Amino Acids (v.44, #4)
Oostatic peptides by Jan Hlaváček (1095-1105).
Oostatic peptides are organic molecules, which influence an insect reproduction due to a regulation of the eggs development. It was proved that decapeptide—H-Tyr-Asp-Pro-Ala-Pro-Pro-Pro-Pro-Pro-Pro-OH (YDPAPPPPPP)—isolated from mosquito Aedes aegypti, inhibits trypsin activity in the midgut of the mosquito. Therefore, it was named trypsin-modulating oostatic factor (Aea-TMOF). Feeding the recombinant cells with cloned and expressed TMOF on the coat protein of tobacco mosaic virus (TMV) to mosquito larvae, caused larval mortality. The TMOF was therefore designed for usage as a new biorational insecticide against mosquito. Similarly, a hexapeptide—H-Asn-Pro-Thr-Asn-Leu-His-OH (NPTNLH)—was isolated from the grey flesh fly Neobellieria bullata. This peptide and some of its analogs inhibited trypsin-like synthesis by the midgut in female flies and was therefore entitled Neb-TMOF. Interestingly, the synthetic Aea-TMOF and mainly its C-terminus shorten analogs, including those containing D-amino acids or methylene-oxy isosteric bond, quickly and strongly inhibited the hatchability and egg development in the flesh fly N. bullata.
Keywords: Insect development regulation; Oostatic activity; TMOF and analogs; Genetic approach; Degradation in ovaries
Dietary requirements of “nutritionally non-essential amino acids” by animals and humans by Guoyao Wu; Zhenlong Wu; Zhaolai Dai; Ying Yang; Weiwei Wang; Chuang Liu; Bin Wang; Junjun Wang; Yulong Yin (1107-1113).
Amino acids are necessary for the survival, growth, development, reproduction and health of all organisms. They were traditionally classified as nutritionally essential or non-essential for mammals, birds and fish based on nitrogen balance or growth. It was assumed that all “non-essential amino acids (NEAA)” were synthesized sufficiently in the body to meet the needs for maximal growth and health. However, there has been no compelling experimental evidence to support this assumption over the past century. NEAA (e.g., glutamine, glutamate, proline, glycine and arginine) play important roles in regulating gene expression, cell signaling, antioxidative responses, neurotransmission, and immunity. Additionally, glutamate, glutamine and aspartate are major metabolic fuels for the small intestine to maintain its digestive function and protect its mucosal integrity. Therefore, based on new research findings, NEAA should be taken into consideration in revising the classical “ideal protein” concept and formulating balanced diets to improve protein accretion, food efficiency, and health in animals and humans.
Keywords: Amino acids; Food efficiency; Health; Metabolism; Nutrition
Tenascins and inflammation in disorders of the nervous system by Igor Jakovcevski; Djordje Miljkovic; Melitta Schachner; Pavle R. Andjus (1115-1127).
In vitro and in vivo studies on the role of tenascins have shown that the two paradigmatic glycoproteins of the tenascin family, tenascin-C (TnC) and tenascin-R (TnR) play important roles in cell proliferation and migration, fate determination, axonal pathfinding, myelination, and synaptic plasticity. As components of the extracellular matrix, both molecules show distinct, but also overlapping dual functions in inhibiting and promoting cell interactions depending on the cell type, developmental stage and molecular microenvironment. They are expressed by neurons and glia as well as, for TnC, by cells of the immune system. The functional relationship between neural and immune cells becomes relevant in acute and chronic nervous system disorders, in particular when the blood brain and blood peripheral nerve barriers are compromised. In this review, we will describe the functional parameters of the two molecules in cell interactions during development and, in the adult, in synaptic activity and plasticity, as well as regeneration after injury, with TnC being conducive for regeneration and TnR being inhibitory for functional recovery. Although not much is known about the role of tenascins in neuroinflammation, we will describe emerging knowledge on the interplay between neural and immune cells in autoimmune diseases, such as multiple sclerosis and polyneuropathies. We will attempt to point out the directions of experimental approaches that we envisage would help gaining insights into the complex interplay of TnC and TnR with the cells that express them in pathological conditions of nervous and immune systems.
Keywords: Extracellular matrix; Nervous system disorders; Neuroinflammation; Regeneration; Tenascin-C; Tenascin-R
Crowdsourcing in proteomics: public resources lead to better experiments by Harald Barsnes; Lennart Martens (1129-1137).
