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BBA - Proteins and Proteomics (v.1794, #2)

Editorial Board (pp. ii).

Proteomics analysis of A375 human malignant melanoma cells in response to arbutin treatment by Jiraporn Nawarak; Rosa Huang-Liu; Shao-Hsuan Kao; Hsien-Hua Liao; Supachok Sinchaikul; Shui-Tein Chen; Sun-Long Cheng (pp. 159-167).

Although the toxicogenomics of A375 human malignant melanoma cells treated with arbutin have been elucidated using DNA microarray, the proteomics of the cellular response to this compound are still poorly understood. In this study, we performed proteomic analyses to investigate the anticancer effect of arbutin on the protein expression profile in A375 cells. After treatment with arbutin (8 μg/ml) for 24, 48 and 72 h, the proteomic profiles of control and arbutin-treated A375 cells were compared, and 26 differentially expressed proteins (7 upregulated and 19 downregulated proteins) were identified by MALDI-Q-TOF MS and MS/MS. Among these proteins, 13 isoforms of six identical proteins were observed. Bioinformatic tools were used to search for protein function and to predict protein interactions. The interaction network of 14 differentially expressed proteins was found to be correlated with the downstream regulation of p53 tumor suppressor and cell apoptosis. In addition, three upregulated proteins (14-3-3G, VDAC-1 and p53) and five downregulated proteins (ENPL, ENOA, IMDH2, PRDX1 and VIME) in arbutin-treated A375 cells were validated by RT-PCR analysis. These proteins were found to play important roles in the suppression of cancer development.

Keywords: Arbutin; A375 cells; Malignant melanoma; Proteomics; Cancer

Identification of interaction partners for individual SH3 domains of Fas ligand associated members of the PCH protein family in T lymphocytes by Andreas Linkermann; Christoph Gelhaus; Marcus Lettau; Jing Qian; Dieter Kabelitz; Ottmar Janssen ⁎ (pp. 168-176).

Pombe Cdc15 homology (PCH) family proteins are regarded as key elements for linking membrane-associated processes to cytoskeletal elements and thus play a major role in exo- and endocytosis and organelle trafficking. We previously reported that, via their SH3 domains, several members of the PCH proteins interact with the proline-rich region of Fas ligand (FasL, CD95L), a key death factor in immune cells. Since protein–protein interactions that govern the storage and transport of FasL-associated vesicles are largely unknown, the present study was performed to identify other potential binding partners for SH3 domains of FasL-interacting PCH proteins. To this end, individual SH3 domains were expressed as GST fusion proteins and used to precipitate associated proteins from leukemic T cell lines and activated human T cell blasts. 87 protein bands representing 34 individual proteins were identified by mass spectrometry. The presented list of candidate interactors not only highlights the role of PCH proteins as adapters between vesicular membranes and the cytoskeleton but also points to an involvement of these proteins in the regulation of signalling events in T lymphocytes.

Keywords: Abbreviations; aa; amino acids; AICD; activation-induced cell death; CME; clathrin mediated endocytosis; EFC; extended Fer/CIP4 homology; FasL; Fas ligand; IS; immunological synapse; MP; metalloproteinase; PBMC; peripheral blood mononuclear cells; SH; Src homology; TCR; T cell receptorPCH protein family; Protein–protein interaction; Signal transduction; Fas ligand; Proteomics; Src homology 3 domain

Proteomic analysis of homocysteine induced proliferation of cultured neonatal rat vascular smooth muscle cells by XiaoHua Liu; Jing Shen; Rui Zhan; XingXing Wang; XiaoMing Wang; ZhiQing Zhang; Xue Leng; ZhiHua Yang; LingJia Qian ⁎ (pp. 177-184).

Hyperhomocysteinemia is a common independent risk factor for cardiovascular diseases. The promoting effect of homocysteine (Hcy) on vascular smooth muscle cell (VSMC) proliferation has been considered as one of the important pathological bases of atherosclerosis. However, the mechanism of VSMC proliferation induced by Hcy remains unclear. The present research used proteomic techniques to globally analyze the protein changes in proliferative VSMCs. After comparing the protein expression profiles of VSMCs between the Hcy-treated and non-treated groups, 11 protein spots were found altered markedly in proliferative VSMCs with expression of eight protein spots increased and three protein spots decreased. In the differentially expressed proteins, eight protein spots were identified successfully including glycolytic metabolism proteins: pyruvate kinase M2 (PKM2), triosephosphate isomerase (TPI) and aldose reductase (AR); cytoskeletal proteins: lamin C and vimentin; and three other proteins: calreticulin; similar to WDR1 protein and LIM and SH3 protein 1. The differentially expressed proteins were further validated by Western blot and confirmed by assay of enzymes' activities and ATP content. These results may provide some clues for comprehensively understanding the mechanism of VSMC proliferation and pathogenesis of atherosclerosis induced by Hcy.

Keywords: Homocysteine; Vascular smooth muscle cell; Proliferation; Two-dimensional gel electrophoresis

Indolicidin-derived antimicrobial peptide analogs with greater bacterial selectivity and requirements for antibacterial and hemolytic activities by Sung-Min Kim; Joung-Min Kim; Bishnu Prasad Joshi; Hyeongjin Cho; Keun-Hyeung Lee (pp. 185-192).

