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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.40, #1)
Expression systems of human β-defensins: vectors, purification and biological activities by L. L. Corrales-Garcia; L. D. Possani; G. Corzo (pp. 5-13).
Human beta-defensins are 2–5 kDa, cationic, microbicidal peptides, which represent the first-line host defense against several Gram-negative and Gram-positive bacteria, fungi and viruses. They contain a conserved disulfide-bridge pattern of three pairs of intramolecular cystine bonds. The well-known public health problem related with the growing number of multiresistant bacteria has driven research to look for novel antibiotics, such beta-defensins and a feasible way to produce them. Heterologous expression of beta-defensins could be one way to generate large quantities of beta-defensins for clinical research; however, heterologous expression of beta-defensins has some biochemical problems, such toxicity toward the host cell, peptide degradation by proteolytic cell enzymes, size, folding constrains and low recombinant peptide yields. In this communication, several heterologous systems for producing human beta-defensins are reviewed.
Keywords: Antibiotic; Bacteria; Defensin; Heterologous expression; Antimicrobial; Peptide
Venomics: a new paradigm for natural products-based drug discovery by Irina Vetter; Jasmine L. Davis; Lachlan D. Rash; Raveendra Anangi; Mehdi Mobli; Paul F. Alewood; Richard J. Lewis; Glenn F. King (pp. 15-28).
The remarkable potency and pharmacological diversity of animal venoms has made them an increasingly valuable source of lead molecules for drug and insecticide discovery. Nevertheless, most of the chemical diversity encoded within these venoms remains uncharacterized, despite decades of research, in part because of the small quantities of venom available. However, recent advances in the miniaturization of bioassays and improvements in the sensitivity of mass spectrometry and NMR spectroscopy have allowed unprecedented access to the molecular diversity of animal venoms. Here, we discuss these technological developments in the context of establishing a high-throughput pipeline for venoms-based drug discovery.
Keywords: Drug discovery; Animal venoms; Venomics; Venom peptides; Natural products
Antimicrobial peptides from Phyllomedusa frogs: from biomolecular diversity to potential nanotechnologic medical applications by Leonardo de Azevedo Calderon; Alexandre de Almeida E. Silva; Pietro Ciancaglini; Rodrigo Guerino Stábeli (pp. 29-49).
Screening for new bioactive peptides in South American anurans has been pioneered in frogs of the genus Phyllomedusa. All frogs of this genus have venomous skin secretions, i.e., a complex mixture of bioactive peptides against potential predators and pathogens that presumably evolved in a scenario of predator–prey interaction and defense against microbial invasion. For every new anuran species studied new peptides are found, with homologies to hormones, neurotransmitters, antimicrobials, and several other peptides with unknown biological activity. From Vittorio Erspamer findings, this genus has been reported as a “treasure store” of bioactive peptides, and several groups focus their research on these species. From 1966 to 2009, more than 200 peptide sequences from different Phyllomedusa species were deposited in UniProt and other databases. During the last decade, the emergence of high-throughput molecular technologies involving de novo peptide sequencing via tandem mass spectrometry, cDNA cloning, pharmacological screening, and surface plasmon resonance applied to peptide discovery, led to fast structural data acquisition and the generation of peptide molecular libraries. Research groups on bioactive peptides in Brazil using these new technologies, accounted for the exponential increase of new molecules described in the last decade, much higher than in any previous decades. Recently, these secretions were also reported as a rich source of multiple antimicrobial peptides effective against multidrug resistant strains of bacteria, fungi, protozoa, and virus, providing instructive lessons for the development of new and more efficient nanotechnological-based therapies for infectious diseases treatment. Therefore, novel drugs arising from the identification and analysis of bioactive peptides from South American anuran biodiversity have a promising future role on nanobiotechnology.
Keywords: Phyllomedusa ; Bioprospection; Antimicrobial peptide; Dermaseptin; Infection disease; New drugs; Nanobiotechnology
Anti-proliferative and cytotoxic activity of pentadactylin isolated from Leptodactylus labyrinthicus on melanoma cells by Michelle S. Libério; Graziella A. Joanitti; Ricardo B. Azevedo; Eduardo M. Cilli; Lanuse C. Zanotta; Anna C. Nascimento; Marcelo V. Sousa; Osmindo R. Pires Júnior; Wagner Fontes; Mariana S. Castro (pp. 51-59).
