HLA‐DQ7 β1 and β2 derived peptides as immunomodulators

Modulation of protein–protein interactions involved in the immune system by using small molecular mimics of the contact interfaces may lead to the blockage of the autoimmune response and the development of drugs for immunotherapy. The nonpolymorphic β‐regions, exposed to the microenvironment, of the modeled HLA‐DQ7, which is genetically linked to autoimmune diseases, were determined. Peptides 132–141 and 58–67, located at the β₁ and β₂ domains of HLA‐DQ7, respectively, were tested for their involvement in the interactions with CD4⁺ T lymphocytes. Linear, cyclic, and dimeric analogs that mimic the exposed surfaces of HLA‐DQ7 were designed and synthesized. Their immunosuppressory activities, found in the secondary, humoral immune response to sheep erythrocytes (SRBC) in mice in vitro, ranged from 11% to 53%. The significance of the total charge of the peptides, the pattern of the hydrogen bonding, and the presence of secondary structure were investigated in relation to the immunomodulatory effect of the peptides. Two dimeric analogs of the HLA‐DQ7 58–67 fragment, consisting of the two monomers covalently linked by a polyethylene glycol (PEG) spacer, able to mimic the superdimers, were also synthesized and studied. As the 58–67 segment is located at the β₁ region of HLA‐DQ7, close to the major histocompatibility complex (MHC) groove, one may assume that the 58–67 peptide could accommodate the association between T‐cell receptor (TCR) and human leukocyte antigen (HLA) by activating a co‐stimulatory molecule of the TCR/HLA interaction. This hypothesis is supported by the confocal laser image of the fluorescein‐labeled 58–67 peptide and by the fact that it is an immunostimulator at low concentration. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Monte Carlo simulation of 4‐α‐glucanotransferase reaction

4‐α‐Glucanotransferase (GTase, D ‐enzyme) catalyzes disproportionation between two short polymers of maltooligosaccharides linked by α‐1,4‐glucoside bonds. Using action modes of the potato GTase for the donor and acceptor substrates, the Monte Carlo method was applied to simulate the GTase reaction. The simulation starts from a single enzyme molecule and a finite number (10⁵) of substrate molecules. All selection processes were performed using random numbers produced by computer. The initial substrates were from trimer to 10‐mer. In every case, the final stage was the steady‐state distribution of polymers. The steady‐state distribution by the potato GTase reaction was different from those by the hypothetical random disproportionation reaction. The simulated data from the reaction of potato GTase and trimer almost quantitatively agreed with experimental data. The mechanism of the GTase reaction was accumulation of probabilistic processes and was well simulated by the Monte Carlo method. GTase randomizes the overall distribution of chain length of the substrate. Therefore the GTase reaction is an entropy‐driven process. © 1999 John Wiley & Sons, Inc. Biopoly 50: 145–151, 1999     

Integrin αvβ3 enhances the suppressive effect of interferon‐γ on hematopoietic stem cells

Hematopoietic homeostasis depends on the maintenance of hematopoietic stem cells (HSCs), which are regulated within a specialized bone marrow (BM) niche. When HSC sense external stimuli, their adhesion status may be critical for determining HSC cell fate. The cell surface molecule, integrin αvβ3, is activated through HSC adhesion to extracellular matrix and niche cells. Integrin β3 signaling maintains HSCs within the niche. Here, we showed the synergistic negative regulation of the pro‐inflammatory cytokine interferon‐γ (IFNγ) and β3 integrin signaling in murine HSC function by a novel definitive phenotyping of HSCs. Integrin αvβ3 suppressed HSC function in the presence of IFNγ and impaired integrin β3 signaling mitigated IFNγ‐dependent negative action on HSCs. During IFNγ stimulation, integrin β3 signaling enhanced STAT1‐mediated gene expression via serine phosphorylation. These findings show that integrin β3 signaling intensifies the suppressive effect of IFNγ on HSCs, which indicates that cell adhesion via integrin αvβ3 within the BM niche acts as a context‐dependent signal modulator to regulate the HSC function under both steady‐state and inflammatory conditions.     

