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: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=712249
• 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。