Hemoglobin Alpha Research Articles

Multiple functions for alpha hemoglobin stabilizing protein (AHSP) in hemoglobin synthesis and homeostasis

Multiple functions for alpha hemoglobin stabilizing protein (AHSP) in hemoglobin synthesis and homeostasis

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.

The Role of EHZF in Erythroid Differentiation of K562 Cells.

Early hematopoietic zinc finger protein (EHZF), the human homolog to mouse Evi3, is a transcription factor with 30 zinc fingers and a highly conserved N-terminal FOG repression motif. The EHZF mRNA expression is abundant in early hematopoietic progenitors and declines during differentiation. In most of the leukemic cells from acute myelogenous leukemia patients, significant levels of EHZF mRNA are detected. These findings suggest that EHZF play an important role in hematopoietic differentiation.In the present study we investigated whether siRNA-mediated depletion of EHZF affected the erythroid differentiation of K562 cells. Three kind of EHZF siRNA were designed and their expression vectors were constructed. After transient expression of these siRNAs by electroporation, hemoglobinization of K562 cells were analyzed by Benzidine staining. In all of these siRNAs introduced cells, the levels of hemoglobinizations were abundant compared to control cells, which suggested that EHZF might influence the erythroid differentiation of K562 cells. To further analyze the effects of EHZF siRNA in erythroid differentiation, we established K562 clones in which EHZF was depleted by siRNA. EHZF siRNA expression vectors were introduced into K562 cells by electroporation and the expression of EHZF mRNA were examined by RT-PCR in G418 resistant clones. The depressions of EHZF mRNA in the clones were confirmed by Northern blotting. We isolated several EHZF depleted clones in each kind of EHZF siRNA introduced cells. Most of the EHZF depleted clones showed marked hemoglobinization by Benzidine staining compared to control clones, and surface expressions of glycophorin A were also increased in these clones. These results confirmed the relevant role of EHZF in erythroid differentiation of K562 cells. Comparison of mRNA expressions between an EHZF depleted clone and a control clone using DNA array showed increase expressions of hemoglobin alpha, beta, delta mRNA in EHZF depleted clone compared to control clone. Several transcription factors which are involved in erythroid differentiation also showed the difference mRNA levels between two clones.Studies are underway to elucidate the mechanisms underlying the role of EHZF in erythroid differentiation, the relationship to other transcription factors (GATA-1, GFi-1B, FOG-1 etc) which involved in erythroid differentiation.

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.

AHSP Promoter Homopolymer in Beta-Thalassemia Syndromes.

Alpha hemoglobin stabilizing protein (AHSP) is an erythroid protein that binds specifically to the alpha globin chains to form a stable complex, preventing their precipitation before the assembly of the hemoglobin tetramere. Studies have shown thatAHSP is required for normal erythropoiesis,knock-out mice has a phenotype similar to beta thalassemia,loss of AHSP worsens the severity of the disease in thalassemic mice.Therefore, altered AHSP expression or function could account for some of the variability in beta-thalassemia phenotype. At position -160bp upstream of exon 1 of the AHSP gene, two main homopolymers (T18 and T15) have been reported that may influence the AHSP expression and hence the hematological phenotype. In particular the frequence of the shorter T15 homopolymer, which is associated with a lower AHSP expression, was much higher than expected (on the basis of the allele frequency in the healthy population) in a group of 9 patients with thalassemia intermedia, due the compound heterozygosity for beta-thalassemia and the triple alpha globin gene (Mei et al, 2006). We have studied the frequency of T15 and T18 homolpolymers in a group of 20 normal subjects and in patients with different thalassemia syndromes, i.e. beta zero 39 homozygotes with thalassemia major (14 patients) and intermedia (19), compound heterozygotes for beta-thalassemia and triple alpha globin gene (13) and simple carriers for beta thalassemia with thalassemia intermedia phenotype (5). We found the following genotype distribution reported in figure1. There is no difference in the frequency of the genotypes among the groups, except in the severe beta-thalassemia carriers, where the short T15 homopolymer is more common. These findings are consistent with the reduced AHSP expression previously reported in the reticulocytes and in the erythroid cultures of these unusually severe beta thalassemia carriers. (Galanello et al, abs 1881. ASH 2003). Therefore, only in the peculiar group of beta-thalassemia carriers with the clinical features of thalassemia intermedia, the AHSP genotype appears to be a relevant contributory factor in the hematological phenotype. [Display omitted]

