ALBERT

Beschreibung: Membrane-spanning proteins may interact with a variety of other integral and peripheral membrane proteins via a diversity of protein-protein interactions. Not surprisingly, defects or mutations in any one of these interacting components can impact the physical and biological properties on the entire complex. Here we use quantum dots to image the diffusion of individual band 3 molecules in the plasma membranes of intact human erythrocytes from healthy volunteers and patients with defects in one of their membrane components, leading to well-known red cell pathologies (hereditary spherocytosis, hereditary elliptocytosis, hereditary hydrocytosis, Southeast Asian ovalocytosis, and hereditary pyropoikilocytosis). After characterizing the motile properties of the major subpopulations of band 3 in intact normal erythrocytes, we demonstrate that the properties of these subpopulations of band 3 change significantly in diseased cells, as evidenced by changes in the microscopic and macroscopic diffusion coefficients of band 3 and in the compartment sizes in which the different band 3 populations can diffuse. Because the above membrane abnormalities largely arise from defects in other membrane components (eg, spectrin, ankyrin), these data suggest that single particle tracking of band 3 might constitute a useful tool for characterizing the general structural integrity of the human erythrocyte membrane.

Beschreibung: Recent advances in understanding the role of JAK2 V617F mutation in Bcr-Abl negative myeloproliferative (MPD) diseases pathogenesis opened up a possibility to develop highly targeted therapies against these debilitating ailments. We used a Ba/F3 cell line expressing the V617F mutant of JAK2 to screen a focused small molecule library for potential inhibitors of JAK2 V617F-dependent proliferation. Further extensive SAR of initial hits resulted in identification of R723, a potent and selective JAK2 inhibitor. This molecule is strongly antiproliferative (IC50 130–200 nM) against mouse BaF3 cells used for initial screening as well as against human UKE1 and SET2 cell lines harboring the same mutation. On the other hand, R723 has only weak activity in IL2-dependent (i.e. JAK1/JAK3-dependent) proliferation assays performed with human primary T (IC50 1300 nM) and mouse T-cell leukaemia CTLL2 cells (IC50 600 nM). A 10 to 20 fold cell-based selectivity of R723 was further confirmed by measuring inhibition of constitutive STAT5 phosphorylation in SET2 and BaF3 cells versus inhibition of IL-2 inducible STAT5 phosphorylation in human primary T and mouse CTLL2 cells using FACS-based approach. Compound R723 has low nonspecific antiproliferative activity against JAK2-independent MOLT4, A549 and H1299 cell lines with an IC50 ranging from 4 to 6 uM. The molecule has been also proven to be potent (IC50 of 2 nM against JAK2 in biochemical assay) and highly selective (window of more than 500 fold over JAK1 and 10 fold over JAK3) inhibitor of JAK2 kinase in vitro. Moreover, when tested in biochemical assay against a panel of more than 200 kinases at a concentration of 20 nM (IC90 for JAK2), R723 inhibited none of them. The selectivity of R723 was further confirmed using a variety of cell-based assays probing T-, B- and mast cell activation. Compound R723 was further evaluated in a stress-induced erythropoiesis mouse model, where kinetics of EPO-dependent hematocrit recovery from phenylhydrazine-induced anemia was assessed. Significant delay in recovery was observed at doses of 75 and 100 mg/kg bid indicating strong compound effect on EPOR signaling in vivo. The result could not be attributed to general toxicity effects as 14 day toxicology study did not identify any abnormalities at doses tested. As a result, R723 could become the basis for next generation of potent and selective compounds targeting JAK2-dependent myeloproliferative diseases.

Beschreibung: The erythrocyte membrane skeleton is the best understood cytoskeleton. Because its protein components have homologs in virtually all other cells, the membrane serves as a fundamental model of biologic membranes. Modern textbooks portray the membrane as a 2-dimensional spectrin-based membrane skeleton attached to a lipid bilayer through 2 linkages: band 3–ankyrin–β-spectrin and glycophorin C–protein 4.1–β-spectrin.1–7 Although evidence supports an essential role for the first bridge in regulating membrane cohesion, rupture of the glycophorin C–protein 4.1 interaction has little effect on membrane stability.8 We demonstrate the existence of a novel band 3–adducin–spectrin bridge that connects the spectrin/actin/protein 4.1 junctional complex to the bilayer. As rupture of this bridge leads to spontaneous membrane fragmentation, we conclude that the band 3–adducin–spectrin bridge is important to membrane stability. The required relocation of part of the band 3 population to the spectrin/actin junctional complex and its formation of a new bridge with adducin necessitates a significant revision of accepted models of the erythrocyte membrane.

