The activin receptor ligand trap ActRIIB:ALK4-Fc ameliorates cardiomyopathy induced by neuromuscular disease and diabetes.

Cardiomyopathies are ascribed to a variety of etiologies, present with diverse clinical phenotypes, and lack disease-modifying treatments. Mounting evidence implicates dysregulated activin receptor signaling in heart disease and highlights inhibition of this pathway as a potential therapeutic target. Here, we explored the effects of activin ligand inhibition using ActRIIB:ALK4-Fc, a heterodimeric receptor fusion protein, in two mechanistically distinct murine models of cardiomyopathy. Treatment with ActRIIB:ALK4-Fc significantly improved systolic or diastolic function in cardiomyopathy induced by neuromuscular disease or diabetes mellitus. Moreover, ActRIIB:ALK4-Fc corrected Ca2+ handling protein expression in diseased heart tissues, suggesting that activin signaling inhibition could alleviate cardiomyopathies in part by rebalancing aberrant intracellular Ca2+ homeostasis-a common underlying pathomechanism in diverse heart diseases.

Functionally diverse heteromeric traps for ligands of the transforming growth factor-ß superfamily.

Ligands of the transforming growth factor-ß (TGF-ß) superfamily are important targets for therapeutic intervention but present challenges because they signal combinatorially and exhibit overlapping activities in vivo. To obtain agents capable of sequestering multiple TGF-ß superfamily ligands with novel selectivity, we generated soluble, heterodimeric ligand traps by pairing the extracellular domain (ECD) of the native activin receptor type IIB (ActRIIB) alternately with the ECDs of native type I receptors activin receptor-like kinase 4 (ALK4), ALK7, or ALK3. Systematic analysis of these heterodimeric constructs by surface plasmon resonance, and comparison with their homodimeric counterparts, revealed that each type I receptor partner confers a distinct ligand-binding profile to the heterodimeric construct. Additional characterization in cell-based reporter gene assays confirmed that the heterodimeric constructs possessed different profiles of signaling inhibition in vitro, which translated into altered patterns of pharmacological activity when constructs were administered systemically to wild-type mice. Our results detail a versatile platform for the modular recombination of naturally occurring receptor domains, giving rise to inhibitory ligand traps that could aid in defining the physiological roles of TGF-ß ligand sets or be directed therapeutically to human diseases arising from dysregulated TGF-ß superfamily signaling.

Smad2/3-pathway ligand trap luspatercept enhances erythroid differentiation in murine ß-thalassaemia by increasing GATA-1 availability.

In ß-thalassaemia, anaemia results from ineffective erythropoiesis characterized by inhibition of late-stage erythroid differentiation. We earlier used luspatercept and RAP-536 protein traps for certain Smad2/3-pathway ligands to implicate Smad2/3-pathway overactivation in dysregulated erythroid differentiation associated with murine ß-thalassaemia and myelodysplasia. Importantly, luspatercept alleviates anaemia and has been shown to reduce transfusion burden in patients with ß-thalassaemia or myelodysplasia. Here, we investigated the molecular mechanisms underlying luspatercept action and pSmad2/3-mediated inhibition of erythroid differentiation. In murine erythroleukemic (MEL) cells in vitro, ligand-mediated overactivation of the Smad2/3 pathway reduced nuclear levels of GATA-1 (GATA-binding factor-1) and its transcriptional activator TIF1γ (transcription intermediary factor 1γ), increased levels of reactive oxygen species, reduced cell viability and haemoglobin levels, and inhibited erythroid differentiation. Co-treatment with luspatercept in MEL cells partially or completely restored each of these. In ß-thalassaemic mice, RAP-536 up-regulated Gata1 and its target gene signature in erythroid precursors determined by transcriptional profiling and gene set enrichment analysis, restored nuclear levels of GATA-1 in erythroid precursors, and nuclear distribution of TIF1γ in erythroblasts. Bone marrow cells from ß-thalassaemic mice treated with luspatercept also exhibited restored nuclear availability of GATA-1 ex vivo. Our results implicate GATA-1, and likely TIF1γ, as key mediators of luspatercept/RAP-536 action in alleviating ineffective erythropoiesis.

Functional characterization of PeIF5B as eIF5B homologue from Pisum sativum.

