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BBA - Proteins and Proteomics (v.1794, #10)

Editorial Board (pp. i).

Conformational plasticity of DM43, a metalloproteinase inhibitor from Didelphis marsupialis: Chemical and pressure-induced equilibrium (un)folding studies by Alex Chapeaurouge ⁎; Samantha M. Martins; Oliver Holub; Surza L.G. Rocha; Richard H. Valente; Ana G.C. Neves-Ferreira; Sérgio T. Ferreira; Gilberto B. Domont; Jonas Perales (pp. 1379-1386).

We have investigated the folding of DM43, a homodimeric metalloproteinase inhibitor isolated from the serum of the South American opossum Didelphis marsupialis. Denaturation of the protein induced by GdnHCl (guanidine hydrochloride) was monitored by extrinsic and intrinsic fluorescence spectroscopy. While the equilibrium (un)folding of DM43 followed by tryptophan fluorescence was well described by a cooperative two-state transition, bis-ANS (4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid) fluorescence measurements revealed an intensity maximum at the midpoint of the unfolding transition (2 M GdnHCl), indicating a partially folded intermediate state. We further investigated the DM43 intermediate stabilized at 2 M GdnHCl using size exclusion chromatography. This analysis revealed that the folding intermediate can be best described as partially folded DM43 monomers. Thermodynamic analysis of the GdnHCl-induced denaturation of DM43 revealed Gibbs free-energy changes of 13.57 kcal/mol for dimer dissociation and 1.86 kcal/mol for monomer unfolding, pointing to a critical role of dimerization as a determinant of the structure and stability of this protein. In addition, by using hydrostatic pressure (up to 3.5 kbar) we were able to stabilize partially folded states different from those stabilized in the presence of GdnHCl. Taken together, these results indicate that the conformational plasticity of DM43 could provide this protein with the ability to adapt its conformation to a variety of different environments and biological partners during its biological lifetime.

Keywords: Snake venom inhibitor; Protein folding; Intermediate; Conformational plasticity

Comparative proteomic analysis of cell cycle-dependent apoptosis induced by transforming growth factor-β by Hong-Yu Tian; Ke-Hua Zhang; Xia Gao; Wei-Wei Lei; Liang Zhang; Mei-Lan Yu; Jian-Guo Song ⁎; Fu-Kun Zhao ⁎ (pp. 1387-1397).

Transforming growth factor-β (TGF-β) can induce G2/M phase-dependent apoptosis and G1/S phase-dependent epithelial–mesenchymal transition (EMT) in hepatocytes, but the underlying mechanism remains poorly understood. In this study, we investigated alterations in the global proteome using two dimensional gel electrophoresis of AML-12 murine hepatocyte cells after treatment with TGF-β at several time points after synchronization in the G2/M or G1/S phase. Upon TGF-β treatment, the expression levels of 44 proteins were found to be significantly changed in cells synchronized in the G2/M phase. These proteins were identified by MALDI-TOF/TOF and classified into seven categories according to function. In addition, TGF-β induced downregulation of glutamine synthetase in cells in G2/M but not G1/S phase, and this was further confirmed by immunoblotting. Moreover, exogenous glutamine completely blocked TGF-β-induced apoptosis in G2/M and non-synchronized cells, whereas it had no effect on EMT, suggesting that the downregulation of glutamine synthetase is involved in G2/M phase-dependent apoptosis. These results provide new insight into the mechanism of the multifunctional effects of TGF-β and how apoptosis and EMT are regulated in the same type of cells.

Keywords: Proteomics; Apoptosis; EMT; Glutamine synthetase

Temperature dependence on structure and dynamics of Bovine Pancreatic Trypsin Inhibitor (BPTI) by M.-S. Appavou; G. Gibrat; M.-C. Bellissent-Funel (pp. 1398-1406).

We have studied the influence of temperature on the structure of BPTI in solution by small angle neutron scattering. We have investigated the variation of the radius of gyration and the modification of the shape of BPTI between ambient temperature and 368 K. Results have shown an increase of the radius of gyration from 10.9 Å at ambient temperature up to 13.3 Å at 368 K. Global and internal dynamics of BPTI in solution were studied by quasielastic neutron scattering. The analysis of neutron data in terms of intermediate scattering function reveals two relaxation times τ₁ and τ₂ related respectively to global translational diffusive motions and to internal motions of protein. Motions of protons belonging to lateral chains of residues located at the surface of the protein have been detected. The results are compared to the recently published results concerning the influence of pressure on structure and dynamics of BPTI in solution [Appavou MS et al. Biochimica et Biophysica Acta, 1764, 2006, pp 414–423].

