Synthesis of 4% Glu-containing Val1 and Ile1-polypentapeptides: Model protein systems for demonstrating mechanochemical coupling

The synthesis of 4% Glu-polypentapeptide (PPP) (i.e., 4 Glu residues per 100 amino acid residues) and 4% Glu-Ile¹-PPP, in which Val¹ is substituted by a more hydrophobic Ile residue, is carried out by copolymerizing thep-nitrophenyl-active esters of GE(OMe)GVP and GE(OMe)GIP with their parent pentamers GVGVP and GVGIP in 1:4 ratios, respectively. After removal of the methyl ester on the side chain of Glu, these polymers exhibited a remarkablepH dependence of the temperature for their inverse temperature transitions, which are followed as turbidity development at 300 nm. On -irradiation crosslinking, the elastomeric bands obtained exhibited apH-mediated contraction and relaxation. Thus, for the first time, mechanochemical coupling is demonstrated in a synthetic polypeptide system. That the basic mechanism involves the hydrophobic effect (chemical modulation of an inverse temperature transition) and not ion-ion electrostatic repulsion is also discussed.     

pH-dependent random coil 1H, 13C,and 15N chemical shifts of the ionizable amino acids: a guide for protein pK a measurements

The pK _a values and charge states of ionizable residues in polypeptides and proteins are frequently determined via NMR-monitored pH titrations. To aid the interpretation of the resulting titration data, we have measured the pH-dependent chemical shifts of nearly all the ¹H, ¹³C, and ¹⁵N nuclei in the seven common ionizable amino acids (X = Asp, Glu, His, Cys, Tyr, Lys, and Arg) within the context of a blocked tripeptide, acetyl-Gly-X-Gly-amide. Alanine amide and N-acetyl alanine were used as models of the N- and C-termini, respectively. Together, this study provides an essentially complete set of pH-dependent intra-residue and nearest-neighbor reference chemical shifts to help guide protein pK _a measurements. These data should also facilitate pH-dependent corrections in algorithms used to predict the chemical shifts of random coil polypeptides. In parallel, deuterium isotope shifts for the side chain ¹⁵N nuclei of His, Lys, and Arg in their positively-charged and neutral states were also measured. Along with previously published results for Asp, Glu, Cys, and Tyr, these deuterium isotope shifts can provide complementary experimental evidence for defining the ionization states of protein residues.     

Single-nucleotide polymorphism associations in common with immune responses to measles and rubella vaccines

Single-nucleotide polymorphisms (SNPs) in candidate immune response genes were evaluated for associations with measles- and rubella-specific neutralizing antibodies, interferon (IFN)-γ, and interleukin (IL)-6 secretion in two separate association analyses in a cohort of healthy immunized subjects. We identified six SNP associations shared between the measles-specific and rubella-specific immune responses, specifically neutralizing antibody titers (DDX58), secreted IL-6 (IL10RB, IL12B), and secreted IFN-γ (IFNAR2, TLR4). An intronic SNP (rs669260) in the antiviral innate immune receptor gene, DDX58, was significantly associated with increased neutralizing antibody titers for both measles and rubella viral antigens post-MMR vaccination (p values 0.02 and 0.0002, respectively). Significant associations were also found between IL10RB (rs2284552; measles study p value 0.006, rubella study p value 0.00008) and IL12B (rs2546893; measles study p value 0.005, rubella study p value 0.03) gene polymorphisms and variations in both measles- and rubella virus-specific IL-6 responses. We also identified associations between individual SNPs in the IFNAR2 and TLR4 genes that were associated with IFN-γ secretion for both measles and rubella vaccine-specific immune responses. These results are the first to indicate that there are SNP associations in common across measles and rubella vaccine immune responses and that SNPs from multiple genes involved in innate and adaptive immune response regulation may contribute to the overall human antiviral response.     

