ALBERT

Description: 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.

Description: 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.

Description: Introduction: Recent data suggest that serum monoclonal free light chain ratio (LCR) may have prognostic significance in monoclonal gammopathies. We routinely assay serum free light chains (FLC) in all new patients with a monoclonal gammopathy with the primary aim of identifying patients with a significant level of free light chain. We made a retrospective analysis of these data in order to establish the range of results observed in our patients and to establish a cohort of patients who will be followed up to evaluate the prognostic significance of LCR abnormalities. Aims: To identify the proportion of patients with monoclonal gammopathies that have abnormal serum LCR at presentation. To identify how many patients have abnormal serum LCR but no other evidence of excess light chain production. To establish the sensitivity of serum LCR for the presence of Bence Jones proteinuria. Method and Setting: We analyzed data from a consecutive cohort of 496 patients with a newly identified paraprotein (by serum or urine protein electrophoresis and immunofixation), who had samples referred to the Immunology Department at Hull Royal Infirmary between 05/03/06 and 07/31/07. Serum FLC analysis was performed by a latex-enhanced immunoassay (The Binding Site, Birmingham, UK on a Beckman-Coulter IMMAGE nephelometric analyzer). Serum (SPE) and urine (UPE) protein electrophoresis were perfomed by SEBIA Hydrasys gel system or Capillarys 2 capillary electrophoresis system. Results: Paraproteins were evident by SPE in 98% of patients (488 of 496). In the remaining 8 patients (2%) the paraprotein was only visible by UPE. Serum LCR was outside the standard reference interval (0.26–1.65) in 272 (55%) of all patients and in 124 (36%) of the 343 patients who had no FLC detected by SPE or UPE. Urines were received from 281 (57%) patients and a paraprotein was detected in 151 (54%) of these by UPE. FLC was detected in 126 (83%) of urines containing a paraprotein and in 89 (59%) was the sole paraprotein present in the urine. Of the 126 patients who had FLC detected by UPE, 124 (98%) had serum LCR outside the standard reference interval, and 119 (94%) had serum LCR outside the mean±3.5 sd range (0.18–2.01). The remaining two patients with normal LCR both had IgG-kappa paraproteins with a small free kappa band in the urine. The molecular weight of these free kappa bands has not yet been confirmed by SDS-PAGE electrophoresis and immunoblotting. Conclusions: Raised serum LCR was present in 55% of patients with a paraprotein at diagnosis. 36% of patients with no other evidence of excess light chain production had abnormal LCR. Using the LCR standard reference interval had a sensitivity of 98% for the presence of Bence Jones proteinuria. This high sensitivity of LCR for Bence Jones proteinuria supports the cessation of UPE and its replacement with serum LCR. Our cohort of newly presenting, unselected patients with monoclonal gammopathies will be followed to determine the prognostic value of LCR for these patients.