Nowadays, the emergence of resistance to the current available chemotherapeutic drugs by cancer cells makes the development of new agents imperative. The skin secretion of amphibians is a natural rich source of antimicrobial peptides (AMP), and researchers have shown that some of these wide spectrum molecules are also toxic to cancer cells. The aim of this study was to verify a putative anticancer activity of the AMP pentadactylin isolated for the first time from the skin secretion of the frog Leptodactylus labyrinthicus and also to study its cytotoxic mechanism to the murine melanoma cell line B16F10. The results have shown that pentadactylin reduces the cell viability of B16F10 cells in a dose-dependent manner. It was also cytotoxic to normal human fibroblast cells; nevertheless, pentadactylin was more potent in the first case. The studies of action mechanism revealed that pentadactylin causes cell morphology alterations (e.g., round shape and shrinkage morphology), membrane disruption, DNA fragmentation, cell cycle arrest at the S phase, and alteration of mitochondrial membrane potential, suggesting that B16F10 cells die by apoptosis. The exact mechanism that causes reduction of cell viability and cytotoxicity after treatment with pentadactylin is still unknown. In conclusion, as cancer cells become resilient to death, it is worthwhile the discovery of new drugs such as pentadactylin that induces apoptosis.
Keywords: Amphibian; Apoptosis; B16F10; Cytotoxicity; Leptodactylus labyrinthicus ; Pentadactylin
Amino acid substitutions in an alpha-helical antimicrobial arachnid peptide affect its chemical properties and biological activity towards pathogenic bacteria but improves its therapeutic index by A. Rodríguez; E. Villegas; H. Satake; L. D. Possani; Gerardo Corzo (pp. 61-68).
Four variants of the highly hemolytic antimicrobial peptide Pin2 were chemically synthesized with the aim to investigate the role of the proline residue in this peptide, by replacing it with the motif glycine-valine-glycine [GVG], which was found to confer low hemolytic activity in a spider antimicrobial peptide. The proline residue in position 14 of Pin2 was substituted by [V], [GV], [VG] and [GVG]. Only the peptide variant with the proline substituted for [GVG] was less hemolytic compared to that of all other variants. The peptide variant [GVG] kept its antimicrobial activity in Muller–Hilton agar diffusion assays, whereas the other three variants were less effective. However, all Pin2 antimicrobial peptide variants, were active when challenged against a Gram-positive bacteria in Muller–Hilton broth assays suggesting that chemical properties of the antimicrobial peptides such as hydrophobicity is an important indication for antimicrobial activity in semi-solid environments.
Keywords: Antimicrobial peptide; Arachnid, pandinin 2; Peptide synthesis; S. aureus
Cupiennin 1a exhibits a remarkably broad, non-stereospecific cytolytic activity on bacteria, protozoan parasites, insects, and human cancer cells by Lucia Kuhn-Nentwig; Jean Willems; Thomas Seebeck; Tarek Shalaby; Marcel Kaiser; Wolfgang Nentwig (pp. 69-76).
Cupiennin 1a, a cytolytic peptide isolated from the venom of the spider Cupiennius salei, exhibits broad membranolytic activity towards bacteria, trypanosomes, and plasmodia, as well as human blood and cancer cells. In analysing the cytolytic activity of synthesised all-d- and all-l-cupiennin 1a towards pro- and eukaryotic cells, a stereospecific mode of membrane destruction could be excluded. The importance of negatively charged sialic acids on the outer leaflet of erythrocytes for the binding and haemolytic activity of l-cupiennin 1a was demonstrated. Reducing the overall negative charges of erythrocytes by partially removing their sialic acids or by protecting them with tri- or pentalysine results in reduced haemolytic activity of the peptide.
Keywords: Cupiennin 1a; M-ctenitoxin-Cs1a; Spider; Cupiennius salei ; Cytolytic activity; Cancer cells; Protozoan parasites; Non-stereospecificity
Investigating the effect of different positioning of lysine residues along the peptide chain of mastoparans for their secondary structures and biological activities by Bibiana Monson de Souza; Marcia Perez dos Santos Cabrera; João Ruggiero Neto; Mario Sergio Palma (pp. 77-90).
In order to investigate the effect of the different positions of the positive charges generated by the ionization of the side-chain of lysine residues, on the structure–activity relationship of the mastoparans, the peptides Protonectarina-MP (INWKALLDAAKKVL-NH2), Parapolybia-MP (INWKKMAATALKMI-NH2) and Asn-2-Polybia-MP I (INWKKLLDAAKQIL-NH2) and MK-578 (INWLKAKKVAGMIL-NH2) were investigated as models. Thus, the four peptides had their secondary structure studied and were submitted to assays of mast cell degranulation, hemolysis, and antibiosis. The results of the bioassays made clear that those peptides bearing the positive charges positioned at the positions 4/5 and/or from 11 to 13 are the most active ones; meanwhile, the localization of the positive charges in the middle of peptide chain resulted in a poorly active peptide. Thus, Protonectarina-MP, Parapolybia-MP, and Asn-2-Polybia-MP I presented physiologically important hemolysis and antibiosis, while MK-578 presented only a reduced antibiotic activity. Circular dichroism analysis were carried-out in different environments revealing that the anionic environment of a mixture of phosphatidylcholine and phosphatidylglycerol (70:30) liposomes favored the higher helical content of the four peptides in this study in relation to the zwiterionic environment of 100% phosphatidylcholine liposomes. The positioning of the lysine residues at the strategic positions (4/5 and 11–13), flanking and maintaining stable α-helix which extends from the 4th to the 13th residue along the peptide chain, seems to contribute to maximal lytic efficiency of the mastoparans, which in turn results in a more homogeneous hydrophobic surface in the amphipathic structure.
