Novel Aspects of Phosphatidylcholine Metabolism

摘要 磷脂酰胆碱(PC)是真核细胞中含量最丰富的磷脂。它作为一种结构 细胞膜的组成和脂质第二信使的储存库。个人电脑是由两个人从头开始制造的 主要途径：肯尼迪途径的CDP-胆碱分支和PE甲基化途径。我们最近 表征了PC分子被改造的第三条路线，因为它的脂肪酰基受到 去酰化/反应途径(PC-DRP)。重要的是，PC合成、周转和重塑的变化 与细胞功能障碍和疾病状态有关。PC-DRP涉及PC通过 磷脂酶(Plb1，NTE1)，产生甘油磷胆碱(GPC)和连续的酰辅酶A依赖 由GPC1和Ale1催化的酰基转移酶反应，将GPC转化为溶解的PC。GPC1，这是 催化反应序列中的承诺步骤，并在我们的实验室中发现，提供 具有i)两步PC再合成路线和ii)两个PC酰基链可以后合成的方法的细胞 改装过的。酰链含量对细胞功能至关重要，因为它影响基本的膜性质，如 流动性和曲率。在之前的资助期间，我们证实了GPC1在S。PC代谢中的作用。 发现GPC1的缺失会导致单不饱和PC物种的减少和增加 在双不饱和PC物种中，从而证实PC-DRP在PC重塑中的作用。未折叠的蛋白质 反应(UPR)是一种信号通路，不仅对未折叠蛋白有反应，而且对内质网双层应激也有反应。我们 已经证明GPC1在内质网双层动态平衡中起关键作用：它的转录增加 UPR的诱导和GPC1的缺失导致UPR的诱导。携带GPC1(Gpc1D)缺失的菌株 突变体)表现出其他表型，包括静止期活性降低和对 在高温下生长。观察到的一些表型可能与酰基转移酶活性无关， 因为GPC1也表现出较低的转酰基酶活性，并且像其他酶一样，可能具有细胞功能 与催化无关。GPC1与已知的酰基转移酶或转酰基酶没有序列相似性，而是 被命名为一个新的蛋白质家族(UniProtKB-P48236)。脊椎动物中缺乏GPC1同源物，但 发现于其他生物中，包括医学上重要的病原真菌。我们公布的研究结果表明， GPC1底物GPC进入白色念珠菌是生物体完全毒力所必需的，强调了重要性 检测利用GPC的酶，如GPC1。我们的目标是使用定向突变来识别关键 GPC1酰基转移酶活性所需的氨基酸残基。催化受损的突变体(S)将是 用来阐明酰基转移酶活性对与蛋白质丢失相关的表型的重要性。 此外，我们将通过检查GPC1的调节来询问PC重塑方面的重要性 以响应膜饱和度的变化。这项研究将在基因易驯化的 酿酒酵母。

Abstract Phosphatidylcholine (PC) is the most abundant phospholipid in eukaryotic cells. It serves as a structural component of cellular membranes and a reservoir of lipid second messengers. PC is made de novo by two primary routes: the CDP-choline branch of the Kennedy pathway and the PE methylation pathway. We recently characterized a third route in which the PC molecule is remodeled, as its fatty acyl groups are subject to a deacylation/reacylation pathway (PC-DRP). Importantly, alterations in PC synthesis, turnover and remodeling are associated with cellular malfunctions and disease states. PC-DRP involves complete deacylation of PC via phospholipases (Plb1, Nte1) that produce glycerophosphocholine (GPC) and successive acyl-CoA-dependent acyltransferase reactions that convert GPC à lysoPC à PC, as catalyzed by Gpc1 and Ale1. Gpc1, which catalyzes the committed step in the reacylation sequence and was discovered in our laboratory, provides the cell with i) a 2-step PC resynthesis route and ii) a means by which both PC acyl chains can be post-synthetically remodeled. Acyl chain content is crucial to cell function, as it affects fundamental membrane properties such as fluidity and curvature. In the previous grant period, we confirmed the role of Gpc1 in PC metabolism in S. cerevisiae and found that loss of Gpc1 results in a decrease in mono-unsaturated PC species and an increase in di-unsaturated PC species, thus confirming the role for PC-DRP in PC remodeling. The Unfolded Protein Response (UPR) is a signaling pathway responsive not only to unfolded proteins, but also ER bilayer stress. We have shown that Gpc1 is a key player in ER membrane bilayer homeostasis: its transcription is increased upon induction of the UPR and deletion of Gpc1 results in UPR induction. Strains bearing a deletion in GPC1 (gpc1D mutants) display other phenotypes, including decreased stationary phase viability and decreased sensitivity to growth at elevated temperature. Some of the observed phenotypes may be unrelated to acyltransferase activity, as Gpc1 also exhibits a lesser transacylase activity and, like other enzymes, may have cellular functions unrelated to catalysis. Gpc1 bears no sequence similarity to known acyltransferases or transacylases, and has been designated a new protein family (UniProtKB - P48236). Gpc1 homologs are lacking in vertebrates but are found in other organisms, including medically important pathogenic fungi. Our published finding that transport of the Gpc1 substrate, GPC, into C. albicans is required for full virulence of the organism, underlines the importance of examining enzymes, like Gpc1, that utilize GPC. Our objective is to use targeted mutagenesis to identify key amino acid residues required for Gpc1 acyltransferase activity. The catalytically-compromised mutant(s) will be used to elucidate the importance of acyltransferase activity to phenotypes associated with loss of the protein. In addition, we will interrogate the importance of the PC remodeling aspect of Gpc1 by examining its regulation in response to changes in membrane saturation. The research will be performed with the genetically tractable S. cerevisiae.