Genomic organisation and regulation of murine alpha haemoglobin stabilising protein by erythroid Kruppel‐like factor

Alpha haemoglobin stabilising protein (AHSP) binds free alpha-globin chains and plays an important role in the protection of red cells, particularly during beta-thalassaemia. Murine ASHP was discovered as a GATA-1 target gene and human AHSP is directly regulated by GATA-1. More recently, AHSP was rediscovered as a highly erythroid Kruppel-like factor (EKLF) -dependent transcript. We have determined the organisation of the murine AHSP gene and compared it to orthologs. There are two CACC box elements in the proximal promoter. The proximal element is absolutely conserved, but does not bind EKLF as it is not a canonical binding site. In rodents, the distal element contains a 3 bp insertion that disrupts the typical EKLF binding consensus region. Nevertheless, EKLF binds this atypical site by gel mobility shift assay, specifically occupies the AHSP promoter in vivo in a chromatin immunoprecipitation assay, and transactivates AHSP through this CACC site in promoter-reporter assays. Our results suggest EKLF can occupy CACC elements in vivo that are not predictable from the consensus binding site inferred from structural studies. We also propose that absence of AHSP in EKLF-null red cells exacerbates the toxicity of free alpha-globin chains, which exist because of the defect in beta-globin gene activation.

Impaired binding of AHSP to α chain variants: Hb Groene Hart illustrates a mechanism leading to unstable hemoglobins with α thalassemic like syndrome

Alpha hemoglobin stabilizing protein (AHSP) is a small protein of 102 residues induced by GATA-1, Oct-1- and EKLF. It is synthesized at a high level in the red blood cell precursors and acts as a chaperone protecting the alpha hemoglobin (α-Hb) chains against precipitation. AHSP and α-Hb form a heterodimer complex. In the absence of AHSP, α-Hb oxidizes and precipitates within the erythrocyte precursors of the bone marrow leading to apoptosis and defective erythropoiesis. In vitro the binding of AHSP to ferrous α-Hb accelerates oxidation of the heme iron in α-Hb, but the complex is more resistant to protein unfolding. AHSP could act as a modulating factor in beta-thalassemia. Recent studies showed more severe thalassemic syndromes in patients with decreased levels of AHSP and in one patient who carried a structurally abnormal AHSP. Some α-Hb variants with structural abnormality located in the contact area between α-Hb and AHSP exhibit an instability and a thalassemic like syndrome. We suggest that this could result from a disturbed interaction between α-Hb variants and AHSP. To study this interaction, we constructed the pGEX-α-AHSP vector that co-expressed human α-Hb and AHSP. Using this approach, we investigated the α42 (C7), α104 (G11) and α119 (H2) sites, where variants with some thalassemic features have been described. Results obtained with recombinant Groene Hart α-Hb and Diamant α-Hb, in which proline 119 is replaced by a serine and a leucine, respectively, showed clearly an impaired interaction with AHSP. In contrast, the α mutants at the sites 42 and 104 exhibit a normal interaction with AHSP. The CO rebinding kinetics of the AHSP/α-Hb 42mutant complexes were similar to those previously obtained with the AHSP/α-Hb WT complex, which shows a modified rate that is intermediate to the classical Hb allosteric states.

Arsenic-induced bladder cancer in an animal model

Dimethylarsinic acid (DMA V) is carcinogenic to the rat urinary bladder, but not in mice. The carcinogenic mode of action involves cytotoxicity followed by regenerative cell proliferation. Dietary DMA V does not produce urinary solids or significant alterations in urinary composition. The cytotoxicity is due to formation of a reactive metabolite, likely dimethylarsinous acid (DMA III), concentrated and excreted in the urine. Urinary concentrations of DMA III are dose-dependent, and the urinary concentrations are at cytotoxic levels based on in vitro studies. The no observed effect level (NOEL) in these rat dietary studies for detectable levels of DMA III, cytotoxicity, and proliferation is 2 ppm, with marginal changes at 10 ppm. The tumorigenic dose is 100 ppm. Recent investigations have demonstrated that arsenicals administered to the rat result in binding to a specific cysteine in the hemoglobin alpha chain as DMA III, regardless of the arsenical being administered. Monomethylarsonic acid (MMA V) is not carcinogenic in rats or mice. In short term experiments (≤ 10 weeks), sodium arsenate in the drinking water induces significant cytotoxicity and regenerative proliferation. There is little evidence that the cytotoxicity produced following administration of arsenicals is caused by oxidative damage, as antioxidants show little inhibitory activity of the cytotoxicity of the various arsenicals either in vitro or in vivo. In summary, the mode of action for DMA V-induced bladder carcinogenesis in the rat involves generation of a reactive metabolite (DMA III) leading to cytotoxicity and regenerative proliferation, is a non-linear process, and likely involves a threshold. Extrapolation to human risk needs to take this into account along with the significant differences in toxicokinetics and toxicodynamics that occur between different species.

Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR

BackgroundControl genes, which are often referred to as housekeeping genes, are frequently used to normalise mRNA levels between different samples. However, the expression level of these genes may vary among tissues or cells and may change under certain circumstances. Thus, the selection of housekeeping genes is critical for gene expression studies. To address this issue, 7 candidate housekeeping genes including several commonly used ones were investigated in isolated human reticulocytes. For this, a simple ΔCt approach was employed by comparing relative expression of 'pairs of genes' within each sample. On this basis, stability of the candidate housekeeping genes was ranked according to repeatability of the gene expression differences among 31 samples.ResultsInitial screening of the expression pattern demonstrated that 1 of the 7 genes was expressed at very low levels in reticulocytes and was excluded from further analysis. The range of expression stability of the other 6 genes was (from most stable to least stable): GAPDH (glyceraldehyde 3-phosphate dehydrogenase), SDHA (succinate dehydrogenase), HPRT1 (hypoxanthine phosphoribosyl transferase 1), HBS1L (HBS1-like protein) and AHSP (alpha haemoglobin stabilising protein), followed by B2M (beta-2-microglobulin).ConclusionUsing this simple approach, GAPDH was found to be the most suitable housekeeping gene for expression studies in reticulocytes while the commonly used B2M should be avoided.

Multiple microarray platforms utilized for hepatic gene expression profiling of GH transgenic coho salmon with and without ration restriction

The objectives of this study are to examine hepatic gene expression changes caused by GH transgenesis and enhanced growth. This is the first use of cDNA microarrays to study the influence of GH transgenesis on liver gene expression in a non-mammalian vertebrate, and the first such study using sexually immature animals. Three groups of coho salmon were examined: GH transgenic on full ration (T), GH transgenic on restricted ration (R), and control non-transgenic (C). Specific growth rates for weight in T were approximately eightfold higher than in C, and fourfold higher than in R. Differential gene expression in T, R, and C samples was determined using approximately 3500 and 16,000 gene microarrays, and R and C samples were compared on a different approximately 4000 gene microarray. The use of multiple microarray platforms increased the overall proportion of the hepatic transcriptome considered in these studies. Cross-platform comparisons identified genes behaving similarly between studies. For example, genes encoding a precerebellin-like protein and complement component C3 were downregulated in R relative to C (R < C) in two microarray studies, and hemoglobins alpha and beta were R > C in all three studies. Comparisons of informative gene lists within and between studies inferred causes of altered gene expression. For example, ten genes, including 78 kDa glucose-regulated protein, glycerol-3-phosphate dehydrogenase, hemoglobins alpha and beta, and a C-type lectin, were likely induced by GH transgenesis due to their presence in both T > C and R > C gene lists. Eleven genes, including hepcidin, nuclear protein p8, precerebellin-like, transketolase, and fatty acid-binding protein, were present in both T < C and R < C gene lists and were, therefore, likely suppressed by GH transgenesis. A large number of salmonid genes identified in these studies are involved in iron homeostasis, mitochondrial function, carbohydrate metabolism, cellular proliferation, and innate immunity. Pentose phosphate pathway genes phosphogluconate dehydrogenase, transaldolase, and transketolase, were dysregulated in GH transgenic samples relative to control samples. Changes in the expression of genes involved in maintaining hemoglobin levels (heme oxygenase, hemoglobins alpha and beta, Kruppel-like globin gene activator, hepcidin) in R and T fish indicate a need for additional hemoglobin in the transgenic fish, perhaps due to higher metabolic rate required for enhanced growth.