Keywords: Mastoparans; Antibiotic peptides; Hemolysis; Lysine-rich peptides; Circular dichroism
The effect of acidic residues and amphipathicity on the lytic activities of mastoparan peptides studied by fluorescence and CD spectroscopy by Natália Bueno Leite; Laiana Cristina da Costa; Dayane dos Santos Alvares; Marcia Perez dos Santos Cabrera; Bibiana Monson de Souza; Mário Sérgio Palma; João Ruggiero Neto (pp. 91-100).
Some mastoparan peptides extracted from social wasps display antimicrobial activity and some are hemolytic and cytotoxic. Although the cell specificity of these peptides is complex and poorly understood, it is believed that their net charges and their hydrophobicity contribute to modulate their biological activities. We report a study, using fluorescence and circular dichroism spectroscopies, evaluating the influence of these two parameters on the lytic activities of five mastoparans in zwitterionic and anionic phospholipid vesicles. Four of these peptides, extracted from the venom of the social wasp Polybia paulista, present both acidic and basic residues with net charges ranging from +1 to +3 which were compared to Mastoparan-X with three basic residues and net charge +4. Previous studies revealed that these peptides have moderate-to-strong antibacterial activity against Gram-positive and Gram-negative microorganisms and some of them are hemolytic. Their affinity and lytic activity in zwitterionic vesicles decrease with the net electrical charges and the dose response curves are more cooperative for the less charged peptides. Higher charged peptides display higher affinity and lytic activity in anionic vesicles. The present study shows that the acidic residues play an important role in modulating the peptides’ lytic and biological activities and influence differently when the peptide is hydrophobic or when the acidic residue is in a hydrophilic peptide.
Keywords: Mastoparans; Antimicrobial peptides; Peptide net charge; Hydrophobicity; Circular dichroism; Fluorescence spectroscopy
Hyperalgesic and edematogenic effects of peptides isolated from the venoms of honeybee (Apis mellifera) and neotropical social wasps (Polybia paulista and Protonectarina sylveirae) by P. Brigatte; Y. Cury; B. M. de Souza; N. B. Baptista-Saidemberg; D. M. Saidemberg; V. P. Gutierrez; Mario Sérgio Palma (pp. 101-111).
Stings by bees and wasps, including Brazilian species, are a severe public health problem. The local reactions observed after the envenoming includes typical inflammatory response and pain. Several studies have been performed to identify the substances, including peptides that are responsible for such phenomena. The aim of the present study is to characterize the possible nociceptive (hyperalgesic) and edematogenic effects of some peptides isolated from the venoms of the honeybee (Apis mellifera) and the social wasps Polybia paulista and Protonectarina sylveirae, in addition to characterize some of the mechanisms involved in these phenomena. For this purpose, different doses of the peptides mellitin (Apis mellifera), Polybia-MP-I, N-2-Polybia-MP-I (Polybia paulista), Protonectarina-MP-NH2 and Protonectarina-MP-OH (Protonectarina sylveirae) were injected into the hind paw of mice. Hyperalgesia and edema were determined after peptide application, by using an electronic von Frey apparatus and a paquimeter. Carrageenin and saline were used as controls. Results showed that melittin, Polybia-MP-I, N-2-Polybia-MP-I, Protonectarina-MP-NH2 and Protonectarina-MP-OH peptides produced a dose- and time-related hyperalgesic and edematogenic responses. Both phenomena are detected 2 h after melittin, Polybia-MP-I, N-2-Polybia-MP-I injection; their effects lasted until 8 h. In order to evaluate the role of prostanoids and the involvement of lipidic mediators in hyperalgesia induced by the peptides, indomethacin and zileuton were used. Results showed that zileuton blocked peptide-induced hyperalgesia and induced a decrease of the edematogenic response. On the other hand, indomethacin did not interfere with these phenomena. These results indicate that melittin, Polybia-MP-I, N-2-Polybia-MP-I, Protonectarina-MP-NH2, and Protonectarina-MP-OH peptides could contribute to inflammation and pain induced by insect venoms.
