Protein Nutrition in Experimental Uremia

项目摘要/摘要 多年来，我们一直对尿毒症刺激蛋白质降解的观察感兴趣，但它也 抑制蛋白质合成，导致蛋白质能量浪费。具体地说，我们记录了这种蛋白质 降解是由caspase-3和泛素-蛋白酶体系统(UPS)的激活引起的，但 尿毒症刺激的蛋白质合成障碍的机制尚不清楚。我们的长期目标是 确定慢性肾脏疾病(CKD)如何激活导致蛋白质-能量的关键调控通路 浪费，因为这是开发治疗干预措施的第一步，以改善发病率和 慢性肾脏病患者的死亡率。在这项应用中，我们计划确定CKD是如何损害蛋白质合成的。为 在这项任务中，我们发现CKD可以刺激一种新的蛋白质--核仁蛋白66(No66)的表达。 No66的表达抑制核糖体DNA(RDNA)转录。No66含有一个带有组蛋白的JmjC结构域 脱甲基酶活性。由于rDNA转录决定了蛋白质合成的速度，我们认为 尿毒症通过NO66依赖的表观遗传学机制损害肌肉蛋白质合成，我们的初步研究 结果支持这一假说：1)我们发现CKD刺激肌肉活检组织中NO66的表达。 病人或老鼠。2)我们已经在小鼠身上创造了No66的全身敲除(No66-/-)并确定它 加速肌肉生长(与对照组相比，No66-/-小鼠的肌肉增加了20%-30% 老鼠)。此外，No66-/-小鼠还能抑制CKD诱导的蛋白质能量浪费。3)我们已经用 我们发现NO66mko具有刺激蛋白质储存的作用。4)我们发现 No66与两种组蛋白修饰蛋白--视网膜母细胞瘤结合蛋白4形成抑制复合体 (RBBP4)+组蛋白脱乙酰酶2(HDAC2)。抑制复合体可能与rDNA和rDNA结合 抑制其转录，导致蛋白质合成减少。5)我们的rna-seq分析使用soleus 在缺少NO66的肌肉中，肌肉发现核糖体双发生信号通路增加。还有一个 与结果相比，No66-/-小鼠肌肉中rRNA和核糖体翻译能力显著增加 来自NO66FLOX/FLOX小鼠。基于这些新的发现，我们假设一种由N66介导的表观遗传学 途径是对长期存在的疑问的答案：尿毒症是如何抑制蛋白质合成的？我们建议 三个具体的目的是检验我们的假设：1)确定CKD刺激NO66的机制 表达会导致蛋白质-能量的浪费。2)确定No66复合体是否与rDNA结合 并通过依赖去甲基酶的机制抑制rDNA转录。3)以确定是否缺乏 肌肉中的66号能增加蛋白质合成，防止尿毒症引起的蛋白质能量浪费。我们的 结果可能揭示改善蛋白质合成的策略，尽管存在CKD或可能的其他 分解代谢状态。

Project Summary/Abstract For years, we have been intrigued by our observation that uremia stimulates protein degradation but it also suppresses protein synthesis, leading to protein-energy wasting. Specifically, we have documented that protein degradation is induced by activation of caspase-3 and the ubiquitin-proteasome system (UPS) but the mechanism underlying uremia-stimulated impairment in protein synthesis is unknown. Our long term goal is to identify how chronic kidney disease (CKD) activates key regulatory pathways that cause protein-energy wasting because this is the first step to developing therapeutic interventions to improve the morbidity and mortality in patients with CKD. In this application, we plan to identify how CKD impairs protein synthesis. For this task, we have found that CKD stimulates the expression of a novel protein, nucleolar protein 66 (NO66). NO66 expression represses ribosomal DNA (rDNA) transcription. NO66 contains a JmjC domain with histone demethylase activity. Since rDNA transcription determines the rate of protein synthesis, we propose that uremia impairs muscle protein synthesis via an NO66-dependent, epigenetic mechanism, Our Preliminary Results support this hypothesis: 1) we show that CKD stimulates NO66 expression in muscle biopsies of patients or mice. 2) We have created whole body knockout of NO66 in mice (NO66-/-) and determined that it accelerates muscle growth (NO66-/- mice experienced a 20-30% increase in muscle vs. changes in control mice). In addition, NO66-/- mice suppress CKD-induced protein-energy wasting. 3) We have created mice with muscle-specific KO of NO66 (NO66mko) and we found that NO66mko stimulates protein stores. 4) We find that NO66 forms a repressive complex with two histone-modifying proteins, retinoblastoma binding protein 4 (RBBP4) plus histone deacetylase 2 (HDAC2). The repressive complex potentially associates with rDNA and represses its transcription, resulting in decreased protein synthesis. 5) Our RNA-seq analysis using soleus muscle identified increased ribosomal bigenesis signaling pathway in muscles lacking NO66. There also is a significant increase in both rRNA and the ribosomal translational capacity in muscle of NO66-/- mice vs. results from NO66flox/flox mice. Based on these novel findings, we hypothesize that a NO66-mediated epigenetic pathway is the answer to the long standing query: how does uremia suppress protein synthesis? We propose three Specific Aims to test our hypotheses: 1) To determine the mechanism by which CKD stimulates NO66 expression contributing to protein-energy wasting. 2) To determine whether the NO66 complex binds to rDNA and represses rDNA transcription via a demethylase-dependent mechanism.3) To determine if the absence of NO66 in muscle will increase protein synthesis and prevent uremia-induced protein-energy wasting. Our results could uncover strategies for improving protein synthesis despite the presence of CKD or possibly, other catabolic conditions.