STRUCTURAL BASIS FOR SCAP/SREBP INTERACTION

SCAP/SREBP 相互作用的结构基础

基本信息

批准号：
8573553
负责人：
Joshua James Ziarek
金额：
$ 5.22万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-01 至 2015-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8573553
关键词：
ADD-1 protein Affect Affinity Amino Acids Architecture Atherosclerosis Binding Binding Proteins Binding Sites Biochemical Biological Assay Boxing C-terminal C-terminal binding protein Cell Nucleus Cell physiology Cells Cholesterol Co-Immunoprecipitations Complex Diabetes Mellitus Disease Embryo Endoplasmic Reticulum Family Fatty Acids Fatty-acid synthase Genes Genetic Transcription Goals Golgi Apparatus Health Heart Diseases Homeostasis Homologous Protein Hormones Hot Spot Importins Kinetics Knock-out Lead Left Length Lipids Liver Location Low Density Lipoprotein Receptor Maintenance Membrane Metabolic Modification Molecular Myocardial Infarction N-terminal Normal Cell Nuclear Import Obesity Oncogenes Pathology Peptide Hydrolases Pharmaceutical Preparations Phosphorylation Site Post-Translational Protein Processing Production Promoter Regions Protein Binding Protein C Protein Family Proteins Proteolysis Proteomics Regulation Regulatory Element Research Running SCAP protein Sequence Analysis Signal Transduction Speed Sterols Stroke Structure Testing Thermodynamics Time Transactivation Variant Vitamins X-Ray Crystallography alpha helix base cholesterol biosynthesis drug discovery lipid biosynthesis neoplastic cell protein complex protein folding public health relevance research study stoichiometry therapeutic development trafficking transcription factor uptake

项目摘要

DESCRIPTION (provided by applicant): Maintenance of cholesterol and fatty acid homeostasis is critical for membrane architecture, protein localization and trafficking, and cellular function. Dysregulation can lead to severe health concerns including obesity, diabetes, heart attack and stroke. Vital to the management of lipid molecules are the sterol regulatory element binding protein (SREBP) family of transcription factors. This family of three proteins transcribes more than 30 genes that control lipid homeostasis. The activation of SREBP is tightly regulated through association with the SREBP cleavage-activating protein (SCAP). SCAP and SREBP bind through their respective C-terminal domains (CTD), but the affinity, stoichiometry, and interface are not known. When sterol levels are high, SCAP maintains SREBP in the endoplasmic reticulum in an inactive form. As sterol concentrations are reduced, SCAP escorts SREBP to the Golgi apparatus where it is activated. Liver-specific knockout of SCAP results in a 70-80% decrease in lipid biosynthesis and germline knockout is hypothesized to be embryonic lethal. Nonetheless, how SCAP recognizes and restricts the location of SREBP is relatively unknown. As a single SCAP variant manages all three SREBP proteins it is reasonable to suspect it may provide an additional level of signaling regulation. Therefore, the overall goal of this proposal is to determine the atomic details of the SCAP CTD, SREBP CTD, and characterize the binding interface. The SCAP CTD contains seven WD40 repeat motifs that are speculated to form a seven-bladed beta-propeller. However, sequence comparison to known structures suggests the existence of an eighth blade following the last WD40 repeat. Aim 1 will test the hypothesis that the SCAP CTD forms an eight-bladed beta-propeller structure. In addition to determining the protein fold and presenting putative binding interfaces, the SCAP CTD would constitute the first NMR characterization of a beta-propeller structure and may reveal general conformational dynamics of this fold. Aim 2 will use a combined approach of NMR and biochemical assays to test the hypothesis that the SREBP-1c CTD possesses a stable tertiary structure in the absence of SCAP. Cell-based assays indicate that the first 200 amino acids of the CTD are sufficient for SCAP binding, but with a reduced affinity compared to the full- length CTD. Our preliminary sequence analysis suggests these residues form a contiguous globular fold followed by two additional ordered regions. The resulting structural and dynamic information will suggest how the full-length and truncated forms can recognize SCAP and may also suggest how post-translational modifications affect complex formation. In aim 3 we will pursue the first characterization of a SCAP/SREBP-1c complex to identify the affinity, stoichiometry, and residues important for binding. We propose to use a combined NMR, biochemical, and cell-based approach to elucidate the thermodynamic, kinetic, and structural parameters underlying this interaction. Our results may also provide a structural basis for abnormal SREBP activity and benefit drug discovery efforts aimed at inhibiting aberrant SREBP signaling.

描述（由申请人提供）：维持胆固醇和脂肪酸稳态对于膜结构、蛋白质定位和运输以及细胞功能至关重要。失调会导致严重的健康问题，包括肥胖、糖尿病、心脏病和中风。对脂质分子的管理至关重要的是转录因子的甾醇调节元件结合蛋白（SREBP）家族。这个由三种蛋白质组成的家族转录 30 多个控制脂质稳态的基因。 SREBP 的激活通过与 SREBP 裂解激活蛋白 (SCAP) 的结合受到严格调节。 SCAP 和 SREBP 通过各自的 C 端结构域 (CTD) 结合，但亲和力、化学计量和界面尚不清楚。当甾醇水平较高时，SCAP 在内质网中维持 SREBP 处于非活性状态。随着甾醇浓度降低，SCAP 护送 SREBP 到达高尔基体，并在那里被激活。肝脏特异性敲除 SCAP 会导致脂质生物合成减少 70-80%，并且推测种系敲除会导致胚胎死亡。尽管如此，SCAP 如何识别和限制 SREBP 的位置相对未知。由于单个 SCAP 变体管理所有三种 SREBP 蛋白，因此有理由怀疑它可能提供额外水平的信号调节。因此，该提案的总体目标是确定 SCAP CTD、SREBP CTD 的原子细节，并表征绑定接口。 SCAP CTD 包含七个 WD40 重复基序，推测它们形成七叶 β 螺旋桨。然而，与已知结构的序列比较表明，在最后一个 WD40 重复之后存在第八个叶片。目标 1 将检验 SCAP CTD 形成八叶片 β 螺旋桨结构的假设。除了确定蛋白质折叠和呈现假定的结合界面之外，SCAP CTD 将构成 β-螺旋桨结构的第一个 NMR 表征，并可能揭示该折叠的一般构象动力学。目标 2 将使用 NMR 和生化检测相结合的方法来检验 SREBP-1c CTD 在没有 SCAP 的情况下具有稳定的三级结构的假设。基于细胞的测定表明，CTD 的前 200 个氨基酸足以结合 SCAP，但与全长 CTD 相比，亲和力降低。我们的初步序列分析表明这些残基形成一个连续的球状折叠，后面跟着两个额外的有序区域。由此产生的结构和动态信息将表明全长和截短形式如何识别 SCAP，还可能表明翻译后修饰如何影响复杂的形成。在目标 3 中，我们将首次表征 SCAP/SREBP-1c 复合物，以确定对结合重要的亲和力、化学计量和残基。我们建议使用核磁共振、生化和细胞相结合的方法来阐明这种相互作用背后的热力学、动力学和结构参数。我们的结果还可能为异常 SREBP 活性提供结构基础，并有利于旨在抑制异常 SREBP 信号传导的药物发现工作。