Folding type-specific secondary structure propensities of amino acids,derived from α-helical, β-sheet, α/β, and α+β proteins of known structures

Folding type-specific secondary structure propensities of 20 naturally occurring amino acids have been derived from α-helical, β-sheet, α/β, and α+β proteins of known structures. These data show that each residue type of amino acids has intrinsic propensities in different regions of secondary structures for different folding types of proteins. Each of the folding types shows markedly different rank ordering, indicating folding type-specific effects on the secondary structure propensities of amino acids. Rigorous statistical tests have been made to validate the folding type-specific effects. It should be noted that α and β proteins have relatively small α-helices and β-strands forming propensities respectively compared with those of α+β and α/β proteins. This may suggest that, with more complex architectures than α and β proteins, α+β and α/β proteins require larger propensities to distinguish from interacting α-helices and β-strands. Our finding of folding type-specific secondary structure propensities suggests that sequence space accessible to each folding type may have differing features. Differing sequence space features might be constrained by topological requirement for each of the folding types. Almost all strong β-sheet forming residues are hydrophobic in character regardless of folding types, thus suggesting the hydrophobicities of side chains as a key determinant of β-sheet structures. In contrast, conformational entropy of side chains is a major determinant of the helical propensities of amino acids, although other interactions such as hydrophobicities and charged interactions cannot be neglected. These results will be helpful to protein design, class-based secondary structure prediction, and protein folding. © 1998 John Wiley & Sons, Inc. Biopoly 45: 35–49, 1998     

Characterization of an Importin α/β‐recognized nuclear localization signal in β‐dystroglycan

β‐dystroglycan (β‐DG) is a widely expressed transmembrane protein that plays important roles in connecting the extracellular matrix to the cytoskeleton, and thereby contributing to plasma membrane integrity and signal transduction. We previously observed nuclear localization of β‐DG in cultured cell lines, implying the existence of a nuclear targeting mechanism that directs it to the nucleus instead of the plasma membrane. In this study, we delineate the nuclear import pathway of β‐DG, characterizing a functional nuclear localization signal (NLS) in the β‐DG cytoplasmic domain, within amino acids 776–782. The NLS either alone or in the context of the whole β‐DG protein was able to target the heterologous GFP protein to the nucleus, with site‐directed mutagenesis indicating that amino acids R⁷⁷⁹ and K⁷⁸⁰ are critical for NLS functionality. The nuclear transport molecules Importin (Imp)α and Impβ bound with high affinity to the NLS of β‐DG and were found to be essential for NLS‐dependent nuclear import in an in vitro reconstituted nuclear transport assay; cotransfection experiments confirmed the dependence on Ran for nuclear accumulation. Intriguingly, experiments suggested that tyrosine phosphorylation of β‐DG may result in cytoplasmic retention, with Y⁸⁹² playing a key role. β‐DG thus follows a conventional Impα/β‐dependent nuclear import pathway, with important implications for its potential function in the nucleus. J. Cell. Biochem. 110: 706–717, 2010. © 2010 Wiley‐Liss, Inc.     

Tertiary structure prediction of mixed α/β proteins via energy minimization

We describe an improved algorithm for protein structure prediction, assuming that the location of secondary structural elements is known, with particular focus on prediction for proteins containing β-strands. Hydrogen bonding terms are incorporated into the potential function, supplementing our previously developed residue-residue potential which is based on a combination of database statistics and an excluded volume term. Two small mixed α/β proteins, 1-CTF and BPTI, are studied. In order to obtain native-like structures, it is necessary to allow the β-strands in BPTI to distort substantially from an ideal geometry, and an automated algorithm to carry this out efficiently is presented. Simulated annealing Monte Carlo methods, which contain a genetic algorithm component as well, are used to produce an ensemble of low-energy structures. For both proteins, a cluster of structures with low RMS deviation from the native structure is generated and the energetic ranking of this cluster is in the top 2 or 3 clusters obtained from simulations. These results are encouraging with regard to the possibility of constructing a robust procedure for tertiary folding which is applicable to β-strand containing proteins. Proteins 33:240–252, 1998. © 1998 Wiley-Liss, Inc.     

