus, mitochondrion, and cytoplasmic vesicles sharing functions in organelle organization, vesicle mediated transport, and protein metabolism. These are unified by the best characterized CC2D1A interactor, the ESCRT III component CHMP4B, and define a pleiotropic role for CC2D1A in membrane trafficking and protein homeostasis. In the hippocampus, using stringent criteria and additional controls, including a hypomorph mouse line, we identified 10 high-confidence interactors in addition to CHMP4B (TNIK, G3BP2, CEP135, MAPKAP1, SHFL, PPT1, PNKD, VAMP5, and PPP6R2) revealing roles for RNA regulation and synaptic function. The HEK studies had also pointed to CC2D1B, the only paralog of CC2D1A, as an interactor. We confirmed that not only the two proteins can bind in the brain, but also localize in different synaptic compartments, showing that CC2D1A is uniquely enriched in the post-synapse. This supports a unique function of CC2D1A in regulation of synaptic transmission that could explain the more severe cognitive deficits in humans and mice upon its loss. To our knowledge these findings provide the most comprehensive characterization of the CC2D1A interactome to date, elucidating novel, multifaceted, and dynamic cellular functions, providing potential implications for its role in neurodevelop-mental disorders.
蛋白质支架卷曲螺旋和含C2结构域蛋白1A（CC2D1A）的缺失会导致人类智力残疾（ID）、自闭症谱系障碍（ASD）和其他神经发育症状。CC2D1A的相互作用已在不同细胞系中进行研究，提出其在内溶酶体成熟和细胞内信号传导中具有多种作用，但CC2D1A相互作用组的组成和功能机制仍知之甚少，尤其是在大脑中。我们进行了全面的蛋白质组学分析以表征CC2D1A结合伴侣，首先比较了在HEK293细胞中使用三种不同抗CC2D1A抗体进行的免疫沉淀，然后对小鼠海马体进行检测。在HEK细胞中，基因本体论（GO）分析揭示了在细胞核、线粒体和细胞质囊泡中的广泛相互作用网络，这些网络在细胞器组织、囊泡介导的运输和蛋白质代谢中具有共同功能。这些由特征最明确的CC2D1A相互作用蛋白、ESCRT III成分CHMP4B统一起来，并确定了CC2D1A在膜运输和蛋白质稳态中的多效性作用。在海马体中，使用严格的标准和额外的对照，包括一个低表达小鼠品系，我们除了CHMP4B之外还鉴定出10个高可信度的相互作用蛋白（TNIK、G3BP2、CEP135、MAPKAP1、SHFL、PPT1、PNKD、VAMP5和PPP6R2），揭示了其在RNA调节和突触功能中的作用。HEK研究还指出CC2D1A的唯一旁系同源物CC2D1B是一种相互作用蛋白。我们证实这两种蛋白不仅能在大脑中结合，还定位于不同的突触区室，表明CC2D1A在突触后独特富集。这支持了CC2D1A在调节突触传递中的独特功能，这可以解释其缺失时人类和小鼠更严重的认知缺陷。据我们所知，这些发现提供了迄今为止对CC2D1A相互作用组最全面的表征，阐明了新的、多方面的和动态的细胞功能，为其在神经发育障碍中的作用提供了潜在的启示。
