AdventHealth, Translational Research Institute (TRI), Orlando, FL 32828, USA.
Tymora Analytical Operations, West Lafayette, IN 47906, USA.
Int J Mol Sci. 2022 May 21;23(10):5779. doi: 10.3390/ijms23105779.
The purpose of this study was to define the proteomic and phosphoproteomic landscape of circulating extracellular vesicles (EVs) in people with normal glucose tolerance (NGT), prediabetes (PDM), and diabetes (T2DM). Archived serum samples from 30 human subjects (n = 10 per group, ORIGINS study, NCT02226640) were used. EVs were isolated using EVtrap®. Mass spectrometry-based methods were used to detect the global EV proteome and phosphoproteome. Differentially expressed features, correlation, enriched pathways, and enriched tissue-specific protein sets were identified using custom R scripts. Phosphosite-centric analyses were conducted using directPA and PhosR software packages. A total of 2372 unique EV proteins and 716 unique EV phosphoproteins were identified among all samples. Unsupervised clustering of the differentially expressed (fold change ≥ 2, p < 0.05, FDR < 0.05) proteins and, particularly, phosphoproteins showed excellent discrimination among the three groups. CDK1 and PKCδ appear to drive key upstream phosphorylation events that define the phosphoproteomic signatures of PDM and T2DM. Circulating EVs from people with diabetes carry increased levels of specific phosphorylated kinases (i.e., AKT1, GSK3B, LYN, MAP2K2, MYLK, and PRKCD) and could potentially distribute activated kinases systemically. Among characteristic changes in the PDM and T2DM EVs, “integrin switching” appeared to be a central feature. Proteins involved in oxidative phosphorylation (OXPHOS), known to be reduced in various tissues in diabetes, were significantly increased in EVs from PDM and T2DM, which suggests that an abnormally elevated EV-mediated secretion of OXPHOS components may underlie the development of diabetes. A highly enriched signature of liver-specific markers among the downregulated EV proteins and phosphoproteins in both PDM and T2DM groups was also detected. This suggests that an alteration in liver EV composition and/or secretion may occur early in prediabetes. This study identified EV proteomic and phosphoproteomic signatures in people with prediabetes and T2DM and provides novel insight into the pathobiology of diabetes.
这项研究的目的是定义具有正常葡萄糖耐量（NGT）、前驱糖尿病（PDM）和 2 型糖尿病（T2DM）的个体循环细胞外囊泡（EVs）的蛋白质组学和磷酸化蛋白质组学图谱。使用了来自 30 个人类受试者的存档血清样本（每组 10 个样本，ORIGINS 研究，NCT02226640）。使用 EVtrap®分离 EVs。使用基于质谱的方法检测全局 EV 蛋白质组和磷酸化蛋白质组。使用自定义 R 脚本鉴定差异表达特征、相关性、富集途径和富集组织特异性蛋白质组。使用 directPA 和 PhosR 软件包进行磷酸化位中心分析。在所有样本中鉴定出 2372 种独特的 EV 蛋白和 716 种独特的 EV 磷酸化蛋白。差异表达（倍数变化≥2，p<0.05， FDR<0.05）蛋白，特别是磷酸化蛋白的无监督聚类在三组之间显示出极好的区分能力。CDK1 和 PKCδ 似乎驱动关键的上游磷酸化事件，这些事件定义了 PDM 和 T2DM 的磷酸化蛋白质组学特征。糖尿病患者的循环 EV 携带特定磷酸化激酶（即 AKT1、GSK3B、LYN、MAP2K2、MYLK 和 PRKCD）的水平升高，并且可能系统地分布激活的激酶。在 PDM 和 T2DM EV 的特征变化中，“整合素转换”似乎是一个核心特征。参与氧化磷酸化（OXPHOS）的蛋白质在糖尿病的各种组织中已知减少，在 PDM 和 T2DM 的 EV 中显著增加，这表明异常升高的 EV 介导的 OXPHOS 成分的分泌可能是糖尿病发展的基础。在 PDM 和 T2DM 组中下调的 EV 蛋白和磷酸化蛋白中还检测到高度富集的肝脏特异性标志物特征。这表明在前驱糖尿病早期，肝脏 EV 组成和/或分泌可能发生改变。这项研究鉴定了前驱糖尿病和 T2DM 患者的 EV 蛋白质组学和磷酸化蛋白质组学特征，并为糖尿病的病理生物学提供了新的见解。
