Identification of novel molecules and pathways that modulate adipogenesis

鉴定调节脂肪生成的新分子和途径

• 批准号: 7593796
• 负责人: Elisabetta Mueller
• 金额: $ 39.29万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7593796
• 关键词: Adipocytes; Affect; Agonist; Bacteria; Biochemical; Biological Assay; Chimeric Proteins; Fatty acid glycerol esters; Goals; In Vitro; Insulin; Laboratories; Ligands; Mass Spectrum Analysis; Molecular; N-terminal; Nuclear Extract; Nuclear Receptors; PPAR gamma; Pathway interactions; Pattern; Phase; Phosphotransferases; Process; Proteins; Purpose; Regulation; Signal Pathway; Signal Transduction Pathway; Transcriptional Activation; adipocyte differentiation; base; cofactor; gain of function; gene induction; in vivo; interest; lipid biosynthesis; novel; research study; transcription factor

The nuclear receptor PPARgamma is a master regulator of adipogenesis. Activation of this transcription factor through specific natural and synthetic agonists leads to the induction of genes involved in fat differentiation and insulin sensitization. Although the mechanisms of PPARgamma's ligand-dependent transcriptional activation have been studied extensively since the discovery of this factor in the early nineties, the ligand-independent function of PPARgamma has not yet been fully elucidated. One of the main projects in the lab is the identification of novel molecules and pathways that can modulate PPARgamma's ligand-independent activity. In order to identify novel PPARgamma interacting molecules we have taken the following approaches: 1)We have analyzed several cofactors candidates that appear to be expressed during adipocyte differentiation and assessed their ability to function as potential transcriptional cofactors for PPARgamma. 2)We have generated several PPARgamma-GST fusion constructs that express distinct domains of PPARgamma. These fusion proteins produced in bacteria have been utilized in biochemical assays to purify potential novel N-terminal interactors. Nuclear extracts obtained from preadipocytes and fully differentiated adipocytes have been analyzed and novel interacting proteins have been identified by mass spectrometry. We are currently confirming that these factors interact directly with PPARgamma and that they can modulate PPARgamma's transcriptional activity and adipogenic function, both in vitro and in vivo. In addition to the characterization of novel PPARgamma interacting molecules, we are interested in defining novel signaling pathways that can modify adipogenesis. For this purpose we have analyzed the pattern of expression of several kinases during different phases of fat differentiation and have identified several potential kinases that could be involved in the regulation of the adipogenic process. We are currently performing gain-of-function experiments in preadipocytes to assess the capacity of these kinases to affect PPARgamma function and, overall, adipocyte differentiation.

核受体PPARgamma是脂肪生成的主要调节因子。 这种转录因子通过特定的天然和合成激动剂的激活导致参与脂肪分化和胰岛素致敏的基因的诱导。 尽管自九十年代早期发现该因子以来，已经广泛研究了PPARgamma的配体依赖性转录激活机制，但PPARgamma的配体非依赖性功能尚未完全阐明。 该实验室的主要项目之一是鉴定可以调节PPARgamma的配体非依赖性活性的新分子和途径。为了鉴定新的PPARgamma相互作用分子，我们采取了以下方法： 1）我们分析了几种在脂肪细胞分化过程中表达的候选辅因子，并评估了它们作为潜在的PPARgamma转录辅因子的能力。 2）我们已经产生了几种表达PPARgamma的不同结构域的PPARgamma-GST融合构建体。这些在细菌中产生的融合蛋白已被用于生物化学测定以纯化潜在的新型N-末端相互作用物。 从前脂肪细胞和完全分化的脂肪细胞中获得的核提取物已被分析，并通过质谱鉴定了新的相互作用蛋白。我们目前正在确认这些因子与PPARgamma直接相互作用，并且它们可以在体外和体内调节PPARgamma的转录活性和脂肪形成功能。 除了新的PPARgamma相互作用分子的特性，我们有兴趣在定义新的信号通路，可以修改脂肪形成。 为此，我们分析了几种激酶在脂肪分化不同阶段的表达模式，并确定了几种可能参与脂肪形成过程调控的潜在激酶。 我们目前正在前脂肪细胞中进行功能获得实验，以评估这些激酶影响PPARgamma功能和脂肪细胞分化的能力。