Hemoglobin Stabilizing Protein Research Articles

Detection of SARS-CoV-2 genome and whole transcriptome sequencing in frontal cortex of COVID-19 patients

SARS-Cov-2 infection is frequently associated with Nervous System manifestations. However, it is not clear how SARS-CoV-2 can cause neurological dysfunctions and which molecular processes are affected in the brain. In this work, we examined the frontal cortex tissue of patients who died of COVID-19 for the presence of SARS-CoV-2, comparing qRT-PCR with ddPCR. We also investigated the transcriptomic profile of frontal cortex from COVID-19 patients and matched controls by RNA-seq analysis to characterize the transcriptional signature.Our data showed that SARS-CoV-2 could be detected by ddPCR in 8 (88%) of 9 examined samples while by qRT-PCR in one case only (11%). Transcriptomic analysis revealed that 11 genes (10 mRNAs and 1 lncRNA) were differential expressed when frontal cortex of COVID-19 patients were compared to controls. These genes fall into categories including hypoxia, hemoglobin-stabilizing protein, hydrogen peroxide processes. This work demonstrated that the quantity of viral RNA in frontal cortex is minimal and it can be detected only with a very sensitive method (ddPCR). Thus, it is likely that SARS-CoV-2 does not actively infect and replicate in the brain; its topography within encephalic structures remains uncertain. Moreover, COVID-19 may have a role on brain gene expression, since we observed an important downregulation of genes associated to hypoxia inducting factor system (HIF) that may inhibit the capacity of defense system during infection and oxigen deprivation, showing that hypoxia, well known multi organ condition associated to COVID-19, also marked the brain.

48 Burn Injury Impedes Late Erythropoiesis via Decreased Alpha Hemoglobin Stabilizing Protein and siglec1

Abstract Introduction Transfusions are of no benefit to burn patients with chronic anemia. We are elucidating the mechanisms of erythropoietin resistant anemia in burn injury using a mouse model in addition to a contrived system with human PBMCs. We have found that Alpha Hemoglobin Stabilizing Protein (AHSP), an erythroblast specific protein essential to effective hemoglobinization, is decreased in PBMC derived erythroblasts (EB) from burn patients (male + female). Hemoglobinization begins at the polychromatic EB (PolyE) stage and intensifies at the orthochromatic EB (OrthoE) stage, culminating in ejection of the nucleus and the cell becoming a reticulocyte (Reti). EB needs the support of a central island macrophage (EBI MØ) until they progress to maturation. We have seen in bone marrow from mice (male) following burn injury that Siglec1, an essential adhesion molecule is decreased in EBI MØ. It is these MØ that provide iron for the assembly of the hemoglobin unit in late erythropoiesis. EB maturation defects seen in burn injury could be due to the intrinsic changes in EB phenotype or the phenotype of supporting EBI MØ or a combination of the two. Our goal was to investigate in the animal model using male and female mice, how myeloid/erythroid intrinsic changes after burn impact the progression of late erythropoiesis from an EB and EBI MØ perspectives. Methods With IACUC approval, 12 male and female mice were anesthetized and subjected to sham burn or scald burn (15% TBSA) upon dorsal immersion in a 100oC water bath for 8 seconds. Animals were sacrificed on post burn day 7 (PBD 7). Total bone marrow cells (TBM) were harvested from bilateral femurs. Cells were characterized using CD71, Ter119, and Syto16 antibodies. EBI MØ were identified using a combination of F4/80, ERHR3, Vcam1, Ly6G and Siglec 1 antibodies. Geometric mean of fluorescence intensity (MFI) and cell counts were obtained by flow cytometric analysis and presented as x103 per million TBM cells. Peripheral blood was harvested via cardiac puncture and hemoglobin concentrations (g/dL) were obtained using a hematology analyzer. Results Among the erythroblasts, EEBs were 2.4 fold higher with a 2.4 fold lower AHSP expression in PBD 7 mice compared to sham as shown in left, top and bottom figures. Similarly, more EEBs were associated with EBIMØ with a lower Siglec1 expression as shown in right, top and bottom figures. Blood Hbg concentrations were lower in burn (sham=15.5±0.7 vs PBD 7= 13.1±0.5; p< 0.01). Conclusions The combined influence of Siglec1 and AHSP expression interferes with effective hemoglobinization impeding late maturation of erythroblasts following burn injury. Whether there is a synergistic action between the two deficiencies is being studied. Applicability of Research to Practice Underpinning the mechanism of erythropoiesis will lead to better options to ineffective transfusions in burn patients with anemia.

Association of alpha hemoglobin-stabilizing protein (AHSP) gene mutation and disease severity among HbE-beta thalassemia patients.

