Key roles of mono-unsaturated fatty acids in lipid metabolism and lipid-dependent signalling

单不饱和脂肪酸在脂质代谢和脂质依赖性信号传导中的关键作用

基本信息

批准号：
RGPIN-2019-06738
负责人：
Mounier, Catherine
金额：
$ 3.64万
依托单位：
Université du Québec à Montréal
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=716370
关键词：
Key roles mono unsaturated fatty

项目摘要

Cellular lipid metabolism as well as multiple cell signalling events are impacted by the intracellular levels of monounsaturated fatty acids (MUFA). However, the mechanisms by which MUFA exert these effects remain poorly understood. Our research program aims to elucidate the role of the stearoyl-CoA desaturase 1 (SCD1) enzyme, which produces MUFA, in these cellular events. We use oleate, a major product of the SCD1 enzyme, as a bioactive MUFA in our studies. OBJECTIVES 1- To determine the role of MUFA in the cellular storage of lipids. We will address the role of SCD1 in intracellular lipid storage, and establish if SCD1 is part of a larger protein complex controlling this process. As oleate preferentially stimulates lipid storage in the form of lipid droplets, we will identify the oleate metabolite responsible for this effect. Further, seipin, a key protein in lipid droplet formation, functionally interacts with SCD1 and, together with SCD1, localizes to the vicinity of lipid droplets; thus, we will examine the role of SCD1 in lipid droplet formation by identifying SCD1 protein interactors under different metabolic states. We will combine information garnered from two cellular models (hepatocytes and yeast) to better understand the contribution of SCD1 to lipid droplet formation. 2- To characterize protein acylation events involving MUFA. We will evaluate the prevalence of MUFA acylation, an emerging type of protein covalent modification, in cellular signalling events. Oleate stimulates lipid synthesis by modulating the activity of SREBP-1, a key lipogenic transcription factor. This modulation of activity likely occurs via direct acylation of SREBP1. Using complementary mass spectrometry analyses, we will identify the lipid moiety on the SREBP-1 protein, as well as its acylated residue(s). We will also use an -alkynyl-biotin pull-down approach to capture palmitoylated and oleylated proteins and identify new MUFA-modified proteins. 3- To determine the impact of MUFA on signalling events associated with cell membranes. We will characterize the impact of oleate on the regulation of signalling events involving lipid second messengers. These events influence cell migration by controlling the formation of cell membrane protrusions. Oleate impacts both cell membrane lipid composition and micro-domain organization, and modulates the activity of membrane signalling complexes. We will characterize the signalling proteins recruited to membrane rafts of oleate-treated cells. Using biosensors, we will also visualize the formation of phospholipase D2 and phosphoinositide 3-kinase products, two enzymes implicated in oleate-stimulated cell migration. We will observe the impact of oleate on the spatial generation of lipid second messengers at the subcellular scale. Our studies are expected to provide understanding of the molecular mechanisms by which MUFA, particularly oleate, act on signalling pathways regulating cellular homeostasis.

细胞脂质代谢以及多个细胞信号事件受单不饱和脂肪酸（MUFA）的细胞内水平的影响。但是，MUFA施加这些作用的机制仍然很少理解。我们的研究计划旨在阐明在这些细胞事件中产生MUFA的stearoyl-COA去饱和酶1（SCD1）酶的作用。在我们的研究中，我们将SCD1酶的主要产物Oleate用作生物活性MUFA。目标 1-确定MUFA在脂质的细胞存储中的作用。我们将解决SCD1在细胞内脂质存储中的作用，并确定SCD1是否是控制此过程的较大蛋白质复合物的一部分。由于Oleate优先以脂质液滴的形式刺激脂质储存，因此我们将识别出负责这种作用的Oleate代谢物。此外，Seipin是一种脂质液滴形成中的关键蛋白，在功能上与SCD1相互作用，并与SCD1相互作用，与脂质液滴附近相互作用。因此，我们将通过在不同代谢状态下识别SCD1蛋白相互作用者来检查SCD1在脂质液滴形成中的作用。我们将结合从两个细胞模型（肝细胞和酵母）获得的信息，以更好地了解SCD1对脂质液滴形成的贡献。 2-表征涉及MUFA的蛋白质酰化事件。我们将评估细胞信号传导事件中新兴蛋白质共价修饰的MUFA酰化的普遍性。 OLEATE通过调节SREBP-1的活性（一种关键的脂肪生成转录因子）刺激脂质合成。这种活性的调节可能是通过直接酰基化SREBP1进行的。使用互补的质谱分析，我们将确定SREBP-1蛋白上的脂质部分及其酰化残基（S）。我们还将使用 - 碱基 - 生物素下拉方法来捕获棕榈酰化和乙酰化蛋白，并鉴定新的MUFA改性蛋白。 3-确定MUFA对与细胞膜相关的信号事件的影响。我们将表征Oleate对涉及脂质第二使者的信号事件调节的影响。这些事件通过控制细胞膜突起的形成来影响细胞迁移。 Oleate会影响细胞膜脂质组成和微域组织，并调节膜信号传导复合物的活性。我们将表征募集到润滑性细胞的膜筏上的信号蛋白。使用生物传感器，我们还将可视化磷脂酶D2和磷酸肌醇3-激酶产物的形成，这两种酶涉及Oleate刺激的细胞迁移。我们将观察Oleate对亚细胞尺度上脂质第二信使的空间产生的影响。我们的研究有望提供对MUFA（尤其是Oleate）作用于调节细胞稳态的信号通路的分子机制的理解。