Red blood cell storage affects the stability of cytosolic native protein complexes.

BACKGROUND: Refrigerated storage of red blood cell (RBC) units promotes the progressive accumulation of the so-called storage lesions, a widespread series of alterations to morphology, metabolism, and proteome integrity of stored RBCs. However, while storage lesions targeting the RBC membrane fraction have been widely documented, the cytosolic fraction is as yet an underinvestigated cause of the technical inconveniences related to the high abundance of hemoglobin. STUDY DESIGN AND METHODS: By exploiting a recently ideated preparative two-dimensional clear native electrophoresis, followed by mass spectrometry analysis, we could monitor the changes of soluble multiprotein complexes (MPCs) in RBCs after 0, 21, and 35 days of storage under standard blood banking conditions. RESULTS: Data indicate a substantial storage-dependent alteration of RBC MPCs, particularly of those involved in energy and redox metabolism, confirming previous evidence about the progressive dysregulation of these pathways in long-stored units. CONCLUSION: The use of native gel-based proteomics to investigate MPCs present in the RBC cytosolic fraction proved to be a powerful tool. Results collected represent a preliminary advance in the knowledge of the key role of native cytosolic MPCs in context of RBC storage lesion. Multiprotein organization and interacting partners of some key enzymes have been found to change during storage duration, suggesting that future studies will be needed to assess whether such alterations could influence their activity and efficiency.

Identification of the interactors of human nibrin (NBN) and of its 26 kDa and 70 kDa fragments arising from the NBN 657del5 founder mutation.

Nibrin (also named NBN or NBS1) is a component of the MRE11/RAD50/NBN complex, which is involved in early steps of DNA double strand breaks sensing and repair. Mutations within the NBN gene are responsible for the Nijmegen breakage syndrome (NBS). The 90% of NBS patients are homozygous for the 657del5 mutation, which determines the synthesis of two truncated proteins of 26 kDa (p26) and 70 kDa (p70). Here, HEK293 cells have been exploited to transiently express either the full-length NBN protein or the p26 or p70 fragments, followed by affinity chromatography enrichment of the eluates. The application of an unsupervised proteomics approach, based upon SDS-PAGE separation and shotgun digestion of protein bands followed by MS/MS protein identification, indicates the occurrence of previously unreported protein interacting partners of the full-length NBN protein and the p26 fragment containing the FHA/BRCT1 domains, especially after cell irradiation. In particular, results obtained shed light on new possible roles of NBN and of the p26 fragment in ROS scavenging, in the DNA damage response, and in protein folding and degradation. In particular, here we show that p26 interacts with PARP1 after irradiation, and this interaction exerts an inhibitory effect on PARP1 activity as measured by NAD+ levels. Furthermore, the p26-PARP1 interaction seems to be responsible for the persistence of ROS, and in turn of DSBs, at 24 h from IR. Since some of the newly identified interactors of the p26 and p70 fragments have not been found to interact with the full-length NBN, these interactions may somehow contribute to the key biological phenomena underpinning NBS.

Storing red blood cells with vitamin C and N-acetylcysteine prevents oxidative stress-related lesions: a metabolomics overview.

BACKGROUND: Recent advances in red blood cell metabolomics have paved the way for further improvements of storage solutions. MATERIALS AND METHODS: In the present study, we exploited a validated high performance liquid chromatography-mass spectrometry analytical workflow to determine the effects of vitamin C and N-acetylcysteine supplementation (anti-oxidants) on the metabolome of erythrocytes stored in citrate-phosphate-dextrose saline-adenine-glucose-mannitol medium under blood bank conditions. RESULTS: We observed decreased energy metabolism fluxes (glycolysis and pentose phosphate pathway). A tentative explanation of this phenomenon could be related to the observed depression of the uptake of glucose, since glucose and ascorbate are known to compete for the same transporter. Anti-oxidant supplementation was effective in modulating the redox poise, through the promotion of glutathione homeostasis, which resulted in decreased haemolysis and less accumulation of malondialdehyde and oxidation by-products (including oxidized glutathione and prostaglandins). DISCUSSION: Anti-oxidants improved storage quality by coping with oxidative stress at the expense of glycolytic metabolism, although reservoirs of high energy phosphate compounds were preserved by reduced cyclic AMP-mediated release of ATP.