17α-Ethyl-5β-estrane-3α,17β-diol, a biological marker for the abuse of norethandrolone and ethylestrenol in slaughter cattle

The metabolism of the illegal growth promoter ethylestrenol (EES) was evaluated in bovine liver cells and subcellular fractions of bovine liver preparations. Incubations with bovine microsomal preparations revealed that EES is extensively biotransformed into norethandrolone (NE), another illegal growth promoter. Furthermore, incubations of monolayer cultures of hepatocytes with NE indicated that NE itself is rapidly reduced to 17α-ethyl-5β-estrane-3α,17β-diol (EED). In vivo tests confirmed that, after administration of either EES or NE, EED is excreted as a major metabolite. Therefore, it was concluded that, both in urine and faeces samples, EED can be used as a biological marker for the illegal use of EES and/or NE. Moreover, by monitoring EED in urine or faeces samples, the detection period after NE administration is significantly prolonged. These findings were further confirmed by three cases of norethandrolone abuse in a routine screening program for forbidden growth promoters.     

Coexistence of β1- and β2-Adrenoceptors in the Melanophore of the Goby Tridentiger obscurus*

The effects of β-adrenergic agonists and antagonists on the pigmentary state of denervated melanophores in isolated, split, caudal fins of the goby Tridentiger obscurus were examined to investigate the function and the subtype of the β-adrenoceptors of the melanophores. Salbutamol, terbutaline, and dobutamine partially inhibited the pigment-aggregating response of melanophores to norepinephrine. The effects of these β-agonists were inhibited by propranolol. It was confirmed that the melanophores possess both α-and β-adrenoceptors, and that the activation of the β-adrenoceptors induces the dispersion of pigment in the melanophores. Norepinephrine, epinephrine, isoproterenol, dobutamine, salbutamol, and terbutaline evoked the dispersion of pigment in the melanophores in which pigment had previously been aggregated by treatment with verapamil in the presence of phentolamine. The pigment-dispersing effects of two β₁-selective agonists, norepinephrine and dobutamine, were effectively inhibited by metoprolol, a selective antagonist of β₁-receptors. By contrast, the pigment-dispersing effects of two β₂-selective agonists, salbutamol and terbutaline, were not inhibited by metoprolol. Both the effects of nonselective agonists, epinephrine and isoproterenol, were partially inhibited by metoprolol. The actions of all of the β-agonists used were effectively inhibited by propranolol, and they were partially inhibited by butoxamine. These results suggest coexistence of β₁- and β₂-adrenoceptors in the melanophores. The relative numbers of β₁- and β₂-adrenoreceptors as a percentage of the total population of β-adrenoceptors were estimated to be 18.6% and 81.4%, respectively, from analyses of Hofstee plots of the effects of the β-agonists on the melanophores in the presence of butoxamine or metoprolol.     

Activation mechanisms of αVβ3 integrin by binding to fibronectin: A computational study

Integrin αVβ3 plays an important role in regulating cellular activities and in human diseases. Although the structure of αVβ3 has been studied by crystallography and electron microscopy, the detailed activation mechanism of integrin αVβ3 induced by fibronectin remains unclear. In this study, we investigated the conformational and dynamical motion changes of Mn²⁺‐bound integrin αVβ3 by binding to fibronectin with molecular dynamics simulations. Results showed that fibronectin binding to integrin αVβ3 caused the changes of the conformational flexibility of αVβ3 domains, the essential mode of motion for the domains of αV subunit and β3 subunit and the degrees of correlated motion of residues between the domains of αV subunit and β3 subunit of integrin αVβ3. The angle of Propeller domain with respect to the Calf‐2 domain of αV subunit and the angle of Hybrid domain with respect to βA domain of β3 subunit significantly increased when integrin αVβ3 was bound to fibronectin. These changes could result in the conformational change tendency of αVβ3 from a bend conformation to an extended conformation and lead to the open swing of Hybrid domain relative to βA domain of β3 subunit, which have demonstrated their importance for αVβ3 activation. Fibronectin binding to integrin αVβ3 significantly decreased the relative position of α1 helix to βA domain and that to metal ion‐dependent adhesion site, stabilized Mn²⁺ ions binding in integrin αVβ3 and changed fibronectin conformation, which are important for αVβ3 activation. Results from this study provide important molecular insight into the “outside‐in” activation mechanism of integrin αVβ3 by binding to fibronectin.     

