Check out our New Publishers' Select for Free Articles

BBA - Proteins and Proteomics (v.1814, #7)

Editorial Board (pp. i).

The yeast two hybrid system in a screen for proteins interacting with axolotl ( Ambystoma mexicanum) Msx1 during early limb regeneration by Mehtap AbuQarn; Christina Allmeling; Inga Amshoff; Bjoern Menger; Inas Nasser; Peter M. Vogt; Kerstin Reimers (pp. 843-849).

Urodele amphibians are exceptional in their ability to regenerate complex body structures such as limbs. Limb regeneration depends on a process called dedifferentiation. Under an inductive wound epidermis terminally differentiated cells transform to pluripotent progenitor cells that coordinately proliferate and eventually redifferentiate to form the new appendage. Recent studies have developed molecular models integrating a set of genes that might have important functions in the control of regenerative cellular plasticity. Among them is Msx1, which induced dedifferentiation in mammalian myotubes in vitro. Herein, we screened for interaction partners of axolotl Msx1 using a yeast two hybrid system. A two hybrid cDNA library of 5-day-old wound epidermis and underlying tissue containing more than 2×10⁶ cDNAs was constructed and used in the screen. 34 resulting cDNA clones were isolated and sequenced. We then compared sequences of the isolated clones to annotated EST contigs of the Salamander EST database (BLASTn) to identify presumptive orthologs. We subsequently searched all no-hit clone sequences against non redundant NCBI sequence databases using BLASTx. It is the first time, that the yeast two hybrid system was adapted to the axolotl animal model and successfully used in a screen for proteins interacting with Msx1 in the context of amphibian limb regeneration.► The yeast two hybrid system was adapted to axolotl limb regeneration model. ► A cDNA library was screened for interaction partners of axolotl Msx1. ► 34 resulting cDNA clones were isolated and sequenced. ► Presumptive orthologs were identified among contigs of the Salamander EST database.

Keywords: Axolotl (; Ambystoma mexicanum; ); Two hybrid system; Limb regeneration; Msx1; Protein interaction; Bioinformatics

An analysis of the phosphoproteome of immune cell lines exposed to the immunomodulatory mycotoxin deoxynivalenol by André Nogueira da Costa; Jeffrey N. Keen; Christopher P. Wild; John B.C. Findlay (pp. 850-857).

The mycotoxin deoxynivalenol (DON) commonly contaminates cereal grains. It is ubiquitous in the Western European diet, although chronic, low-dose effects in humans are not well described, but immunotoxicity has been reported. In this study, two-dimensional gel electrophoresis was used to identify phosphoproteomic changes in human B (RPMI1788) and T (Jurkat E6.1) lymphocyte cell lines after exposure to modest concentrations of DON (up to 500ng/mL) for 24h. Proteins identified as having altered phosphorylation state post-treatment (C-1-tetrahydrofolate synthase, eukaryotic elongation factor 2, nucleoside diphosphate kinase A, heat shock cognate 71kDa protein, eukaryotic translation initiation factor 3 subunit I and growth factor receptor-bound protein 2) are involved in regulation of metabolic pathways, protein biosynthesis and signaling transduction. All exhibited a greater than 1.4-fold change, reproducible in three separate experiments consisting of 36 gels in total. Flow cytometry validated the observations for eukaryotic elongation factor 2 and growth factor receptor-bound protein 2. These findings provide further insights as to how low dose exposure to DON may affect human immune function and may have potential as mechanism-based phosphoprotein biomarkers for DON exposure.► Deoxynivalenol contaminates cereal grains worldwide. ► Low-dose exposure induces immunotoxicity. ► Deoxynivalenol treatment alters immune cell lines phosphoproteome. ► We identified potential mechanism-based phosphoprotein biomarkers.

Keywords: Abbreviations; C1-THF synthase; C-1-tetrahydrofolate synthase; DON; deoxynivalenol; eEF2; eukaryotic elongation factor 2; eIF3i; eukaryotic translation initiation factor 3 subunit I; ERK; extracellular signal-regulated kinase; GMPS; GMP synthase; GRB2; growth factor receptor-bound protein 2; Hsc70; heat shock cognate 71; kDa protein; IMDH2; Inosine-5′-monophosphate dehydrogenase 2; MFI; mean fluorescence intensity; MTT; 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; NDKA; nucleoside diphosphate kinase A; PE; phycoerythrin; PKR; protein kinase R; PMTDI; provisional maximum tolerable daily intakeBiomarker; Deoxynivalenol; Mycotoxin; Phosphoproteomics

Site-directed mutations and kinetic studies show key residues involved in alkylammonium interactions and reveal two sites for phosphorylcholine in Pseudomonas aeruginosa phosphorylcholine phosphatase by Paola R. Beassoni; Lisandro H. Otero; Cristhian Boetsch; Carlos E. Domenech; Fernado D. González-Nilo; Ángela T. Lisa (pp. 858-863).

