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BBA - Proteins and Proteomics (v.1774, #9)

Editorial Board (pp. ii).

Structural similarities and functional diversity of eukaryotic discoidin-like domains by A. Kiedzierska; K. Smietana; H. Czepczynska; J. Otlewski (pp. 1069-1078).

The discoidin domain is a ∼150 amino acid motif common in both eukaryotic and prokaryotic proteins. It is found in a variety of extracellular, intracellular and transmembrane multidomain proteins characterized by a considerable functional diversity, mostly involved in developmental processes. The biological role of the domain depends on its interactions with different molecules, including growth factors, phospholipids and lipids, galactose or its derivatives, and collagen. The conservation of the motif, as well as the serious physiological consequences of discoidin domain disorders underscore the importance of the fold, while the ability to accommodate such an extraordinarily broad range of ligand molecules makes it a fascinating research target. In present review we characterize the distinctive features of discoidin domains and briefly outline the biological role of this module in various eukaryotic proteins.

Keywords: Abbreviations; ACLP; aortic carboxypeptidase-like protein; BTB; bric-a-brac, tramtrack, broad-complex; Carb; carboxypeptidase; CFV/VIII; coagulation factors V and VIII; CNS; central nervous system; CTLH; C-terminal domain to LisH; CUB; complement factor C1s/C1r, urchin embryonic growth factor, bone morphogenetic protein homology; DEL1; developmental endothelial locus-1; DDR; discoidin domain receptor; DS domain; discoidin-like domain (FA58C, F5/8C, C2 domain of coagulation factors V and VIII); DS; discoidin; ECM; extracellular matrix; EGF; epidermal growth factor; ER; endoplasmatic reticulum; GO; galactose oxidase; LamG; laminin G; LisH; N-terminal domain in Lissencephaly 1; LH; lisH with CTLH; MAM; meprin, A5, tyrosine phosphatase (mu) homology domain; mkln1; muskelin; MMP; matrix metalloproteinse; Npn1; neuropilin-1; Nrx IV; neurexin IV; PKC; protein kinase C; RS1; retinoschisin; RTK; tyrosine kinase receptor; Sco; scospondin; SED1/MFG; sperm–egg adhesion protein/milk fat globule; SMART; Simple Modular Architecture Research Toolβ-barrel; Cell adhesion; Coagulation factor; Discoidin

The interplay of processivity, substrate inhibition and a secondary substrate binding site of an insect exo-β-1,3-glucanase by Fernando A. Genta; Alexandra F. Dumont; Sandro R. Marana; Walter R. Terra; Clélia Ferreira (pp. 1079-1091).

Abracris flavolineata midgut contains a processive exo-β-glucanase (ALAM) with lytic activity against Saccharomyces cerevisiae, which was purified (yield, 18%; enrichment, 37 fold; specific activity, 1.89 U/mg). ALAM hydrolyses fungal cells or callose from the diet. ALAM (45 kDa; pI 5.5; pH optimum 6) major products with 0.6 mM laminarin as substrate are β-glucose (61%) and laminaribiose (39%). Kinetic data obtained with laminaridextrins and methylumbelliferyl glucoside suggest that ALAM has an active site with at least six subsites. The best fitting of kinetic data to theoretical curves is obtained using a model where one laminarin molecule binds first to a high-affinity accessory site, causing active site exposure, followed by the transference of the substrate to the active site. The two-binding-site model is supported by results from chemical modifications of amino acid residues and by ALAM action in MUβGlu plus laminarin. Low laminarin concentrations increase the modification of His, Tyr and Asp or Glu residues and MUβGlu hydrolysis, whereas high concentrations abolish modification and inhibit MUβGlu hydrolysis. Our data indicate that processivity results from consecutive transferences of substrate between accessory and active site and that substrate inhibition arises when both sites are occupied by substrate molecules abolishing processivity.

Keywords: Enzyme processivity; Substrate inhibition; Chemical modification; Accessory site; Substrate affinitiesAbbreviations; ALAM; A. flavolineata; laminarinase; CFUs; colony forming units; DPC; diethyl pyrocarbonate; EDC; N-(3-dimethylaminopropyl)-; N; ′-ethylcarbodiimide; GGE; 2-glyceroyl-3-glucosyl ethylglycol mixed acetal; MES; 2-Morpholinoethanesulfonic acid; MUβGlu; 4-methylumbelliferyl β-; d; -glucopyranoside; NBS; N-bromosuccinimide; NPβGlu; p-nitrophenyl-β-; d; -glucopiranoside; or-laminarin; periodate-oxidized and borohydride reduced laminarin; PG; 1-phenylglyoxal; pHMB; 4-(hydroxymercuri) benzoic acid; red-laminarin; borohydride reduced laminarin; TAPS; N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid; TEMED; N,N,N′,N′; -tetramethylethylenediamine; TNM; tetranitromethane; YPDA; medium with 1% yeast extract, 1% peptone, 1% dextrose and 2% agar

Identification of a novel glyoxylate reductase supports phylogeny-based enzymatic substrate specificity prediction by Maarten Fauvart; Kristien Braeken; Ruth Daniels; Karen Vos; Maxime Ndayizeye; Jean-Paul Noben; Johan Robben; Jos Vanderleyden; Jan Michiels (pp. 1092-1098).

