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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.44, #5)

Interaction proteomics of the AMPA receptor: towards identification of receptor sub-complexes by Ka Wan Li; Ning Chen; August B. Smit (pp. 1247-1251).

AMPA receptors (AMPAR) are the main ligand-gated ion channels responsible for the fast excitatory synaptic transmission in the mammalian brain. Whereas a number of proteins that interact with AMPAR are known to be involved in the trafficking and localization of the receptor and/or the regulation of receptor channel properties, the protein composition of the AMPAR supra-complexes are largely unclear. Recent interaction proteomics report the presence of up to 34 proteins as high-confidence constituents of the AMPAR. It was proposed that the inner core of the receptor complex consists of the GluA tetramer and four auxiliary proteins comprising transmembrane AMPA receptor regulatory proteins and/or cornichons. The other AMPAR interactors, present in lower amount, may form the outer shell of the AMPAR with a range in size and variability.

Keywords: Synapse; Excitatory neurotransmission; AMPA receptor; Protein complex; Proteomics

Phylogenetic aspects of the sulfate assimilation genes from Thalassiosira pseudonana by Mariusz A. Bromke; Rainer Hoefgen; Holger Hesse (pp. 1253-1265).

Diatoms are unicellular algae responsible for approximately 20 % of global carbon fixation. Their evolution by secondary endocytobiosis resulted in a complex cellular structure and metabolism compared to algae with primary plastids. In the last years the interest on unicellular algae increased. On the one hand assessments suggest that diatom-mediated export production can influence climate change through uptake and sequestration of atmospheric CO₂. On the other hand diatoms are in focus because they are discussed as potential producer of biofuels. To follow the one or other idea it is necessary to investigate the diatoms biochemistry in order to understand the cellular regulatory mechanisms. The sulfur assimilation and methionine synthesis pathways provide S-containing amino acids for the synthesis of proteins and a range of metabolites such as dimethylsulfoniopropionate (DMSP) in order to provide basic metabolic precursors needed for the diatoms metabolism. To obtain an insight into the localization and organization of the sulfur metabolism pathways, the genome of Thalassiosira pseudonana—a model organism for diatom research—might help to understand the fundamental questions on adaptive responses of diatoms to dynamic environmental conditions such as nutrient availability in a broader context.

Keywords: Sulfate; Cysteine; Homocysteine; Thalassiosira pseudonana

Fate of ferrisiderophores after import across bacterial outer membranes: different iron release strategies are observed in the cytoplasm or periplasm depending on the siderophore pathways by Isabelle J. Schalk; Laurent Guillon (pp. 1267-1277).

Siderophore production and utilization is one of the major strategies deployed by bacteria to get access to iron, a key nutrient for bacterial growth. The biological function of siderophores is to solubilize iron in the bacterial environment and to shuttle it back to the cytoplasm of the microorganisms. This uptake process for Gram-negative species involves TonB-dependent transporters for translocation across the outer membranes. In Escherichia coli and many other Gram-negative bacteria, ABC transporters associated with periplasmic binding proteins import ferrisiderophores across cytoplasmic membranes. Recent data reveal that in some siderophore pathways, this step can also be carried out by proton-motive force-dependent permeases, for example the ferrichrome and ferripyochelin pathways in Pseudomonas aeruginosa. Iron is then released from the siderophores in the bacterial cytoplasm by different enzymatic mechanisms depending on the nature of the siderophore. Another strategy has been reported for the pyoverdine pathway in P. aeruginosa: iron is released from the siderophore in the periplasm and only siderophore-free iron is transported into the cytoplasm by an ABC transporter having two atypical periplasmic binding proteins. This review presents recent findings concerning both ferrisiderophore and siderophore-free iron transport across bacterial cytoplasmic membranes and considers current knowledge about the mechanisms involved in iron release from siderophores.

