Proteomics and Phosphoproteomics of Circulating Extracellular Vesicles Provide New Insights into Diabetes Pathobiology

Abstract

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.

Extracellular vesicle proteomics and phosphoproteomics identify pathways for increased risk in patients hospitalized with COVID-19 and type 2 diabetes mellitus

“…EV purification was conducted as described by Nunez Lopez and colleagues [19] , using EVtrap™ (Tymora Analytical, Lafayette, IN, USA), a non-antibody- bead-based affinity technology developed to specifically and quantitatively isolate EVs. The EVtrap™ technology has been further described and validated in detail elsewhere [19] , [20] , [21] , [22] , [23] , [24] , [25] , [26] , [27] , [28] . In short, frozen plasma samples were thawed and large debris removed by centrifugation at 2500×g for 10 min.…”

“…LC-MS was conducted as described by Nunez Lopez and colleagues [19] . Briefly, EVs were solubilized in lysis solution then diluted fivefold with 50 mM triethylammonium bicarbonate and digested with Lys-C (Wako) at 1:100 (wt/wt) enzyme-to-protein ratio for 3 h at 37°C.…”

“…Raw MS/MS data was analyzed as described by Nunez Lopez and colleagues [19] . In short, peptides were searched directly against the human Uniprot database with no redundant entries, using Byonic (Protein Metrics, Cupertino, CA, USA) and Sequest search engines loaded into Proteome Discoverer 2.3 software (Thermo Fisher Scientific, Waltham, MA, USA).…”

“…Beyond COVID-19, EVs have been more extensively studied in T2D. Several studies suggest that both the proteome and the mirnome (miRNAs) of EVs from patients with diabetes have proinflammatory proprieties, which we reason, could indirectly contribute to the severity of COVID-19 when both diseases co-occur [11] , [12] , [13] , [14] , [15] , [16] , [17] , [18] , [19] . Prattichizzo and colleagues, for example, recently provided evidence that a specific subpopulation of EVs characterized by the expression of the surface marker platelet endothelial cell adhesion molecule 1 (PECAM1/CD31), derived from platelets, immune cells, and endothelial cells, can promote proinflammatory pathways in targeted endothelial cells [16] , [17] .…”

“…In a study of the proteomic and phosphoproteomic landscape of circulating EVs in subjects with prediabetes, T2D and normal glucose tolerance we identified a potential role of protein kinase C as a driver of upstream phosphorylation events that define phosphoproteomic signatures in prediabetes and T2D. [19] We reasoned that circulating EVs from patients with dysglycemia distribute activated kinases that alter relevant signaling pathways in target tissues and ultimately contribute to diabetes development and progression.…”