Keywords: Hyperalgesia; Inflammation; Apis mellifera ; Polybia paulista ; Protonectarina sylveirae ; Peptides
Peptidomic analysis of the skin secretions of the frog Pachymedusa dacnicolor by Erika P. Meneses; Oscar Villa-Hernández; Lorena Hernández-Orihuela; Ruben Castro-Franco; Victoria Pando; Manuel B. Aguilar; Cesar Vicente Ferreira Batista (pp. 113-122).
High-resolution mass spectrometry-based peptidomics has been used to characterize several components in electro-stimulated skin secretions of the endemic Mexican frog Pachymedusa dacnicolor. Peptide mass screening performed in an Orbitrap-XL mass spectrometer showed that P. dacnicolor skin secretions possess 194 different components with molecular masses ranging mainly from 500 to 6,000 Da. Dozens of molecules were partially sequenced including two novel protease inhibitors. Additionally, one posttranslationally modified bradykinin and two novel dermaseptin-like antimicrobial peptides were fully sequenced. The novel peptide named here DMS-DA5 was fully characterized and showed potent antibacterial activity against various bacteria such as Escherichia coli, Bacillus subtilis, Salmonella enterica serovar typhimurium, and Pseudomonas aeruginosa with minimal inhibitory concentrations from 3.10 to 25.0 μM.
Keywords: Frog skin; Antibacterial peptides; Peptidomics; Mass spectrometry
The structural parameters for antimicrobial activity, human epithelial cell cytotoxicity and killing mechanism of synthetic monomer and dimer analogues derived from hBD3 C-terminal region by L. Zhou; S. P. Liu; L. Y. Chen; J. Li; L. B. Ong; L. Guo; T. Wohland; C. C. Tang; R. Lakshminarayanan; J. Mavinahalli; C. Verma; R. W. Beuerman (pp. 123-133).
Understanding the molecular mechanisms of antimicrobial peptide–membrane interactions is crucial in predicting the design of useful synthetic antimicrobial peptide analogues. Defensins are small (3–5 kDa) cysteine-rich cationic proteins which constitute the front line of host innate immunity. In this study, a series of eight 10 AA C-terminal analogues of hBD3 [sequence: RGRKXXRRKK, X = W, F, Y, V, L, I, H, C(Acm); net charge = +7, coded as W2, F2, Y2, V2, L2, I2, H2, and C2] and covalent V2-dimer [(RGRKVVRR)2KK] (18 AA, net charge = +11) were synthesized using solid phase peptide synthesis (SPPS) in Fmoc chemistry. Wild-type hBD3 was used as a control in all analyses. W2, V2, and especially Y2 showed high activity selectively against Gram-negative bacteria Pseudomonas aeruginosa in the concentration range of 4.3–9.7 μM. The covalent dimeric form of V2-monomer, V2-dimer, showed increased antibacterial killing compared to the monomeric form, V2-monomer. Cytotoxicity assays on a human conjunctival epithelial cell line (IOBA-NHC cells) showed that no change in viable cell number 24 h after constant exposure to all the eight peptide analogues even at concentrations up to 200 μg/ml. Fluorescence correlation spectroscopy (FCS) was used to study the interaction of these peptides against POPC vesicles (neutral; mammalian cell membrane mimic) and POPG vesicles (negatively charged; bacterial cell membrane mimic). Using FCS, significant aggregation and some leakage of Rhodamine dye were observed with POPG with Y2, W2 and V2 at the concentration of 5–10 μM and no significant aggregation or disruption of vesicles was observed for all peptide analogues tested against POPC. V2-dimer induced more leakage and aggregation than the monomeric form. Overall, V2-dimer is the most effective antimicrobial peptide, with aggregation of POPG vesicles observed at concentrations as low as 1 μM. The concentration of 5–10 μM for Y2 from FCS correlated with the concentration of 5 μM (6.25 μg/ml), at which Y2 showed a cooperative increase in the activity. This suggests a structural transition of Y2 in the 2.5–5 μM concentration range resulting in the correlated increased antimicrobial activity. These results and the FCS together with previous NMR and molecular dynamics (MD) suggested that the charge density-based binding affinity, stable covalent dimerization, the ability to dimerize or even oligomerize and adopt a well-defined structure are important physicochemical properties distinguishing more effective cationic antimicrobial peptides.
Keywords: Antimicrobial peptides (AMPs); Human β-defensin 3 (hBD-3); Covalent dimerization; Cytotoxicity; Fluorescence correlation spectroscopy (FCS)
Labaditin, a cyclic peptide with rich biotechnological potential: preliminary toxicological studies and structural changes in water and lipid membrane environment by S. C. Barbosa; E. M. Cilli; Luis G. Dias; Rodrigo G. Stabeli; P. Ciancaglini (pp. 135-144).