Indolicidin (ILPWKWPWWPWRR-NH₂) has received attention due to its unique primary structure and biological activities. In this study, amide bonds at various positions in indolicidin were replaced with the reduced amide bonds ψ[CH₂NH] and the effect of the secondary structure on the biological activity was investigated. The circular dichroism spectra revealed that the rigidity and hydrogen bond of the amide bond between Trp⁸ and Trp⁹ were important for stabilizing the turn structure of indolicidin. A structure–activity study revealed that the turn structure of indolicidin was not required for antimicrobial activity and leakage activity for LUVs with a negatively charged surface. The pseudopeptide containing two reduced amide bonds showed less hemolytic activity as well as improved stability without a decrease in its antimicrobial activity. These results will provide valuable information for designing indolicidin analogs with greater bacterial selectivity and increased stability and for elucidating the role of the secondary structure of membrane-active peptides for antimicrobial and hemolytic activities.

Keywords: Indolicidin; Secondary structure; Antibacterial peptide; Antimicrobial activity; Stability; Selectivity; Reduced amide bond

DNA dependent recruitment of DDX17 and other interacting proteins by the human androgen receptor by Hao Yun Wong ⁎; Jeroen A.A. Demmers; Karel Bezstarosti; J. Anton Grootegoed; Albert O. Brinkmann (pp. 193-198).

An oligonucleotide-based assay (OBA) was used to identify novel co-factors that can be recruited by the deoxyribonucleic acid (DNA)-bound androgen receptor (AR). Nuclear extracts obtained from LNCaP cells, after incubation with R1881, were incubated with biotinylated oligonucleotides bound to streptavidin coated beads. The oligonucleotides contain 3 copies in tandem of the androgen responsive element ARE1 from the prostate specific antigen ( PSA) gene promoter. As control incubation, a scrambled version of the tandem ARE1 was used. Immunoblots of the eluents revealed that the AR was bound to the ARE1 oligonucleotide and to a much lesser extent to the scrambled oligonucleotide. Proteins eluted from the oligonucleotides, were separated on a 5–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gradient gel, followed by identification using mass spectrometry. Identified proteins were scored for having one or more of the following known properties: nuclear localization, involved in transcription regulation, involvement in steroid hormone receptor (SHR) function, or specifical involvement in AR function. A total number of 85 nuclear proteins were found in two separate OBAs. Based on peptide counting, we found enrichment of 7 proteins eluted from the ARE1 oligonucleotide, compared to the scrambled oligonucleotide. Taken together with the obtained scores, these proteins are considered putative AR co-factors. One of these proteins, DDX17, is known to be a co-factor for estrogen receptor α (ERα), but has never been associated with AR function. The results indicate that the ARE oligonucleotide-based assay may allow enrichment of new candidate DNA-bound AR interacting proteins.

Keywords: Abbreviations; aa; amino acid; AD; activation domain; AF-2; activation function 2; ARE; androgen responsive element; DHT; dihydrotestosterone; LBD; ligand binding domain; N-CoR; nuclear receptor co-repressor; NTD; NH2-terminal domain; OBA; oligonucleotide-based assay; PSA; prostate specific antigen; T; testosterone; wt; wild-typeMass spectrometry; Androgen responsive element; Transcription factors; DNA-bound; Co-factor

Coenzyme binding and hydride transfer in Rhodobacter capsulatus ferredoxin/flavodoxin NADP(H) oxidoreductase by Ana Bortolotti; Inmaculada Pérez-Dorado; Guillermina Goñi; Milagros Medina; Juan A. Hermoso; Néstor Carrillo; Néstor Cortez ⁎ (pp. 199-210).

Ferredoxin-NADP(H) reductases catalyse the reversible hydride/electron exchange between NADP(H) and ferredoxin/flavodoxin, comprising a structurally defined family of flavoenzymes with two distinct subclasses. Those present in Gram-negative bacteria (FPRs) display turnover numbers of 1–5 s−¹ while the homologues of cyanobacteria and plants (FNRs) developed a 100-fold activity increase. We investigated nucleotide interactions and hydride transfer in Rhodobacter capsulatus FPR comparing them to those reported for FNRs. NADP(H) binding proceeds as in FNRs with stacking of the nicotinamide on the flavin, which resulted in formation of charge-transfer complexes prior to hydride exchange. The affinity of FPR for both NADP(H) and 2′-P-AMP was 100-fold lower than that of FNRs. The crystal structure of FPR in complex with 2′-P-AMP and NADP⁺ allowed modelling of the adenosine ring system bound to the protein, whereas the nicotinamide portion was either not visible or protruding toward solvent in different obtained crystals. Stabilising contacts with the active site residues are different in the two reductase classes. We conclude that evolution to higher activities in FNRs was partially favoured by modification of NADP(H) binding in the initial complexes through changes in the active site residues involved in stabilisation of the adenosine portion of the nucleotide and in the mobile C-terminus of FPR.

Keywords: Abbreviations; FNR; plastidic ferredoxin NADP(H) oxidoreductase; FPR; bacterial ferredoxin/flavodoxin NADP(H) oxidoreductaseFerredoxin(flavodoxin)-NADP(H) oxidoreductase; Rhodobacter capsulatus; Hydride transfer; Coenzyme binding; NADP; ⁺; -complex structure

Proteomic profiling of phosphoproteins and glycoproteins responsive to wild-type alpha-synuclein accumulation and aggregation by Jayanarayan Kulathingal; Li-wen Ko; Bernadette Cusack; Shu-Hui Yen ⁎ (pp. 211-224).

