Underlying mechanisms of regulation of glycosphingolipid synthesis in cells

细胞中鞘糖脂合成调节的潜在机制

• 批准号: 3470-2012
• 负责人: Lingwood, Clifford
• 金额: $ 2.48万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2012
• 资助国家: 加拿大
• 起止时间: 2012-01-01 至 2013-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=506018
• 关键词: Underlying; mechanisms; regulation; glycosphingolipid; synthesis

Glycosphingolipids (GSLs) are sugar-lipid conjugates which play a key role in cell membrane processes and their deficiency or accumulation can result in many diseases. However, a central regulatory step in the synthesis of GSLs is essentially unknown. GSLs are made in a vesicular structure in cells called the Golgi. However, the one sugar GSL glucosyl ceramide(GlcCer), from which all more complex GSLs are derived, is made on the outside membrane of the Golgi. An enzyme called a 'flippase' must translocate glucosyl ceramide from the outside to the inside membrane of the Golgi. This regulates GSL synthesis but no GlcCer flippase has yet been identified. This is a major missing step in understanding GSL synthesis. We found the multiple drug resistance pump, MDR1, mediates ~50% of Golgi GlcCer flipping in spleen, kidney and liver. We will define the other GlcCer flippase(s) which translocate GlcCer into the Golgi in these, and the other, mouse organs. We will use molecular inhibitory technology for candidate flippases we identified by sequence alignment, and crosslinking to a photoaffinity derivative of GlcCer we will make, to identify these missing GlcCer flippases. Synthesis of acidic GSLs (containing sialic acid but also derived from GlcCer) is unaffected by MDR1, suggesting acidic GSLs can be made from a separate GlcCer source in the Golgi (translocated by a different flippase?). We showed cholesterol-GSL binding changes membrane GSL sugar from exposed/protruding, to flat/obscured. This change could inhibit enzymes making certain GSLs from GlcCer. As cholesterol content increases from early to late Golgi, the position of GlcCer in the Golgi stacks could define the type of GSL made. Different flippases may translocate GlcCer into different parts of the Golgi to provide different GlcCer pools (with different conformations).We showed loss of cell cholesterol induces synthesis of a new GSL, consistent with cholesterol control of GlcCer availability as a GSL precursor. Effect of increasing/decreasing cell cholesterol on GSL synthesis and action of the GlcCer flippases we identify will be determined. These studies will reveal a completely new regulation of cellular GSLs and define the major missing step in GSL metabolism.

鞘糖脂（Glycosphingolipids，GSL）是一类糖脂结合物，在细胞膜过程中起着重要作用，其缺乏或积累可导致多种疾病。然而，GSL合成中的中心调节步骤基本上是未知的。GSL是在称为高尔基体的细胞中以泡状结构制成的。然而，一种糖GSL葡萄糖基神经酰胺（GlcCer）是在高尔基体的外膜上产生的，所有更复杂的GSL都是由它衍生的。一种叫做“翻转酶”的酶必须将葡萄糖基神经酰胺从高尔基体的外膜转移到内膜。这调节GSL合成，但尚未鉴定出GlcCer翻转酶。 这是理解GSL合成过程中缺少的一个重要步骤。我们发现多药耐药泵，MDR 1，介导脾脏，肾脏和肝脏中约50%的高尔基体GlcCer翻转。我们将定义在这些和其他小鼠器官中将GlcCer转位到高尔基体中的其他GlcCer翻转酶。我们将使用分子抑制技术对我们通过序列比对鉴定的候选翻转酶进行抑制，并与我们将制备的GlcCer的光亲和衍生物交联，以鉴定这些缺失的GlcCer翻转酶。酸性GSL（含有唾液酸，但也来源于GlcCer）的合成不受MDR 1的影响，表明酸性GSL可以由高尔基体中的单独GlcCer来源制成（通过不同的翻转酶转运？）。我们发现胆固醇-GSL结合改变膜GSL糖从暴露/突出，到平坦/模糊。这种变化可以抑制由GlcCer产生某些GSL的酶。随着胆固醇含量从早期到晚期高尔基体的增加，GlcCer在高尔基体堆叠中的位置可以定义所产生的GSL的类型。不同的翻转酶可以将GlcCer转位到高尔基体的不同部分以提供不同的GlcCer池（具有不同的构象）。我们表明细胞胆固醇的损失诱导新的GSL的合成，这与胆固醇控制GlcCer作为GSL前体的可用性一致。将确定增加/减少细胞胆固醇对GSL合成和我们鉴定的GlcCer翻转酶的作用的影响。这些研究将揭示细胞GSL的全新调节，并定义GSL代谢中缺失的主要步骤。