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

• 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: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=765225
• 关键词: Key; roles; mono; unsaturated; fatty

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.

细胞内单不饱和脂肪酸(MUFA)水平影响细胞内脂代谢和多种细胞信号转导事件。然而，多不饱和脂肪酸发挥这些作用的机制仍然知之甚少。我们的研究计划旨在阐明产生MUFA的硬脂酰辅酶A脱饱和酶1(SCD1)在这些细胞事件中的作用。在我们的研究中，我们使用SCD1酶的主要产物油酸作为具有生物活性的多不饱和脂肪酸。目的1-确定MUFA在细胞储存脂质中的作用。我们将讨论SCD1在细胞内脂肪储存中的作用，并确定SCD1是否是控制这一过程的更大蛋白质复合体的一部分。由于油酸盐优先以脂滴的形式刺激脂肪储存，我们将确定与这一效应有关的油酸盐代谢物。此外，Seipin是脂滴形成的关键蛋白，它与SCD1在功能上相互作用，并与SCD1一起定位于脂滴附近；因此，我们将通过识别不同代谢状态下的SCD1蛋白相互作用来研究SCD1在脂滴形成中的作用。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在脂滴形成中的作用。2-表征涉及MUFA的蛋白质酰化事件。我们将评估MUFA酰化，一种新兴的蛋白质共价修饰，在细胞信号事件中的流行程度。油酸通过调节SREBP-1的活性来刺激脂质的合成，SREBP-1是关键的造脂转录因子。这种活性的调节很可能是通过SREBP1的直接酰化发生的。利用互补质谱分析，我们将鉴定SREBP-1蛋白上的脂肪部分，以及它的酰化残基(S)。我们还将使用？-炔基-生物素下拉方法来捕获棕榈酰化和油基化的蛋白质，并识别新的MUFA修饰的蛋白质。3-确定多不饱和脂肪酸对与细胞膜相关的信号事件的影响。我们将表征油酸对涉及脂质第二信使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的迁移。油酸影响细胞膜脂质组成和微区结构，并调节膜信号复合体的活性。我们将对招募到油酸处理细胞膜筏上的信号蛋白进行表征。使用生物传感器，我们还将可视化磷脂酶D2和磷脂酰肌醇3-激酶产物的形成，这两种酶与油酸刺激的细胞迁移有关。我们将在亚细胞水平上观察油酸对脂质第二信使空间生成的影响。我们的研究有望提供对MUFA，特别是油酸，作用于调节细胞内稳态的信号通路的分子机制的理解。