We earlier reported 'PeIF5B' as a novel factor from Pisum sativum that has sequence similarity to eIF5B (S. Rasheedi, S. Ghosh, M. Suragani et al., P. sativum contains a factor with strong homology to eIF5B, Gene 399 (2007) 144-151). The main aim of the present study was to perform functional characterization of PeIF5B as an eIF5B homologue from plant system. PeIF5B shows binding to Met - tRNA(f)(Met), hydrolyses GTP and interacts with ribosomes. In vivo growth complementation analysis shows that PeIF5B partially complements its yeast homologue. Interestingly, PeIF5B mainly localizes in the nucleus as confirmed by nuclear localization signal (NLS) prediction, confocal imaging and immunoblots of cellular fractions. Similar to the yeast eIF5B but unlike the human orthologue, PeIF5B is an intron-less gene. This study highlights PeIF5B's role as a functional eIF5B homologue possibly participating in nuclear translation in plant system.

Modified activin receptor IIB ligand trap mitigates ineffective erythropoiesis and disease complications in murine ß-thalassemia.

In ß-thalassemia, unequal production of α- and ß-globin chains in erythroid precursors causes apoptosis and inhibition of late-stage erythroid differentiation, leading to anemia, ineffective erythropoiesis (IE), and dysregulated iron homeostasis. Here we used a murine model of ß-thalassemia intermedia (Hbb(th1/th1) mice) to investigate effects of a modified activin receptor type IIB (ActRIIB) ligand trap (RAP-536) that inhibits Smad2/3 signaling. In Hbb(th1/th1) mice, treatment with RAP-536 reduced overactivation of Smad2/3 in splenic erythroid precursors. In addition, treatment of Hbb(th1/th1) mice with RAP-536 reduced α-globin aggregates in peripheral red cells, decreased the elevated reactive oxygen species present in erythroid precursors and peripheral red cells, and alleviated anemia by promoting differentiation of late-stage erythroid precursors and reducing hemolysis. Notably, RAP-536 treatment mitigated disease complications of IE, including iron overload, splenomegaly, and bone pathology, while reducing erythropoietin levels, improving erythrocyte morphology, and extending erythrocyte life span. These results implicate signaling by the transforming growth factor-ß superfamily in late-stage erythropoiesis and reveal potential of a modified ActRIIB ligand trap as a novel therapeutic agent for thalassemia syndrome and other red cell disorders characterized by IE.

Transforming growth factor-ß superfamily ligand trap ACE-536 corrects anemia by promoting late-stage erythropoiesis.

Erythropoietin (EPO) stimulates proliferation of early-stage erythrocyte precursors and is widely used for the treatment of chronic anemia. However, several types of EPO-resistant anemia are characterized by defects in late-stage erythropoiesis, which is EPO independent. Here we investigated regulation of erythropoiesis using a ligand-trapping fusion protein (ACE-536) containing the extracellular domain of human activin receptor type IIB (ActRIIB) modified to reduce activin binding. ACE-536, or its mouse version RAP-536, produced rapid and robust increases in erythrocyte numbers in multiple species under basal conditions and reduced or prevented anemia in murine models. Unlike EPO, RAP-536 promoted maturation of late-stage erythroid precursors in vivo. Cotreatment with ACE-536 and EPO produced a synergistic erythropoietic response. ACE-536 bound growth differentiation factor-11 (GDF11) and potently inhibited GDF11-mediated Smad2/3 signaling. GDF11 inhibited erythroid maturation in mice in vivo and ex vivo. Expression of GDF11 and ActRIIB in erythroid precursors decreased progressively with maturation, suggesting an inhibitory role for GDF11 in late-stage erythroid differentiation. RAP-536 treatment also reduced Smad2/3 activation, anemia, erythroid hyperplasia and ineffective erythropoiesis in a mouse model of myelodysplastic syndromes (MDS). These findings implicate transforming growth factor-ß (TGF-ß) superfamily signaling in erythroid maturation and identify ACE-536 as a new potential treatment for anemia, including that caused by ineffective erythropoiesis.

Heme-regulated eIF2α kinase activated Atf4 signaling pathway in oxidative stress and erythropoiesis.