In this study, we aimed to investigate the pattern and association of genetic mutations occurring within the alpha hemoglobin-stabilizing protein (AHSP) gene among HbE beta thalassemia patients with varying phenotypic expressions. Fifty-four diagnosed cases of HbE beta thalassemia (transfusion dependent and independent) were included in the study. Among them, 38 patients with similar genotypes (IVS 1-5, alpha gene deletion and triplication, Xmn polymorphism) were selected for further analysis. AHSP gene sequencing was done for these 38 samples to study associated mutations in AHSP gene. HbE beta thalassemia patients with similar genotypes but different phenotypic expressions were found to have mutations in the AHSP gene. There were five mutations found most prevalent among the samples analyzed for AHSP gene sequencing. Among these, two mutations were from intron 1 region of AHSP and three mutations were found in exon 3. The most prevalent mutation was found at the Oct binding site at intron 1 of AHSP. The mutations in exon 3 were more prevalent among the TDT groups. A mutation in exon 3 changing the amino acid (33rd) from serine to phenylalanine was found to be associated with only TDT group. This study documents that among the HbE beta thalassemia patients with varying severity, an exon mutation in AHSP is significantly prevalent only among the TDT group. Further understanding of the mechanism will shed light upon the impact of AHSP in modifying the disease severity in thalassemia.

Role of Alpha Hemoglobin Stabilizing Protein Expression in Beta Thalassemia Patients

AbstractBackground: Beta (b)-Thalassemia is an autosomal reces-sive inherited disease causing variable degrees of anemia. The molecular defects are owing to point mutations or small deletions leading to diminution or absence of b-globin chain synthesis. The unmatched a-Hb is destructive to itself and other cellular components, resulting in ineffective erythropoi-esis and hemolysis. Alpha-Hemoglobin Stabilizing Protein (AHSP) is a scavenger protein of erythroid that fastly and reversibly attach the a subunit of hemoglobin molecule preventing its precipitation and subsequent hemolysis.Aim of Work: Is to assess AHSP gene expression in thalassemia major and intermedia and its relation to clinical features, laboratory investigations and to determine its role on the severity of thalassemia.Subjects and Methods: This case-control study is consisted of 120 studied subjects categorized into 40 thalassemia major patients, 40 thalassemia intermedia patients and 40 age healthy individuals as a control group. All subjects underwent the following; history, clinical picture, complete blood picture and Hb electrophoresis, serum ferritin and measurement of AHSP, alpha globin and beta globin genes expression by real time PCR.Results: AHSP gene in thal. intermedia group was higher than both thal. major (p<0.001). Beside thal. intermedia group had higher a globin/AHSP ratio versus thal. major group (p=0.011). b globin/AHSP ratio was not significant between both thal. groups. AHSP in thal.major group correlated with a globin and b globin expression and negatively correlated with organomegally, ferritin and no. of blood transfusion. Whereas in thal. intermedia group, was positively correlated with Hb, HCT, MCV, MCH, a globin gene expression, b globin gene expression and b globin/a globin genes ratio.Conclusion: AHSP expression was higher in thalassemia intermedia group than thalassemia major group. Consequently, AHSP can be considered genetic modifier in b thalassemia.

Escorting α-globin to eNOS: α-globin-stabilizing protein paves the way.

In the vascular wall, endothelial nitric oxide synthase (eNOS) produces NO to regulate peripheral vascular resistance, tissue perfusion, and blood pressure. In resistance arteries, eNOS couples with α-globin and, through chemical reactions, modulates NO diffusion needed for vascular smooth muscle relaxation. While α-globin protein alone is known to be unstable, the mechanisms that enable α-globin protein expression remain elusive. Here, Lechauve et al. report that arterial endothelium expresses α hemoglobin-stabilizing protein, which acts as a critical chaperone protein for α-globin expression and vascular function.

Kinetics of α-Globin Binding to α-Hemoglobin Stabilizing Protein (AHSP) Indicate Preferential Stabilization of Hemichrome Folding Intermediate

Human α-hemoglobin stabilizing protein (AHSP) is a conserved mammalian erythroid protein that facilitates the production of Hemoglobin A by stabilizing free α-globin. AHSP rapidly binds to ferrous α with association (k'(AHSP)) and dissociation (k(AHSP)) rate constants of ≈10 μm(-1) s(-1) and 0.2 s(-1), respectively, at pH 7.4 at 22 °C. A small slow phase was observed when AHSP binds to excess ferrous αCO. This slow phase appears to be due to cis to trans prolyl isomerization of the Asp(29)-Pro(30) peptide bond in wild-type AHSP because it was absent when αCO was mixed with P30A and P30W AHSP, which are fixed in the trans conformation. This slow phase was also absent when met(Fe(3+))-α reacted with wild-type AHSP, suggesting that met-α is capable of rapidly binding to either Pro(30) conformer. Both wild-type and Pro(30)-substituted AHSPs drive the formation of a met-α hemichrome conformation following binding to either met- or oxy(Fe(2+))-α. The dissociation rate of the met-α·AHSP complex (k(AHSP) ≈ 0.002 s(-1)) is ∼100-fold slower than that for ferrous α·AHSP complexes, resulting in a much higher affinity of AHSP for met-α. Thus, in vivo, AHSP acts as a molecular chaperone by rapidly binding and stabilizing met-α hemichrome folding intermediates. The low rate of met-α dissociation also allows AHSP to have a quality control function by kinetically trapping ferric α and preventing its incorporation into less stable mixed valence Hemoglobin A tetramers. Reduction of AHSP-bound met-α allows more rapid release to β subunits to form stable fully, reduced hemoglobin dimers and tetramers.