Keywords: BPTI; Small angle neutron scattering; Quasielastic neutron scattering; Temperature

Sequence and structural analysis of artemin based on ferritin: A comparative study by Behnam Rasti; S. Shirin Shahangian; Reza H. Sajedi ⁎; Majid Taghdir; Sadegh Hasannia; Bijan Ranjbar (pp. 1407-1413).

Artemia cysts can tolerate extreme environments, partly due to a heat-stable protein called artemin. According to previous studies, artemin shares structural similarity with ferritins. Actually, there is still no strong structural information about artemin three-dimensional (3-D) structure. In this research, the artemin encoding gene from Artemia urmiana was cloned and sequenced. A reliable 3-D model of artemin was initially built using ferritin as template and refined using Molecular Dynamic (MD) Simulation. It is interesting that the proposed model, confirmed by circular dichroism (CD), shows significant differences in secondary structure contents with ferritin. Three conserved regions (ferroxidase center, iron nucleation center and 3-fold channel) in ferritins, cooperating in iron-interaction, have been substantially changed in artemin. Analysis of C-terminal region of the model revealed its major role in preventing artemin from iron-binding due to some suitable interactions. Finally, it is concluded that significant differences between artemin and ferritin, both in conserved regions related to iron-interaction and three-dimensional structure, can justify their functional differences.

Keywords: Artemia; Artemin; Ferritin; Circular dichroism; Homology modeling

Methionine γ-lyase: Mechanistic deductions from the kinetic pH-effects by Nicolai G. Faleev; Kirill V. Alferov; Marina A. Tsvetikova; Elena A. Morozova; Svetlana V. Revtovich; Elena N. Khurs; Mikhail M. Vorob'ev; Robert S. Phillips; Tatyana V. Demidkina; Radii M. Khomutov (pp. 1414-1420).

We have studied and compared the pH-dependencies of the main kinetic parameters for the α,γ-elimination reactions of methionine γ-lyase (MGL) of Citrobacter intermedius with natural substrate,l-methionine, with its phosphinic analogue, and for α,β-elimination reaction with S-methyl-l-cysteine. From the pH-dependency of k_cat/ K_m for the reaction withl-methionine we have concluded that MGL is selective with respect to the zwitterionic form of its natural substrate. For the reaction of MGL with 1-amino-3-methylthiopropylphosphinic acid the p K_a of the substrate's amino group, equal to 7.55, is not reflected in the pH-profile of k_cat/ K_m. Consequently, the enzyme does not manifest well-defined selectivity with respect to the zwitterion and anion ionic forms of the substrate. The ascending limbs of pH-dependencies of k_cat/ K_m for reactions withl-methionine and S-methyl-l-cysteine are controlled by a single p K_a equal to 7.1–7.2, while for the reaction with 1-amino-3-methylthiopropylphosphinic acid two equal p K_as of 6.2 were found in the respective pH-profile. The descending limbs of pH-dependencies of k_cat/ K_m for the reactions with S-methyl-l-cysteine and racemic 1-amino-3-methylthiopropylphosphinic acid are very similar and are controlled by two acidic groups having average p K_a values of 8.7. On the basis of these results we suggest a mechanism of catalytic action of MGL. According to this mechanism Tyr 113, in its conjugated base form, acts as an acceptor of the proton from the amino group of the substrate upon its binding in the active site. Elimination of the leaving thiol groups during both α,γ- and α,β-elimination reactions is assisted by the acidic groups of Tyr 113 and Tyr 58. Both tyrosyl residues are able to fulfill this catalytic function with different efficiencies depending on the type of elimination reaction. Tyr 113 residue plays the determining role in the α,γ-elimination, and Tyr 58 — in the α,β-elimination process.

Keywords: Methionine γ-lyase; Kinetics; Mechanism; pH-dependency; Phosphinic amino acid; 1-Amino-3-methylthiopropylphosphinic acid

Major proteins and antigens of Treponema denticola by Paul D. Veith; Stuart G. Dashper; Neil M. O'Brien-Simpson; Rita A. Paolini; Rebecca Orth; Katrina A. Walsh; Eric C. Reynolds (pp. 1421-1432).

