Phospholipid-induced structural changes to an erythroid β spectrin ankyrin-dependent lipid-binding site

The region of β-spectrin that is responsible for interactions with ankyrin was shown to comprise an ankyrin-sensitive lipid-binding site. Structural studies indicate that it exhibits a mixed 3₁₀/α helical conformation and is highly amphipathic. These features together with the distinctively conserved sequence of the lipid-binding site motivated us to explore the mechanism of its interactions with biological membranes. A series of singly and doubly spin-labeled erythroid β-spectrin-derived peptides was constructed, and the spin-label mobility and spin-spin distances were analyzed via electron paramagnetic resonance spectroscopy and two different calculation methods. The results indicate that in β-spectrin, the lipid-binding domain, which is part of the 14^th segment, has the topology of typical triple-helical spectrin repeat. However, it undergoes significant changes when interacting with phospholipids or detergents. A mechanism for these interactions is proposed in this paper.     

Cutting edge: tubulin α functions as an adaptor in NFAT-importin β interaction

Upon T cell stimulation, NFAT is dephosphorylated by calcineurin, leading to nuclear translocation via NFAT-importin β interaction. Whereas the process of NFAT dephosphorylation has been well researched, the molecular mechanism of NFAT-importin β interaction remains unknown. In contrast to NF-κB and STAT, no importin α family members have been reported as adaptor proteins for NFAT. Our study shows that tubulin α, but not tubulin β, binds to the N-terminal region of NFAT containing the regulatory and Rel homology domains. Importin β interacts with the NFAT-tubulin α complex rather than NFAT or tubulin α alone, resulting in cotranslocation of NFAT and tubulin α into the nucleus. Furthermore, the interaction is suppressed by acetate-induced tubulin α acetylation at lysine 40. In conclusion, tubulin α functions as an adaptor in NFAT-importin β interaction, and this function is regulated by acetate-induced acetylation.     

Pdx1 Maintains β Cell Identity and Function by Repressing an α Cell Program

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Ectodomain Shedding of Interleukin-2 Receptor β and Generation of an Intracellular Functional Fragment

Interleukin-2 (IL-2) regulates different functions of various lymphoid cell subsets. These are mediated by its binding to the IL-2 receptor (IL-2R) composed of three subunits (IL2-Rα, -β, and -γ_c). IL-2Rβ is responsible for the activation of several signaling pathways. Ectodomain shedding of membrane receptors is thought to be an important mechanism for down-regulation of cell surface receptor abundance but is also emerging as a mechanism that cell membrane-associated molecules require for proper action in vivo. Here, we demonstrate that IL-2Rβ is cleaved in cell lines of different origin, including T cells, generating an intracellular 37-kDa fragment (37βic) that comprises the full intracellular C-terminal and transmembrane domains. Ectodomain shedding of IL-2Rβ decreases in a mutant deleted of the juxtamembrane region, where cleavage is predicted to occur, and is inhibited by tissue inhibitor of metalloproteases-3. 37βic is tyrosine-phosphorylated and associates with STAT-5, a canonic signal transducer of IL-2R. Finally, lymphoid cell transfection with a truncated form of IL-2Rβ mimicking 37βic increases their proliferation. These data indicate that IL-2Rβ is subject to ectodomain shedding generating an intracellular fragment biologically functional, because (i) it is phosphorylated, (ii) it associates with STAT5A, and (iii) it increases cell proliferation.     

Isolation of an Arabidopsis thaliana casein kinase II β subunit by complementation in Saccharomyces cerevisiae

Casein kinase II is thought to play an essential role in the control of cell division and differentiation in all eukaryotes. Through complementation of a defective casein kinase II catalytic subunit gene from Saccharomyces cerevisiae, we isolated an Arabidopsis thaliana casein kinase II regulatory subunit homologue, CKB1. A second regulatory subunit was identified by low-stringency hybridization with CKB1.Casein kinase II from S. cerevisiae is composed of two catalytic () and two regulatory () subunits. Simultaneous disruption of the genes for the and subunits, CKA1 and CKA2, respectively, is lethal. Strain YDH8 has disruptions of CKA1 and CKA2; its viability depends on a temperature-sensitive allele of CKA2, cka2–8, carried on a centromeric plasmid. We screened an A. thaliana cDNA library, whose inserts are under the control of the galactose-inducible GAL10 promoter, for cDNAs which enabled YDH8 cells to grow at the restrictive temperature. One cDNA, CKB1, was isolated by this screen which had homology to cDNAs of casein kinase II subunits. A second cDNA, CKB2, was isolated by hybridization and was also able to suppress the YDH8 mutant phenotype.The proteins encoded by CKB1 and CKB2 are 80% identical. The carboxy-terminal two thirds of both proteins is ca. 54% identical to the regulatory subunits of casein kinase II from other species. The amino termini are unrelated to any other known proteins. CKB1 and CKB2 lack the conserved autophosphorylation site characteristic of animal subunits, but have potential casein kinase II phosphorylation sites in the same region. Suppression of the cka1 cka2–8 mutant phenotype occurs by interaction of CKB1 with the defective, cka2–8-encoded, catalytic subunit. Cells with disruptions in CKA1 and CKA2 are not rescued by expression of CKB1.     