Keywords: Siderophore; Iron uptake; Iron homeostasis; TonB-dependent transporters; ABC transporters

Quinacrine reactivity with prion proteins and prion-derived peptides by Zbigniew Zawada; Martin Šafařík; Eva Dvořáková; Olga Janoušková; Anna Březinová; Ivan Stibor; Karel Holada; Petr Bouř; Jan Hlaváček; Jaroslav Šebestík (pp. 1279-1292).

Quinacrine is a drug that is known to heal neuronal cell culture infected with prions, which are the causative agents of neurodegenerative diseases called transmissible spongiform encephalopathies. However, the drug fails when it is applied in vivo. In this work, we analyzed the reason for this failure. The drug was suggested to “covalently” modify the prion protein via an acridinyl exchange reaction. To investigate this hypothesis more closely, the acridine moiety of quinacrine was covalently attached to the thiol groups of cysteines belonging to prion-derived peptides and to the full-length prion protein. The labeled compounds were conveniently monitored by fluorescence and absorption spectroscopy in the ultraviolet and visible spectral regions. The acridine moiety demonstrated characteristic UV–vis spectrum, depending on the substituent at the C-9 position of the acridine ring. These results confirm that quinacrine almost exclusively reacts with the thiol groups present in proteins and peptides. The chemical reaction alters the prion properties and increases the concentration of the acridine moiety in the prion protein.

Keywords: Quinacrine; Prion protein and peptide model reactions; Solid phase and recombinant synthesis

Hydrophilic interaction ultra-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry for highly rapid and sensitive analysis of underivatized amino acids in functional foods by Guisheng Zhou; Hanqing Pang; Yuping Tang; Xin Yao; Xuan Mo; Shaoqing Zhu; Sheng Guo; Dawei Qian; Yefei Qian; Shulan Su; Li Zhang; Chun Jin; Yong Qin; Jin-ao Duan (pp. 1293-1305).

This work presented a new analytical methodology based on hydrophilic interaction ultra-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry in multiple-reaction monitoring mode for analysis of 24 underivatized free amino acids (FAAs) in functional foods. The proposed method was first reported and validated by assessing the matrix effects, linearity, limit of detections and limit of quantifications, precision, repeatability, stability and recovery of all target compounds, and it was used to determine the nutritional substances of FAAs in ginkgo seeds and further elucidate the nutritional value of this functional food. The result showed that ginkgo seed turned out to be a good source of FAAs with high levels of several essential FAAs and to have a good nutritional value. Furthermore, the principal component analysis was performed to classify the ginkgo seed samples on the basis of 24 FAAs. As a result, the samples could be mainly clustered into three groups, which were similar to areas classification. Overall, the presented method would be useful for the investigation of amino acids in edible plants and agricultural products.

Keywords: Underivatized amino acids analysis; HILIC-UPLC–TQ-MS/MS; PCA; Functional foods; Ginkgo seed

S-linked protein homocysteinylation: identifying targets based on structural, physicochemical and protein–protein interactions of homocysteinylated proteins by Yumnam Silla; Elayanambi Sundaramoorthy; Puneet Talwar; Shantanu Sengupta (pp. 1307-1316).

An elevated level of homocysteine, a thiol-containing amino acid is associated with a wide spectrum of disease conditions. A majority (>80 %) of the circulating homocysteine exist in protein-bound form. Homocysteine can bind to free cysteine residues in the protein or could cleave accessible cysteine disulfide bonds via thiol disulfide exchange reaction. Binding of homocysteine to proteins could potentially alter the structure and/or function of the protein. To date only 21 proteins have been experimentally shown to bind homocysteine. In this study we attempted to identify other proteins that could potentially bind to homocysteine based on the criteria that such proteins will have significant 3D structural homology with the proteins that have been experimentally validated and have solvent accessible cysteine residues either with high dihedral strain energy (for cysteine–cysteine disulfide bonds) or low pKa (for free cysteine residues). This analysis led us to the identification of 78 such proteins of which 68 proteins had 154 solvent accessible disulfide cysteine pairs with high dihedral strain energy and 10 proteins had free cysteine residues with low pKa that could potentially bind to homocysteine. Further, protein–protein interaction network was built to identify the interacting partners of these putative homocysteine binding proteins. We found that the 21 experimentally validated proteins had 174 interacting partners while the 78 proteins identified in our analysis had 445 first interacting partners. These proteins are mainly involved in biological activities such as complement and coagulation pathway, focal adhesion, ECM-receptor, ErbB signalling and cancer pathways, etc. paralleling the disease-specific attributes associated with hyperhomocysteinemia.

