荧光探针 XY-69 在脂质体中用于测量磷酯酶 C 同工酶的活性。
Fluorogenic XY-69 in Lipid Vesicles for Measuring Activity of Phospholipase C Isozymes.
机构信息
Division of Chemical Biology and Medicinal Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
出版信息
Methods Mol Biol. 2021;2251:225-236. doi: 10.1007/978-1-0716-1142-5_17.
Abstract
Mammalian phospholipase C (PLC) isozymes are major signaling nodes that regulate a wide range of cellular processes. Dysregulation of PLC activity has been associated with a growing list of human diseases such as cancer and Alzheimer's disease. However, methods to directly and continuously monitor PLC activity at membranes with high sensitivity and throughput are still lacking. We have developed XY-69, a fluorogenic PIP analog, which can be efficiently hydrolyzed by PLC isozymes either in solution or at membranes. Here, we describe the optimized assay conditions and protocol to measure the activity of PLC-γ1 (D1165H) with XY-69 in lipid vesicles. The described protocol also applies to other PLC isozymes.
摘要
哺乳动物磷酯酶 C（PLC）同工酶是调节广泛细胞过程的主要信号节点。PLC 活性的失调与越来越多的人类疾病有关，如癌症和阿尔茨海默病。然而，直接且持续地以高灵敏度和高通量监测膜上 PLC 活性的方法仍然缺乏。我们开发了 XY-69，一种荧光 PIP 类似物，可在溶液或膜中被 PLC 同工酶有效水解。在此，我们描述了使用 XY-69 在脂质小泡中测量 PLC-γ1（D1165H）活性的优化测定条件和方案。该方案也适用于其他 PLC 同工酶。
相似文献
1
Fluorogenic XY-69 in Lipid Vesicles for Measuring Activity of Phospholipase C Isozymes.荧光探针 XY-69 在脂质体中用于测量磷酯酶 C 同工酶的活性。
Methods Mol Biol. 2021;2251:225-236. doi: 10.1007/978-1-0716-1142-5_17.
2
A fluorogenic, small molecule reporter for mammalian phospholipase C isozymes.一种用于哺乳动物磷酯酶 C 同工酶的荧光小分子报告分子。
ACS Chem Biol. 2011 Mar 18;6(3):223-8. doi: 10.1021/cb100308n. Epub 2011 Jan 10.
3
Dynamics of allosteric regulation of the phospholipase C-γ isozymes upon recruitment to membranes.PLC-γ 同工酶在被招募到膜上时的变构调节动力学。
Elife. 2022 Jun 16;11:e77809. doi: 10.7554/eLife.77809.
4
Phospholipase C beta2 association with phospholipid interfaces assessed by fluorescence resonance energy transfer. G protein betagamma subunit-mediated translocation is not required for enzyme activation.通过荧光共振能量转移评估磷脂酶Cβ2与磷脂界面的关联。酶激活不需要G蛋白βγ亚基介导的易位。
J Biol Chem. 1996 Oct 11;271(41):25071-8. doi: 10.1074/jbc.271.41.25071.
5
Binding of phosphoinositide-specific phospholipase C-zeta (PLC-zeta) to phospholipid membranes: potential role of an unstructured cluster of basic residues.磷酸肌醇特异性磷脂酶C-ζ（PLC-ζ）与磷脂膜的结合：碱性残基无结构簇的潜在作用
J Biol Chem. 2007 Jun 1;282(22):16644-53. doi: 10.1074/jbc.M701072200. Epub 2007 Apr 12.
6
A High-Throughput Assay to Identify Allosteric Inhibitors of the PLC-γ Isozymes Operating at Membranes.一种高通量测定法，用于鉴定作用于膜的 PLC-γ同工酶的别构抑制剂。
Biochemistry. 2020 Oct 20;59(41):4029-4038. doi: 10.1021/acs.biochem.0c00511. Epub 2020 Oct 7.
7
Membrane-induced allosteric control of phospholipase C-β isozymes.膜诱导的磷脂酶C-β同工酶的变构调控
J Biol Chem. 2014 Oct 24;289(43):29545-57. doi: 10.1074/jbc.M114.586784. Epub 2014 Sep 5.
8
A membrane-associated, fluorogenic reporter for mammalian phospholipase C isozymes.一种膜相关的、荧光报告物，用于哺乳动物的 PLC 同工酶。
J Biol Chem. 2018 Feb 2;293(5):1728-1735. doi: 10.1074/jbc.RA117.000926. Epub 2017 Dec 20.
9
Fluorescent phosphatidylinositol 4,5-bisphosphate derivatives with modified 6-hydroxy group as novel substrates for phospholipase C.荧光磷脂酰肌醇 4,5-二磷酸衍生物，6-羟基被修饰，作为新型的磷脂酶 C 的底物。
Biochemistry. 2012 Jul 3;51(26):5300-6. doi: 10.1021/bi300637h. Epub 2012 Jun 22.
10
Phospholipase C expression and activity in smooth muscle cells of renal arterioles and aorta of young, spontaneously hypertensive rats during culture.培养过程中年轻自发性高血压大鼠肾小动脉和主动脉平滑肌细胞中磷脂酶C的表达与活性
Am J Hypertens. 2007 May;20(5):520-6. doi: 10.1016/j.amjhyper.2006.12.014.