Phylogenetic analysis of the superfamily ofD-2-hydroxyacid dehydrogenases identified the previously unrecognized cluster of glyoxylate/hydroxypyruvate reductases (GHPR). Based on the genome sequence of Rhizobium etli, the nodulating endosymbiont of the common bean plant, we predicted a putative 3-phosphoglycerate dehydrogenase to exhibit GHPR activity instead. The protein was overexpressed and purified. The enzyme is homodimeric under native conditions and is indeed capable of reducing both glyoxylate and hydroxypyruvate. Other substrates are phenylpyruvate and ketobutyrate. The highest activity was observed with glyoxylate and phenylpyruvate, both having approximately the same k_cat/ K_m ratio. This kind of substrate specificity has not been reported previously for a GHPR. The optimal pH for the reduction of phenylpyruvate to phenyllactate is pH 7. These data lend support to the idea of predicting enzymatic substrate specificity based on phylogenetic clustering.

Keywords: Phenylpyruvate; Phenyllactate; Glyoxylate; Glycolate; Hydroxypyruvate; D; -glycerate; D; -2-hydroxyacid dehydrogenase; Substrate specificity; Phylogeny; Rhizobium etli

Identification of a novel endochitinase from a marine bacterium Vibrio proteolyticus strain No. 442 by Shiro Itoi; Yuna Kanomata; Yuki Koyama; Kazunari Kadokura; Shinsuke Uchida; Toshiyuki Nishio; Tadatake Oku; Haruo Sugita (pp. 1099-1107).

Chitin binding proteins prepared from Vibrio proteolyticus were purified and the N-terminal amino-acid sequence of a protein from a 110-kDa band on SDS-PAGE was found to be 85–90% identical to the 22nd–41st residues of the N-termini of chitinase A precursor proteins from other vibrios. We cloned the corresponding gene, which encodes a putative protein of 850 amino acids containing a 26-residue signal sequence. The chitinase precursor from V. proteolyticus was 78–80% identical to those from Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio carchariae. However, the proteolytic cleavage site for C-terminal processing between R⁵⁹⁷ and K⁵⁹⁸ in the chitinase precursor of other vibrios was not observed in the amino acid sequence of V. proteolyticus, which instead had the sequence R⁶⁰⁰ and A⁶⁰¹. Subsequently, full-length and truncated chitinases were generated in Escherichia coli. The specific activity of full-length chitinase expressed in E. coli was 17- and 20-folds higher for colloidal and α-chitins (insoluble substrate), respectively, than that of the C-terminal truncated enzyme. However, both recombinants showed similar hydrolysis patterns of hexa- N-acetyl-chitohexaose (soluble substrate), producing di- N-acetyl-chitobiose as major product on TLC analysis. We showed that the C-terminus of the V. proteolyticus chitinase A was important for expression of high specific activity against insoluble chitins.

Keywords: Chitinase; Chitinolytic activity; Marine bacteria; Vibrio proteolyticus

Soluble factors released by ATDC5 cells affect the formation of calcium phosphate crystals by Leonie F.A. Huitema; P. René van Weeren; Bas W.M. van Balkom; Tom Visser; Chris H.A. van de Lest; Ab Barneveld; J. Bernd Helms; Arie B. Vaandrager (pp. 1108-1117).

During biomineralization the organism controls the nature, orientation, size and shape of the mineral phase. The aim of this study was to investigate whether proteins or vesicles that are constitutively released by growing ATDC5 cells have the ability to affect the formation of the calcium phosphate crystal. Therefore, subconfluent cultured ATDC5 cells were incubated for 1 h in medium without serum. Subsequently, medium was harvested and incubated for 24 h in the presence of additional Pi. This resulted in the formation of flat mineralizing structures (FMS), consisting of complex irregularly shaped flat crystals, which occasionally contained fiber-like structures (∼40 μm in size). Without pre-incubation of medium with cells, only small punctate (dot like) calcium phosphate precipitates were observed. The formation of FMS was shown to be caused by soluble factors released by subconfluent ATDC5 cells. Proteomic analysis by mass spectrometry showed that FMS contained a specific set intracellular proteins, serum proteins, and extracellular matrix proteins. Bulk cytosolic proteins derived from homogenized cells or serum proteins did, however, not induce the formation of FMS. Conditioned medium from HeLa, CHO K1, RAW 264.7 and MDCK cells was also capable to form FMS under our experimental conditions. Therefore the formation of FMS seems to be caused by specific soluble factors constitutively released by ADTC5 and other cells. This in vitro model system can be used as a tool to identify factors that affect the shape of the biomineral phase.