Keywords: Homocysteine; Homologous structure; Disulfide; Dihedral strain energy; pKa; Protein–protein interaction

WRF-TMH: predicting transmembrane helix by fusing composition index and physicochemical properties of amino acids by Maqsood Hayat; Asifullah Khan (pp. 1317-1328).

Membrane protein is the prime constituent of a cell, which performs a role of mediator between intra and extracellular processes. The prediction of transmembrane (TM) helix and its topology provides essential information regarding the function and structure of membrane proteins. However, prediction of TM helix and its topology is a challenging issue in bioinformatics and computational biology due to experimental complexities and lack of its established structures. Therefore, the location and orientation of TM helix segments are predicted from topogenic sequences. In this regard, we propose WRF-TMH model for effectively predicting TM helix segments. In this model, information is extracted from membrane protein sequences using compositional index and physicochemical properties. The redundant and irrelevant features are eliminated through singular value decomposition. The selected features provided by these feature extraction strategies are then fused to develop a hybrid model. Weighted random forest is adopted as a classification approach. We have used two benchmark datasets including low and high-resolution datasets. tenfold cross validation is employed to assess the performance of WRF-TMH model at different levels including per protein, per segment, and per residue. The success rates of WRF-TMH model are quite promising and are the best reported so far on the same datasets. It is observed that WRF-TMH model might play a substantial role, and will provide essential information for further structural and functional studies on membrane proteins. The accompanied web predictor is accessible at http://111.68.99.218/WRF-TMH/ .

Keywords: Transmembrane helix; Physicochemical properties; Compositional index; Weighted random forest; Structures of membrane proteins

An expression system for the efficient incorporation of an expanded set of tryptophan analogues by Dejan M. Petrović; Kees Leenhouts; Maarten L. van Roosmalen; Jaap Broos (pp. 1329-1336).

Biosynthetic incorporation of tryptophan (Trp) analogues in recombinant proteins using an E. coli Trp auxotroph expression host is limited to analogues modified with a small substituent like a fluoro atom or a hydroxyl or amine group. We report here the efficient incorporation (>89 %) of chloro- and bromo atoms containing Trp analogues in alloproteins at high expression levels using a Lactococcus lactis Trp auxotroph strain. This result was only obtained after coexpression of the enzyme tryptophanyl-tRNA synthetase (TrpRS) of L. lactis, an enzyme believed to show a more relaxed substrate specificity than TrpRS from E. coli. Chloro- and bromo-Trps are attractive intrinsic phosphorescence probes as these Trp analogues are much less sensitive for quenchers in the medium, like oxygen, than Trp. Coexpression of TrpRS was also essential for the biosynthetic incorporation (94 %) of the Trp analogue 5,6 difluoroTrp. This makes our expression system ideally suited to generate a set of methyl- and fluoro-substituted Trp analogue-containing alloproteins in high yield for investigating the involvement of the Trp residue in cation-pi or pi–pi interactions. Taken together, the presented Trp auxotroph expression system features the most relaxed specificity for Trp analogue structures reported to date and gives a high alloprotein yield.

Keywords: Lactococcus lactis ; Trp auxotroph; Tryptophanyl-tRNA synthetase; Bromotryptophan; Chlorotryptophan; Difluorotryptophan

^99mTc-labeled monomeric and dimeric NGR peptides for SPECT imaging of CD13 receptor in tumor-bearing mice by Wenhui Ma; Fei Kang; Zhe Wang; Weidong Yang; Guiyu Li; Xiaowei Ma; Guoquan Li; Kai Chen; Yingqi Zhang; Jing Wang (pp. 1337-1345).