With the growing interest in the field of proteomics, the amount of publicly available proteome resources has also increased dramatically. This means that there are many useful resources available for almost all aspects of a proteomics experiment. However, it remains vital to use the right resource, for the right purpose, at the right time. This review is therefore meant to aid the reader in obtaining an overview of the available resources and their application, thus providing the necessary background to choose the appropriate resources for the experiment at hand. Many of the resources are also taking advantage of so-called crowdsourcing to maximize the potential of the resource. What this means and how this can improve future experiments will also be discussed. The text roughly follows the steps involved in a proteomics experiment, starting with the planning of the experiment, via the processing of the data and the analysis of the results, to the community-wide sharing of the produced data.
Keywords: Proteomics; Mass spectrometry; Bioinformatics; Databases; Repositories
Potency of GABA at human recombinant GABAA receptors expressed in Xenopus oocytes: a mini review by Nasiara Karim; Petrine Wellendorph; Nathan Absalom; Graham A. R. Johnston; Jane R. Hanrahan; Mary Chebib (1139-1149).
GABAA receptors are members of the ligand-gated ion channel superfamily that mediate inhibitory neurotransmission in the central nervous system. They are thought to be composed of 2 alpha (α), 2 beta (β) subunits and one other such as a gamma (γ) or delta (δ) subunit. The potency of GABA is influenced by the subunit composition. However, there are no reported systematic studies that evaluate GABA potency on a comprehensive number of subunit combinations expressed in Xenopus oocytes, despite the wide use of this heterologous expression system in structure–function studies and drug discovery. Thus, the aim of this study was to conduct a systematic characterization of the potency of GABA at 43 human recombinant GABAA receptor combinations expressed in Xenopus oocytes using the two-electrode voltage clamp technique. The results show that the α-subunits and to a lesser extent, the β-subunits influence GABA potency. Of the binary and ternary combinations with and without the γ2L subunit, the α6/γ2L-containing receptors were the most sensitive to GABA, while the β2- or β3-subunit conferred higher sensitivity to GABA than receptors containing the β1-subunit with the exception of the α2β1γ2L and α6β1γ2L subtypes. Of the δ-subunit containing GABAA receptors, α4/δ-containing GABAA receptors displayed highest GABA sensitivity, with mid-nanomolar concentrations activating α4β1δ and α4β3δ receptors. At α4β2δ, GABA had low micromolar activity.
Keywords: GABA; Synaptic and extrasynaptic GABAA receptors; GABA potency; Xenopus oocytes; Two-electrode voltage clamp
Astroglial amino acid-based transmitter receptors by Vladimir Parpura; Alexei Verkhratsky (1151-1158).
Amino acids appear in prebiotic period being one of the first organic molecules on Earth. For neurobiologists, it is of importance that AAs are not only representing building blocks of life, but are also the essential part of metabolism and cellular signaling. In the mammalian brain, the most common excitatory and inhibitory transmitters acting upon cellular plasmalemmal receptors are the amino acid glutamate and its derivative γ-aminobutyric acid, respectively. Other amino acids, i.e. aspartate, glycine, d-serine, and homocysteic acid, as well as the sulfonic acid taurine, are also active compounds involved in receptor-mediated brain signaling. Receptors for these amino acid-based transmitters are either ion channels, also referred to as ionotropic receptors, or metabotropic, i.e. seven transmembrane domain G-protein coupled receptors. In this mini-review, we focus our interest on amino acid-based transmitter receptors on neuroglia, astrocytes in particular.
Keywords: Amino acids; Astrocytes; Glia; Transmitters; Receptors
A novel approach in potential anticoagulants from peptides epitope 558–565 of A2 subunit of factor VIII by C. Anastasopoulos; Y. Sarigiannis; G. Stavropoulos (1159-1165).
Factor VIII, a human blood plasma protein, plays an important role during the intrinsic pathway of blood coagulation cascade after its activation by thrombin. The activated form of FVIII acts as cofactor to the serine protease Factor IXa, in the conversion of the zymogen Factor X to the active enzyme Factor Xa. The Ser558–Gln565 region of the A2 subunit of Factor VIII has been shown to be crucial for FVIIIa–FIXa interaction. Based on this, a series of linear peptides, analogs of the 558–565 loop of the A2 subunit of the heavy chain of Factor VIII were synthesized using the acid labile 2-chlorotrityl chloride resin and biologically evaluated in vitro by measuring the chronic delay of activated partial thromboplastin time and the inhibition of Factor VIII activity, as potential anticoagulants.