Modulation of expression and polymerization of hemoglobin Polytaur, a potential blood substitute

Chemically or genetically modified hemoglobins are a therapeutic class indicated for the treatment of a variety of hypo-oxygenation pathologies, severe trauma-related hemorrhages or elective surgery when blood transfusions are refused or not available. Recombinant heterologous hemoglobins offer the possibility of a potentially unlimited production and genetically optimized properties in terms of oxygen affinity, NO reactivity and resistance to autoxidation. Hemoglobin Polytaur is an autopolymerizing human-bovine hybrid mutant, previously obtained as a 500 kDa polymer, shown to reduce the infarct volume from focal cerebral ischemia in in vivo animal models. In this work, hemoglobin Polytaur polymerization, carried out under conditions to minimize heme oxidation and modification, resulted in a 180 kDa cyclic homogeneous trimer of hemoglobin tetramers. This novel oligomer was characterized by electrophoresis, MALDI-TOF mass spectrometry and gel filtration. The size and the oxygen binding properties were shown to be ideally suited for its use as a blood substitute. Co-expression with the human α hemoglobin-stabilizing protein (AHSP), a chaperone that assists hemoglobin folding in vivo, resulted in an unexpected decrease in yield and in unusual spectroscopic and functional properties, suggesting the formation of strong protein–protein interactions that reduce the expression, hinder the tetramer assembly and prevent purification.

Unstable Alpha Hemoglobin Stabilizing Protein as a Cause of Thalassemia: Proof of Concept.

Abstract 462Alpha hemoglobin stabilizing protein (AHSP), a small 102-residue protein previously known as erythroid differentiation-related factor (EDRF), is present only in erythroid cells, where it acts as a chaperone of α-Hb by forming a stable complex which prevents the aggregation of excess α-Hb that by precipitation would damage membrane structure and trigger cell death. AHSP knock-out mice exhibit ineffective erythropoiesis with reticulocytosis, abnormal erythrocyte morphology, intracellular inclusion bodies, and increased production of reactive oxygen species (ROS) with subsequent cellular oxidative damage as observed in thalassemias. A similar disorder involving AHSP has not yet been found in humans as a cause of thalasssemia. We report here a first case presenting such a syndrome, for a family originating from SE Asia. Their second child was referred to the hospital one month after birth, because of palor and anemia. Biological tests demonstrated a hemolytic microcytic anemia (Hb 6.9 g/dl, MCV : 73.2 fL and MCH 25 pg) and 3% Hb Bart's detected by CE-HPLC. No abnormality was revealed for the molecular screening for α-thal, including detection of common and rare α locus deletions using Gap-PCR and MLPA analysis, the sequencing of α1 and α2 genes, and the sequencing of the HS-40 core sequence. We considered thus the possibility for an abnormality of a modulator gene; the AHSP gene and its promoter region were sequenced. The proband was found to be homozygous for a mutation in exon 3 (c.167 T>G), while the parents and the first son were heterozygous for it. This mutation changes Val 56 to a Gly: the modified residue is not in direct contact with α-Hb but located in the corner between the three helices of the AHSP molecule and could play some role in the stability of the protein. To demonstrate that this abnormal AHSP could be the cause of the thalassemic syndrome, this molecule was produced as a Glutathione-S-transferase (GST) fusion protein by protein engineering in E.coli using both the pGEX6P-AHSP and the pGEX6P-α-AHSP co-expression vectors after site-directed mutagenesis. The yield of the mutated AHSP was lower than that of the wild type when solubilized suggesting that it was less stable than the normal. The kinetics of protein interaction indicate a factor of 2-3 decrease in the affinity. In addition a 20%-30% decrease in capture within GST micro-column of normal α-globin by the mutated recombinant GST-AHSP was observed. Studying the intragenic polymorphisms of the AHSP gene in this family, we observe that the mutated allele belongs to more weakly expressed “clade B” group and the two normal alleles to the “clade A” group as defined by Lai et al (BJH, 2006, 133, 675-682). Up to now, only 3 mutants have been described in the AHSP gene but none was found to impair the association with normal α-Hb chain. This case is thus the first evidence of an isolated α-thal not linked with the α locus but rather with a mutated AHSP. Heterozygous carriers for this AHSP mutation are clinically normal, but the homozygous patient having only the low expressed unstable AHSP presents a more severe α-thal syndrome than that caused by simple α-gene deletion(s). Disclosures:No relevant conflicts of interest to declare.

Context-Specific KLF-1 Mediated Transcriptional Activation at the Alpha Hemoglobin-Stabilizing Protein and Dematin Promoters in Erythroid Cells.