Purification of an amide hydrolase DamH from Delftia sp. T3-6 and its gene cloning,expression, and biochemical characterization

A highly active amide hydrolase (DamH) was purified from Delftia sp. T3-6 using ammonium sulfate precipitation, diethylaminoethyl anion exchange, hydrophobic interaction chromatography, and Sephadex G-200 gel filtration. The molecular mass of the purified enzyme was estimated to be 32 kDa by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis. The sequence of the N-terminal 15 amino acid residues was determined to be Gly-Thr-Ser-Pro-Gln-Ser-Asp-Phe-Leu-Arg-Ala-Leu-Phe-Gln-Ser. Based on the N-terminal sequence and results of peptide mass fingerprints, the gene (damH) was cloned by PCR amplification and expressed in Escherichia coli BL21(DE3). DamH was a bifunctional hydrolase showing activity to amide and ester bonds. The specific activities of recombinant DamH were 5,036 U/mg for 2′-methyl-6′-ethyl-2- chloroacetanilide (CMEPA) (amide hydrolase function) and 612 U/mg for 4-nitrophenyl acetate (esterase function). The optimum substrate of DamH was CMEPA, with K _m and k _cat values of 0.197 mM and 2,804.32 s^?1, respectively. DamH could also hydrolyze esters such as 4-nitrophenyl acetate, glycerol tributyrate, and caprolactone. The optimal pH and temperature for recombinant DamH were 6.5 and 35 °C, respectively; the enzyme was activated by Mn²⁺ and inhibited by Cu²⁺, Zn²⁺, Ni²⁺, and Fe²⁺. DamH was inhibited strongly by phenylmethylsulfonyl and SDS and weakly by ethylenediaminetetraacetic acid and dimethyl sulfoxide.     

Interaction of synthetic glycopeptides carrying clusters ofO-glycosidic disaccharide chains (β-d-Gal(1–3)-α-d-GalNAc) withβ-d-galactose-binding lectins

The specificity of severald-galactose-binding lectins including Agaricus bisporus (mushroom),Arachis hypogaea (peanut),Bauhinia purpurea andVicia graminea has been examined by inhibition of hemagglutination using a series of synthetic oligopeptides representing the N-terminal end of glycophorin A from N and M individuals, all carrying one or several disaccharide chains,d-Galβ1–3-d-GalNAcα-(T-hapten). Peanut lectin was inhibited by T-hapten-carrying glycopeptides, but the presence of a cluster of disaccharide chains had no effect on the lectin specificity. On the contrary, bothAgaricus bisporus andBauhinia purpurea lectins exhibited an enhanced reactivity with polyglycosylated peptides suggesting that their combining site might include two proximal galactose residues. All synthetic glycopeptides inhibitingVicia graminea lectin carry a cluster of T-disaccharide chains and the leucine residue at the N-terminal end, and the presence of a Glu residue at position 5 slightly increased the lectin activity. It is concluded that the binding ofVicia graminea is dependent upon a specific spatial conformation including a cluster of T-hapten chains in close vicinity of a hydrophobic surface represented by an appropriate N-terminal amino acid residue.     

Understanding the Molecular Dynamics of Type-2 Diabetes Drug Target DPP-4 and its Interaction with Sitagliptin and Inhibitor Diprotin-A

