Insulin/TZD regulation of protein structure in fat cells

胰岛素/TZD 对脂肪细胞蛋白质结构的调节

• 批准号: 6556578
• 负责人: Fred J SCHAUFELE
• 金额: $ 15.12万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-01 至 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6556578
• 关键词: adipocytes; biological signal transduction; conformation; diabetes mellitus; fluorescence microscopy; fluorescence resonance energy transfer; fluorescent dye /probe; gene expression; green fluorescent proteins; hormone regulation /control mechanism; in situ hybridization; insulin; ionophores; protein protein interaction; protein structure; thiazoles; tissue /cell culture; transcription factor

DESCRIPTION (provided by applicant): Diabetes results from deficiencies in insulin production and/or insulin signaling. Insulin signals adipocytes to alter the expression of genes for enzymes and hormones that regulate energy balance. Transcription factors that control the expression of these genes include peroxisome proliferator activated receptor gamma (PPARy) and CCAAT enhancer binding protein alpha (C/EBPa). Insulin signaling alters the phosphorylation status of C/EBPa. PPARy is the receptor for thiazolidenediones (TZDs). PPARy heterodimerizes with RXR, the receptor for 9-cis retinoic acid, which enhances the insulin-sensitizing actions of TZDs. The interactions of these factors with themselves, their ligands and co-activators in response to insulin are poorly defined. Understanding these interactions in the cellular milieu will lead to improved insulin-sensitizing therapies.We uniquely have developed powerful fluorescence microscopy techniques that measure the amounts, structure and interactions of proteins at tens of thousands of locations within cells. Transcription factors and co-factors involved in insulin regulation will be tagged with spectrally distinct derivatives of green fluorescent protein (GFP), and expressed pairwise in 3T3-L1 pre-adipocyte/adipocyte model cells. The relative locations of each factor will be determined microscopically within the living cell by comparing the locations of fluorescence emitted from each fluorophore-factor fusion. Factor location will be compared to gene location by in situ hybridizations in fixed cells. Direct interactions between, and conformations within, the factors will be measured at each subcellular location as the degree to which fluorescence energy excited in the fluorophore attached to one factor (or factor domain) is transferred to a fluorophore attached to the second factor (or domain). Thus, regulation of these cooperative factors will be determined, in 3T3-L1 cells, by measuring the separate and combined effects of insulin, the TZD rosiglitazone and 9-cis retinoic acid on the:1. location, dimerization and interactions of C/EBPa, PPARg, RXRa and their co-activators PGC-1, PGC-2, SRC-la, CBP and TRAP2202. conformation of C/EBPa, PPARg, RXRa and the same co-activators, and3. conformations, dimers and interactions of the above factors specifically in the neighborhood of the genes that they regulate.

描述（由申请人提供）：糖尿病是由于胰岛素产生和/或胰岛素信号传导不足引起的。胰岛素向脂肪细胞发出信号，改变调节能量平衡的酶和激素的基因表达。控制这些基因表达的转录因子包括过氧化物酶体增殖物激活受体γ（PPARy）和CCAAT增强子结合蛋白α（C/EBPa）。胰岛素信号传导改变C/EBPa的磷酸化状态。PPARy是噻唑烷二酮（TZD）的受体。PPARy与RXR（9-顺式视黄酸的受体）异二聚化，其增强TZD的胰岛素增敏作用。这些因子与其自身、其配体和共激活因子在胰岛素应答中的相互作用尚不清楚。了解细胞环境中的这些相互作用将有助于改善胰岛素增敏疗法。我们独特地开发了强大的荧光显微镜技术，可以测量细胞内数万个位置的蛋白质的数量，结构和相互作用。参与胰岛素调节的转录因子和辅因子将用绿色荧光蛋白（GFP）的光谱上不同的衍生物标记，并在3 T3-L1前脂肪细胞/脂肪细胞模型细胞中成对表达。通过比较从每个荧光团-因子融合物发射的荧光的位置，在活细胞内用显微镜确定每个因子的相对位置。将通过固定细胞中的原位杂交将因子定位与基因定位进行比较。将在每个亚细胞位置测量因子之间的直接相互作用和因子内的构象，作为在连接到一个因子（或因子结构域）的荧光团中激发的荧光能量转移到连接到第二因子（或结构域）的荧光团的程度。因此，在3 T3-L1细胞中，将通过测量胰岛素、TZD罗格列酮和9-顺式视黄酸对以下的单独和组合作用来确定这些协同因子的调节：1. C/EBPa、PPARg、RXRa及其共活化剂PGC-1、PGC-2、SRC-Ia、CBP和TRAP 2202的位置、二聚化和相互作用。C/EBPa、PPARg、RXRa和相同的共活化剂的构象;构象，二聚体和相互作用的上述因素，特别是在附近的基因，他们调节。