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.

摘要 磷脂酰胆碱（PC）是真核细胞中含量最丰富的磷脂。它作为一个结构性的 它是细胞膜的组成部分和脂质第二信使的储存库。PC是由两个从头开始 主要途径：Kennedy途径的CDP-胆碱分支和PE甲基化途径。我们最近 其特征在于第三种途径，其中PC分子被重塑，因为其脂肪酰基基团受到 脱酰/再酰化途径（PC-DRP）。重要的是，PC合成、周转和重塑的改变 与细胞功能障碍和疾病状态有关。PC-DRP涉及PC的完全脱酰， 磷脂酶（Plb 1，Nte 1）产生甘油磷酸胆碱（GPC）和连续的酰基辅酶A依赖性 由Gpc 1和Ale 1催化的将GPC转化为lysoPC的酰基转移酶反应。GPC1， 催化再酰化序列中的关键步骤，并在我们的实验室中发现， 细胞具有i）两步PC再合成路线和ii）两条PC酰基链可以合成后的方法 改造过了酰基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。在前期研究中，我们证实了Gpc 1在S. 发现Gpc 1的缺失导致单不饱和PC种类的减少和单不饱和PC种类的增加。 在双不饱和PC物种中，从而证实了PC-DRP在PC重塑中的作用。未折叠蛋白 反应（UPR）是一个信号通路，不仅响应于未折叠的蛋白质，但ER双层应力。我们 已经表明Gpc 1是ER膜双层稳态的关键参与者： UPR的诱导和Gpc 1的缺失导致UPR诱导。GPC 1（gpc 1D）缺失菌株 突变体）显示其他表型，包括稳定期活力降低和对 在高温下生长。一些观察到的表型可能与酰基转移酶活性无关， 因为Gpc 1也表现出较低的转酰酶活性，并且像其它酶一样，可能具有细胞功能 与催化作用无关。Gpc 1与已知的酰基转移酶或转酰基酶没有序列相似性， 命名为一个新的蛋白质家族（UniProtKB-P48236）。gpc 1同源物在脊椎动物中缺乏，但在 在其他生物体中发现，包括医学上重要的致病真菌。我们发表的研究发现， Gpc 1底物GPC转化为C.白念珠菌是生物体完全毒力所必需的，强调了 检测酶，如Gpc 1，利用GPC。我们的目的是利用靶向突变来确定关键的 Gpc 1酰基转移酶活性所需的氨基酸残基。催化受损的突变体将是 用于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，我们将通过研究Gpc 1的调节来询问其PC重塑方面的重要性 以响应膜饱和度的变化。这项研究将用遗传上易于控制的 S.啤酒。

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

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

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

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