Treponema denticola is a Gram-negative, motile, asaccharolytic, anaerobic spirochaete which along with Porphyromonas gingivalis and Tannerella forsythia has been shown to form a bacterial consortium called the Red Complex that is strongly associated with the clinical progression of chronic periodontitis. T. denticola was grown in continuous culture in a complex medium with a mean generation time of 15.75 h. Samples from two different membrane-enriched preparations and a cytoplasm-enriched preparation were separated by two-dimensional gel electrophoresis and the proteins identified by MALDI-TOF/TOF mass spectrometry. In total, 219 non-redundant proteins were identified including numerous virulence factors, lipoproteins, ABC transporter proteins and enzymes involved in the metabolism of nine different amino acids of which glycine seems to be of particular importance. Novel findings include the identification of several abundant peptide uptake systems, and the identification of three flagellar filament outer layer proteins. Two-dimensional Western blot analysis using sera from mice immunized with formalin-killed T. denticola cells suggested that Msp, PrcA, OppA, OppA10, MglB, TmpC and several flagellar filament proteins are antigenic.

Keywords: Treponema denticola; Major protein; Antigen; Mass spectrometry

Comparative proteomic analysis of colon cancer cells in response to Oxaliplatin treatment by Yi Yao; Xiao-Yuan Jia; Hong-Yu Tian; Yu-Xiang Jiang; Gen-Jun Xu; Qi-Jun Qian ⁎; Fu-Kun Zhao ⁎ (pp. 1433-1440).

Colon cancer is one of the most common malignancies in the world. Oxaliplatin, a third-generation platinum compound, is widely used in clinical chemotherapy of colon cancer. Although the mechanisms of the antitumor effect of Oxaliplatin have been investigated in recent years, the proteomic changes that are associated with the cellular response to this compound are poorly understood. In this study, we performed a comparative proteomic analysis to survey the global changes in protein expression levels after Oxaliplatin treatment in three colon cancer cell lines: HT29, SW620, and LoVo. Two-dimensional gel electrophoresis coupled with MALDI-TOF/TOF mass spectrometry revealed 57, 48, and 53 differentially expressed proteins in the three cell lines (HT29, SW620 and LoVo, respectively) after Oxaliplatin treatment. Of these proteins, 21 overlapped among all three cell lines. These overlapping proteins participate in many cellular processes, such as apoptosis, signal transduction, transcription and translation, cell structural organization, and metabolism. Additionally, the expression levels of ezrin (EZRI), heat-shock protein beta-1 (HSPB1), translationally controlled tumor protein (TCTP), and cell division control protein 2 homolog (CDC2) were confirmed by immunoblotting. This is the first direct proteomic analysis of Oxaliplatin-treated colon cancer cells. Several interesting proteins that we found warrant further investigation owing to their potential significant functions in the antitumor effect of Oxaliplatin.

Keywords: 2-DE; Apoptosis; Cell cycle; Colon cancer; Mass spectrometry; Oxaliplatin

HCV-NS3 inhibitors: Determination of their kinetic parameters and mechanism by María Victoria Flores; Joanne Strawbridge; Giuseppe Ciaramella; Romuald Corbau (pp. 1441-1448).

Existing HCV protease inhibitors fall into two categories: reversible and non-covalent, such as BILN-2061, and covalent and reversible, exemplified by SCH-503034 and VX-950. In this work, the characterization of the kinetics of these three inhibitors is presented. SCH-503034 and VX-950 initially bind to the genotype 1b HCV NS3/4A protease to form a low affinity complex, with K_i values of 5 and 5.8 μM respectively. The ability of those two compounds to form a second covalent complex (EI⁎) results in a potency increase, with overall K_i⁎ values of 20 and 45 nM, respectively. The increase in potency can be explained by their slow dissociation rate, forming complexes with half-lives of 2 h (VX-950) and 5 h (SCH-503034). Although BILN-2061 has been described as a fast reversible, non-covalent inhibitor, our results show a slow binding two-step mechanism. Contrary to SCH-503034 and VX-950, BILN-2061 can form a high affinity first complex with a K_i value of 3.9 nM, and an overall K_i⁎ of 0.14 nM. The half-life of the BILN-2061 EI⁎ complex is shorter ( t_1/2 ∼0.7 h) than that of the other two compounds. The potency of these compounds is genotype dependent, and a kinetic analysis using NS3/4A from genotype 3a indicates that the loss of potency of SCH-503034 and VX-950 relative to genotype 1 is mainly due to the slow on-rate and faster off-rate for the formation of the EI⁎ complex. In the case of BILN-2061, a better fit is obtained using a one-step model, indicating that the loss of potency is due to an increase in the off-rate of the EI complex.

