Red cell proteasome modulation by storage, redox metabolism and transfusion

Blood Transfus. 2020 Nov 27. doi: 10.2450/2020.0179-20. Online ahead of print.

Abstract

Background: Proteasomes are proteolytic complexes with prominent roles in the control of protein homeostasis and cellular viability. However, little is known about the effects of storage and glucose-6-phosphate dehydrogenase deficiency (G6PD-) on the activity and topology of red blood cell (RBC) proteasomes.

Materials and methods: We investigated the concentration (by GeLC-MS proteomics analysis and immunoblotting), activity (by using peptide substrates and proteasome inhibitors), and subcellular/extracellular distribution (following cell fractionation and isolation of extracellular vesicles, respectively) of RBC proteasomes in fresh blood and RBCs from control and G6PD- donors following storage in leukoreduced units. RBC proteasome activity was also tested in transfusion-mimicking conditions in vitro.

Results: Stored RBCs were characterised by decreased cytosolic proteasome activity compared to fresh RBCs but increased membrane activity and protein concentration levels. Active proteasomes along with other "repair or destroy" proteins are recruited to the membrane during storage. A proportion of them is released in the supernatant in soluble form or inside extracellular vesicles. Significantly increased enzymatic activity and release of proteasomes were observed in G6PD- vs control RBCs. Similar variations were observed in stress protein biomarkers at the G6PD- membrane. The proteasome profile (mainly the caspase-like activity) had significant correlations with the G6PD- metabolome and quality markers of the RBC units. The storage-induced modifications in the proteasome activities were only partly restored in transfusion-mimicking conditions.

Discussion: Storage conditions and G6PD deficiency affect (individually and in synergy) the abundance, distribution, activity, and release of RBC proteasomes. The partial irreversibility of these effects in transfusion-mimicking conditions demands further investigation of their clinical impact on transfusion outcomes.