The occurrence of type 2 diabetes (T2D) accounts for 90–95 % of all diabetes. Intestine hormone glucagon-like peptide-1 (GLP-1) has an antidiabetic role that enhances insulin secretion and pancreatic β-cell proliferation. GLP-1 is degraded by the enzyme dipeptidyl peptidase-4 (DPP-4) rapidly. Hence, the DPP-4 inhibition has been preferred not only for the treatment but also as a major drug target. Sitagliptin and Diprotin-A are antihyperglycemic agents for the treatment of T2D. However, little is known on the molecular dynamics of DPP-4 and the interaction properties with its ligands, namely Sitagliptin and Diprotin-A. This study has used the latest bioinformatic tools to understand the molecular dynamics and its interaction properties of DPP-4. This study has explored the number of α helices, β strands, β hairpins, Ψ loop, β bulges, β turns, and ? turns and they were 19, 46, 25, 1, 14, 70, and 4, respectively. The highest number of H-bonds was recorded in α helix of domain-1, and the lowest number H-bonds were noted in α helix of domain-2. During interaction between residues, in A- and B-chain, 47 and 48 residues are involved for interaction, and interaction interface area was more in A-Chain (2176 Å²). From DPP-4 and Sitagliptin interaction, three residues in active sites such as Try226, Glu205, and Glu206 were involved in three H-bond formation, while 10 other amino acids (Try547, Try667, Asn710, Val711, His740, Ser630, Ser209, Arg358, Phe357, and Val207) were involved in hydrophobic interactions. In this review, we have shown the importance of bioinformatics as an excellent tool for a rapid method to assess the molecular dynamics and its interaction properties of DPP-4. Our predictions highlighted in this review will help researchers to understand the interaction properties and recognition of interactive sites to design more DPP-4 inhibitors for the treatment of T2D and drug discovery.     

The specific response of gypsy moth A1 neurosecretory neurons to different environmental stressors

Changes in the number and morphometric parameters of A1 neurosecretory neurons (nsn) were analyzed in Lymantria dispar 4^th instar caterpillars, exposed for 3 days to different stressors: cadmium, high temperature and tannic acid. The relative cytoplasm density of A1 nsn was also estimated. Caterpillars reared on a diet supplemented with cadmium exhibited increased size of A1 nuclei (10 and 250 μg Cd per g of dry food weight), increased number of nucleolii in nuclei and raised relative cytoplasm density in all experimental groups. Cadmium obviously induces intensive synthetic activity in A1 nsn. The second stressor was high environmental temperature of 35°C. Decrease of all analyzed morphometric parameters suggests that acute exposure of 4^th instar caterpillars to 35°C, as well as 12 h recovery at optimal temperature of 23°C, reduced the activity of A1 nsn. Tannic acid was added to the artificial diet in the following concentrations: 1%, 2.5% and 5%. All estimated morphological parameters did not change after addition 1 and 2.5% of tannic acid. After addition of 5% of tannic acid, the activity of A1 nsn declined.     

Studies on the status of tyrosyl residues in phospholipases A2 fromNaja naja atra andNaja nigricollis snake venoms

Two phospholipases A₂ (PLA₂) fromNaja naja atra andNaja nigricollis snake venoms were subjected to tyrosine modification withp-nitrobenzenesulfonyl fluoride (NBSF) atpH 8.0. Three major NBS derivatives from each PLA₂ were separated by high-performance liquid chromatography. The results of amino acid analysis showed that only two Tyr residues out of nine were modified, and the modified residues were identified to be Tyr-3 and Tyr-63 (or Tyr-62) in the sequence. Spectrophotometric titration indicated that the phenolic group of Tyr-3 and Tyr-63 (or Tyr-62) had apK of 10.1 and 11.0, respectively. The reactivity of Tyr-3 toward NBSF was not affected in the presence or absence of Ca ²⁺; however, the reactivity of Tyr-63 (or Tyr-62) toward NBSF was greatly enhanced by Ca²⁺. Modification of Tyr-63 (or Tyr-62) resulted in a marked decrease in both lethality and enzymatic activity. Conversely, modification of Tyr-3 inN. naja atra PLA₂ could cause more than a sixfold increase in lethal potency, in sharp contrast to the loss of enzymatic activity. Tyrosine-63-modifiedN. naja atra PLA₂ exhibited the same Ca²⁺-induced difference spectra as that of native PLA₂, indicating that the Ca²⁺-binding ability of Tyr-63-modifiedN. naja atra PLA₂ was not impaired. However, Tyr-3-modified PLA₂ and all Tyr-modifiedN. nigricollis CMS-9 were not perturbed by Ca²⁺, revealing that the Ca²⁺-binding ability have been lost after tyrosine modification. These results suggest that Tyr-62 inN. nigricollis CMS-9 and Tyr-3 in both enzymes are involved in Ca²⁺ binding. AtpH 8.0, both native PLA₂ enzymes enhance the emission intensity of 8-anilinonaphthalene sulfonate (ANS) dramatically, while all of the Tyr-modified derivatives did not enhance the emission intensity at all either in the presence or absence of Ca²⁺, suggesting that the hydrophobic pocket that interacts with ANS might be the substrate binding site, in which Tyr-3 and Tyr-63 (or Tyr-62) are involved.     

