Hydroxychloroquine binding to cytoplasmic domain of Band 3 in human erythrocytes: Novel mechanistic insights into drug structure, efficacy and toxicity.

Hydroxychloroquine (HCQ) is a widely used drug in the treatment of autoimmune diseases, such as arthritis and systemic lupus erythematosus. It has also been prescribed for the treatment of malaria owing to its lower toxicity compared to its closely related compound chloroquine (CQ). However, the mechanisms of action of HCQ in erythrocytes (which bind preferentially this drug) have not been documented and the reasons underlying the lower side effects of HCQ compared to CQ remain unclear. Here we show that, although the activity of erythrocyte lactate dehydrogenase (LDH), but not GAPDH, was inhibited by both HCQ and CQ in vitro, LDH activity in erythrocytes incubated with 20 mM HCQ was not significantly reduced within 5 h in contrast to CQ did. Using HCQ coupled Sepharose chromatography (HCQ-Sepharose), we identified Band 3, spectrin, ankyrin, protein 4.1R and protein 4.2 as HCQ binding proteins in human erythrocyte plasma membrane. Recombinant cytoplasmic N-terminal 43 kDa domain of Band 3 bound to HCQ-Sepharose and was eluted with 40 mM (but not 20 mM) HCQ. Band 3 transport activity was reduced by only 23% in the presence of 20 mM HCQ. Taken together, these data demonstrate that HCQ binds to the cytoplasmic N-terminal domain of Band 3 in human erythrocytes but does not inhibit dramatically its transport activity. We hypothesize that the trapping of HCQ on Band 3 contributes to the lower side effects of the drug on energy production in erythrocytes.

Enzymatic and thermodynamic profiles of a heterotetramer lactate dehydrogenase isozyme in swine.

Lactate dehydrogenase (LDH) is a glycolytic enzyme that catalyzes the final step of glycolysis and produces NAD+. In somatic cells, LDH forms homotetramers and heterotetramers that are encoded by two different genes: LDHA (skeletal muscle type, M) and LDHB (heart type, H). Analysis of LDH isozymes is important for understanding the physiological role of homotetramers and heterotetramers and for optimizing inhibition of their enzymatic activity as it may result in distinct effects. Previously, we reported that hydroxychloroquine (HCQ) inhibited LDH activity, but we did not examine isozyme specificity. In the present study, we isolated heterotetrameric LDH (H2M2) from swine brain, determined its kinetic and thermodynamic properties, and examined the effect of HCQ on its activity compared to homotetrameric LDH isozymes. We show that: (1) the Km values for H2M2-mediated catalysis of pyruvate or lactate were intermediate compared to those for the homotetrameric isozymes, M4 and H4 whereas the Vmax values were similar; (2) the Km and Vmax values for H2M2-mediated catalysis of NADH were not significantly different among LDH isozymes; (3) the values for activation energy and van't Hoff enthalpy changes for pyruvate reduction of H2M2 were intermediate compared to those for the homotetrameric isozymes; (4) the temperature for half residual activity of H2M2 was closer to that for M4 than for H4. We also show that HCQ had different affinities for various LDH isozymes.

Phylogenic insights into the enucleation of erythroblasts in human.

Two key questions remain unanswered in the erythropoiesis field: Why and how do erythroblasts enucleate in mammalian species? Recent studies have unveiled the roles of various molecules, cytoskeletal proteins, motor proteins, vesicle transport, signaling pathways, lipid rafts and actomyosin ring contraction in the enucleation process. However, few reports provide insights into the fitness benefit for mammalian species of having anucleate erythrocytes. Herein, we discuss the biological significance of enucleation of human erythroblasts based on our recent results and on evolutionary considerations related to the biology of hemoglobin and the comparative biochemistry of erythrocyte membrane cytoskeletal proteins, such as protein 4.1R. We specifically focus on the Mesozoic era, a geological period during which dinosaurs and the ancestors of mammalian species coexisted. Approximately 200 million years ago, at the beginning of this era, the earth's atmosphere was hypoxic. Interestingly, animals adopted different respiration systems to adapt to this hypoxic environment. Recent studies using state-of-the-art technologies have shown that dinosaurs might have had nucleated erythrocytes. After dinosaurs became extinct about 65.5 million years ago, their respiration system was maintained by birds. We propose a new adaptive theory that establishes a correlation between evolution towards nucleated or anucleate erythrocytes depending on organism respiration systems during the Mesozoic era.

