Roles of endosomal lipid sorting in membrane degradation

生物膜的脂质组成对其功能特性至关重要。它在溶酶体的膜降解过程中也起着重要作用。众所周知，除了适当的脂质组成外，位于内体/溶酶体腔室的不同脂质转移蛋白的存在是膜分解和降解所必需的。在上一个资助期内，我们开发了一种体外检测系统，用于检测双分子层之间蛋白质介导的脂质转移，以及自发和蛋白质介导的膜融合。我们发现，在单酰基甘油磷酸酯（BMP）存在的情况下，神经酰胺刺激由尼曼-匹克c2蛋白（NPC2）介导的胆固醇转移，而鞘磷脂则发挥强烈的抑制作用。BMP是一种酸性脂质，存在于体内和溶酶体膜中。基于这些体外数据，我们开发了一个脂质分选和膜降解模型，根据（除其他特征外），内体膜的组成由快速作用的酶（酸性鞘磷脂酶）调节降解，酸鞘磷脂酶将抑制脂质鞘磷脂转化为刺激脂质神经酰胺，进而在更下游的溶酶体中以一种非常受控的方式降解。为了证明细胞和组织内吞过程中脂质成分的变化，我们计划进行亚分离实验，分离质膜组分、早期内体、晚期内体和溶酶体，并通过薄层色谱和质谱分析确定这些细胞器中的膜成分。我们还将进行标记实验，然后用显微技术来确认脂质分析的结果。我们假设膜降解的最佳脂质组成是在这个途径中建立的。随后，我们将对具有实验确定的脂质组成的膜进行转移和融合分析，以生成溶酶体膜降解过程的详细分子模型。

The lipid composition of biological membranes is crucial for their functional properties. It also plays an important role during membrane degradation within the lysosomes. It is known that in addition to an appropriate lipid composition, the presence of different lipid transfer proteins, localized in the endosomal / lysosomal compartment, is required for membrane disintegration and degradation.Within the last funding period, we developed an in vitro assay-system for protein-mediated lipid transfer between bilayers and for spontaneous as well as for protein-mediated membrane fusion. We showed that in the presence of bis(monoacylglycero)phosphate (BMP), an acidic lipid characteristic for intraendosomal and intralysosomal membranes, ceramide stimulates cholesterol-transfer mediated by the Niemann-Pick C2-protein (NPC2), while sphingomyelin exerts a drastic inhibitory effect. Based on these in vitro data we developed a model for lipid sorting and membrane degradation, according to that (among other features), the composition of intraendosomal membranes is adjusted for degradation by the rapidly acting enzyme, acid sphingomyelinase, which converts the inhibiting lipid sphingomyelin to the stimulating lipid ceramide, which, in turn, is degraded more downstream in lysosomes in a very controlled way. To demonstrate that this change in lipid composition during the endocytic pathway occurrs in cells and tissues, we plan subfractionation experiments to isolate plasma membrane fractions, early endosomes, late endosomes, and lysosomes and to determine the membrane composition found in these organelles by thin layer chromatography and mass spectrometry. We will also perform labeling experiments followed by microscopical techniques to confirm the results from lipid analysis. We assume that lipid compositions optimal for membrane degradation are established in this pathway. Later on, we will conduct transfer and fusion assays with membranes having the experimentally determined lipid composition to generate a detailed molecular model for the process of lysosomal membrane degradation.