Quantitative phospho-proteomics reveals the Plasmodium merozoite triggers pre-invasion host kinase modification of the red cell cytoskeleton

The invasive blood-stage malaria parasite - the merozoite - induces rapid morphological changes to the target erythrocyte during entry. However, evidence for active molecular changes in the host cell that accompany merozoite invasion is lacking. Here, we use invasion inhibition assays, erythrocyte resealing and high-definition imaging to explore red cell responses during invasion. We show that although merozoite entry does not involve erythrocyte actin reorganisation, it does require ATP to complete the process. Towards dissecting the ATP requirement, we present an in depth quantitative phospho-proteomic analysis of the erythrocyte during each stage of invasion. Specifically, we demonstrate extensive increased phosphorylation of erythrocyte proteins on merozoite attachment, including modification of the cytoskeletal proteins beta-spectrin and PIEZO1. The association with merozoite contact but not active entry demonstrates that parasite-dependent phosphorylation is mediated by host-cell kinase activity. This provides the first evidence that the erythrocyte is stimulated to respond to early invasion events through molecular changes in its membrane architecture.</p

The role of charged residues in the transmembrane helices of monocarboxylate transporter 1 and its ancillary protein basigin in determining plasma membrane expression and catalytic activity

Monocarboxylate transporters MCT1-MCT4 require basigin (CD147) or embigin (gp70), ancillary proteins with a glutamate residue in their single transmembrane (TM) domain, for plasma membrane (PM) expression and activity. Here we use site-directed mutagenesis and expression in COS cells or Xenopus oocytes to investigate whether this glutamate (Glu218 in basigin) may charge-pair with a positively charged TM-residue of MCT1. Such residues were predicted using a new molecular model of MCT1 based upon the published structure of the E. coli glycerol-3-phosphate transporter. No evidence was obtained for Arg306 (TM 8) of MCT1 and Glu218 of basigin forming a charge-pair; indeed E218Q-basigin could replace WT-basigin, although E218R-basigin was inactive. No PM expression of R306E-MCT1 or D302R-MCT1 was observed but D302R/R306D-MCT1 reached the PM, as did R306K-MCT1. However, both were catalytically inactive suggesting that Arg306 and Asp302 form a charge-pair in either orientation, but their precise geometry is essential for catalytic activity. Mutation of Arg86 to Glu or Gln within TM3 of MCT1 had no effect on plasma membrane expression or activity of MCT1. However, unlike WT-MCT1, these mutants enabled expression of E218R-basigin at the plasma membrane of COS cells. We propose that TM3 of MCT1 lies alongside the TM of basigin with Arg86 adjacent to Glu218 of basigin. Only when both these residues are positively charged (E218R-basigin with WT-MCT1) is this interaction prevented; all other residue pairings at these positions may be accommodated by charge-pairing or stabilization of unionized residues through hydrogen bonding or local distortion of the helical structure

MTAP-related increased erythroblast proliferation as a mechanism of polycythaemia vera

Polycythaemia vera (PV) is a haematological disorder caused by an overproduction of erythroid cells. To date, the molecular mechanisms involved in the disease pathogenesis are still ambiguous. This study aims to identify aberrantly expressed proteins in erythroblasts of PV patients by utilizing mass spectrometry-based proteomic analysis. Haematopoietic stem cells (HSCs) were isolated from newly-diagnosed PV patients, PV patients who have received cytoreductive therapy, and healthy subjects. In vitro erythroblast expansion confirmed that the isolated HSCs recapitulated the disease phenotype as the number of erythroblasts from newly-diagnosed PV patients was significantly higher than those from the other groups. Proteomic comparison revealed 17 proteins that were differentially expressed in the erythroblasts from the newly-diagnosed PV patients compared to those from healthy subjects, but which were restored to normal levels in the patients who had received cytoreductive therapy. One of these proteins was S-methyl-5′-thioadenosine phosphorylase (MTAP), which had reduced expression in PV patients’ erythroblasts. Furthermore, MTAP knockdown in normal erythroblasts was shown to enhance their proliferative capacity. Together, this study identifies differentially expressed proteins in erythroblasts of healthy subjects and those of PV patients, indicating that an alteration of protein expression in erythroblasts may be crucial to the pathology of PV

Secretory factors from OP9 stromal cells delay differentiation and increase the expansion potential of adult erythroid cells in vitro