Abstract 461Krüppel-like factor 1 (KLF1) is essential for erythroid gene expression. Key molecular mechanisms modulated by this transacting factor have been elucidated at the b-globin locus. KLF1 has been associated with recruitment of SWI/SNF and RNA polymerase (PolII) complexes necessary for chromatin remodeling and gene transcription respectively, and for facilitating the apposition of the promoter with the far-upstream locus control region. More recently, KLF1 has been implicated in the regulation of an erythroid-specific gene program unlinked to the b-globin locus. Coordinated expression of these genes, including Alpha Hemoglobin-Stabilizing Protein (AHSP), a factor required for globin tetramer stability, and the red cell membrane protein Dematin, are critical for erythroid ontogeny. To compare the role(s) of KLF1 at these loci, we have used a unique 4-OH-Tamoxifen (4-OHT) inducible erythroid cell line, which facilitates the characterization of the temporal kinetics of KLF1-dependent erythroid gene activation. In preliminary experiments, we observed that KLF1 binding was maximal at the three loci within 60 minutes of 4-OHT induction. AHSP and dematin primary RNA transcripts followed similar kinetics, being maximal at 60-90 minutes post-induction. In contrast, b-globin gene transcription reached a plateau 4-6 hours post-induction. From these observations, we hypothesized that transcriptional activation at AHSP and dematin differs from that observed at the b-globin cluster. Consistent with this hypothesis, we observed significant differences in chromatin remodeling at the three loci. At the b-globin promoter, we observed a small but statistically significant increase in DNaseI sensitivity, a measure of chromatin remodeling, with KLF1 binding. In contrast, we observed a complete loss of DNaseI resistance after KLF1 binding at the AHSP and dematin promoters. Consistent with these findings, we observed a five-fold reduction in histone H3 occupancy at the AHSP and dematin promoters, contrasting with no significant change in occupancy at the b-promoter. Importantly, these differences were not observed in regions 1-5 kb upstream of the promoters. These observations, coupled with similar differences in DNaseI hypersensitivity and histone occupancy in fetal liver erythroblasts from wild type and KLF1-null mice, suggest a profound difference in the mechanisms of chromatin remodeling at KLF1-dependent erythroid gene loci. To explore the potential mechanisms underlying these differences in chromatin accessibility, we examined the kinetics of recruitment of other transacting factors and co-activators to the three loci. We observed similar increases in binding of serine-5 phosphorylated PolII, GATA-1, and p45NF-E2 at the promoters. In contrast, binding of BRG1, the core ATPase component of the SWI/SNF complex differed between the b–promoter and the other erythroid genes. Although BRG1 binding was co-incident with KLF1 binding to the b-gene, we observed significant albeit weak binding of this complex to the AHSP and Dematin promoters only after maximal gene transcription had occurred. Our results suggest that different KLF1 multiprotein complexes are recruited to remodel target gene promoters in vivo. Furthermore, we propose that KLF1's chromatin remodeling capabilities are not limited to the recruitment of the SWI/SNF complexes Disclosures:No relevant conflicts of interest to declare.

292: Alpha hemoglobin stabilizing protein (AHSP) in recurrent miscarriage

MONICA EMANUELLI, PIERGIORGIO STORTONI, DAVIDE SARTINI, MONIA CECATI, ALESSANDRA CORRADETTI, STEFANO RAFFAELE GIANNUBILO, ANDREA TRANQUILLI, Marche Polytechnic University, Biochemical biotechnologies, Ancona, Italy, Marche Polytechnic University, Maternal and Child Sciences, Ancona, Italy OBJECTIVE: Alpha hemoglobin-stabilizing protein (AHSP) inhibits the production of reactive oxygen species in various cells, including erythrocytes. Reduced AHSP indicates reduced protection from oxidative stressors. Our objective was to investigate if and how AHSP varies in recurrent miscarriage. STUDY DESIGN: Forty specimens of trophoblast were collected from women at 10 weeks= gestation. Five were having recurrent miscarriage. Twenty women were experiencing a first spontaneous miscarriage. Those were followed up to their next pregnancy. Five of them experienced a repeated miscarriage. Ten women who had decided for voluntary abortion served as controls. At the end of the collection we had four groups: VA (Voluntary abortion-Controls; n 10); SA (Spontaneous first miscarriage with subsequent normal pregnancy, n 15); SASA (Spontaneous first miscarriage subsequent miscarriage; n 5); RSA (Recurrent miscarriage; not previously investigated n 5); RSAPS (Recurrent miscarriage previously studied, n 5). AHSP mRNA was determined using real time quantitative PCR. All statistical analyses were performed by using the GraphPad Prism Software. Differences were considered significant at p 0.05. RESULTS: All patients had AHSP mRNA levels significantly lower than controls. AHSP mRNA levels were maximally reduced in RSA and RSAPS (8.0E10-6 1.3 and 8.1E10-6 0.7, respectively) when compared to first miscarriage and controls. (16.1E10-6 2.37 and 26.1E10-6 2.7, respectively). Women experiencing first spontaneous miscarriage followed by subsequent repeated miscarriage showed levels significantly reduced, as well (9.0.0E10-6 2.3). CONCLUSION: Recurrent miscarriage is charachterized by a status of reduced defense from oxifdative cell stressors. Such status may be evidenced already at a first miscarriage, thus identifying women who are at high risk for subsequent eventful pregnancy.

