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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.39, #3)
Transglutaminase 2: a multi-functional protein in multiple subcellular compartments by Donghyun Park; Sun Shim Choi; Kwon-Soo Ha (pp. 619-631).
Transglutaminase 2 (TG2) is a multifunctional protein that can function as a transglutaminase, G protein, kinase, protein disulfide isomerase, and as an adaptor protein. These multiple biochemical activities of TG2 account for, at least in part, its involvement in a wide variety of cellular processes encompassing differentiation, cell death, inflammation, cell migration, and wound healing. The individual biochemical activities of TG2 are regulated by several cellular factors, including calcium, nucleotides, and redox potential, which vary depending on its subcellular location. Thus, the microenvironments of the subcellular compartments to which TG2 localizes, such as the cytosol, plasma membrane, nucleus, mitochondria, or extracellular space, are important determinants to switch on or off various TG2 biochemical activities. Furthermore, TG2 interacts with a distinct subset of proteins and/or substrates depending on its subcellular location. In this review, the biological functions and molecular interactions of TG2 will be discussed in the context of the unique environments of the subcellular compartments to which TG2 localizes.
Keywords: TG2; Subcellular localization; Subcellular compartment; Organelle; Transglutaminase; Gh ; Protein disulfide isomerase; Kinase
Nutrition and health relevant regulation of intestinal sulfur amino acid metabolism by Zhengfeng Fang; Kang Yao; Xiaoling Zhang; Shengjun Zhao; Zhihong Sun; Gang Tian; Bing Yu; Yan Lin; Biquan Zhu; Gang Jia; Keying Zhang; Daiwen Chen; De Wu (pp. 633-640).
Sulfur amino acids (SAA), particularly methionine and cysteine, are critical for the gut to maintain its functions including the digestion, absorption and metabolism of nutrients, the immune surveillance of the intestinal epithelial layer and regulation of the mucosal response to foreign antigens. However, the metabolism of SAA in the gut, specifically the transmethylation of methionine, will result in a net release of homocysteine, which is shown to be associated with cardiovascular disease and stroke. Furthermore, the extensive catabolism of dietary methionine by the intestine or by luminal microbes may result in a decrease in nutritional efficiency. Therefore, the regulation of SAA metabolism in the gut is not only nutritionally relevant, but also relevant to the overall health and well-being. The superiority of dl-2-hydroxy-4-methylthiobutyrate to dl-methionine in decreasing homocysteine production, alleviating stress responses, and reducing the first-pass intestinal metabolism of dietary methionine may provide a promising implication for nutritional strategies to manipulate SAA metabolism and thus to improve the nutrition and health status of animals and perhaps humans.
Keywords: Gut; Health; Metabolism; Nutrition; Sulfur amino acids
Synthetic study on cystinyl peptides using solution and solid phase methodology: human IgG1 hinge region by P. Niederhafner; V. Gut; J. Ježek; M. Buděšínský; V. Kašička; E. Wünsch; J. Hlaváček (pp. 641-650).
Synthetic study on cystinyl peptides using solution and solid phase methodology was carried out with the central hinge region of immunoglobulin IgG1. In the solid phase synthesis of hexadecapeptide 1c, the time necessary for the formation of disulfide bonds between linear precursors was shortened four times by the action of pure oxygen in buffered solution, in comparison with air oxidation. The product was thus obtained devoid of impurities from side reactions. In the preparation of the shortened bis-cystinyl analogs 2k and 3d of the natural hexadecapeptide 1c, both the classical and polyethylene glycol (PEG6000) solution methods were utilized using a disulfide synthon (Boc-Cys-OPfp)2 to obtain peptide chains in a natural parallel alignment. In the PEG6000 strategy, lysine as a linker on both sides of the polymer was attached to enhance the loading capacity. The leucine residue, instead of proline one, was introduced to the carboxy terminus to facilitate a specific enzymatic cleavage of the peptides from PEG6000 by thermolysine.
Keywords: Hinge region; Immunoglobulin; Prion protein; Solution synthesis; Solid phase synthesis; PEG; Disulfide synthon; Enzymatic cleavage; Thermolysin
Exploring the cause of drug resistance by the detrimental missense mutations in KIT receptor: computational approach by R. Rajasekaran; Rao Sethumadhavan (pp. 651-660).
In this work, we computationally identified the most detrimental missense mutations of KIT receptor causing gastrointestinal stromal tumors and analyzed the drug resistance of these missense mutations. Out of 31 missense mutations, 19 variants were commonly found less stable, deleterious and damaging by I-Mutant 2.0, SIFT and PolyPhen programs, respectively. Subsequently, we performed modeling of these 19 variants to understand their change in conformations with respect to native KIT receptor by computing their RMSD. Further, the native and 19 mutants were docked with the drug ‘Imatinib’ to explain the drug resistance of these detrimental missense mutations. Among the 19 mutants, we found by docking studies that 12 mutants, namely, F584C, F584L, V654A, L656P, T670I, R804W, D816F, D816V, D816Y, N822K, Y823D and E839K had less binding affinity with Imatinib than the native type. Finally, we analyzed that the loss of binding affinity of these 12 mutants, was due to altered flexibility in their binding amino acids with Imatinib as compared with native type by normal mode analysis. In our work, we found the novel data that the majority of the drug-binding amino acids in those 12 mutants had encountered loss of flexibility, which could be the theoretical basis for the cause of drug insensitivity.