31P nuclear magnetic resonance study of enzymatically phosphorylated glycophorin A

Glycophorin A was phosphorylated using protein kinases and the new protein was investigated using³¹P NMR spectroscopy. Most of these ～30 moles of phosphate were found to be attached to Ser and Thr. Some of these phosphate residues appear to be affected by the carbohydrate residues present. The phosphorylated protein appears to be in a severe state of aggregation, with the degree of aggregationpH-dependent.     

Conformation study of poly(γ-methyl-L-glutamate) in helicogenic solvents by small-angle X-ray scattering

Small-angle x-ray scattering of poly(γ-methyl-L -glutamate), [Glu(OMe)]_n, in m-cresol and in pyridine was measured to determine the mass per unit length, M_q, and the radius of gyration of the cross section, 〈S〉^1/2. It was confirmed from the values of M_q that [Glu(OMe)]_n exists in an α-helical conformation in these solvents. It was elucidated from the calculations on 〈S〉^1/2 that the side chains come in moderately close contact with the main chain in these solvents. It was indicated from the analysis of the outer portion of the scattering curves that the side-chain conformation varied depending on the solvent.     

HLA alleles associated with the adaptive immune response to smallpox vaccine: a replication study

We previously reported HLA allelic associations with vaccinia virus (VACV)-induced adaptive immune responses in a cohort of healthy individuals (n = 1,071 subjects) after a single dose of the licensed smallpox (Dryvax) vaccine. This study demonstrated that specific HLA alleles were significantly associated with VACV-induced neutralizing antibody (NA) titers (HLA-B*13:02, *38:02, *44:03, *48:01, and HLA-DQB1*03:02, *06:04) and cytokine (HLA-DRB1*01:03, *03:01, *10:01, *13:01, *15:01) immune responses. We undertook an independent study of 1,053 healthy individuals and examined associations between HLA alleles and measures of adaptive immunity after a single dose of Dryvax-derived ACAM2000 vaccine to evaluate previously discovered HLA allelic associations from the Dryvax study and determine if these associations are replicated with ACAM2000. Females had significantly higher NA titers than male subjects in both study cohorts [median ID50 discovery cohort 159 (93, 256) vs. 125 (75, 186), p < 0.001; replication cohort 144 (82, 204) vs. 110 (61, 189), p = 0.024]. The association between the DQB1*03:02 allele (median ID₅₀ discovery cohort 152, p = 0.015; replication cohort 134, p = 0.010) and higher NA titers was replicated. Two HLA associations of comparable magnitudes were consistently found between DRB1*04:03 and DRB1*08:01 alleles and IFN-γ ELISPOT responses. The association between the DRB1*15:01 allele with IFN-γ secretion was also replicated (median pg/mL discovery cohort 182, p = 0.052; replication cohort 203, p = 0.014). Our results suggest that smallpox vaccine-induced adaptive immune responses are significantly influenced by HLA gene polymorphisms. These data provide information for functional studies and design of novel candidate smallpox vaccines.     