Focused Proteomics in Post-Mortem Human Spinal Cord

With a highly sensitive electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS) system, proteins were identified in minimal amounts of spinal cord from patients with the neurodegenerative disease amyotrophic lateral sclerosis (ALS) and compared to proteins in spinal cord from control subjects. The results show 18 versus 16 significantly identified (p < 0.05) proteins, respectively, all known to be found in the central nervous system. The most abundant protein in both groups was the glial fibrillary acidic protein, GFAP. Other proteins were, for example, hemoglobin alpha- and beta chain, myelin basic protein, thioredoxin, alpha enolase, and choline acetyltransferase. This study also includes the technique of laser microdissection in combination with pressure catapulting (LMPC) for the dissection of samples and specific neurons. Furthermore, complementary experiments with nanoLC-matrix assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF MS) confirmed the results of the ESI-FTICR MS screening and provided additional results of further identified proteins.

Gene expression profiles in Finnish twins with multiple sclerosis

BackgroundSince genetic alterations influencing susceptibility to multiple sclerosis (MS), the most common autoimmune demyelinating disease of the central nervous system (CNS), are as yet poorly understood, the purpose of this study was to identify genes responsible for MS by studying monozygotic (MZ) twin pairs discordant for MS.MethodsIn order to identify genes involved in MS development, the gene expression profiles in blood mononuclear cells obtained from eight MZ twin pairs discordant for MS were analyzed by cDNA microarray technology detecting the expression of 8 300 genes. The twins were collected from the Finnish Twin Cohort Study and both affected subjects and their healthy siblings underwent neurological evaluation and cerebral and spinal magnetic resonance imaging. Gene expressions were confirmed by relative quantitative reverse transcription PCR.ResultsIt appeared that 25 genes were at least two-fold up-regulated and 15 genes down-regulated in 25% (2/8) of twins with MS when compared to their healthy siblings. Moreover, 6/25 genes were up-regulated in 40% of MS twins and one gene, interferon alpha-inducible protein (clone IFI-6-16) (G1P3), in 50% of them. The six most constantly expressed genes are (1) G1P3, (2) POU domain, class 3, transcription factor 1, (3) myxovirus resistance 2, (4) lysosomal-associated multispanning membrane protein-5, (5) hemoglobin alpha 2 and (6) hemoglobin beta.ConclusionOver two-fold up-regulation of these six genes in almost half of MZ twins with MS suggests their role in MS pathogenesis. Studies using MZ MS twins obtained from genetically homogeneous population offer a unique opportunity to explore the genetic nature of MS.

A Natural Variant Sequence in the AHSP Gene May Impact Severity of Beta-Thalassemia.

Alpha-hemoglobin stabilizing protein (AHSP) is an erythroid-specific molecular chaperone that binds the alpha chains of hemoglobin, preventing their precipitation and deleterious effects. Loss of AHSP exacerbates alpha globin precipitation and anemia in a murine model for beta thalassemia. In vitro, recombinant AHSP inhibits the production of reactive oxygen species (ROS) by alpha hemoglobin. To further define the role of AHSP as a modifier of beta-thalassemia, we analyzed AHSP sequences for mutations in a large population of beta-thalassemic and control subjects. A single nucleotide change, from A to T at position 12888 (Genebank accession number AC106.730.2) in the third exon of the AHSP gene was identified, converting asparagine 75 to leucine (N75I). We detected a patient who was heterozygous for both beta-thalassemia (b39/bA) and the AHSP-N75I mutation. She presented with an unusually severe anemia that required regular blood transfusion. The father is hematologically normal, without any thalassemia mutation and heterozygous for AHSP-N751. The mother is heterozygous for the beta 39 mutation with classical mild hypochromic microcytic anemia and no mutation in the AHSP gene. Of 282 unrelated control subjects tested, one (0.35%) contained the N75I mutation. Analysis of red blood cells from this subject revealed normal hemoglobin indices but a small number of Heinz bodies, suggesting hemoglobin precipitation. This finding is similar to what we observed previously in mice that are heterozygous for an AHSP null allele. To investigate how the N75I mutation affects AHSP function, we studied the biochemical properties of the recombinant mutant protein. Surface plasmon resonance showed that the binding affinity of AHSP N75I for alpha hemoglobin is normal. Importantly, compared to wild type AHSP, the N75I mutant protein exhibited significantly reduced capacity to inhibit ROS production by alpha hemoglobin. Hence, AHSP N75I may be less effective at conferring protection from oxidative-mediated damage by free alpha hemoglobin in erythrocytes. These effects, when coupled with beta thalassemia, could result in more severe anemia, implicating AHSP N75I as a potential genetic modifier.