AHSP Knockdown in Human Erythroleukemia Cell Line and Human Hematopoietic Stem Cells Results in Alpha Hemoglobin Chain Precipitation, Decreased Hemoglobin Levels and Increased Cell Death.

Alpha Hemoglobin Stabilizing Protein (AHSP) binds alpha hemoglobin chain (αHb), avoiding its precipitation and its pro-oxidant activity. In the presence of beta hemoglobin chain (βHb), the αHb-AHSP complex is dismembered and βHb displaces AHSP to generate the quaternary structure of hemoglobin. These data have been obtained in vitro and in mouse cells, but strongly suggest the importance of AHSP for normal hemoglobin synthesis in humans. To the best of our knowledge, the relationship between hemoglobin formation and alterations in AHSP expression has not yet been described in human red cells. Hence, to investigate the consequences of a reduced AHSP synthesis in human red cells, we established the RNA interference-mediated knockdown of AHSP expression in human erythroleukemia cell line (K562 cells) and human hematopoietic stem cells (CD34+ cells) induced to erythroid differentiation, and analyzed the consequent cellular and molecular aspects of AHSP knockdown in these cells. shRNA expression vectors, aimed at the AHSP mRNA target sequence, were cloned and transfected into K562 and CD34+ cells using a non-liposomal lipid reagent. Following transfection, K562 cells that stably expressed AHSP-shRNA and CD34+ cells that transiently expressed AHSP-shRNA were selected. K562 and CD34+ cells were stimulated to erythroid differentiation by hemin and erythropoietin (EPO) respectively. The cells were examined in terms of gene expression using quantitative real-time PCR; production of reactive oxygen species (ROS), apoptosis and hemoglobin production through flow cytometry assays; and immunofluorescence assays for globin chains. AHSP-shRNA hemin-induced K562 cells and AHSP-shRNA EPO-induced CD34+ cells presented 71% and 75% decreases in AHSP expression levels, respectively. The RNAi-mediated knockdown of AHSP expression resulted in a considerable αHb precipitation, as well as in a significant decrease in fetal hemoglobin formation. In addition, AHSP-knockdown cells demonstrated an increased ROS production and increased rate of apoptosis. These findings strengthen the hypothesis that AHSP stabilizes the alpha hemoglobin chain, avoiding its precipitation and its ability to generate ROS which implicate in cell death. Moreover, data indicate that AHSP may be highly significant for human hemoglobin formation and suggest that AHSP is a key chaperone protein during human erythropoiesis.

Identification of Two New Synthetic Histone Deacetylase Inhibitors That Modulate Globin Gene Expression in Erythroid Cells from Healthy Donors and Patients with Thalassemia

We have identified two new histone deacetylase (HDAC) inhibitors (9 and 24) capable of inducing the expression of gamma-globin and/or beta-globin promoter-driven reporter genes in a synthetic model of Hb switch. Both compounds also increased, with different mechanisms, the gamma/(gamma+beta) ratio expressed in vitro by normal human erythroblasts. Compound 9 increased the levels of gamma-globin mRNA and the gamma/(gamma+beta) ratio (both by 2-fold). Compound 24 increased by 3-fold the level of gamma-globin and decreased by 2-fold that of beta-globin mRNA, increasing the gamma/(gamma+beta) ratio by 6-fold, and raising (by 50%) the cell HbF content. Both compounds raised the acetylation state of histone H4 in primary cells, an indication that their activity was mediated through HDAC inhibition. Compounds 9 and 24 were also tested as gamma/(gamma+beta) mRNA inducers in erythroblasts obtained from patients with beta(0) thalassemia. Progenitor cells from patients with beta(0) thalassemia generated in vitro morphologically normal proerythroblasts that, unlike normal cells, failed to mature in the presence of EPO and expressed low beta-globin levels but 10 times higher-than-normal levels of the alpha hemoglobin-stabilizing protein (AHSP) mRNA. Both compounds ameliorated the impaired in vitro maturation in beta(0) thalassemic erythroblasts, decreasing AHSP expression to normal levels. In the case of two patients (of five analyzed), the improved erythroblast maturation was associated with detectable increases in the gamma/(gamma+beta) mRNA ratio. The low toxicity exerted by compounds 9 and 24 in all of the assays investigated suggests that these new HDAC inhibitors should be considered for personalized therapy of selected patients with beta(0) thalassemia.

Role of the alpha hemoglobin-stabilizing protein in HELLP syndrome, intrauterine growth restriction and fetal death

Role of the alpha hemoglobin-stabilizing protein in HELLP syndrome, intrauterine growth restriction and fetal death

AHSP (Alpha Hemoglobin Stabilizing Protein) Gene Expression during Normal and β-Thalassemic Erythroid Differentiation.