The expression of α2β1 integrin and α smooth muscle actin in fibroblasts grown on collagen

The maturation of connective tissue involves the organization of collagen fibres by resident fibroblasts. Fibroblast attachment to collagen has been demonstrated to involve cell surface receptors, integrins of the β₁ family. Integrins are associated with cytoplasmic actin of microfilaments either directly or through focal adhesions. The major actin isoform of fibroblast microfilaments is β actin and to a lesser extent α smooth muscle (α SM) actin. Cultured human dermal fibroblasts derived from adult dermis, newborn foreskin or keloid scar were grown on either uncoated or collagen-coated surfaces. The expression and synthesis of both α₂β₁ integrin and α SM actin were followed by immunohistology and immunoprecipitation. Fibroblasts on uncoated surfaces expressed little α₂β₁ integrin on their surface, while 20 per cent of them demonstrated α SM actin within microfilaments. Fibroblasts grown on a collagen-coated surface minimally expressed α SM actin in microfilament structures and a majority of the cells were positive for α₂β₁ integrin on their membranes. Using [³⁵S]-methionine incorporation and immunoprecipitation, it was shown that fibroblasts grown in uncoated dishes synthesized more α SM actin than fibroblasts grown on collagen-coated dishes. In contrast, fibroblasts grown on collagen coated dishes synthesized more α₂β₁ integrin compared to the same cells grown on uncoated dishes. Fibroblasts maintained on a type I collagen upregulate the expression and synthesis of α₂β₁ integrin, and downregulate the expression and synthesis of α SM actin. © 1998 John Wiley & Sons, Ltd.     

Synthesis and receptor binding of opioid peptide analogues containing β3‐homo‐amino acids

β‐Amino acids containing hybrid peptides and β‐peptides show great potential as peptidomimetics. In this paper we describe the synthesis and affinity toward the µ‐ and δ‐opioid receptors of β‐peptides, analogues of Leu‐enkephalin, deltorphin I, dermorphin and α,β‐hybrides, analogues of deltorphin I. Substitution of α‐amino acid residues with β³‐homo‐amino acid residues, in general resulted in decrease of affinity to opioid receptors. However, the incorporation β³h‐D ‐Ala in position 2 or β³hPhe in position 3 of deltorphin I resulted in potent and selective ligand for δ‐opioid receptor. The NMR studies of β‐deltorphin I analogue suggest that conformational motions in the central part of the peptide backbone are partially restricted and some conformational preferences can be expected. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Studies on the 5α-androstane-3β, 17β-diol-6α-hydroxylase and on the 5α-androstane-3β, 17β-diol-7α-hydroxylase in anterior hypophysis of male rats.

In anterior pituitaries from male rats, it appeared that 5α-androstane-3β, 17β-diol was quickly metabolized into 5α-androstane-3β,6α-17β-triol and 5α-androstane-3β,7α, 17β-triol by action of 6α- and 7α-hydroxylases. Hydroxysteroid hydroxylases were located in endoplasmic reticulum and were dependent on NADPH⁺. Their optimum pH was 8.0, optima temperature, 37°C, and their apparent Km was 2.7 μM. Hydroxylative reactions were not reversible and not modified by gonadectomy. Hydroxylation seemed an efficient control of the pituitary level of 5α-andros-tane-3β, 17β-diol.     

The role of calcium in apoptosis induced by 7β‐hydroxycholesterol and cholesterol‐5β,6β‐epoxide