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.

The Role of Alpha Hemoglobin Stabilizing Protein (AHSP) in the Formation of Hemoglobin A.

Alpha hemoglobin stabilizing protein (AHSP) binds α hemoglobin (Hb) to stabilize its structure and limit its chemical reactivity in vitro and in vivo. Addition of β Hb to α Hb-AHSP displaces AHSP to form HbA (α2β2) tetramer. Recently, we determined that binding of AHSP to free α Hb generates a novel structure in which the helix F of α Hb is disordered and the heme iron is reduced to the ferrous form and coordinated by the distal histidine (ferrous α Hb-AHSP). Over time, the heme iron within this complex oxidizes to the ferric form (ferric α Hb-AHSP) and the F helix reorders, generating a bis-histidyl heme iron that exhibits decreased capacity to catalyze the formation of reactive oxygen species. At room temperature in ambient oxygen, the conversion of ferrous to ferric α Hb-AHSP occurs over several hours. We proposed that in vivo, these complexes could serve as transient intermediates to maintain α Hb in a stable state until β Hb is available to form tetrameric HbA. The current study addresses two questions related to this hypothesis:Does bis-histidyl ferric α Hb-AHSP form more readily under physiologic conditions?Can ferrous and ferric α Hb-AHSP complexes form functional tetrameric HbA upon reaction with β Hb?The conversion of ferrous to ferric α Hb-AHSP is temperature-dependent with complete conversion occurring in 40 minutes at 37°C, as opposed to 5 hours at 22°C. Furthermore, the conversion is accelerated by partial oxygen saturation, increasing the likelihood that ferric-AHSP Hb will form in vivo. Using cellulose acetate gel electrophoresis, we demonstrated that β Hb displaces AHSP to form tetrameric HbA from both α Hb-AHSP complexes. The resulting tetramer from ferrous α Hb-AHSP is similar to HbA, both spectroscopically and in terms of oxygen binding. In contrast, the Hb tetramer formed from ferric α Hb-AHSP and β Hb is similar to HbY (αFeIII2βFeII2) spectroscopically. However, the α globin heme iron within this tetrameric complex appears to be occluded, as the rate of cyanide binding is reduced. Furthermore, it is resistant to reduction by sodium dithionite or methemoglobin reductase, suggesting that the α Hb retains its bis-histidyl nature. Together, these studies show that both α Hb-AHSP complexes are likely to form under physiological conditions. Upon interaction with β Hb, ferrous α Hb-AHSP forms functional Hb A tetramer. In contrast, ferric α Hb-AHSP is more chemically inert, but may not be able to form viable HbA.

AHSP Is a Quantitative Trait Gene That Modifies the Phenotype of β Thalassemia.

Alpha hemoglobin stabilizing protein (AHSP) was identified in a screen for genes that are activated by the erythroid transcription factor, GATA-1. Studies have shown that:AHSP binds specifically to the α chain of hemoglobin (Hb) but not to β Hb or Hb A;AHSP knock-out mice demonstrated pathological features similar to β thalassemia; andloss of AHSP exacerbates severity of disease in β thalassemia mice.The evidence suggests that AHSP acts as a chaperone for free α Hb and that altered AHSP expression levels could modify the severity of β thalassemia in humans.To assess variation of AHSP expression, mRNA levels were measured in peripheral blood reticulocytes of 103 healthy individuals by quantitative real-time RT-PCR. The sample was approximately 90% (91/103) female with an average age of 52 years (SD=0.14, min=18, max=76). AHSP expression relative to GAPDH, varied up to 3-fold, and did not correlate with age or sex. A systematic survey of the genomic region encompassing the AHSP locus revealed 8 sequence variants of which six were common - five single nucleotide polymorphisms (SNPs), and one homopolymer (Tn) at position 160 bp upstream of exon 1. Four variants (c.-69–237A, c.-69–160 T18, c.-4–27G, and c. 337T) showed strong association with AHSP expression and had nearly equal frequencies. The four variants are in near complete linkage disequilibrium with the minor alleles in coupling. The haplotype consisting of the four minor alleles termed clade B, was associated with relatively lower expression of AHSP. Relative expression of AHSP and AHSP/α globin ratio were both significantly higher (p<0.001) in homozygotes for clade A haplotypes compared to heterozygotes. A potential functional role of one of the variable sites, a T-homopolymer (the T18 variant being part of clade A) in the promoter was investigated in-vitro using luciferase reporter assays in K562 cells. Luciferase activity was 1.30±0.08 times higher in the T18 promoter compared to T14, consistent with genetic studies. We investigated if a shorter homopolymer could have an adverse effect on β thalassemia. Nine patients with thalassemia intermedia and a genotypic combination of heterozygous β thalassemia and 5 α globin genes (aaa/aa) were studied. Based on the allele frequencies in the healthy population, 6–7 of the 9 patients are expected to be homozygous for T18 but only two were observed. In contrast, while 2–3 patients are expected to be heterozygous for the shorter homopolymer, 3 patients were homozygous and 4 heterozygous.In conclusion, AHSP gene expression is variable, with cis control accounting for some of its variance. The subtle altered expression might precipitate a phenotype of thalassemia intermedia in β thalassemia heterozygotes with α globin overload.

