Genetic Regulation of Alcohol Metabolism in Yeast

描述(申请人提供)：生物体以多种方式对不断变化的环境做出反应。动物们可以走开。对于植物和单细胞微生物来说，离开一个充满压力的环境不是一个选择。它也不是多细胞生物体中单个细胞的一个选择。在每一种情况下，细胞都必须适应。也许最常见的环境变化是细胞沐浴的营养物质的变化。这影响了它们最基本的属性：产生ATP以维持代谢动态平衡的能力。在简单的单细胞微生物中，萌芽酵母酿酒酵母中，最简单的营养转移实验范式是对葡萄糖损失的反应，葡萄糖是这一过程中的首选能量和碳源，就像在所有生物体中一样。在自然界中，酵母主要从可发酵的糖中获得营养供应，因此进化出了非常有效的途径来在广泛的浓度范围内吸收和代谢糖。它们的糖酵解途径是如此有效，以至于它们可以完全省去呼吸，使它们成为所谓的“小阴性”酵母，这意味着它们可以在没有正常运作的线粒体呼吸链的情况下生长。然而，当糖耗尽时，会诱导强劲的呼吸新陈代谢，使它们能够利用各种次要碳源，从乙醇和甘油到复杂的储存碳水化合物和脂肪。为了激活这些通路，蛋白激酶Snf1及其附属蛋白启动了复杂的细胞内信号级联反应。SNF1是在所有多细胞生物体中发现的普遍存在的AMP激活的蛋白激酶(AMPKs)的同源物。AMPK作为细胞内的能量感应器，根据代谢产物和能量的供应和需要改变代谢活动的方向。AMPKs通过磷酸化直接改变酶的活性，并通过启动复杂的转录级联反应间接地改变酶的活性，转录因子激活转录因子，进而激活下游靶基因。我们的主要兴趣是在生化和分子水平上了解Snf1通过其下游的两个效应因子--转录因子Adr1和Cat8作用于酵母中激活基因表达的机制。在上一个资助期，我们发现了一个不活跃的预启动复合体，它是当染色质允许结合Adr1和Cat8时形成的，但对转录激活仍然是抑制的。我们建议分离并鉴定不活跃的“稳定的”RNA PolII复合体。在过去的一年里，我们发现Adr1的活性受到一种抑制物的调节，这种抑制物在酵母中被称为BMH。BMH与Adr1的磷酸化调控结构域结合。我们建议表征BMH的结合部位，并确定BMH抑制Adr1活性的机制。 与公共健康相关：AMP激活蛋白激酶(AMPK)活性的改变导致转录调控的改变被认为发生在心脏病、代谢综合征、糖尿病、发育和癌症中。我们的主要目标是了解AMPK如何影响酵母中受营养胁迫调控的一大组基因的转录。了解酵母AMPK(Snf1复合体)是如何调节下游基因的，可以揭示AMPK改变人类基因转录以应对营养胁迫的机制。这些信息反过来可能导致对与糖代谢相关的病理条件所带来的代谢紊乱的治疗和诊断有新的见解。

DESCRIPTION (provided by applicant): Organisms respond to a changing environment in multiple ways. Animals can walk away. For plants and single-celled microbes, leaving a stressful environment is not an option. Nor is it an option for individual cells within a multicellular organism. In each of these cases the cells have to adapt. Perhaps the most common environmental change is a change in the nutrients the cell is bathed in. This influences their most basic property: the ability to generate ATP to maintain metabolic homeostasis. In the simple single celled microbe, the budding yeast Saccharomyces cerevisiae, the simplest experimental paradigm for a nutrient shift is the response to loss of glucose, the preferred energy and carbon source in this as in all organisms. In nature, yeast derive their nutrient supply primarily from fermentable sugars and have thus evolved very efficient pathways to take up and metabolize sugars over a wide range of concentrations. Their glycolytic pathway is so efficient that they can dispense altogether with respiration, making them a so-called "petite-negative" yeast, meaning they can grow without a functioning mitochondrial respiratory chain. However, when sugar is exhausted a robust respiratory metabolism is induced that allows them to utilize a variety of secondary carbon sources, ranging from ethanol and glycerol to complex stored carbohydrates and lipids. To activate these pathways an intricate intracellular signaling cascade is initiated by the protein kinase Snf1 and its accessory proteins. Snf1 is a homolog of the ubiquitous AMP-activated protein kinases (AMPKs) found in all multicellular organisms. AMPK functions as an intracellular energy sensor, re-directing metabolic activity to correspond to the availability and need for metabolites and energy. AMPKs modify enzyme activities directly by phosphorylation and indirectly by setting in motion a complex transcriptional cascade that activates transcription factors that in turn activate downstream target genes. Our primary interest is in understanding at a biochemical and molecular level the mechanisms by which Snf1 activates gene expression in yeast, acting through two of its downstream effectors, the transcription factors Adr1 and Cat8. In the last funding period we discovered an inactive pre-initiation complex that was formed when chromatin had become permissive for binding Adr1 and Cat8, but remained repressive for transcription activation. We propose to isolate and characterize the inactive "poised" RNA pol II complex. In the last year we discovered that Adr1 activity is regulated by a repressor, a 14-3-3 protein called Bmh in yeast. Bmh binds to a phosphorylated Regulatory Domain of Adr1. We propose to characterize the binding site for Bmh and to determine the mechanism whereby Bmh represses Adr1 activity. PUBLIC HEALTH RELEVANCE: Alterations in transcriptional regulation brought about by changes in AMP-activated protein kinase (AMPK) activity are thought to occur in heart disease, metabolic syndrome, diabetes, development, and cancer. Our major goal is to understand how AMPK influences the transcription of a large set of genes that are regulated by nutrient stress in yeast. Understanding how the yeast AMPK, the Snf1 complex, regulates downstream genes could shed light on the mechanisms by which AMPK alters the transcription of human genes in response to nutrient stress. This information in turn, might lead to new insights into treatment and diagnosis of metabolic disorders brought about by pathological conditions related to glucose metabolism.