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

适当的蛋白质亚细胞定位是大脑发育的基础。然而，决定蛋白质定位的全局规则仍然知之甚少。一些证据表明，蛋白质SUMO化（一个小的泛素样修饰物[SUMO]对赖氨酸的共价修饰）不仅对蛋白质定位很重要，而且对脑功能也很重要。具体来说，无Sumo2（但不包括其类似物Sumo1和Sumo3）的小鼠在发育早期死亡。因此，我们推测，确定Sumo2底物将为大脑功能提供重要的线索。为了验证这一想法，我们培育了携带HA表位标签的Sumo2敲入小鼠，并从小鼠的大脑中提取HA-Sumo2偶联物，通过质谱鉴定底物，并将其与Sumo1敲入小鼠进行比较（Tirard et al, 2012）。当比较驻留在大脑中的Sumo2-和sumo1 -客户端时，我们发现只有28%的蛋白重叠。免疫荧光染色显示Sumo1（严格核）与Sumo2（广泛分布）的亚细胞定位截然不同，支持了这一点。在Sumo2和Sumo1下拉下调中，选择性地挑选出的最强有力的候选蛋白之一是CaMKII激酶。这是令人兴奋的，因为CaMKII是记忆形成的关键介质，是一种受到严格调控的蛋白质。已知其水平、磷酸化和与其他蛋白质的相互作用介导其活性。然而，其监管的程度尚未完全了解。鉴于SUMOylation蛋白在发育中的关键作用，这一机制可能是介导CaMKII水平、定位、活性以及最终其神经功能的强大调节剂。此外，我们发现sumo2共轭CaMKII是内源性发生的，这表明一种新的蛋白质调控层可能适用于许多脑内蛋白底物，从而允许深入研究蛋白质sumo酰化在大脑发育中的作用。在这个项目的短期内，我们将测试SUMOylation如何介导CaMKII的功能。具体来说，我们将测试这些如何影响CaMKII蛋白水平，亚细胞定位和记忆形成的神经基质。此外，我们将在体外测试神经元活动是否调节CaMKII SUMOylation。最后，我们将在空间和时间上解构CaMKII在发育过程中不同大脑区域的summoylation。未来的研究将研究这种修饰如何影响小鼠的行为，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，这个项目将使神经科学界受益，因为它提供了sumo2结合神经基质的图谱，从而揭示了这种修饰如何影响它们在大脑中的功能。鉴于在蛋白质组学筛选中鉴定的多种底物，几个项目和HQP将从其成果中受益。

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.