Dissecting the Energies that Stabilize Sickle Hemoglobin Polymers

Sickle hemoglobin forms long, multistranded polymers that account for the pathophysiology of the disease. The molecules in these polymers make significant contacts along the polymer axis (i.e., axial contacts) as well as making diagonally directed contacts (i.e., lateral contacts). The axial contacts do not engage the mutant β6 Val and its nonmutant receptor region on an adjacent molecule, in contrast to the lateral contacts which do involve the mutation site. We have studied the association process by elastic light scattering measurements as a function of temperature, concentration, and primary and quaternary structure, employing an instrument of our own construction. Even well below the solubility for polymer formation, we find a difference between the association behavior of deoxy sickle hemoglobin molecules (HbS), which can polymerize at higher concentration, in comparison to COHbS, COHbA, or deoxygenated Hemoglobin A (HbA), none of which have the capacity to form polymers. The nonpolymerizable species are all quite similar to one another, and show much less association than deoxy HbS. We conclude that axial contacts are significantly weaker than the lateral ones. All the associations are entropically favored, and enthalpically disfavored, typical of hydrophobic interactions. For nonpolymerizable Hemoglobin, ΔH_o was 35 ± 4 kcal/mol, and ΔS was 102.7 ± 0.5 cal/(mol−K). For deoxyHbS, ΔH_o was 19 ± 2 kcal/mol, and ΔS was 56.9 ± 0.5 cal/(mol−K). The results are quantitatively consistent with the thermodynamics of polymer assembly, suggesting that the dimer contacts and polymer contacts are very similar, and they explain a previously documented significant anisotropy between bending and torsional moduli. Unexpectedly, the results also imply that a substantial fraction of the hemoglobin has associated into dimeric species at physiological conditions.     

The Microrheology of Sickle Hemoglobin Gels

Sickle cell disease is a rheological disease, yet no quantitative rheological data exist on microscopic samples at physiological concentrations. We have developed a novel method for measuring the microrheology of sickle hemoglobin gels, based on magnetically driven compression of 5- to 8-μm-thick emulsions containing hemoglobin droplets ∼80 μm in diameter. Using our method, by observing the expansion of the droplet area as the emulsion is compressed, we were able to resolve changes in thickness of a few nanometers with temporal resolution of milliseconds. Gels were formed at various initial concentrations and temperatures and with different internal domain structure. All behaved as Hookean springs with Young's modulus from 300 to 1500 kPa for gels with polymerized hemoglobin concentration from 6 g/dl to 12 g/dl. For uniform, multidomain gels, Young's modulus mainly depended on the terminal concentration of the gel rather than the conditions of formation. A simple model reproduced the quadratic dependence of the Young's modulus on the concentration of polymerized hemoglobin. Partially desaturated samples also displayed quadratic concentration dependence but with a smaller proportionality coefficient, as did samples that were desaturated in steps; such samples were significantly less rigid than gels formed all at once. The magnitude of the Young's modulus provides quantitative support for the dominant models of sickle pathophysiology.     

The Neurotoxic Effect of Sickle Cell Hemoglobin

A growing body of experimental evidence suggests that the oxidative neurotoxicity of hemoglobin A may contribute to neuronal loss after CNS hemorrhage. Several hemoglobin variants, including hemoglobin S, are more potent oxidants in cell-free systems. However, despite the increased incidence of hemorrhagic stroke associated with sickle cell disease, little is known of the effect of hemoglobin S on cells of neural origin. In the present study, its toxicity was quantified and directly compared with that of hemoglobin A in murine cortical cell cultures. Reactive oxygen species production, as assessed by cellular fluorescence after treatment with dihydrorhodamine 123, was significantly increased by exposure to 10?μM hemoglobin S for 2–4?h. Neuronal death, as measured by propidium iodide staining and lactate dehydrogenase release, commenced at 4?h; for a 20-h exposure, the EC⁵⁰ was approximately 0.71?μm. Glial cells were not injured. Cell death was completely blocked by iron chelation with deferoxamine or phenanthroline. Direct comparison of sister cultures exposed to either hemoglobin A or hemoglobin S revealed a similar amount of cell injury in both groups. A significant difference was consistently observed only after treatment with 1?μM hemoglobin for 20?h, which resulted in death of approximately one third more neurons with hemoglobin S than with hemoglobin A. The results of this study suggest that sickle cell hemoglobin is neurotoxic at physiologically relevant concentrations. This toxicity is iron-dependent, oxidative, and quantitatively similar to that produced by hemoglobin A.     

