Novel Aspects of Phosphatidylcholine Metabolism

磷脂酰胆碱代谢的新方面

• 批准号: 10578470
• 负责人: JANA L VOGT
• 金额: $ 41.4万
• 依托单位: DUQUESNE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-12-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10578470
• 关键词: Acyl Coenzyme A; Acyltransferase; Affect; Amino Acids; Biochemical; Biological Process; Candida albicans; Catalysis; Cell physiology; Cells; Cellular Membrane; Cytidine Diphosphate Choline; Defect; Development; Diabetes Mellitus; Disease; Enzymes; Essential Fatty Acids; Eukaryotic Cell; Exhibits; Fatty Acid Desaturases; Fatty Acids; Gene Family; Genetic Transcription; Grant; Growth; High temperature of physical object; Homeostasis; Laboratories; Lecithin; Lipids; Malignant Neoplasms; Mass Spectrum Analysis; Medical; Membrane; Membrane Fluidity; Membrane Lipids; Metabolic; Metabolism; Methods; Methylation; Modernization; Molecular Genetics; Monitor; Mutagenesis; Mutation; Obesity; Organism; Pathway interactions; Phase; Phenotype; Phospholipase; Phospholipids; Property; Protein Family; Proteins; Publishing; Radiolabeled; Reaction; Regulation; Research; Role; Route; Saccharomyces cerevisiae; Second Messenger Systems; Signal Pathway; Stress; Techniques; Temperature; Testing; Transacylase; Unsaturated Fatty Acids; Ursidae Family; Vertebrates; Virulence; acyl group; deacylation; feeding; fluidity; graduate student; in vivo; lipid metabolism; lipidomics; member; mutant; novel; pathogenic fungus; promoter; response; undergraduate student

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.

抽象的 磷脂酰胆碱（PC）是真核细胞中最丰富的磷脂。它充当结构 PC由两个从头开始 主要路线：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分子重塑的第三个途径，因为其脂肪酰基均受到A的约束 脱酰化/重酰化途径（PC-DRP）。重要的是，PC合成，周转和重塑的改变 与细胞故障和疾病状态有关。 PC-DRP涉及PC通过 磷脂酶（PLB1，NTE1）产生甘油磷胆碱（GPC）和成功依赖于酰基-COA的磷脂酶 通过GPC1和ALE1催化的转化GPCàLysopcàPC的酰基转移酶反应。 GPC1，哪个 催化恢复序列中所致力的步骤，并在我们的实验室中发现，提供了 带有i）2步PC Res-Resynsis途径的单元格和ii）两种PC酰基链可以在三个方向上进行的手段 重塑。酰基链含量对细胞功能至关重要，因为它影响了基本的膜特性，例如 流动性和曲率。在上一个赠款期间，我们证实了GPC1在PC代谢中的作用。 酿酒酵母，发现GPC1的损失导致单不饱和PC物种降低，并增加 在DI-Unstrature PC物种中，因此证实了PC-DRP在PC重塑中的作用。展开的蛋白质 反应（UPR）是一种信号通路，不仅对展开的蛋白质有反应，而且对ER双层应力有反应。我们 已经证明GPC1是ER膜双层体内平衡的关键参与者：其转录增加了 GPC1的UPR诱导和缺失导致UPR诱导。带有GPC1（GPC1D）中缺失的菌株 突变体）显示其他表型，包括固定相的生存能力降低和对 在升高温度下的生长。某些观察到的表型可能与酰基转移酶活性无关， 由于GPC1还表现出较小的交易基酶活性，并且与其他酶一样，可能具有细胞功能 与催化无关。 GPC1与已知的酰基转移酶或Transylases没有序列相似，并且具有 设计了一个新的蛋白质家族（UniprotkB -P48236）。 GPC1同源物缺乏脊椎动物，但 在其他生物体中发现，包括医学上重要的致病真菌。我们发表的发现，运输 GPC1底物GPC进入白色念珠菌需要该生物体的全部病毒，强调了重要性 利用GPC的GPC1等酶。我们的目标是使用靶向诱变来识别密钥 GPC1酰基转移酶活性所需的氨基酸保留。催化作用的突变体将是 用于阐明酰基转移酶活性对与蛋白质丧失相关的表型的重要性。 此外，我们将通过检查其调节的PC重塑方面的重要性 响应膜安全的变化。这项研究将使用一般的处理 S. cerevisiae。

项目成果

The glycerophosphocholine acyltransferase Gpc1 contributes to phosphatidylcholine biosynthesis, long-term viability, and embedded hyphal growth in Candida albicans.

• DOI: 10.1016/j.jbc.2023.105543
• 发表时间: 2024-01
• 期刊: The Journal of biological chemistry
• 作者: King WR;Singer J;Warman M;Wilson D;Hube B;Lager I;Patton-Vogt J
• 通讯作者: Patton-Vogt J

Phospholipid turnover and acyl chain remodeling in the yeast ER.

• DOI: 10.1016/j.bbalip.2019.05.006
• 发表时间: 2020-01
• 期刊: Biochimica et biophysica acta. Molecular and cell biology of lipids

The acyltransferase Gpc1 is both a target and an effector of the unfolded protein response in Saccharomyces cerevisiae.

• DOI: 10.1016/j.jbc.2023.104884
• 发表时间: 2023-07
• 期刊: JOURNAL OF BIOLOGICAL CHEMISTRY
• 影响因子: 4.8
• 作者: Hrach, Victoria Lee;King, William R.;Nelson, Laura D.;Conklin, Shane;Pollock, John A.;Patton-Vogt, Jana
• 通讯作者: Patton-Vogt, Jana

Glycerophosphocholine provision rescues Candida albicans growth and signaling phenotypes associated with phosphate limitation.

• DOI: 10.1128/msphere.00231-23
• 发表时间: 2023-12-20
• 期刊: mSphere
• 影响因子: 4.8

Overproduction of Phospholipids by the Kennedy Pathway Leads to Hypervirulence in Candida albicans.

肯尼迪途径磷脂的过量产生导致白色念珠菌的高毒力。

• DOI: 10.3389/fmicb.2019.00086
• 发表时间: 2019
• 期刊: Frontiers in microbiology
• 影响因子: 5.2
• 作者: Tams,RobertN;Cassilly,ChelsiD;Anaokar,Sanket;Brewer,WilliamT;Dinsmore,JustinT;Chen,Ying-Lien;Patton-Vogt,Jana;Reynolds,ToddB
• 通讯作者: Reynolds,ToddB