Keywords: Abbreviations; E; free enzyme concentration; E; _t; total enzyme concentration; ES; enzyme–substrate complex; EI; enzyme–inhibitor complex; EI⁎; high affinity enzyme–inhibitor complex; S; substrate concentration; P; reaction product; K; _M; Michaelis Menten constant; K; _i; initial inhibition constant; K; _i; _app; apparent inhibition constant; K; _i; ⁎; overall binding constant; IC; ₅₀; maximal half inhibitory concentration; t; _1/2; dissociation half-life of the enzyme–inhibitor complex; V; steady-state reaction rate in the presence of inhibitor; Vo; steady-state rate in absence of inhibitor; RFU; relative fluorescence units; RP; product response factorHCV-NS3 protease; Enzyme kinetics; Enzyme inhibition; Slow binding; Progress curve

Influence of non-enzymatic post-translation modifications on the ability of human serum albumin to bind iron by Andre M.N. Silva; Robert C. Hider (pp. 1449-1458).

Human serum albumin (HSA) is known as a low affinity iron binding protein and it has been proposed as a ligand for the non-transferrin-bound iron (NTBI) pool existing in the sera of iron-overload patients, but definitive evidence is still lacking. In this study, gel filtration linked to inductively coupled plasma mass spectrometry (GF-ICP-MS) was employed to assess iron speciation in solutions containing physiological concentrations of citrate and HSA at physiological values of pH and ionic strength. The influence of common non-enzymatic post-translation modifications on the ability of HSA to bind iron was also studied. HSA was found to bind between 60 and 20% of the available iron when iron concentration was varied over the common clinical range of NTBI concentrations (1 to 10 µM), thus proving to be a relevant ligand for NTBI speciation. Analysis of modified albumins showed that both glycation and oxidation increase the albumin iron binding capacity. The results presented indicate that non-enzymatic modifications of albumin, that are prevalent in diabetes and increased oxidative stress, may have a fundamental role in NTBI speciation. Experiments with the nitrilotriacetic acid (NTA) based assay for NTBI determination demonstrate the inability of NTA to quantitatively mobilize albumin-bound iron, especially after the albumin had undergone oxidation or glycation. This result would indicate that the low NTBI levels so far measured in diabetic sera may be understated and that different analytical techniques are required to determine NTBI levels in diabetic patients.

Keywords: Abbreviations; HSA; human serum albumin; BSA; bovine serum albumin; AGE-HSA; advanced glycation end-products of HSA; CML; N; ^ɛ; -carboxymethyllysine; HSA-Ox; oxidized albumin; HSA-Cys; albumin with a cystine residue at position 34; GF-ICP-MS; Gel filtration linked to inductively coupled plasma mass spectrometry; NTBI; Non-transferrin-bound iron; NTA; nitrilotriacetic acid; ROOH; protein bound hydroperoxides; FOX1; ferrous oxidation with xylenol orange assayNTBI; Human serum albumin; Post-translation modification; Glycation; Oxidation; Albumin-bound iron

Role of S114 in the NADH-induced conformational change and catalysis of 3α-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni by Yi-Hsun Chang; Tzu-Jung Huang; Lea-Yea Chuang; Chi-Ching Hwang ⁎ (pp. 1459-1466).

3α-Hydroxysteroid dehydrogenase/carbonyl reductase reversibly catalyzes the oxidation of androsterone with NAD⁺ to form androstanedione and NADH. In this study, we characterize the role of the conserved residue S114 in cofactor binding and catalysis, using site-directed mutagenesis, steady-state kinetics, fluorescence quenching and anisotropy measurements. The catalytic efficiency of V/K_NADH Et for wild-type and S114A is 1.5×10⁷ and 3.8×10³ M−¹ s−¹, respectively, suggesting that NADH association to wild-type and S114A mutant enzymes involves two steps, a bimolecular binding step and isomerization. The binding of NADH into a hydrophobic pocket in the active site of wild-type and S114A mutant enzymes restricts its motion and shields the fluorescence quenching from solvent, with an increase in the fluorescence intensity and a blue shift at the maximum wavelength. Furthermore, the binding of NADH leads to the protein fluorescence quenching, mainly due to fluorescence resonance energy transfer to NADH. S114A mutant enzyme decreases 3100-fold in V/Et with no apparent change in K_m for substrates. Addition of NADH to S114A mutant enzyme induces a secondary structural change. These results suggest that S114 is important to maintain the correct conformation for the nucleotide binding and facilitate the reaction. Substitution of alanine for S114 eliminates the hydrogen bonding interaction with P185, causing a conformational change in a nonproductive binding of NADH and a significant loss of activity.

