Check out our New Publishers' Select for Free Articles

BBA - Proteins and Proteomics (v.1774, #6)

Editorial Board (pp. ii).

Remarkable enhancement in PLD activity from Streptoverticillium cinnamoneum by substituting serine residue into the GG/GS motif by Chiaki Ogino; Hidenori Daido; Yuka Ohmura; Namiko Takada; Yoshiki Itou; Akihiko Kondo; Hideki Fukuda; Nobuaki Shimizu (pp. 671-678).

The gene that encodes phospholipase D (PLD) from Streptoverticillium cinnamoneum contains three consensus regions (Region I, II and IV as shown in Fig. 1A) that are conserved among the PLD superfamily. The glycine–glycine (GG) motif in Region I and the glycine–serine (GS) motif in Region IV are also conserved in the PLD superfamily. These (GG and GS) motifs are located 7 residues downstream from each HKD motif. In an investigation of fifteen GG/GS motif mutants, generated as fusion proteins with maltose-binding protein (MBP–PLDs), three highly active mutants were identified. Three of the mutants (G215S, G216S, and G216S–S489G) contained a serine residue in the GG motif, and exhibited approximately a 9–27-fold increased transphosphatidylation activity to DPPC compared with recombinant wild type MBP–PLD. When heat stability was compared between three mutants and the recombinant wild type, only G216S–S489G showed heat labile properties. It appears that the 489th serine residue in the GS motif also contributes to the thermal stability of the enzyme. In addition, the GG/GS motif was very close to the active center residue, including two HKD motifs, as shown by computer modeling. The findings suggest that the GG/GS motif of PLD is a key motif that affects catalytic function and enzymatic stability.

Keywords: Phospholipase D; Transphosphatidylation; Phospholipid conversion; Site-directed mutation; HKD motif

Engineered disulfide bonds increase active-site local stability and reduce catalytic activity of a cold-adapted alkaline phosphatase by Bjarni Ásgeirsson; Björn Vidar Adalbjörnsson; Gudjón Andri Gylfason (pp. 679-687).

Alkaline phosphatase is an extracellular enzyme that is membrane-bound in eukaryotes but resides in the periplasmic space of bacteria. It normally carries four cysteine residues that form two disulfide bonds, for instance in the APs of Escherichia coli and vertebrates. An AP variant from a Vibrio sp. has only one cysteine residue. This cysteine is second next to the nucleophilic serine in the active site. We have individually modified seven residues to cysteine that are on two loops predicted to be within a 5 Å radius. Four of them formed a disulfide bond to the endogenous cysteine. Thermal stability was monitored by circular dichroism and activity measurements. Global stability was similar to the wild-type enzyme. However, a significant increase in heat-stability was observed for the disulfide-containing variants using activity as a measure, together with a large reduction in catalytic rates ( k_cat) and a general decrease in K_m values. The results suggest that a high degree of mobility near the active site and in the helix carrying the endogenous cysteine is essential for full catalytic efficiency in the cold-adapted AP.

Keywords: Cold-adaptation; Site-directed mutagenesis; Catalytic efficiency; Cysteine; Disulfide bond

An integrated analysis of the effects of Esculentin 1–21 on Saccharomyces cerevisiae by Tania Gamberi; Duccio Cavalieri; Francesca Magherini; Maria L. Mangoni; Carlotta De Filippo; Marina Borro; Giovanna Gentile; Maurizio Simmaco; Alessandra Modesti (pp. 688-700).

The antimicrobial peptide esculentin 1–21 (Esc 1–21) is a shorter synthetic version of the 46-residue peptide occurring in the Rana esculenta skin secretion. Here we propose an integrated proteomic and transcriptomic approach to interpret the biological effects of this peptide on Saccharomyces cerevisiae. We further investigated the response to this peptide by correlating the results of the transcriptome and proteome analysis with phenotypic effects. The results show that S. cerevisiae adapts to Esc 1–21 using the High Osmolarity Glycerol (HOG) pathway involved in osmotic tolerance and cell wall maintenance. Comparative proteomics reveals that Esc 1–21 causes downregulation of enzymes of the lower glycolytic pathway and in genes involved in spindle body formation and remodelling of cell-wall synthesis. Moreover the peptide induces downexpression of protein actin within 45 min and cells pre-treated with peptide show less sensitivity to osmotic stress and increased sensitivity to heat shock stress. The results obtained with the two different methodologies are in agreement at the cellular process levels. A combined approach may help elucidate the main aspects related to the effects of this peptide on the eukaryotic cell. The employment of different technologies may reveal the potential and limitations of each adapted approach in a prospective application for drug screening.

Keywords: S. cerevisiae; Systems biology; Proteomic; Transcriptomic; Antimicrobial peptide

Peptide binding specificity of the chaperone calreticulin by Noreen Sandhu; Karen Duus; Charlotte S. Jørgensen; Paul R. Hansen; Susanne W. Bruun; Lars Ø. Pedersen; Peter Højrup; Gunnar Houen (pp. 701-713).

Calreticulin is a molecular chaperone with specificity for polypeptides and N-linked monoglucosylated glycans. In order to determine the specificity of polypeptide binding, the interaction of calreticulin with polypeptides was investigated using synthetic peptides of different length and composition. A large set of available synthetic peptides ( n=127) was tested for binding to calreticulin and the results analysed by multivariate data analysis. The parameter that correlated best with binding was hydrophobicity while β-turn potential disfavoured binding. Only hydrophobic peptides longer than 5 amino acids showed binding and a clear correlation with hydrophobicity was demonstrated for oligomers of different hydrophobic amino acids. Insertion of hydrophilic amino acids in a hydrophobic sequence diminished or abolished binding. In conclusion our results show that calreticulin has a peptide-binding specificity for hydrophobic sequences and delineate the fine specificity of calreticulin for hydrophobic amino acid residues.