Cyclic peptides isolated from the plants of the Euphorbiaceae family have been largely studied due to their rigid conformation, which is considered significant for biologic activity. The peptide Labaditin (L0) and its open chain analogs (L1) were synthesized by the solid-phase peptide synthesis technique (Fmoc/tBu), and purified to elucidate its interaction with membrane models. A shift in λmax emission and Stern–Volmer constants values indicate that both tryptophans migrate to a more apolar environment, with L1 decreasing less than L0. A circular dichroism (CD) study revealed that L0 was kept unstructured in aqueous media as much as in the presence of dipalmitoilphosphatidylcholine liposomes. The thermodynamic studies by differential calorimetry (DSC) show a ΔH increase (50 and 18 kcal/mol, for L0 and L1, respectively) with peptide concentrations, which is indicative of lipids associating with peptides, resulting in the inability of the lipids to participate in the main transition. Therefore, all CD, DSC, and fluorescence data suggest a greater L0 membrane insertion. A probable mechanism for Labaditin interaction is based initially on the hydrophobic interaction of the peptide with the lipid membrane, conformational change, peptide adsorption on the lipid surface, and internalization process. Peptide’s antibacterial effect was also evaluated and revealed that only L0 showed reduction in viability in Gram-positive bacteria while no effects to the Gram-negative.
Keywords: Labaditin; Peptide synthesis; Liposome; Fluorescence; Antibacterial activity; Membrane interaction
High-abundance proteins depletion for serum proteomic analysis: concomitant removal of non-targeted proteins by Elisa Bellei; Stefania Bergamini; Emanuela Monari; Luca Isaia Fantoni; Aurora Cuoghi; Tomris Ozben; Aldo Tomasi (pp. 145-156).
In clinical and pharmaceutical proteomics, serum and plasma are frequently used for detection of early diagnostic biomarkers for therapeutic targets. Although obtaining these body fluid samples is non-invasive and easy, they contain some abundant proteins that mask other protein components present at low concentrations. The challenge in identifying serum biomarkers is to remove the abundant proteins, uncovering and enriching at the same time the low-abundance ones. The depletion strategies, however, could lead to the concomitant removal of some non-targeted proteins that may be of potential interest. In this study, we compared three different methods aimed to deplete high-abundance proteins from human serum, focusing on the identification of non-specifically bound proteins which might be eventually removed. A Cibacron blue-dye-based method for albumin removal, an albumin and IgG immunodepletion method and an immunoaffinity column (Multiple Affinity Removal System) that simultaneously removes a total of six high-abundance proteins, were investigated. The bound proteins were eluted, separated by two-dimensional gel electrophoresis and identified by Nano LC-CHIP-MS system. Flow-through fractions and bound fractions were also analysed with the ProteinChip technology SELDI-TOF-MS. Our results showed that the methods tested removed not only the targeted proteins with high efficiency, but also some non-targeted proteins. We found that the Multiple Affinity Removal Column improved the intensity of low-abundance proteins, displayed new protein spots and increased resolution. Notably, the column showed the lowest removal of untargeted proteins, proved to be the most promising depletion approach and a reliable method for serum preparation prior to proteomic studies.
Keywords: High-abundance proteins; Human serum; Nano LC-CHIP-MS; Non-targeted proteins; SELDI-TOF-MS; Two-dimensional gel electrophoresis
Differential effects of long-term leucine infusion on tissue protein synthesis in neonatal pigs by Fiona A. Wilson; Agus Suryawan; Renán A. Orellana; María C. Gazzaneo; Hanh V. Nguyen; Teresa A. Davis (pp. 157-165).
Leucine is unique among the amino acids in its ability to promote protein synthesis by activating translation initiation via the mammalian target of rapamycin (mTOR) pathway. Previously, we showed that leucine infusion acutely stimulates protein synthesis in fast-twitch glycolytic muscle of neonatal pigs but this response cannot be maintained unless the leucine-induced fall in amino acids is prevented. To determine whether leucine can stimulate protein synthesis in muscles of different fiber types and in visceral tissues of the neonate in the long-term if baseline amino acid concentrations are maintained, overnight fasted neonatal pigs were infused for 24 h with saline, leucine (400 μmol kg−1 h−1), or leucine with replacement amino acids to prevent the leucine-induced hypoaminoacidemia. Changes in the fractional rate of protein synthesis and activation of mTOR, as determined by eukaryotic initiation factor 4E binding protein (4E-BP1) and S6 kinase 1 (S6K1) phosphorylation, in the gastrocnemius and masseter muscles, heart, liver, jejunum, kidney, and pancreas were measured. Leucine increased mTOR activation in the gastrocnemius and masseter muscles, liver, and pancreas, in both the absence and presence of amino acid replacement. However, protein synthesis in these tissues was increased only when amino acids were infused to maintain baseline levels. There were no changes in mTOR signaling or protein synthesis in the other tissues we examined. Thus, long-term infusion of leucine stimulates mTOR signaling in skeletal muscle and some visceral tissues but the leucine-induced stimulation of protein synthesis in these tissues requires sustained amino acid availability.