Keywords: Abbreviations; 3α-HSD/CR; 3α-hydroxysteroid dehydrogenase/carbonyl reductase; SDR; short chain dehydrogenase/reductase; CD; circular dichroism; FRET; fluorescence resonance energy transfer; Hepes; N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]3α-hydroxysteroid dehydrogenase/carbonyl reductase; Cofactor binding; Fluorescence titration; Mutagenesis; Conformational change

Functional role of a non-active site residue Trp²³ on the enzyme activity of Escherichia coli thioesterase I/protease I/lysophospholipase L₁ by Li-Chiun Lee; Yi-Li Chou; Hong-Hwa Chen; Ya-Lin Lee; Jei-Fu Shaw (pp. 1467-1473).

Escherichia coli possesses a versatile protein with the enzyme activities of thioesterase I, protease I, and lysophospholipase L₁. The protein is dubbed as TAP according to the chronological order of gene discovery ( TesA/ ApeA/ PldC). Our previous studies showed that TAP comprises the catalytic triad Ser¹⁰, Asp¹⁵⁴, and His¹⁵⁷ as a charge relay system, as well as Gly⁴⁴ and Asn⁷³ residues devoted to oxyanion hole stabilization. Geometrically, about 10 Å away from the enzyme catalytic cleft, Trp²³ showed a stronger resonance shift than the backbone amide resonance observed in the nuclear magnetic resonance (NMR) analyses. In the present work, we conducted site-directed mutagenesis to change Trp into alanine (Ala), phenylalanine (Phe), or tyrosine (Tyr) to unveil the role of the Trp²³ indole ring. Biochemical analyses of the mutant enzymes in combination with TAP's three-dimensional structures suggest that by interlinking the residues participating in this catalytic machinery, Trp²³ could effectively influence substrate binding and the following turnover number. Moreover, it may serve as a contributor to both H-bond and aromatic–aromatic interaction in maintaining the cross-link within the interweaving framework of protein.

Keywords: Enzyme kinetics; Hydrogen bond; Aromatic–aromatic interaction; Protein structure; Tryptophan

Leishmania donovani trypanothione reductase: Role of urea and guanidine hydrochloride in modulation of functional and structural properties by Smita Rai; Upendra Nath Dwivedi; Neena Goyal ⁎ (pp. 1474-1484).

Trypanothione reductase [TR], an NADPH-dependent disulfide oxidoreductase, unique to kinetoplastid parasites including Trypanosoma and Leishmania, is a validated target for the design of improved drugs. TR is a stable homodimer with a FAD molecule tightly bound to each subunit. In this paper, structure, function, stability properties and cofactor protein interactions of recombinant TR from Leishmania donovani were investigated under equilibrium unfolding/denaturing conditions. Urea induced unfolding was non-reductive in nature and led to the formation of partially folded intermediate. This intermediate species lacks catalytic activity and characteristic conformation of native LdTR but has significant secondary structure and could be partially reactivated. Guanidine hydrochloride-induced irreversible denaturation was marked by the presence of molten globule intermediate. Reactivation and cross-linking experiments clearly demonstrated that the loss of activity at lower denaturant concentrations was not coincided by dimer dissociation or structural unfolding. The studies demonstrate that functional conformation and stability are largely governed by ionic interactions and active site disulfide plays a vital role in maintaining functional conformation. The results obtained from this study provide intriguing insight into the possible mechanism/s of modulation of structure, function and stability of LdTR induced by the cationic, guanidine hydrochloride and the neutral denaturant, urea.

Keywords: Leishmania donovani; Trypanothione reductase; Unfolding; Intermediate; CD; Fluorescence; Molten globule

Proteomic characterization of the dynamic KSR-2 interactome, a signaling scaffold complex in MAPK pathway by Lin Liu; Padma L. Channavajhala; Vikram R. Rao; Ioannis Moutsatsos; Leeying Wu; Yuhua Zhang; Lih-ling Lin; Yongchang Qiu (pp. 1485-1495).