Keywords: Factor VIII; Anticoagulant activity; Activated partial thromboplastin time; Synthetic peptides; Blood coagulation cascade; FVIII-FIX interaction
4-[18F]Fluoro-N-methyl-N-(propyl-2-yn-1-yl)benzenesulfonamide ([18F]F-SA): a versatile building block for labeling of peptides, proteins and oligonucleotides with fluorine-18 via Cu(I)-mediated click chemistry by Theres Ramenda; Jörg Steinbach; Frank Wuest (1167-1180).
Cu(I)-mediated [3+2]cycloaddition between azides and alkynes has evolved into a valuable bioconjugation tool in radiopharmaceutical chemistry. We have developed a simple, convenient and reliable radiosynthesis of 4-[18F]fluoro-N-methyl-N-(propyl-2-yn-1-yl)benzenesulfonamide ([ 18 F]F-SA) as a novel aromatic sulfonamide-based click chemistry building block. [ 18 F]F-SA could be prepared in a remotely controlled synthesis unit in 32 ± 5 % decay-corrected radiochemical yield in a total synthesis time of 80 min. The determined lipophilicity of [ 18 F]F-SA (logP = 1.7) allows handling of the radiotracer in aqueous solutions. The versatility of [ 18 F]F-SA as click chemistry building block was demonstrated by the labeling of a model peptide (phosphopeptide), protein (HSA), and oligonucleotide (L-RNA). The obtained radiochemical yields were 77 % (phosphopeptide), 55–60 % (HSA), and 25 % (L-RNA), respectively. Despite the recent emergence of a multitude of highly innovative novel bioconjugation methods for 18F labeling of biopolymers, Cu(I)-mediated click chemistry with [ 18 F]F-SA represents a reliable, robust and efficient radiolabeling technique for peptides, proteins, and oligonucleotides with the short-lived positron emitter 18F.
Keywords: Cu(I)-mediated click chemistry; Fluorine-18; Peptides; Proteins; Oligonucleotides; Positron emission tomography (PET)
Chemical rescue of the post-translationally carboxylated lysine mutant of allantoinase and dihydroorotase by metal ions and short-chain carboxylic acids by Ya-Yeh Ho; Yen-Hua Huang; Cheng-Yang Huang (1181-1191).
Bacterial allantoinase (ALLase) and dihydroorotase (DHOase) are members of the cyclic amidohydrolase family. ALLase and DHOase possess similar binuclear metal centers in the active site in which two metals are bridged by a post-translationally carboxylated lysine. In this study, we determined the effects of carboxylated lysine and metal binding on the activities of ALLase and DHOase. Although DHOase is a metalloenzyme, purified DHOase showed high activity without additional metal supplementation in a reaction mixture or bacterial culture. However, unlike DHOase, ALLase had no activity unless some specific metal ions were added to the reaction mixture or culture. Substituting the metal binding sites H59, H61, K146, H186, H242, or D315 with alanine completely abolished the activity of ALLase. However, the K146C, K146D and K146E mutants of ALLase were still active with about 1–6 % activity of the wild-type enzyme. These ALLase K146 mutants were found to have 1.4–1.7 mol metal per mole enzyme subunit, which may indicate that they still contained the binuclear metal center in the active site. The activity of the K146A mutant of the ALLase and the K103A mutant of DHOase can be chemically rescued by short-chain carboxylic acids, such as acetic, propionic, and butyric acids, but not by ethanol, propan-1-ol, and imidazole, in the presence of Co2+ or Mn2+ ions. However, the activity was still ~10-fold less than that of wild-type ALLase. Overall, these results indicated that the 20 natural basic amino acid residues were not sufficiently able to play the role of lysine. Accordingly, we proposed that during evolution, the post-translational modification of carboxylated lysine in the cyclic amidohydrolase family was selected for promoting binuclear metal center self-assembly and increasing the nucleophilicity of the hydroxide at the active site for enzyme catalysis. This kind of chemical rescue combined with site-directed mutagenesis may also be used to identify a binuclear metal center in the active site for other metalloenzymes.