A tetracycline inducible transfectant cell line (3D5) capable of producing soluble and sarkosyl-insoluble assemblies of wild-type human alpha-synuclein (α-Syn) upon differentiation with retinoic acid was used to study the impact of α-Syn accumulation on protein phosphorylation and glycosylation. Soluble proteins from 3D5 cells, with or without the induced α-Syn expression were analyzed by two-dimensional gel electrophoresis and staining of gels with dyes that bind to proteins (Sypro ruby), phosphoproteins (Pro-Q diamond) and glycoproteins (Pro-Q emerald). Phosphoproteins were further confirmed by binding to immobilized metal ion affinity column. α-Syn accumulation caused differential phosphorylation and glycosylation of 16 and 12, proteins, respectively, whose identity was revealed by mass spectrometry. These proteins, including HSP90, have diverse biological functions including protein folding, signal transduction, protein degradation and cytoskeletal regulation. Importantly, cells accumulating α-Syn assemblies with different abilities to bind thioflavin S displayed different changes in phosphorylation and glycosylation. Consistent with the cell-based studies, we demonstrated a reduced level of phosphorylated HSP90 α/β in the substantia nigra of subjects with Parkinson's disease as compared to normal controls. Together, the results indicate that α-Syn accumulation causes complex cellular responses, which if persist may compromise cell viability.

Keywords: Abbreviations; ACN; acetonitrile; α-Syn; wild-type alpha-synuclein; AD; Alzheimer's disease; BCA; bicinchoninic acid assay; 2-D; two-dimensional; FA; formic acid; IEF; isoelectric focusing; IMAC; immobilized metal ion affinity chromatography; PAGE; polyacrylamide gel electrophoresis; PD; Parkinson's disease; SDS; sodium dodecyl sulfate; Skp; sarkosyl-insoluble pellet; SN1; buffer-extractable supernatant; Tet; tetracycline; TetOff; tetracycline-off; ThS; thioflavin S; TBS; Tris-buffered saline; WT; wild-typeα-Synuclein; Phosphoprotein; Glycoprotein; Proteomic

A proteomic approach to paclitaxel chemoresistance in ovarian cancer cell lines by Michela Di Michele; Anna Della Corte; Lucia Cicchillitti; Piero Del Boccio; Andrea Urbani; Cristiano Ferlini; Giovanni Scambia; Maria Benedetta Donati; Domenico Rotilio (pp. 225-236).

Ovarian cancer is the leading cause of gynaecological cancer mortality. Paclitaxel is used in the first line treatment of ovarian cancer, but acquired resistance represents the most important clinical problem and a major obstacle to a successful therapy. Several mechanisms have been implicated in paclitaxel resistance, however this process has not yet been fully explained. To better understand molecular resistance mechanisms, a comparative proteomic approach was undertaken on the human epithelial ovarian cancer cell lines A2780 (paclitaxel sensitive), A2780TC1 and OVCAR3 (acquired and inherently resistant). Proteins associated with chemoresistance process were identified by DIGE coupled with mass spectrometry (MALDI-TOF and LC-MS/MS). Out of the 172 differentially expressed proteins in pairwise comparisons among the three cell lines, 151 were identified and grouped into ten main functional classes. Most of the proteins were related to the category of stress response (24%), metabolism (22%), protein biosynthesis (15%) and cell cycle and apoptosis (11%), suggesting that alterations of those processes might be involved in paclitaxel resistance mechanisms. This is the first direct proteomic comparison of paclitaxel sensitive and resistant ovarian cancer cells and may be useful for further studies of resistance mechanisms and screening of resistance biomarkers for the development of tailored therapeutic strategies.

Keywords: Abbreviations; 2D-DIGE; two-dimensional in gel differential electrophoresis; MALDI-TOF MS; matrix assisted laser desorption/ionization time of flight mass spectrometry; SDS PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresis2D-DIGE; Drug resistance; Ovarian cancer; Paclitaxel; Proteomics

Effect of actin C-terminal modification on tropomyosin isoforms binding and thin filament regulation by Radosław Skórzewski; Małgorzata Śliwińska; Danuta Borys; Apolinary Sobieszek; Joanna Moraczewska ⁎ (pp. 237-243).

Tropomyosins, a family of actin-binding regulatory proteins, are present in muscle and non-muscle cells. Multiple tropomyosin (TM) isoforms differ in actin affinity and regulatory properties, but little is known about the molecular bases of these differences. The C-terminus of actin stabilizes contacts between actin subunits in the filament and interacts with myosin and regulatory proteins. The goal of this work was to reveal how structural changes in actin and differences between TM isoforms affect binding between these proteins and affect thin filament regulation. Actin proteolytically truncated by three C-terminal amino acids exhibited 1.2–1.5 fold reduced affinity for non-muscle and smooth muscle tropomyosin isoforms. The truncation increased the cooperativity of myosin S1-induced tropomyosin binding for short tropomyosins (TM5a and TM1b9a), but it was neutral for long isoforms (smTM and TM2). Actin modification affected regulation of actomyosin ATPase activity in the presence of all tropomyosins by shifting the filament into a more active state. We conclude that the integrity of the actin C-terminus is important for actin–tropomyosin interactions, however the increased affinity of tropomyosin binding in the S1-induced state of the filament appears not to be involved in the tropomyosin isoform-dependent mechanism of the actomyosin ATPase activation.

Keywords: Actin; Truncated actin; Tropomyosin; Smooth muscle; Non-muscle; Regulation

Stereospecific inactivation of tyrosinase byl- andd-ascorbic acid by Jose Luis Munoz-Munoz; Francisco Garcia-Molina; Pedro Antonio García-Ruiz; Ramon Varon; Jose Tudela; Francisco García-Cánovas ⁎; Jose Neptuno Rodriguez-Lopez (pp. 244-253).