Muscarinic receptor subtypes mediate stimulatory and paradoxical inhibitory effects on an insulin-secreting β cell line

Acetylcholine (ACh), a major neurotransmitter from the autonomic nervous system, regulates the cholinergic stimulation of insulin secretion, through interactions with muscarinic receptors. The present study has characterised the individual involvement of muscarinic receptor subtypes in ACh-induced insulin secretion, using clonal β cells and selective muscarinic receptor antagonists. BRIN BD11 cells clearly expressed mRNA encoding m1–m4 whereas m5 was not detected by RT-PCR. Insulin release was measured from BRIN BD11 cells treated with ACh in the presence of muscarinic receptor antagonists at concentrations ranging from 3 nM to 1 μM. 300 nM of muscarinic toxin-3 (M4 antagonist) and 1 μM of methoctramine (M2 antagonist) increased ACh (100 μM) stimulated insulin secretion by 168% and 50% respectively (ANOVA, P<0.05). The antagonists alone had no effect on insulin secretion. In contrast, 300 nM of pirenzepine (M1 antagonist) and 30 nM of hexahydro-sila-difenidol p-fluorohydrochloride (M3 antagonist) inhibited ACh stimulation by 91% and 84% respectively (ANOVA, P<0.01). It is concluded that ACh acts on different receptor subtypes producing both a stimulatory and an inhibitory action on insulin release.     

Equilibrium,kinetic and structural properties of hemoglobin Cranston,an elongated β chain variant

Hemoglobin Cranston has an elongated β subunit owing to a frame shift mutation. Oxygen equilibrium measurements of stripped Hb Cranston³ at 20 °C in the absence of phosphate revealed a high affinity (P₅₀ = 0·2 mm Hg at pH 7), non-co-operative hemoglobin variant with markedly reduced Böhr effect ($\text{?Δ}\text{log}\text{P}{}_{50}\text{Δ}\text{pH}{}_{\mathrm{7–8}}\text{= 0·2}$). The addition of inositol hexaphosphate resulted in an overall decrease in oxygen affinity (P₅₀ = 0·7 mm Hg at pH 7), as well as an increase in co-operativity and Böhr effect ($\text{?Δ}\text{log}\text{P}{}_{50}\text{Δ}\text{pH}{}_{\mathrm{7–8}}\text{= 0·2}$). Rapid mixing and flash photolysis experiments reflected the equilibrium results. Over a pH range from 6 to 9 in the absence of phosphate, the rate of combination of carbon monoxide with Hb Cranston measured by a stopped-flow technique and following full or partial flash photolysis was extremely rapid (l′, l′₄, of ～ 6 × 10⁶m^?1s^?1). In rapid kinetic experiments the addition of inositol hexaphosphate lowered the value of l′ to ～ 0·5 × 10⁶m^?1s^?1 only after prior incubation with the deoxygenated protein. Inositol hexaphosphate had no effect on the rate of recombination of carbon monoxide following either full or partial flash photolysis. Overall oxygen dissociation and oxygen dissociation with carbon monoxide replacement, were measured and found to be slow (k, k₄～ 11 s^?1), consistent with a high affinity hemoglobin. Sedimentation equilibrium experiments revealed that Hb Cranston, at concentrations used in the functional studies, is somewhat less tetrameric than Hb A but nonetheless does not exist solely as a non-co-operative dimer. These kinetic and centrifugational findings in conjunction with X-ray diffraction evidence suggested that a high affinity tetramer of Hb Cranston exists which may equilibrate slowly with inositol hexaphosphate. Oxygen equilibrium measurements, ligand binding kinetics and X-ray diffraction studies on equivalent mixtures of Hb Cranston and Hb A revealed an interaction between these two hemoglobins in vitro that most probably exists in vivo. The presence of asymmetric hybrid molecules, α₂β^Aβ^Cranston, in the difference Fourier maps indicated that the hydrophobic tail of Hb Cranston is accommodated in the central cavity of the hybrid molecule between the two β chains and is relatively protected from the water environment, thus aiding in the stability of Hb Cranston in the red cell.     