Native protein complexes in the cytoplasm of red blood cells.

Despite decades of advancements, the investigation of the red blood cell (RBC) cytosolic proteome still represents a challenging task because of the overwhelming abundance of hemoglobin. Besides, the separation method is one of the main limiting factors when investigating protein complexes. In this study, we performed for the first time a 2D-clear native (CN)-SDS-PAGE followed by mass spectrometry-based identification to screen multiprotein complexes (MCPs) in the cytosol of human RBCs. Upstream to 2D-CN-SDS-PAGE, we applied a recently developed native pre-enrichment strategy that allows discriminating and separately collecting three distinct fractions, one of which is highly enriched for hemoglobin. Such prefractionation strategy is conservative, in that it makes soluble native-complex analyses amenable without loss of biological information. Because of the resolution of native gel electrophoresis techniques, we could observe and describe 55 potential hetero-oligomeric MPCs from the RBC native cytosolic proteome, among which ultratetrameric hemoglobin. The detected protein complexes were characterized by proteins mainly involved in oxygen transport, antioxidant responses, metabolism, and protein degradation cascades, in agreement with recent in silico models. Metabolic enzyme oligomers also interacted with complexes of proteins involved in oxidative stress responses, thus suggesting a functional relationship between metabolic modulation and antioxidant defenses.

Red blood cell populations and membrane levels of peroxiredoxin 2 as candidate biomarkers to reveal blood doping.

BACKGROUND: Blood doping represents one main trend in doping strategies. Blood doping refers to the practice of boosting the number of red blood cells (RBCs) in the bloodstream in order to enhance athletic performance, by means of blood transfusions, administration of erythropoiesis-stimulating substances, blood substitutes, natural or artificial altitude facilities, and innovative gene therapies. While detection of recombinant EPO and homologous transfusion is already feasible through electrophoretic, mass spectrometry or flow cytometry-based approaches, no method is currently available to tackle doping strategies relying on autologous transfusions. MATERIALS AND METHODS: We exploited an in vitro model of autologous transfusion through a 1:10 dilution of concentrated RBCs after 30 days of storage upon appropriate dilution in freshly withdrawn RBCs from the same donor. Western blot towards membrane Prdx2 and Percoll density gradients were exploited to assess their suitability as biomarkers of transfusion. RESULTS: Membrane Prdx2 was visible in day 30 samples albeit not in day 0, while it was still visible in the 1:10 dilution of day 30 in day 0 RBCs. Cell gradients also highlighted changes in the profile of the RBC subpopulations upon dilution of stored RBCs in the fresh ones. DISCUSSION: From this preliminary in vitro investigation it emerges that Prdx2 and RBC populations might be further tested as candidate biomarkers of blood doping through autologous transfusion, though it is yet to be assessed whether the kinetics in vivo of Prdx2 exposure in the membrane of transfused RBCs will endow a sufficient time-window to allow reliable anti-doping testing.

Red blood cell processing for cryopreservation: from fresh blood to deglycerolization.

BACKGROUND: Cryostorage of red blood cells (RBCs) represents a valid alternative to liquid storage, since units can be preserved safely for at least a decade while conserving RBC viability. While cryostorage has attracted a great deal of attention clinically, little is known about the biochemistry and physiology of cryostored erythrocyte concentrates. STUDY DESIGN AND METHOD: In the present study, we investigated cryostorage of RBCs through monitoring of cell processing steps (from fresh blood, to glycerolization, thawing and deglycerolization/washing) through repeated assays of standard parameters (MCV, RDW-SD) and scanning electron microscopy. RESULTS: Cell processing for cryostorage resulted in increased RBC volumes. Shape alterations caused an increase in osmotic fragility and permeability to ions. A significant pH drop was observed which could not to be attributed to a higher metabolic rate, since the levels of lactate did not show substantial fluctuation during the cell processing steps tested in this study. Membrane anomalies are likely related to the hemolysis observed which preferentially affected the densest and oldest cell sub-populations, as confirmed by means of discontinuous density gradients. CONCLUSION: Our results indicate that cryostorage itself in presence of glycerol does not significantly affect RBCs. Most of the alterations observed were related to cell processing and, in particular, to the increase of cytosolic glycerol as a consequence of the glycerolyzation step. Further studies might profitably investigate replacing glycerol with non-penetrating cryoprotectants.