A kinetic study of the inactivation of tyrosinase byl- andd-ascorbic acid isomers has been carried out. In aerobic conditions, a suicide inactivation mechanism operates, which was attributed to the enzymatic form oxytyrosinase. This suicide inactivation is stereospecific as regards the affinity of the enzyme for the substrate but not as regards the speed of the process, which is the same for both isomers, reflecting the influence of the chemical shift of the carbon C-2 ( δ₂) and C-3 ( δ₃) as seen by¹³C-NMR. The inactivation of deoxytyrosinase and mettyrosinase observed in anaerobic conditions, is irreversible and faster than the suicide inactivation process, underlining the fact that the presence of oxygen protects the enzyme against inactivation.

Keywords: Ascorbic acid; Mushroom tyrosinase; Suicide; Irreversible; Inactivation

O-GlcNAcylation disrupts glyceraldehyde-3-phosphate dehydrogenase homo-tetramer formation and mediates its nuclear translocation by Jungeun Park; Dohyun Han; Kyunggon Kim; Yup Kang; Youngsoo Kim (pp. 254-262).

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a typical glycolytic enzyme comprised of four identical 37 kDa subunits. In addition to its glycolytic function, GAPDH has a number of biological functions which are related to its subcellular localization. Generally, protein O-linked N-acetylglucosamine modification ( O-GlcNAcylation) is considered, among other effects, to mediate the nuclear transportation of cytosolic proteins. To elucidate the effect of O-GlcNAcylation on GAPDH, we determined the location of the O-GlcNAcylation site by tandem mass spectrometry, and subsequently examined the biological significance of this derivatization. The site involved was identified to be Thr227 by β-elimination and Michael addition. Transient transfection assays demonstrated that the T227A mutation induced the cytoplasmic accumulation of GAPDH, whereas the wild type was present in the cytoplasm and nuclei. Structural modeling, mutagenesis of Thr227 to Lys and Arg, and gel filtration chromatography of mutated and wild type GAPDH, together suggested that O-GlcNAcylation at Thr227 interrupts the hydrophobic interfaces formed between GAPDH monomers in its tetrameric state. The present study identified Thr227 as the major GAPDH O-GlcNAcylation site, which suggests that this modification mediates the nuclear translocation of GAPDH, presumably by disrupting the conformation of tetrameric GAPDH.

Keywords: β-elimination and Michael addition; GAPDH; Structural biology; Post-translational modification; O; -GlcNAcylation

Effects of recombinant protein misfolding and aggregation on bacterial membranes by D. Ami; A. Natalello; T. Schultz; P. Gatti-Lafranconi; M. Lotti; S.M. Doglia ⁎; A. de Marco ⁎ (pp. 263-269).

The expression of recombinant proteins is known to induce a metabolic rearrangement in the host cell. We used aggregation-sensitive model systems to study the effects elicited in Escherichia coli cells by the aggregation of recombinant glutathione-S-transferase and its fusion with the green fluorescent protein that, according to the expression conditions, accumulate intracellularly as soluble protein, or soluble and insoluble aggregates. We show that the folding state of the recombinant protein and the complexity of the intracellular aggregates critically affect the cell response. Specifically, protein misfolding and aggregation induce changes in specific host proteins involved in lipid metabolism and oxidative stress, a reduction in the membrane permeability, as well as a rearrangement of its lipid composition. The temporal evolution of the host cell response and that of the aggregation process pointed out that the misfolded protein and soluble aggregates are responsible for the membrane modifications and the changes in the host protein levels. Interestingly, native recombinant protein and large insoluble aggregates do not seem to activate stress markers and membrane rearrangements.

Keywords: Abbreviations; DPH; 1,6-diphenyl-1,3,5-hexatriene; E; ₃; dihidrolipoyl dehydrogenase E; ₃; EM; electron microscopy; FT–IR; Fourier transform infrared spectroscopy; GFP–GST; green fluorescent protein-glutathione-S-transferase; GST; glutathione-S-transferase; IB; inclusion body; IPTG; isopropyl β-; d; -1-thiogalactopyranoside; NPN; 1-N-phenylnaphtylamine; pETM33-GST vector; GST under T7 promoter; PFL; Pseudomonas fragi; lipase; pQE30-GST vector; GST under T5 promoter; SOD; superoxide dismutase; TLC; thin-layer chromatographyFourier transform infrared spectroscopy; Membrane lipid; Protein aggregation; Recombinant protein; Stress metabolism; Small chaperone

Creatine kinase structural changes induced by substrates by Daniela Hornikova; Petr Herman; Jiri Mejsnar ⁎; Jaroslav Vecer; Jitka Zurmanova (pp. 270-274).

Myofibrillar creatine kinase (CK) buffers the cellular ATP concentration during fluctuating ATP turnover in a muscle. In order to detect structural changes of the CK molecule due to bound substrates, the dynamics of free, ATP-bound, and ATP+creatine-bound CK were examined, using steady-state and time-resolved fluorescence spectroscopy. The intrinsic tryptophan fluorescence of non-labelled CK presented the smaller fluorescence lifetime 2.38 ns and rotation correlation time 27 ns for the CK–ATP (in comparison with the times 2.72 ns and 35 ns for the free CK), and their moderate return to the longer times 2.42 ns and 29 ns for the CK–ATP+creatine complex. Three conformations for the non-labelled CK were indicated also by different quenching of fluorescence by acrylamide. Data were confirmed by anisotropy experiments with CK-(FITC labelled), providing the same substrate dependence of the rotation times (34 ns, 27 ns and returning 30 ns). The results indicate the existence of three conformations arranged according to the “energy minimizing principle” by ligated substrates. In this way the data implicate another essential component of physiological control at the subcellular level in the transition of the nonreactive CK–ATP+creatine complex to the reactive enzyme molecule.