Keywords: Allantoinase; Dihydroorotase; Hydantoinase; Imidase; Chemical rescue; Post-translationally carboxylated lysine; Metalloenzyme
Ant 4,4, a polyamine-anthracene conjugate, induces cell death and recovery in human promyelogenous leukemia cells (HL-60) by Rui Traquete; Radiah A. Ghani; Otto Phanstiel; Heather M. Wallace (1193-1203).
One of the major problems in cancer therapy is the lack of specificity of chemotherapeutic agents towards cancer cells, resulting in adverse side effects. One means to counter this is to selectively deliver the drug to the cancer cell. Cancer cells accumulate increased concentrations of polyamines compared to normal cells, mainly through an increased uptake of preformed polyamines via the polyamine transport system (PTS). Furthermore, the non-stringent structural requirements of the PTS enable the transport of a range of polyamine-based molecules. Thus, the PTS can be used to transport compounds linked to polyamines selectively to cancer cells. In our laboratory, polyamine–anthracene conjugates have shown potent anti-tumour activity towards HL-60 cells. The aim of this study was to determine the cytotoxicity of Ant-4,4, a homospermidine–anthracene conjugate, and assess the long-term effects by determining whether cancer cells were able to recover from treatment. During exposure, Ant-4,4 was an effective growth-inhibitory agent in HL-60 cells decreasing viable cell number, protein and polyamine content. Evidence indicates concomitant cell-cycle arrest and increased apoptosis. Once the drug was removed, HL-60 cells recovered gradually over time. Increasing cell number, protein content and polyamine content, as well as diminished effects on cell-cycle and apoptotic stimuli were observed over time. These data suggest that, despite being an effective way of delivering anthracene, these polyamine conjugates do not exert long-lasting effects on HL-60 cells.
Keywords: Cancer; Targeted drug delivery; Polyamine conjugate; Recovery of cancer cells
A quantitative analysis of spontaneous isoaspartate formation from N-terminal asparaginyl and aspartyl residues by Bert H.-O. Güttler; Holger Cynis; Franziska Seifert; Hans-Henning Ludwig; Andrea Porzel; Stephan Schilling (1205-1214).
The formation of isoaspartate (isoAsp) from asparaginyl or aspartyl residues is a spontaneous post-translational modification of peptides and proteins. Due to isopeptide bond formation, the structure and possibly function of peptides and proteins is altered. IsoAsp modifications within the peptide chain have been reported for many cytosolic proteins. Amyloid peptides (Aβ) deposited in Alzheimer’s disease may carry an N-terminal isoAsp-modification. Here, we describe a quantitative investigation of isoAsp-formation from N-terminal Asn and Asp using model peptides similar to the Aβ N-terminus. The study is based on a newly developed separation of peptides using capillary electrophoresis (CE). 1H NMR was employed to validate the basic finding of N-terminal isoAsp-formation from Asp and Asn. Thereby, the isomerization of Asn at neutral pH (0.6 day−1, peptide NGEF) is approximately six times faster than that within the peptide chain (AANGEF). The difference in velocity between Asn and Asp isomerization is approximately 50-fold. In contrast to N-terminal Asn, Asp isomerization is significantly accelerated at acidic pH. The kinetic solvent isotope (k D2O/k H2O) effect of 2.46 suggests a rate-limiting proton transfer in isoAsp-formation. The proton inventory is consistent with transfer of one proton in the transition state, supporting the previous notion of rate-limiting deprotonation of the peptide backbone amide during succinimide-intermediate formation. The study provides evidence for a spontaneous N-terminal isoAsp-formation within peptides and might explain the accumulation of N-terminal isoAsp in amyloid deposits.
Keywords: Alzheimer’s disease; PIMT; Capillary electrophoresis; 1H NMR; Isoaspartate
Rational design of cationic antimicrobial peptides by the tandem of leucine-rich repeat by Q. Q. Ma; Y. F. Lv; Y. Gu; N. Dong; D. S. Li; A. S. Shan (1215-1224).