Keywords: Growth; Muscle; Translation initiation; Mammalian target of rapamycin; Eukaryotic initiation factor 4E binding protein; Ribosome protein S6 kinase
Favored and disfavored pathways of protein crosslinking by glucose: glucose lysine dimer (GLUCOLD) and crossline versus glucosepane by Ina Nemet; Christopher M. Strauch; Vincent M. Monnier (pp. 167-181).
We describe the isolation and molecular characterization of a novel glucose-lysine dimer crosslink 1,3-bis-(5-amino-5-carboxypentyl)-4-(1′,2′,3′,4′-tetrahydroxybutyl)-3H-imidazolium salt, named GLUCOLD. GLUCOLD was easily formed from the Amadori product (fructose–lysine). However, when BSA was incubated with 100 mM glucose for 25 days, the levels of the lysine-lysine glucose crosslinks GLUCOLD and CROSSLINE were only 21 and <1 pmol/mg, respectively, compared to 611 pmol/mg protein for the lysine-arginine GLUCOSEPANE crosslink, in spite of more than 20 potential lysine-lysine crosslinking sites in the protein. Mechanistic investigation revealed that metal-free phosphate ions catalyzed formation of fructose–lysine and all three crosslinks from amino acids, while cationic MOPS buffer had an opposite effect. This together with the rapid formation of N 6-1,4-dideoxy-5,6-dioxoglucosone derivatives by dicarbonyl trapping agents, such as 1,2-diaminobenzene or γ-guanidinobutyric acid, strongly suggests that enolization of the Amadori product and trapping of the 5,6-dioxo derivative by arginine residues constitutes the major pathway for glucose-mediated crosslinking in proteins.
Keywords: Advanced glycation end-product; Maillard reaction; Glycation; Crosslink; Diabetes
C-terminal amidation of PMAP-23: translocation to the inner membrane of Gram-negative bacteria by Jin-Young Kim; Seong-Cheol Park; Moon-Young Yoon; Kyung-Soo Hahm; Yoonkyung Park (pp. 183-195).
PMAP-23 is a member of the cathelicidin family derived from pig myeloid cells and has potent antimicrobial activity. Amidation of the carboxyl terminus (C-terminus) of an antimicrobial peptide generally enhances its structural stability and antimicrobial activity or decreases its cytotoxicity. The aim of the present study was to investigate the effect of amidation on the mode of action in PMAP-23. Irrespective of amidation, PMAP-23 adopts a helix–hinge–helix structure in a membrane-mimetic environment. The antibacterial activities of PMAP-23C, which had a free C-terminus, and PMAP-23N, which had an amidated C-terminus, were similar against Gram-negative bacteria, reflecting a similar ability to neutralize lipopolysaccharide. However, PMAP-23N assumed a perpendicular orientation across the outer to the inner leaflet of the bacterial inner membrane, while PMAP-23C was orientated parallel to the lipid bilayer, as determined by following the blue shift in tryptophan fluorescence, as well as calcein release from liposomes and SYTOX Green uptake assays. These results suggest that N-terminal amidation of PMAP-23 provides structural stability and increases the peptide’s cationic charge, facilitating translocation into the bacterial inner membrane.
Keywords: PMAP-23; Antimicrobial peptide; Lipopolysaccharide; Amidation; Translocation; Membrane bilayer
Synthesis of potentially bioactive PABA-related N-(aminoalkyl)lactamic amino acids and esters via selective SNAr reactions by Renato S. Gonçalves; Patrícia V. Abdelnur; Vanessa G. Santos; Rosineide C. Simas; Marcos N. Eberlin; Alviclér Magalhães; Eduardo R. Pérez González (pp. 197-204).
Potentially bioactive N-(aminoalkyl)lactamic amino acids and esters were synthesized in satisfactory to good yields by SNAr reactions of aromatic acids with N-(3-aminopropyl)lactams followed by esterification with tertiary amino alcohols. The addition–elimination SNAr mechanism was confirmed by NMR and MS measurements.