Beschreibung: Abstract 2009 Poster Board I-1031 Peroxiredoxin 2 (Prx2) is the third most abundant cytoplasmic protein in red blood cells (RBCs) and is involved in defence against oxidative stress. Although much is known regarding the structure and function of Prx2 in healthy RBCs, its role in protecting pathological RBCs remains largely unexplored. Here, we studied Prx2 dimerization and membrane translocation in mouse RBCs (wild-type mice, n=20) in response to in vitro oxidative stress by either phenylhydrazine-PHZ (50 μM) or diamide (2mM) or H2O2 (75-100 μM) and in vivo in β thalassemic (β thal) mouse RBCs (Hbbth/th and Hbbth3/+ mouse strains; n=16 from each group). In RBCs lysates we observed (i) slightly increased Prx2 dimerization in PHZ treated RBCs; (ii) complete Prx2 dimerization in presence of diamide; (iii) not apparent Prx2 dimerization in H2O2, suggesting a different sensitivity of Prx2 to the various oxidant agents. In RBCs membrane, Prx2 association was (i) reduced in PHZ treated RBCs; (ii) increased in diamide treated RBCs as both monomers and dimers and (iii) reduced to almost undetectable amount in H2O2 treated RBCs. In order to evaluate whether Prx2 native or from RBCs exposed to oxidative stress possess different degrees of affinity to native or oxidized membrane, we compared Prx2 membrane association in control, diamide, PHZ treated membranes with the cytoplasmic RBC fractions obtained from control, diamide and PHZ treated RBCs. We observed a remarkable loss of interactions only between Prx2 and the membranes obtained from PHZ treated red cells (n=3), suggesting that PHZ may inhibit Prx2 binding to the membrane through the masking of its docking sites. Using multiple techniques (immunofluorescence confocal microscopy, immunoblot and flow cytometric analysis), we showed that Prx2 was displaced from PHZ treated RBC membrane in a dose dependent manner. We then investigated the connection between Prx2 membrane displacement and hemicrome binding in time course experiments with PHZ treated RBCs. Prx2 was displaced from the membrane concomitantly to hemichrome membrane binding, indicating that PHZ blocks the Prx2 binding to the membrane through the masking of its docking sites by hemichromes. Since PHZ membrane oxidative damage mimics that of b thal RBCs we have examined Prx2 in RBCs from two mouse models of b thalassemia. We showed that Prx2 content was higher in b thal mouse RBCs than in controls and correlated with the clinical severity of b thalassemia, but the amount of Prx2 associated to the membrane was markedly reduced as observed in PHZ-treated RBCs. These data suggest that in b thal RBCs the masking of Prx2 membrane binding sites by hemichromes and the accumulation of its oxidized/dimerized state in the cytoplasm may affect Prx2 catalytic efficiency on b thal membrane proteins, therefore contributing to their lack of repair from oxidative insults. Thus, the abnormally low membrane association of Prx2 in b thal mouse RBCs represents a new additional factor amplifying the oxidative damage characterizing b thal RBCs and responsible for their reduced lifespan in the periferal circulation. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Peroxiredoxin 2 (Prx2), a thiol-dependent peroxidase, is the third most abundant protein in the erythrocyte, and its absence in knock-out mice gives rise to hemolytic anemia. We have found that in human erythrocytes, Prx2 was extremely sensitive to oxidation by H2O2, as dimerization was observed after exposure of 5 × 106 cells/mL to 0.5 μM H2O2. In contrast to Prx2 in Jurkat T lymphocytes, Prx2 was resistant to overoxidation (oxidation of the cysteine thiol to a sulfinic/sulfonic acid) in erythrocytes. Reduction of dimerized Prx2 in the erythrocyte occurred very slowly, with reversal occurring gradually over a 20-minute period. Very low thioredoxin reductase activity was detected in hemolysates. We postulate that this limits the rate of Prx2 regeneration, and this inefficiency in recycling prevents the overoxidation of Prx2. We also found that Prx2 was oxidized by endogenously generated H2O2, which was mainly derived from hemoglobin autoxidation. Our results demonstrate that in the erythrocyte Prx2 is extremely efficient at scavenging H2O2 noncatalytically. Although it does not act as a classical antioxidant enzyme, its high concentration and substrate sensitivity enable it to handle low H2O2 concentrations efficiently. These unique redox properties may account for its nonredundant role in erythrocyte defense against oxidative stress.

