STRUCTURAL BASIS FOR SCAP/SREBP INTERACTION

SCAP/SREBP 相互作用的结构基础

• 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
• 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

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。失调可能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素结合蛋白（SREBP）家族。这个三种蛋白质家族转录了控制脂质稳态的30多个基因。 SREBP的激活通过与SREBP裂解激活蛋白（SCAP）的关联而受到严格调节。 SCAP和SREBP通过其各自的C末端结构域（CTD）结合，但是尚不清楚亲和力，化学计量和界面。当固醇水平较高时，SCAP会以非活性形式保持内质网中的SREBP。随着固醇浓度的降低，SCAP护送SREBP被激活的Golgi设备。 SCAP的肝特异性敲除导致脂质生物合成和种系敲除降低70-80％是胚胎致死的。但是，SCAP如何识别和限制SREBP的位置相对尚不清楚。由于单个SCAP变体管理了所有三种SREBP蛋白，因此可以合理地怀疑它可能会提供额外的信号调节水平。因此，该提案的总体目标是确定SCAP CTD，SREBP CTD的原子细节，并表征结合界面。 SCAP CTD包含七个WD40重复基序，这些基序被认为是形成七叶β-螺旋桨的。然而，与已知结构进行比较的序列表明，在上次WD40重复后，存在第八刀片。 AIM 1将检验SCAP CTD形成八叶β螺旋桨结构的假设。除了确定蛋白质折叠和提出推定的结合界面外，SCAP CTD还构成了β-螺旋体结构的第一个NMR表征，并可能揭示该折叠的一般构象动力学。 AIM 2将使用NMR和生化测定的联合方法来检验以下假设：在没有SCAP的情况下，SREBP-1C CTD具有稳定的三级结构。基于细胞的测定法表明，CTD的前200个氨基酸足以用于SCAP结合，但与全长CTD相比，亲和力降低。我们的初步序列分析表明，这些残基形成连续的球状褶皱，然后是两个其他有序区域。由此产生的结构和动态信息将表明全长和截断的形式如何识别SCAP，还可能表明翻译后修饰如何影响复杂的形成。在AIM 3中，我们将追求SCAP/SREBP-1C复合物的首次表征，以识别亲和力，化学计量和对结合重要的残基。我们建议使用基于NMR，生化和基于细胞的组合方法来阐明这种相互作用的热力学，动力学和结构参数。我们的结果还可能为SREBP活性异常提供结构性基础，并有益于旨在抑制异常SREBP信号传导的药物发现工作。