Phosphatidylinositol-4,5 bisphosphate (PIP(2)) inhibits apo-calmodulin binding to protein 4.1.

Membrane skeletal protein 4.1R(80) plays a key role in regulation of erythrocyte plasticity. Protein 4.1R(80) interactions with transmembrane proteins, such as glycophorin C (GPC), are regulated by Ca(2+)-saturated calmodulin (Ca(2+)/CaM) through simultaneous binding to a short peptide (pep11; A(264)KKLWKVCVEHHTFFRL) and a serine residue (Ser(185)), both located in the N-terminal 30 kDa FERM domain of 4.1R(80) (H·R30). We have previously demonstrated that CaM binding to H·R30 is Ca(2+)-independent and that CaM binding to H·R30 is responsible for the maintenance of H·R30 ß-sheet structure. However, the mechanisms responsible for the regulation of CaM binding to H·R30 are still unknown. To investigate this, we took advantage of similarities and differences in the structure of Coracle, the Drosophila sp. homologue of human 4.1R(80), i.e. conservation of the pep11 sequence but substitution of the Ser(185) residue with an alanine residue. We show that the H·R30 homologue domain of Coracle, Cor30, also binds to CaM in a Ca(2+)-independent manner and that the Ca(2+)/CaM complex does not affect Cor30 binding to the transmembrane protein GPC. We also document that both H·R30 and Cor30 bind to phosphatidylinositol-4,5 bisphosphate (PIP2) and other phospholipid species and that that PIP2 inhibits Ca(2+)-free CaM but not Ca(2+)-saturated CaM binding to Cor30. We conclude that PIP2 may play an important role as a modulator of apo-CaM binding to 4.1R(80) throughout evolution.

Enucleation of human erythroblasts involves non-muscle myosin IIB.

Mammalian erythroblasts undergo enucleation, a process thought to be similar to cytokinesis. Although an assemblage of actin, non-muscle myosin II, and several other proteins is crucial for proper cytokinesis, the role of non-muscle myosin II in enucleation remains unclear. In this study, we investigated the effect of various cell-division inhibitors on cytokinesis and enucleation. For this purpose, we used human colony-forming unit-erythroid (CFU-E) and mature erythroblasts generated from purified CD34(+) cells as target cells for cytokinesis and enucleation assay, respectively. Here we show that the inhibition of myosin by blebbistatin, an inhibitor of non-muscle myosin II ATPase, blocks both cell division and enucleation, which suggests that non-muscle myosin II plays an essential role not only in cytokinesis but also in enucleation. When the function of non-muscle myosin heavy chain (NMHC) IIA or IIB was inhibited by an exogenous expression of myosin rod fragment, myosin IIA or IIB, each rod fragment blocked the proliferation of CFU-E but only the rod fragment for IIB inhibited the enucleation of mature erythroblasts. These data indicate that NMHC IIB among the isoforms is involved in the enucleation of human erythroblasts.

Characterization of cytoskeletal protein 4.1R interaction with NHE1 (Na(+)/H(+) exchanger isoform 1).

NHE1 (Na(+)/H(+) exchanger isoform 1) has been reported to be hyperactive in 4.1R-null erythrocytes [Rivera, De Franceschi, Peters, Gascard, Mohandas and Brugnara (2006) Am. J. Physiol. Cell Physiol. 291, C880-C886], supporting a functional interaction between NHE1 and 4.1R. In the present paper we demonstrate that 4.1R binds directly to the NHE1cd (cytoplasmic domain of NHE1) through the interaction of an EED motif in the 4.1R FERM (4.1/ezrin/radixin/moesin) domain with two clusters of basic amino acids in the NHE1cd, K(519)R and R(556)FNKKYVKK, previously shown to mediate PIP(2) (phosphatidylinositol 4,5-bisphosphate) binding [Aharonovitz, Zaun, Balla, York, Orlowski and Grinstein (2000) J. Cell. Biol. 150, 213-224]. The affinity of this interaction (K(d) = 100-200 nM) is reduced in hypertonic and acidic conditions, demonstrating that this interaction is of an electrostatic nature. The binding affinity is also reduced upon binding of Ca(2+)/CaM (Ca(2+)-saturated calmodulin) to the 4.1R FERM domain. We propose that 4.1R regulates NHE1 activity through a direct protein-protein interaction that can be modulated by intracellular pH and Na(+) and Ca(2+) concentrations.