Oxysterols, such as 7β‐hydroxy‐cholesterol (7β‐OH) and cholesterol‐5β,6β‐epoxide (β‐epoxide), may have a central role in promoting atherogenesis. This is thought to be predominantly due to their ability to induce apoptosis in cells of the vascular wall and in monocytes/macrophages. Although there has been extensive research regarding the mechanisms through which oxysterols induce apoptosis, much remains to be clarified. Given that experimental evidence has long associated alterations of calcium (Ca²⁺) homeostasis to apoptotic cell death, the aim of the present study was to determine the influence of intracellular Ca²⁺ changes on apoptosis induced by 7β‐OH and β‐epoxide. Ca²⁺ responses in differentiated U937 cells were assessed by epifluorescence video microscopy, using the ratiometric dye fura‐2. Over 15‐min exposure of differentiated U937 cells to 30 μM of 7β‐OH induced a slow but significant rise in fura‐2 ratio. The Ca²⁺ channel blocker nifedipine and the chelating agent EGTA blocked the increase in cytoplasmic Ca²⁺. Moreover, dihydropyridine (DHP) binding sites identified with BODIPY‐FLX‐DHP were blocked following pretreatment with nifedipine, indicating that the influx of Ca²⁺ occurred through L‐type channels. However, following long‐term incubation with 7β‐OH, elevated levels of cytoplasmic Ca²⁺ were not maintained and nifedipine did not provide protection against apoptotic cell death. Our results indicate that the increase in Ca²⁺ may be an initial trigger of 7β‐OH–induced apoptosis, but following chronic exposure to the oxysterol, the influence of Ca²⁺ on apoptotic cell death appears to be less significant. In contrast, Ca²⁺ did not appear to be involved in β‐epoxide–induced apoptosis. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:324–332, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20295     

The stress response neuropeptide CRF increases amyloid‐β production by regulating γ‐secretase activity

The biological underpinnings linking stress to Alzheimer's disease (AD) risk are poorly understood. We investigated how corticotrophin releasing factor (CRF), a critical stress response mediator, influences amyloid‐β (Aβ) production. In cells, CRF treatment increases Aβ production and triggers CRF receptor 1 (CRFR1) and γ‐secretase internalization. Co‐immunoprecipitation studies establish that γ‐secretase associates with CRFR1; this is mediated by β‐arrestin binding motifs. Additionally, CRFR1 and γ‐secretase co‐localize in lipid raft fractions, with increased γ‐secretase accumulation upon CRF treatment. CRF treatment also increases γ‐secretase activity in vitro, revealing a second, receptor‐independent mechanism of action. CRF is the first endogenous neuropeptide that can be shown to directly modulate γ‐secretase activity. Unexpectedly, CRFR1 antagonists also increased Aβ. These data collectively link CRF to increased Aβ through γ‐secretase and provide mechanistic insight into how stress may increase AD risk. They also suggest that direct targeting of CRF might be necessary to effectively modulate this pathway for therapeutic benefit in AD, as CRFR1 antagonists increase Aβ and in some cases preferentially increase Aβ42 via complex effects on γ‐secretase.     

Human α1β3γ2L gamma‐aminobutyric acid type A receptors: High‐level production and purification in a functional state

Gamma‐aminobutyric acid type A receptors (GABA_ARs) are the most important inhibitory chloride ion channels in the central nervous system and are major targets for a wide variety of drugs. The subunit compositions of GABA_ARs determine their function and pharmacological profile. GABA_ARs are heteropentamers of subunits, and (α1)₂(β3)₂(γ2L)₁ is a common subtype. Biochemical and biophysical studies of GABA_ARs require larger quantities of receptors of defined subunit composition than are currently available. We previously reported high‐level production of active human α1β3 GABA_AR using tetracycline‐inducible stable HEK293 cells. Here we extend the strategy to receptors containing three different subunits. We constructed a stable tetracycline‐inducible HEK293‐TetR cell line expressing human (N)–FLAG–α1β3γ2L–(C)–(GGS)₃GK–1D4 GABA_AR. These cells achieved expression levels of 70–90 pmol [³H]muscimol binding sites/15‐cm plate at a specific activity of 15–30 pmol/mg of membrane protein. Incorporation of the γ2 subunit was confirmed by the ratio of [³H]flunitrazepam to [³H]muscimol binding sites and sensitivity of GABA‐induced currents to benzodiazepines and zinc. The α1β3γ2L GABA_ARs were solubilized in dodecyl‐d ‐maltoside, purified by anti‐FLAG affinity chromatography and reconstituted in CHAPS/asolectin at an overall yield of ～30%. Typical purifications yielded 1.0–1.5 nmoles of [³H]muscimol binding sites/60 plates. Receptors with similar properties could be purified by 1D4 affinity chromatography with lower overall yield. The composition of the purified, reconstituted receptors was confirmed by ligand binding, Western blot, and proteomics. Allosteric interactions between etomidate and [³H]muscimol binding were maintained in the purified state.     

