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.     

Candidacidal mechanism of a Leu/Lys‐rich α‐helical amphipathic model antimicrobial peptide and its diastereomer composed of D,L‐amino acids

We investigated the mechanism of candidacidal action of a Lys/Leu‐rich α‐helical model antimicrobial peptide (K₉L₈W) and its diastereomeric peptide (D₉‐K₉L₈W) composed of D ,L ‐amino acids. K₉L₈W killed completely Candida albicans within 30 min, but D₉‐K₉L₈W killed only 72% of C. albicans even after 100 min. Tryptophan fluorescence spectroscopy indicated that the fungal cell selectivity of D₉‐K₉L₈W is closely correlated with a selective interaction with the negatively charged PC/PE/PI/ergosterol (5:2.5:2.5:1, w/w/w/w) phospholipids, which mimic the outer leaflet of the plasma membrane of C. albicans. K₉L₈W was able to induce almost 100% calcein leakage from PC/PE/PI/ergosterol (5:2.5:2.5:1, w/w/w/w) liposomes at a peptide:lipid molar ratio of 1:16, whereas D₉‐K₉L₈W caused only 25% dye leakage even at a peptide:lipid molar ratio of 1:2. Confocal laser‐scanning microscopy revealed that FITC‐labeled D₉‐K₉L₈W penetrated the cell wall and cell membrane and accumulated inside the cells, whereas FITC‐labeled K₉L₈W did not penetrate but associated with the membranes. Collectively, our results demonstrated that the candidacidal activity of K₉L₈ W and D₉‐K₉L₈W may be due to the transmembrane pore/channel formation or perturbation of the fungal cytoplasmic membranes and the inhibition of intracellular functions, respectively. Finally, D₉‐K₉L₈W with potent anti‐Candida activity but no hemolytic activity may be potentially a useful lead compound for the development of novel antifungal agents. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Nanostructures from the self‐assembly of α‐helical peptide amphiphiles

Self‐assembly of PAs composed of palmitic acid and several repeated heptad peptide sequences, C₁₅H₃₁CO‐(IEEYTKK)_n‐NH₂ (n = 1–4, represented by PA1–PA4), was investigated systematically. The secondary structures of the PAs were characterized by CD. PA3 and PA4 (n = 3 and 4, respectively) showed an α‐helical structure, whereas PA1 and PA2 (n = 1 and 2, respectively) did not display an α‐helical conformations under the tested conditions. The morphology of the self‐assembled peptides in aqueous medium was studied by transmission electron microscopy. As the number of heptad repeats in the PAs increased, the nanostructure of the self‐assembled peptides changed from nanofibers to nanovesicles. Changes of the secondary structures and the self‐assembly morphologies of PA3 and PA4 in aqueous medium with various cations were also studied. The critical micelle concentrations were determined using a pyrene fluorescence probe. In conclusion, this method may be used to design new peptide nanomaterials. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

IFN‐γ inhibits basal and α‐MSH‐induced melanogenesis

Inflammatory cytokines are closely related to pigmentary changes. In this study, the effects of IFN‐γ on melanogenesis were investigated. IFN‐γ inhibits basal and α‐MSH‐induced melanogenesis in B16 melanoma cells and normal human melanocytes. MITF mRNA and protein expressions were significantly inhibited in response to IFN‐γ. IFN‐γ inhibited CREB binding to the MITF promoter but did not affect CREB phosphorylation. Instead, IFN‐γ inhibited the association of CBP and CREB through the increased association between CREB binding protein (CBP) and STAT1. These findings suggest that IFN‐γ inhibits both basal and α‐MSH‐induced melanogenesis by inhibiting MITF expression. The inhibitory action of IFN‐γ in α‐MSH‐induced melanogenesis is likely to be associated with the sequestration of CBP via the association between CBP and STAT1. These data suggest that IFN‐γ plays a role in controlling inflammation‐ or UV‐induced pigmentary changes.     

Conformations of peptides containing a chiral cyclic α, α‐disubstituted α‐amino acid within the sequence of Aib residues

A single chiral cyclic α,α‐disubstituted amino acid, (3S,4S)‐1‐amino‐(3,4‐dimethoxy)cyclopentanecarboxylic acid [(S,S)‐Ac₅c^dOM], was placed at the N‐terminal or C‐terminal positions of achiral α‐aminoisobutyric acid (Aib) peptide segments. The IR and ¹H NMR spectra indicated that the dominant conformations of two peptides Cbz‐[(S,S)‐Ac₅c^dOM]‐(Aib)₄‐OEt ( 1) and Cbz‐(Aib)₄‐[(S,S)‐Ac₅c^dOM]‐OMe (2) in solution were helical structures. X‐ray crystallographic analysis of 1 and 2 revealed that a left‐handed (M) 3₁₀‐helical structure was present in 1 and that a right‐handed (P) 3₁₀‐helical structure was present in 2 in their crystalline states. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and antimicrobial activity of α‐aminoboronic‐containing peptidomimetics

