Single cell assay for protein palmitoylation dynamics

蛋白质棕榈酰化动力学的单细胞测定

• 批准号: 7155104
• 负责人: LAURA M BORLAND
• 金额: $ 4.06万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7155104
• 关键词: acyl coA; capillary electrophoresis; fatty acylation; fluorescence microscopy; fluorescent dye /probe; insulin; palmitates; pharmacokinetics; postdoctoral investigator; posttranslational modifications; protein localization; protein structure function; protein transport; single cell analysis; stimulant /agonist

DESCRIPTION (provided by applicant): Palmitoylation, the dynamic and reversible posttranslational modification of proteins, is suggested to play a role in protein signaling by promoting movement and localization of proteins to different sites of action in vivo. A capillary electrophoresis (CE)-based single cell assay will be developed to assess palmitoylation of fluorescently-labeled peptide substrates in vivo. Four peptides will be synthesized and fluorescently-labeled for use as palmitoylation substrates: N-ras, H-ras, GAP-43, and Gi-alpha1. First, palmitoylated and non-palmitoylated versions of these substrates will be separated by micellar electrokinetic capillary chromatography (MEKC) in vitro. Subsequently, single cells loaded with fluorescently-labeled peptides will be lysed and sampled using a laser micropipette system, followed by separation and quantification by MEKC-CE. Substrate palmitoylation will be assessed following pharmacological stimulation with acyl-CoA, cerulenin, 2-bromopalmitate and insulin. Palmitoylation kinetics, before and after pharmacological manipulation, will also be assessed for each of the labeled substrates. Finally, fluorescence microscopy will be used to correlate substrate location to palmitoylation state before and after pharmacological manipulation.

描述（由申请人提供）：棕榈酰化是蛋白质的动态且可逆的翻译后修饰，被认为通过促进蛋白质在体内的不同作用位点的运动和定位在蛋白质信号传导中发挥作用。将开发基于毛细管电泳（CE）的单细胞测定法来评估体内荧光标记肽底物的棕榈酰化。将合成四种肽并进行荧光标记，用作棕榈酰化底物：N-ras、H-ras、GAP-43 和 Gi-alpha1。首先，这些底物的棕榈酰化和非棕榈酰化版本将通过胶束电动毛细管色谱 (MEKC) 在体外进行分离。随后，使用激光微量移液器系统裂解负载荧光标记肽的单细胞并取样，然后通过 MEKC-CE 进行分离和定量。在用酰基辅酶A、浅蓝素、2-溴棕榈酸酯和胰岛素进行药理学刺激后，将评估底物棕榈酰化。还将评估每种标记底物在药理学操作之前和之后的棕榈酰化动力学。最后，荧光显微镜将用于在药理学操作之前和之后将底物位置与棕榈酰化状态相关联。