Beschreibung: Glycolytic enzymes (GEs) including aldolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphofructokinase (PFK), pyruvate kinase (PK) and lactate dehydrogenase (LDH) are known to associate with the inner surface of the human erythrocyte membrane. Previous studies have suggested that the N-terminus of the cytoplasmic domain of band 3 (cdb3) constitutes the likely binding site for aldolase, GAPDH and PFK, however, the membrane docking sites for PK and LDH have not been elucidated. In this study, we demonstrate that PK and LDH exhibit no affinity for band 3, regardless of whether the association is measured by co-immunoprecipitation assay, binding competition studies, or catalytic inhibition analyses. We further find that the binding sites for GAPDH, aldolase and PFK on band 3 are distinct but partially overlapping, as evidenced by the fact that: 1) deletion of residues 1–11 of cdb3 eliminates the binding of aldolase, but not PFK or GAPDH, 2) fusion of thioredoxin (Trx) to the N-terminus of residues cdb3 blocks aldolase binding, but not the association of GAPDH or PFK, 3) deletion of sequences 1–50, 1–40, 1–31, or 1–23 of cdb3 blocks cdb3 association with all three GEs, whereas deletion of residues 12-23 only abrogates aldolase binding (while reducing the affinity of PFK and GAPDH), 4) the presence of both sequences, 6–DDYED-10 and 19-EEYED-23, are necessary for cdb3 association with aldolase, whereas the presence of either sequence alone (especially 19-EEYED-23) is sufficient to maintain association with GAPDH and PFK, 5) mutation of all of the acidic residues in the above two sequences to their corresponding amides (E→Q and D→N) results in loss of affinity for all GEs. Because i) kidney cdb3 (which lacks residues 1-65 of cdb3) shows no affinity for any of the GEs, ii) residues 1–55 of cdb3 show near normal affinity for aldolase, GAPDH and PFK, and iii) GAPDH, aldolase, and PFK all compete with each other for cdb3, we conclude that the binding sites for aldolase, GAPDH and PFK are all located within the first 23 residues of cdb3, with the docking site for aldolase likely residing somewhat more N-terminal than the binding sites for GAPDH and PFK. Finally, because each band 3 monomer contains three homologous sequences (6-DDYED-10, 19-EEYED-23 and 902-DEYDE-906) that are found in other proteins that bind GEs (e.g. actin, β-tubulin, troponin T), and since the first two of these sequences were shown to be required for cdb3 binding, the question naturally arose whether different GEs might also associate with residues 902-DEYDE-906 at the extreme C-terminus of band 3. Similar binding studies demonstrate that such an interaction does not occur. Thus, our data show that GAPDH, aldolase, and PFK all bind near the N-terminus of band 3 and that PK and LDH must dock somewhere else on the red cell membrane.

Beschreibung: Band 3, the major protein of the human erythrocyte membrane, associates with multiple metabolic, ion transport, and structural proteins of the cell. Functional studies demonstrate that the oxygenation state of the erythrocyte regulates metabolic, ion transport and mechanical properties of the cell. Because deoxyhemoglobin, but not oxyhemoglobin, binds band 3 with high affinity, these observations raise the hypothesis that hemoglobin (Hb) might regulate erythrocyte properties through its oxygenation-dependent association with band 3. To explore how reversible association of deoxyHb with band 3 might modulate erythrocyte properties, we have characterized the binding site of deoxyHb on human erythrocyte band 3. We report that: the deoxyHb binding site on the erythrocyte membrane comprises residues 12–23 of band 3; mutation of residues on either side of this sequence greatly enhances affinity of deoxyHb for band 3, suggesting that evolution of a higher affinity interaction was possible had it been beneficial; Hb does not bind to two similar sequences in band 3 despite their high sequence homology to residues 12–23, and the Hb binding site on band 3 lies proximal to binding sites for glycolytic enzymes, band 4.1 and ankyrin, suggesting possible mechanisms through which multifarious erythrocyte properties might be regulated by Hb oxygenation. We conclude that nature has evolved a deoxyHb binding site on band 3 whose affinity is regulated by O2, enabling the reversible association of deoxyHb with band 3 to serve as a regulator of the oxygenation-dependent properties of the cell.

