Understanding the mechanisms of UBE3A regulation in neuronal development

抽象的。UBE 3A是编码HECT（Homopolymerase to E6 AP C-末端）结构域E3泛素连接酶的基因 与许多发育和精神疾病有关。母源性UBE 3A功能丧失 酶引起Angelman综合征，而由于UBE 3A的复制或三倍， 与包括Dup 15 q综合征、精神分裂症和情绪障碍在内的广泛疾病有关。这些 观察结果强烈表明，UBE 3A活性的双向变化有助于神经精神疾病的发生。 病理然而，调节UBE 3A活性的机制仍然知之甚少。首要目标 我们提出的一项研究是揭示UBE 3A调节的机制，当出现故障时，可能导致异常的 UBE 3A功能的获得或丧失。在前期工作中，我们开发了一种高通量检测方法来评估 非截短UBE 3A错义变体的功能后果。这个屏幕识别了许多小说 功能缺失突变，以及功能获得突变，过度激活UBE 3A活性， 野生型（WT）酶水平。这些结果提供了深层的结构-功能信息， 利用杠杆来揭示UBE 3A监管机制。该提案旨在：1）建立一个完整的功能 在个体中鉴定的已知错义变体的目录，2）利用结构-功能分析来鉴定 可导致酶功能异常获得和丧失的机制，以及3）利用生物化学 我们的目标是构建能够靶向UBE 3A活性的蛋白质。我们工作产生的分子 应用于检查UBE 3A活性的改变是否可以挽救在小鼠中观察到的突触表型 在UBE 3A中携带超活化突变。如果成功，我们的工作将提供新的生物化学见解， 这些工具将使靶向UBE 3A用于各种神经精神疾病的治疗干预成为可能。

Abstract. UBE3A is a gene that encodes a HECT (Homologous to E6AP C-terminus) domain E3 ubiquitin ligase linked to numerous developmental and psychiatric disorders. Loss of function of the maternally derived UBE3A enzyme causes Angelman syndrome whereas gain of function, due to duplication or triplication of UBE3A, is linked to a broad range of disorders including Dup15q syndrome, schizophrenia, and mood disorders. These observations strongly suggest that bi-directional changes in UBE3A activity contribute to neuropsychiatric pathology. However, the mechanisms that regulate UBE3A activity remain poorly understood. The primary goal of our proposed research is to uncover mechanisms of UBE3A regulation, that when faulty, can lead to aberrant gain or loss of UBE3A function. In preliminary work, we developed a high-throughput assay to assess the functional consequence of non-truncating UBE3A missense variants. This screen identified numerous novel loss of function mutations, as well as gain of function mutations that hyperactivate UBE3A activity well above wild type (WT) enzyme levels. These results provide deep structure-function information that we can now leverage to uncover mechanisms of UBE3A regulation. This proposal aims to, 1) create a complete functional catalogue of known missense variants identified in individuals, 2) utilize structure-function analyses to identify mechanisms that can lead to both aberrant gain and loss of enzyme function, and 3) leverage biochemical insights to engineer proteins that can target UBE3A activity. The molecules generated from our work will be applied to examine whether alteration of UBE3A activity can rescue synaptic phenotypes observed in mice harboring hyperactivating mutations in UBE3A. If successful, our work will provide new biochemical insights and tools that will make it possible to target UBE3A for therapeutic intervention in various neuropsychiatric disorders.