Homeostatic Responses to Amino Acid Insufficiency

项目摘要 膳食氨基酸不足（AAI）可产生各种各样的结果，这些结果可能是不利的， 有益于健康。更清楚地了解指导稳态的分子因素和过程 AAI期间蛋白质组的控制（蛋白质稳态）对于确定改善应激的新原因至关重要 抵抗力和营养健康。总的来说，蛋白质合成、蛋白质水解和靶向是 蛋白质稳态和AAI触发对细胞适应至关重要的应激信号传导途径。总体 该建议的假设是，这些响应AAI的关键信号网络的结果对于 控制蛋白质稳定和抗病性。AAI的细胞感知涉及重叠信号 从酵母到人类的大部分转导机制都是保守的。我们已公布的和初步的 数据表明，这些网络中的关键是多部分综合应激反应（ISR），它 功能翻译和转录基因表达网络指导磷酸化的真核生物 起始因子2.在ISR网络中，转录因子ATF 4是指导基因表达的核心 帮助改善AAI的项目。此应用程序的目的是定义ISR对以下方面的贡献 早期分子和生理反应，在饮食AAI期间维持蛋白质稳态。到 为了实现本申请的目的，我们提出了三个具体目标：1）定义AAI中的变化如何 2）确定ATF 4在转录网络和蛋白质抑制反应中的作用， AAI;和3）评估AAI期间ISR对蛋白水解的新控制。为了实现这些目标， 研究将在ISR靶向缺陷的小鼠的细胞系和遗传品系中进行。 分析将利用复杂的分子生物学和稳定同位素技术相结合， 并比较ISR在细胞裂解物中以及在肝脏和骨骼中的活化和增殖机制 AAI期间小鼠肌肉。该提案的创新之处在于，它将首次揭示 转录组和翻译组在体内AAI期间由ISR指导，并提供ISR如何 与其他营养传感网络协调以调节蛋白质平衡。建议的工作意义重大 因为对AAI激活机制的更深入理解将导致新的分子靶点， 更好地预防或治疗人类疾病。

PROJECT SUMMARY Dietary amino acid insufficiency (AAI) can yield a wide variety of outcomes that can be either adverse or beneficial to health. A clearer understanding of the molecular factors and processes guiding homeostatic control of the proteome (proteostasis) during AAI is essential to identify novel causes of improved stress resistance and nutritional health. Collectively protein synthesis, proteolysis, and targeting are central to proteostasis and AAI triggers stress signaling pathways that are central for cell adaptation. The overarching hypothesis of this proposal is that the outcome of these key signaling networks that respond to AAI are vital to controlling proteostasis and disease resistance. Cellular sensing of AAI involves overlapping signal transduction mechanisms that are largely conserved from yeast to humans. Our published and preliminary data demonstrate that key among these networks is the multi-part Integrated Stress Response (ISR), which features translational and transcriptional gene expression networks directed by phosphorylation of eukaryotic initiation factor 2. In the ISR network, the transcription factor ATF4 is central to directing the gene expression programs that help ameliorate AAI. The objective of this application is to define the contribution of the ISR to the early molecular and physiological responses that function to maintain proteostasis during dietary AAI. To achieve the objective of this application we propose three Specific Aims: 1) Define how variations in AAI activate the ISR; 2) Determine the role of ATF4 in the transcriptional networks and proteostatic responses to AAI; and 3) Assess novel control of proteolysis by the ISR during AAI. To accomplish these aims, time course studies will be conducted in cell lines and genetic strains of mice with targeted deficiencies in the ISR. Analyses will utilize a combination of sophisticated molecular biology and stable isotope techniques to assess and compare the mechanism of ISR activation and propagation in cell lysates and in the liver and skeletal muscle of mice during AAI. The proposal is innovative in that it will reveal for the first time how the transcriptome and translatome are guided by the ISR during AAI in vivo and provide insight into how the ISR coordinates with other nutrient sensing networks to regulate protein balance. The work proposed is significant because a greater understanding of the mechanisms activated by AAI will lead to new molecular targets and approaches to better prevent or treat human diseases.