STRUCTURAL BASIS FOR SCAP/SREBP INTERACTION

SCAP/SREBP 相互作用的结构基础

• 批准号: 8730192
• 负责人: Joshua James Ziarek
• 金额: $ 5.51万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 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/8730192
• 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

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）：胆固醇和脂肪酸稳态的维持对于膜结构、蛋白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、心脏病发作和中风。对于脂质分子的管理至关重要的是转录因子的固醇调节元件结合蛋白（SREBP）家族。这三个蛋白质家族转录超过30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）的结合而受到严格调节。SCAP和SREBP通过其各自的C-末端结构域（CTD）结合，但亲和力、化学计量和界面尚不清楚。当固醇水平高时，SCAP使内质网中的SREBP以非活性形式维持。当固醇浓度降低时，SCAP护送SREBP到高尔基体，在那里它被激活。肝脏特异性敲除SCAP导致脂质生物合成减少70-80%，并且假设种系敲除是胚胎致死的。尽管如此，SCAP如何识别和限制SREBP的位置是相对未知的。由于一个单一的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的信号调节水平。因此，本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WD 40重复基序，推测其形成七叶β-螺旋桨。然而，与已知结构的序列比较表明，在最后一个WD 40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推定的结合界面，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。目的2将使用NMR和生化测定的组合方法来测试SREBP-1c CTD在不存在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个氨基酸足以用于SCAP结合，但与全长CTD相比具有降低的亲和力。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，其次是两个额外的有序区域。由此产生的结构和动态信息将提示全长和截短形式如何识别SCAP，也可能提示翻译后修饰如何影响复合物的形成。在目标3中，我们将进行SCAP/SREBP-1c复合物的首次表征，以确定亲和力，化学计量和对结合重要的残基。我们建议使用相结合的核磁共振，生物化学和细胞为基础的方法来阐明这种相互作用的热力学，动力学和结构参数。我们的研究结果也可能为异常SREBP活性提供结构基础，并有助于旨在抑制异常SREBP信号传导的药物发现工作。