Relations structure-fonction de l'alpha-hémoglobine et de sa protéine chaperon l'AHSP (octamères d'hémoglobine recombinante)

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Rôle du chaperon moléculaire de l’alpha-hémoglobine dans la formation de l’hémoglobine et l’expression clinique de certaines hémoglobinopathies

International audienceAlpha-hemoglobin stabilizing protein (AHSP), described as a chaperone of alpha-hemoglobin (α-Hb), is synthesized at a high concentration in the erythroid precursors. AHSP specifically recognizes the G and H helices of α-Hb and forms a stable complex with free α-Hb until its association with the partner β-subunits. Unlike the free β-Hb which are soluble and form homologous tetramers, freshly synthesized α-Hb chains are highly unstable molecular species which precipitate and generate reactive oxygen species within the erythrocyte precursors of the bone marrow leading to apoptosis and ineffective erythropoiesis. AHSP protects the free α-Hb chains in maintaining it in the soluble state. In this review, we report data from the literature and our laboratory concerning the key role of AHSP in the biosynthesis of Hb and its possible involvement in some disorders of the red blood cell as well as the hemoglobinopathies and we discuss its use as a prognostic tool in thalassemia syndromes

α-Haemoglobin pool measurement: a useful biomarker for evaluation of β-thalassaemia intermedia? - response to Huang and Li

International audienceNo abstract availabl

Alpha haemoglobin‐stabilising protein concentration in the red blood cells of patients with sickle cell anaemia with and without hydroxycarbamide treatment

International audienceAlpha haemoglobin-stabilising protein (AHSP) is a key chaperone synthesised in red blood cell (RBC) precursors. Many studies have reported AHSP as a potential biomarker of various diseases. AHSP gene expression has been studied in detail, but little is known about AHSP protein levels in RBCs. We investigated the AHSP concentration of RBC lysates from control subjects (n = 10) and patients with sickle cell anaemia (SCA) with (n = 10) and without (n = 12) hydroxycarbamide (HC) treatment, to evaluate the clinical relevance of AHSP in SCA. We developed a sandwich enzyme-linked immunosorbent assay method, with which we were able, for the first time, to determine the mean AHSP concentration in control RBC lysates (0·82 µg/ml). The AHSP concentration (2·23 µg/ml) was significantly higher in untreated patients with the SS genotype than in controls. The AHSP concentration decreased significantly on HC treatment (1·50 µg/ml) but remained significantly higher than that in controls. A strong positive correlation was observed between the AHSP concentration and the α-haemoglobin pool with the three groups of subjects pooled into a single group. Our present findings indicate that AHSP concentration can be considered a candidate biomarker for monitoring HC responses in patients with SCA and suggest a role for AHSP in various RBC diseases

Interaction de l’hémoglobine octamérique recombinante avec les cellules endothéliales

International audienceHemoglobin-based oxygen carriers (HBOCs) may generate oxidative stress, vasoconstriction and inflammation. To reduce these undesirable vasoactive properties, we increased hemoglobin (Hb) molecular size by genetic engineering with octameric Hb, recombinant (r) HbβG83C. We investigate the potential side effects of rHbβG83C on endothelial cells. The rHbβG83C has no impact on cell viability, and induces a huge repression of endothelial nitric oxide synthase gene transcription, a marker of vasomotion. No induction of Intermolecular-Adhesion Molecule 1 and E-selectin (inflammatory markers) transcription was seen. In the presence of rHbβG83C, the transcription of heme oxygenase-1 (oxidative stress marker) is weakly increased compared to the two other HBOCs (references) or Voluven (control). This genetically engineered octameric Hb, based on a human Hb βG83C mutant, leads to little impact at the level of endothelial cell inflammatory response and thus appears as an interesting molecule for HBOC development.Les transporteurs d’oxygène à base d’hémoglobine (HBOCs) peuvent induire stress oxydant, vasoconstriction et inflammation. Afin de réduire ces propriétés vasoactives indésirables, nous avons augmenté, par génie génétique, la taille moléculaire de l’hémoglobine (Hb) produisant une Hb octamérique recombinante (r), la rHbβG83C. La rHbβG83C n’a pas d’impact sur la viabilité des cellules endothéliales et induit une répression très importante de la transcription du gène de la NO synthase (marqueur de vasoactivité). Aucune induction de la transcription de gènes des molécules d’adhésion ICAM-1 et E-sélectine (marqueurs d’inflammation) n’a été mise en évidence. En présence de rHbβG83C, la transcription du gène de l’hème oxygénase-I (marqueur de stress oxydant) est faiblement augmentée en comparaison du cas des deux autres HBOCs références et au Voluven (témoin). La rHbβG83C, basée sur un mutant de l’Hb humaine, présente moins d’impact au niveau de la réponse inflammatoire des cellules endothéliales et semble donc être une molécule intéressante pour le développement d’un HBOC

