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     

Computational study of the conformational preferences of the (R)-8-amino-pentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecane-8-carboxylic acid monopeptide.

alpha-Amino acids are important building blocks for the synthesis of a large number of bioactive compounds and pharmaceutical drugs. However, a literature survey revealed that no theoretical conformational study of alpha-amino acids with cage carbon frameworks has been performed to date. This paper reports the results of a conformational study on the (R)-8-amino-pentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecane-8-carboxylic acid monopeptide (cage monopeptide), using molecular mechanics and ab initio methods. The in vacuo Ramachandran maps computed using the different parameterizations of the AMBER force field show the C7eq structure as the most favourable conformation, in contrast to the C7ax structure, that is the lowest energy conformation at the ab initio level. Analysis of these maps reveals the helical preference for the monopeptide and provides the potential for the cage residue to be incorporated into constrained peptide analogues.     

Effect of external electrostatic field on the stability of β sheet structures

To explore the effect of an external electrostatic field (EEF) on the stability of protein conformations, the molecular dynamic modeling approach was applied to evaluate the effect of an EEF along the x or y direction on a water cluster containing a parallel or antiparallel β sheet structure. The β sheet structure contained two strands with a (Gly)₃ sequence separated by a distance d along the x direction. The mean forces between the two strands along the x direction were computed from the trajectories of molecular dynamics simulations. In the absence of the EEF, the forces between the two strands in vacuum were repulsive and attractive in the parallel and antiparallel β sheet structures, respectively. In contrast, the mean forces between the two strands in water were attractive in both the parallel and antiparallel β sheet structures. This is because the electric interactions between the two strands were shielded by water, and the hydrophobic effect dominated the interaction between the two strands. When an EEF >50 MV/cm was applied to the water cluster, the attractive force between the two strands in the parallel and antiparallel β sheet structures decreased and increased, respectively. Further, the binding affinity between the two strands in the parallel and antiparallel β sheet structures also decreased and increased, respectively. This is because the large EEF leads to dielectric saturation, and consequently reduces the effects of the dielectric shielding and hydrophobic interactions. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 861–870, 2014.     

A Factor Found in Aged Tungstate Solution Enhanced the Antibacterial Effect of β-Lactams on Methicillin-Resistant Staphylococcus aureus

We have found a factor (Factor T) in aged mixtures of tungstate and phosphate which greatly enhances the antibacterial effects of β-lactams on both inducible and constitutive methicillin-resistant Staphylococcus aureus, but not on methicillin-susceptible S. aureus. Factor T alone did not strongly inhibit bacterial growth. There was no synergism of Factor T with other classes of antibiotics, nor with other groups of bacteria, and it reduced the efficacy of aminoglycosides and tetracycline. Upon preparation of Factor T, acidifying and heating the mixture of tungstate and phosphate resulted in a high yield and reproducibility, and no enhancing effect was observed when other anions such as sulfate or molybdate were used instead. Factor T is heat- and acid-stable but labile to alkalization, and is probably a complex of phosphate and tungstate.     

Direct folding simulation of alpha-helices and beta-hairpins based on a single all-atom force field with an implicit solvation model

Recently, we have shown that a modified energy model based on the param99 force field with the generalized Born (GB) solvation model produces reliable free energy landscapes of mini-proteins with a betabetaalpha motif (BBA5, 1FSD, and 1PSV), with the native structures of the mini-proteins located in their lowest free energy minimum states. One of the main features in the modified energy model is a significant improvement for more balanced treatments of alpha and beta strands in proteins. In this study, using the replica exchange molecular dynamics (REMD) simulation method with this new force field, we have carried out extensive ab initio folding studies of several well-known peptides with alpha or beta strands (C-peptide, EK-peptide, le0q, and gbl). Starting from fully extended conformations as the initial conditions, all of the native-like structures of the target peptides were successfully identified by REMD, with reasonable representations of free energy surfaces. The present simulation results with the modified energy model are consistent with experiments, demonstrating an extended applicability of the energy model to folding studies of a variety of alpha-helices, beta-strands, and alpha/beta proteins.     

Exploring the free energy landscape of a model β‐hairpin peptide and its isoform

Secondary structural transitions from α‐helix to β‐sheet conformations are observed in several misfolding diseases including Alzheimer's and Parkinson's. Determining factors contributing favorably to the formation of each of these secondary structures is therefore essential to better understand these disease states. β‐hairpin peptides form basic components of anti‐parallel β‐sheets and are suitable model systems for characterizing the fundamental forces stabilizing β‐sheets in fibrillar structures. In this study, we explore the free energy landscape of the model β‐hairpin peptide GB1 and its E2 isoform that preferentially adopts α‐helical conformations at ambient conditions. Umbrella sampling simulations using all‐atom models and explicit solvent are performed over a large range of end‐to‐end distances. Our results show the strong preference of GB1 and the E2 isoform for β‐hairpin and α‐helical conformations, respectively, consistent with previous studies. We show that the unfolded states of GB1 are largely populated by misfolded β‐hairpin structures which differ from each other in the position of the β‐turn. We discuss the energetic factors contributing favorably to the formation of α‐helix and β‐hairpin conformations in these peptides and highlight the energetic role of hydrogen bonds and non‐bonded interactions. Proteins 2014; 82:2394–2402. © 2014 Wiley Periodicals, Inc.     

