Underlying mechanisms of regulation of glycosphingolipid synthesis in cells

鞘糖脂（GSLs）是糖脂结合物，在细胞膜过程中起着关键作用，其缺乏或积累可导致许多疾病。然而，GSLs合成的中心调控步骤基本上是未知的。gsl是在被称为高尔基体的细胞的水泡结构中产生的。然而，所有更复杂的GSL都是由一种糖GSL葡萄糖基神经酰胺（GlcCer）衍生而来，它是在高尔基体的外膜上产生的。一种叫做“翻转酶”的酶必须将葡萄糖基神经酰胺从外部转运到高尔基体膜的内部。这调节GSL合成，但尚未发现glcer翻转酶。这是理解GSL合成过程中缺失的一个重要步骤。我们发现多重耐药泵MDR1介导约50%的高尔基糖蛋白在脾脏、肾脏和肝脏的翻转。我们将定义其他glcer翻转酶，将glcer转移到这些和其他小鼠器官的高尔基体中。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，并与我们将制作的GlcCer光亲和衍生物交联，以鉴定这些缺失的GlcCer翻转酶。酸性GSLs（含有唾液酸，但也来源于GlcCer）的合成不受MDR1的影响，这表明酸性GSLs可以由高尔基体中单独的GlcCer来源合成（通过不同的翻转酶进行易位）。我们发现胆固醇-GSL结合改变了膜GSL糖从暴露/突出到平坦/模糊。这种变化可能会抑制从葡萄糖中生成某些gsl的酶。随着胆固醇含量从高尔基体早期到晚期的增加，glcer在高尔基体中的位置可以定义GSL的类型。不同的翻转可能会将glcer转移到高尔基体的不同部位，从而提供不同的glcer池（具有不同的构象）。我们发现细胞胆固醇的损失诱导了新的GSL的合成，这与胆固醇控制GlcCer作为GSL前体的有效性一致。增加/减少细胞胆固醇对GSL合成和我们鉴定的glcer翻转酶的作用的影响将被确定。这些研究将揭示细胞GSL的全新调控，并确定GSL代谢的主要缺失步骤。

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.