KSR-1 is a scaffold protein that is essential for Ras-induced activation of the highly conserved RAF-MEK-ERK kinase module. Previously, we identified a close homolog of KSR-1, called KSR-2, through structural homology-based data mining. In order to further understand the role of KSR-2 in MAPK signaling, we undertook a functional proteomics approach to elucidate the dynamic composition of the KSR-2 functional complex in HEK-293 cells under conditions with and without TNF-α stimulation. We found nearly 100 proteins that were potentially associated with KSR-2 complex and 43 proteins that were likely recruited to the super molecular complex after TNF-α treatment. Our results indicate that KSR-2 may act as a scaffold protein similar as KSR-1 to mediate the MAPK core (RAF-MEK-ERK) signaling but with a distinct RAF isoform specificity, namely KSR-2 may only mediate the A-RAF signaling while KSR-1 is responsible for transducing signals only from c-RAF. In addition, KSR-2 may be involved in the activation of many MAPK downstream signaling molecules such as p38 MAPK, IKAP, AIF, and proteins involved in ubiquitin–proteasome, apoptosis, cell cycle control, and DNA synthesis and repair pathways, as well as mediating crosstalks between MAPK and several other signaling pathways, including PI3K and insulin signaling. While interactions with these molecules are not known for KSR-1, it's reasonable to hypothesize that KSR-1 may also play a similar role in mediating these downstream signaling pathways.

Keywords: Abbreviations; KSR; kinase suppressor of Ras; MAPK; mitogen-activated protein kinase; ERK; extracellular signal-regulated kinase; MEK; MAPK/ERK kinase; MAP3K; MAPK kinase kinase; HEK; human embryonic kidney; TNF-α; tumor necrosis factor-α; HSP; heat shock protein; TPL-2; tumor progression locus 2; TRAF; TNF receptor-associated factor; PI3K; phosphatidylinositol-3 kinase; AMPK; AMP-activated protein kinase; DNA-PK; DNA-dependent protein kinase; IKAP; I kappa B kinase complex-associated proteinKinase suppressor of Ras (KSR); MAPK signaling; Scaffold protein signaling complex; Mass spectrometry; Proteomics

Refolding, characterization and crystal structure of ( S)-malate dehydrogenase from the hyperthermophilic archaeon Aeropyrum pernix by Ryushi Kawakami; Haruhiko Sakuraba; Shuichiro Goda; Hideaki Tsuge; Toshihisa Ohshima (pp. 1496-1504).

Tartrate oxidation activity was found in the crude extract of an aerobic hyperthermophilic archaeon Aeropyrum pernix, and the enzyme was identified as ( S)-malate dehydrogenase (MDH), which, when produced in Escherichia coli, was mainly obtained as an inactive inclusion body. The inclusion body was dissolved in 6 M guanidine–HCl and gradually refolded to the active enzyme through dilution of the denaturant. The purified recombinant enzyme consisted of four identical subunits with a molecular mass of about 110 kDa. NADP was preferred as a coenzyme over NAD for ( S)-malate oxidation and, unlike MDHs from other sources, this enzyme readily catalyzed the oxidation of (2 S,3 S)-tartrate and (2 S,3 R)-tartrate. The tartrate oxidation activity was also observed in MDHs from the hyperthermophilic archaea Methanocaldococcus jannaschii and Archaeoglobus fulgidus, suggesting these hyperthermophilic MDHs loosely bind their substrates. The refolded A. pernix MDH was also crystallized, and the structure was determined at a resolution of 2.9 Å. Its overall structure was similar to those of the M. jannaschii, Chloroflexus aurantiacus, Chlorobium vibrioforme and Cryptosporidium parvum [lactate dehydrogenase-like] MDHs with root-mean-square-deviation values between 1.4 and 2.1 Å. Consistent with earlier reports, Ala at position 53 was responsible for coenzyme specificity, and the next residue, Arg, was important for NADP binding. Structural comparison revealed that the hyperthermostability of the A. pernix MDH is likely attributable to its smaller cavity volume and larger numbers of ion pairs and ion-pair networks, but the molecular strategy for thermostability may be specific for each enzyme.

Keywords: Aeropyrum pernix; [LDH-like] MDH; Malate dehydrogenase; Hyperthermophilic archaea; Refolding; Crystal structure

Profiling of calpain activity with a series of FRET-based substrates by Jacqueline C. Kelly; Dominic Cuerrier; Laurie A. Graham; Robert L. Campbell; Peter L. Davies (pp. 1505-1509).