Dynamic secondary structural changes in Ca²âº-saturated calmodulin upon interaction with the antagonist, W-7.

Although the 3D structure of the Ca(2+)-bound CaM (Ca(2+)/CaM) complex with the antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7), has been resolved, the dynamic changes in Ca(2+)/CaM structure upon interaction with W-7 are still unknown. We investigated time- and temperature-dependent dynamic changes in Ca(2+)/CaM interaction with W-7 in physiological conditions using one- and two-dimensional Fourier-transformed infrared spectroscopy (2D-IR). We observed changes in the α-helix secondary structure of Ca(2+)/CaM when complexed with W-7 at a molar ratio of 1:2, but not at higher molar ratios (between 1:2 and 1:5). Kinetic studies revealed that, during the initial 125s at 25°C, Ca(2+)/CaM underwent formation of secondary coil and turn structures upon binding to W-7. Variations in temperature that induced significant changes in the structure of the Ca(2+)/CaM complex failed to do so when Ca(2+)/CaM was complexed with W-7. We concluded that W-7 induced stepwise conformational changes in Ca(2+)/CaM that resulted in a rigidification of the complex and its inability to interact with target proteins and/or polypeptides.

Structural stabilization of protein 4.1R FERM domain upon binding to apo-calmodulin: novel insights into the biological significance of the calcium-independent binding of calmodulin to protein 4.1R.

In erythrocytes, 4.1R80 (80 kDa isoform of protein 4.1R) binds to the cytoplasmic tail of the transmembrane proteins band 3 and GPC (glycophorin C), and to the membrane-associated protein p55 through the N- (N-terminal), α- (α-helix-rich) and C- (C-terminal) lobes of R30 [N-terminal 30 kDa FERM (4.1/ezrin/radixin/moesin) domain of protein 4.1R] respectively. We have shown previously that R30 binds to CaM (calmodulin) in a Ca2+-independent manner, the equilibrium dissociation constant (Kd) for R30-CaM binding being very similar (in the submicromolar range) in the presence or absence of Ca2+. In the present study, we investigated the consequences of CaM binding on R30's structural stability using resonant mirror detection and FTIR (Fourier-transform IR) spectroscopy. After a 30 min incubation above 40° C, R30 could no longer bind to band 3 or to GPC. In contrast, R30 binding to p55, which could be detected at a temperature as low as 34° C, was maintained up to 44° C in the presence of apo-CaM. Dynamic light scattering measurements indicated that R30, either alone or complexed with apo-CaM, did not aggregate up to 40° C. FTIR spectroscopy revealed that the dramatic variations in the structure of the ß-sheet structure of R30 observed at various temperatures were minimized in the presence of apo-CaM. On the basis of Kd values calculated at various temperatures, ΔCp and ΔG° for R30 binding to apo-CaM were determined as -10 kJ · K(-1) · mol-1 and ~ -38 kJ · mol(-1) at 37° C (310.15 K) respectively. These data support the notion that apo-CaM stabilizes R30 through interaction with its ß-strand-rich C-lobe and provide a novel function for CaM, i.e. structural stabilization of 4.1R80.

High affinity of interaction between superantigen and T cell receptor Vbeta molecules induces a high level and prolonged expansion of superantigen-reactive CD4+ T cells.