Keywords: Muscle creatine kinase; Protein–substrates structural interaction; Enzyme activity control; Fluorescence and absorption spectroscopy

Stability of oxidized, reduced and copper bound forms of Bacillus subtilis Sco by David E. Davidson; Bruce C. Hill (pp. 275-281).

Sco is an accessory protein required for assembly of the Cu_A center of cytochrome c oxidase. Functions proposed for Sco include as a copper chaperone and as a thiol–disulfide exchange protein. Differential scanning calorimetry (DSC) is used here to assess the interaction between the Bacillis subtilis version of Sco (BsSco) and Cu(II). When BsSco binds Cu(II) its melting temperature increases by 23 °C, which corresponds to an equilibrium dissociation constant of 3.50 pM. In contrast BsSco exhibits a much weaker affinity for Cu(I) ( K_D=10 μM). BsSco–Cu(II) is stable over days indicating an extremely slow dissociation for BsSco–Cu(II). However, at high ionic strength in the presence of excess copper, BsSco–Cu(II) returns to its oxidized, disulfide-bonded state and loses its copper binding capacity with a half time of 100 s. DSC of BsSco at high ionic strength indicates an increase in stability of metal free, reduced BsSco combined with a small destabilization of BsSco–Cu(II). It is proposed that BsSco undergoes an ionic strength induced conformational change that promotes electron transfer from the thiol groups on BsSco to Cu(II) to effect copper release. Such a redox transformation could be an important aspect of the copper transfer role proposed for BsSco in Cu_A assembly.

Keywords: Abbreviations; BCS; bathocuproine disulfonate; BsSco; Sco1 homolog in; Bacillus subtilis; DPDS; 4,4′-dipyridyl disulfide; DSC; differential scanning calorimetry; DTT; dithiothreitol; EPR; electron paramagnetic resonanceSco; Cu; _A; assembly; Cytochrome; c; oxidase assembly; Protein-metal binding; Metalloprotein DSC

Smoking and Parkinson's disease: Does nicotine affect α-synuclein fibrillation? by Dong-Pyo Hong; Anthony L. Fink; Vladimir N. Uversky ⁎ (pp. 282-290).

α-synuclein is a small presynaptic protein (14,460 D) that is abundantly distributed in the brain. Although, its function is unknown, the aggregated form of α-synuclein is a pathological hallmark of several neurodegenerative diseases, including Parkinson's disease (PD). Epidemiological studies have shown that smoking can lessen the incidence of Parkinson's disease, indicating that smoke may contain chemicals that are neuro-protective. The fibrillation of α-synuclein was studied in relation to five different compounds found in cigarette smoke: anabasine, cotinine, hydroquinone, nicotine and nornicotine. Thioflavin T assays, gel electrophoresis, size exclusion chromatography–high performance liquid chromatography (SEC–HPLC) and atomic force microscopy (AFM) were utilized to monitor the rate of α-synuclein fibrillation and the inhibitory effects of the cigarette smoke components. We show that nicotine and hydroquinone inhibit α-synuclein fibril formation in a concentration-dependent manner, with nicotine being more effective. The SEC–HPLC data show that nicotine and hydroquinone stabilize soluble oligomers. The morphology of the oligomers stabilized by nicotine was evaluated by AFM, which showed the presence of three stable oligomers with an average height of 16 nm, 10 nm and 4 nm. Comparable results were obtained for the effect of the cigarette smoke components on the A53T mutant fibrillation. These results show that nicotine and hydroquinone inhibit α-synuclein fibrillation and stabilize soluble oligomeric forms. This information can be used to understand the molecular mechanism of the nicotine and hydroquinone action to develop therapeutic solutions for PD.

Keywords: α-synuclein; Intrinsically disordered protein; Smoking; Parkinson's disease; Nicotine; Hydroquinone; Fibrillation; Misfolding

Evidence for pentagalloyl glucose binding to human salivary α-amylase through aromatic amino acid residues by Gyöngyi Gyémánt; Ágnes Zajácz; Bálint Bécsi; Chandran Ragunath; Narayanan Ramasubbu; Ferenc Erdődi; Gyula Batta; Lili Kandra (pp. 291-296).

We demonstrate here that pentagalloyl glucose (PGG), a main component of gallotannins, was an effective inhibitor of HSA and it exerted similar inhibitory potency to Aleppo tannin used in this study. The inhibition of HSA by PGG was found to be non-competitive and inhibitory constants of K_EI=2.6 µM and K_ESI=3.9 µM were determined from Lineweaver–Burk secondary plots. PGG as a model compound for gallotannins was selected to study the inhibitory mechanism and to characterize the interaction of HSA with this type of molecules. Surface plasmon resonance (SPR) binding experiments confirmed the direct interaction of HSA and PGG, and it also established similar binding of Aleppo tannin to HSA. Saturation transfer difference (STD) experiment by NMR clearly demonstrated the aromatic rings of PGG may be involved in the interaction suggesting a possible stacking with the aromatic side chains of HSA. The role of aromatic amino acids of HSA in PGG binding was reinforced by kinetic studies with the W58L and Y151M mutants of HSA: the replacement of the active site aromatic amino acids with aliphatic ones decreased the PGG inhibition dramatically, which justified the importance of these residues in the interaction.

Keywords: Pentagalloyl glucose; Human salivary α-amylase inhibition; Surface plasmon resonance; Saturation transfer difference; NMR

The 1.4 Å crystal structure of the large and cold-active Vibrio sp. alkaline phosphatase by Ronny Helland; Renate Lie Larsen; Bjarni Ásgeirsson (pp. 297-308).