Keywords: Missense mutation; KIT receptor; Imatinib; Flexibility
γ-Glutamyl 16-diaminopropane derivative of vasoactive intestinal peptide: a potent anti-oxidative agent for human epidermoid cancer cells by Paola Stiuso; Gaia Giuberti; Angela Lombardi; Alessandra Dicitore; Vittorio Limongelli; Maria Cartenì; Alberto Abbruzzese; Michele Caraglia (pp. 661-670).
We previously demonstrated that the γ-glutamyl 16 amine derivative of vasoactive intestinal peptide (VIP) acts as structural VIP agonist with affinity and potency higher than VIP. Herein, we have evaluated the effects of VIP and γ-Gln16-diaminopropane derivative of VIP (VIP-DAP3) on the proliferation and protection from oxidative stress induced by hydrogen peroxide (H2O2) on epidermoid carcinoma cell lines. We have found that 10−11 M VIP-DAP3 completely antagonized the inhibition induced by H2O2 on both cell proliferation and S-phase distribution while these effects were only partially antagonized by equimolar concentrations of VIP. Moreover, both oxidative stress and intracellular lipid oxidation induced by H2O2 were reduced by VIP and completely antagonized by VIP-DAP3. Thereafter, we have found that H2O2 increased p38 kinase activity and both HSP70 and HSP27 expression. VIP and VIP-DAP3 again antagonized these effects partially or totally, respectively. H2O2 reduced the activity of extracellular signal-regulated kinases Erk-1/2 and Akt, signalling proteins involved in proliferation/survival pathways. Again VIP restored the activity of both kinases while VIP-DAP3 caused indeed an increase of their activity as compared to untreated cells. These data suggest that VIP-DAP3 has a stronger anti-oxidative activity as compared to VIP likely based on its super-agonistic binding on the putative receptor.
Keywords: Akt; Extracellular signal-regulated kinases Erk-1/2; p38 kinase; Oxidative stress; Proliferation; Vasoactive intestinal peptide (VIP); γ-Glutamyl 16 amine vasoactive intestinal peptide (VIP-DAP3)
Laccase-catalyzed cross-linking of amino acids and peptides with dihydroxylated aromatic compounds by Annett Mikolasch; Veronika Hahn; Katrin Manda; Judith Pump; Nicole Illas; Dirk Gördes; Michael Lalk; Manuela Gesell Salazar; Elke Hammer; Wolf-Dieter Jülich; Stephan Rawer; Kerstin Thurow; Ulrike Lindequist; Frieder Schauer (pp. 671-683).
In order to design potential biomaterials, we investigated the laccase-catalyzed cross-linking between l-lysine or lysine-containing peptides and dihydroxylated aromatics. l-Lysine is one of the major components of naturally occurring mussel adhesive proteins (MAPs). Dihydroxylated aromatics are structurally related to 3,4-dihydroxyphenyl-l-alanine, another main component of MAPs. Mass spectrometry and nuclear magnetic resonance analyses show that the ε-amino group of l-lysine is able to cross-link dihydroxylated aromatics. Additional oligomer and polymer cross-linked products were obtained from di- and oligopeptides containing l-lysine. Potential applications in medicine or industry for biomaterials synthesised via the three component system consisting of the oligopeptide [Tyr-Lys]10, dihydroxylated aromatics and laccase are discussed.
Keywords: Laccase; Mussel adhesive proteins; Cross-link; Biomaterial; ε-Amino group
The influences of water solvent on the structures and stabilities of Na+–AD conformers by J. F. Fan; M. Tang; L. G. Qiao; J. Liu; L. J. He (pp. 685-697).
The influences of water solvent on the structures and stabilities of the complex ion conformers formed by the coordination of alanine dipeptide (AD) and Na+ have been investigated using supramolecular and polarizable continuum solvation models at the level of B3LYP/6-311++G**, respectively; 12 monohydrated and 12 dihydrated structures of Na+–AD complex ion were obtained after full geometrical optimization. The results showed that H2O molecules easily bind with Na+ of Na+–AD complex ion, forming an ion-lone pair interaction with the Na–O bond length of 2.1–2.3 Å. Besides, H2O molecules also can form hydrogen bonds OW–HW···O(1), OW–HW···O(2), N(1)–H(1)···OW or N(2)–H(2)···OW with O or N groups of the Na+–AD backbone. The most stable gaseous bidentate conformer C7AB of Na+–AD is still the most stable one in the solvent of water. However, the structure of the most unstable gaseous conformer α′B of Na+–AD collapses under the attack of H2O molecules and changes into C7AB conformation. Computations with IEFPCM solvation model of self-consistent reaction field theory give that aqueous C5A is more stable than C7eqB and that the stabilization energies of water solvent on monodentate conformers of Na+–AD complex ion (about 272–294 kJ/mol) are more than those on bidentate ones (about 243 kJ/mol).