The amide III vibrational circular dichroism band as a probe to detect conformational preferences of alanine dipeptide in water

The conformational preferences of blocked alanine dipeptide (ADP), Ac‐Ala‐NHMe, in aqueous solution were studied using vibrational circular dichroism (VCD) together with density functional theory (DFT) calculations. DFT calculations of three most representative conformations of ADP surrounded by six explicit water molecules immersed in a dielectric continuum have proven high sensitivity of amide III VCD band shape that is characteristic for each conformation of the peptide backbone. The polyproline II (P_II) and α_R conformation of ADP are associated with a positive VCD band while β conformation has a negative VCD band in amide III region. Knowing this spectral characteristic of each conformation allows us to assign the experimental amide III VCD spectrum of ADP. Moreover, the amide III region of the VCD spectrum was used to determine the relative populations of conformations of ADP in water. Based on the interpretation of the amide III region of VCD spectrum we have shown that dominant conformation of ADP in water is P_II which is stabilized by hydrogen bonded water molecules between CO and NH groups on the peptide backbone. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 814–818, 2014.     

Different conversion metabolic rates of testosterone are associated to hormone-sensitive status and -response of human prostate cancer cells

The main goal of the present work was to compare the ability of human prostate cancer (PCa) cells to metabolize testosterone (T) in living conditions. To this end we studied three different human PCa cell lines (LNCaP, DU145 and PC3) having different hormone-sensitive status and capability of response to androgens. We used an original approach which allows the evaluation of conversion metabolic rates in growing cells after administration of labeled steroid precursor (presently T), at physiological concentrations (1–10 nM). Analysis of both precursor degradation and formation of several products was carried out using reverse phase-high performance liquid chromatography (RP-HPLC) and “on line” radioactive detection. Comparison of the three human PCa cells revealed that their metabolic aptitude differed in many respects: (i) rates of precursor degradation, (ii) different products' formation, and (iii) extent of conjugate production. In detail, PC3 cells quickly degraded T and exhibited high formation rates of androstenedione (A-4-ene-Ad); both DU145 and LNCaP cells mostly retained high levels of unconverted T, with a limited production of A-4-ene-Ad and its 17-keto derivatives (if any). Either LNCaP or DU145 cells generated a relatively high amount of dihydrotestosterone (DHT). In contrast, neither DHT nor its main metabolites were detected in PC3 cells at both short and longer incubation times. As expected, T degradation and A-4-ene-Ad production were highly correlated (r = 0.97; P < 0.03); similarly, A-4-ene-Ad and DHT formation showed a negative, significant correlation. Negligible production of conjugates was noted in both PC3 and DU145 cells, whilst it was remarkable in LNCaP cells (ranging from 43 to 57%). Overall, our data indicate that human PCa cells degrade T quite differently, favoring alternatively reductive or oxidative patterns of androgen metabolism.     

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.     

Chemical cross-linking between lysine groups in vimentin oligomers is dependent on local peptide conformations

Previous studies have shown that cytoplasmic intermediate filaments, other than the keratins, are each constructed from a single type of polypeptide chain. Studies involving chemical crosslinking between lysine groups have shown that assembly of the filaments begins with the formation of dimers in which the peptide chains are parallel and in exact register, and that these dimers further associate in antiparallel patterns having specific degrees of overlap. In the present study, molecular modeling of the conformations of vimentin molecules indicated that lysine side chains in identical positions in regions of α-helix in parallel chains might be unable to be linked because they are on opposite sides of the coiled coil hydrophobic core. Examination of published data on chemical crosslinking of lysines in vimentin confirmed that there were no instances of linkage within dimers between the nine pairs of identical lysines that lie more than one position within α-helical regions in parallel chains. Even among linkages that apparently were between dimers, only one of the 11 linkage products identified involved lysines that were both within an α-helical region. In 10 of the 11 identified linkages between dimers, one or both of the linked lysines were in regions of random coil conformation. These results of molecular modeling indicate that relative motion between polypeptide chains in oligomers of intermediate filament proteins is not sufficient to overcome an orientation of lysine groups that is unfavorable for their chemical linkage. This finding supports the interpretations of keratin cross-linking data indicating that parallel homodimers are the basis for keratin intermediate filament assembly. © 1996 Wiley-Liss, Inc.     