Free Heme and the Polymerization of Sickle Cell Hemoglobin

In search of novel control parameters for the polymerization of sickle cell hemoglobin (HbS), the primary pathogenic event of sickle cell anemia, we explore the role of free heme, which may be excessively released in sickle erythrocytes. We show that the concentration of free heme in HbS solutions typically used in the laboratory is 0.02-0.04 mole heme/mole HbS. We show that dialysis of small molecules out of HbS solutions arrests HbS polymerization. The addition of 100-260 μM of free heme to dialyzed HbS solutions leads to rates of nucleation and polymer fiber growth faster by two orders of magnitude than before dialysis. Toward an understanding of the mechanism of nucleation enhancement by heme, we show that free heme at a concentration of 66 μM increases by two orders of magnitude the volume of the metastable clusters of dense HbS liquid, the locations where HbS polymer nuclei form. These results suggest that spikes of the free heme concentration in the erythrocytes of sickle cell anemia patients may be a significant factor in the complexity of the clinical manifestations of sickle cell anemia. The prevention of free heme accumulation in the erythrocyte cytosol may be a novel avenue to sickle cell therapy.     

Heterogeneous Nucleation and Crowding in Sickle Hemoglobin: An Analytic Approach

Sickle hemoglobin nucleation occurs in solution as a homogeneous process or on existing polymers in a heterogeneous process. We have developed an analytic formulation to describe the solution crowding and large nonideality that affects the heterogeneous nucleation of sickle hemoglobin by using convex particle theory. The formulation successfully fits the concentration and temperature dependence of the heterogeneous nucleation process over 14 orders of magnitude. Unlike previous approaches, however, the new formulation can also accurately describe the effects of adding nonpolymerizing agents to the solution. Without additional adjustable parameters, the model now describes the data of M. Ivanova, R. Jasuja, S. Kwong, R. W. Briehl, and F. A. Ferrone, (Biophys. J. 2000, 79:1016-1022), in which up to 50% of the sickle hemoglobin is substituted by cross-linked hemoglobin A, which does not polymerize, and which substitution causes the rates to decrease by 10⁵. The success of this approach provides insight into the polymerization process: from the size-dependence of the contact energy deduced here, it also appears that various contacts of unknown origin are energetically significant in the heterogeneous nucleation process.     

Sickle Cell Hemoglobin: Molecular Basis Of Sickling Phenomenon Theory And Therap

Abstract

Thirty years ago, Linus Pauling suggested that sickle cell disease might be due to an abnormal hemoglobin molecule, and I think this was the beginning of molecular medicine. This is what he said about it in his Harvey Lecture^69a which he delivered in 1953     

Incidence of Sickle Cell Disease and Other Hemoglobin Variants in 10,095 Lebanese Neonates

Hemoglobinopathies are highly prevalent diseases and impose a public health burden. Early diagnosis and treatment can ameliorate the course of these diseases and improve survival. Despite purported high incidence of hemoglobinopathies in Lebanon, there are no nationwide screening programs. In this study, newborn screening utilizing high pressure liquid chromatography was executed in all public hospitals across Lebanon between 2010 and 2013. All newborns with an abnormal hemoglobin (Hb) were offered genetic counseling and all those with disease were enrolled in comprehensive hemoglobinopathy clinics. Among newborns, 2.1% were found to have an abnormal Hb variant with sickle Hb being the most common while 0.1% were found to have sickle cell disease (SCD). The majority of those with SCD had non-Lebanese origins. The most common causes of hospitalizations in infants with SCD were acute splenic sequestration and pain crises. No bacteremia or other life threatening infections were noted. At a median follow up 14 months (follow up range 7 to 34 months), all children with disease are alive and compliant with treatment. Systematic screening for SCD and other Hb variants was shown to be feasible, cost effective, and of accurate predictive value. This program was also clinically effective because it led to the identification of babies with disease and to providing them with free early multidisciplinary care. Conclusively, a newborn screening program should be implemented across Lebanon to detect hemoglobinopathies and initiate early therapeutic and preventive strategies and genetic counseling.     

Genome Wide Association Study of Fetal Hemoglobin in Sickle Cell Anemia in Tanzania

Background

Fetal hemoglobin (HbF) is an important modulator of sickle cell disease (SCD). HbF has previously been shown to be affected by variants at three loci on chromosomes 2, 6 and 11, but it is likely that additional loci remain to be discovered.

Methods and Findings

We conducted a genome-wide association study (GWAS) in 1,213 SCA (HbSS/HbSβ⁰) patients in Tanzania. Genotyping was done with Illumina Omni2.5 array and imputation using 1000 Genomes Phase I release data. Association with HbF was analysed using a linear mixed model to control for complex population structure within our study. We successfully replicated known associations for HbF near BCL11A and the HBS1L-MYB intergenic polymorphisms (HMIP), including multiple independent effects near BCL11A, consistent with previous reports. We observed eight additional associations with P<10⁻⁶. These associations could not be replicated in a SCA population in the UK.