Keywords: Na+–AD; Water; Solvation effect; B3LYP/6-311++G**
Light-induced cleavage of model phenylalanine conjugates based on coumarins and quinolones by Andrea S. C. Fonseca; M. Sameiro T. Gonçalves; Susana P. G. Costa (pp. 699-712).
In order to evaluate the application of quinolone as a new photocleavable protecting group, in comparison with coumarin, a series of model phenylalanine conjugates were prepared by reaction with chloromethylated O and N heterocycles. The photophysical properties of the resulting ester conjugates were evaluated as well as the photosensitivity under irradiation at 250, 300, 350, and 419 nm. The results obtained showed that the quinolone conjugates were readily photolysed, with complete release of the amino acid in short irradiation times and could be considered a new addition to the family of photocleavable protecting groups for the carboxylic acid function of amino acids.
Keywords: Coumarin; Quinolone; Bioconjugates; Amino acids; Photocleavable protecting groups
DomSVR: domain boundary prediction with support vector regression from sequence information alone by Peng Chen; Chunmei Liu; Legand Burge; Jinyan Li; Mahmood Mohammad; William Southerland; Clay Gloster; Bing Wang (pp. 713-726).
Protein domains are structural and fundamental functional units of proteins. The information of protein domain boundaries is helpful in understanding the evolution, structures and functions of proteins, and also plays an important role in protein classification. In this paper, we propose a support vector regression-based method to address the problem of protein domain boundary identification based on novel input profiles extracted from AAindex database. As a result, our method achieves an average sensitivity of ∼36.5% and an average specificity of ∼81% for multi-domain protein chains, which is overall better than the performance of published approaches to identify domain boundary. As our method used sequence information alone, our method is simpler and faster.
Keywords: Domain boundary prediction; Support vector regression; AAindex; Principal component analysis
Glutamate-induced metabolic changes in Lactococcus lactis NCDO 2118 during GABA production: combined transcriptomic and proteomic analysis by Roberto Mazzoli; Enrica Pessione; Magali Dufour; Valérie Laroute; Maria Gabriella Giuffrida; Carlo Giunta; Muriel Cocaign-Bousquet; Pascal Loubière (pp. 727-737).
GABA is a molecule of increasing nutraceutical interest due to its modulatory activity on the central nervous system and smooth muscle relaxation. Potentially probiotic bacteria can produce it by glutamate decarboxylation, but nothing is known about the physiological modifications occurring at the microbial level during GABA production. In the present investigation, a GABA-producing Lactococcus lactis strain grown in a medium supplemented with or without glutamate was studied using a combined transcriptome/proteome analysis. A tenfold increase in GABA production in the glutamate medium was observed only during the stationary phase and at low pH. About 30 genes and/or proteins were shown to be differentially expressed in glutamate-stimulated conditions as compared to control conditions, and the modulation exerted by glutamate on entire metabolic pathways was highlighted by the complementary nature of transcriptomics and proteomics. Most glutamate-induced responses consisted in under-expression of metabolic pathways, with the exception of glycolysis where either over- or under-expression of specific genes was observed. The energy-producing arginine deiminase pathway, the ATPase, and also some stress proteins were down-regulated, suggesting that glutamate is not only an alternative means to get energy, but also a protective agent against stress for the strain studied.
Keywords: GABA; Glutamate decarboxylase; Branched chain amino acids; ADI route; Stress; ATPase
The interaction of zinc with membrane-associated 18.5 kDa myelin basic protein: an attenuated total reflectance-Fourier transform infrared spectroscopic study by Graham S. T. Smith; Lin Chen; Vladimir V. Bamm; John R. Dutcher; George Harauz (pp. 739-750).
Myelin basic protein (MBP) is an essential structural protein required for tight compaction of the myelin sheath of the central nervous system, and belongs to the family of intrinsically disordered proteins. It contains a high proportion of polar and charged amino acids, and has an adaptive conformation depending on its environment and binding surfaces (membranes) or partners (other proteins or small ligands including divalent cations). Zinc is an important stabilizing component of myelin and its concentration is substantially higher than that of any other trace element in the brain. In this study, we investigate the effect of zinc on different variants of 18.5 kDa MBP, including new recombinant forms lacking hexahistidine tags which would interfere with the binding of the cation. Isothermal titration calorimetry showed the dissociation constant to be in the micromolar range for all variants. Circular dichroism spectroscopy showed that there was minimal effect of zinc on the secondary structure on MBP in aqueous solution. When MBP was reconstituted with myelin-mimetic membranes, attenuated total reflectance-Fourier transform infrared spectroscopy revealed that there was a rearrangement of secondary structure components upon addition of zinc that was subtly different for each variant, indicative of a synergistic protein–membrane–cation interaction.
