Protein kinase A (PKA) plays an important role in cellular life activities. Recently, PKA was found to bind to inhibitor of nuclear factor-kappaB (IκB), a key protein in the nuclear factor-kappaB (NF-κB) pathway, to form a complex involved in the regulation of inflammatory response. However, the role of PKA in the anti-inflammatory of goose fatty liver is still unclear. A total of 14 healthy 70-day-old male Lander geese were randomly divided into a control group and an overfeeding group. Inflammation level was analyzed by histopathological method in the liver. The mRNA and protein abundance of PKA and tumor necrosis factor-alpha (TNFα), as well as ubiquitination level of PKA were detected. Moreover, goose primary hepatocytes were co-treated with glucose, harringtonine and carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132). Finally, the co-immunoprecipitated samples of PKA from the control and overfeeding group were used for protein mass spectrometry. The results showed that no difference in PKA mRNA expression was observed (P > 0.05), while the PKA protein level in overfed group was significantly reduced (P < 0.05) when compared with the control group. The ubiquitination level of PKA was higher than that of the control group in fatty liver. The mRNA expression of PKA was elevated but protein abundance was reduced in goose primary hepatocytes with 200 mmol/L glucose treatment (P < 0.05). The PKA protein abundance was dramatically reduced in hepatocytes treated with harringtonine (P < 0.01) when compared with the glucose supplemented group. Nevertheless, MG132 tended to alleviate the inhibitory effect of harringtonine on PKA protein abundance (P = 0.081). There was no significant difference on TNFα protein level among glucose-treated groups and control (P > 0.05). Protein mass spectrometry analysis showed that 29 and 76 interacting proteins of PKA were screened in goose normal and fatty liver, respectively. Validation showed that PKA interacted with the E3 ubiquitination ligases ring finger protein 135 (RNF135) and potassium channel modulatory factor 1 (KCMF1). In summary, glucose may inhibit the inflammatory response in goose fatty liver by increasing the ubiquitination level of PKA. Additionally, RNF135 and KCMF1 may be involved in the regulation of PKA ubiquitination level as E3 ubiquitination ligases.

中文翻译：

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葡萄糖通过增加PKA泛素化水平抑制鹅脂肪肝炎症反应


蛋白激酶A（PKA）在细胞生命活动中发挥着重要作用。最近，人们发现PKA与核因子κB（IκB）抑制剂（核因子κB（NF-κB）通路中的关键蛋白）结合，形成参与炎症反应调节的复合物。但PKA在鹅脂肪肝抗炎中的作用尚不清楚。 14只70日龄健康雄性兰德鹅随机分为对照组和过量饲喂组。通过组织病理学方法分析肝脏的炎症水平。检测PKA和肿瘤坏死因子-α（TNFα）的mRNA和蛋白丰度以及PKA的泛素化水平。此外，用葡萄糖、三尖杉酯碱和苯甲氧酯-L-亮氨酰-L-亮氨酰-L-亮氨醛（MG132）共同处理鹅原代肝细胞。最后，将对照组和过量喂养组的PKA免疫共沉淀样品用于蛋白质谱分析。结果显示，与对照组相比，过喂组PKA mRNA表达量无差异（P><0.05），而过量喂养组PKA蛋白水平显着降低（P<<0.05）。脂肪肝组PKA泛素化水平高于对照组。 200 mmol/L葡萄糖处理的鹅原代肝细胞中PKA mRNA表达升高，但蛋白丰度降低（P<<0.05）。与补充葡萄糖的组相比，三尖杉酯碱处理的肝细胞中 PKA 蛋白丰度显着降低 (P< 0.01)。然而，MG132 倾向于减轻三尖杉酯碱对 PKA 蛋白丰度的抑制作用（P = 0.081）。葡萄糖处理组与对照组之间TNFα蛋白水平无显着差异（P><0.05）。 蛋白质谱分析显示，在鹅正常肝和脂肪肝中分别筛选出29个和76个PKA相互作用蛋白。验证表明 PKA 与 E3 泛素化连接酶环指蛋白 135 (RNF135) 和钾通道调节因子 1 (KCMF1) 相互作用。综上所述，葡萄糖可能通过提高PKA泛素化水平来抑制鹅脂肪肝的炎症反应。此外，RNF135和KCMF1可能作为E3泛素化连接酶参与PKA泛素化水平的调节。