Alkaline phosphatase (AP) from the cold-adapted Vibrio strain G15-21 is among the AP variants with the highest known k_cat value. Here the structure of the enzyme at 1.4 Å resolution is reported and compared to APs from E. coli, human placenta, shrimp and the Antarctic bacterium strain TAB5. The Vibrio AP is a dimer although its monomers are without the long N-terminal helix that embraces the other subunit in many other APs. The long insertion loop, previously noted as a special feature of the Vibrio AP, serves a similar function. The surface does not have the high negative charge density as observed in shrimp AP, but a positively charged patch is observed around the active site that may be favourable for substrate binding. The dimer interface has a similar number of non-covalent interactions as other APs and the “crown"-domain is the largest observed in known APs. Part of it slopes over the catalytic site suggesting that the substrates may be small molecules. The catalytic serines are refined with multiple conformations in both monomers. One of the ligands to the catalytic zinc ion in binding site M1 is directly connected to the crown-domain and is closest to the dimer interface. Subtle movements in metal ligands may help in the release of the product and/or facilitate prior dephosphorylation of the covalent intermediate. Intersubunit interactions may be a major factor for promoting active site geometries that lead to the high catalytic activity of Vibrio AP at low temperatures.

Keywords: Abbreviations; ECAP; E coli; alkaline phosphatase; PLAP; human placental alkaline phosphatase, VAP,; Vibrio; G15-21 alkaline phosphatase; SAP; Northern shrimp alkaline phosphatase; TAP; alkaline phosphatase from Antarctic bacterium strain TAB5Cold-adaptation; Metalloenzyme; Dimer; Psychrophilic bacteria; Crystallography

α-Synuclein enhances bioluminescent activity of firefly luciferase by facilitating luciferin localization by Jehoon Kim; Chung Hee Moon; Seunho Jung; Seung R. Paik (pp. 309-314).

α-Synuclein, the pathological component of Parkinson's disease, has been demonstrated to be highly interactive with various protein partners. α-Synuclein has been shown to exert a novel effect on the bioluminescence of firefly luciferase by stimulating the oxyluciferin formation from its substrate of luciferin, which results in a significant enhancement of the spike of flashing light via concomitant augmentation for both rapid rise and quick decay of the luminescence. Binding affinity between α-synuclein and luciferase was evaluated with K_d of 8.1 µM based on a dose-dependent enhancement of the luciferase activity by α-synuclein. Kinetic analyses indicated that α-synuclein has facilitated luciferin localization to the luciferase by decreasing apparent K_m, which makes the maximum rate of bioluminescence no longer dependent upon ATP concentration. Catalytic consequences of the α-synuclein binding to luciferase have led to a delayed onset of the coenzyme A-mediated retardation of the quick decay of flashing light as well as a shift in the emission spectra of bioluminescence. Taken together, the novel effects of α-synuclein toward the bioluminescence of luciferase have been demonstrated to be initiated by the specific molecular interaction between the proteins which has influenced the substrate (luciferin) localization to the enzyme.

Keywords: Abbreviations; BSA; bovine serum albumin; CoA; coenzyme A; DTT; dithiothreitol; EDTA; ethylenediaminetetraacetic acid; PD; Parkinson's diseaseFirefly luciferase; α-Synuclein; Bioluminescence; Luciferin; ATP assay

Effects of inherited mutations on catalytic activity and structural stability of human glucose-6-phosphate isomerase expressed in Escherichia coli by Hua-Yang Lin; Ya-Heui Kao; Shu-Ting Chen; Menghsiao Meng ⁎ (pp. 315-323).

Glucose-6-phosphate isomerase (GPI), a homodimeric enzyme, catalyzes the interconversion between glucose-6-phosphate and fructose-6-phosphate. In mammals, it can also act as an autocrine motility factor, neuroleukin, and maturation factor. Deficiency of the enzymatic activity in red blood cells causes nonspherocytic hemolytic anemia in human. To gain a more complete understanding of the molecular basis for the hemolytic anemia due to the GPI-deficiency, the wild-type enzyme and sixteen genetic variants were expressed in Escherichia coli and functionally characterized. Conclusions are as follows: (1) mutations usually have negative influences on catalytic parameters, particularly k_cat, as well as structure stability; (2) mutations at or close to the active site, including R273H, H389R, and S278L, cause great damage to the catalytic function, yet those at distance can still reduce the magnitude of k_cat, despite lesser extents; (3) mutations decrease the enzyme tolerance to heat or SDS by mechanisms of decreasing packing efficiency (V101M, T195I, S278L, L487F, L339P, T375R, I525T), weakening network bonding (R75G, R347C, R347H, R472H, E495K), increasing water-accessible hydrophobic surface (R83W), and destabilizing the ternary structure (T195I, R347C, R347H, and I525T); (4) A300P, L339P, and E495K mutations may also negatively affect the protein folding efficiency.

Keywords: Glucose-6-phosphate isomerase; Phosphoglucose isomerase; Nonspherocytic hemolytic anemia; HNSHA; Blue native PAGE; Thermostability

Biochemical characterization of Alr1529, a novel SGNH hydrolase variant from Anabaena sp. PCC 7120 by Kiranmayee Bakshy; Sathyanarayana N. Gummadi ⁎; Narayanan Manoj ⁎ (pp. 324-334).