Keywords: Myelin basic protein (MBP); Intrinsically disordered protein; Induced folding; Isothermal titration calorimetry (ITC); Circular dichroism (CD); Fourier transform infrared (FTIR) spectroscopy; Attenuated total reflectance (ATR) spectroscopy
Shotgun strategy-based proteome profiling analysis on the head of silkworm Bombyx mori by Jianying Li; S. Hossein Hosseini Moghaddam; Xiang Chen; Ming Chen; Boxiong Zhong (pp. 751-761).
Insect head is comprised of important sensory systems to communicate with internal and external environment and endocrine organs such as brain and corpus allatum to regulate insect growth and development. To comprehensively understand how all these components act and interact within the head, it is necessary to investigate their molecular basis at protein level. Here, the spectra of peptides digested from silkworm larval heads were obtained from liquid chromatography tandem mass spectrometry (LC–MS/MS) and were analyzed by bioinformatics methods. Totally, 539 proteins with a low false discovery rate (FDR) were identified by searching against an in-house database with SEQUEST and X!Tandem algorithms followed by trans-proteomic pipeline (TPP) validation. Forty-three proteins had the theoretical isoelectric point (pI) greater than 10 which were too difficult to separate by two-dimensional gel electrophoresis (2-DE). Four chemosensory proteins, one odorant-binding protein, two diapause-related proteins, and a lot of cuticle proteins, interestingly including pupal cuticle proteins were identified. The proteins involved in nervous system development, stress response, apoptosis and so forth were related to the physiological status of head. Pathway analysis revealed that many proteins were highly homologous with the human proteins which involved in human neurodegenerative disease pathways, probably implying a symptom of the forthcoming metamorphosis of silkworm. These data and the analysis methods were expected to be of benefit to the proteomics research of silkworm and other insects.
Keywords: Bombyx mori ; Insect head; Proteomics; LTQ-Orbitrap; Gene Ontology; Pathway
Novel antimicrobial peptides from the venom of the eusocial bee Halictus sexcinctus (Hymenoptera: Halictidae) and their analogs by Lenka Monincová; Miloš Buděšínský; Jiřina Slaninová; Oldřich Hovorka; Josef Cvačka; Zdeněk Voburka; Vladimír Fučík; Lenka Borovičková; Lucie Bednárová; Jakub Straka; Václav Čeřovský (pp. 763-775).
Two novel antimicrobial peptides, named halictines, were isolated from the venom of the eusocial bee Halictus sexcinctus. Their primary sequences were established by ESI-QTOF mass spectrometry, Edman degradation and enzymatic digestion as Gly-Met-Trp-Ser-Lys-Ile-Leu-Gly-His-Leu-Ile-Arg-NH2 (HAL-1), and Gly-Lys-Trp-Met-Ser-Leu-Leu-Lys–His-Ile-Leu-Lys-NH2 (HAL-2). Both peptides exhibited potent antimicrobial activity against Gram-positive and Gram-negative bacteria but also noticeable hemolytic activity. The CD spectra of HAL-1 and HAL-2 measured in the presence of trifluoroethanol or SDS showed ability to form an amphipathic α-helical secondary structure in an anisotropic environment such as bacterial cell membrane. NMR spectra of HAL-1 and HAL-2 measured in trifluoroethanol/water confirmed formation of helical conformation in both peptides with a slightly higher helical propensity in HAL-1. Altogether, we prepared 51 of HAL-1 and HAL-2 analogs to study the effect of such structural parameters as cationicity, hydrophobicity, α-helicity, amphipathicity, and truncation on antimicrobial and hemolytic activities. The potentially most promising analogs in both series are those with increased net positive charge, in which the suitable amino acid residues were replaced by Lys. This improvement basically relates to the increase of antimicrobial activity against pathogenic Pseudomonas aeruginosa and to the mitigation of hemolytic activity.
Keywords: Antimicrobial peptides; Analogs; Wild-bee venom; Hemolytic activity; NMR spectroscopy; CD spectroscopy
Identification of functionally diverse lipocalin proteins from sequence information using support vector machine by Ganesan Pugalenthi; Krishna Kumar Kandaswamy; P. N. Suganthan; G. Archunan; R. Sowdhamini (pp. 777-783).