β-thalassemia severity is stricktly related to the amount of free a-chains depending at least in part from different b-thalassemic genotype (β0 /β0, β0/β+, β+ /β+). Recently a new erythroid-specific protein, named AHSP (Alpha Hemoglobin Stabilizing Protein), able to form a stable complex with free α-hemoglobin, has been identified. This protein probably acts as a molecular chaperone to prevent the harmful aggregation and precipitation of α-globin that occurs during normal erythropoiesis and to a greater extent in different β-thalassemias. The aim of this study was to evaluate if the AHSP expression in erythroid cells depends on free α-chains amount. In order to verify this hypothesis, we studied the relative expression of AHSP and globin genes during differentiation of erythroid progenitors derived from peripheral blood of both normal subjects and β-thalassemic patients with different genotypes. Erythroid cultures were set up accordingly to the two phases liquid colture method described by Fibach. Total cytoplasmatic mRNA was extracted by the method of Chomczynsky-Sacchi and quantitative real-time PCR was performed using SYBRGreen to evaluate AHSP, α, β and γ globin genes expression. To quantify gene expression a mathematical model using calibration data (2-ΔΔct) was used and quantification of the house-keeping gene S14 was the internal control. In normal subjects AHSP expression was first detected at proerythroblast stage (on day 3 of culture), peaked at orthocromatic stage (on day 9) and declined to low levels by polychromatophilic-reticulocytes stage (day 13) (a decrease of 65% vs day 9 expression). Globin genes expression reached a steady state (a/non-a ratio value of 0.78 very near to a clinical equilibrium) on day 13. In this situation few free α-globin are detectable explaning the lower need of AHSP expression. In β-thalassemic subjects we observed a persistance of globin gene unbalance with an excess of free α-chains also in the advanced differentiation steps: interestingly the worst was the genotype, the highest was the globin ratio and higher was AHSP expression. In β+/β+ thalassemic patients AHSP expression declined on day 13 (a decrease of 15% vs day 9 expression) and globin gene ratio was 2.4, expression of a persistance of free α-chains excess. In β0 /β+ patients globin gene ratio on day 13 was 2.8 and AHSP expression increased of 2.5 times. Finally in β0/β0 patients globin gene ratio was very high with values up to 9–10; AHSP expression profiles instead were different between different subiects and related not only to globin unbalance but also to ineffective erythropoiesis rate and γ-globin chains persistance. All these findings underline an evident relation between AHSP and globin genes expression, suggesting a probable function of this protein during erythropoiesis.

Regulation of Alpha Hemoglobin Stabilizing Protein (AHSP) mRNA Stability by a 3′ UTR Iron Response Element.

AHSP is an erythroid protein that binds the alpha globin subunit of hemoglobin (Hb) to maintain its structure and limit its oxidative activity. Biochemical and genetic studies demonstrate that AHSP is essential for normal Hb homeostasis and may act via at least two mechanisms: First, as a molecular chaperone to promote the folding and stability of alpha globin prior to its incorporation into HbA (alpha2beta2). Second, to bind and detoxify excess alpha globin that accumulates in normal erythroid precursors, and to a larger extent, in beta thalassemic ones. The existence of a potential iron responsive element (IRE) in the 3′-UTR of the AHSP mRNA raises the interesting possibility that iron homeostasis impacts on alpha globin stability via AHSP. IREs form stem-loop structures that bind cytosolic iron-sensing proteins (IRPs). Typically, IRP binding to the 3′ UTR stabilizes mRNA. Association with iron releases the IRP, enhancing mRNA degradation. Computational algorithms identified IRE-like stem-loop structures in AHSP mRNA of multiple species, yet the primary sequences deviate significantly from canonical IRE consensus sequences determined by studies of classical IREs, such as Transferrin receptor and Ferritin. Several lines of evidence now show that the AHSP IRE binds IRPs to regulate mRNA stability in an iron-dependent fashion:1) in vitro gelshift competition assays demonstrate that human and mouse AHSP IREs bind tightly to IRPs.2) AHSP mRNA co-immunoprecipitates with IRPs in erythroid cells in a fashion that depends on an intact IRE.3) AHSP mRNA is destabilized by iron in both erythroid and heterologous cells; disruption of the IRE renders the mRNA constitutively unstable.To study how iron regulates AHSP expression in vivo, we treated mice with iron dextran for 10 days and then examined AHSP mRNA in Ter119+ erythroid progenitors by RT-PCR. We found that short-term iron overload reduced AHSP mRNA levels by about 50% (p > 0.05). Our findings indicate that AHSP mRNA stability is regulated by iron via an atypical 3′-UTR IRE. These findings extend the potential repertoire for functional IREs that do not conform to previously defined canonical consensus sequences. In addition, they provide a potential mechanism by which erythroid cells can regulate globin stability according to iron status. For example, induction of AHSP during iron deficiency might stabilize apo- alpha globin that could accumulate from lack of heme. Conversely, iron overload could destabilize alpha globin by reducing AHSP levels. As such, iron overload, which occurs in patients with beta thalassemia, might aggravate the disease by further elevating the levels of toxic free alpha globin.

Alpha Hemoglobin Stabilizing Protein (AHSP) Optimizes Hemoglobin A Synthesis by Maintaining a Pool of Viable Alpha Globin Subunits.