In mice implanted with an osmotic pump filled with the superantigen (SAG) staphylococcal enterotoxin A (SEA), the Vß3(+)CD4(+) T cells exhibited a high level of expansion whereas the Vß11(+)CD4(+) T cells exhibited a mild level of expansion. In contrast, in mice implanted with an osmotic pump filled with SE-like type P (SElP, 78.1% homologous with SEA), the Vß11(+)CD4(+) T cells exhibited a high level of expansion while the Vß3(+)CD4(+) T cells exhibited a low level of expansion, suggesting that the level of the SAG-induced response is determined by the affinities between the TCR Vß molecules and SAG. Analyses using several hybrids of SEA and SElP showed that residue 206 of SEA determines the response levels of Vß3(+)CD4(+) and Vß11(+)CD4(+) T cells both in vitro and in vivo. Analyses using the above-mentioned hybrids showed that the binding affinities between SEA and the Vß3/Vß11 ß chains and between SEA-MHC class II-molecule complex and Vß3(+)/Vß11(+) CD4(+) T cells determines the response levels of the SAG-reactive T cells both in vitro and in vivo.

Similarities and differences in the structure and function of 4.1G and 4.1R135, two protein 4.1 paralogues expressed in erythroid cells.

Membrane skeletal protein 4.1R is the prototypical member of a family of four highly paralogous proteins that include 4.1G, 4.1N and 4.1B. Two isoforms of 4.1R (4.1R135 and 4.1R80), as well as 4.1G, are expressed in erythroblasts during terminal differentiation, but only 4.1R80 is present in mature erythrocytes. Although the function of 4.1R isoforms in erythroid cells has been well characterized, there is little or no information on the function of 4.1G in these cells. In the present study, we performed detailed characterization of the interaction of 4.1G with various erythroid membrane proteins and the regulation of these interactions by calcium-saturated calmodulin. Like both isoforms of 4.1R, 4.1G bound to band 3, glycophorin C, CD44, p55 and calmodulin. While both 4.1G and 4.1R135 interact with similar affinity with CD44 and p55, there are significant differences in the affinity of their interaction with band 3 and glycophorin C. This difference in affinity is related to the non-conserved N-terminal headpiece region of the two proteins that is upstream of the 30 kDa membrane-binding domain that harbours the binding sites for the various membrane proteins. The headpiece region of 4.1G also contains a high-affinity calcium-dependent calmodulin-binding site that plays a key role in modulating its interaction with various membrane proteins. We suggest that expression of the two paralogues of protein 4.1 with different affinities for band 3 and glycophorin C is likely to play a role in assembly of these two membrane proteins during terminal erythroid differentiation.

Two different unique cardiac isoforms of protein 4.1R in zebrafish, Danio rerio, and insights into their cardiac functions as related to their unique structures.

Protein 4.1R (4.1R) has been identified as the major component of the human erythrocyte membrane skeleton. The members of the protein 4.1 gene family are expressed in a tissue-specific alternative splicing manner that increases their functions in each tissue; however, the exact roles of cardiac 4.1R in the developing myocardium are poorly understood. In zebrafish (ZF), we identified two heart-specific 4.1R isoforms, ZF4.1RH2 and ZF4.1RH3, encoding N-terminal 30 kDa (FERM) domain and spectrin-actin binding domain (SABD) and C-terminal domain (CTD), separately. Applying immunohistochemistry using specific antibodies for 30 kDa domain and CTD separately, the gene product of ZF4.1RH2 and ZF4.1RH3 appeared only in the ventricle and in the atrium, respectively, in mature hearts. During embryogenesis, both gene expressions are expressed starting 24 h post-fertilization (hpf). Following whole-mount in situ hybridization, ZF4.1RH3 gene expression was detected in the atrium of 37 hpf embryos. These results indicate that the gene product of ZF4.1RH3 is essential for normal morphological shape of the developing heart and to support the repetitive cycles of its muscle contraction and relaxation.

Marked difference in membrane-protein-binding properties of the two isoforms of protein 4.1R expressed at early and late stages of erythroid differentiation.