Antimicrobial peptides represent ancient host defense effector molecules present in organisms across the evolutionary spectrum. Lots of antimicrobial peptides were synthesized based on well-known structural motif widely existed in a variety of lives. Leucine-rich repeats (LRRs) are sequence motifs present in over 60,000 proteins identified from viruses, bacteria, and eukaryotes. To elucidate if LRR motif possesses antimicrobial potency, two peptides containing one or two LRRs were designed. The biological activity and membrane–peptide interactions of the peptides were analyzed. The results showed that the tandem of two LRRs exhibited similar antibacterial activity and significantly weaker hemolytic activity against hRBCs than the well-known membrane active peptide melittin. The peptide with one LRR was defective at antimicrobial and hemolytic activity. The peptide containing two LRRs formed α-helical structure, respectively, in the presence of membrane-mimicking environment. LRR-2 retained strong resistance to cations, heat, and some proteolytic enzymes. The blue shifts of the peptides in two lipid systems correlated positively with their biological activities. Other membrane-peptide experiments further provide the evidence that the peptide with two LRRs kills bacteria via membrane-involving mechanism. The present study increases our new understanding of well-known LRR motif in antimicrobial potency and presents a potential strategy to develop novel antibacterial agents.
Keywords: Antimicrobial peptides; Leucine-rich repeat; Membrane–peptide interaction
Simultaneous analysis of the non-canonical amino acids norleucine and norvaline in biopharmaceutical-related fermentation processes by a new ultra-high performance liquid chromatography approach by Michael Biermann; Bettina Bardl; Sebastian Vollstädt; Julia Linnemann; Uwe Knüpfer; Guido Seidel; Uwe Horn (1225-1231).
In this study, a precise and reliable ultra-high performance liquid chromatography (UHPLC) method for the simultaneous determination of non-canonical (norvaline and norleucine) and standard amino acids (aspartic acid, glutamic acid, serine, histidine, glycine, threonine, arginine, tyrosine, methionine, valine, phenylalanine, isoleucine, leucine) in biopharmaceutical-related fermentation processes was established. After pre-column derivatization with ortho-phthaldialdehyde and 2-mercaptoethanol, the derivatives were separated on a sub-2 μm particle C18 reverse-phase column. Identification and quantification of amino acids were carried out by fluorescence detection. To test method feasibility on standard HPLC instruments, the assay was properly transferred to a core–shell particle C18 reverse-phase column. The limits of detection showed excellent sensitivity by values from 0.06 to 0.17 pmol per injection and limits of quantification between 0.19 and 0.89 pmol. In the present study, the newly established UHPLC method was applied to a recombinant antibody Escherichia coli fermentation process for the analysis of total free amino acids. We were able to specifically detect and quantify the unfavorable amino acids in such complex samples. Since we observed trace amounts of norvaline and norleucine during all fermentation phases, an obligatory process monitoring should be considered to improve quality of recombinant protein drugs in future.
Keywords: Recombinant antibody; Norleucine; Norvaline; Ultra-high performance liquid chromatography; Biopharmaceutical fermentation; Escherichia coli ; Bioprocess design
Phosphorylation of nuclear and cytoplasmic pools of ribosomal protein S6 during cell cycle progression by Margit Rosner; Katharina Schipany; Markus Hengstschläger (1233-1240).
Of all known ribosomal proteins, the 40S ribosomal protein S6 is by far the most extensively studied. Still, little is known about some basic aspects of S6 regulation including its cell cycle-related expression and localization. Using a flow cytometric single cell approach applied to whole cells and isolated nuclei, we monitored nucleocytoplasmic expression of total and S240/4 phosphorylated S6 during unperturbed cell cycle progression, providing first evidence for a S6-specific spatiotemporal pattern and its deregulation under conditions of hyperactivated mTOR.
Keywords: S6 serine 240/4; mTOR; TSC1/2; Cell cycle; Nucleocytoplasmic; Phospho-flow
Contributions of aminoacyl-tRNA synthetase-interacting multifunctional protein-3 to mammalian translation initiation by Min Jeong Ku; Sang Yeol Lee (1241-1245).
Aminoacyl-tRNA synthetase-interacting multifunctional protein-3 (AIMP3) stabilizes and protects mammalian methionyl-tRNA synthetase (MRS) and eukaryotic initiation factor 2 subunit gamma (eIF2γ), factors involved in the formation and the delivery of Met-tRNA i Met respectively, through the binding interactions. Due to the protections that MRS and eIF2γ are provided from the interactions with AIMP3, cellular levels of MRS and eIF2γ may be able to be maintained high enough for their canonical and/or non-canonical functions.
Keywords: Aminoacyl-tRNA synthetase-interacting multifunctional protein-3 (AIMP3); Methionyl-tRNA synthetase (MRS); Eukaryotic initiation factor 2 subunit gamma (eIF2γ); Protein–protein interaction; Translation initiation