Red Blood Cell Proteasome in Beta-Thalassemia Trait: Topology of Activity and Networking in Blood Bank Conditions

Abstract

Proteasomes are multi-catalytic complexes with important roles in protein control. Their activity in stored red blood cells (RBCs) is affected by both storage time and the donor's characteristics. However, apart from their abundancy in the membrane proteome, not much is known about their topology, activity, and networking during the storage of RBCs from beta-thalassemia trait donors (βThal⁺). For this purpose, RBC units from fourteen βThal⁺ donors were fractionated and studied for proteasome activity distribution and interactome through fluorometric and correlation analyses against units of sex- and aged-matched controls. In all the samples examined, we observed a time-dependent translocation and/or activation of the proteasome in the membrane and a tight connection of activity with the oxidative burden of cells. Proteasomes were more active in the βThal⁺ membranes and supernatants, while the early storage networking of 20S core particles and activities showed a higher degree of connectivity with chaperones, calpains, and peroxiredoxins, which were nonetheless present in all interactomes. Moreover, the βThal⁺ interactomes were specially enriched in kinases, metabolic enzymes, and proteins differentially expressed in βThal⁺ membrane, including arginase-1, piezo-1, and phospholipid scramblase. Overall, it seems that βThal⁺ erythrocytes maintain a considerable "proteo-vigilance" during storage, which is closely connected to their distinct antioxidant dynamics and membrane protein profile.

Keywords: activity; beta thalassemia trait; interactome; membrane localization; proteasome; proteostasis; red blood cell; regulation; storage.

Figure 1

Proteasome activities in red blood cells of beta-thalassemia minor and control donors. Chymotrypsin (CH)-, caspase (CASP)-, and trypsin (TR)-like activities in (A) RBC cytosol, (B) RBC membrane, (C) RBC units’ supernatant, and (D) RBC-derived extracellular vesicles on day 42. Horizontal axis: days of storage; F: freshly drawn blood; (*) p < 0.05 between βThal⁺ and controls. Created with BioRender.com.

Figure 2

Correlation analysis between reactive oxygen species (ROS) generation and proteasome activities. Indicative scatter plots between (A) intrinsic ROS levels and cytosolic proteasome activities, (B) tert-butyl-hydroperoxide (tBHP)-induced ROS levels and cytosolic proteasome activities, and (C) intrinsic ROS levels and membrane proteasome activities. CASP: caspase, TR: trypsin, CH: chymotrypsin. R ² values > 0.284 are statistically significant at p < 0.05.

Figure 3

Network analysis of proteomic and physiological parameters in beta-thalassemia minor and control stored red blood cells during early (A) or late (B) storage. The connections represent statistically significant correlations (p < 0.01) between RBC proteins found on the membrane/cytoskeleton and physiological parameters. Solid lines: positive correlation; dashed lines: negative correlation. Node numbers correspond to the definitions provided in Table S1.

Figure 4

Network analysis for core 20S proteasome proteins in beta-thalassemia minor and control stored red blood cells during early storage. Interactomes showing statistically significant correlations (p < 0.01) between RBC parameters and 20S proteasome proteins. Solid lines: positive correlation; dashed lines: negative correlation. Node numbers correspond to the definitions provided in Table S1.

Figure 5

Network analysis for core 20S proteasome proteins in beta-thalassemia minor and control stored red blood cells during late storage. Interactomes showing statistically significant correlations (p < 0.01) between RBC parameters and 20S proteasome proteins. Solid lines: positive correlation; dashed lines: negative correlation. Node numbers correspond to the definitions provided in Table S1.

Figure 6

Network analysis for proteasomic activities in beta-thalassemia minor and control stored red blood cells during early storage. Interactomes showing statistically significant correlations (p < 0.01) between RBC parameters and proteasome activities. Solid lines: positive correlation; dashed lines: negative correlation. Node numbers correspond to the definitions provided in Table S1. Prdx2: peroxiredoxin-2; tBHP: tert-butyl-hydroperoxide.

Figure 7

Network analysis for proteasomic activities in beta-thalassemia minor and control stored red blood cells during late storage. Interactomes showing statistically significant correlations (p < 0.01) between RBC parameters and proteasome activities. Solid lines: positive correlation; dashed lines: negative correlation. Node numbers correspond to the definitions provided in Table S1. Prdx2: peroxiredoxin-2; GLUT1: glucose transporter-1; PHZ: phenylhydrazine.