Underlying mechanisms of regulation of glycosphingolipid synthesis in cells

鞘糖脂(Glycspingolipid，GSLS)是在细胞膜过程中起关键作用的糖脂结合物，其缺乏或积聚可导致多种疾病。然而，GSLS合成的中心调控步骤基本上是未知的。GSL是在细胞中一种叫做高尔基体的泡状结构中形成的。然而，一种糖GSL葡萄糖神经酰胺(GlcCer)，所有更复杂的GSLs是从它衍生出来的，是在高尔基体的外膜上制造的。一种名为“Flippase”的酶必须将葡萄糖神经酰胺从高尔基体的外膜转移到内膜。这调节GSL的合成，但尚未鉴定出GlcCer翻转酶。这是理解GSL合成的一个主要缺失步骤。我们发现，多药耐药泵MDR1介导了大约50%的Golgi GlcCer在脾、肾和肝脏的翻转。我们将定义另一个GlcCer翻转酶(S)，它将GlcCer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确定的候选Flppase，并将其交联到我们将制作的GlcCer的光亲和衍生物上，以鉴定这些缺失的GlcCer Flppase。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可以来自高尔基体中单独的GlcCer来源(通过不同的Flppase？转运)。我们发现胆固醇-GSL结合使膜上的GSL糖从暴露/突出变为平坦/模糊。这种变化可能会抑制从GlcCer产生某些GSLS的酶。随着胆固醇含量从早期到晚期的增加，GlcCer在高尔基体堆叠中的位置可以定义所制造的GSL的类型。不同的翻转酶可以将GlcCer转移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)。我们发现细胞胆固醇的丢失诱导了新的GSL的合成，这与胆固醇控制GlcCer作为GSL前体的作用是一致的。增加/降低细胞胆固醇对GSL合成的影响以及我们确定的GlcCer翻转酶的作用将被确定。这些研究将揭示细胞GSLS的一个全新的调节机制，并确定GSL代谢中缺失的主要步骤。

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.