Amounts of an estrogen receptor β isoform increased in the theca of preovulatory follicles of sheep

Determination of the specific roles of the estrogen receptor (ER) forms in reproductive processes of different species remains incomplete. In the present experiment, cellular localization and changes in relative amounts of the ERα and ERβ in late developing ovarian follicles, oviduct, and uterus were determined during the follicular phase of the estrous cycle in sheep. Ewes in mid-luteal phase were treated with prostaglandin F(2α) (PG) to induce luteolysis and control the onset of the follicular phase. The oviducts, uterus, and the ovaries were collected at 0 (ewes not treated with PG), 4, 18, and 36 h after PG treatment (early, mid, and late follicular phase, respectively) and processed to evaluate the ERs using immunohistochemical (IHC) procedures. The ERα was localized to nuclei of granulosa cells of late developing follicles and most cells of the oviduct and uterus. The ERβ was detected only in ovarian follicles using two antibodies directed to different regions of the ERβ. Western immunoblotting demonstrated that the antibody directed against the N-terminal region of the ERβ detected one isoform (approximately 53 kDa) whereas the antibody directed against the C-terminus detected two ERβ isoforms (approximately 53 kDa and 59 kDa). Western and IHC results combined indicated presence of the 59 kDa ERβ in granulosa cells and the 53 kDa ERβ in both granulosa and theca cells. Relative amounts (immunostaining intensity) of the ERα increased (P<.05) in granulosa cells of preovulatory follicles and in the isthmian muscularis of the oviduct at the late follicular phase. Amounts of the ERα in the mucosal epithelium of the oviductal regions (isthmus, ampulla, and infundibulum), and in various uterine cell types (glandular and luminal epithelia, endometrial stromal cells, and myometrium) did not change (P>.05) throughout the follicular phase. A major increase (four-fold) in expression of the 53 kDa ERβ in the theca and a less pronounced increase in the granulosa occurred at the late follicular phase. The ERα is broadly expressed in reproductive organs of sheep and is upregulated only in few cell types during the late follicular phase. Immunoreactive ERβ was detected only in the ovary. Important estrogen actions in theca cells during preovulatory follicular development likely occur in association with a major increase in expression of an ERβ isoform.     

Low dose β blockade in acute stroke (“BEST” trial): an evaluation

The β blocker stroke (“BEST”) trial was designed to see if the apparent protective effect of propranolol on cerebral function in patients with subarachnoid haemorrhage applied also to patients suffering from acute stroke. Three hundred and two conscious patients with clinically diagnosed hemispheric strokes sustained within the past 48 hours were randomly assigned to receive atenolol, propranolol, or matching placebo capsules for three weeks. More early deaths occurred among the patients allocated to receive β blockers, but this was largely explained by differences in the initial characteristics of the patients among the different treatment groups. By contrast, the outcome in a further 60 patients, who had been taking β blockers at the time of their stroke but were otherwise similar to the patients in the trial, was considerably better, suggesting that prior treatment with β blockers might be protective.The search for an effective medical treatment for acute stroke must continue. The approach used here, in which neurological outcome was assessed in a modest number of patients with a view to proceeding subsequently to a full scale trial of functional outcome, allows practical benefits of a treatment to be evaluated under realistic conditions and an ineffective treatment to be eliminated without undue cost.     

The Metalloprotease Meprin β Generates Amino Terminal-truncated Amyloid β Peptide Species

The amyloid β (Aβ) peptide, which is abundantly found in the brains of patients suffering from Alzheimer disease, is central in the pathogenesis of this disease. Therefore, to understand the processing of the amyloid precursor protein (APP) is of critical importance. Recently, we demonstrated that the metalloprotease meprin β cleaves APP and liberates soluble N-terminal APP (N-APP) fragments. In this work, we present evidence that meprin β can also process APP in a manner reminiscent of β-secretase. We identified cleavage sites of meprin β in the amyloid β sequence of the wild type and Swedish mutant of APP at positions p1 and p2, thereby generating Aβ variants starting at the first or second amino acid residue. We observed even higher kinetic values for meprin β than BACE1 for both the wild type and the Swedish mutant APP form. This enzymatic activity of meprin β on APP and Aβ generation was also observed in the absence of BACE1/2 activity using a β-secretase inhibitor and BACE knock-out cells, indicating that meprin β acts independently of β-secretase.     