Conclusions

This is the largest GWAS study in SCA in Africa. We have confirmed known associations and identified new genetic associations with HbF that require further replication in SCA populations in Africa.     

Fiber Depolymerization: Fracture, Fragments, Vanishing Times, and Stochastics in Sickle Hemoglobin

The well-characterized rates, mechanisms, and stochastics of nucleation-dependent polymerization of deoxyhemoglobin S (HbS) are important in governing whether or not vaso-occlusive sickle cell crises will occur. The less well studied kinetics of depolymerization may also be important, for example in achieving full dissolution of polymers in the lungs, in resolution of crises and/or in minimizing gelation-induced cellular damage. We examine depolymerization by microscopic observations on depolymerizing HbS fibers, by Monte Carlo simulations and by analytical characterization of the mechanisms. We show that fibers fracture. Experimental scatter of rates is consistent with stochastic features of the analytical model and Monte Carlo results. We derive a model for the distribution of vanishing times and also show the distribution of fracture-dependent fiber fragment lengths and its time dependence. We describe differences between depolymerization of single fibers and bundles and propose models for bundle dissolution. Our basic model can be extended to dissolution of gels containing many fibers and is also applicable to other reversible linear polymers that dissolve by random fracture and end-depolymerization. Under the model, conditions in which residual HbS polymers exist and facilitate repolymerization and thus pathology can be defined; whereas for normal polymers requiring cyclic polymerization and depolymerization for function, conditions for rapid cycling due to residual aggregates can be identified.     

Candidate Sequence Variants and Fetal Hemoglobin in Children with Sickle Cell Disease Treated with Hydroxyurea

Background

Fetal hemoglobin level is a heritable complex trait that strongly correlates swith the clinical severity of sickle cell disease. Only few genetic loci have been identified as robustly associated with fetal hemoglobin in patients with sickle cell disease, primarily adults. The sole approved pharmacologic therapy for this disease is hydroxyurea, with effects largely attributable to induction of fetal hemoglobin.

Methodology/Principal Findings

In a multi-site observational analysis of children with sickle cell disease, candidate single nucleotide polymorphisms associated with baseline fetal hemoglobin levels in adult sickle cell disease were examined in children at baseline and induced by hydroxyurea therapy. For baseline levels, single marker analysis demonstrated significant association with BCL11A and the beta and epsilon globin loci (HBB and HBE, respectively), with an additive attributable variance from these loci of 23%. Among a subset of children on hydroxyurea, baseline fetal hemoglobin levels explained 33% of the variance in induced levels. The variant in HBE accounted for an additional 13% of the variance in induced levels, while variants in the HBB and BCL11A loci did not contribute beyond baseline levels.

Conclusions/Significance

These findings clarify the overlap between baseline and hydroxyurea-induced fetal hemoglobin levels in pediatric disease. Studies assessing influences of specific sequence variants in these and other genetic loci in larger populations and in unusual hydroxyurea responders are needed to further understand the maintenance and therapeutic induction of fetal hemoglobin in pediatric sickle cell disease.     

Dissecting the forces that shape crops: an interview with Ryohei Terauchi

Ryohei Terauchi is a Professor at Kyoto University and a Group Leader at the Iwate Biotechnology Research Center, Japan, studying the evolution of crops and their pathogens. In this interview, Ryohei describes his research interests, how the revolution in sequencing technology helped improve our understanding of orphan crops, and who are the scientists that inspire him.     

Dissecting the molecular bridges that mediate the function of Frizzled in planar cell polarity

Many epithelia have a common planar cell polarity (PCP), as exemplified by the consistent orientation of hairs on mammalian skin and insect cuticle. One conserved system of PCP depends on Starry night (Stan, also called Flamingo), an atypical cadherin that forms homodimeric bridges between adjacent cells. Stan acts together with other transmembrane proteins, most notably Frizzled (Fz) and Van Gogh (Vang, also called Strabismus). Here, using an in vivo assay for function, we show that the quintessential core of the Stan system is an asymmetric intercellular bridge between Stan in one cell and Stan acting together with Fz in its neighbour: such bridges are necessary and sufficient to polarise hairs in both cells, even in the absence of Vang. By contrast, Vang cannot polarise cells in the absence of Fz; instead, it appears to help Stan in each cell form effective bridges with Stan plus Fz in its neighbours. Finally, we show that cells containing Stan but lacking both Fz and Vang can be polarised to make hairs that point away from abutting cells that express Fz. We deduce that each cell has a mechanism to estimate and compare the numbers of asymmetric bridges, made between Stan and Stan plus Fz, that link it with its neighbouring cells. We propose that cells normally use this mechanism to read the local slope of tissue-wide gradients of Fz activity, so that all cells come to point in the same direction.     