Oxidation reduces the fibrillation but not the neurotoxicity of the prion peptide PrP106–126 by Ann-Louise Bergström; Joëlle Chabry; Lone Bastholm; Peter M.H. Heegaard (pp. 1118-1127).

There is increasing evidence that soluble oligomers of misfolded protein may play a role in the pathogenesis of protein misfolding diseases including the transmissible spongiform encephalopathies (TSE) where the protein involved is the prion protein, PrP. The effect of oxidation on fibrillation tendency and neurotoxicity of different molecular variants of the prion peptide PrP106–126 was investigated. It was found that methionine oxidation significantly reduced amyloid fibril formation and proteinase K resistance, but it did not reduce (but rather increase slightly) the neurotoxicity of the peptides in vivo (electroretinography after intraocular injections in mice) and in vitro (in primary neuronal cultures). We furthermore found that the bovine variant of PrP106–126, containing only one methionine residue, showed both reduced fibril forming capacity and in vivo and in vitro neurotoxicity. The findings imply (I) that there is not a simple relation between the formation of amyloid fibrils and neurotoxicity of PrP106–126 derived peptides, (II) that putative, soluble, non-amyloid protofibrils, presumed to be present in increased proportions in oxidized PrP106–126, could play a role in the pathogenesis of TSE and III) that the number of methionine residues in the PrP106–126 peptide seems to have a pivotal role in determining the physical and biological properties of PrP106–126.

Keywords: Abbreviations; TSE; Transmissible spongiform encephalopathy; MetO; Methionine sulfoxide; ERG; Electroretinography; PK; Proteinase K; ThT; Thioflavin T; TUNEL; Terminal dUTP nick end labeling; EM; Electron microscopy; wt; Wild typePrion peptide; Oxidation; Electroretinography; Apoptosis; Oligomers; Protofibrils

Aggregation and fibrillation of bovine serum albumin by Nikolaj K. Holm; Stine K. Jespersen; Lise V. Thomassen; Tine Y. Wolff; Pankaj Sehgal; Line A. Thomsen; Gunna Christiansen; Christian Beyschau Andersen; Anders D. Knudsen; Daniel E. Otzen (pp. 1128-1138).

The all-α helix multi-domain protein bovine serum albumin (BSA) aggregates at elevated temperatures. Here we show that these thermal aggregates have amyloid properties. They bind the fibril-specific dyes Thioflavin T and Congo Red, show elongated although somewhat worm-like morphology and characteristic amyloid X-ray fiber diffraction peaks. Fibrillation occurs over minutes to hours without a lag phase, is independent of seeding and shows only moderate concentration dependence, suggesting intramolecular aggregation nuclei. Nevertheless, multi-exponential increases in dye-binding signal and changes in morphology suggest the existence of different aggregate species. Although β-sheet content increases from 0 to ca. 40% upon aggregation, the aggregates retain significant amounts of α-helix structure, and lack a protease-resistant core. Thus BSA is able to form well-ordered β-sheet rich aggregates which nevertheless do not possess the same structural rigidity as classical fibrils. The aggregates do not permeabilize synthetic membranes and are not cytotoxic. The ease with which a multidomain all-α helix protein can form higher-order β-sheet structure, while retaining significant amounts of α-helix, highlights the universality of the fibrillation mechanism. However, the presence of non-β-sheet structure may influence the final fibrillar structure and could be a key component in aggregated BSA's lack of cytotoxicity.

Keywords: Albumin; Fibrillation; Aggregation; Amyloid; Proteolysis; Nucleation

Kinetic analysis of butyrylcholinesterase-catalyzed hydrolysis of acetanilides by Patrick Masson; Marie-Thérèse Froment; Emilie Gillon; Florian Nachon; Sultan Darvesh; Lawrence M. Schopfer (pp. 1139-1147).