Type II phosphatidylinositol 4-kinase β is an integral signaling component of early T cell activation mechanisms

The early signaling events in T cell activation through CD3 receptor include a rapid change in intra cellular free calcium concentration and reorganization of actin cytoskeleton. Phosphatidylinositol 4-kinases (PtdIns 4-kinases) are implicated as key components in these early signaling events. The role of type II PtdIns 4-kinase β in CD3 receptor signaling was investigated with the help of short hairpin RNA sequences. Cross-linking of CD3 receptors on Jurkat T Cells with monoclonal antibodies showed an early increase in type II PtdIns 4-kinase activity and co-localization of type II PtdIns 4-kinase β with CD3 ζ. Transfection of Jurkat T Cells with shRNAs inhibited CD3 receptor mediated type II PtdIns 4-kinase activation with a concomitant reduction in intra cellular calcium release, suggesting a role for type II PtdIns 4-kinase β in CD3 receptor signal transduction. Knock-down of type II PtdIns 4-kinase β with shRNAs also correlated with a decrease in PtdIns 4-kinase activity in cytoskeleton fractions and reduced adhesion to matrigel surfaces. These results indicate that type II PtdIns 4-kinase β is a key component in early T cell activation signaling cascades.     

Brucella β 1,2 Cyclic Glucan Is an Activator of Human and Mouse Dendritic Cells

Bacterial cyclic glucans are glucose polymers that concentrate within the periplasm of alpha-proteobacteria. These molecules are necessary to maintain the homeostasis of the cell envelope by contributing to the osmolarity of Gram negative bacteria. Here, we demonstrate that Brucella β 1,2 cyclic glucans are potent activators of human and mouse dendritic cells. Dendritic cells activation by Brucella β 1,2 cyclic glucans requires TLR4, MyD88 and TRIF, but not CD14. The Brucella cyclic glucans showed neither toxicity nor immunogenicity compared to LPS and triggered antigen-specific CD8⁺ T cell responses in vivo. These cyclic glucans also enhanced antigen-specific CD4⁺ and CD8⁺ T cell responses including cross-presentation by different human DC subsets. Brucella β 1,2 cyclic glucans increased the memory CD4⁺ T cell responses of blood mononuclear cells exposed to recombinant fusion proteins composed of anti-CD40 antibody and antigens from both hepatitis C virus and Mycobacterium tuberculosis. Thus cyclic glucans represent a new class of adjuvants, which might contribute to the development of effective antimicrobial therapies.     

The hairpin conformation of the amyloid β peptide is an important structural motif along the aggregation pathway

The amyloid β (Aβ) peptides are 39–42 residue-long peptides found in the senile plaques in the brains of Alzheimer’s disease (AD) patients. These peptides self-aggregate in aqueous solution, going from soluble and mainly unstructured monomers to insoluble ordered fibrils. The aggregation process(es) are strongly influenced by environmental conditions. Several lines of evidence indicate that the neurotoxic species are the intermediate oligomeric states appearing along the aggregation pathways. This minireview summarizes recent findings, mainly based on solution and solid-state NMR experiments and electron microscopy, which investigate the molecular structures and characteristics of the Aβ peptides at different stages along the aggregation pathways. We conclude that a hairpin-like conformation constitutes a common motif for the Aβ peptides in most of the described structures. There are certain variations in different hairpin conformations, for example regarding H-bonding partners, which could be one reason for the molecular heterogeneity observed in the aggregated systems. Interacting hairpins are the building blocks of the insoluble fibrils, again with variations in how hairpins are organized in the cross-section of the fibril, perpendicular to the fibril axis. The secondary structure propensities can be seen already in peptide monomers in solution. Unfortunately, detailed structural information about the intermediate oligomeric states is presently not available. In the review, special attention is given to metal ion interactions, particularly the binding constants and ligand structures of Aβ complexes with Cu(II) and Zn(II), since these ions affect the aggregation process(es) and are considered to be involved in the molecular mechanisms underlying AD pathology.     

