Identification and characterization of proteins regulating the metabolic network of cellular fat storage organelles

几乎所有的细胞都将代谢能量以脂肪的形式储存在专门的细胞器--脂滴(LDS)中。作为脂质代谢的关键分子，LDS为多种细胞过程储存和提供过多的代谢物，在肥胖症和2型糖尿病等代谢性疾病中发挥重要作用。LDS被整合到一个整体的细胞代谢网络中，该网络依赖于LDS和其他细胞细胞器的功能和物理耦合。有效耦合所需的因素和调节这些因素的机制目前尚不清楚。该项目的目的是确定和描述LDS在其实体中的代谢网络。我们开发了一个高通量的流式细胞仪筛选平台，用于监测模式生物酿酒酵母的LD动态。将其应用于全基因组筛选，我们确定了LD消耗过程中LDS与其他细胞器功能偶联所需的候选蛋白质。在这里，我们的目标是在分子水平上分析鉴定出的蛋白质的功能。为了实现这一目标，我们将应用以下研究方法：1)使用细胞生物学、生化和显微镜方法分析识别出的蛋白质的分子功能。2)应用基于质谱学的方法分析细胞内的脂质成分。这将揭开已识别的蛋白质如何影响LD衍生的脂质代谢物的利用和分布。3)分析哺乳动物肝细胞中保守候选蛋白在调节LD动态中的作用。这种翻译方法将破译LDS复杂代谢网络背后的进化保守机制。鉴于全球肥胖症的流行和相关疾病的日益流行，如2型糖尿病，我们认为这一研究项目对于了解潜在的细胞机制和将基础研究迅速转化为未来的预防和治疗策略至关重要。

Almost all cells store metabolic energy as fat in specialized organelles, the lipid droplets (LDs). As key players in lipid metabolism, LDs store and provide a plethora of metabolites for a variety of cellular processes and play a major role in metabolic diseases, such as obesity and type 2 diabetes. LDs are integrated into an overall cellular metabolic network, which relies on the functional and physical coupling of LDs and other cellular organelles. Factors required for the efficient coupling and mechanisms regulating them are currently unknown. The aim of this project is to identify and characterize the metabolic network of LDs in its entity. We developed a high-throughput flow cytometry screening-platform to monitor LD dynamics in the model organisms yeast, Saccharomyces cerevisiae. Applying it to a genome-wide screen, we identified candidate proteins which are required for the functional coupling of LDs and other organelles during LD consumption. Here, we aim to analyze the function of the identified proteins at a molecular level. To achieve this, we will apply the following research approaches: 1) Analysis of the molecular function of identified proteins using cell biological, biochemical, and microscopy-based methods. 2) Application of mass spectrometry-based methods to analyze the cellular lipid composition. This will unravel how the identified proteins affect the utilization and distribution of LD-derived lipid metabolites. 3) Analysis of the role of conserved candidate proteins in regulating LD dynamics in mammalian hepatocytes. This translational approach will decipher evolutionary conserved mechanisms underlying the complex metabolic network of LDs. In the light of a global obesity pandemic and an ever increasing prevalence of related diseases, such as type 2 diabetes, we consider this research project essential for the understanding of the underlying cellular mechanisms and the rapid translation of basic research into future prevention- and treatment strategies.