The aryl-acylamidase (AAA) activity of butyrylcholinesterase (BuChE) has been known for a long time. However, the kinetic mechanism of aryl-acylamide hydrolysis by BuChE has not been investigated. Therefore, the catalytic properties of human BuChE and its peripheral site mutant (D70G) toward neutral and charged aryl-acylamides were determined. Three neutral (o-nitroacetanilide, m-nitroacetanilide, o-nitrophenyltrifluoroacetamide) and one positively charged (3-(acetamido) N,N,N-trimethylanilinium, ATMA) acetanilides were studied. Hydrolysis of ATMA by wild-type and D70G enzymes showed a long transient phase preceding the steady state. The induction phase was characterized by a hysteretic “burst”. This reflects the existence of two enzyme states in slow equilibrium with different catalytic properties. Steady-state parameters for hydrolysis of the three acetanilides were compared to catalytic parameters for hydrolysis of esters giving the same acetyl intermediate. Wild-type BuChE showed substrate activation while D70G displayed a Michaelian behavior with ATMA as with positively charged esters. Owing to the low affinity of BuChE for amide substrates, the hydrolysis kinetics of neutral amides was first order. Acylation was the rate-determining step for hydrolysis of aryl-acetylamide substrates. Slow acylation of the enzyme, relative to that by esters may, in part, be due suboptimal fit of the aryl-acylamides in the active center of BuChE. The hypothesis that AAA and esterase active sites of BuChE are non-identical was tested with mutant BuChE. It was found that mutations on the catalytic serine, S198C and S198D, led to complete loss of both activities. The silent variant (FS117) had neither esterase nor AAA activity. Mutation in the peripheral site (D70G) had the same effect on esterase and AAA activities. Echothiophate inhibited both activities identically. It was concluded that the active sites for esterase and AAA activities are identical, i.e. S198. This excludes any other residue present in the gorge for being the catalytic nucleophile pole.

Keywords: Abbreviations; AAA; aryl-acylamidase; AChE; acetylcholinesterase; ASCh; acetylthiocholine; ATMA; 3-(acetamido); N,N,N; -trimethylanilinium; BuChE; butyrylcholinesterase; BuSCh; butyrylthiocholine; DFP; diisopropylfluorophosphate; echo; echothiophate; m-NAC; m-nitroacetanilide; m-NA; m-nitroaniline; o-NAC; o-nitroacetanilide; o-NA; o-nitroaniline; o-NTFNAC; o-nitrophenyltrifluoroacetanilide; o-NPA; o-nitrophenylacetate; o-NP; o-nitrophenol; p-NPA; p-nitrophenylacetate; p-nitrophenol; p-NP; o-TMA; trimethylammonioaniline; PAS; peripheral anionic siteButyrylcholinesterase; Aryl-acylamidase; Acetanilide; Hysteretic behavior; Burst; Active site

The connection between metal ion affinity and ligand affinity in integrin I domains by Thomas Vorup-Jensen; Travis T. Waldron; Nathan Astrof; Motomu Shimaoka; Timothy A. Springer (pp. 1148-1155).

Integrins are cell-surface heterodimeric proteins that mediate cell–cell, cell–matrix, and cell–pathogen interactions. Half of the known integrin α subunits contain inserted domains (I domains) that coordinate ligand through a metal ion. Although the importance of conformational changes within isolated I domains in regulating ligand binding has been reported, the relationship between metal ion binding affinity and ligand binding affinity has not been elucidated. Metal and ligand binding by several I domain mutants that are stabilized in different conformations are investigated using isothermal titration calorimetry and surface plasmon resonance studies. This work suggests an inverse relationship between metal ion affinity and ligand binding affinity (i.e. constructs with a high affinity for ligand exhibit a low affinity for metal). This trend is discussed in the context of structural studies to provide an understanding of interplay between metal ion binding and ligand affinities and conformational changes.

Keywords: Integrin; Metal ion; LFA-1; ICAM-1; Isothermal calorimetry; Surface plasmon resonance

Phosphorylation sites of Arabidopsis MAP kinase substrate 1 (MKS1) by Mikael B. Caspersen; Jin-Long Qiu; Xumin Zhang; Erik Andreasson; Henrik Næsted; John Mundy; Birte Svensson (pp. 1156-1163).

The Arabidopsis MAP kinase 4 (MPK4) substrate MKS1 was expressed in Escherichia coli and purified, full-length, 6x histidine (His)-tagged MKS1 was phosphorylated in vitro by hemagglutinin (HA)-tagged MPK4 immuno-precipitated from plants. MKS1 phosphorylation was initially verified by electrophoresis and gel-staining with ProQ Diamond and the protein was digested by either trypsin or chymotrypsin for maximum sequence coverage to facilitate identification of phosphorylated positions. Prior to analysis by mass spectrometry, samples were either desalted, passed over TiO₂ or both for improved phosphopeptide detection. As MAP kinases generally phosphorylate serine or threonine followed by proline (Ser/Thr–Pro), theoretical masses of potentially phosphorylated peptides were calculated and mass spectrometric peaks matching these masses were fragmented and searched for a neutral-loss signal at ∼98 Da indicative of phosphorylation. Additionally, mass spectrometric peaks present in the MPK4-treated MKS1, but not in the control peptide map of untreated MKS1, were fragmented. Fragmentation spectra were subjected to a MASCOT database search which identified three of the twelve Ser–Pro serine residues (Ser72, Ser108, Ser120) in the phosphorylated form.