Lipocalins are functionally diverse proteins that are composed of 120–180 amino acid residues. Members of this family have several important biological functions including ligand transport, cryptic coloration, sensory transduction, endonuclease activity, stress response activity in plants, odorant binding, prostaglandin biosynthesis, cellular homeostasis regulation, immunity, immunotherapy and so on. Identification of lipocalins from protein sequence is more challenging due to the poor sequence identity which often falls below the twilight zone. So far, no specific method has been reported to identify lipocalins from primary sequence. In this paper, we report a support vector machine (SVM) approach to predict lipocalins from protein sequence using sequence-derived properties. LipoPred was trained using a dataset consisting of 325 lipocalin proteins and 325 non-lipocalin proteins, and evaluated by an independent set of 140 lipocalin proteins and 21,447 non-lipocalin proteins. LipoPred achieved 88.61% accuracy with 89.26% sensitivity, 85.27% specificity and 0.74 Matthew’s correlation coefficient (MCC). When applied on the test dataset, LipoPred achieved 84.25% accuracy with 88.57% sensitivity, 84.22% specificity and MCC of 0.16. LipoPred achieved better performance rate when compared with PSI-BLAST, HMM and SVM-Prot methods. Out of 218 lipocalins, LipoPred correctly predicted 194 proteins including 39 lipocalins that are non-homologous to any protein in the SWISSPROT database. This result shows that LipoPred is potentially useful for predicting the lipocalin proteins that have no sequence homologs in the sequence databases. Further, successful prediction of nine hypothetical lipocalin proteins and five new members of lipocalin family prove that LipoPred can be efficiently used to identify and annotate the new lipocalin proteins from sequence databases. The LipoPred software and dataset are available at http://www3.ntu.edu.sg/home/EPNSugan/index_files/lipopred.htm .
Keywords: Lipocalin; Diverse function; Odorant binding; Support vector machine; Ligand binding; Allergenic proteins; Salivary proteins
Protective effect of isoflavones against homocysteine-mediated neuronal degeneration in SH-SY5Y cells by Youn-Jin Park; Yumi Jang; Young Hye Kwon (pp. 785-794).
Previously, we reported that isoflavones exert a protective effect against the endoplasmic reticulum (ER) stress-mediated neuronal degeneration, and ER stress-mediated homocysteine toxicity may play an important role in the pathogenesis of neurodegeneration. Therefore, in this study we investigated the effects of isoflavones (genistein and daidzein) against homocysteine-mediated neurotoxicity in SH-SY5Y human neuroblastoma cells. The treatment of cells with either 17β-estradiol or isoflavones significantly protected the cells against homocysteine-mediated apoptosis. Isoflavones repressed homocysteine-mediated ER stress, reflected in the reduced expression of the immunoglobin heavy chain-binding protein mRNA, spliced X-box-protein-1 mRNA and the phosphorylated form of eukaryotic translation initiation factor 2α protein. Homocysteine caused significant increases in intracellular S-adenosylhomocysteine (SAH) and DNA damage. Isoflavones significantly alleviated DNA damage, but did not change SAH levels. Furthermore, the treatment of cells with isoflavones significantly reduced the microtubule-associated protein tau hyperphosphorylation by inactivating glycogen synthase kinase-3β and activating serine/threonine-protein phosphatase 2A. These results clearly demonstrate that isoflavones alleviate the ER stress- and DNA damage-mediated neurodegeneration caused by homocysteine.
Keywords: DNA damage; ER stress; Homocysteine; Isoflavone; Neurodegeneration; SH-SY5Y cells
Synthesis of a novel Fmoc-protected nucleoaminoacid for the solid phase assembly of 4-piperidyl glycine/l-arginine-containing nucleopeptides and preliminary RNA interaction studies by Giovanni N. Roviello; Claudia Crescenzo; Domenica Capasso; Sonia Di Gaetano; Simona Franco; Enrico M. Bucci; Carlo Pedone (pp. 795-800).
In this work, we report the synthesis of a novel Fmoc-protected nucleoaminoacid, based on 4-piperidinyl glycine, carrying the DNA nucleobase on the secondary amino group, suitable for the solid-phase synthesis of nucleopeptides. After ESI–MS and NMR characterization this building block was used for the assembly of a thymine-functionalized tetrapeptide, composed of 4-piperidinyl glycine and l-arginine moieties alternated in the backbone. The ability to interact with RNA and the efficiency in interfering with the reverse transcription of eukaryotic mRNA of the novel nucleo-tetrapeptide found in this study are in favour of the employment of chiral nucleopeptides based on alternate 4-piperidinyl glycine/l-arginine backbone in biomedicine.
Keywords: Nucleopeptide; RNA; CD; RT–PCR
Integration of insulin and amino acid signals that regulate hepatic metabolism-related gene expression in rainbow trout: role of TOR by Marine Lansard; Stéphane Panserat; Elisabeth Plagnes-Juan; Iban Seiliez; Sandrine Skiba-Cassy (pp. 801-810).
Amino acids are considered to be regulators of metabolism in several species, and increasing importance has been accorded to the role of amino acids as signalling molecules regulating protein synthesis through the activation of the TOR transduction pathway. Using rainbow trout hepatocytes, we examined the ability of amino acids to regulate hepatic metabolism-related gene expression either alone or together with insulin, and the possible involvement of TOR. We demonstrated that amino acids alone regulate expression of several genes, including glucose-6-phosphatase, phosphoenolpyruvate carboxykinase, pyruvate kinase, 6-phospho-fructo-1-kinase and serine dehydratase, through an unknown molecular pathway that is independent of TOR activation. When insulin and amino acids were added together, a different pattern of regulation was observed that depended upon activation of the TOR pathway. This pattern included a dramatic up-regulation of lipogenic (fatty acid synthase, ATP-citrate lyase and sterol responsive element binding protein 1) and glycolytic (glucokinase, 6-phospho-fructo-1-kinase and pyruvate kinase) genes in a TOR-dependent manner. Regarding gluconeogenesis genes, only glucose-6-phosphatase was inhibited in a TOR-dependent manner by combination of insulin and amino acids and not by amino acids alone. This study is the first to demonstrate an important role of amino acids in combination with insulin in the molecular regulation of hepatic metabolism.