Heme-regulated eIF2α kinase (Hri) is necessary for balanced synthesis of heme and globin. In addition, Hri deficiency exacerbates the phenotypic severity of ß-thalassemia intermedia in mice. Activation of Hri during heme deficiency and in ß-thalassemia increases eIF2α phosphorylation and inhibits globin translation. Under endoplasmic reticulum stress and nutrient starvation, eIF2α phosphorylation also induces the Atf4 signaling pathway to mitigate stress. Although the function of Hri in regulating globin translation is well established, its role in Atf4 signaling in erythroid precursors is not known. Here, we report the role of the Hri-activated Atf4 signaling pathway in reducing oxidative stress and in promoting erythroid differentiation during erythropoiesis. On acute oxidative stress, Hri(-/-) erythroblasts suffered from increased levels of reactive oxygen species (ROS) and apoptosis. During chronic iron deficiency in vivo, Hri is necessary both to reduce oxidative stress and to promote erythroid differentiation. Hri(-/-) mice developed ineffective erythropoiesis during iron deficiency with inhibition of differentiation at the basophilic erythroblast stage. This inhibition is recapitulated during ex vivo differentiation of Hri(-/-) fetal liver erythroid progenitors. Importantly, the Hri-eIF2αP-Atf4 pathway was activated and required for erythroid differentiation. We further demonstrate the potential of modulating Hri-eIF2αP-Atf4 signaling with chemical compounds as pharmaceutical therapies for ß-thalassemia.

Haem-regulated eIF2alpha kinase is necessary for adaptive gene expression in erythroid precursors under the stress of iron deficiency.

Haem-regulated eIF2alpha kinase (HRI) is essential for the regulation of globin gene translation and the survival of erythroid precursors in iron/haem deficiency. This study found that that in iron deficiency, fetal definitive erythropoiesis is inhibited at the basophilic erythroblast stage with increased proliferation and elevated apoptosis. This hallmark of ineffective erythropoiesis is more severe in HRI deficiency. Microarray gene profiling analysis showed that HRI was required for adaptive gene expression in erythroid precursors during chronic iron deficiency. The number of genes with expression affected more than twofold increased, from 213 in iron deficiency and 73 in HRI deficiency, to 3135 in combined iron and HRI deficiencies. Many of these genes are regulated by Gata1 and Fog1. We demonstrate for the first time that Gata1 expression in developing erythroid precursors is decreased in iron deficiency, and is decreased further in combined iron and HRI deficiencies. Additionally, Fog1 expression is decreased in combined deficiencies, but not in iron or HRI deficiency alone. Our results indicate that HRI confers adaptive gene expression in developing erythroblasts during iron deficiency through maintaining Gata1/Fog1 expression.

Intersubunit and interprotein interactions of alpha- and beta-subunits of human eIF2: Effect of phosphorylation.

Purified recombinant human subunits of eukaryotic initiation factor 2 (eIF2) expressed in bacteria are found to interact with each other to form alphabeta, alphagamma, and betagamma complexes in a pull-down experiment. Recombinant phosphorylated human eIF2alpha that cannot interact with purified eIF2B, the GDP/GTP exchange factor of eIF2, however interacts efficiently with eIF2B along with the beta-subunit of eIF2 of the rabbit reticulocyte lysates and also with the purified recombinant beta-subunit. These findings therefore suggest that the beta-subunit of eIF2 mediates the productive and non-productive interactions between eIF2 and 2B. Recombinant alpha and beta-subunits serve as substrates for not only kinases but also for caspase 3 and interestingly phosphorylated subunits resist caspase action. Phosphorylation also modifies the beta-subunit's interaction with Nck1, a cofactor of eIF2alpha phosphatase, but not with eIF5, the GTPase activating protein. These findings suggest that subunits of mammalian eIF2 interact with each other and the beta-subunit plays a critical role both in the regulation and function of eIF2.

Deficiency of heme-regulated eIF2alpha kinase decreases hepcidin expression and splenic iron in HFE-/- mice.

Heme-regulated eIF2alpha kinase (HRI) is essential for regulating globin translation in iron deficiency and in beta-thalassemia. We investigated the role of heme-regulated eIF2alpha kinase in hemoglobin and red blood cell production as well as in iron homeostasis in a mouse model of iron overload. We show that HRI deficiency does not significantly affect red cell parameters of hemochromatosis (HFE(-)(/)(-)) mice. Importantly, heme-regulated eIF2alpha kinase deficiency exacerbates decreases in hepcidin expression and splenic macrophage iron in HFE(-)(/)(-) mice. Furthermore, the serum level of bone morphogenic protein 2, which positively regulates hepcidin, is reduced in heme-regulated eIF2alpha kinase deficiency, but not in HFE deficiency.