CD13 receptor plays a critical role in tumor angiogenesis and metastasis. We therefore aimed to develop ^99mTc-labeled monomeric and dimeric NGR-containing peptides, namely, NGR1 and NGR2, for SPECT imaging of CD13 expression in HepG2 hepatoma xenografts. Both NGR-containing monomer and dimer were synthesized and labeled with ^99mTc. In vivo receptor specificity was demonstrated by successful blocking of tumor uptake of ^99mTc-NGR dimer in the presence of 20 mg/kg NGR2 peptide. Western blot and immunofluorescence staining confirmed the CD13 expression in HepG2 cells. The NGR dimer showed higher binding affinity and cell uptake in vitro than the NGR-containing monomer, presumably due to a multivalency effect. ^99mTc-Labeled monomeric and dimeric NGR-containing peptides were subjected to SPECT imaging and biodistribution studies. SPECT scans were performed in HepG2 tumor-bearing mice at 1, 4, 12, and 24 h post-injection of ~7.4 MBq tracers. The metabolism of tracers was determined in major organs at different time points after injection which demonstrated rapid, significant tumor uptake and slow tumor washout for both traces. Predominant clearance from renal and hepatic system was also observed in ^99mTc-NGR1 and ^99mTc-NGR2. In conclusion, monomeric and dimeric NGR peptide were developed and labeled with ^99mTc successfully, while the high integrin avidity and long retention in tumor make ^99mTc-NGR dimer a promising agent for tumor angiogenesis imaging.

Keywords: ^99mTc; NGR; CD13; Angiogenesis; SPECT

Senegalin: a novel antimicrobial/myotropic hexadecapeptide from the skin secretion of the African running frog, Kassina senegalensis by Hui Wang; Renjie Li; Xinping Xi; Teng Meng; Mei Zhou; Lei Wang; Yingqi Zhang; Tianbao Chen; Chris Shaw (pp. 1347-1355).

Amphibian skin is a rich and unique source of novel bioactive peptides most of which are endowed with either antimicrobial or pharmacological properties. Here, we report the identification and structural characterization of a novel peptide, named senegalin, which possesses both activities. Senegalin is a hexadecapeptide amide (FLPFLIPALTSLISSL-NH₂) of unique primary structure found in the skin secretion of the African running frog, Kassina senegalensis. The structure of the biosynthetic precursor of senegalin, deduced from cloned skin cDNA, consists of 76 amino acid residues and displays the typical domain organization of an amphibian skin peptide precursor. Both natural senegalin and its synthetic replicate displayed antimicrobial and myotropic activities. Senegalin was active against Staphylococcus aureus (MIC 50 μM) and Candida albicans (MIC 150 μM) but was non-haemolytic at concentrations up to and including 150 μM. In contrast, senegalin induced a dose-dependent contraction of rat urinary bladder smooth muscle (EC₅₀ 2.9 nM) and a dose-dependent relaxation of rat tail artery smooth muscle (EC₅₀ 37.7 nM). Senegalin thus represents a prototype biologically active amphibian skin peptide and illustrates the fact that amphibian skin secretion peptidomes continue to be unique sources of such molecules.

Keywords: Amphibian; Peptide; Antimicrobial; Smooth muscle; Molecular cloning

Side reactions in the SPPS of Cys-containing peptides by Panagiotis Stathopoulos; Serafim Papas; Charalambos Pappas; Vassilios Mousis; Nisar Sayyad; Vassiliki Theodorou; Andreas G. Tzakos; Vassilios Tsikaris (pp. 1357-1363).