Keywords: Amino acids; Insulin; TOR; Hepatic gene expression; Metabolism
A novel method for promoting heterologous protein expression in Escherichia coli by fusion with the HIV-1 TAT core domain by Yonghong Wu; Changhong Ren; Yan Gao; Bing Hou; Tingfang Chen; Chenggang Zhang (pp. 811-820).
The human immunodeficiency virus type 1 (HIV-1) transactivator of transcription (TAT) protein, a member of the protein transduction domain (PTD) superfamily, can deliver heterologous proteins across most biomembranes without losing bioactivity. However, there is no report on whether the TAT core domain containing the sequence ‘YGRKKRRQRRR’ has other functions. As the TAT core domain is most basic (pI = 12.8) and has biomembrane crossing ability, we hypothesized it might probably influence the protein expression level due to subcellular redistribution of target proteins in the cells. To address this issue, we constructed the prokaryotic expression vector pET28b-TAT-EGFP (using the vector pET28b-EGFP for control) containing the core domain coding region, and transformed the vector into E. coli BL21 (DE3) cells for expression of the enhanced green fluorescent protein (EGFP) with the inducer isopropyl-β-d-thiogalactopyranoside (IPTG). Equal amount of the total proteins were fractionated using 15% SDS-PAGE and identified by western blot, and the plasmid copy number was assayed by Southern blot. In order to further study the subcellular localization of heterologous proteins in E. coli cells, the cytoplasmic and periplasmic components were extracted by chloroform and osmotic shock techniques. Interestingly, our data showed that the TAT core domain was not only able to promote the heterologous protein expression in E. coli, but also improve the yields and the solubility of heterologous proteins, while the plasmid copy number of TAT-containing clones and TAT-free clones was not affected by the TAT core domain. In addition, the TAT-tagged protein was mainly localized in the cytoplasm and also accumulated in the periplasmic space along with the time for protein expression, while in contrast, the TAT-free protein was mainly expressed in the periplasm and only a few in cytoplasm. A further examination on the distribution of the expressed proteins in cytoplasm and periplasm suggested that the TAT core domain might promote protein expression in the cytoplasm initially and then partially deliver them across the cytomembrane to the periplasmic space in a concentration-dependent manner. Taken together, our current data have provided a novel method for improving heterologous protein expression in prokaryotic cells by fusion with the TAT core domain, which will promote expression efficiency of bioactive proteins for protein engineering.
Keywords: HIV-1 TAT domain; Prokaryotic expression; Plasmid copy number; Cytoplasm; Periplasm; E. coli
l-Arginine reduces thioflavin T fluorescence but not fibrillation of bovine serum albumin by Kuan-Nan Liu; Hsiang-Yun Wang; Chih-Yuan Chen; Steven S.-S. Wang (pp. 821-829).
This work examines the effects of l-arginine (l-Arg) on the aggregation and amyloid fibrillation of bovine serum albumin (BSA). We demonstrate that l-Arg dose-dependently reduces thioflavin T (ThT) fluorescence of BSA within the l-Arg concentration range used (0–1.4 M). However, as revealed by electron microscopy, size exclusion chromatography, and dynamic light scattering results, l-Arg does not prevent amyloid-like fibril formation by BSA. We conclude that l-Arg competes against ThT for binding sites on BSA amyloid-like fibrils, leading to biased results in ThT fluorescence measurements. Moreover, the use of ThT fluorescence assay to screen for potential inhibitors against amyloid fibrillation can give misleading results.
Keywords: Amyloid fibril; Aggregate; Arginine; ThT fluorescence; Bovine serum albumin
Dietary supplementation with l-arginine or N-carbamylglutamate enhances intestinal growth and heat shock protein-70 expression in weanling pigs fed a corn- and soybean meal-based diet by Xin Wu; Zheng Ruan; Yunling Gao; Yulong Yin; Xihong Zhou; Lei Wang; Meimei Geng; Yongqing Hou; Guoyao Wu (pp. 831-839).