Pseudomonas aeruginosa phosphorylcholine phosphatase (PchP) catalyzes the hydrolysis of phosphorylcholine (Pcho) to produce choline and inorganic phosphate. PchP belongs to the haloacid dehalogenase superfamily (HAD) and possesses the three characteristic motifs of this family: motif I (³¹D and³³D), motif II (¹⁶⁶S), and motif III (²⁴²K,²⁶¹G,²⁶²D and²⁶⁷D), which fold to form the catalytic site that binds the metal ion and the phosphate moiety of Pcho. Based on comparisons to the PHOSPHO1 and PHOSPHO2 human enzymes and the choline-binding proteins of Gram-(+) bacteria, we selected residues⁴²E and⁴³E and the aromatic triplet⁸²YYY⁸⁴ for site-directed mutagenesis to study the interactions with Pcho and p-nitrophenylphosphate as substrates of PchP. Because mutations in⁴²E,⁴³E and the three tyrosine residues affect both the substrate affinity and the inhibitory effect produced by high Pcho concentrations, we postulate that two sites, one catalytic and one inhibitory, are present in PchP and that they are adjacent and share residues.► With in silico studies we selected candidate residues for alkylammonium interaction. ► We present kinetic data with p-NPP and Pcho for the wtPchP and mutants. ► We propose the presence of two binding sites for Pcho. ► We present a model with both substrates docked in the active site. ► We propose some residues of PchP in the interaction with the choline moiety of Pcho.

Keywords: Pseudomonas aeruginosa; Phosphorylcholine; Choline; Site-directed mutagenesis; HAD superfamily; Substrate inhibition

m-Calpain activation in vitro does not require autolysis or subunit dissociation by Jordan S. Chou; Francis Impens; Kris Gevaert; Peter L. Davies (pp. 864-872).

Calpains are Ca²⁺-dependent, intracellular cysteine proteases involved in many physiological functions. How calpains are activated in the cell is unknown because the average intracellular concentration of Ca²⁺ is orders of magnitude lower than that needed for half-maximal activation of the enzyme in vitro. Two of the proposed mechanisms by which calpains can overcome this Ca²⁺ concentration differential are autoproteolysis (autolysis) and subunit dissociation, both of which could release constraints on the core by breaking the link between the anchor helix and the small subunit to allow the active site to form. By measuring the rate of autolysis at different sites in calpain, we show that while the anchor helix is one of the first targets to be cut, this occurs in the same time-frame as several potentially inactivating cleavages in Domain III. Thus autolytic activation would overlap with inactivation. We also show that the small subunit does not dissociate from the large subunit, but is proteolyzed to a 40–45k heterodimer of Domains IV and VI. It is likely that this autolysis-generated heterodimer has previously been misidentified as the small subunit homodimer produced by subunit dissociation. We propose a model for m-calpain activation that does not involve either autolysis or subunit dissociation.►Autolysis of anchor peptide and inactivating cuts in Domain III are contemporaneous. ►Calpain small subunit stays associated with the large subunit when Ca²⁺ is present. ►We propose an activation model independent of autolysis and subunit dissociation.

Keywords: Calpain activation model; Autolysis; Subunit dissociation; Mass spectrometry; Calcium requirement; Chromatography

NMR relaxation unravels interdomain crosstalk of the two domain prolyl isomerase and chaperone SlyD by Michael Kovermann; Robert Zierold; Caroline Haupt; Low Christian Löw; Jochen Balbach (pp. 873-881).