Keywords: Arabidopsis; MAP kinase substrate; Recombinant MKS1; Protein phosphorylation site; Mass spectrometry

Lactoperoxidase folding and catalysis relies on the stabilization of the α-helix rich core domain: A thermal unfolding study by Barbara Boscolo; Sónia S. Leal; Elena M. Ghibaudi; Cláudio M. Gomes (pp. 1164-1172).

Lactoperoxidase (LPO) belongs to the mammalian peroxidase family and catalyzes the oxidation of halides, pseudo-halides and a number of aromatic substrates at the expense of hydrogen peroxide. Despite the complex physiological role of LPO and its potential involvement in carcinogenic mechanisms, cystic fibrosis and inflammatory processes, little is known on the folding and structural stability of this protein. We have undertaken an investigation of the conformational dynamics and catalytic properties of LPO during thermal unfolding, using complementary biophysical techniques (differential scanning calorimetry, electron spin resonance, optical absorption, fluorescence and circular dichroism spectroscopies) together with biological activity assays. LPO is a particularly stable protein, capable of maintaining catalysis and structural integrity up to a high temperature, undergoing irreversible unfolding at 70 °C. We have observed that the first stages of the thermal denaturation involve a minor conformational change occurring at 40 °C, possibly at the level of the protein β-sheets, which nevertheless does not result in an unfolding transition. Only at higher temperature, the protein hydrophobic core, which is rich in α-helices, unfolds with concomitant disruption of the catalytic heme pocket and activity loss. Evidences concerning the stabilizing role of the disulfide bridges and the covalently bound heme cofactor are shown and discussed in the context of understanding the structural stability determinants in a relatively large protein.

Keywords: Abbreviations; LPO; Lactoperoxidase; MPO; Myeloperoxidase; EPO; Eosinophil peroxidase; ESR; Electron Spin Resonance; CD; Circular Dichroism; DSC; Differential Scanning Calorimetry; GdmCl; Guanidinium Chloride; MTSL; 1-oxyl-2,2,5,5,-tetramethyl-Δ; ³; -pyrroline-3-methyl)-methanethiosulfonate Spin Label; DMF; Dimethyl Formamide; DMAB; 3-dimethylamino-benzoic acid; MBTH; 3-methyl-2-benzothiazolinone hydrazone hydrochloride; ANS; 1-anilinonaphalene-8-sulphonateProtein folding; Structural stability; Mammalian peroxidase; Heme proteins; Circular dichroism spectroscopy; Fluorescence spectroscopy

Differentially expressed cytosolic proteins in human leukemia and lymphoma cell lines correlate with lineages and functions by Swetlana Gez; Ben Crossett; Richard I. Christopherson (pp. 1173-1183).

Identification of cytosolic proteins differentially expressed between types of leukemia and lymphoma may provide a molecular basis for classification and understanding their cellular properties. Two-dimensional fluorescence difference gel electrophoresis (DIGE) and mass spectrometry have been used to identify proteins that are differentially expressed in cytosolic extracts from four human leukemia and lymphoma cell lines: HL-60 (acute promyelocytic leukemia), MEC1 (B-cell chronic lymphocytic leukemia), CCRF-CEM (T-cell acute lymphoblastic leukemia) and Raji (B-cell Burkitt's lymphoma). A total of 247 differentially expressed proteins were identified between the four cell lines. Analysis of the data by principal component analysis identified 22 protein spots (17 different protein species) differentially expressed at more than a 95% variance level between these cell lines. Several of these proteins were differentially expressed in only one cell line: HL-60 (myeloperoxidase, phosphoprotein 32 family member A, ras related protein Rab-11B, protein disulfide-isomerase, ran-specific GTPase-activating protein, nucleophosmin and S-100 calcium binding protein A4), and Raji (ezrin). Several of these proteins were differentially expressed in two cell lines: Raji and MEC1 (C-1-tetrahydrofolate synthase, elongation factor 2, α- and β-tubulin, transgelin-2 and stathmin). MEC1 and CCRF-CEM (γ-enolase), HL-60 and CCRF-CEM (ubiquitin-conjugating enzyme E2 N). The differentially expressed proteins identified in these four cell lines correlate with cellular properties and provide insights into the molecular basis of these malignancies.