Proteomic analysis of differential protein expression in response to epidermal growth factor in neonatal porcine pancreatic cell monolayers

We have proposed that porcine neonatal pancreatic cell clusters (NPCCs) may be a useful alternative source of cells for islet transplantation, and that monolayer cultures might provide an opportunity to manipulate the cells before transplantation. In addition we previously identified 10 genes up-regulated by epidermal growth factor (EGF) in cultured porcine NPCC monolayers. We have now analyzed the intracellular signaling pathways activated by EGF and searched for proteins differentially expressed following EGF treatment of the monolayers, using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). EGF treatment resulted in phosphorylation of both Erk 1/2 and Akt, as well as increased cell proliferation. Five unknown and 13 previously identified proteins were differentially expressed in response to EGF. EGF treatment increased the expression of several structural proteins of epithelial cells, such as cytokeratin 19 and plakoglobin, whereas vimentin, the intermediate filament protein of mesenchymal cells, and non-muscle myosin alkali chain isoform 1, decreased. Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 factor, which promotes epithelial cell proliferation, and hemoglobin alpha I & II also increased, whereas cyclin A1, immunoglobulin heavy chain, apolipoprotein A1, 5,10-ethylenetetrahydrofolated reductase (5,10-MTHFR), angiotensin-converting enzyme 2 (ACE2), co-lipase II precursor, and NAD+ isocitrate dehydrogenase (NAD+ IDH) alpha chain proteins decreased. Our results show that EGF stimulates proliferation of pancreatic epithelial cells by simultaneously activating the MAPK and PI-3K pathways. HnRNP A2/B1, hemoglobin, cyclin A1, and ACE2 may play roles in the proliferation of epithelial cells in response to EGF.

Oral Infections and Glycemic Control in Pregnant Type 2 Diabetics

The relationship between diabetes mellitus and infection is bidirectional. Diabetes favors infection, while infections make controlling diabetes much more difficult. The most frequent infections encountered in pregnant diabetics are those of the urinary tract (UTI) and cervicovaginal (CVI) area. Periodontal diseases (PD) and active caries (AC) are infectious diseases frequently found in these cases often remaining as hidden entities of low intensity. The aim of this study was to assess whether there is an association between PD and caries with lack of glycemic control often encountered in pregnant type 2 diabetic women. A single skilled researcher performed the periodontal evaluation of PD and AC cases. Glycated alpha hemoglobin (HbA(1c)) over 6.5% was used to diagnose metabolic glucose disturbances. The controlled variables were UTI, CVI and adherence to treatment. The statistical tests used were chi-square adjusted for continuity, analysis of variance, odds ratio with 95% confidence intervals and logistic regression with a significance level of p < 0.05. One hundred twenty-seven type 2 diabetic women were seen during the second trimester of their pregnancies. Mean age was 31.3 years; 55% had lack of glycemic control associated with CVI (chi2 21.07, p < 0.000), PD (chi2 5.72, p < 0.005) and UTI (chi2 13.77, p < 0.000) with therapeutic adherence (TA) (chi2 14.80, p < 0.00). No association was found with AC. The logistic regression results showed that UTI, CIV, PD and TA are associated with lack of glycemic control. Periodontal diseases may act as "hidden" infections in pregnant diabetics and be associated with lack of glycemic control. This situation should be taken into consideration by healthcare teams in charge of prenatal care.