Identification and characterization of TCRγ and TCRδ chains in channel catfish,Ictalurus punctatus

Channel catfish, Ictalurus punctatus, T cell receptors (TCR) γ and δ were identified by mining of expressed sequence tag databases, and full-length sequences were obtained by 5′-RACE and RT-PCR protocols. cDNAs for each of these TCR chains encode typical variable (V), diversity (D), joining (J), and constant (C) regions. Three TCRγ V families, seven TCRγ J sequences, and three TCRγ C sequences were identified from sequencing of cDNA. Primer walking on bacterial artificial chromosomes (BACs) confirmed that the TRG locus contained seven TRGJ segments and indicated that the locus consists of (Vγ3-Jγ6-Cγ2)–(Vγ1_n-Jγ7-Cγ3)–(Vγ2-Jγ5-Jγ4-Jγ3-Jγ2-Jγ1-Cγ1). In comparison for TCRδ, two V families, four TCRδ D sequences, one TCRδ J sequence, and one TCRδ C sequence were identified by cDNA sequencing. Importantly, the finding that some catfish TCRδ cDNAs contain TCR Vα-D-Jδ rearrangements and some TCRα cDNAs contain Vδ-Jα rearrangements strongly implies that the catfish TRA and TRD loci are linked. Finally, primer walking on BACs and Southern blotting suggest that catfish have four TRDD gene segments and a single TRDJ and TRDC gene. As in most vertebrates, all three reading frames of each of the catfish TRDD segments can be used in functional rearrangements, and more than one TRDD segment can be used in a single rearrangement. As expected, catfish TCRδ CDR3 regions are longer and more diverse than TCRγ CDR3 regions, and as a group they utilize more nucleotide additions and contain more nucleotide deletions than catfish TCRγ rearrangements.     

Purification and characterization of aβ-galactosidase fromTreponema phagedenis (Reiter strain)

A β-galactosidase was highly purified from a cellular extract ofTreponema phagedenis (Reiter strain) by ammonium sulfate precipitation and successive chromatography on Sepharose 6B and DEAE-Sephadex. The purified enzyme was homogeneous in SDS-polyacrylamide gel electrophoresis. The molecular weight estimated was 580,000. The optimal pH, ionic strength, and temperature were 6.5, 0.1, and 50°C, respecitvely. The enzyme was stable only at around pH 6.5 and at temperatures lower than 35°C. The enzyme was irreversibly inhibited byp-chloromercuribenzoate and divalent cations such as Hg²⁺, Cu²⁺, Zn²⁺, Cd²⁺, and Pb²⁺. The Km values forp-nitrophenyl-β-D-galactopyranoside,o-nitrophenyl-β-D-galactopyranoside, and lactose were 0.29, 0.36, and 5.4 mM, respectively.     

Aspartame downregulates 3T3-L1 differentiation

Aspartame is an artificial sweetener used as an alternate for sugar in several foods and beverages. Since aspartame is 200 times sweeter than traditional sugar, it can give the same level of sweetness with less substance, which leads to lower-calorie food intake. There are reports that consumption of aspartame-containing products can help obese people lose weight. However, the potential role of aspartame in obesity is not clear. The present study investigated whether aspartame suppresses 3T3-L1 differentiation, by downregulating phosphorylated peroxisome proliferator-activated receptor γ (p-PPARγ), peroxisome proliferator-activated receptor γ (PPARγ), fatty acid-binding protein 4 (FABP4), CCAAT/enhancer-binding protein α (C/EBPα), and sterol regulatory element-binding protein 1 (SREBP1), which are critical for adipogenesis. The 3T3-L1 adipocytes were cultured and differentiated for 6 d in the absence and presence of 10 μg/ml of aspartame. Aspartame reduced lipid accumulation in differentiated adipocytes as evidenced by Oil Red O staining. qRT-PCR analysis showed that the PPARγ, FABP4, and C/EBPα mRNA expression was significantly reduced in the aspartame-treated adipocytes. Western blot analysis showed that the induction of p-PPARγ, PPARγ, SREBP1, and adipsin was markedly reduced in the aspartame-treated adipocytes. Taken together, these data suggest that aspartame may be a potent substance to alter adipocyte differentiation and control obesity.     