The dynamics of the two domain prolyl-peptidyl cis/trans isomerase and chaperone SlyD was studied on a ps-to-ns time scale to correlate dynamic changes with the catalytic function.¹⁵N transversal and longitudinal relaxation rates as well as heteronuclear Overhauser effects were determined at different temperatures for Escherichia coli SlyD ( EcSlyD) and for Thermus thermophilus SlyD ( TtSlyD). With the well established extended Lipari–Szabo approach, the order parameter, S², the internal correlation time, τ_e, the exchange rate, R_ex, of the backbone amide protons, and the overall molecular tumbling time, τ_m, were determined. The study was extended to a relaxation analysis of the peptide bound state for both SlyD species. We found highly different relaxation and dynamic behavior of the two domains for free SlyD. Surprisingly, in the presence of a substrate for the chaperone domain, the ps-to-ns dynamics in the remote center of the prolyl-peptidyl cis/ trans isomerization domain increases. We observed this crosstalk between the two domains for both EcSlyD and TtSlyD.►The domain communication of the 2-domain protein SlyD has been examined by NMR relaxation. ►An extended set of relaxation and diffusion data of mesophilic and thermophilic SlyD has been analyzed. ►Dynamic parameter could be derived from the free and substrate bound state. ►Surprisingly, substrate binding in the remote IF domain increases the local flexibility of the active site in the FKBP domain. ►Chemical shift changes are not sensitive enough to reveal this interdomain communication.

Keywords: Abbreviations; SlyD; product of the; slyD; (sensitive-to-lysis) gene; Ec; SlyD*; 1–165 fragment of; Escherichia coli; SlyD; h; FKBP12; human FKBP12; IF; insert in flap; Ec; SlyD*ΔIF; variant of; Ec; SlyD* in which the IF domain is replaced by the flap of human FKBP12; Tt; SlyD; full-length protein of; Thermus thermophilus; SlyD; Tt; SlyDΔIF; variant of; Tt; SlyD in which the IF domain is replaced by the flap of human FKBP12; Tat; twin-arginine-translocationProtein folding; Prolyl isomerase; Molecular chaperone; FKBP; NMR dynamics; Diffusion

Conformational, receptor interaction and alanine scan studies of glucose-dependent insulinotropic polypeptide by Kalyana C. Venneti; J. Paul G. Malthouse; Finbarr P.M. O'Harte; Chandralal M. Hewage (pp. 882-888).

Glucose-dependent insulinotropic polypeptide (GIP) is an insulinotropic incretin hormone that stimulates insulin secretion during a meal. GIP has glucose lowering abilities and hence is considered as a potential target molecule for type 2 diabetes therapy. In this article, we present the solution structure of GIP in membrane-mimicking environments by proton NMR spectroscopy and molecular modelling. GIP adopts an α-helical conformation between residues Phe⁶–Gly³¹ and Ala¹³–Gln²⁹ for micellar and bicellar media, respectively. Previously we examined the effect of N-terminal Ala substitution in GIP, but here eight GIP analogues were synthesised by replacing individual residues within the central 8–18 region with alanine. These studies showed relatively minor changes in biological activity as assessed by insulin releasing potency. However, at higher concentration, GIP(Ala¹⁶), and GIP(Ala¹⁸) showed insulin secreting activity higher than the native GIP (P<0.01 to P<0.001) in cultured pancreatic BRIN-BD11 cells. Receptor interaction studies of the native GIP with the extracellular domain of its receptor were performed by using two different docking algorithms. At the optimised docking conformation, the complex was stabilised by the presence of hydrophobic interactions and intermolecular hydrogen bonding. Further, we have identified some potentially important additional C-terminal interactions of GIP with its N-terminal extracellular receptor domain.Structural studies of GIP and the receptor interaction studies of GIP with the extracellular domain of its receptor were performed by using two different docking algorithms. Docking model displays molecular interactions of GIP ligand with its N-terminal extra cellular domain at the receptor binding pocket.Display Omitted► Solution structural studies of GIP hormone in micellar and bicellar media. ► GIP adopts an alpha helical conformation in membrane mimicking environments. ► N-terminal receptor interaction studies of GIP receptor. ► Advanced docking studies of GIP with its N-terminal domain. ► Molecular recognition studies of GIP ligand and its receptor interactions.