AHSP binds alpha hemoglobin (Hb) to maintain its structure and limit its prooxidant activities. In addition, AHSP binds and stabilizes apo-alpha globin, which lacks heme. Previously, we demonstrated that Ahsp−/− mice exhibit hemolytic anemia with Hb precipitation in erythroid cells. Through interbreeding of mutant strains, we also showed that loss of AHSP exacerbates beta thalassemia. Together, these studies indicate that AHSP participates in Hb homeostasis and may act to neutralize potentially toxic excess alpha globin that is known to accumulate in normal erythroid precursors, and to a greater extent, in beta thalassemic ones. However, additional functions for AHSP may exist. In particular, AHSP-alpha globin complexes may also promote HbA synthesis. To test this, we depleted the pool of excess alpha globin in Ahsp−/− mice by interbreeding with alpha thalassemic ones. Compared to mice with either mutation alone, compound mutants missing both AHSP and one alpha globin allele (genotype Ahsp−/− //alpha globin*α/αα) exhibited more severe erythroid defects, including worsened anemia, hypochromia, Hb instability and ineffective erythropoiesis. Pulse-labeling of double-mutant reticulocytes showed that alpha to beta globin synthetic ratios were unaffected by loss of AHSP, but precipitation of both alpha and beta nascent chains into cell membranes was strongly enhanced. These data indicate that AHSP is important for erythropoiesis and Hb production even when alpha globin is not produced in excess. In vitro translation studies using wheat germ extracts showed that recombinant AHSP present during the synthesis of alpha globin improved its ability to become incorporated into HbA. Moreover, AHSP conferred protease resistance to nascent alpha globin, suggesting enhanced folding into the native state. Further supporting this interpretation, circular dichroism studies showed that AHSP accelerated refolding of purified denatured alpha Hb. Finally, through pulse labeling of reticulocytes followed by isoelectric focusing of soluble cytosolic fractions, we identified transient pools of free alpha Hb and AHSP-alpha Hb in vivo and showed that Ahsp gene ablation fully depleted both pools. Together, our studies indicate that AHSP acts as a molecular chaperone to promote alpha globin folding and stability prior to its incorporation into HbA. In addition, it is possible that alterations in AHSP gene function or expression could modulate alpha thalassemia severity in patients.

Biochemical Fates of α Hemoglobin Bound to α Hemoglobin-stabilizing Protein AHSP

Alpha hemoglobin-stabilizing protein (AHSP) is an erythroid protein that binds free alpha hemoglobin (alphaHb) to maintain its structure and limit its pro-oxidant activity. Prior studies have defined two different alphaHb.AHSP complexes. Binding of AHSP to Fe(II) alphaHb induces an unusual configuration in which the F helix of the globin becomes disordered and the heme ring becomes solvent-exposed. Over time, this intermediate oxidizes to form a stable hemichrome in which the proximal (F8) and distal (E7) histidines are coordinated to the heme iron atom. The addition of betaHb to either Fe(II) or Fe(III) alphaHb.AHSP displaces AHSP to generate tetrameric (alpha(2)beta(2)) HbA species. The biochemical properties and in vivo significance of the two alphaHb.AHSP complexes are poorly understood. Here we show that Fe(III) alphaHb.AHSP forms from auto-oxidation of oxygenated alphaHb bound to AHSP and that this process is greatly accelerated at physiologic temperature and oxygen pressures. In contrast to free Fe(III) alphaHb hemichromes, AHSP-bound Fe(III) alphaHb does not precipitate and can be recycled into functional HbA. This requires enzymatic reduction of AHSP-bound alphaHb, either prior to or after extraction by beta subunits. In contrast, reaction of Fe(II) alphaHb-AHSP with betaHb generates functional HbA directly. Our findings support a model in which AHSP can either stabilize alphaHb transiently en route to HbA formation during normal erythropoiesis or convert excessive free alphaHb into a more chemically inert state from which recovery of alphaHb is possible by redox cycling.

Alterations in Expression and Chromatin Configuration of the Alpha Hemoglobin-Stabilizing Protein Gene in Erythroid Krüppel-Like Factor-Deficient Mice

Erythroid Krüppel-like factor (EKLF) is an erythroid zinc finger protein identified by its interaction with a CACCC sequence in the beta-globin promoter, where it establishes local chromatin structure permitting beta-globin gene transcription. We sought to identify other EKLF target genes and determine the chromatin status of these genes in the presence and absence of EKLF. We identified alpha hemoglobin-stabilizing protein (AHSP) by subtractive hybridization and demonstrated a 95 to 99.9% reduction in AHSP mRNA and the absence of AHSP in EKLF-deficient cells. Chromatin at the AHSP promoter from EKLF-deficient cells lacked a DNase I hypersensitive site and exhibited histone hypoacetylation across the locus compared to hyperacetylation of wild-type chromatin. Wild-type chromatin demonstrated a peak of EKLF binding over a promoter region CACCC box that differs from the EKLF consensus by a nucleotide. In mobility shift assays, the AHSP promoter CACCC site bound EKLF in a manner comparable to the beta-globin promoter CACCC site, indicating a broader recognition sequence for the EKLF consensus binding site. The AHSP promoter was transactivated by EKLF in K562 cells, which lack EKLF. These results support the hypothesis that EKLF acts as a transcription factor and a chromatin modulator for the AHSP and beta-globin genes and indicate that EKLF may play similar roles for other erythroid genes.