Alkylation of sensitive amino acids during synthesis of biologically important peptides is a common and well-documented problem in Fmoc-based strategy. Herein, we probed for the first time an unexpected S-alkylation of Cys-containing peptides that occur during the final TFA cleavage of peptides from the Wang solid support. Through a battery of approaches (NMR, UV and LC–MS) the formed by-product was assigned as the alkylation of the cysteine sulfydryl group by the p-hydroxyl benzyl group derived from the acidic Wang linker decomposition. Factors affecting this side reaction were monitored and a protocol that minimizes the presence of the by-product is reported.

Keywords: Solid phase peptide synthesis; Wang resin decomposition; Cysteine; S-alkylation; TFA cleavage

Learning protein multi-view features in complex space by Dong-Jun Yu; Jun Hu; Xiao-Wei Wu; Hong-Bin Shen; Jun Chen; Zhen-Min Tang; Jian Yang; Jing-Yu Yang (pp. 1365-1379).

Protein attribute prediction from primary sequences is an important task and how to extract discriminative features is one of the most crucial aspects. Because single-view feature cannot reflect all the information of a protein, fusing multi-view features is considered as a promising route to improve prediction accuracy. In this paper, we propose a novel framework for protein multi-view feature fusion: first, features from different views are parallely combined to form complex feature vectors; Then, we extend the classic principal component analysis to the generalized principle component analysis for further feature extraction from the parallely combined complex features, which lie in a complex space. Finally, the extracted features are used for prediction. Experimental results on different benchmark datasets and machine learning algorithms demonstrate that parallel strategy outperforms the traditional serial approach and is particularly helpful for extracting the core information buried among multi-view feature sets. A web server for protein structural class prediction based on the proposed method (COMSPA) is freely available for academic use at: http://www.csbio.sjtu.edu.cn/bioinf/COMSPA/ .

Keywords: Protein attribute prediction; Feature extraction; Serial feature fusion; Parallel feature fusion; Complex space

The use of native gels for the concomitant determination of protein sequences and modifications by mass spectrometry with subsequent conformational and functional analysis of native proteins following electro-elution by Wei-Qiang Chen; Elena Karnaukhova; Gert Lubec (pp. 1381-1389).

The protocol consists of running a native gel with in-gel digestion by proteases, subsequent mass spectrometrical determination of protein sequence and modifications, followed by electro-elution and conformational analysis using melting point and circular dichroism. Finally, the eluted protein is tested for preserved function. Herein, C1 esterase inhibitor is applied on a native gel; in-gel digestion by proteases is carried out and peptides are identified by nano-LC-ESI-CID/ETD-MS/MS using an ion trap for generation of peptide sequences and protein modifications. Protein from replicate bands from the same gel is electro-eluted and used for determination of the melting point and used for circular dichroism analysis. Additional bands from the native gel are either in-gel digested with asparaginase to generate deamidation or PNGase F for deglycosylation, followed by mass spectrometry, conformational and functional studies. Preserved conformation and function of the C1 esterase inhibitor was shown. This protocol can be completed in 1 week.

Keywords: Conformation; CD; Protein structure; Native gel

Increasing effects of S-methyl-l-cysteine on the extracellular d-serine concentrations in the rat medial frontal cortex by Sayuri Ishiwata; Shigehiro Ogata; Asami Umino; Hirohisa Shiraku; Yoshitaka Ohashi; Yasushi Kajii; Toru Nishikawa (pp. 1391-1395).

In an in vivo dialysis experiment, the intra-medial frontal cortex infusion of a system A and Asc-1 transporter inhibitor, S-methyl-l-cysteine, caused a concentration-dependent increase in the dialysate contents of an endogenous coagonist for the N-methyl-d-aspartate (NMDA) type glutamate receptor, d-serine, in the cortical portion. These results suggest that these neutral amino acid transporters could control the extracellular d-serine signaling in the brain and be a target for the development of a novel threapy for neuropsychiatric disorders with an NMDA receptor dysfunction.

Keywords: Asc-1 transporter; In vivo microdialysis; Medial frontal cortex; S-Methyl-l-cysteine; d-Serine

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.44, #5)

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.44, #5)