Silymarin attenuated the amyloid β plaque burden and improved behavioral abnormalities in an Alzheimer's disease mouse model

Alzheimer's disease (AD) is characterized by progressive cognitive impairment and the formation of senile plaques. Silymarin, an extract of milk thistle, has long been used as a medicinal herb for liver diseases. Here we report marked suppression of amyloid β-protein (Aβ) fibril formation and neurotoxicity in PC12 cells after silymarin treatment in vitro. In vivo studies had indicated a significant reduction in brain Aβ deposition and improvement in behavioral abnormalities in amyloid precursor protein (APP) transgenic mice that had been preventively treated with a powdered diet containing 0.1% silymarin for 6 months. The silymarin-treated APP mice also showed less anxiety than the vehicle-treated APP mice. These behavioral changes were associated with a decline in Aβ oligomer production induced by silymarin intake. These results suggest that silymarin is a promising agent for the prevention of AD.     

Chemical Shifts Provide Fold Populations and Register of β Hairpins and β Sheets

A detailed analysis of peptide backbone amide (H_N) and Hα chemical shifts reveals a consistent pattern for β hairpins and three-stranded β sheets. The Hα’s at non-hydrogen-bonded strand positions are inwardly directed and shifted downfield ~1 ppm due largely to an anisotropy contribution from the cross-strand amide function. The secondary structure associated Hα shift deviations for the H-bonded strand positions are also positive but much smaller (0.1–0.3 ppm) and the turn residues display negative Hα chemical shift deviations (CSDs). The pattern of (H_N) shift deviations is an even better indicator of both hairpin formation and register, with the cross-strand H-bonded sites shifted downfield (also by ~1 ppm) and with diagnostic values for the first turn residue and the first strand position following the turn. These empirical observations, initially made for [2:2]/[2:4]-type-I' and -II' hairpins, are rationalized and can be extended to the analysis of other turns, hairpin classes ([3:5], [4:4]/[4:6]), and three-stranded peptide β-sheet models. The Hα’s at non-hydrogen-bonded sites and (H_N)’s in the intervening H-bonded sites provide the largest and most dependable measures of hairpin structuring and can be used for melting studies; however the intrinsic temperature dependence of (H_N) shifts deviations needs to reflect the extent of solvent sequestration in the folded state. Several observations made in the course of this study provide insights into β-sheet folding mechanisms: (1) The magnitude of the (H_N) shifts suggests that the cross-strand H-bonds in peptide hairpins are as short as those in protein β sheets. (2) Even L-Pro-Gly turns, which are frequently used in unfolded controls for hairpin peptides, can support hairpin populations in aqueous fluoroalcohol media. (3) The good correlation between hairpin population estimates from cross-strand H-bonded (H_N) shift deviations, Hα shift deviations, and structuring shifts at the turn locus implies that hairpin folding transitions approximate two-state behavior. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Substrate-induced DNA Polymerase β Activation

DNA polymerases and substrates undergo conformational changes upon forming protein-ligand complexes. These conformational adjustments can hasten or deter DNA synthesis and influence substrate discrimination. From structural comparison of binary DNA and ternary DNA-dNTP complexes of DNA polymerase β, several side chains have been implicated in facilitating formation of an active ternary complex poised for chemistry. Site-directed mutagenesis of these highly conserved residues (Asp-192, Arg-258, Phe-272, Glu-295, and Tyr-296) and kinetic characterization provides insight into the role these residues play during correct and incorrect insertion as well as their role in conformational activation. The catalytic efficiencies for correct nucleotide insertion for alanine mutants were wild type ∼ R258A > F272A ∼ Y296A > E295A > D192A. Because the efficiencies for incorrect insertion were affected to about the same extent for each mutant, the effects on fidelity were modest (<5-fold). The R258A mutant exhibited an increase in the single-turnover rate of correct nucleotide insertion. This suggests that the wild-type Arg-258 side chain generates a population of non-productive ternary complexes. Structures of binary and ternary substrate complexes of the R258A mutant and a mutant associated with gastric carcinomas, E295K, provide molecular insight into intermediate structural conformations not appreciated previously. Although the R258A mutant crystal structures were similar to wild-type enzyme, the open ternary complex structure of E295K indicates that Arg-258 stabilizes a non-productive conformation of the primer terminus that would decrease catalysis. Significantly, the open E295K ternary complex binds two metal ions indicating that metal binding cannot overcome the modified interactions that have interrupted the closure of the N-subdomain.