Dynamics of α-Hb chain binding to its chaperone AHSP depends on heme coordination and redox state

International audienceBackground: AHSP is an erythroid molecular chaperone of the α-hemoglobin chains (α-Hb). Upon AHSP binding, native ferric α-Hb undergoes an unprecedented structural rearrangement at the heme site giving rise to a 6th coordination bond with His(E7).Methods: Recombinant AHSP, WT α-Hb:AHSP and α-Hb(HE7Q):AHSP complexes were expressed in Escherichia coli. Thermal denaturation curves were measured by circular dichroism for the isolated α-Hb and bound to AHSP. Kinetics of ligand binding and redox reactions of α-Hb bound to AHSP as well as α-Hb release from the α-Hb:AHSP complex were measured by time-resolved absorption spectroscopy.Results: AHSP binding to α-Hb is kinetically controlled to prevail over direct binding with β-chains and is also thermodynamically controlled by the α-Hb redox state and not the liganded state of the ferrous α-Hb. The dramatic instability of isolated ferric α-Hb is greatly decreased upon AHSP binding. Removing the bis-histidyl hexacoordination in α-HbH58(E7)Q:AHSP complex reduces the stabilizing effect of AHSP binding. Once the ferric α-Hb is bound to AHSP, the globin can be more easily reduced by several chemical and enzymatic systems compared to α-Hb within the Hb-tetramer.Conclusion: α-Hb reduction could trigger its release from AHSP toward its final Hb β-chain partner producing functional ferrous Hb-tetramers. This work indicates a preferred kinetic pathway for Hb-synthesis.General significance: The cellular redox balance in Hb-synthesis should be considered as important as the relative proportional synthesis of both Hb-subunits and their heme cofactor. The in vivo role of AHSP is discussed in the context of the molecular disorders observed in thalassemia

Role of α-globin H helix in the building of tetrameric human hemoglobin: interaction with α-hemoglobin stabilizing protein (AHSP) and heme molecule.

Alpha-Hemoglobin Stabilizing Protein (AHSP) binds to α-hemoglobin (α-Hb) or α-globin and maintains it in a soluble state until its association with the β-Hb chain partner to form Hb tetramers. AHSP specifically recognizes the G and H helices of α-Hb. To investigate the degree of interaction of the various regions of the α-globin H helix with AHSP, this interface was studied by stepwise elimination of regions of the α-globin H helix: five truncated α-Hbs α-Hb1-138, α-Hb1-134, α-Hb1-126, α-Hb1-123, α-Hb1-117 were co-expressed with AHSP as two glutathione-S-transferase (GST) fusion proteins. SDS-PAGE and Western Blot analysis revealed that the level of expression of each truncated α-Hb was similar to that of the wild type α-Hb except the shortest protein α-Hb1-117 which displayed a decreased expression. While truncated GST-α-Hb1-138 and GST-α-Hb1-134 were normally soluble; the shorter globins GST-α-Hb1-126 and GST-α-Hb1-117 were obtained in very low quantities, and the truncated GST-α-Hb1-123 provided the least material. Absorbance and fluorescence studies of complexes showed that the truncated α-Hb1-134 and shorter forms led to modified absorption spectra together with an increased fluorescence emission. This attests that shortening the H helix leads to a lower affinity of the α-globin for the heme. Upon addition of β-Hb, the increase in fluorescence indicates the replacement of AHSP by β-Hb. The CO binding kinetics of different truncated AHSPWT/α-Hb complexes showed that these Hbs were not functionally normal in terms of the allosteric transition. The N-terminal part of the H helix is primordial for interaction with AHSP and C-terminal part for interaction with heme, both features being required for stability of α-globin chain