Calpains are intracellular proteases that selectively cleave proteins in response to calcium signals. Although calpains cut many different sequences, residue preferences within peptide substrates were recently determined and incorporated into a superior FRET (fluorescence resonance energy transfer)-based substrate (PLFAER). Here we show PLFAER is cleaved by calpain at the intended F-A scissile bond. Sequential replacement of individual residues by alanine reduced activity except with PLFAAR, which is cleaved 2.3 times faster than PLFAER. The rates of hydrolysis of the alanine-substituted substrates were used to compare substrate preferences of calpain, papain and cathepsins B and L. The preferences of the two major isoforms, calpains 1 and 2, were virtually indistinguishable and were very similar to those of the calpain 1 protease core and papain. However, the activity profiles with the FRET substrate series were significantly different for the cathepsins, particularly cathepsin B.

Keywords: Abbreviations; DABCYL; 4-((4-(dimethylamino)phenyl)azo)benzoic acid; EDANS; 5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid; FRET; fluorescence resonance energy transfer; MALDI-MS; matrix-assisted laser desorption/ionization-mass spectrometry; mC105S; calpain 2 with an inactivating Cys105Ser mutationCalpain; Protease; Calcium; Peptide substrate; FRET; Peptidase

Characterisation of dihydrodipicolinate synthase (DHDPS) from Bacillus anthracis by L.J. Domigan; S.W. Scally; M.J. Fogg; C.A. Hutton; M.A. Perugini; R.C.J. Dobson; A.C. Muscroft-Taylor; J.A. Gerrard; S.R.A. Devenish (pp. 1510-1516).

Bacillus anthracis is a Gram-positive spore-forming bacterium that is the causative agent of anthrax disease. The use of anthrax as a bioweapon has increased pressure for the development of an effective treatment. Dihydrodipicolinate synthase (DHDPS) catalyses the first committed step in the biosynthetic pathway yielding two essential bacterial metabolites, meso-diaminopimelate (DAP) and ( S)-lysine. DHDPS is therefore a potential antibiotic target, as microbes require either lysine or DAP as a component of the cell wall. This paper is the first biochemical description of DHDPS from B. anthracis. Enzyme kinetic analyses, isothermal titration calorimetry (ITC), mass spectrometry and differential scanning fluorimetry (DSF) were used to characterise B. anthracis DHDPS and compare it with the well characterised Escherichia coli enzyme. B. anthracis DHDPS exhibited different kinetic behaviour compared with E. coli DHDPS, in particular, substrate inhibition by ( S)-aspartate semi-aldehyde was observed for the B. anthracis enzyme ( K_si(ASA)=5.4±0.5 mM), but not for the E. coli enzyme. As predicted from a comparison of the X-ray crystal structures, the B. anthracis enzyme was not inhibited by lysine. The B. anthracis enzyme was thermally stabilised by the first substrate, pyruvate, to a greater extent than its E. coli counterpart, but has a weaker affinity for pyruvate based on enzyme kinetics and ITC studies. This characterisation will provide useful information for the design of inhibitors as new antibiotics targeting B. anthracis.

Keywords: Abbreviations; ASA; (; S; ); -; aspartate semi-aldehyde; DAP; meso-; diaminopimelate; DHDPR; dihydrodipicolinate reductase; DHDPS; dihydrodipicolinate synthase; ITC; isothermal titration calorimetry; DSF; differential scanning fluorimetry; dRFU/dt; change in fluorescence (relative fluorescence units) with respect to timeDihydrodipicolinate synthase; Lysine biosynthesis; Bacillus anthracis; Isothermal titration calorimetry; Enzyme kinetics

Amyloid fibrils formed by selective N-, C-terminal sequences of mouse apolipoprotein A-II by Jinko Sawashita; Fuyuki Kametani; Kazuhiro Hasegawa; Shinobu Tsutsumi-Yasuhara; Beiru Zhang; Jingmin Yan; Masayuki Mori; Hironobu Naiki; Keiichi Higuchi (pp. 1517-1529).