Alr1529, a serine hydrolase from the cyanobacteria Anabaena sp. strain PCC 7120 is a member of the SGNH hydrolase superfamily. Biochemical characterization of the purified enzyme revealed that the protein is a dimer in solution and is specific for aryl esters of short chain carboxylic acids. The enzyme was regio-selective for α-naphthyl esters with maximum activity at pH 7.5 and has a broad optimal temperature range (25–45 °C). A structure based comparison of Alr1529 with other superfamily members confirmed the presence of the catalytic triad (Ser17-Asp179-His182) and oxyanion hole (Ser17-Arg54-Asn87) residues. Alr1529 exhibits a previously undescribed variation in the active site wherein a conserved Gly, a proton donor making up the oxyanion hole in the SGNH hydrolases, is substituted by Arg54. Site-directed mutagenesis studies suggest that Arg54 is crucial for substrate binding and catalytic activity. Ser17 plays a very crucial role in catalysis as evident from the 50-fold lower activity of the S17A mutant.

Keywords: Alr1529; Arylesterase; Variant SGNH hydrolase; Anabaena; sp.; Kinetics

Identification of the lysine residue responsible for coenzyme A binding in the heterodimeric 2-oxoacid:ferredoxin oxidoreductase from Sulfolobus tokodaii, a thermoacidophilic archaeon, using 4-fluoro-7-nitrobenzofurazan as an affinity label by Jing Luo; Eriko Fukuda; Hirofumi Takase; Shinya Fushinobu; Hirofumi Shoun; Takayoshi Wakagi ⁎ (pp. 335-340).

The heterodimeric 2-oxoacid:ferredoxin oxidoreductase (StOFOR) from Sulfolobus tokodaii, a thermoacidophilic archaeon, was inactivated by low concentrations of 4-fluoro-7-nitrobenzofurazan (NBD-F), with concomitant increase in fluorescence in subunit-b. The inactivation was prevented by CoA, suggesting that NBD-F covalently bound to the Lys which is responsible for CoA binding. The NBD-labeled subunit-b was isolated and digested with endoproteinase Lys-C. The resulting polypeptide mixture was separated by reverse phase HPLC and the fluorescent fraction was isolated. Amino acid sequencing of the fraction revealed that it comprised a mixture of two polypeptides containing Lys125 and Lys173, respectively. Two StOFOR mutants, K125A and K173A, were constructed, expressed and purified. K125A showed a large increase in the K_m value for CoA and showed poor inactivation by NBD-F, compared with K173A and wild type StOFOR, indicating Lys125 in subunit-b is the critical residue that interacts with CoA.

Keywords: Abbreviations; OFOR; 2-oxoacid:ferredoxin oxidoreductase; StOFOR; 2-oxoacid:ferredoxin oxidoreductase from; Sulfolobus tokodaii; DaPFOR; pyruvate:ferredoxin oxidoreductase from; Desulfovibrio africanus; NBD-F; 4-fluoro-7-nitrobenzofurazan; NBD-CL; 4-chloro-7-nitrobenzofurazan; ODH; 2-oxoacid dehydrogenase multienzyme complex; CoA; coenzyme A; TPP; thiamine pyrophosphate2-Oxoacid:ferredoxin oxidoreductase; Affinity label; NBD-F; CoA; Archaea; Sulfolobus

Effects of polyhydroxy compounds on beetle antifreeze protein activity by Natapol Amornwittawat; Sen Wang; Joseph Banatlao; Melody Chung; Efrain Velasco; John G. Duman; Xin Wen ⁎ (pp. 341-346).

Antifreeze proteins (AFPs) noncolligatively depress the nonequilibrium freezing point of a solution and produce a difference between the melting and freezing points termed thermal hysteresis (TH). Some low-molecular-mass solutes can affect the TH values. The TH enhancement effects of selected polyhydroxy compounds including polyols and carbohydrates on an AFP from the beetle Dendroides canadensis were systematically investigated using differential scanning calorimetry (DSC). The number of hydroxyl groups dominates the molar enhancement effectiveness of polyhydroxy compounds having one to five hydroxyl groups. However, the above rule does not apply for polyhydroxy compounds having more than five hydroxyl groups. The most efficient polyhydroxy enhancer identified is trehalose. In a combination of enhancers the strongest enhancer plays the major role in determining the TH enhancement. Mechanistic insights into identification of highly efficient AFP enhancers are discussed.

Keywords: Abbreviations; AFP; antifreeze protein; TH; thermal hysteresis; DSC; differential scanning calorimetry; DAFP; Dendroides canadensis; antifreeze proteinAntifreeze protein; Thermal hysteresis activity; Polyhydroxy compounds; Antifreeze protein activity enhancement; Differential scanning calorimetry

Two non-redundant fragments in the N-terminal peptide of human cytosolic methionyl-tRNA synthetase were indispensable for the multi-synthetase complex incorporation and enzyme activity by Ran He; Li-Dong Zu; Peng Yao; Xin Chen; En-Duo Wang ⁎ (pp. 347-354).

In human cytoplasm, nine aminoacyl-tRNA synthetases (aaRSs) and three protein factors form a multi-synthetase complex (MSC). Human cytosolic methionyl-tRNA synthetase (hcMetRS) is a component of the MSC. Sequence alignment revealed that hcMetRS has an N-terminal extension of 267 amino acid residues. This extension can be divided into three sub-domains: GST-like, GN, and GC sub-domains. The effect of each sub-domain in the N-terminal extension of hcMetRS on enzymatic activity and incorporation into the MSC was studied. The results of cellular assay showed that the GST-like sub-domain was responsible for the incorporation of hcMetRS into the MSC. The entire N-terminal extension of hcMetRS is indispensable for the enzymatic activity. Deletion mutagenesis revealed that a seven-amino acid motif within the sub-domain GC was important for the activity of amino acid activation. A conserved proline residue within the seven-amino acid motif was crucial, while the other six residues were moderately important for the amino acid activation activity. Thus, the last 15 residues of previously defined N-terminal extension of hcMetRS was a part of the catalytic domain; whereas the first 252 residues of hcMetRS constitute the N-terminal extended domain of hcMetRS. The formerly defined N-terminal extension of hcMetRS possesses two functions of two different domains.