A library of 175 dipeptidomimetics and tripeptidomimetics containing an α‐amino boronic acid or boronate has been synthesized, and the activity toward Mycobacterium tuberculosis, Candida albicans, Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli and Pseudomonas aeruginosa has been screened. Although there is no clear structure–activity relationship, several compounds exhibit promising activity against different pathogens. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Basic conformers in β‐peptides

The conformation of oligomers of β‐amino acids of the general type Ac‐[β‐Xaa]_n‐NHMe (β‐Xaa = β‐Ala, β‐Aib, and β‐Abu; n = 1–4) was systematically examined at different levels of ab initio molecular orbital theory (HF/6‐31G*, HF/3‐21G). The solvent influence was considered employing two quantum‐mechanical self‐consistent reaction field models. The results show a wide variety of possibilities for the formation of characteristic elements of secondary structure in β‐peptides. Most of them can be derived from the monomer units of blocked β‐peptides with n = 1. The stability and geometries of the β‐peptide structures are considerably influenced by the side‐chain positions, by the configurations at the C^α‐ and C^β‐atoms of the β‐amino acid constituents, and especially by environmental effects. Structure peculiarities of β‐peptides, in particular those of various helix alternatives, are discussed in relation to typical elements of secondary structure in α‐peptides. © 1999 John Wiley & Sons, Inc. Biopoly 50: 167–184, 1999     

Efficient synthesis of polypeptide‐α‐thioester by the method combining polypeptide expression and chemical activation for the semi‐synthesis of interferon‐γ having oligosaccharides

In order to synthesize interferon‐γ glycoform having an oligosaccharide at the 97 position by a semi‐synthetic method, interferon‐γ‐polypeptide‐(1–94)‐α‐hydrazide was prepared by the specific Cys‐cyanylation of polypeptide‐(1–94)‐Cys‐His₆ expressed from E. coli and subsequent hydrazinolysis in 22% yield (two steps). This polypeptide‐α‐hydrazide was then converted into corresponding polypeptide‐α‐thioester under NaNO₂/acid conditions followed by thiolysis in 83% yield. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

5α‐Androst‐16‐en‐3α‐ol β‐D‐Glucuronide,Precursor of 5α‐Androst‐16‐en‐3α‐ol in Human Sweat

5α‐Androst‐16‐en‐3α‐ol (α‐androstenol) is an important contributor to human axilla sweat odor. It is assumed that α‐andostenol is excreted from the apocrine glands via a H₂O‐soluble conjugate, and this precursor was formally characterized in this study for the first time in human sweat. The possible H₂O‐soluble precursors, sulfate and glucuronide derivatives, were synthesized as analytical standards, i.e., α‐androstenol, β‐androstenol sulfates, 5α‐androsta‐5,16‐dien‐3β‐ol (β‐androstadienol) sulfate, α‐androstenol β‐glucuronide, α‐androstenol α‐glucuronide, β‐androstadienol β‐glucuronide, and α‐androstenol β‐glucuronide furanose. The occurrence of α‐androstenol β‐glucuronide was established by ultra performance liquid chromatography (UPLC)/MS (heated electrospray ionization (HESI)) in negative‐ion mode in pooled human sweat, containing eccrine and apocrine secretions and collected from 25 female and 24 male underarms. Its concentration was of 79 ng/ml in female secretions and 241 ng/ml in male secretions. The release of α‐androstenol was observed after incubation of the sterile human sweat or α‐androstenol β‐glucuronide with a commercial glucuronidase enzyme, the urine‐isolated bacteria Streptococcus agalactiae, and the skin bacteria Staphylococcus warneri DSM 20316, Staphylococcus haemolyticus DSM 20263, and Propionibacterium acnes ATCC 6919, reported to have β‐glucuronidase activities. We demonstrated that if α‐ and β‐androstenols and androstadienol sulfates were present in human sweat, their concentrations would be too low to be considered as potential precursors of malodors; therefore, the H₂O‐soluble precursor of α‐androstenol in apocrine secretion should be a β‐glucuronide.     

