Pre-transfusion storage alters the proteostasis function and capacity to circulate of a morphologically- and metabolically-altered red cell subpopulation, but preserves other red cells
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Erythrocyte, Blood
SUBMITTER: 
Emilie-Fleur GAUTIER
LAB HEAD: Dr Pascal Amireault
PROVIDER: PXD049411 | Pride | 2025-04-02
REPOSITORIES: Pride
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Project description:Background: Plasmodium falciparum causes the majority of malaria mortality worldwide, and the disease occurs during the asexual red blood cell (RBC) stage of infection. In the absence of an effective and available vaccine, and with increasing drug resistance, asexual RBC stage parasites are an important research focus. In recent years, mass spectrometry-based proteomics using Data Dependent Acquisition (DDA) has been extensively used to understand the biochemical processes within the parasite. However, DDA is problematic for the detection of low abundance proteins, protein coverage, and has poor run-to-run reproducibility. Results: Here, we present a comprehensive P. falciparum-infected RBC (iRBC) spectral library to measure the abundance of 44,449 peptides from 3,113 P. falciparum and 1,617 RBC proteins using a Data Independent Acquisition (DIA) approach. The spectral library includes proteins expressed in the three morphologically distinct RBC stages (ring, trophozoite, schizont), the RBC compartment of trophozoite-iRBCs, and the cytosolic fractions from uninfected RBCs (uRBC). This spectral library contains 87% of P. falciparum proteins with previously blood-stage protein-level evidence as well as 692 previously unidentified proteins. The P. falciparum spectral library was successfully applied to generate semi-quantitative proteomics datasets that characterise the three distinct asexual parasite stages in RBCs, and compare artemisinin resistant (Cam3.IIR539T) and sensitive (Cam3.IIrev) parasites. Conclusion: A reproducible, high-coverage proteomics spectral library and analysis method has been generated for investigating sets of proteins expressed in the iRBC stage of P. falciparum malaria. This will provide a foundation for an improved understanding of parasite biology, pathogenesis, drug mechanisms and vaccine candidate discovery for malaria.
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Project description:Circulating human red blood cells (RBCs) consist of mature erythrocytes, and immature reticulocytes. As being anucleated cells, RBCs lack typical, abundant transcriptomes but are known to contain low amounts of diverse long transcripts and microRNAs. The exact role and importance of these RNAs is, however, lacking. To study this further, we have explored the RNA content of RBCs as well as extracellular vesicles of RBCs using next generation sequencing (NGS). Furthermore, to understand the dynamics of the RBC transcriptome we performed single cell RNA sequencing on RBCs both from fresh blood and blood unit. Analysis of the single cell transcriptomes revealed that while the majority of the cells don’t have detectable RNA the remaining, approximately 10% of the cells fall into three sub-populations based on their RNA content. Overall decrease in the RNA quantity over the populations is observed. Qualitative changes include the differences in the hemoglobin (Hb) content of the cells, and the changes in the expression of ribosomal and mitochondrial genes. A previously unknown transcript of a long non-coding RNA, MALAT1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) is the most enriched marker gene for one subpopulation of RBCs suggesting a role for MALAT1 in reticulocyte maturation. Enriched pathways in RBC transcriptome, such as autophagy, reflect the processes required for reticulocyte maturation to erythrocytes. RBC transcriptome is transferred to extracellular vesicles (EVs) suggesting the vesiculation as the mechanism to remove the cellular contents at the final stages of erythrocyte maturation.
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Project description:Phosphorylation of proteins is involved in cell functions, of which cytoskeleton organization. In particular, the phosphotyrosine (pY) has been reported to play a role in red blood cell (RBC) functions. During the storage of RBC in the context of transfusion medicine, cells suffer from storage lesions that affect energy metabolism as well as cell morphology. In the present research work, RBCs were treated (in absence of calcium) with a protein tyrosine phosphatase inhibitor (orthovanadate, OV) to trigger phosphorylation allowing to investigate this effect on RBCs during the storage. Erythrocytes were studied by phosphoproteomics, Western blot analyses and cell morphology by digital holographic microscopy in presence of kinase inhibitors. The OV treatment triggered phosphorylation events, that disappeared during the storage. As a result, 609 phosphoproteins containing 5’298 phosphosites were detected in the membrane extract, of which 43 pY were up- or down-regulated. Following these phosphorylation processes, shape of RBCs shifted from discocytes to spherocytes, and the addition of kinase inhibitors (KIs) inhibited this transition by counteracting the OV treatment. In addition, the different KIs used highlighted potentially two mechanisms involving membrane proteins and playing a role in RBC shape. The capacity of RBCs to maintain their function is central in transfusion medicine and the presented results contribute to a better understanding of RBC biology.
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