Post-Translational Control of Neuroplasticity Effector Arc/Arg3.1

神经元具有非凡的能力来重组它们的结构、功能和连接以响应刺激。在支持这些神经可塑性变化的许多信号分子和蛋白质效应物中，活性调节的细胞因子相关蛋白（Arc）可以被认为是中心的，也是最通用的参与者之一。过去的研究强调了Arc在神经元中的多种作用;然而，是什么允许这种蛋白质承担一种细胞功能，或者与适当的分子效应器相互作用以响应特定信号，仍然知之甚少。这项发现补助金提案的首要目标是检验以下假设：Arc蛋白在精确位点的赖氨酸乙酰化对其亚细胞分布、功能和/或与不同伴侣的亲和力产生强烈的调节作用。最终，更好地了解这种类型的翻译后修饰（PTM）如何影响Arc生物学将促进我们对大脑可塑性分子基础的全面理解。在我的博士后培训期间，我使用多学科方法来搜索Arc表达和功能的药理学修饰剂。作为这项研究的一部分，我收集了第一个证据弧赖氨酸乙酰化使用串联质谱和其他互补的生化策略。对四个Arc赖氨酸残基（我将其鉴定为乙酰化的候选靶标）的初步分析显示，这些位点中的至少一个（K24）的修饰可以通过限制其多聚泛素化和随后的蛋白酶体降解来显著促进Arc蛋白积累（Lalonde et al.，Nature Communications出版）。总的来说，这一发现表明赖氨酸乙酰化对Arc生物学的强烈影响，并支持对神经元中这种修饰的功能影响以及分子基础进行更广泛研究的必要性。为了实现这一目标，我建议：1）确定先前鉴定的Arc赖氨酸残基的乙酰化如何在培养的神经元中形成Arc依赖性过程，如AMPA受体运输和树突棘形态发生; 2）借助蛋白质组学技术建立赖氨酸乙酰化如何改变Arc亚细胞分布及其与其他蛋白质的相互作用;和3）使用靶向RNAi筛选策略揭示神经元细胞中介导Arc乙酰化的特异性乙酰转移酶和脱乙酰转移酶。总之，这三条互补的研究路线将提供赖氨酸乙酰化对Arc神经生物学影响的清晰写照，并为将来扩展我们对Arc乙酰化对大脑发育，行为控制以及学习和记忆机制的研究提供坚实的经验基础。

Neurons have the remarkable capacity to reorganize their structure, function, and connections in response to stimuli. Among the many signaling molecules and protein effectors found supporting these neuroplastic changes, the Activity-regulated cytoskeleton-associated protein (Arc) can be considered one of the central, as well as most versatile, players. Past research highlight the multiple roles of Arc in neurons; however, what allows this protein to assume one cellular function over another, or interact with the appropriate molecular effectors in response to a specific signal, remains poorly understood. The overarching goal of this Discovery Grant proposal is to test the hypothesis that lysine acetylation of Arc protein at precise sites produces a strong modulatory effect on its subcellular distribution, functions, and/or affinity with different partners. Ultimately, a better understanding of how this type of post-translational modification (PTM) is influencing Arc biology will advance our general understanding of the molecular basis of brain plasticity into new territories. During my postdoctoral training, I used a multidisciplinary approach to search for pharmacological modifiers of Arc expression and function. As part of this investigation, I collected the first evidence of Arc lysine acetylation using tandem mass spectrometry and other complementary biochemical strategies. An initial analysis of the four Arc lysine residues, which I identified as candidate targets for acetylation, revealed that modification of at least one of these sites (K24) could strikingly promote Arc protein accumulation by limiting its polyubiquitination and subsequent proteasomal degradation (Lalonde et al., accepted by Nature Communications). Overall, this finding suggests a strong influence of lysine acetylation on Arc biology and supports the need for a broader investigation into the functional impact, as well as molecular underpinnings, of this modification in neurons. In order to fulfill that goal, I propose to: 1) determine how the individual acetylation of previously identified Arc lysine residues could shape Arc-dependent processes, like AMPA receptor trafficking and dendritic spine morphogenesis, in cultured neurons; 2) establish with the help of proteomic techniques how lysine acetylation changes Arc subcellular distribution and its interaction with other proteins; and 3) uncover the specific acetyltransferase and deacetyltransferase enzymes mediating Arc acetylation in neuronal cells using a targeted RNAi screening strategy. Together, these three complementary lines of research will offer a clear portrait of the influence of lysine acetylation on the neurobiology of Arc, and provide a solid empirical foundation for extending, in the future, our examination of Arc acetylation towards brain development, behavioral control, and the mechanisms of learning and memory.