β‐Amino acids containing peptides and click‐cyclized peptide as β‐turn mimics: a comparative study with ‘conventional’ lactam‐ and disulfide‐bridged hexapeptides

The increasing interest in click chemistry and its use to stabilize turn structures led us to compare the propensity for β‐turn stabilization of different analogs designed as mimics of the β‐turn structure found in tendamistat. The β‐turn conformation of linear β‐amino acid‐containing peptides and triazole‐cyclized analogs were compared to ‘conventional’ lactam‐ and disulfide‐bridged hexapeptide analogs. Their 3D structures and their propensity to fold in β‐turns in solution, and for those not structured in solution in the presence of α‐amylase, were analyzed by NMR spectroscopy and by restrained molecular dynamics with energy minimization. The linear tetrapeptide Ac‐Ser‐Trp‐Arg‐Tyr‐NH₂ and both the amide bond‐cyclized, c[Pro‐Ser‐Trp‐Arg‐Tyr‐D ‐Ala] and the disulfide‐bridged, Ac‐c[Cys‐Ser‐Trp‐Arg‐Tyr‐Cys]‐NH₂ hexapeptides adopt dominantly in solution a β‐turn conformation closely related to the one observed in tendamistat. On the contrary, the β‐amino acid‐containing peptides such as Ac‐(R)‐β³‐hSer‐(S)‐Trp‐(S)‐β³‐hArg‐(S)‐β³‐hTyr‐NH₂, and the triazole cyclic peptide, c[Lys‐Ser‐Trp‐Arg‐Tyr‐βtA]‐NH₂, both specifically designed to mimic this β‐turn, do not adopt stable structures in solution and do not show any characteristics of β‐turn conformation. However, these unstructured peptides specifically interact in the active site of α‐amylase, as shown by TrNOESY and saturation transfer difference NMR experiments performed in the presence of the enzyme, and are displaced by acarbose, a specific α‐amylase inhibitor. Thus, in contrast to amide‐cyclized or disulfide‐bridged hexapeptides, β‐amino acid‐containing peptides and click‐cyclized peptides may not be regarded as β‐turn stabilizers, but can be considered as potential β‐turn inducers. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Dehydroepiandrosterone 7α-hydroxylation in human tissues: Possible interference with type 1 11β-hydroxysteroid dehydrogenase-mediated processes

Dehydroepiandrosterone (DHEA) is 7α-hydroxylated by the cytochome P450 7B1 (CYP7B1) in the human brain and liver. This produces 7α-hydroxy-DHEA that is a substrate for 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) which exists in the same tissues and carries out the inter-conversion of 7α- and 7β-hydroxy-DHEA through a 7-oxo-intermediary. Since the role of 11β-HSD1 is to transform the inactive cortisone into active cortisol, its competitive inhibition by 7α-hydroxy-DHEA may support the paradigm of native anti-glucocorticoid arising from DHEA. Therefore, our objective was to use human tissues to assess the presences of both CYP7B1 and 11β-HSD1. Human skin was selected then and used to test its ability to produce 7α-hydroxy-DHEA, and to test the interference of 7α- and 7β-hydroxy-DHEA and 7-oxo-DHEA with the 11β-HSD1-mediated oxidoreduction of cortisol and cortisone. Immuno-histochemical studies showed the presence of both CYP7B1 and 11β-HSD1 in the liver, skin and tonsils. DHEA was readily 7α-hydroxylated when incubated using skin slices. A S9 fraction of dermal homogenates containing the 11β-HSD1 carried out the oxidoreduction of cortisol and cortisone. Inhibition of the cortisol oxidation by 7α-hydroxy-DHEA and 7β-hydroxy-DHEA was competitive with a K_i at 1.85 ± 0.495 and 0.255 ± 0.005 μM, respectively. Inhibition of cortisone reduction by 7-oxo-DHEA was of a mixed type with a K_i at 1.13 ± 0.15 μM. These findings may support the previously proposed native anti-glucocorticoid paradigm and suggest that the 7α-hydroxy-DHEA production is a key for the fine tuning of glucocorticoid levels in tissues.     

Kinetics of the competitive reactions of isomerization and peptide bond cleavage at l‐α‐ and d‐β‐aspartyl residues in an αA‐crystallin fragment