This study determined effects of dietary supplementation with l-arginine (Arg) or N-carbamylglutamate (NCG) on intestinal health and growth in early-weaned pigs. Eighty-four Landrace × Yorkshire pigs (average body weight of 5.56 ± 0.07 kg; weaned at 21 days of age) were fed for 7 days one of the three isonitrogenous diets: (1) a corn- and soybean meal-based diet (CSM), (2) CSM + 0.08% NCG (0.08%), and (3) CSM + 0.6% Arg. There were four pens of pigs per diet (7 pigs/pen). At the end of a 7-day feeding period, six piglets were randomly selected from each treatment for tissue collections. Compared with the control group, Arg or NCG supplementation increased (P < 0.05): (1) Arg concentrations in plasma, (2) small-intestinal growth, (3) villus height in duodenum, jejunum and ileum, (4) crypt depth in jejunum and ileum, (5) goblet cell counts in intestinal mucosae, and (6) whole-body weight gain in pigs. Real-time polymerase chain reaction and western blotting analyses revealed that both mRNA and protein levels for heat shock protein-70 (HSP70) were higher (P < 0.05) in the intestinal mucosae of Arg- or NCG-supplemented pigs than in the control group. Furthermore, the incidence of diarrhea in the NCG group was 18% lower (P < 0.01) than that in the control group. Collectively, these results indicate that dietary supplementation with 0.6% Arg or 0.08% NCG enhances intestinal HSP70 gene expression, intestinal growth and integrity, and the availability of dietary nutrients for whole-body weight gain in postweaning pigs fed a CSM-based diet. Thus, Arg or NCG is a functional ingredient in the weaning diet to improve nutrition, health, and growth performance of these neonates.
Keywords: l-Arginine; N-carbamylglutamate; Growth performance; Diarrhea; Intestinal morphology; Weaned pigs
Construction of chiral polyesters from polycondensation of multifunctional monomer containing both flexible amino acid and rigid pendant groups with aromatic diols by Shadpour Mallakpour; Marziyeh Khani (pp. 841-848).
A number of chiral wholly aromatic polyesters (PEs) with phthalimido and flexible chiral unit in the backbone were prepared from a chiral synthesized diacid monomer, 5-(3-methyl-2-phthalimidylpentanoylamino)isophthalic acid (1), and various aromatic diols via the polyesterification reaction. The tosyl chloride/pyridine/N,N-dimethylformamide (DMF) system was used as a condensing agent. All of the these polymers having bulky phthalimido and amino acid functionalities in the side chain showed excellent solubility and readily dissolved in various solvents such as N-methyl-2-pyrrolidinone, N,N-dimethylacetamide and DMF. Since, these chiral polymers have natural amino acids in the polymer architecture, they are expected to be biodegradable and therefore may be classified under eco-friendly polymers. They had useful levels of thermal stability associated with excellent solubility. Thermogravimetric analysis (TGA) showed that the obtained PEs are rather thermally stable, 10% weight loss temperatures in excess of 317°C, and char yields at 700°C in the nitrogen atmosphere higher than 24%. The resulting polymers were obtained in good yields with inherent viscosities ranging between 0.22 and 0.56 dL/g and were characterized with FT-IR, 1H-NMR, elemental and TGA techniques.
Keywords: Isoleucine; Inherent viscosity; Optically active polymers; Polycondensation; Polyesters
A stereoselective synthesis of α-deuterium-labelled (S)-α-amino acids by Elaine O’Reilly; Daniele Balducci; Francesca Paradisi (pp. 849-858).
An atom-efficient and stereoselective synthesis has been developed for the preparation of α-2H-labelled (S)-α-amino acids, starting from a novel chiral diketopiperazine scaffold. Efficient mono-alkylation of the chiral template afforded the (S)-substituted adducts with the nature of the electrophile significantly effecting the stereochemical outcome. Subsequent alkylation was totally selective producing the 1,4-cis adduct as the sole diastereoisomer. The deprotection was carried out using cerium ammonium nitrate followed by acid hydrolysis affording the enantipure α-amino acids.
Keywords: (S)-Deuterated-α-amino acids; Diketopiperazine; Asymmetric synthesis
Purification of recombinant growth hormone by clear native gels for conformational analyses: preservation of conformation and receptor binding by Wei-Qiang Chen; Anita Salmazo; Matti Myllykoski; Björn Sjöblom; Martin Bidlingmaier; Arnold Pollak; Peter Baumgärtel; Kristina Djinovic-Carugo; Petri Kursula; Gert Lubec (pp. 859-869).
Most protein preparations require purification steps prior to biophysical analysis assessing protein stability, secondary structure and degree of folding. It was, therefore, the aim of this study to develop a system to separate and purify a protein from a commercially available medicinal product, recombinant human growth hormone (rhGH) and show preservation of conformation and function following the gel-based procedure. The rhGH was run on clear native (CN) gels and recovered from the gels by electroelution using D-Tube Dialyzer Midi under rigorous cooling. Melting point studies indicated preservation of the structural integrity. This finding was confirmed by synchrotron radiation circular dichroism spectroscopy (SRCD) revealing an identical folding pattern for the sample before and after electrophoretic separation and purification. Synchrotron small-angle X-ray scattering (SAXS) indicated that the sample was folded and monomeric, both before and after separation and purification, and that its shape corresponded well to the known crystal structure of GH. Binding properties of rhGH to a receptor-model system before and after clear native electrophoresis were comparable. This analytical and preparative approach to purify and concentrate a protein preserving conformation and function may be helpful for many applications in analytical, protein and stereochemistry.