TAPP analogs containing β3‐homo‐amino acids: synthesis and receptor binding

β‐Amino acids containing α,β‐hybrid peptides show great potential as peptidomimetics. In this paper, we describe the synthesis and affinity to μ‐opioid and δ‐opioid receptors of α,β‐hybrids, analogs of the tetrapeptide Tyr‐ d ‐Ala‐Phe‐Phe‐NH₂ (TAPP). Each amino acid was replaced with an l ‐ or d ‐β³‐h‐amino acid. All α,β‐hybrids of TAPP analogs were synthesized in solution and tested for affinity to μ‐opioid and δ‐opioid receptors. The analog Tyr‐β³h‐ d ‐Ala‐Phe‐PheNH₂ was found to be as active as the native tetrapeptide. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Induction of mouse β integrin expression following transfection with human α4 chain

We report here an analysis of the expression and function of the α chain of human VLA-4 in stable mouse L cell transfectants and the requirement for the β chain in these processes. L cells were transfected with human α4 cDNA or α4 and human β1 cDNA. Unexpectedly, human α4 cDNA, when transfected alone, could induce de novo surface expression of host β7 and increased expression of host β1. Induction of mouse β7 and β1 surface expression was not due to de novo gene activation, but instead represented α4/β intracellular subunit association and transport to the cell surface. Transfection with human β1 prevented surface expression of mouse β integrins. Whereas human α4 and human β1 subunits associated very tightly in anti-α4 immunoprecipitates, human α4 and mouse β subunits were only partially associated. Furthermore, binding of human/mouse chimeric receptors to recombinant VCAM, a major ligand for α4β7 and α4β1, was very poor, whereas human α4/human β1 receptors bound strongly to VCAM. One α4 transfectant, which exhibited a tight human α4/mouse β1 association, could be induced, but only after PMA activation, to bind strongly to VCAM. These results indicate that α4 subunits have specific affinity for β7 and β1 integrins and require β subunits for surface expression as well as high affinity ligand binding activity. Our results indicate that a tight association between the α4 and β subunit appears to be critical for ligand binding, consistent with a direct as well as regulatory role for the β subunit in ligand binding. Furthermore, these studies demonstrate that expression of foreign recombinant proteins can alter host cell protein expression resulting in de novo surface protein expression. © 1996 Wiley-Liss, Inc.     

Effects of P450 inhibition and induction on the olfactory toxicity of β, β′-iminodipropionitrile (IDPN) in the rat

In addition to the neurotoxic effects of β, β′-iminodipropionitrile (IDPN) which have been previously reported by other investigators, the olfactory toxicity of this compound has recently been uncovered in this laboratory. Due to the apparently conflicting observations that the IDPN-induced lesion in the olfactory mucosa is very focal in nature (suggesting site-specific activation) and the observation by other investigators that the behavioral effects of IDPN appear to be due to the parent compound, we initiated studies into the possible role of the cytochrome P450 enzymes in the olfactory toxicity of IDPN. Immunohistochemical studies with antibodies raised against several different P450 isoforms revealed good correlation between IDPN-induced olfactory mucosal degeneration and the localization of a protein immunoreacting with an antibody to P450 2E1. Enzymatic studies revealed that there is approximately fivefold more ρ-nitrophenol hydroxylation activity in the olfactory mucosa than in the liver on a per milligram microsomal protein basis. Administration of 1% acetone in the drinking water increased the levels of olfactory mucosal 2E1, and the increase in enzyme levels corresponded to increased olfactory toxicity of IDPN; inhibition of P450 activities with either metyrapone or carbon tetrachloride eliminated or significantly decreased the olfactory toxicity of IDPN, respectively. These studies suggest a role for cytochrome P450, specifically the 2E1 isoform, in the activation of IDPN within the nasal mucosa.