The function of heme-regulated eIF2alpha kinase in murine iron homeostasis and macrophage maturation.

Heme-regulated eIF2alpha kinase (HRI) plays an essential protective role in anemias of iron deficiency, erythroid protoporphyria, and beta-thalassemia. In this study, we report that HRI protein is present in murine macrophages, albeit at a lower level than in erythroid precursors. Hri-/- mice exhibited impaired macrophage maturation and a weaker antiinflammatory response with reduced cytokine production upon LPS challenge. The level of production of hepcidin, an important player in the pathogenesis of the anemia of inflammation, was significantly decreased in Hri-/- mice, accompanied by decreased splenic macrophage iron content and increased serum iron content. Hepcidin expression was also significantly lower, with a concomitant increase in serum iron in Hri-/- mice upon LPS treatment. We also demonstrated an impairment of erythrophagocytosis by Hri-/- macrophages both in vitro and in vivo under chronic hemolytic anemia, providing evidence for the role of HRI in recycling iron from senescent red blood cells. This work demonstrates that HRI deficiency attenuates hepcidin expression and iron homeostasis in mice, indicating a potential role for HRI in the anemia of inflammation.

Expression and purification of the subunits of human translational initiation factor 2 (eIF2): phosphorylation of eIF2 alpha and beta.

Eukaryotic initiation factor 2 (eIF2) is a GDP-binding protein with three subunits: alpha, beta, and gamma. It delivers initiator tRNA (Met-tRNAi) to 40S ribosomes in a GTP-dependent manner. The factor regulates the translation of messenger RNAs through the phosphorylation of serine 51 residue in the small or alpha-subunit of eIF2 (eIF2alpha) and modulation of its interaction with a rate-limiting heteropentameric protein eIF2B. To understand the structural, functional, and regulatory roles of each of these subunits in the various activities of phosphorylated and unphosphorylated eIF2, such, as its ability to interact with GTP, Met-tRNAi, 40S ribosomes and with various proteins, we have for the first time over expressed all the three subunits of human eIF2 independently, and, also together in Sf9 cells using pFast Bac HT vector of baculovirus expression system. The expression of all subunits increased with increase in infection time up to 72 h. We have also over expressed three mutant forms of eIF2alpha viz, S51A, S51D, and S48A in which the serine at 51 or 48 position is replaced by an alanine or aspartic acid with 6x histidine tag at the N-terminus. Further, any of the two subunits or all the three subunits of eIF2 were coexpressed by multiple infection of cells with recombinant viruses. Purified alpha (wt and mutants) and beta subunits were found suitable to serve as substrates for different kinases. The recombinant subunits of eIF2alpha and beta-subunits were also phosphorylated in cultured insect cells. Phosphorylation of eIF2alpha in vitro was not significantly different in the presence and absence of the other subunits.

Interaction of recombinant human eIF2 subunits with eIF2B and eIF2alpha kinases.

The heterotrimeric eukaryotic initiation factor 2 (eIF2) plays a critical role in the mechanics and regulation of protein synthesis. Unlike yeast and archaeal eIF2, the purified baculovirus-expressed recombinant human eIF2 subunits used in these studies reveal that the alpha- and beta-subunits interact with each other. Consistent with this observation, the beta-subunit specifically interacts with the purified eIF2B in ELISA studies and this interaction is enhanced when wt eIF2alpha in the recombinant trimeric complex is phosphorylated or replaced by a mutant phosphomimetic eIF2alpha (S51D). These findings together with other observations raise the possibility that the beta-subunit plays a key role in the regulation and function of mammalian eIF2 complex. PERK, an eIF2alpha kinase, is found to interact with wt and mutants of eIF2alpha in which the serine 51 or 48 residue is replaced by alanine or aspartic acid thereby suggesting that the phosphorylation site in the substrate is not important for interaction. Fluorescence spectroscopic and fluorescence resonance energy transfer analyses reveal that the energy transfer occurs from PERK to eIF2alpha. The dissociation constant of alpha-subunit-PERK complex (Kd alpha-subunit) is 0.74 microM and the interaction is stoichiometric.