Two major isoforms of protein 4.1R, a 135 kDa isoform (4.1R(135)) and an 80 kDa isoform (4.1R(80)), are expressed at distinct stages of terminal erythroid differentiation. The 4.1R(135) isoform is exclusively expressed in early erythroblasts and is not present in mature erythrocytes, whereas the 4.1R(80) isoform is expressed at late stages of erythroid differentiation and is the principal component of mature erythrocytes. These two isoforms differ in that the 4.1R(135) isoform includes an additional 209 amino acids designated as the HP (head-piece) at the N-terminus of 4.1R(80). In the present study, we performed detailed characterization of the interactions of the two 4.1R isoforms with various membrane-binding partners and identified several isoform-specific differences. Although both 4.1R(135) and 4.1R(80) bound to cytoplasmic domains of GPC (glycophorin C) and band 3, there is an order of magnitude difference in the binding affinities. Furthermore, although both isoforms bound CaM (calmodulin), the binding of 4.1R(80) was Ca2+-independent, whereas the binding of 4.1R(135) was strongly Ca2+-dependent. The HP of 4.1R(135) mediates this Ca2+-dependent binding. Ca2+-saturated CaM completely inhibited the binding of 4.1R(135) to GPC, whereas it strongly reduced the affinity of its binding to band 3. Interestingly, in spite of the absence of spectrin-binding activity, the 4.1R(135) isoform was able to assemble on to the membrane of early erythroblasts suggesting that its ability to bind to membrane proteins is sufficient for its membrane localization. These findings enable us to offer potential new insights into the differential contribution of 4.1R isoforms to membrane assembly during terminal erythroid differentiation.

ATP produced by anaerobic glycolysis is essential for enucleation of human erythroblasts.

More than 2million human erythroblasts extrude their nuclei every second in bone marrow under hypoxic conditions (<7% O2). Enucleation requires specific signal transduction pathways and the local assembly of contractile actomyosin rings. However, the energy source driving these events has not yet been identified. We examined whether different O2 environments (hypoxic [5% O2] and normoxic [21% O2] conditions) affected human CD34+ cell erythroblast differentiation. We also investigated the regulatory mechanisms underlying energy production in erythroblasts during terminal differentiation under 5% or 21% O2 conditions. The results obtained revealed that the enucleation ratio and intracellular levels of adenosine triphosphate (ATP), lactate dehydrogenase (LDH) M3H, and hypoxia-inducible factor 1α in erythroblasts during terminal differentiation were higher under the 5% O2 condition than under the 21% O2 condition. We also found that the enzymatic inhibition of glyceraldehyde 3-phosphate dehydrogenase and LDH, key enzymes in anaerobic glycolysis, blocked the proliferation of colony-forming units-erythroid and enucleation of erythroblasts, and also reduced ATP levels in erythroblasts under both hypoxic and normoxic conditions. Under both conditions, phosphorylation of the Ser232, Ser293, and Ser300 residues in pyruvate dehydrogenase (inactive state of the enzyme) in erythroblasts was involved in regulating the pathway governing energy metabolism during erythroid terminal differentiation. This reaction may be mediated by pyruvate dehydrogenase kinase (PDK) 4, the major PDK isozyme expressed in erythroblasts undergoing enucleation. Collectively, these results suggest that ATP produced by anaerobic glycolysis is the main source of energy for human erythroblast enucleation in the hypoxic bone marrow environment.

Interactions of Plasmodium falciparum erythrocyte membrane protein 3 with the red blood cell membrane skeleton.

Plasmodium falciparum parasites express and traffick numerous proteins into the red blood cell (RBC), where some associate specifically with the membrane skeleton. Importantly, these interactions underlie the major alterations to the modified structural and functional properties of the parasite-infected RBC. P. falciparum Erythrocyte Membrane Protein 3 (PfEMP3) is one such parasite protein that is found in association with the membrane skeleton. Using recombinant PfEMP3 proteins in vitro, we have identified the region of PfEMP3 that binds to the RBC membrane skeleton, specifically to spectrin and actin. Kinetic studies revealed that residues 38-97 of PfEMP3 bound to purified spectrin with moderately high affinity (K(D(kin))=8.5 x 10(-8) M). Subsequent deletion mapping analysis further defined the binding domain to a 14-residue sequence (IFEIRLKRSLAQVL; K(D(kin))=3.8 x 10(-7) M). Interestingly, this same domain also bound to F-actin in a specific and saturable manner. These interactions are of physiological relevance as evidenced by the binding of this region to the membrane skeleton of inside-out RBCs and when introduced into resealed RBCs. Identification of a 14-residue region of PfEMP3 that binds to both spectrin and actin provides insight into the potential function of PfEMP3 in P. falciparum-infected RBCs.

