SUMOylation guides synaptic protein localization and function in mammalian brain development.

SUMOylation 指导哺乳动物大脑发育中的突触蛋白定位和功能。

• 批准号: RGPIN-2019-04133
• 负责人: Rousseaux, Maxime
• 金额: $ 2.7万
• 依托单位: University of Ottawa
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=714643
• 关键词: SUMOylation; guides; synaptic; protein; localization

The proper subcellular localization of proteins is fundamental for brain development. The global rules that dictate protein localization, however, remain poorly understood. Several lines of evidence suggest that protein SUMOylation (covalent modification of a small ubiquitin-like modifier [SUMO] to lysines) is not only important for protein localization but also for brain function. Specifically, Sumo2 (but not its paralogues Sumo1 and Sumo3) null mice die early on in development. Therefore, we theorized that identifying Sumo2 substrates would shed important light into brain function. To test this idea, we generated Sumo2 knockin mice which carry an HA epitope tag and pulled down HA-Sumo2-conjugates from the brains of mice, identifying the substrates via mass-spectrometry and comparing them to Sumo1 knockin mice (Tirard et al, 2012). When comparing the brain-resident Sumo2- and Sumo1-clients, we found only 28% overlapping proteins. This is supported by immunofluorescence staining showing drastically different subcellular localization of Sumo1 (strictly nuclear) vs. Sumo2 (broadly distributed). One of the most robust candidates picked up selectively in Sumo2 vs. Sumo1 pulldowns is the kinase CaMKII. This is exciting because CaMKII is a critical mediator of memory formation and is a tightly regulated protein. Its levels, phosphorylation and interactions with other proteins are known to mediate its activity. Nevertheless, the extent of its regulation is not completely understood. Given the critical role for protein SUMOylation in development, this mechanism may be a strong modulator in mediating CaMKII levels, localization, activity and ultimately its neural function. Moreover, our finding that Sumo2-conjugated CaMKII occurs endogenously, suggests a new tier of protein regulation which may be applied across numerous brain-resident protein substrates, thus allowing for an in-depth research program on the role of protein SUMOylation in the context of brain development. In the short term of this program, we will test how SUMOylation mediates CaMKII function. Specifically, we will test how these affect CaMKII protein levels, subcellular localization and on neural substrates of memory formation. Moreover, we will test whether neuronal activity modulates CaMKII SUMOylation in vitro. Lastly, we will spatially and temporally deconstruct CaMKII SUMOylation across different brain regions during development. Future studies will investigate how this modification affects mouse behaviours to ultimately understand the impact of SUMOylation on dictating protein function and the developmental fate of the brain. Long term, this program stands to benefit the neuroscience community as it provides an atlas of Sumo2-bound neural substrates and thus sheds important light into how this modification affects their function in the brain. Given the multiple substrates identified in this proteomics screen, several projects and HQP stand to benefit from its fruit.

蛋白质的正确亚细胞定位是大脑发育的基础。然而，人们对决定蛋白质定位的全球规则仍然知之甚少。多项证据表明，蛋白质 SUMO 化（小型泛素样修饰剂 [SUMO] 与赖氨酸的共价修饰）不仅对蛋白质定位很重要，而且对大脑功能也很重要。具体来说，Sumo2（但不是其旁系同源 Sumo1 和 Sumo3）无效小鼠在发育早期死亡。因此，我们推测识别 Sumo2 底物将为了解大脑功能提供重要线索。为了测试这个想法，我们生成了携带 HA 表位标签的 Sumo2 敲入小鼠，并从小鼠大脑中拉下 HA-Sumo2 缀合物，通过质谱法识别底物并将其与 Sumo1 敲入小鼠进行比较（Tirard 等人，2012）。当比较大脑驻留的 Sumo2 和 Sumo1 客户端时，我们发现只有 28% 的重叠蛋白质。免疫荧光染色显示 Sumo1（严格位于核内）与 Sumo2（广泛分布）的亚细胞定位完全不同，这也支持了这一点。在 Sumo2 与 Sumo1 下拉中选择性选取的最强大的候选者之一是激酶 CaMKII。这是令人兴奋的，因为 CaMKII 是记忆形成的关键介质，并且是一种受到严格调控的蛋白质。已知其水平、磷酸化以及与其他蛋白质的相互作用介导其活性。然而，其监管范围尚不完全清楚。鉴于蛋白质 SUMO 化在发育中的关键作用，该机制可能是介导 CaMKII 水平、定位、活性及其神经功能的强大调节剂。此外，我们发现 Sumo2 缀合的 CaMKII 是内源性发生的，这表明了一种新的蛋白质调节机制，可以应用于多种大脑驻留蛋白质底物，从而可以对蛋白质 SUMO 化在大脑发育中的作用进行深入研究。 在这个项目的短期内，我们将测试 SUMOylation 如何介导 CaMKII 功能。具体来说，我们将测试它们如何影响 CaMKII 蛋白水平、亚细胞定位以及记忆形成的神经基质。此外，我们将在体外测试神经元活性是否调节 CaMKII SUMOylation。最后，我们将在空间和时间上解构发育过程中不同大脑区域的 CaMKII SUMOylation。未来的研究将调查这种修饰如何影响小鼠行为，以最终了解 SUMOylation 对决定蛋白质功能和大脑发育命运的影响。从长远来看，该计划将使神经科学界受益，因为它提供了 Sumo2 结合神经底物的图谱，从而为了解这种修饰如何影响其在大脑中的功能提供了重要线索。鉴于此蛋白质组学筛选中确定了多种底物，多个项目和 HQP 将从其成果中受益。