REGULATION OF PROTEIN TURNOVER IN SEPSIS

描述(由申请人提供)：本文描述的研究目标是通过了解蛋白质合成障碍的生化基础来开发增加肌肉质量的策略，蛋白质合成障碍是脓毒症代谢反应的特征。持续的骨骼肌萎缩与脓毒症相关的发病率和死亡率有关。我们发现，脓毒症，而不是无菌炎症，在至少两个细胞信号通路中诱导特定的失调，最终通过限制骨骼肌中mRNA翻译起始的过程而抑制蛋白质合成。一种包括哺乳动物靶标雷帕霉素(MTOR)，一种丝氨酸/苏氨酸激酶，控制另外两个蛋白因子S6K1和4E-BP1的磷酸化，这两个蛋白调节mRNA翻译和活性elF4E-elF4G复合体的形成和elF4G磷酸化。第二个途径是通过激活糖原合成酶激酶-3p(GSKSFI)使elF2Be磷酸化。以下具体目的是使用转基因、药理学和/或营养学方法来验证这些信号通路在抑制蛋白质合成方面的相对贡献和潜在的治疗相关性，因此骨骼肌蛋白质对败血症侮辱的抑制作用。具体目标1将研究败血症时mTOR活性降低对骨骼肌蛋白质合成减少的贡献，以及亮氨酸(或去甲亮氨酸)逆转mTOR和elF4G磷酸化抑制的能力。我们还将确定，用营养物质刺激mTOR活性是否能逆转脓毒症引起的骨骼肌蛋白质合成的抑制，以及mTOR的药理学抑制和遗传消融在营养刺激的蛋白质合成中的作用。进入BCAT2基因敲除小鼠将使我们能够确定亮氨酸或亮氨酸代谢物是否负责亮氨酸激活mTOR信号。具体目标2将探讨使用基因依赖性抗炎治疗、药物抑制GSK-3(3)和非磷酸化elF2Be的过度表达对脓毒症时肌肉蛋白质合成的影响，逆转脓毒症时由脓毒症引起的elF2Be磷酸化增加和细胞含量下降的影响。我们将验证这样一种假设，即用GSK3P抑制剂LICI、SB216763和SB415286处理脓毒症大鼠的肌肉或表达活性的、非磷酸化的elF2Be将消除脓毒症引起的骨骼肌蛋白质合成抑制。

DESCRIPTION (provided by applicant): The objective of the studies described herein are directed at developing strategies for enhancing muscle mass through understanding the biochemical basis for the impairment in protein synthesis that characterizes the metabolic response to sepsis. Sustained skeletal muscle wasting contributes to the morbidity and mortality associated with sepsis. We established that sepsis, but not sterile inflammation, induces specific maladjustments in at least two cell signaling pathways culminating in the inhibition of protein synthesis by limiting the process of mRNA translation initiation in skeletal muscle. One includes the mammalian target of rapamycin (mTOR), a serine/threonine kinase that controls phosphorylation of two other protein factors S6K1 and 4E-BP1 that regulate mRNA translation and formation of an active elF4E-elF4G complex and elF4G phosphorylation. The second is the phosphorylation of elF2Be through activation of glycogen synthase kinase-3p (GSKSfi). The following specific aims use transgenic, pharmacologic and/or nutritional approaches to validate the relative contribution and potential therapeutic relevance of these signaling pathways in the restraint in protein synthesis, and hence skeletal muscle protein, to a septic insult. Specific Aim 1 will investigate the contribution of lowered mTOR activity to the decrease in skeletal muscle protein synthesis and the ability of leucine (or norleucine) to reverse that inhibition of mTOR and elF4G phopshorylation during sepsis. We will also determine if stimulating mTOR activity with nutrients reverses the sepsis-induced inhibition of skeletal muscle protein synthesis and the role of pharacolgical inhibition and genetic ablation of mTOR in nutrient stimulated protein synthesis. Access to the BCAT2 knockout mouse will allow us to establish whether leucine or a leucine metabolite is responsible for leucine's activation of mTOR signaling. Specific Aim 2 will investigate the impact of reversing the septic-induced increase in phosphorylation and decrease in cellular content of elF2Be using genetically-dependent anti-inflammatory therapy, pharmacological inhibition of GSK-3(3 and over expression of non-phosphorylatable elF2Be on muscle protein synthesis during sepsis. We will test the hypothesis that treatment of muscles from septic rats with the GSK3P inhibitors LiCI, SB216763 and SB415286 or expression of active, non-phosphorylatable elF2Be will abrogate the sepsis-induced inhibition of protein synthesis in skeletal muscle.