Keywords: Abbreviations; ALL; acute lymphoblastic leukemia; AML; acute myeloid leukemia; CLL; chronic lymphocytic leukemia; PCA; principal component analysisDIGE; Leukemia; Lymphoma; Cytosolic proteins; Expression profile

Cofactor-specific modulation of 11β-hydroxysteroid dehydrogenase 1 inhibitor potency by Bhavana Sahni-Arya; Michael J. Flynn; Laurie Bergeron; Mary Ellen K. Salyan; Donna L. Pedicord; Rajasree Golla; Zhengping Ma; Haixia Wang; Ramakrishna Seethala; Shung C. Wu; James J. Li; Akbar Nayeem; Cynthia Gates; Lawrence G. Hamann; David A. Gordon; Yuval Blat (pp. 1184-1191).

11β-hydroxysteroid dehydrogenase 1 regulates the tissue availability of cortisol by interconverting cortisone and cortisol. It is capable of functioning as both a reductase and a dehydrogenase depending upon the surrounding milieu. In this work, we have studied the reaction mechanism of a soluble form of human 11β-hydroxysteroid dehydrogenase 1 and its mode of inhibition by potent and selective inhibitors belonging to three different structural classes. We found that catalysis follows an ordered addition with NADP(H) binding preceding the binding of the steroid. While all three inhibitors tested bound to the steroid binding pocket, they differed in their interactions with the cofactor NADP(H). Compound A, a pyridyl amide bound more efficiently to the NADPH-bound form of 11β-hydroxysteroid dehydrogenase 1. Compound B, an adamantyl triazole, was unaffected by NADP(H) binding and the sulfonamide, Compound C, showed preferential binding to the NADP⁺-bound form of 11β-hydroxysteroid dehydrogenase 1. These differences were found to augment significant selectivity towards inhibition of the reductase reaction versus the dehydrogenase reaction. This selectivity may translate to differences in the in vivo effects of 11β-hydroxysteroid dehydrogenase 1 inhibitors.

Keywords: Abbreviations; 11β-HSD1; 11β-hydroxysteroid dehydrogenase 1; 11β-HSD2; 11β-hydroxysteroid dehydrogenase 2; CE; collision energy; ER; endoplasmic reticulum; GR; glucocorticoid receptor; H6PD; hexose-6-phosphate dehydrogenase; HEPES; N; -(2-hydroxyethyl)piperaszine-; N′; -(2-ethanosulfonic acid); IPTG; isopropyl β-; d; -thiogalactopyranoside; LC/MS; liquid chromatography/mass spectroscopy; MES; 2-morpholinoethansulfonic acid; NADPH; reduced nicotinamide adenine dinucleotide phosphate; NADP; ⁺; oxidized nicotinamide adenine dinucleotide phosphate; SDR; short chain dehydrogenase11β-HSD1; Inhibitor; Glucocorticoid

Variation in relative substrate specificity of bifunctional β-d-xylosidase/α-l-arabinofuranosidase by single-site mutations: Roles of substrate distortion and recognition by Douglas B. Jordan; Xin-Liang Li (pp. 1192-1198).

To probe differential control of substrate specificities for 4-nitrophenyl-α-l-arabinofuranoside (4NPA) and 4-nitrophenyl-β-d-xylopyranoside (4NPX), residues of the glycone binding pocket of GH43 β-d-xylosidase/α-l-arabinofuranosidase from Selenomonas ruminantium were individually mutated to alanine. Although their individual substrate specificities ( k_cat/ K_m)^4NPX and ( k_cat/ K_m)^4NPA are lowered 330 to 280,000 fold, D14A, D127A, W73A, E186A, and H248A mutations maintain similar relative substrate specificities as wild-type enzyme. Relative substrate specificities ( k_cat/ K_m)^4NPX/( k_cat/ K_m)^4NPA are lowered by R290A, F31A, and F508A mutations to 0.134, 0.407, and 4.51, respectively, from the wild type value of 12.3 with losses in ( k_cat/ K_m)^4NPX and ( k_cat/ K_m)^4NPA of 18 to 163000 fold. R290 and F31 reside above and below the C4 OH group of 4NPX and the C5 OH group of 4NPA, where they can serve as anchors for the two glycone moieties when their ring systems are distorted to transition-state geometries by raising the position of C1. Thus, whereas R290 and F31 provide catalytic power for hydrolysis of both substrates, the native residues are more important for 4NPX than 4NPA as the xylopyranose ring must undergo greater distortion than the arabinofuranose ring. F508 borders C4 and C5 of the two glycone moieties and can serve as a hydrophobic platform having more favorable interactions with xylose than arabinofuranose.