Keywords: Growth hormone; Electroelution; Conformation; SRCD; Function
Olfactory bulb proteins linked to olfactory memory in C57BL/6J mice by Lin Li; Veronika Mauric; Jun-Fang Zheng; Sung Ung Kang; Sudarshan Patil; Harald Höger; Gert Lubec (pp. 871-886).
Information on systematic analysis of olfactory memory-related proteins is poor. In this study, the odor discrimination task to investigate olfactory recognition memory of adult male C57BL/6J mice was used. Subsequently, olfactory bulbs (OBs) were taken, proteins extracted, and run on two-dimensional gel electrophoresis with in-gel-protein digestion, followed by mass spectrometry and quantification of differentially expressed proteins. Dual specificity mitogen-activated protein kinase kinase 1 (MEK1), dihydropyrimidinase-related protein 1 (DRP1), and fascin are related with Lemon odor memory. Microtubule-associated protein RP/EB family member 3 is related to Rose odor memory. Hypoxanthine-guanine phosphoribosyltransferase is related with both Lemon and Rose odors memory. MEK1 and DRP1 levels were increased, while microtubule-associated protein RP/EB family member 3, fascin and hypoxanthine-guanine phosphoribosyltransferase levels were decreased during olfactory memory. In summary, neurogenesis, signal transduction, cytoskeleton, and nucleotide metabolism are involved in olfactory memory formation and storage of C57BL/6J mice.
Keywords: Olfactory bulb; Olfactory memory; C57BL/6J mice; Olfactory discrimination task
Use of the Burrows–Wheeler similarity distribution to the comparison of the proteins by Lianping Yang; Guisong Chang; Xiangde Zhang; Tianming Wang (pp. 887-898).
In this paper, we present an approach based on Burrows–Wheeler transform to compare the protein sequences. The strings representing amino acid sequences do not reflect the chemical physical properties better, and it is very hard to extract any key features by reading these long character strings directly. The use of the Burrows–Wheeler similarity distribution needs a suitable representation which can reflect some interesting properties of the proteins. For the comparison of the primary protein sequences we convert the protein sequences into digital codes by the Ponnuswamy hydrophobicity index, and for the comparison of the structure of the proteins we adjust the topology of protein structure strings, which are simple but useful representation of the secondary structure of proteins to match the Burrows–Wheeler similarity distribution. At last, some experiments show that the approach proposed in this paper is a powerful and useful tool for the comparison of proteins.
Keywords: Burrows–Wheeler transform; Burrows–Wheeler similarity distribution; Phylogenetic tree; Similarity; Hydrophobicity; TOPS string
Acid-induced unfolding of didecameric keyhole limpet hemocyanin: detection and characterizations of decameric and tetrameric intermediate states by Ankita Varshney; Basir Ahmad; Gulam Rabbani; Vijay Kumar; Savita Yadav; Rizwan Hasan Khan (pp. 899-910).
Keyhole limpet hemocyanin (KLH) is widely used as an immune stimulant and hapten carrier derived from a marine mollusc Megathura crenulata. To provide details of the stability and equilibrium of KLH, different intermediate species were investigated with a series of biophysical techniques: circular dichroism, binding of hydrophobic dye, 1-anilino-8-naphthalene sulfonic acid, acrylamide-induced fluorescence quenching, thermal stability and dynamic light scattering. KLH in its native state at pH 7.4 exists in the stable didecameric form with hydrodynamic radii (R h) of 28.22 nm, which is approximately equal to a molecular mass of 8.8 ± 0.6 MDa. The experimental results demonstrated the presence of two structurally distinct species in the conformational transition of KLH under acidic conditions. One species populates at pH 2.8, characterized as decameric (4.8 ± 0.2 MDa; R h = 22.02 nm), molten globule-like state, while the other accumulates at pH 1.2 and is characterized as a tetramer (2.4 ± 0.8 MDa; R h = 16.47 nm) with more organized secondary and tertiary structures. Our experimental manipulation of the oligomeric states of KLH has provided data that correlate well with the known oligomeric forms obtained from total KLH formed in vivo and extends our understanding of multimer formation by KLH. The results are of particular interest in light of the important role of the mechanistic pathway of pH-dependent structural changes of Hc stability in the biochemical and medical applications of these respiratory proteins.
Keywords: Acidic pH; Decamer state; Dynamic light scattering; Keyhole limpet hemocyanin; Multimeric protein; Tetramer state
l-Histidine and l-arginine promote Knoevenagel reaction in water by Abbas Rahmati; Kobra Vakili (pp. 911-916).
Histidine and arginine were applied to the synthesis of trisubstituted alkenes through a condensation of an aldehyde with an activated CH-acid such as ethyl cyanoacetate, malononitrile, acetyl acetone or ethyl acetoacetate during 5–12 h in water at room temperature.
Keywords: Amino acids; Knoevenagel condensation reaction; Water; Histidine; Arginine