Molecular modeling indicates that homodimers form the basis for intermediate filament assembly from human and mouse epidermal keratins

Mammalian epidermal keratin molecules adopt rod-shaped conformations that aggregate to form cytoplasmic intermediate filaments. To investigate these keratin conformations and the basis for their patterns of molecular association, graphical methods were developed to relate known amino acid sequences to probable spacial configurations. The results support the predominantly α-helical conformation of keratin chains, interrupted by short non-α-helical linkages. However, it was found that many of the linkages have amino acid sequences typical of β-strand conformations. Space-filling atomic models revealed that the β-strand sequences would permit the formation of 2-chain and 4-chain cylindrical β-helices, fully shielding the hydrophobic amino acid chains that alternate with hydrophilic residues in these sequences. Because of the locations of the β-helical regions in human and mouse stratum corneum keratin chains, only homodimers of the keratins could interact efficiently to form 2-chain and 4-chain β-helices. Tetramers having the directions and degrees of overlap of constituent dimers that have been identified by previous investigators are also predicted from the interactions of β-helical motifs. Heterotetramers formed from dissimilar homodimers could combine, through additional β-helical structures, to form higher oligomers having the dimensions seen in electron microscopic studies. Previous results from chemical crosslinking studies can be interpreted to support the concept of homodimers rather than heterodimers as the basis for keratin filament assembly. © 1995 Wiley-Liss, Inc.     

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.     

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     

Effects of endocrine disrupters on sex steroid synthesis and metabolism pathways in fish

The interactions of estrogenic (nonylphenol, dicofol, atrazine), androgenic (organotins, phthalates, fenarimol) and anti-androgenic compounds (vinclozolin, diuron, p,p'-DDE) with key enzymatic activities involved in both synthesis and metabolism of sex hormones was investigated. Carp testicular microsomes incubated in the presence of androstenedione and different xenobiotics evidenced higher sensitivity of 5alpha-reductase activity than 17beta-hydroxysteroid dehydrogenase activity towards those chemicals. Dicofol, organotins and phthalates were among the most effective inhibitors. In contrast, ovarian synthesis of maturation-inducing hormones (20alpha- and 20beta-hydroxysteroid dehydrogenase activities) were enhanced by nonylphenol, dicofol, fenarimol and p,p'-DDE. Metabolic clearance pathways of hormones were also affected. Fenarimol, nonylphenol and triphenyltin inhibited the glucuronidation of testosterone and estradiol at concentrations as low as 10, 50 and 100 microM, respectively. Triphenyltin, tributyltin and nonylphenol were also inhibitors of estradiol sulfation with IC(50) values of 17, 18 and 41 microM. Overall, the data indicates the interaction of selected chemicals with key enzymatic pathways involved in both synthesis and metabolism of sex hormones. This interference might be one of the underlying mechanisms for the reported hormonal disrupting properties of the tested compounds, and might finally affect physiological processes such as gamete growth and maturation.     

Protective effects of cilengitide on inflammation in chondrocytes under excessive mechanical stress

Chondrocytes constantly receive external stimuli, which regulates remodeling. An optimal level of mechanical stress is essential for maintaining chondrocyte homeostasis, however, excessive mechanical stress induces inflammatory cytokines and protease, such as matrix metalloproteinases (MMPs). Therefore, excessive mechanical stress is considered to be one of the main causes to cartilage destruction leading to osteoarthritis (OA). Integrins are well‐known as cell adhesion molecules and act as receptors for extracellular matrix (ECM), and are believed to control intracellular signaling pathways both physically and chemically as a mechanoreceptor. However, few studies have focused on the roles and functions of integrins in inflammation caused by excessive mechanical stress. In this study, we examined the relationship between integrins (αVβ3 and αVβ5) and the expression of inflammatory factors under mechanical loading in chondrocytes by using an integrin receptor antagonist (cilengitide). Cilengitide suppressed the gene expression of interleukin‐1β (IL‐1β), tumor necrosis factor‐α (TNF‐α), matrix metalloproteinase‐3 (MMP‐3), and MMP‐13 induced by excessive mechanical stress. In addition, the protein expression of IL1‐β and MMP‐13 was also inhibited by the addition of cilengitide. Next, we investigated the involvement of intracellular signaling pathways in stress‐induced integrin signaling in chondrocytes by using western blotting. The levels of p‐FAK, p‐ERK, p‐JNK, and p‐p38 were enhanced by excessive mechanical stress and the enhancement was suppressed by treatment with cilengitide. In conclusion, this study revealed that excessive mechanical stress may activate integrins αVβ3 and αVβ5 on the surface of chondrocytes and thereby induce an inflammatory reaction by upregulating the expression of IL‐1β, TNF‐α, MMP‐3, and MMP‐13 through phosphorylation of FAK and MAPKs.     

From methyl -glucopyranoside to methyl -allopyranoside via the Mitsunobu reaction

Methyl β- -glucopyranoside reacted with a 4-molar excess of the Mitsunobu reagents (triphenylphosphine–diethyl azodicarboxylate–benzoic acid) under Weinges et al. [Carbohydr. Res., 164 (1987) 453–458] conditions to furnish four differently benzoylated methyl β- -allopyranosides in a very good overall yield. The same reaction applied to methyl α- -glucopyranoside yielded allosides in a low yield and nine other sugar products. These results give an insight into the course of the Mitsunobu esterification–inversion reaction.     

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.