Keywords: Glycoside hydrolase; GH43; Stereoelectronic effect; Near attack conformation; Transition state

Purification and characterization of a novel glucosyltransferase specific to 27β-hydroxy steroidal lactones from Withania somnifera and its role in stress responses by Bhaskara Reddy Madina; Lokendra Kumar Sharma; Pankaj Chaturvedi; Rajender Singh Sangwan; Rakesh Tuli (pp. 1199-1207).

Sterol glycosyltransferases catalyze the synthesis of diverse glycosterols in plants. Withania somnifera is a medically important plant, known for a variety of pharmacologically important withanolides and their glycosides. In this study, a novel 27β-hydroxy glucosyltransferase was purified to near homogeneity from cytosolic fraction of W. somnifera leaves and studied for its biochemical and kinetic properties. The purified enzyme showed activity with UDP-glucose but not with UDP-galactose as sugar donor. It exhibited broad sterol specificity by glucosylating a variety of sterols/withanolides with β-OH group at C-17, C-21 and C-27 positions. It transferred glucose to the alkanol at C-25 position of the lactone ring, provided an α-OH was present at C-17 in the sterol skeleton. A comparable enzyme has not been reported earlier from plants. The enzyme is distinct from the previously purified W. somnifera 3β-hydroxy specific sterol glucosyltransferase and does not glucosylate the sterols at C-3 position; though it also follows an ordered sequential bisubstrate reaction mechanism, in which UDP-glucose and sterol are the first and second binding substrates. The enzyme activity with withanolides suggests its role in secondary metabolism in W. somnifera. Results on peptide mass fingerprinting showed its resemblance with glycuronosyltransferase like protein. The enzyme activity in the leaves of W. somnifera was enhanced following the application of salicylic acid. In contrast, it decreased rapidly on exposure of the plants to heat shock, suggesting functional role of the enzyme in biotic and abiotic stresses.

Keywords: Glucosyltransferase; Substrate specificity; Withanoside; Stress response; Saponin; Kinetic mechanism; Salicylic acid signal

PinA from Aspergillus nidulans binds to pS/pT-P motifs using the same Loop I and XP groove as mammalian Pin1 by Yusuke Kato; Chai Ann Ng; Robert T.C. Brownlee; Masaru Tanokura (pp. 1208-1212).

Binding of the Cdc25c-T48 ligand to PinA from Aspergillus nidulans has been characterised by the identification of¹⁵N and¹H resonances from¹H–¹⁵N HSQC NMR titration experiments using previous backbone assignments. It is shown that the binding site for the Cdc25c-T48 ligand with PinA is the same as in the mammalian protein Pin1, although with a reduced binding affinity. It had previously been proposed that the arginine residue (R17) in the loop I region of the Pin1 WW domain is essential for binding to the pSer/pThr-Pro motifs of phosphorylated ligands such as Cdc25c. In PinA, a fungal homologue of Pin1, the arginine residue (R17) is replaced with an asparagine residue (N17). The effect of substitution of R17 by N17 in Pin1 has been investigated via a computational study, which predicted that changing R17 to N17 in Pin1 lowers the ligand binding affinity as a result of reduced hydrogen bonding between the protein and the phosphate group of the ligand.

Keywords: WW domain; XP groove; p patch; PinA; NMR

The RGS protein inhibitor CCG-4986 is a covalent modifier of the RGS4 Gα-interaction face by Adam J. Kimple; Francis S. Willard; Patrick M. Giguère; Christopher A. Johnston; Viorel Mocanu; David P. Siderovski (pp. 1213-1220).

Regulator of G-protein signaling (RGS) proteins accelerate GTP hydrolysis by Gα subunits and are thus crucial to the timing of G protein-coupled receptor (GPCR) signaling. Small molecule inhibition of RGS proteins is an attractive therapeutic approach to diseases involving dysregulated GPCR signaling. Methyl- N-[(4-chlorophenyl)sulfonyl]-4-nitrobenzenesulfinimidoate (CCG-4986) was reported as a selective RGS4 inhibitor, but with an unknown mechanism of action [D.L. Roman, J.N. Talbot, R.A. Roof, R.K. Sunahara, J.R. Traynor, R.R. Neubig, Identification of small-molecule inhibitors of RGS4 using a high-throughput flow cytometry protein interaction assay, Mol. Pharmacol. 71 (2007) 169–75]. Here, we describe its mechanism of action as covalent modification of RGS4. Mutant RGS4 proteins devoid of surface-exposed cysteine residues were characterized using surface plasmon resonance and FRET assays of Gα binding, as well as single-turnover GTP hydrolysis assays of RGS4 GAP activity, demonstrating that cysteine-132 within RGS4 is required for sensitivity to CCG-4986 inhibition. Sensitivity to CCG-4986 can be engendered within RGS8 by replacing the wildtype residue found in this position to cysteine. Mass spectrometry analysis identified a 153-Dalton fragment of CCG-4986 as being covalently attached to the surface-exposed cysteines of the RGS4 RGS domain. We conclude that the mechanism of action of the RGS protein inhibitor CCG-4986 is via covalent modification of Cys-132 of RGS4, likely causing steric hindrance with the all-helical domain of the Gα substrate.