The localization of α-synuclein in the process of differentiation of human erythroid cells.

Although the neuronal protein α-synuclein (α-syn) is thought to play a central role in the pathogenesis of Parkinson's disease (PD), its physiological function remains unknown. It is known that α-syn is also abundantly expressed in erythrocytes. However, its role in erythrocytes is also unknown. In the present study, we investigated the localization of α-syn in human erythroblasts and erythrocytes. Protein expression of α-syn increased during terminal differentiation of erythroblasts (from day 7 to day 13), whereas its mRNA level peaked at day 11. α-syn was detected in the nucleus, and was also seen in the cytoplasm and at the plasma membrane after day 11. In erythroblasts undergoing nucleus extrusion (day 13), α-syn was detected at the periphery of the nucleus. Interestingly, we found that recombinant α-syn binds to trypsinized inside-out vesicles of erythrocytes and phosphatidylserine (PS) liposomes. The dissociation constants for binding to PS/phosphatidylcholine (PC) liposomes of N-terminally acetylated (NAc) α-syn was lower than that of non NAc α-syn. This suggests that N-terminal acetylation plays a significant functional role. The results of the present study collectively suggest that α-syn is involved in the enucleation of erythroblasts and the stabilization of erythroid membranes.

Characterization of protein 4.1R in erythrocytes of zebrafish (Danio rerio): unique binding properties with transmembrane proteins and calmodulin.

Cytoskeletal protein 4.1R is instrumental in regulating erythrocyte plasticity. 4.1R is comprised of four domains identified after chymotryptic digestion: an N-terminal 30 kDa domain responsible for interaction with membrane proteins, a unique domain, a spectrin-actin binding (SAB) domain, and a C-terminal domain (CTD). 4.1R 30 kDa domain interactions with transmembrane proteins are regulated by the Ca(2+)/calmodulin (CaM) complex. Unlike mature mammalian erythrocytes, fish erythrocytes remain nucleated. Comparing their cytoskeleton architecture and functional properties is therefore of great interest. Here we characterized the recently cloned zebrafish (Danio rerio, ZF) 4.1R and compared its properties with human 4.1R. We identified three ZF4.1R mRNA transcripts in erythrocytes, all characterized by exclusion of the central domains. The major transcript, referred to as BL31, included a full length 30 kDa domain (ZFR30) and parts of the unique region Ua and of CTD. Two minor transcripts, referred to as BL42 and BL56, expressed parts of ZFR30 and of the unique region Ub and full length SAB and CTD domains. Antibodies to ZFR30, ZF4.1R CTD and ZF glycophorin C (GPC) labeled the ZF erythrocyte plasma membrane. ZFR30 bound to CaM in presence or absence of Ca(2+). Resonant mirror detection binding assays revealed that ZFR30 bound to human Band3 with low K((D)) ( approximately 10nM), and to GPC with higher K((D)) ( approximately 1nM). The Ca(2+)/CaM complex did not affect ZFR30 binding to Band3 and GPC. Finally, we confirmed ZFR30 binding to erythrocyte plasma membrane proteins by pulling down ZFR30 with human erythrocyte inside-out vesicles (IOV). This study defines unique structural and functional properties for ZF4.1R.

Regulation of protein 4.1R interactions with membrane proteins by Ca2+ and calmodulin.

