Structural Investigation of Lipid Storage Protein by Solid-State NMR

描述(申请人提供)：本研究项目的长期目标是通过研究脂肪储存蛋白的原子分辨结构来了解细胞脂肪代谢。中性脂的异常储存与严重的健康影响有关，包括肥胖、糖尿病、脂肪肝和心脏病。中性甘油三酯是动物体内脂肪和能量储备的主要形式。甘油三酯储存在称为脂滴的细胞器中，脂滴聚集在脂肪细胞的胞浆中。在脂滴表面发现的几种蛋白质对调节甘油三酯的储存和释放至关重要。然而，这些蛋白质的结构以及蛋白质活性如何受到蛋白质-膜相互作用的影响的细节还知之甚少。脂类储存液滴蛋白1(LSD1)在甘油三酯水解酶的激活过程中起关键作用。这也是第一个成功纯化并重组到脂滴状颗粒中的脂滴蛋白。结构信息对于阐明其活性的详细机制至关重要。固体核磁共振为LSD1在功能天然类脂环境中提供重要的三维结构和动力学数据提供了最好的机会，这对传统的结构测定技术构成了巨大的挑战。我们将使用新型固体核磁共振技术和高场光谱仪，结合圆二色谱和其他互补的生物物理和生化方法，获得关于LSD1脂蛋白复合体的重要结构和功能数据。新的质子探测、魔角旋转核磁共振方法由于其高灵敏度和额外的质子化学位移信息的优点将被应用。我们能够将LSD1提纯为几十毫克的脂蛋白颗粒。初步的核磁共振数据表明，可以获得高质量的多维核磁共振谱，具有良好的灵敏度和光谱分辨率，并且经过几周的实验，脂蛋白样品表现出结构的均一性和稳定性。这项研究包括以下具体目标：(1)实现LSD1的残基特异性共振指定，这是使用核磁共振技术确定结构不可或缺的一步。(2)利用核磁共振测量的距离约束和角度约束计算LSD1的三维结构。(3)通过相关的核磁共振实验，了解蛋白质-脂质相互作用的机制。由于LSD1与甘油三酯脂肪酶和其他蛋白质相互作用，本研究获得的LSD1的结构信息对于理解其功能和未来脂肪代谢领域的进展至关重要。 公共卫生相关性：这项拟议的研究旨在提高我们对细胞脂肪代谢的理解，建立在脂质储存蛋白的三维结构的基础上。这项研究的知识有望为如何影响脂肪储存和燃烧之间的平衡提供线索，并最终发现新的治疗策略来预防/治疗肥胖和相关的健康问题，如糖尿病、脂肪肝和心脏病。

DESCRIPTION (provided by applicant): The long-term objective of this research project is to understand cellular fat metabolism by investigating the atomic-resolution structure of lipid storage proteins. Abnormal storage of neutral lipids has been implicated in conditions of serious health impacts, including obesity, diabetes, fatty liver, and heart diseases. Neutral lipid triglycerides are a main form of fat and energy reserve in animals. Triglycerides are stored in organelles called lipid droplets, which accumulate in the cytosol of adipocytes. Several proteins found on the surface of the lipid droplets are critical to he regulation of storage and release of triglycerides. However, structures of these proteins and details of how the protein activities are affected by protein-membrane interactions are poorly understood. Lipid storage droplet protein 1 (Lsd1) is known to play a key role in the activation of triglyceride hydrolysis. It is also the first lipid droplet protein that has been successfully purified and reconstituted in lipid droplet-like particles. Structural information is critical to the elucidation of the detailed mechanism of its activity. Solid-state NMR offers the best opportunity to provide important three-dimensional structure and dynamics data for Lsd1 in the functional native-like lipid environment, which poses a significant challenge to conventional structure determination techniques. We will employ novel solid-state NMR techniques with high- field spectrometers, along with circular dichroism and other complementary biophysical and biochemical methods, to obtain important structural and functional data on the Lsd1 lipoprotein complexes. Novel proton-detected, magic-angle spinning NMR methods will be applied for their advantages of high sensitivity and additional proton chemical shift information. We were able to purify Lsd1 as lipoprotein particles in tens of milligrams. Preliminary NMR data demonstrated that high quality multidimensional NMR spectra can be obtained with excellent sensitivity and spectral resolution, and that the lipoprotein samples have shown structural homogeneity and stability after weeks of experiments. This study encompasses these specific aims: (1) To achieve residue-specific resonance assignment for Lsd1, which is an indispensible step toward structure determination using NMR techniques. (2) To calculate three-dimensional structure for Lsd1 using distance and angle restraints measured by NMR. (3) To understand the mechanism of protein-lipid interactions using NMR experiments correlating these two types of molecules. The structural information of Lsd1 accomplished in this study is central to the understanding of its function and to the future progress in the field of fat metabolism, since Lsd1 interacts with the triglyceride lipase and other proteins. PUBLIC HEALTH RELEVANCE: The proposed study aims to improve our understanding of cellular fat metabolism on the basis of three-dimensional structure of lipid storage proteins. Knowledge derived from this study is expected to provide clues for how to influence the balance between fat storage and burning, and to eventually discover new therapeutic strategies to prevent/treat obesity and related health problems such as diabetes, fatty liver, and heart diseases.