引用本文的文献
1
A high-throughput assay platform to discover small molecule activators of the phospholipase PLC-γ2 to treat Alzheimer's disease.一种用于发现磷脂酶PLC-γ2的小分子激活剂以治疗阿尔茨海默病的高通量检测平台。
J Biol Chem. 2025 Apr;301(4):108356. doi: 10.1016/j.jbc.2025.108356. Epub 2025 Feb 25.
2
The crystal and cryo-EM structures of PLCγ2 reveal dynamic interdomain recognitions in autoinhibition.PLCγ2 的晶体和冷冻电镜结构揭示了自身抑制中的结构域动态识别。
Sci Adv. 2024 Nov 29;10(48):eadn6037. doi: 10.1126/sciadv.adn6037.
3
A novel micellular fluorogenic substrate for quantitating the activity of 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma (PLCγ) enzymes.一种新型胶束荧光底物，用于定量测定 1-磷酸肌醇 4,5-二磷酸磷酸二酯酶 γ（PLCγ）酶的活性。
PLoS One. 2024 Mar 29;19(3):e0299541. doi: 10.1371/journal.pone.0299541. eCollection 2024.
4
The fusiform gyrus exhibits differential gene-gene co-expression in Alzheimer's disease.梭状回在阿尔茨海默病中表现出不同的基因-基因共表达。
Front Aging Neurosci. 2023 May 15;15:1138336. doi: 10.3389/fnagi.2023.1138336. eCollection 2023.
5
A novel fluorogenic reporter substrate for 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma-2 (PLCγ2): Application to high-throughput screening for activators to treat Alzheimer's disease.一种新型荧光报告底物用于 1-磷酸肌醇 4,5-二磷酸磷酸二酯酶 γ-2（PLCγ2）：用于高通量筛选治疗阿尔茨海默病的激活剂的应用。
SLAS Discov. 2023 Jun;28(4):170-179. doi: 10.1016/j.slasd.2023.03.003. Epub 2023 Mar 17.
本文引用的文献
1
Structural basis for the activation of PLC-γ isozymes by phosphorylation and cancer-associated mutations.磷酸化和癌症相关突变激活 PLC-γ 同工酶的结构基础。
Elife. 2019 Dec 31;8:e51700. doi: 10.7554/eLife.51700.
2
Alzheimer's disease phospholipase C-gamma-2 (PLCG2) protective variant is a functional hypermorph.阿尔茨海默病磷脂酶 C-γ-2 (PLCG2) 保护性变体是一种功能超变体。
Alzheimers Res Ther. 2019 Feb 2;11(1):16. doi: 10.1186/s13195-019-0469-0.
3
A membrane-associated, fluorogenic reporter for mammalian phospholipase C isozymes.一种膜相关的、荧光报告物，用于哺乳动物的 PLC 同工酶。
J Biol Chem. 2018 Feb 2;293(5):1728-1735. doi: 10.1074/jbc.RA117.000926. Epub 2017 Dec 20.
4
Rare coding variants in PLCG2, ABI3, and TREM2 implicate microglial-mediated innate immunity in Alzheimer's disease.PLCG2、ABI3和TREM2中的罕见编码变异表明小胶质细胞介导的先天性免疫与阿尔茨海默病有关。
Nat Genet. 2017 Sep;49(9):1373-1384. doi: 10.1038/ng.3916. Epub 2017 Jul 17.
5
A Comprehensive Gene Expression Meta-analysis Identifies Novel Immune Signatures in Rheumatoid Arthritis Patients.一项全面的基因表达荟萃分析确定了类风湿性关节炎患者新的免疫特征。
Front Immunol. 2017 Feb 2;8:74. doi: 10.3389/fimmu.2017.00074. eCollection 2017.
6
Integrated molecular analysis of adult T cell leukemia/lymphoma.成人 T 细胞白血病/淋巴瘤的综合分子分析。
Nat Genet. 2015 Nov;47(11):1304-15. doi: 10.1038/ng.3415. Epub 2015 Oct 5.
7
Dysfunction of phospholipase Cγ in immune disorders and cancer.磷脂酶 Cγ 在免疫紊乱和癌症中的功能障碍。
Trends Biochem Sci. 2014 Dec;39(12):603-11. doi: 10.1016/j.tibs.2014.09.004. Epub 2014 Oct 30.
8
Tumor suppressor role of phospholipase C epsilon in Ras-triggered cancers.PLCε 在 Ras 触发的癌症中的肿瘤抑制作用。
Proc Natl Acad Sci U S A. 2014 Mar 18;111(11):4239-44. doi: 10.1073/pnas.1311500111. Epub 2014 Mar 3.
9
Phosphoinositides: tiny lipids with giant impact on cell regulation.磷酸肌醇：调控细胞的微小脂质，却具有巨大影响。
Physiol Rev. 2013 Jul;93(3):1019-137. doi: 10.1152/physrev.00028.2012.
10
Autoinhibition and phosphorylation-induced activation of phospholipase C-γ isozymes.PLC-γ 同工酶的自动抑制和磷酸化诱导激活。
Biochemistry. 2013 Jul 16;52(28):4810-9. doi: 10.1021/bi400433b. Epub 2013 Jul 9.
Zotero 插件