Abstract Development of in vitro culture systems for the generation of red blood cells is a goal of scientists globally with the aim of producing clinical grade products for transfusion. Although mature reticulocytes can be efficiently generated by such systems, the numbers produced fall short of that required for therapeutics, due to limited proliferative capacity of the erythroblasts. To overcome this hurdle, approaches are required to increase the expansion potential of such culture systems. The OP9 mouse stromal cell line is known to promote haematopoietic differentiation of pluripotent stem cells, however an effect of OP9 cells on erythropoiesis has not been explored. In this study, we show not only OP9 co-culture, but factors secreted by OP9 cells in isolation increase the proliferative potential of adult erythroid cells by delaying differentiation and hence maintaining self-renewing cells for an extended duration. The number of reticulocytes obtained was increased by approximately 3.5-fold, bringing it closer to that required for a therapeutic product. To identify the factors responsible, we analysed the OP9 cell secretome using comparative proteomics, identifying 18 candidate proteins. These data reveal the potential to increase erythroid cell numbers from in vitro culture systems without the need for genetic manipulation or co-culture

Quantitative proteomics of plasma vesicles identify novel biomarkers for Hemoglobin E/ β-Thalassemic patients

Key PointsChaperones, antioxidants, iron-sequestering proteins, and cathepsin S exhibited increased abundance in thalassemic EVs. Haptoglobin and hemopexin are reduced in thalassemic patients’ EVs, reflecting hemolysis. These could be used as clinical biomarkers.</jats:p

The inhibition of monocarboxylate transporter 2 (MCT2) by AR-C155858 is modulated by the associated ancillary protein

In mammalian cells, MCTs (monocarboxylate transporters) require association with an ancillary protein to enable plasma membrane expression of the active transporter. Basigin is the preferred binding partner for MCT1, MCT3 and MCT4, and embigin for MCT2. In rat and rabbit erythrocytes, MCT1 is associated with embigin and basigin respectively, but its sensitivity to inhibition by AR-C155858 was found to be identical. Using RT (reverse transcription)–PCR, we have shown that Xenopus laevis oocytes contain endogenous basigin, but not embigin. Co-expression of exogenous embigin was without effect on either the expression of MCT1 or its inhibition by AR-C155858. In contrast, expression of active MCT2 at the plasma membrane of oocytes was significantly enhanced by co-expression of exogenous embigin. This additional transport activity was insensitive to inhibition by AR-C155858 unlike that by MCT2 expressed with endogenous basigin that was potently inhibited by AR-C155858. Chimaeras and C-terminal truncations of MCT1 and MCT2 were also expressed in oocytes in the presence and absence of exogenous embigin. L-Lactate Km values for these constructs were determined and revealed that the TM (transmembrane) domains of an MCT, most probably TM7–TM12, but not the C-terminus, are the major determinants of L-lactate affinity, whereas the associated ancillary protein has little or no effect. Inhibitor titrations of lactate transport by these constructs indicated that embigin modulates MCT2 sensitivity to AR-C155858 through interactions with both the intracellular C-terminus and TMs 3 and 6 of MCT2. The C-terminus of MCT2 was found to be essential for its expression with endogenous basigin

LEF-1 drives aberrant β-catenin nuclear localization in myeloid leukemia cells

Canonical Wnt/β-catenin signaling is frequently dysregulated in myeloid leukemias and is implicated in leukemogenesis. Nuclear-localized β-catenin is indicative of active Wnt signaling and is frequently observed in acute myeloid leukemia patients; however, some patients exhibit little or no nuclear β-catenin even where cytosolic β-catenin is abundant. Control of the subcellular localization of β-catenin therefore represents an additional mechanism regulating Wnt signaling in hematopoietic cells. To investigate the factors mediating the nuclear-localization of β-catenin we carried out the first nuclear/cytoplasmic proteomic analysis of the β-catenin interactome in myeloid leukemia cells and identified putative novel β-catenin interactors. Comparison of interacting factors between Wnt-responsive cells (high nuclear β-catenin) versus Wnt-unresponsive cells (low nuclear β-catenin) suggested the transcriptional partner, LEF-1, could direct the nuclear-localization of β-catenin. The relative levels of nuclear LEF-1 and β-catenin were tightly correlated in both cell lines and in primary AML blasts. Furthermore, LEF-1 knockdown perturbed β-catenin nuclear-localization and transcriptional activation in Wnt-responsive cells. Conversely, LEF-1 overexpression was able to promote both nuclear-localization and β-catenin-dependent transcriptional responses in previously Wnt-unresponsive cells. This is the first β-catenin interactome study in hematopoietic cells and reveals LEF-1 as a mediator of nuclear β-catenin level human myeloid leukemia