Keywords: Abbreviations; CCG-4986; methyl-; N; -[(4-chlorophenyl)sulfonyl]-4-nitrobenzenesulfinimidoate; DTT; dithiothreitol (Cleland's reagent); ESI-MS; electrospray ionization mass spectrometry; GAP; GTPase-accelerating protein; FRET; Förster resonance energy transfer; GDP; guanosine diphosphate; GPCR; G protein-coupled receptor; G protein; guanine nucleotide-binding protein; GTP; guanosine triphosphate; GTPase; GTP hydrolysis activity; PCR; polymerase chain reaction; RGS; regulator of G-protein signaling; SPR; surface plasmon resonanceCCG-4986; RGS4; RGS protein inhibitor; Regulator of G-protein signaling; Thiol adduct

Purification and antimicrobial activity studies of the N-terminal fragment of ubiquitin from human amniotic fluid by Jin-Young Kim; Sun Young Lee; Seong-Cheol Park; Song Yub Shin; Sang Joon Choi; Yoonkyung Park; Kyung-Soo Hahm (pp. 1221-1226).

A 4.3-kDa antimicrobial peptide was isolated from human amniotic fluid by dialysis, ultrafiltration, and C18 reversed-phase high performance liquid chromatography. This peptide, which we named Amniotic Fluid Peptide-1 (AFP-1), possessed antimicrobial activity but lacked hemolytic activity. In addition, AFP-1 potently inhibited the growth of a variety of bacteria ( Escherichia coli, Salmonella typhimurium, Listeria monocytogenes and Staphylococcus aureus), filamentous fungi ( Botrytis cinerea, Aspergillus fumigatus, Neurospora crassa and Fusarium oxysporum) and yeast cells ( Candida albicans and Cryptococcus neoformans). Automated Edman degradation showed that the N-terminal sequence of AFP-1 was NH₂-Met-Gln-Ile-Phe-Val-Lys-Thr-Leu-Thr-Gly-Lys-Thr-Ile-Thr-Leu-Glu-Val-Glu-. The partial sequence had 100% homology to the N-terminal sequence of ubiquitin. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry revealed that the molecular mass of AFP-1 was 4280.2 Da. Our data show an antimicrobial activity of ubiquitin N-terminal derived peptide that makes it suitable for use as an antimicrobial agent.

Keywords: Abbreviations; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; RP-HPLC; reverse phase-high performance liquid chromatography; BCA; bicinchoninic acid; CFU; colony forming unitAmniotic Fluid Peptide-1 (AFP-1); Antimicrobial activity; Ubiquitin; Antimicrobial agent

Functional motifs outside the kinase domain of yeast Srb10p. Their role in transcriptional regulation and protein-interactions with Tup1p and Srb11p by Laura Núñez; Ma Isabel González-Siso; Manuel Becerra; Ma Esperanza Cerdán (pp. 1227-1235).

Several derivatives of the native Srb10 proteins from Saccharomyces cerevisiae and Kluyveromyces lactis, with removed selected motifs, have been constructed in order to test their role in Srb10p function. It has been demonstrated that the ATP binding site is necessary for repression of FLO11, CYC7 and SPI1. Yeast Srb10p specific motifs CM-I and CM-II, outside the kinase domain, are also necessary to complement two mutant phenotypes in S. cerevisiae Δsrb10 strains, the failure to growth in galactose at 37 °C and flocculation. They are also required to keep transcriptional repression of FLO11 in non-flocculants, and for aerobic repression of CYC7 and SPI1. Two-hybrid analyses revealed that, in Srb10p derivatives, the absence of these motifs decreases the interaction of Srb10p with its cyclin partner Srb11p and with the component Tup1p of the general co-repressor complex Tup1p–Ssn6p.

Keywords: Abbreviations; BS, binding site; CTD, carboxy-terminal domain; CDK, cyclin-dependent kinase; GAD, Gal4p Activation domain; GBD, Gal4p DNA-binding domainSrb10p motif; Yeast; Transcription of hypoxic gene; SRB11; TUP1

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BBA - Proteins and Proteomics (v.1774, #9)