Farrell Kayla, Musaus Madeline, Navabpour Shaghayegh, Martin Kiley, Ray W Keith, Helm Richard F, Jarome Timothy J
Department of Animal and Poultry Science, Virginia Polytechnic Institute and State University, Blacksburg, VA, United States.
School of Neuroscience, Virginia Polytechnic Institute and State University, Blacksburg, VA, United States.
Front Mol Neurosci. 2021 Sep 29;14:716284. doi: 10.3389/fnmol.2021.716284. eCollection 2021.
Ubiquitin-proteasome mediated protein degradation has been widely implicated in fear memory formation in the amygdala. However, to date, the protein targets of the proteasome remain largely unknown, limiting our understanding of the functional significance for protein degradation in fear memory formation. Additionally, whether similar proteins are targeted by the proteasome between sexes has yet to be explored. Here, we combined a degradation-specific K48 Tandem Ubiquitin Binding Entity (TUBE) with liquid chromatography mass spectrometry (LC/MS) to identify the target substrates of the protein degradation process in the amygdala of male and female rats following contextual fear conditioning. We found that males (43) and females (77) differed in the total number of proteins that had significant changes in K48 polyubiquitin targeting in the amygdala following fear conditioning. Many of the identified proteins (106) had significantly reduced levels in the K48-purified samples 1 h after fear conditioning, suggesting active degradation of the substrate due to learning. Interestingly, only 3 proteins overlapped between sexes, suggesting that targets of the protein degradation process may be sex-specific. In females, many proteins with altered abundance in the K48-purified samples were involved in vesicle transport or are associated with microtubules. Conversely, in males, proteins involved in the cytoskeleton, ATP synthesis and cell signaling were found to have significantly altered abundance. Only 1 protein had an opposite directional change in abundance between sexes, LENG1, which was significantly enhanced in males while lower in females. This suggests a more rapid degradation of this protein in females during fear memory formation. Interestingly, GFAP, a critical component of astrocyte structure, was a target of K48 polyubiquitination in both males and females, indicating that protein degradation is likely occurring in astrocytes following fear conditioning. Western blot assays revealed reduced levels of these target substrates following fear conditioning in both sexes, confirming that the K48 polyubiquitin was targeting these proteins for degradation. Collectively, this study provides strong evidence that sex differences exist in the protein targets of the degradation process in the amygdala following fear conditioning and critical information regarding how ubiquitin-proteasome mediated protein degradation may contribute to fear memory formation in the brain.
泛素 - 蛋白酶体介导的蛋白质降解在杏仁核恐惧记忆形成中具有广泛影响。然而，迄今为止，蛋白酶体的蛋白质靶点仍大多未知，这限制了我们对恐惧记忆形成中蛋白质降解功能意义的理解。此外，两性之间蛋白酶体靶向的蛋白质是否相似尚待探索。在此，我们将降解特异性的K48串联泛素结合实体（TUBE）与液相色谱 - 质谱联用（LC/MS），以鉴定情境恐惧条件反射后雄性和雌性大鼠杏仁核中蛋白质降解过程的目标底物。我们发现，恐惧条件反射后，雄性（43种）和雌性（77种）杏仁核中K48多聚泛素靶向发生显著变化的蛋白质总数存在差异。许多鉴定出的蛋白质（106种）在恐惧条件反射后1小时的K48纯化样本中水平显著降低，表明由于学习导致底物的主动降解。有趣的是，两性之间只有3种蛋白质重叠，这表明蛋白质降解过程的靶点可能具有性别特异性。在雌性中，K48纯化样本中丰度改变的许多蛋白质参与囊泡运输或与微管相关。相反，在雄性中，发现参与细胞骨架、ATP合成和细胞信号传导的蛋白质丰度有显著改变。只有1种蛋白质在两性之间丰度变化方向相反，即LENG1，它在雄性中显著增强而在雌性中较低。这表明在恐惧记忆形成过程中，该蛋白质在雌性中降解更快。有趣的是，星形胶质细胞结构的关键成分GFAP在雄性和雌性中都是K48多聚泛素化的靶点，表明恐惧条件反射后蛋白质降解可能发生在星形胶质细胞中。蛋白质印迹分析显示，恐惧条件反射后两性中这些目标底物的水平均降低，证实K48多聚泛素靶向这些蛋白质进行降解。总体而言，本研究提供了有力证据，表明恐惧条件反射后杏仁核中降解过程的蛋白质靶点存在性别差异，并提供了关于泛素 - 蛋白酶体介导的蛋白质降解如何可能促进大脑恐惧记忆形成的关键信息。