Beschreibung: Abstract 3019 Poster Board II-995 A compartment/pool of ATP that is generated by glycolytic enzymes has been reported to exist on the erythrocyte membrane where it channels ATP directly to the Na+/K+-ATPase (Proverbio F and Hoffman JF. J. Gen. Physiol. 1977, 69:605-632. Mercer RW and Dunham PB. J. Gen. Physiol. 1981, 78:547-567). In order to identify the protein components enclosing this compartment of ATP, a photoactivatable probe, 8-azido-[αa-32P]ATP, was loaded into porous erythrocytes under conditions that fill the compartment with ATP, and the radiolabeled ATP was induced to label proximal proteins by illumination with UV light. Analysis of radiolabeled bands reveals that spectrin, adducin, protein 4.1, and actin constitute major components of the compartment. To further verify the involvement of these proteins in the ATP compartment, antibodies against the aforementioned proteins were pre-incubated with porous erythrocytes before attempting to load the compartment with ATP. Analysis of the efficiency of ATP loading in the presence of these blocking antibodies reveals that antibody binding prevents normal filling of the ATP pool. These findings confirm Hoffman's earlier hypothesis that a membrane-bound compartment of ATP exists. The data also suggest that the location of the compartment might reside at the junctional complex, and that the complex of enzymes that fill the ATP pool is different from the complex organized around band 3. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Activated macrophages over-express a receptor for the vitamin, folic acid. Conjugation of folic acid to low molecular weight drugs, genes, liposomes, nanoparticles, and imaging agents does not significantly compromise the vitamin’s affinity for its receptor, thus facilitating both therapeutic and imaging agents to be targeted as folate conjugates to activated macrophages. In these studies the in vivo kinetics and binding characteristics of the folate receptor on macrophages has been characterized with the objective of optimizing targeted drug delivery. Our results show that saturation of the receptor in inflamed tissues can be reached at a dose of 100 nmol/kg. Furthermore, the rapid recycling characteristics of the receptor (every 10–20 minutes) suggests that frequent dosing will allow for maximal uptake of folate conjugates by activated macrophages accumulated in inflamed tissues. Finally, the short circulation half-life (30 minutes) of the water-soluble folate conjugate used in this study minimizes the possibility of nonspecific uptake by receptor-negative tissues. The kinetic observations made in this study can be utilized to optimize the therapeutic efficacy and imaging agent delivery of folate conjugates to activated macrophages in vivo.

Beschreibung: In a process termed “hop diffusion”, membrane proteins diffuse randomly within a cytoskeletally-enclosed compartment over short time scales, periodically hopping from one compartment to the next. Band 3 diffuses in the erythrocyte membrane bilayer in a manner that is thought to be restricted by the size and stability of the spectrin network. Although a fraction of band 3 may exhibit reduced mobility due to its association with spectrin (mediated by proteins such as ankyrin and adducin), another subpopulation of band 3 is thought to diffuse freely within the spectrin compartments, hopping from one corral to the next by transient opening of the barrier (presumably a spectrin tetramer) separating adjacent corrals. Based on this hypothesis, measurement of the compartment size and hopping frequency of a band 3 molecule in the erythrocyte membrane can provide information on the structure and stability of the spectrin network. We have combined single particle tracking (SPT) techniques with high-speed video microscopy to determine the average compartment size and diffusion coefficient of the mobile fraction of band 3 in both normal and diseased red blood cells (RBCs). Streptavidin-linked quantum dots (525nm emission) and 40 nm fluorescent beads were attached to band 3 via a DIDS-biotin-linker, and fluorescence microscopy coupled to a high speed camera was used to determine the position of band 3 with a time resolution of 8 ms (120 fps). Labeling specificity was demonstrated by blotting with streptavidin-horseradish peroxidase, and flow cytometry studies established the binding constant of DIDS-biotin for band 3 in the intact cell of ∼1-2*10−5 M. For diffusion studies, RBCs were incubated with 10−11 M conjugate in order to attach only one or two streptavidin-quantum dots per cell. SPT data were collected on both healthy and pathologic human RBCs, including HbSS (sickle cells), HbSC (sickle cell hemoglobin C), HbSBo (sickle cell zero-beta-thalasamia), HbSB+ (sickle cell beta-plus-thalasamia), HS (hereditary spherocytosis), and HPP (hereditary pyropoikilocytosis). Movement within the observed compartments was found to be random, with an average diagonal length for the mobile fraction in healthy cells of ∼100nm and a diffusion coefficient of ∼1–2*10−10cm2/s. HbSS cells, however, displayed a comparatively smaller compartment size (∼60–80nm) with a lower diffusion coefficient (0.1−1*10−10cm2/s). In contrast, HPP cells exhibited an average compartment size of ∼133nm with a diffusion coefficient of 6*10−10cm2/s. SPT data further suggest the presence of multiple populations of band 3, exhibiting both free diffusion and restricted mobilities with different characteristics. Details of band 3 diffusion in all of the aforementioned diseased and normal blood cells will be provided, and a model accounting for the multiple populations of band 3 will be presented.