A cis-Proline in ��-Hemoglobin Stabilizing Protein Directs the Structural Reorganization of ��-Hemoglobin

α-Hemoglobin (αHb) stabilizing protein (AHSP) is expressed in erythropoietic tissues as an accessory factor in hemoglobin synthesis. AHSP forms a specific complex with αHb and suppresses the heme-catalyzed evolution of reactive oxygen species by converting αHb to a conformation in which the heme is coordinated at both axial positions by histidine side chains (bis-histidyl coordination). Currently, the detailed mechanism by which AHSP induces structural changes in αHb has not been determined. Here, we present x-ray crystallography, NMR spectroscopy, and mutagenesis data that identify, for the first time, the importance of an evolutionarily conserved proline, Pro³⁰, in loop 1 of AHSP. Mutation of Pro³⁰ to a variety of residue types results in reduced ability to convert αHb. In complex with αHb, AHSP Pro³⁰ adopts a cis-peptidyl conformation and makes contact with the N terminus of helix G in αHb. Mutations that stabilize the cis-peptidyl conformation of free AHSP, also enhance the αHb conversion activity. These findings suggest that AHSP loop 1 can transmit structural changes to the heme pocket of αHb, and, more generally, highlight the importance of cis-peptidyl prolyl residues in defining the conformation of regulatory protein loops.Mammalian adult hemoglobin (HbA)⁵ is a tetramer of two αHb and two βHb subunits, which is produced to extremely high concentrations (∼340 mg/ml) in red blood cells. Numerous mechanisms exist to balance and coordinate HbA synthesis in normal erythropoiesis, and problems with the production of either HbA subunit give rise to thalassemia, a common cause of anemia worldwide. Previously, we identified α-hemoglobin stabilizing protein (AHSP) as an accessory factor in normal HbA production (1). AHSP forms a dimeric complex with αHb (see Fig. 1A) (2) but does not interact with βHb or HbA. AHSP also binds heme-free (apo) αHb (3) and may serve functions in both the folding of nascent αHb (4) and the detoxification of excess αHb that remains following HbA assembly (2, 5). Mice carrying an Ahsp gene knock-out display mild anemia, ineffective erythropoiesis, and enhanced sensitivity to oxidative stress (1, 6), features also observed in β-thalassemia patients due to the cytotoxic effects of free αHb.Open in a separate windowFIGURE 1.Summary of αHb·AHSP interactions. A, the αHb·AHSP complex(PDB code 1Z8U) (2). The interface is formed from helices 1 and 2 and the intervening loop 1 (green) of AHSP, together with helices G-H and the B-C corner of αHb (cyan). B, detailed views of the heme binding site of αHb as it appears in oxy-HbA (PDB code 1GZX) (69) and the final bis-histidyl αHb·AHSP complex (PDB code 1Z8U) with two histidine ligands to the iron. Typical visible absorption spectra in the region 450–700 nm are shown.Free αHb promotes the formation of harmful reactive oxygen species as a result of reduction/oxidation reactions involving the heme iron (7, 8). Reactive oxygen species can damage heme, αHb, and other cellular structures, resulting in hemoglobin precipitates and death of erythroid precursor cells (9–12). The presence of AHSP may explain how cells tolerate the slight excess of αHb that is observed in normal erythropoiesis, which is postulated to inhibit the formation of non-functional βHb tetramers, thus providing a robust mechanism for achieving the correct subunit stoichiometry during HbA assembly (13).Structural and biochemical studies have begun to elucidate the molecular mechanism by which AHSP detoxifies αHb. AHSP binds to oxygenated αHb to generate an initial complex that retains the oxy-heme, as evidenced by a characteristic visible absorption spectrum (see Fig. 1B, middle) and resonance Raman spectrum (5). This initial oxy-αHb·AHSP complex then converts to a low spin Fe³⁺ complex (2), in which the heme iron is bound at both axial positions by the side chains of His⁵⁸ and His⁸⁷ from αHb (see Fig. 1B, right). The formation of this complex inhibits αHb peroxidase activity and heme loss (2). Bis-histidyl heme coordination is becoming increasingly recognized as a feature of numerous vertebrate and non-vertebrate globins (14) and has been shown previously to confer a relative stabilization of the Fe³⁺ over the Fe²⁺ oxidation state (15–17). Although bis-histidyl heme coordination has previously been detected in solutions of met-Hb, formed through spontaneous autoxidation of Hb (18–21), the bishis-αHb·AHSP complex provides the first evidence that the bis-histidyl heme may play a positive functional role in Hb biochemistry by inhibiting the production of harmful reactive oxygen species.Despite its potential importance, the mechanism by which AHSP influences heme coordination in its binding partner is still unknown. As shown in Fig. 1A, AHSP binds αHb at a surface away from the heme pocket, and thus structural changes must somehow be transmitted through the αHb protein. It is intriguing that the free AHSP protein switches between two alternative conformations linked to cis/trans isomerization of the Asp²⁹-Pro³⁰ peptide bond in loop 1 (22) and that, in complex with αHb, this loop is located at the αHb·AHSP interface (see Fig. 1A). Peptide bonds preceding proline residues are unique in that the cis or trans bonding conformations have relatively similar stabilities (23), allowing an interconversion between these conformations that can be important for protein function (24, 25). Previous x-ray crystal structures of αHb·AHSP complexes have been obtained only with a P30A mutant of AHSP, in which isomerization is abolished and the Asp²⁹-Ala³⁰ peptide bond adopts a trans conformation, leaving the potential structural and functional significance of the evolutionarily conserved Pro³⁰ undisclosed. Here, we demonstrate a functional role for AHSP Pro³⁰ in conversion of oxy-αHb to the bis-histidyl form and identify a specific structural role for a cis Asp²⁹-Pro³⁰ peptide bond in this process. From a mechanistic understanding of how AHSP promotes formation of bis-histidyl αHb, we may eventually be able to engineer AHSP function as a tool in new treatments for Hb diseases such as β-thalassemia.     