Red blood cell protein 4.1 (4.1R) is essential for maintaining erythrocyte shape and controlling membrane mechanical properties, such as deformability and stability. The importance of 4.1R has been demonstrated by the dramatic erythrocyte alterations observed in patients lacking this protein. Indeed, 4.1R null red blood cells adopt an elliptical shape and are characterized by unstable membranes. The key role of 4.1R likely results from multiple protein-protein interactions: lateral interactions with the spectrin/actin network and vertical interactions with the cytoplasmic domain of transmembrane proteins glycophorin C (GPC), Band 3 (anion exchanger 1, AE1), and CD44. 4.1R promotes the formation of a ternary complex with GPC and p55 through its 30 kDa membrane-binding domain. Based on the primary structure of the prototypical 80 kDa isoform of 4.1R, functional domains and sites for binding partners have been identified. The others and we have been focusing on the structure and function of the 30 kDa NH2-terminal domain of 4.1R, which is responsible for 4.1R interaction with the transmembrane proteins described above. A major finding is that Ca2+, in association with calmodulin (CaM), plays a critical role in regulation of the interaction of the 30 kDa domain with its various binding partners. This review is a detailed report of our current knowledge regarding 4.1R, and more specifically, 4.1R 30 kDa domain: its primary structure, functions and modulation by Ca2+ and CaM. Emphasis is given on the relationships between structure and function that we have been able to establish through X-ray crystal structure analysis of the 30 kDa membrane-binding domain in 4.1R. Finally, we give insights into the potential roles of 4.1R in the dynamic organization of the membrane skeleton viewed as a complex system.

Conserved and unique thermodynamic properties of lactate dehydrogenases in an ectothermic organism, the teleost Microstomus achne, and an endothermic organism, bovine.

It is widely believed that enzymatic activities in ectothermic organisms adapt to environmental temperatures. However, to date, no study has thoroughly compared multiple thermodynamic enzymatic characteristics across species living in dramatically different environments. To start to address this gap, we compared the characteristics of lactate dehydrogenase (LDH) purified from the muscles from slime flounder Microstomus achne white muscle and bovine skeletal muscle (bM4) and heart. The K m and V max for pyruvate reduction were about three times higher for M. achne LDH than bM4 Surprisingly, maximum LDH activity was observed at ∼30 °C and ∼50 °C for M. achne and bovine LDHs, respectively, suggesting that the maximum enzymatic activity of LDH is set at a temperature ∼20 °C higher than environmental or body temperature across species. Although K m and V max values of these LDHs increased with temperature, the V max/K m ratio for M. achne LDH and bM4 was independent. Differential scanning calorimetry and enthalpy change measurements confirmed that M. achne and bovine muscle-specific LDHs shared similar properties. Based on the present findings and previous reports, we hypothesize that the function and thermodynamic properties of muscle LDH are highly conserved between a teleost adapted to cold, M. achne, and bovine.

Erythroblast enucleation is a dynein-dependent process.

Mammalian erythroblasts undergo enucleation through a process thought to be similar to cytokinesis. Microtubule-organizing centers (MTOCs) mediate organization of the mitotic spindle apparatus that separates the chromosomes during mitosis and are known to be crucial for proper cytokinesis. However, the role of MTOCs in erythroblast enucleation remains unknown. We therefore investigated the effect of various MTOC inhibitors on cytokinesis and enucleation using human colony-forming units-erythroid (CFU-Es) and mature erythroblasts generated from purified CD34(+) cells. We found that erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA), a dynein inhibitor, and monastrol, a kinesin Eg5 inhibitor, as well as various inhibitors of MTOC regulators, including ON-01910 (Plk-1), MLN8237 (aurora A), hesperadin (aurora B), and LY294002 (PI3K), all inhibited CFU-E cytokinesis. Among these inhibitors, however, only EHNA blocked enucleation. Moreover, terminally differentiated erythroblasts expressed only dynein; little or none of the other tested proteins was detected. Over the course of the terminal differentiation of human erythroblasts, the fraction of cells with nuclei at the cell center declined, whereas the fraction of polarized cells, with nuclei shifted to a position near the plasma membrane, increased. Dynein inhibition impaired nuclear polarization, thereby blocking enucleation. These data indicate that dynein plays an essential role not only in cytokinesis but also in enucleation. We therefore conclude that human erythroblast enucleation is a process largely independent of MTOCs, but dependent on dynein.