Modification of Axial Fiber Contact Residues Impact Sickle Hemoglobin Polymerization by Perturbing a Network of Coupled Interactions

The identity of intermolecular contact residues in sickle hemoglobin (HbS) fiber is largely known. However, our knowledge about combinatorial effects of two or more contact sites or the mechanistic basis of such effects is rather limited. Lys16, His20, and Glu23 of the α-chain occur in intra-double strand axial contacts in the sickle hemoglobin (HbS) fiber. Here we have constructed two novel double mutants, HbS (K16Q/E23Q) and (H20Q/E23Q), with a view to delineate cumulative impact of interactions emanating from the above contact sites. Far-UV and visible region CD spectra of the double mutants were similar to the native HbS indicating the presence of native-like secondary and tertiary structure in the mutants. The quaternary structures in both the mutants were also preserved as judged by the derivative UV spectra of liganded (oxy) and unliganded (deoxy) forms of the double mutants. However, the double mutants displayed interesting polymerization behavior. The polymerization behaviour of the double mutants was found to be non-additive of the individual single mutants. While HbS (H20Q/E23Q) showed inhibitory effect similar to that of HbS (E23Q), the intrinsic inhibitory propensity of the associated single mutants was totally quelled in HbS (K16Q/E23Q) double mutant. Molecular dynamics (MD) simulations studies of the isolated α-chains as well as a module of the fiber containing the double and associated single mutants suggested that these contact sites at the axial interface of the fiber impact HbS polymerization through a coupled interaction network. The overall results demonstrate a subtle role of dynamics and electrostatics in the polymer formation and provide insights about interaction-linkage in HbS fiber assembly.     

Dissecting the Forces that Dominate Dimerization of the Nucleotide Binding Domains of ABCB1

P-glycoprotein, also known as multidrug resistance protein 1 or ABCB1, can export a wide range of chemically unrelated compounds, including chemotherapeutic drugs. ABCB1 consists of two transmembrane domains that form the substrate binding and translocation domain, and of two cytoplasmic nucleotide binding domains (NBDs) that energize substrate transport by ATP binding and hydrolysis. ATP binding triggers dimerization of the NBDs, which switches the transporter from an inward facing to an outward facing transmembrane domain conformation. We performed MD simulations to study the dynamic behavior of the NBD dimer in the presence or absence of nucleotides. In the apo configuration, the NBDs were overall attractive to each other as shown in the potential of mean force profile, but the energy well was shallow and broad. In contrast, a sharp and deep energy minimum (～?42 kJ/mol) was found in the presence of ATP, leading to a well-defined conformation. Motif interaction network analyses revealed that ATP stabilizes the NBD dimer by serving as the central hub for interdomain connections. Simulations showed that forces promoting dimerization are multilayered, dominated by electrostatic interactions between the nucleotide and conserved amino acids of the signature sequence and the Walker A motif. In addition, direct and water-bridged hydrogen bonds between NBDs provided conformation-defining interactions. Importantly, we characterized a largely unrecognized but essential contribution from hydrophobic interactions between the adenine moiety of the nucleotides and a hydrophobic surface of the X-loop to the stabilization of the nucleotide-bound NBD dimer. These hydrophobic interactions lead to a sharp energy minimum, thereby conformationally restricting the nucleotide-bound state.     

Dissecting signaling pathways that govern self-renewal of rabbit embryonic stem cells

The pluripotency and self-renewal of embryonic stem cells (ESC) are regulated by a variety of cytokines/growth factors with some species differences. We reported previously that rabbit ESC (rESC) are more similar to primate ESC than to mouse ESC. However, the signaling pathways that regulate rESC self-renewal had not been identified. Here we show that inhibition of the transforming growth factor beta (TGFbeta), fibroblast growth factor (FGF), and canonical Wnt/beta-catenin (Wnt) pathways results in enhanced differentiation of rESC accompanied by down-regulation of Smad2/3 phosphorylation and beta-catenin expression and up-regulation of phosphorylation of Smad1 and beta-catenin. These results imply that the TGFbeta, FGF, and Wnt pathways are required for rESC self-renewal. Inhibition of the MAPK/ERK and PI3K/AKT pathways, which lie downstream of the FGF pathway, led to differentiation of rESC accompanied by down-regulation of phosphorylation of ERK1/2 or AKT, respectively. Long-term self-renewal of rESC could be achieved by adding a mixture of TGFbeta ligands (activin A, Nodal, or TGFbeta1) plus basic FGF (bFGF) and Noggin in the absence of serum and feeder cells. Our findings also suggest that there is a regulatory network consisting of the FGF, Wnt, and TGFbeta pathways that controls rESC pluripotency and self-renewal. We conclude that bFGF controls the stem cell properties of rESC both directly and indirectly through TGFbeta or other pathways, whereas the effect of Wnt on rESC might be mediated by the TGFbeta pathway.