Keywords: Abbreviations; aaRS; aminoacyl-tRNA synthetase; hctRNA; _i; ^Met; human cytosolic initiator tRNA; ^Met; (ATG); WB; western blot; NTA; nitrilotriacetic acid; GST; glutathione S-transferase; hcMetRS/MRS; human cytosolic methionyl-tRNA synthetase (other aminoacyl-tRNA synthetases are abbreviated by the three-letter code of the appropriate amino acid followed by “RS”); MSC; multi-synthetase complexMethionyl-tRNA synthetase; Multi-synthetase complex; Human cytoplasm; Incorporation; Activity

Design and expression of human α7 nicotinic acetylcholine receptor extracellular domain mutants with enhanced solubility and ligand-binding properties by Marios Zouridakis; Paraskevi Zisimopoulou; Elias Eliopoulos; Konstantinos Poulas; Socrates J. Tzartos ⁎ (pp. 355-366).

In order to facilitate structural studies of the extracellular domain (ECD) of human α7 nicotinic acetylcholine receptor (nAChR), we designed several mutants, since the wild-type-ECD forms large oligomers and microaggregates, and expressed them in the yeast Pichia pastoris. Mutant design was based on a 3D model of human α7-nAChR-ECD, constructed using as templates the X-ray crystal structure of the homologous acetylcholine-binding protein (AChBP) and the electron microscopy structure of the Torpedo α-nAChR-ECD. At least one mutant, mut10, carrying six single-point mutations (Phe3Tyr, Val69Thr, Cys116Ser, Ile165Thr, Val177Thr, Phe187Tyr) and the replacement of its Cys-loop with the corresponding and more hydrophilic AChBP Cys-loop, was expressed with a 4-fold higher expression yield (1.2 mg/L) than the wild-type α7-ECD, existing exclusively as a soluble oligomeric, probably pentameric, form, at concentrations up to at least 10 mg/mL, as judged by gel filtration and dynamic light scattering. This mutant displayed a significantly improved¹²⁵I-α-bungarotoxin-binding affinity ( K_d=24 nM) compared to the wild-type-ECD ( K_d=70 nM), the binding being inhibited by unlabelled α-bungarotoxin,d-tubocurarine or nicotine ( K_i of 21.5 nM, 127 μM and 17.5 mM, respectively). Circular dichroism studies of mut10 revealed (a) a similar secondary structure composition (∼5% α-helix, ∼45% β-sheet) to that of the AChBP, Torpedo α-nAChR-ECD, and mouse α1-nAChR-ECD, (b) a well-defined tertiary structure and (c) binding of small cholinergic ligands at micromolar concentrations. Furthermore, electron microscopy showed well-assembled, probably pentameric, particles of mut10. Finally, since deglycosylation did not alter its solubility or ligand-binding properties, mut10, in either its glycosylated or deglycosylated form, is a promising α7-ECD mutant for structural studies, useful for the rational drug design to treat α7-nAChR-related diseases.

Keywords: Abbreviations; α-bgtx; α-bungarotoxin; α7-dm-ECD; double mutant of the α7-ECD; α7-wt-ECD; wild-type of the α7-ECD; ACh; acetylcholine; AChBP; acetylcholine-binding protein; BSA; bovine serum albumin; CCh; carbamylcholine; CD; circular dichroism; DLS; dynamic light scattering; ECD; extracellular domain; EM; electron microscopy; LGICs; ligand-gated ion channels; Ls-AChBP; Lymnaea stagnalis; acetylcholine-binding protein; mAb; monoclonal antibody; nAChR; nicotinic acetylcholine receptor; nAChR-ECD; extracellular domain of the nicotinic acetylcholine receptor; PB; phosphate bufferHuman α7 nicotinic acetylcholine receptor extracellular domain; 3D model; Circular dichroism spectroscopy; Ligand-binding; Dynamic light scattering; Electron microscopy

Kinetic analysis of salting activation of a subtilisin-like halophilic protease by Débora N. Okamoto; Marcia Y. Kondo; Jorge A.N. Santos; Sawa Nakajima; Kazumi Hiraga; Kohei Oda; Maria A. Juliano; Luiz Juliano; Iuri E. Gouvea ⁎ (pp. 367-373).

The secreted extracellular subtilase SR5-3 from Halobacillus sp. bacterium, isolated from the high-salt environment of Thai fish sauce, was utilized as a model halophilic serine protease. The dependence of salt activation on the size and structure of substrates was evaluated assaying the enzyme with Suc-AAPF-MCA and with the Fluorescence Resonance Energy Transfer (FRET) peptide Abz-AAPFSSKQ-EDDnp. Solvent isotope effects (SIE) and the thermodynamic parameters for activation of the hydrolysis of Suc-AAPF-MCA and Abz-AAPFSSKQ-EDDnp by SR5-3 protease in the presence of salts were also performed. All the obtained results support the notion that the salting out effect is responsible for the halophilic character of SR5-3, and the magnitude of its hydrolytic activity is mainly derived from the improvement of catalytic and/or interaction steps depending on the nature and size of the substrates, principally if they occupy the substrate prime subsites.

Keywords: Peptidase; Serine protease; Proton inventory

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BBA - Proteins and Proteomics (v.1794, #2)