Keywords: Abbreviations; Nt-ECD; N-terminal extracellular receptor domain; GIP; glucose-dependent insulinotropic polypeptide; NOE; nuclear Overhauser effect; NOESY; nuclear Overhauser effect spectroscopy; RMSD; root mean square deviation; TOCSY; total correlation spectroscopyGIP; Glucose dependent insulinotropic polypeptide; NMR; Docking; Ala-scan; Type 2 diabetes

The interactions of calreticulin with immunoglobulin G and immunoglobulin Y by Mollegaard Karen Mai Møllegaard; Karen Duus; Traeholt Sofie Dietz Træholt; Morten Thaysen-Andersen; Yan Liu; Angelina S. Palma; Ten Feizi; Paul R. Hansen; Hojrup Peter Højrup; Gunnar Houen (pp. 889-899).

Calreticulin is a chaperone of the endoplasmic reticulum (ER) assisting proteins in achieving the correctly folded structure. Details of the binding specificity of calreticulin are still a matter of debate. Calreticulin has been described as an oligosaccharide-binding chaperone but data are also accumulating in support of calreticulin as a polypeptide binding chaperone. In contrast to mammalian immunoglobulin G (IgG), which has complex type N-glycans, chicken immunoglobulin Y (IgY) possesses a monoglucosylated high mannose N-linked glycan, which is a ligand for calreticulin. Here, we have used solid and solution-phase assays to analyze the in vitro binding of calreticulin, purified from human placenta, to human IgG and chicken IgY in order to compare the interactions. In addition, peptides from the respective immunoglobulins were included to further probe the binding specificity of calreticulin. The experiments demonstrate the ability of calreticulin to bind to denatured forms of both IgG and IgY regardless of the glycosylation state of the proteins. Furthermore, calreticulin exhibits binding to peptides (glycosylated and non-glycosylated) derived from trypsin digestion of both immunoglobulins. Additionally, calreticulin peptide binding was examined with synthetic peptides covering the IgG Cγ2 domain demonstrating interaction with approximately half the peptides. Our results show that the dominant binding activity of calreticulin in vitro is toward the polypeptide moieties of IgG and IgY even in the presence of the monoglucosylated high mannose N-linked oligosaccharide on IgY.► Calreticulin binds denatured forms of both IgG and IgY regardless of the glycosylation state of the proteins. ► Calreticulin exhibits binding to peptides (glycosylated and non-glycosylated) derived from trypsin digestion of both immunoglobulins. ► The dominant binding activity of calreticulin in vitro is toward the polypeptide moieties of IgG and IgY even in the presence of the monoglucosylated high mannose N-linked oligosaccharide on IgY.

Keywords: Abbreviations; AP; Alkaline phosphatase; ConA; concanavalin A; DIC; N,N′-diisopropylcarbodiimide; DMSO; dimethylsulfoxide; ER; endoplasmic reticulum; ELISA; enzyme-linked immunosorbent assay; eq; equivalent; Fmoc; 9-fluorenylmethoxycarbonyl; Fuc; fucose; Gal; galactose; Glc; glucose; HOAt; 1-hydroxy-7-aza-benzotriazole; IgG; immunoglobulin G; IgY; immunoglobulin Y; IVIG; intravenous immunoglobulin; Man; mannose; MS; mass spectrometry; GlcNAc; N-acetylglucosamine; NeuAc; N-acetylneuraminic acid; NMP; N-methyl-2-pyrrolidone; NGL; neoglycolipid; PNGase F; N-glycosidase F; OVA; ovalbumin; PIP; piperidine; p; NPP; para; -nitrophenylphosphate; PBS; phosphate buffered saline; RP-HPLC; reverse phase high-preformance liquid chromatography; SDS-PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresis; SPPS; solid-phase peptide synthesis; THIO; thioanisole; TIS; triisopropylsilane; TOF; time-of-flight; TFA; trifluoroacetic acidCalreticulin; Chaperone; Immunoglobulin; Peptide specificity

Characterization of a novel posttranslational modification in neuronal nitric oxide synthase by small ubiquitin-related modifier-1 by Masatomo Watanabe; Kouichi Itoh (pp. 900-907).