Genomic Organization, Chromatin Structure, and Transcriptional Regulation of the Murine Alpha Hemoglobin Stabilizing Protein (AHSP) Gene.

Alpha Hemoglobin Stabilizing Protein (AHSP, Eraf) is an abundant erythroid protein that binds and stabilizes alpha globin and alpha hemoglobin (Hb). In mice, loss of AHSP causes hemolytic anemia, with elevated levels of reactive oxygen species and Hb precipitation in erythrocytes. Loss of AHSP exacerbates beta thalassemia phenotypes in mice, presumably by enhancing the toxicity of excessive free alpha Hb. Based on these findings, AHSP is a candidate modifier gene for beta thalassemia in humans. No mutations in the AHSP coding region have been identified in patients to date. However, several groups reported an inverse correlation between beta thalassemia severity and erythroid AHSP expression levels, raising the possibility that AHSP is a quantitative trait modifier of beta thalassemia. To address this possibility, it is important to define the mechanisms that control expression of the AHSP gene. Transcripts of murine Ahsp are inducible by GATA-1. The goals of the current studies are to investigate the mechanisms of this induction and to define the DNA domain that regulates the locus. Using phylogenetic comparisons, we identified a hotspot for mammalian chromosomal rearrangement just downstream of the Ahsp gene. This hotspot is located at the end of a syntenic block of approximately 350 kb that is conserved in mammals and likely marks the 3′ end of the gene regulatory domain. We focused our initial functional studies on a 7 kb genomic region bounded at the 5′ (centromeric) end of Ahsp by the nearest adjacent gene, an EST expressed in multiple tissues, and at the 3′ (telomeric) end by the rearrangement hotspot. In transient transfection assays, the Ahsp promoter region conferred erythroid-specific expression to a linked reporter gene. In heterologous cells, GATA-1 transactivated the Ahsp promoter in a dose-dependent fashion. To examine GATA-1 binding and its subsequent effects on the Ahsp gene in vivo, we used G1E-ER4 cells, a GATA-1 null erythroblast line that undergoes terminal erythroid maturation after activation of an estradiol-inducible form of GATA-1. We made several findings with regards to the role of GATA-1 in Ahsp gene regulation. First, GATA-1 and its cofactor, Friend of GATA-1 (FOG-1), bind directly to the Ahsp locus at regions that contain conserved GATA consensus motifs and are predicted to be important erythroid regulatory elements by our bioinformatic studies. Second, GATA-1 induces epigenetic changes in chromatin structure that are associated with gene activation, including formation of a DNase I hypersensitive site, hyperacetylation of histones H3 and H4, and methylation of histone H3 lysine-4. Together, these findings begin to establish the DNA region and mechanisms that control Ahsp transcription, allowing for further studies to map the cis elements responsible for population variations in gene expression.

A Stem-Loop in the 3′-UTR Mediates Iron-Dependent Regulation of Alpha Hemoglobin Stabilizing Protein mRNA Stability.

Alpha-hemoglobin stabilizing protein (AHSP) acts as a molecular chaperone that binds and stabilizes alpha globin, minimizing the formation of cytotoxic alpha-globin precipitates in red blood cells. It has been proposed that variations in AHSP protein levels could influence disease severity in patients with beta-thalassemia. A number of genomic sequences that could be involved with AHSP transcriptional regulation have been studied. Multiple consensus GATA-1 binding motifs and a single CACCC-binding site have been identified and characterized. In vivo studies demonstrated that AHSP gene transcription is dependent on both EKLF and GATA-1 transcription factors. Here, we identify an IRE (iron response-like element) in the AHSP 3′ untranslated region that is predicted to regulate mRNA stability. IREs are regulatory elements that encode a phylogenetically defined sequence-structure motif that provide a specific recognization site for the IRE-binding proteins (IRE-BP1 or IRE-BP2). Iron starvation of cells induces high affinity binding of cytoplasmic IRE-BP to an IRE, which has at least two distinct known biological consequences. IRE in the 5′-UTR of an mRNA provides for iron-dependent regulation of translation, whereas an IRE in the 3′-UTR confers iron-dependent regulation of mRNA stability. Computer modeling identified a potential IRE stem-loop structure near the 3′-end of murine and human AHSP mRNAs. In vitro binding studies indicate that this structure recognizes an IRE-BP. Moreover, the affinity of this interaction was reduced by mutations that disrupt base pairing within the stem-loop. Iron supplementation, which is predicted to decrease IRE-BP affinity for the IRE, destabilizes AHSP in human and mouse erythroleukemia cells. These results indicate that AHSP expression is regulated by RNA stability in an iron-dependent fashion. Our findings have implications for the control of AHSP expression in beta-thalassemia.