Keywords: PABA derivatives; Lactams; Potential bio-activity; SNAr reaction
Adsorption of cysteine on hematite, magnetite and ferrihydrite: FT-IR, Mössbauer, EPR spectroscopy and X-ray diffractometry studies by Alessandra P. Vieira; Graciele Berndt; Ivan G. de Souza Junior; Eduardo Di Mauro; Andrea Paesano Jr; Henrique de Santana; Antonio Carlos S. da Costa; Cássia T. B. V. Zaia; Dimas A. M. Zaia (pp. 205-214).
In the present paper, the adsorption of cysteine on hematite, magnetite and ferrihydrite was studied using FT-IR, electron paramagnetic resonance (EPR), Mössbauer spectroscopy and X-ray diffractometry. Cysteine was dissolved in artificial seawater (two different pHs) which contains the major constituents. There were two main findings described in this paper. First, after the cysteine adsorption, the FT-IR spectroscopy and X-ray diffractometry data showed the formation of cystine. Second, the Mössbauer spectroscopy did not show any increase in the amount of Fe2+ as expected due the oxidation of cysteine to cystine. An explanation could be that Fe2+ was oxidized by the oxygen present in the seawater or there occurred a reduction of cystine by Fe2+ generating cysteine and Fe3+. The specific surface area and pH at point of zero charge of the iron oxides were influenced by adsorption of cysteine. When compared to other iron oxides, ferrihydrite adsorbed significantly (p < 0.05) more cysteine. The pH has a significant (p < 0.05) effect only on cysteine adsorption on hematite. The FT-IR spectroscopy results showed that cystine remains adsorbed on the surface of the iron oxides even after being mixed with KCl and the amine and carboxylic groups are involved in this interaction. X-ray diffractometry showed no changes on iron oxides mineralogy and the following precipitated substances were found along with the iron oxides after drying the samples: cysteine, cystine and seawater salts. The EPR spectroscopy showed that cysteine interacts with iron oxides, changing the relative amounts of iron oxides and hydroxide.
Keywords: Iron oxides; Amino acids; Surface complexation; Spectroscopy; Prebiotic chemistry
Preparation of optically active β-hydroxy-α-amino acid by immobilized Escherichia coli cells with serine hydroxymethyl transferase activity by Gen-Hai Zhao; Hui Li; Wei Liu; Wei-Guo Zhang; Fei Zhang; Qian Liu; Qing-Cai Jiao (pp. 215-220).
In this research, an improved method for preparation of optically pure β-hydroxy-α-amino acids, catalyzed by serine hydroxymethyl transferase with threonine aldolase activity, is reported. Using recombinant serine hydroxymethyl transferase (SHMT), an enzymatic resolution process was established. A series of new substrates, β-phenylserine, β-(nitrophenyl) serine and β-(methylsulfonylphenyl) serine were used in the resolution process catalyzed by immobilized Escherichia coli cells with SHMT activity. It was observed that the K m for l-threonine was 28-fold higher than that for l-allo-threonine, suggesting that this enzyme can be classified as a low-specificity l-allo-threonine aldolase. The results also shows that SHMT activity with β-phenylserine as substrate was about 1.48-fold and 1.25-fold higher than that with β-(methylsulfonylphenyl) serine and β-(nitrophenyl) serine as substrate, respectively. Reaction conditions were optimized by using 200 mmol/l β-hydroxy-α-amino acid, and 0.1 g/ml of immobilized SHMT cells at pH 7.5 and 45°C. Under these conditions, the immobilized cells were continuously used 10 times, yielding an average conversion rate of 60.4%. Bead activity did not change significantly the first five times they were used, and the average conversion rate during the first five instances was 84.1%. The immobilized cells exhibited favourable operational stability.
Keywords: d-threo-Amino acid; Enzymatic resolution; Immobilized cells; Serine hydroxymethyl transferase
Characterization, using comparative proteomics, of differentially expressed proteins in the hippocampus of the mesial temporal lobe of epileptic rats following treatment with valproate by Liwen Wu; Jing Peng; Chaoping Wei; Gu Liu; Guoli Wang; Kongzhao Li; Fei Yin (pp. 221-238).
