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: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=659387
• 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.

Opi 1是一种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Opi 1 ER结合。从ER释放后，Opi 1进入细胞核，在细胞核中充当过多磷脂生物合成基因的转录抑制因子。目标1.确定Opi 1 LZ结构域在膜结合、定位和活性中的作用。我们已经进行了一种新的生化遗传学筛选，并确定了独特的等位基因的两亲性蛋白Opi 1的组成型活性。我们分离的六个新的Opi 1突变体中有五个位于LZ结构域，而一个位于LZ结构域和相邻PA结合区的交界处。我们建议，这种串联结构域阵列可以作为一个换能器的膜状态到收购的阻遏活动。我们将使用纯化的Opi 1以PA依赖性方式确定Opi 1 LZ突变体对膜的亲和力。还将在体内评估LZ区域的作用，以解决LZ对膜结合和磷脂生物合成基因转录和ER膜扩张的调节的作用。目标二。确定Opi 1 LZ结构域在Opi 1自身相互作用中的作用。我们假设Opi 1的LZ区域介导Opi 1-Opi 1相互作用，并且增加Opi 1二聚化将驱动Opi 1对磷脂生物合成基因的转录抑制。使用纯化的Opi 1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滴定Opi 1膜相互作用的生长条件来解决Opi 1体内二聚化如何调节磷脂生物合成基因转录和ER膜扩张。目标3。Opi 1与Hac 1相互作用以协同膜合成与UPR。我们假设Opi 1可以通过与含LZ的蛋白Hac 1相互作用将磷脂合成与UPR整合。Hac 1在ER合成以响应安装UPR的需要。我们已经成功地将Opi 1与Hac 1免疫共沉淀。我们将使用纯化的蛋白质和蛋白质下拉来确定Opi 1和Hac 1是否相互作用，使用我们的Opi 1 LZ突变体来评估LZ在这种相互作用中的作用。我们将继续进一步绘制Opi 1-Hac 1相互作用结构域，并确定这种相互作用是否是UPR和Opi 1介导的磷脂生物合成和ER膜扩张所必需的。** 这个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UPR整合的知识。这将是我们对细胞生物学理解的一个重大进步，我们预计这将成为本科生物化学教科书中普遍存在的元素。