Japanese Encephalitis��A Pathological and Clinical Perspective

Japanese encephalitis (JE) is the leading form of viral encephalitis in Asia. It is caused by the JE virus (JEV), which belongs to the family Flaviviridae. JEV is endemic to many parts of Asia, where periodic outbreaks take hundreds of lives. Despite the catastrophes it causes, JE has remained a tropical disease uncommon in the West. With rapid globalization and climatic shift, JEV has started to emerge in areas where the threat was previously unknown. Scientific evidence predicts that JEV will soon become a global pathogen and cause of worldwide pandemics. Although some research documents JEV pathogenesis and drug discovery, worldwide awareness of the need for extensive research to deal with JE is still lacking. This review focuses on the exigency of developing a worldwide effort to acknowledge the prime importance of performing an extensive study of this thus far neglected tropical viral disease. This review also outlines the pathogenesis, the scientific efforts channeled into develop a therapy, and the outlook for a possible future breakthrough addressing this killer disease.     

Binding of the Molecular Chaperone ��B-Crystallin to A�� Amyloid Fibrils Inhibits Fibril Elongation

The molecular chaperone αB-crystallin is a small heat-shock protein that is upregulated in response to a multitude of stress stimuli, and is found colocalized with Aβ amyloid fibrils in the extracellular plaques that are characteristic of Alzheimer''s disease. We investigated whether this archetypical small heat-shock protein has the ability to interact with Aβ fibrils in vitro. We find that αB-crystallin binds to wild-type Aβ₄₂ fibrils with micromolar affinity, and also binds to fibrils formed from the E22G Arctic mutation of Aβ₄₂. Immunoelectron microscopy confirms that binding occurs along the entire length and ends of the fibrils. Investigations into the effect of αB-crystallin on the seeded growth of Aβ fibrils, both in solution and on the surface of a quartz crystal microbalance biosensor, reveal that the binding of αB-crystallin to seed fibrils strongly inhibits their elongation. Because the lag phase in sigmoidal fibril assembly kinetics is dominated by elongation and fragmentation rates, the chaperone mechanism identified here represents a highly effective means to inhibit fibril proliferation. Together with previous observations of αB-crystallin interaction with α-synuclein and insulin fibrils, the results suggest that this mechanism is a generic means of providing molecular chaperone protection against amyloid fibril formation.     

Membrane Partitioning: ��Classical�� and ��Nonclassical�� Hydrophobic Effects

The free energy of transfer of nonpolar solutes from water to lipid bilayers is often dominated by a large negative enthalpy rather than the large positive entropy expected from the hydrophobic effect. This common observation has led to the idea that membrane partitioning is driven by the "nonclassical" hydrophobic effect. We examined this phenomenon by characterizing the partitioning of the well-studied peptide melittin using isothermal titration calorimetry (ITC) and circular dichroism (CD). We studied the temperature dependence of the entropic (-TΔS) and enthalpic (ΔH) components of free energy (ΔG) of partitioning of melittin into lipid membranes made of various mixtures of zwitterionic and anionic lipids. We found significant variations of the entropic and enthalpic components with temperature, lipid composition and vesicle size but only small changes in ΔG (entropy-enthalpy compensation). The heat capacity associated with partitioning had a large negative value of about -0.5 kcal mol(-1) K(-1). This hallmark of the hydrophobic effect was found to be independent of lipid composition. The measured heat capacity values were used to calculate the hydrophobic-effect free energy ΔG (hΦ), which we found to dominate melittin partitioning regardless of lipid composition. In the case of anionic membranes, additional free energy comes from coulombic attraction, which is characterized by a small effective peptide charge due to the lack of additivity of hydrophobic and electrostatic interactions in membrane interfaces [Ladokhin and White J Mol Biol 309:543-552, 2001]. Our results suggest that there is no need for a special effect-the nonclassical hydrophobic effect-to describe partitioning into lipid bilayers.     