Aryldiazenido derivatives: A new entry to the functionalization of Keggin polyoxometalates

A series of aryldiazenido polyoxomolybdates of the type (nBu₄N)₂[Mo₅O₁₃(OMe)₄(NNAr){Na(MeOH)}] (Ar = C₆F₅, 1; Ar = O₂N-o-C₆H₄, 2; Ar = O₂N-m-C₆H₄, 3; Ar = O₂N-p-C₆H₄, 4a; Ar = (O₂N)₂-o,p-C₆H₃, 5) have been obtained by controlled degradation of the parent compounds (nBu₄N)₃[Mo₆O₁₈(NNAr)] with NaOH in methanol. They have been characterized by elemental analysis and UV-Vis and IR spectroscopy. In addition, 4a has been characterized by ⁹⁵Mo NMR spectroscopy and the crystal structure of (nBu₄N)₂[Mo₅O₁₃(OMe)₄(NNC₆H₄-p-NO₂){Na(H₂O))]·H₂O (4b) has been determined by X-ray diffraction. The molecular structure of the anion of 4b features a lacunary Lindqvist-type anion [Mo₅O₁₃(OMe)₄(NNC₆H₄-p-NO₂)]³⁻ interacting with a sodium cation through the four terminal axial oxygen atoms. The 1:1 sodium complexes react with BaCl₂ and BiCl₃ to yield 2:1 complexes which have been isolated as (nBu₄N)₄[Ba{Mo₅O₁₃(OMe)₄(NNAr)}₂] (Ar = C₆F₅, 6; Ar = O₂N-p-C₆H₄, 7) and (nBu₄N)₃[Bi{Mo₅O₁₃(OMe)₄(NNAr)}₂] (Ar = C₆F₅, 8; Ar = O₂N-p-C₆H₄, 9). X-ray crystallography analysis of 9·Me₂CO has shown that the tetradentate [Mo₅O₁₃(OMe)₄(N₂C₆H₄-p-NO₂)]³⁻ anions provide a square-antiprismatic environment for Bi. In contrast, IR spectroscopy provides evidence for a square-prismatic environment of Ba in 6 and 7. In acetonitrile-methanol mixed solvent, [Mo₅O₁₃(OMe)₄(NNAr)]³⁻ and [PW₁₁O₃₉]⁷⁻, generated in situ by alkaline degradation of their respective parents, [Mo₆O₁₈(NNAr)]³⁻ and [PW₁₂O₄₀]³⁻, react together to give the Keggin-type diazenido compounds (nBu₄N)₄[PW₁₁O₃₉(MoNNAr)] (Ar = O₂N-o-C₆H₄, 10; Ar = O₂N-m-C₆H₄, 11; Ar = O₂N-p-C₆H₄, 12), which have been characterized by ³¹P and ¹⁸³W NMR spectroscopy.     

Self-association of glutamic acid-rich fusion peptide analogs of influenza hemagglutinin in the membrane-mimic environments: Effects of positional difference of glutamic acids on side chain ionization constant and intra- and inter-peptide interactions deduced from NMR and gel electrophoresis measurements

Two glutamic acid-rich fusion peptide analogs of influenza hemagglutinin were synthesized to study the organization of the charged peptides in the membranous media. Fluorescence and gel electrophoresis experiments suggested a loose association between the monomers in the vesicles. A model was built which showed that a positional difference of 3, 7 and 4, 8 results in the exposure of Glu3 and Glu7 side chains to the apolar lipidic core. Supportive results include: first, pK_a values of two pH units higher than reference value in aqueous medium for Glu3 and Glu7 CγH, whereas the deviation of pK_a from the reference value for Glu4 and Glu8 CγH is substantially smaller; second, Hill coefficients of titration shift of these protons indicate anti-cooperativity for Glu3 and Glu7 side chain protons but less so for Glu4 and Glu8, implying a strong electrostatic interaction between Glu3 and Glu7 possibly resulting from their localization in an apolar environment; third, positive and larger titration shift for NH of Glu3 is observed compared to that of Glu4, suggesting stronger hydrogen bond between the NH and the carboxylic group of Glu3 than that of Glu4, consistent with higher degree of exposure to hydrophobic medium for the side chain of Glu3.