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

细胞内单不饱和脂肪酸（MUFA）水平影响细胞脂质代谢以及多种细胞信号传导事件。然而，MUFA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硬脂酰辅酶A去饱和酶1（SCD 1）的作用，产生MUFA，在这些细胞事件。我们使用油酸，SCD 1酶的主要产物，作为我们研究中的生物活性MUFA。 目标 1-确定单不饱和脂肪酸在细胞储存脂质中的作用。我们将讨论SCD 1在细胞内脂质储存中的作用，并确定SCD 1是否是控制这一过程的较大蛋白质复合物的一部分。由于油酸盐优先刺激脂滴形式的脂质储存，因此我们将鉴定负责该效应的油酸盐代谢物。此外，seipin，在脂滴形成的关键蛋白质，功能上与SCD 1相互作用，并与SCD 1一起，定位到脂滴附近，因此，我们将检查SCD 1在脂滴形成中的作用，通过确定SCD 1蛋白相互作用在不同的代谢状态。我们将结合联合收割机从两种细胞模型（肝细胞和酵母）获得的信息，以更好地了解SCD 1对脂滴形成的贡献。 2-表征涉及MUFA的蛋白酰化事件。我们将评估MUFA酰化，一种新兴类型的蛋白质共价修饰，在细胞信号事件的患病率。油酸通过调节SREBP-1（一种关键的脂肪生成转录因子）的活性来刺激脂质合成。这种活性调节可能通过SREBP 1的直接酰化发生。使用互补质谱分析，我们将鉴定SREBP-1蛋白上的脂质部分及其酰化残基。我们还将使用一个-炔基-生物素下拉的方法来捕获棕榈酰化和oleylated蛋白质，并确定新的MUFA修饰的蛋白质。 3-确定MUFA对细胞膜相关信号事件的影响。我们将描述油酸对涉及脂质第二信使的信号传导事件的调节的影响。这些事件通过控制细胞膜突起的形成来影响细胞迁移。油酸影响细胞膜脂质组成和微区组织，并调节膜信号复合物的活性。我们将表征信号蛋白招募到油酸酯处理的细胞膜筏。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了解单不饱和脂肪酸，特别是油酸，作用于调节细胞稳态的信号通路的分子机制。

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.