d ‐β‐aspartyl (Asp) residue has been found in a living body such as aged lens crystallin, although l ‐α‐amino acids are constituents in natural proteins. Isomerization from l ‐α‐ to d ‐β‐Asp probably modulates structures to affect biochemical reactions. At Asp residue, isomerization and peptide bond cleavage compete with each other. To gain insight into how fast each reaction proceeds, the analysis requires the consideration of both pathways simultaneously and independently. No information has been provided, however, about these competitive processes because each reaction has been studied separately. The contribution of Asp isomers to the respective pathways has still been veiled. In this work, the two competitive reactions, isomerization and spontaneous peptide bond cleavage at Asp residue, were simultaneously observed and compared in an αA‐crystallin fragment, S⁵¹LFRTVLD⁵⁸SG⁶⁰ containing l ‐α‐ and d ‐β‐Asp58 isomers. The kinetics showed that the formation of l ‐ and d ‐succinimide (Suc) intermediate, as a first step of isomerization, was comparable at l ‐α‐ and d ‐β‐Asp. Although l ‐Suc was converted to l ‐β‐Asp, d ‐Suc was liable to return to the original d ‐β‐Asp, the reverse reaction marked enough to consider d ‐β‐Asp as apparently stable. d ‐β‐Asp was also resistant to the peptide bond cleavage. Such apparent less reactivity is probably the reason for gradual and abnormal accumulation of d ‐β‐Asp in a living body under physiological conditions. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

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     

α1 adrenoceptor activation by norepinephrine inhibits LPS‐induced cardiomyocyte TNF‐α production via modulating ERK1/2 and NF‐κB pathway

Cardiomyocyte tumour necrosis factor α (TNF‐α) production contributes to myocardial depression during sepsis. This study was designed to observe the effect of norepinephrine (NE) on lipopolysaccharide (LPS)‐induced cardiomyocyte TNF‐α expression and to further investigate the underlying mechanisms in neonatal rat cardiomyocytes and endotoxaemic mice. In cultured neonatal rat cardiomyocytes, NE inhibited LPS‐induced TNF‐α production in a dose‐dependent manner. α₁‐ adrenoceptor (AR) antagonist (prazosin), but neither β₁‐ nor β₂‐AR antagonist, abrogated the inhibitory effect of NE on LPS‐stimulated TNF‐α production. Furthermore, phenylephrine (PE), an α₁‐AR agonist, also suppressed LPS‐induced TNF‐α production. NE inhibited p38 phosphorylation and NF‐κB activation, but enhanced extracellular signal‐regulated kinase 1/2 (ERK1/2) phosphorylation and c‐Fos expression in LPS‐treated cardiomyocytes, all of which were reversed by prazosin pre‐treatment. To determine whether ERK1/2 regulates c‐Fos expression, p38 phosphorylation, NF‐κB activation and TNF‐α production, cardiomyocytes were also treated with U0126, a selective ERK1/2 inhibitor. Treatment with U0126 reversed the effects of NE on c‐Fos expression, p38 mitogen‐activated protein kinase (MAPK) phosphorylation and TNF‐α production, but not NF‐κB activation in LPS‐challenged cardiomyocytes. In addition, pre‐treatment with SB202190, a p38 MAPK inhibitor, partly inhibited LPS‐induced TNF‐α production in cardiomyocytes. In endotoxaemic mice, PE promoted myocardial ERK1/2 phosphorylation and c‐Fos expression, inhibited p38 phosphorylation and IκBα degradation, reduced myocardial TNF‐α production and prevented LPS‐provoked cardiac dysfunction. Altogether, these findings indicate that activation of α₁‐AR by NE suppresses LPS‐induced cardiomyocyte TNF‐α expression and improves cardiac dysfunction during endotoxaemia via promoting myocardial ERK phosphorylation and suppressing NF‐κB activation.     

Inflammation has synergistic effect with nicotine in periodontitis by up‐regulating the expression of α7 nAChR via phosphorylated GSK‐3β

Periodontitis is the leading cause of adult tooth loss, and those who smoke are at an increased risk of developing periodontitis. α7 nicotinic acetylcholine receptor (α7 nAChR) is proposed to mediate the potential synergistic effect of nicotine and inflammation in smoking‐related periodontitis. However, this has not been experimentally demonstrated. We isolated and cultured human periodontal ligament stem cells (PDLSCs) from healthy and inflamed tissues. PDLSCs were treated with either inflammatory factors or nicotine. We measured expression of genes that are associated with osteogenic differentiation and osteoclast formation using RT‐qPCR and Western blot analyses. Besides, immunohistochemical staining, micro‐CT analysis and tartaric acid phosphatase staining were used to measure α7 nAChR expression and function. Inflammation up‐regulated α7 nAChR expression in both periodontal ligament tissues and PDLSCs. The up‐regulated α7 nAChR contributed to the synergistic effect of nicotine and inflammation, leading to a decreased capability of osteogenic differentiation and increased capability of osteoclast formation‐induction of PDLSCs. Moreover, the inflammation‐induced up‐regulation of α7 nAChR was partially dependent on the level of phosphorylated GSK‐3β. This study provides experimental evidence for the pathological development of smoking‐related periodontitis and sheds new light on developing inflammation and α7 nAChR‐targeted therapeutics to treat and prevent the disease.