Li Qiuling, Lim Kayla Y, Altawell Raad, Verderose Faith, Li Xiling, Dong Wanying, Martinez Joshua, Dickman Dion, Stavropoulos Nicholas
NYU Neuroscience Institute, Department of Neuroscience and Physiology, New York University School of Medicine, New York, New York, United States of America.
Waksman Institute, Rutgers University, Piscataway, New Jersey, United States of America.
PLoS Genet. 2025 Jan 22;21(1):e1011574. doi: 10.1371/journal.pgen.1011574. eCollection 2025 Jan.
Mutations of the Cullin-3 (Cul3) E3 ubiquitin ligase are associated with autism and schizophrenia, neurological disorders characterized by sleep disturbances and altered synaptic function. Cul3 engages dozens of adaptor proteins to recruit hundreds of substrates for ubiquitination, but the adaptors that impact sleep and synapses remain ill-defined. Here we implicate Insomniac (Inc), a conserved protein required for normal sleep and synaptic homeostasis in Drosophila, as a Cul3 adaptor. Inc binds Cul3 in vivo, and mutations within the N-terminal BTB domain of Inc that weaken Inc-Cul3 associations impair Inc activity, suggesting that Inc function requires binding to the Cul3 complex. Deletion of the conserved C-terminus of Inc does not alter Cul3 binding but abolishes Inc activity in the context of sleep and synaptic homeostasis, indicating that the Inc C-terminus has the properties of a substrate recruitment domain. Mutation of a conserved, disease-associated arginine in the Inc C-terminus also abolishes Inc function, suggesting that this residue is vital for recruiting Inc targets. Inc levels are negatively regulated by Cul3 in neurons, consistent with Inc degradation by autocatalytic ubiquitination, a hallmark of Cullin adaptors. These findings link Inc and Cul3 in vivo and support the notion that Inc-Cul3 complexes are essential for normal sleep and synaptic function. Furthermore, these results indicate that dysregulation of conserved substrates of Inc-Cul3 complexes may contribute to altered sleep and synaptic function in autism and schizophrenia associated with Cul3 mutations.
Cullin-3（Cul3）E3泛素连接酶的突变与自闭症和精神分裂症有关，这两种神经疾病的特征是睡眠障碍和突触功能改变。Cul3与数十种衔接蛋白结合，以招募数百种底物进行泛素化，但影响睡眠和突触的衔接蛋白仍不明确。在这里，我们发现失眠蛋白（Inc）是果蝇正常睡眠和突触稳态所需的一种保守蛋白，它是一种Cul3衔接蛋白。Inc在体内与Cul3结合，Inc的N端BTB结构域内的突变会削弱Inc-Cul3的结合，从而损害Inc的活性，这表明Inc的功能需要与Cul3复合物结合。删除Inc保守的C端不会改变与Cul3的结合，但会在睡眠和突触稳态的背景下消除Inc的活性，这表明Inc的C端具有底物招募结构域的特性。Inc C端一个保守的、与疾病相关的精氨酸发生突变也会消除Inc的功能，这表明该残基对于招募Inc的靶点至关重要。在神经元中，Inc的水平受到Cul3的负调控，这与Inc通过自催化泛素化降解一致，这是Cullin衔接蛋白的一个标志。这些发现将Inc和Cul3在体内联系起来，并支持了Inc-Cul3复合物对正常睡眠和突触功能至关重要的观点。此外，这些结果表明，Inc-Cul3复合物保守底物的失调可能导致与Cul3突变相关的自闭症和精神分裂症患者睡眠和突触功能的改变。
