Regulation of Phospholipid Synthesis by the Master Transcription Factor Opi1

主转录因子 Opi1 调节磷脂合成

• 批准号: RGPIN-2017-06491
• 负责人: Mcmaster, Christopher
• 金额: $ 2.91万
• 依托单位: Dalhousie University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=687998
• 关键词: Regulation; Phospholipid; Synthesis; Master; Transcription

Opi1 is an amphipathic transcriptional repressor that regulates phospholipid synthesis. Phosphatidic acid (PA) is a phospholipid biosynthetic intermediate whose level in the endoplasmic reticulum (ER) regulate Opi1 ER binding. Upon release from the ER Opi1 enters the nucleus where is acts as a transcriptional repressor of a plethora of phospholipid biosynthetic genes.******Aim 1. Determining the Role of the Opi1 LZ Domain in Membrane binding, Localization, and Activity. We have performed a novel biochemical-genetics screen and identified unique alleles of the amphipathic protein Opi1 that are constitutively active. Five of the six new Opi1 mutants we isolated reside in the LZ domain, while one is at the junction of the LZ domain and the adjacent PA binding region. We propose that this tandem domain array could act as a transducer of membrane status into acquisition of repressing activity. We will use purified Opi1 to determine the affinity of the Opi1 LZ mutants for membranes in a PA dependent manner. The role of the LZ region will also be assessed in vivo to address the role of the LZ on membrane binding and regulation of phospholipid biosynthetic gene transcription and ER membrane expansion.******Aim 2. Determining the Role of the Opi1 LZ Domain in Opi1 Self-interaction. We hypothesize that the LZ region of Opi1 mediates Opi1-Opi1 interaction, and increased Opi1 dimerization will drive Opi1 transcriptional repression of phospholipid biosynthetic genes. Using purified Opi1 and our battery of LZ mutants, we will assess protein dimerization and the role of the LZ in mediating this interaction. We will also use growth conditions known to titer Opi1 membrane interaction to address Opi1 dimerization in vivo how dimerization regulates phospholipid biosynthetic gene transcription and ER membrane expansion.******Aim 3. Opi1 Interaction with Hac1 to Co-coordinate Membrane Synthesis with the UPR. We hypothesized that Opi1 could integrate phospholipid synthesis with the UPR via interaction with the LZ containing protein Hac1. Hac1 is synthesized at the ER in response to the need to mount an UPR. We have successfully co-immunoprecipitated Opi1 with Hac1. We will to determine if Opi1 and Hac1 interact using purified proteins and protein pull-downs, using our Opi1LZ mutants to assess the role of the LZ in this interaction. We will go on to further map the Opi1-Hac1 interaction domain and determine if this interaction is necessary for the UPR and Opi1 mediated phospholipid biosynthesis and ER membrane expansion. ******Completion of this research project will increase our knowledge on how how cells sense and coordinate phospholipid synthesis and its integration with the UPR. This would be a major advance in our understanding of cell biology that we expect would become elements prevalent in undergraduate biochemistry textbooks.

Opi1是调节磷脂合成的两亲性转录抑制因子。磷脂酸（PA）是一种磷脂生物合成中间体，其在内质网（ER）中的水平调节opi1er的结合。从内质网释放后，Opi1进入细胞核，在那里它作为过多的磷脂生物合成基因的转录抑制因子。* * * * * *的目标1。确定Opi1 LZ结构域在膜结合、定位和活性中的作用。我们进行了一项新的生化遗传学筛选，并鉴定了两亲性蛋白Opi1的独特等位基因，这些等位基因具有组成性活性。我们分离的6个新的Opi1突变体中有5个位于LZ结构域，而一个位于LZ结构域和邻近的PA结合区域的交界处。我们提出这种串联结构域阵列可以作为膜状态的传感器来获取抑制活性。我们将使用纯化的Opi1来确定Opi1 LZ突变体以PA依赖的方式对膜的亲和力。我们还将在体内评估LZ区域的作用，以解决LZ在膜结合、磷脂生物合成基因转录和内质网膜扩张调控中的作用。* * * * * *的目标2。确定Opi1 LZ结构域在Opi1自相互作用中的作用。我们假设Opi1的LZ区介导了Opi1-Opi1的相互作用，增加的Opi1二聚化将驱动Opi1对磷脂生物合成基因的转录抑制。使用纯化的Opi1和我们的LZ突变体电池，我们将评估蛋白质二聚化和LZ在介导这种相互作用中的作用。我们还将使用已知的生长条件来滴度Opi1膜相互作用，以研究Opi1二聚化在体内是如何调节磷脂生物合成基因转录和内质网膜扩张的。* * * * * *的目标3。Opi1与Hac1相互作用协同膜合成与UPR。我们假设Opi1可以通过与含有LZ的Hac1蛋白相互作用将磷脂合成与UPR结合。Hac1是在急诊室合成的，以响应安装普遍定期审查的需要。我们成功地与Hac1共免疫沉淀了Opi1。我们将使用纯化蛋白和蛋白下拉来确定Opi1和Hac1是否相互作用，使用我们的Opi1LZ突变体来评估LZ在这种相互作用中的作用。我们将进一步绘制Opi1- hac1相互作用域，并确定这种相互作用对于UPR和Opi1介导的磷脂生物合成和ER膜扩张是否必要。******这项研究项目的完成将增加我们对细胞如何感知和协调磷脂合成及其与普遍定期审议的整合的了解。这将是我们对细胞生物学理解的一个重大进步，我们希望这将成为本科生物化学教科书中普遍存在的元素。