The Caenorhabditis elegans A��1�C42 Model of Alzheimer Disease Predominantly Expresses A��3�C42

Transgenic expression of human amyloid β (Aβ) peptide in body wall muscle cells of Caenorhabditis elegans has been used to better understand aspects of Alzheimer disease (AD). In human aging and AD, Aβ undergoes post-translational changes including covalent modifications, truncations, and oligomerization. Amino truncated Aβ is increasingly recognized as potentially contributing to AD pathogenesis. Here we describe surface-enhanced laser desorption ionization-time of flight mass spectrometry mass spectrometry of Aβ peptide in established transgenic C. elegans lines. Surprisingly, the Aβ being expressed is not full-length 1–42 (amino acids) as expected but rather a 3–42 truncation product. In vitro analysis demonstrates that Aβ_3–42 self-aggregates like Aβ_1–42, but more rapidly, and forms fibrillar structures. Similarly, Aβ_3–42 is also the more potent initiator of Aβ_1–40 aggregation. Seeded aggregation via Aβ_3–42 is further enhanced via co-incubation with the transition metal Cu(II). Although unexpected, the C. elegans model of Aβ expression can now be co-opted to study the proteotoxic effects and processing of Aβ_3–42.Numerous studies support a role for aggregating Aβ³ in mediating the toxicity that underlies AD (1, 2). However, several key questions remain central to understanding how AD and Aβ pathology are related. What is the connection between Aβ aggregation and toxicity? Is there a specific toxic Aβ conformation or species? How and why does aging impact on Aβ precipitation? Significant effort to address these questions has been invested in the use of vertebrate and simple invertebrate model organisms to simulate neurodegenerative diseases through transgenic expression of human Aβ (3). From these models, several novel insights into the proteotoxicity of Aβ have been gained (4–7).Human Aβ (e.g. in brain, cerebrospinal fluid, or plasma) is not found as a single species but rather as diverse mixtures of various modified, truncated, and cross-linked forms (8–10). Specific truncations, covalent modifications, and cross-linked oligomers of Aβ have potentially important roles in determining Aβ-associated neurotoxicity. For example, N-terminal truncations of Aβ have increased abundance in AD, rapidly aggregate, and are neurotoxic (9, 11). Furthermore, the N-terminal glutamic acid residue of Aβ_3–42 can be cyclized to pyroglutamate (Aβ_3(pE)-42) (12), which may be particularly important in AD pathogenesis (13, 14). Aβ_3(pE)-42 is a significant fraction of total Aβ in AD brain (15), accounting for more than 50% of Aβ accumulated in plaques (16). Aβ_3(pE)-42 seeds Aβ aggregation (17), confers proteolytic resistance, and is neurotoxic (13). Recently, glutaminyl cyclase (QC) has been proposed to catalyze, in vivo, pyroglutamate formation of Aβ_3(pE)-40/42 (14, 18). Aβ_1–42 itself cannot be cyclized by QC to Aβ_3(pE)-42 (19), unlike Aβ that commences with an N-terminal glutamic acid-residue (e.g. Aβ_3–42 and Aβ_11–42) (20). QC has broad expression in mammalian brain (21, 22), and its inhibition attenuates accumulation of Aβ_3(pE)-42 into plaques and improves cognition in a transgenic mouse model of AD that overexpresses human amyloid precursor protein (14). N-terminal truncations at position 3 have been reported in senile plaques (23, 24); however, the process that generates Aβ_3–42 is unknown. Currently there are no reported animal models of Aβ_3–42 expression.Advances in surface-enhanced laser desorption ionization-time of flight mass spectrometry (SELDI-TOF MS) analysis now facilitate accurate identification of particular Aβ species. Using this technology, we examined well characterized C. elegans transgenic models of AD that develop amyloid aggregates (25, 26) to see whether the human Aβ they express is post-translationally modified.     