The objective of the study was to explore the pathogenesis of mesial temporal lobe epilepsy (MTLE) and the mechanism of valproate administration in the early stage of MTLE development. We performed a global comparative analysis and function classification of differentially expressed proteins using proteomics. MTLE models of developmental rats were induced by lithium-pilocarpine. Proteins in the hippocampus were separated by 2-DE technology. PDQuest software was used to analyze 2-DE images, and MALDI-TOF-MS was used to identify the differentially expressed proteins. Western blot was used to determine the differential expression levels of synapse-related proteins synapsin-1, dynamin-1 and neurogranin in both MTLE rat and human hippocampus. A total of 48 differentially expressed proteins were identified between spontaneous and non-spontaneous MTLE rats, while 41 proteins between MTLE rats and post valproate-treatment rats were identified. All of the proteins can be categorized into several groups by biological functions: synaptic and neurotransmitter release, cytoskeletal structure and dynamics, cell junctions, energy metabolism and mitochondrial function, molecular chaperones, signal regulation and others. Western blot results were similar to the changes noted in 2-DE. The differentially expressed proteins, especially the proteins related to synaptic and neurotransmitter release function, such as synapsin-1, dynamin-1 and neurogranin, are probably involved in the mechanism of MTLE and the pharmacological effect of valproate. These findings may provide important clues to elucidate the mechanism of chronic MTLE and to identify an optimum medication intervention time and new biomarkers for the development of pharmacological therapies targeted at epilepsy.
Keywords: Mesial temporal lobe epilepsy; Hippocampus; Valproate; Proteomics; Mechanism
Identification of RNA-binding sites in proteins by integrating various sequence information by Cui-cui Wang; Yaping Fang; Jiamin Xiao; Menglong Li (pp. 239-248).
RNA–protein interactions play a pivotal role in various biological processes, such as mRNA processing, protein synthesis, assembly, and function of ribosome. In this work, we have introduced a computational method for predicting RNA-binding sites in proteins based on support vector machines by using a variety of features from amino acid sequence information including position-specific scoring matrix (PSSM) profiles, physicochemical properties and predicted solvent accessibility. Considering the influence of the surrounding residues of an amino acid and the dependency effect from the neighboring amino acids, a sliding window and a smoothing window are used to encode the PSSM profiles. The outer fivefold cross-validation method is evaluated on the data set of 77 RNA-binding proteins (RBP77). It achieves an overall accuracy of 88.66% with the Matthew’s correlation coefficient (MCC) of 0.69. Furthermore, an independent data set of 39 RNA-binding proteins (RBP39) is employed to further evaluate the performance and achieves an overall accuracy of 82.36% with the MCC of 0.44. The result shows that our method has good generalization abilities in predicting RNA-binding sites for novel proteins. Compared with other previous methods, our method performs well on the same data set. The prediction results suggest that the used features are effective in predicting RNA-binding sites in proteins. The code and all data sets used in this article are freely available at http://cic.scu.edu.cn/bioinformatics/Predict_RBP.rar .
Keywords: RNA–protein interactions; RNA-binding sites prediction; Support vector machines; Position-specific scoring matrix (PSSM)
Chronic treatment with zinc and antidepressants induces enhancement of presynaptic/extracellular zinc concentration in the rat prefrontal cortex by Magdalena Sowa-Kućma; Magdalena Kowalska; Marek Szlósarczyk; Krystyna Gołembiowska; Włodzimierz Opoka; Bogusław Baś; Andrzej Pilc; Gabriel Nowak (pp. 249-258).
Zinc exhibits antidepressant-like activity in preclinical tests/models. Moreover, zinc homeostasis is implicated in the pathophysiology of affective disorders. The aim of the present study was to examine the effect of chronic zinc, citalopram and imipramine intraperitoneal administration on the presynaptic and extracellular zinc concentration in the rat prefrontal cortex and hippocampus. We used two methods: zinc–selenium histochemistry (which images the pool of presynaptic-vesicle zinc) and anodic stripping voltammetry (ASV) for zinc determination in microdialysate (which assays the extracellular zinc concentration). We report that chronic (14×) zinc (hydroaspartate, 10 and 65 mg/kg) and citalopram (20 mg/kg) administration increased the pool of presynaptic zinc (by 34, 50 and 37%, respectively) in the rat prefrontal cortex. The 21% increase induced by imipramine (20 mg/kg) was marginally significant. Likewise, zinc (hydroaspartate, 65 mg/kg), citalopram and imipramine increased the extracellular zinc (although with a different pattern: time point, area under the curve and/or basal level) in this brain region. Furthermore, zinc induced an increase in presynaptic (by 65%) and extracellular zinc (by 90%) in the hippocampus, while both citalopram and imipramine did not. These results indicate that all of the treatments increase presynaptic/extracellular zinc concentrations in the rat prefrontal cortex, which may then contribute to their antidepressant mechanisms. Alterations induced by zinc (but not antidepressants) administration in the hippocampus may be related to specific zinc mechanisms. All the data (previous and present) on the effect of antidepressant treatments on the presynaptic/extracellular zinc concentrations suggest the involvement of this biometal presynaptic/synaptic homeostasis in the antidepressant mechanism(s).
Keywords: Zinc; Antidepressants; Zinc–selenium histochemistry; Microdialysis; Stripping voltammetry