The multifaceted functions of nitric oxide (NO) in the CNS are defined by the activity of neuronal NO synathase (nNOS). The activities of nNOS are modulated by posttranslational modifications, such as phosphorylation and ubiquitination, but whether it is modified by small ubiquitin-related modifier (SUMO) remains unknown. The aim of this study was to elucidate whether nNOS is posttranslationally modified by SUMO proteins. Bioinformatic analyses using SUMOplot and SUMOFI predicted that nNOS had potential SUMO modification sites. When HEK293T cells were transiently co-expressed with nNOS and SUMO-1, two bands corresponding to nNOS-SUMO-1 conjugates were detected. In addition, two nNOS-SUMO-1 conjugates were confirmed by an in vitro sumoylation assay using recombinant proteins. Furthermore, nNOS-SUMO-1 conjugates were identified by MALDI-QIT/TOF mass spectrometry. These findings indicate that nNOS is clearly defined as a SUMO-1 target protein both in vitro and at the cellular level. We next characterized specific enzymes in the nNOS-SUMO-1 conjugation cycle at the cellular level. SUMO-1 conjugation of nNOS depended on Ubc9 (E2). The interaction between nNOS and Ubc9 was facilitated by PIASxβ (E3). On the other hand, SUMO-1 was deconjugated from nNOS by SENP1 and SENP2. Overall, this study has newly identified that nNOS is posttranslationally modified by SUMO-1.►nNOS is covalently modified by SUMO-1 protein at the cellular level. ►Sumoylation of nNOS was confirmed by an in vitro assay and identified by MS analysis. ►nNOS sumoylation is catalyzed by Ubc9 as the E2 SUMO-conjugating enzyme. ►nNOS sumoylation by Ubc9 is facilitated by PIASxβ as the E3 SUMO-ligase. ►Sumoylated-nNOS is deconjugated by SENP1 and SENP2, which have isopeptidase activity.

Keywords: Abbreviations; HEK293T cells; human embryonic kidney 293T cells; MALDI-QIT/TOF; Matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight; MS; mass spectrometry; NMDA; N-methyl-; d; -aspartate; NO; nitric oxide; nNOS; neuronal nitric oxide synthase; PIAS; protein inhibitor of activated STAT; SENP; sentrin-specific protease; SUMO; small ubiquitin-related modifierNeuronal nitric oxide synthase; Nitric oxide; Small ubiquitin-related modifier; SUMO-1; Sumoylation; Posttranslational modification

Impact of site-specific N-glycosylation on cellular secretion, activity and specific activity of the plasma phospholipid transfer protein by John J. Albers; Joseph R. Day; Gertrud Wolfbauer; Hal Kennedy; Simona Vuletic; Marian C. Cheung (pp. 908-911).

The plasma phospholipid transfer protein (PLTP) plays a key role in lipid and lipoprotein metabolism. It has six potential N-glycosylation sites. To study the impact of these sites on PLTP secretion and activity, six variants containing serine to alanine point mutations were prepared by site-directed mutagenesis and expressed in Chinese hamster ovary Flp-In cells. The apparent size of each of the six PLTP mutants was slightly less than that of wild type by Western blot, indicating that all six sites are glycosylated or utilized. The size of the carbohydrate at each N-glycosylation site ranged from 3.14 to 4.2kDa. The effect of site-specific N-glycosylation removal on PLTP secretion varied from a modest enhancement (15% and 60%), or essentially no effect, to a reduction in secretion (8%, 14% and 32%). Removal of N-glycosylation at any one of the six glycosylation sites resulted in a significant 35-78% decrease in PLTP activity, and a significant 29-80% decrease in PLTP specific activity compared to wild type. These data indicate that although no single N-linked carbohydrate chain is a requirement for secretion or activity, the removal of the carbohydrate chains had a quantitative impact on cellular secretion of PLTP and its phospholipid transfer activity.► Six PLTP sites are N-glycosylated at residues 47, 77, 100, 126, 228 and 381. ► The size of these six carbohydrate (CHO) chains ranges from 3.14 to 4.20kDa. ► CHO removal at each site reduced PLTP activity to 22-65% of wild type. ► CHO removal at each site reduced PLTP specific activity to 20-71% of wild type.

Keywords: Abbreviations; BPI; bactericidal permeability increasing protein; CETP; cholesteryl ester transfer protein; CHO; carbohydrate; FBS; fetal bovine serum; LBP; lipopolysaccharide binding protein; PC; phosphatidylcholine; PLTA; PLTP phospholipid transfer activity; PLTP; phospholipid transfer protein; WT; wild typePhospholipid transfer protein; N-glycosylation; Protein secretion; Lipid transfer

Sections

Personal tools

BBA - Proteins and Proteomics (v.1814, #7)

Sections

Personal tools

Local resources

BBA - Proteins and Proteomics (v.1814, #7)