A��42-to-A��40- and Angiotensin-converting Activities in Different Domains of Angiotensin-converting Enzyme

Amyloid β-protein 1–42 (Aβ42) is believed to play a causative role in the development of Alzheimer disease (AD), although it is a minor part of Aβ. In contrast, Aβ40 is the predominant secreted form of Aβ and recent studies have suggested that Aβ40 has neuroprotective effects and inhibits amyloid deposition. We have reported that angiotensin-converting enzyme (ACE) converts Aβ42 to Aβ40, and its inhibition enhances brain Aβ42 deposition (Zou, K., Yamaguchi, H., Akatsu, H., Sakamoto, T., Ko, M., Mizoguchi, K., Gong, J. S., Yu, W., Yamamoto, T., Kosaka, K., Yanagisawa, K., and Michikawa, M. (2007) J. Neurosci. 27, 8628–8635). ACE has two homologous domains, each having a functional active site. In the present study, we identified the domain of ACE, which is responsible for converting Aβ42 to Aβ40. Interestingly, Aβ42-to-Aβ40-converting activity is solely found in the N-domain of ACE and the angiotensin-converting activity is found predominantly in the C-domain of ACE. We also found that the N-linked glycosylation is essential for both Aβ42-to-Aβ40- and angiotensin-converting activities and that unglycosylated ACE rapidly degraded. The domain-specific converting activity of ACE suggests that ACE inhibitors could be designed to specifically target the angiotensin-converting C-domain, without inhibiting the Aβ42-to-Aβ40-converting activity of ACE or increasing neurotoxic Aβ42.     

A Synthetic Combinatorial Strategy for Developing ��-Conotoxin Analogs as Potent ��7 Nicotinic Acetylcholine Receptor Antagonists

α-Conotoxins are peptide neurotoxins isolated from venomous cone snails that display exquisite selectivity for different subtypes of nicotinic acetylcholine receptors (nAChR). They are valuable research tools that have profound implications in the discovery of new drugs for a myriad of neuropharmacological conditions. They are characterized by a conserved two-disulfide bond framework, which gives rise to two intervening loops of extensively mutated amino acids that determine their selectivity for different nAChR subtypes. We have used a multistep synthetic combinatorial approach using α-conotoxin ImI to develop potent and selective α₇ nAChR antagonists. A positional scan synthetic combinatorial library was constructed based on the three residues of the n-loop of α-conotoxin ImI to give a total of 10,648 possible combinations that were screened for functional activity in an α₇ nAChR Fluo-4/Ca²⁺ assay, allowing amino acids that confer antagonistic activity for this receptor to be identified. A second series of individual α-conotoxin analogs based on the combinations of defined active amino acid residues from positional scan synthetic combinatorial library screening data were synthesized. Several analogs exhibited significantly improved antagonist activity for the α₇ nAChR compared with WT-ImI. Binding interactions between the analogs and the α₇ nAChR were explored using a homology model of the amino-terminal domain based on a crystal structure of an acetylcholine-binding protein. Finally, a third series of refined analogs was synthesized based on modeling studies, which led to several analogs with refined pharmacological properties. Of the 96 individual α-conotoxin analogs synthesized, three displayed ≥10-fold increases in antagonist potency compared with WT-ImI.     

A Protein-Protein Interaction Map of Trypanosome ��20S Editosomes

Mitochondrial mRNA editing in trypanosomatid parasites involves several multiprotein assemblies, including three very similar complexes that contain the key enzymatic editing activities and sediment at ∼20S on glycerol gradients. These ∼20S editosomes have a common set of 12 proteins, including enzymes for uridylyl (U) removal and addition, 2 RNA ligases, 2 proteins with RNase III-like domains, and 6 proteins with predicted oligonucleotide binding (OB) folds. In addition, each of the 3 distinct ∼20S editosomes contains a different RNase III-type endonuclease, 1 of 3 related proteins and, in one case, an additional exonuclease. Here we present a protein-protein interaction map that was obtained through a combination of yeast two-hybrid analysis and subcomplex reconstitution with recombinant protein. This map interlinks ten of the proteins and in several cases localizes the protein region mediating the interaction, which often includes the predicted OB-fold domain. The results indicate that the OB-fold proteins form an extensive protein-protein interaction network that connects the two trimeric subcomplexes that catalyze U removal or addition and RNA ligation. One of these proteins, KREPA6, interacts with the OB-fold zinc finger protein in each subcomplex that interconnects their two catalytic proteins. Another OB-fold protein, KREPA3, appears to link to the putative endonuclease subcomplex. These results reveal a physical organization that underlies the coordination of the various catalytic and substrate binding activities within the ∼20S editosomes during the editing process.