Comprehensive analysis of ROS-induced protein phosphorylation and oxidation in physiology and pathophysiology of pancreatic beta cells

ROS诱导的胰腺β细胞生理和病理生理中蛋白质磷酸化和氧化的综合分析

• 批准号: 326234036
• 负责人: Professorin Dr. Andrea Henze
• 金额: --
• 依托单位: Department of Biochemistry and Molecular Biology
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2017-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/326234036?language=en
• 关键词: Comprehensive; analysis; ROS; induced; protein

Dysfunction of pancreatic beta-cells is a hallmark of pathogenesis and pathophysiology of type 2 diabetes mellitus and characterised by an inappropriate insulin metabolism. Several reasons for beta-cell dysfunction have been identified; however, the increased formation of reactive oxygen species (ROS) due to chronic hyperglycemia has been identified as a major pathologic trigger. On the contrary, low amounts of ROS production in beta-cells are mandatory to maintain an appropriate insulin secretion. In either instance ROS-signalling seems to be mediated by the oxidation of cysteine residues resulting in changes of protein structure and function. Proteins that are frequently affected by ROS oxidation are protein kinases and phosphatases that are also essentially involved in the regulation of insulin synthesis, processing and secretion. The present project aims to analyse the effects of ROS on the physiology and pathophysiology of insulin secretion mediated by protein oxidation and phosphorylation and the interaction of both modification types in cell signalling. The effect of ROS on protein phosphorylation and oxidation will be analysed in vitro using primary rat beta-cells and immortalised INS-1E cells. Insulin secretion will be stimulated by glucose, glucose-like peptide 1 and its agonists as well as calcium with and without exposure to oxidants (i.e. hydrogen peroxide, menadion) and high-glucose stress. Cells will be harvested and lysed and proteins will be digested enzymatically. Subsequently, peptides will be labelled by isobaric tags and phosphorylated and cysteine containing peptides will be enriched by different chromatography techniques. Finally, peptide analysis will be accomplished by LC-MS/MS. Additionally, the posttranslational modification patterns of subcellular fractions (i.e. insulin vesicles and mitochondria) will be characterised after separation e.g. by ultracentrifugation. Finally, proteins that have been identified by this procedure as potent candidates will be confirmed and quantified by ELISA and immunoblotting. The systemic and comprehensive analysis of the association of protein phosphorylation and oxidation will considerably contribute to the understanding of physiological and pathophysiological processes in beta-cells associated with diabetes mellitus. Additionally, the results reveal great potential for the development of new therapeutic strategies and pharmacological intervention.

胰岛β细胞功能障碍是2型糖尿病发病机制和病理生理的标志，以胰岛素代谢异常为特征。β细胞功能障碍的几个原因已被确认；然而，慢性高血糖导致的活性氧物种(ROS)的增加已被确定为主要的病理触发因素。相反，在β细胞中产生少量的ROS是维持适当的胰岛素分泌所必需的。在这两种情况下，ROS信号似乎都是通过半胱氨酸残基的氧化来调节的，从而导致蛋白质结构和功能的改变。经常受ROS氧化影响的蛋白质是蛋白激酶和磷酸酶，它们也主要参与胰岛素的合成、加工和分泌的调节。本项目旨在分析ROS对蛋白质氧化和磷酸化介导的胰岛素分泌的生理和病理生理学的影响，以及这两种修饰类型在细胞信号转导中的相互作用。ROS对蛋白质磷酸化和氧化的影响将在体外利用原代大鼠β细胞和永生化的INS-1E细胞进行分析。胰岛素的分泌将受到葡萄糖、葡萄糖样肽1及其激动剂以及钙的刺激，无论是否暴露于氧化剂(如过氧化氢、甲二酮)和高糖应激。细胞将被收获和裂解，蛋白质将被酶消化。随后，多肽将被等压标记，磷酸化和含半胱氨酸的多肽将通过不同的层析技术进行富集化。最后，多肽分析将通过LC-MS/MS完成。此外，亚细胞部分(即胰岛素小泡和线粒体)的翻译后修饰模式将在分离后进行表征，例如通过超速离心法。最后，通过这一程序被鉴定为有效候选的蛋白质将通过ELISA和免疫印迹进行确认和定量。对蛋白质磷酸化和氧化之间关系的系统和全面的分析将有助于理解糖尿病相关的β细胞的生理和病理生理过程。此外，研究结果还揭示了开发新的治疗策略和药物干预的巨大潜力。