Novel molecules and pathways that modulate adipogenesis

转录因子PPARgamma是核受体超家族的成员，是脂肪形成的关键调节因子。已经证明，PPARgamma的配体激活会导致参与脂肪分化和胰岛素致敏的基因激活。虽然PPARgamma？自1994年以来，已经广泛研究了在配体结合时激活转录的能力，但是该转录因子的配体非依赖性功能尚未完全阐明。该实验室的目标之一是鉴定可以调节这种转录因子的配体非依赖性活性的新分子和途径。 我们目前正在采取不同的方法来研究PPARgamma是如何调节的，以及在没有配体的情况下它是如何发挥作用的。 1）我们感兴趣的是鉴定能够与PPARgamma结合并以配体非依赖性方式修饰其转录活性的特异性辅因子。为此，我们正在生成PPARgamma？表达PPARgamma的不同结构域的GST融合构建体。这些构建体将用于生物化学测定，以从前脂肪细胞和完全分化的脂肪细胞的核提取物中潜在地纯化能够与未配体的PPARgamma相互作用的新型辅因子。 2)我们目前正在确定是否各种信号通路可以改变PPARgamma功能独立的配体的存在。我们正在评估一些激酶直接磷酸化PPARgamma及其辅激活因子的能力，并可能改变其脂肪形成能力。我们正在建立与瞬时转染和分化试验相结合的体外激酶试验，以确定这些候选激酶在没有配体的情况下直接影响基础PPARgamma转录活性的能力。

The transcription factor PPARgamma, a member of the nuclear receptor superfamily, is a key regulator of adipogenesis. It has been demonstrated that ligand activation of PPARgamma leads to activation of genes involved in fat differentiation and insulin sensitization. Although PPARgamma?s ability to activate transcription upon ligand binding has been studied extensively since 1994, the ligand-independent function of this transcription factor has not been fully elucidated. One of the goals of the lab is the identification of novel molecules and pathways that can modulate the ligand-independent activity of this transcription factor. We are currently taking different approaches to study how PPARgamma is regulated and how it functions in the absence of ligand. 1)We are interested in the identification of specific cofactors that are able to bind to PPARgamma and modify its transcriptional activity in a ligand-independent manner. For this purpose, we are in the process of generating PPARgamma ?GST fusion constructs expressing different domains of PPARgamma. These constructs will be utilized in biochemical assays to potentially purify novel cofactors, from nuclear extracts of preadipocytes and fully differentiated adipocytes, that are able to interact with unliganded PPARgamma. 2) We are currently determining whether various signaling pathways can alter PPARgamma function independently of the presence of ligand. We are assessing the ability of a number of kinases to directly phosphorylate PPARgamma and its coactivators and potentially alter their adipogenic capacity. We are in the process of setting up in vitro kinase assays coupled to transient transfection and differentiation assays to determine the ability of these candidate kinases to directly influence the basal PPARgamma transcriptional activity in the absence of ligand.