In mice, amyloidogenic type C apolipoprotein A-II (apoA-II) forms amyloid fibrils in age-associated amyloidosis. To understand the mechanism of amyloid fibril formation by apoA-II, we examined the polymerization of synthetic partial peptides of apoA-II in vitro. None of the partial apoA-II peptides polymerized into amyloid fibrils when tested as a single species mixture. We found a unique mechanism in which N- and C-terminal peptides associated into amyloid fibrils in a 1:1 ratio at pH 2.5. The 11-residue amino acid sequence (6–16), which is a common sequence of type B apoA-II and type C apoA-II proteins in amyloidosis-resistant mice and amyloidosis-susceptible mice, respectively, was critical for polymerization into amyloid fibrils. The 18-residue-long amino acid sequence (48–65) is also necessary for nucleation, but not for the extension phase. These findings suggest that there may be different mechanisms underlying the nucleation and extension phases of apoA-II amyloid fibril formation. We also found that amino acid substitutions between type B apoA-II (Pro5, Val38) and type C apoA-II (Gln5, Ala38) did not affect either phase. The strategy of using synthetic partial peptides of amyloidogenic proteins in vitro is a useful system for understanding amyloid fibril formation and for the development of novel therapies.

Keywords: Abbreviations; apoA-I; apolipoprotein A-I; apoA-II; apolipoprotein A-II; AApoAII; amyloid fibrils derived from apoA-II; CD; circular dichroism; DMSO; dimethylsulfoxide; HDL; high-density lipoprotein; HPLC; high-performance liquid chromatography; LC/MS/MS; liquid chromatography/mass spectrometry/mass spectrometry; LM; light microscopy; TEM; transmission electron microscopy; ThT; thioflavin TApolipoprotein A-II; Mouse senile amyloidosis; Fibril conformation

The origin of Listeria monocytogenes 4b isolates is signified by subproteomic profiling by Emilie Dumas; Bruno Meunier; Jean-Louis Berdagué; Christophe Chambon; Mickaël Desvaux; Michel Hébraud ⁎ (pp. 1530-1536).

Among the 13 known serovars of Listeria monocytogenes, strains exhibiting the serovar 4b are the most prevalently involved in epidemics of human listeriosis. The molecular reasons for the major involvement of serovar 4b strains in all major foodborne outbreaks in contrast to the lower prevalence of this serovar among food isolates remain indefinite. In order to provide further insight in the protein expression of L. monocytogenes 4b strains, the cytoplasmic and extracellular proteomes of L. monocytogenes 4b strains from different origins, i.e. environmental, clinical and asymptomatical carriage, were investigated by two-dimensional gel electrophoresis. Statistical hierarchical clustering analysis on subproteomic profiles clearly discriminated the strains according to their origin. Protein spots differentiating the subproteome patterns were identified using MALDI-TOF MS through their peptide mass fingerprint.

Keywords: Listeria monocytogenes; Serovar 4b; Cytoplasmic proteome; Extracellular proteome; MALDI-TOF mass spectrometry; Biodiversity

Ca²⁺, within the physiological concentrations, selectively accelerates Aβ42 fibril formation and not Aβ40 in vitro by Atta Ahmad; Mahvish Muzaffar; Vernon M. Ingram (pp. 1537-1548).

Alzheimer's disease (AD) in humans is a common progressive neurodegenerative disease, associated with cognitive dysfunction, memory loss and neuronal loss. Alzheimer peptides Aβ40 and Aβ42 are precursors of the amyloid fibers that accumulate in the brain of patients. These peptides misfold and the monomers aggregate to neurotoxic oligomers and fibrils. Thus, the aggregation kinetics of these peptides is central to understanding the etiology of AD. Using size exclusion chromatography as well as filtration methods, we report here that Ca²⁺ ions at physiological concentrations greatly accelerate the rate of aggregation of Aβ42 to form intermediate soluble associated species and fibrils. In the presence of 1 or 2 mM Ca²⁺, CD spectra indicated that the secondary structure of Aβ42 changed from an unfolded to a predominantly β-sheet conformation. These concentrations of Ca²⁺ greatly decreased the lag time for Aβ42 fibril formation, measured with thioflavin T. However, the elongation rate was apparently unaffected. Ca²⁺ appears to predominantly accelerate the nucleation stage of Aβ42 on pathway to the Alzheimer's fibril formation. Unlike Aβ42, Ca²⁺ was not observed to trigger similar effect at any stage during the study of fibrillation kinetics of Aβ40 by any techniques. Aβ40 and Aβ42 seem to have distinct aggregation pathways.

Keywords: Abbreviations; ThT; Thioflavin T; Aβ40; Amyloid beta peptide 1–40; Aβ42; Amyloid beta peptide 1–42; CD; Circular dichroism; EM; Electron micrograph; AD; Alzheimer's diseaseAβ40; Aβ42; Calcium; Amyloid; Alzheimer's

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BBA - Proteins and Proteomics (v.1794, #10)

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BBA - Proteins and Proteomics (v.1794, #10)