Keywords: Calreticulin; Chaperone; Peptide specificity

Functional duplication of ligand-binding domains within low-density lipoprotein receptor-related protein for interaction with receptor associated protein, α₂-macroglobulin, factor IXa and factor VIII by Alexander B. Meijer; Jakub Rohlena; Carmen van der Zwaan; Anton-Jan van Zonneveld; Ria C. Boertjes; Peter J. Lenting; Koen Mertens (pp. 714-722).

The low-density lipoprotein receptor-related protein (LRP) binds a range of proteins including receptor associated protein (RAP), activated α₂-macroglobulin (α₂M*), factor IXa (FIXa), and factor VIII (FVIII) light chain. The binding is mediated by the complement-type repeats, which are clustered in four distinct regions within LRP. Cluster II of 8 repeats (CR3–10) and cluster IV of 11 repeats (CR21–31) have been implicated in ligand-binding. Previous studies have aimed to identify the cluster II repeats involved in binding α₂M* and RAP. We now evaluated the binding to RAP, α₂M*, FIXa and FVIII light chain of triplicate repeat-fragments of not only clusters II but also of cluster IV. Employing surface plasmon resonance analysis, we found that most efficient ligand-binding was displayed by the repeats within region CR4–8 of cluster II and within region CR24–28 of cluster IV. Whereas the binding to RAP could be attributed to two consecutive repeats (CR5–6, CR26–27), combinations of three repeats showed most efficient binding to FIXa (CR6–8, CR26–28), FVIII light chain (CR5–7, CR6–8, CR24–26), and α₂M* (CR4–6, CR24–26). The results imply that there is an internal functional duplication of complement-type repeats within LRP resulting in two clusters that bind the same ligands.

Keywords: LRP; Complement-type repeats; Factor VIII; Factor IX; RAP; Alpha-2-macroglubulin

Disulphide bond reduction and S-carboxamidomethylation of PSP94 affects its conformation but not the ability to bind immunoglobulin by Dhanashree D. Jagtap; Akkaladevi Narahari; Musti J. Swamy; Smita D. Mahale (pp. 723-731).

Prostate secretory protein of 94 amino acids (PSP94) is a small non-glycosylated, cysteine rich protein with a molecular mass of 10 kDa. It has also been referred to as β-microseminoprotein (β-MSP) and proteins homologous to it have been reported in a number of species. Comparison of the amino acid sequence of these proteins suggests that, it is a rapidly evolving protein. However, all the ten cysteine residues are well conserved in these homologues, indicating their possible role in maintaining the structure and function of these proteins. In the present study, PSP94 was purified from human seminal plasma and characterized further and it showed the presence of five disulfide bonds. Reduction of disulphide bonds of PSP94 led to significant changes in the secondary and tertiary structure of PSP94. CD of disulphide bond reduced PSP94 indicates an overall decrease in the beta sheet content from 79.8% to 46.4%. Tertiary structural changes as monitored by fluorescence quenching reveal that reduction of disulphide bonds of PSP94 followed by the modification of the free thiol groups leads to complete exposure of Trp32 and Trp92 and that one or more side chain carboxyl groups move closer to their indole side chains. Antibodies against native and modified PSP94 demonstrated that the changes following reduction of disulphide linkages are within the immunodominant region of the protein. Changes induced in the functional properties of PSP94, if any, by modification were investigated with respect to IgG binding as PSP94 has been reported to be similar to immunoglobulin binding factor purified from seminal plasma. A novel finding from this study is that both native PSP94 as well as modified protein have the ability to bind human IgG, suggesting the involvement of sequential epitopes of PSP94 in IgG binding.

Keywords: β-Microseminoprotein; Disulphide bonds; Fluorescence quenching; Human seminal plasma inhibin; Prostate secretory protein 94

Structure of full-length transcription regulator CcpA in the apo form by Bernhard Loll; Wolfram Saenger; Jacek Biesiadka (pp. 732-736).

The catabolite control protein A (CcpA) from Bacillus megaterium is a member of the bacterial repressor protein family GalR–LacI. CcpA functions as master transcriptional regulator of carbon catabolite repression/regulation in firmicutes. Here we present the crystal structure of full-length apo CcpA at 2.5 Å resolution from B. megaterium. The structure reveals the location of the helix–turn–helix domain as well as the hinge region, which were not visible due to their high flexibility in earlier crystallographic studies on CcpA molecules. The structure of the apo CcpA homodimer in the present form is in contrast to other reported structures for CcpA.

Keywords: Abbreviations; CcpA; catabolite control protein A; cre; catabolite responsive element; HPr; histidine-containing protein; HTH; helix–turn–helix; LacI; lactose repressor; PBP; periplasmic binding protein domain; PurR; purine repressor; rmsd; root-mean-square deviation Bacillus megaterium; Carbon catabolite repression; Catabolite control protein A; DNA-binding protein; Crystal structure; Domain movement

On translocation mechanism of ring-shaped helicase along single-stranded DNA by Ping Xie (pp. 737-748).

The ring-shaped helicases represent one important group of helicases that can translocate along single-stranded (ss) DNA and unwinding double-stranded (ds) DNA by using the energy derived from NTP binding and hydrolysis. Despite intensive studies, the mechanism by which the ring-shaped helicase translocates along ssDNA and unwinds dsDNA remains undetermined. In order to understand their chemomechanical-coupling mechanism, two models on NTPase activities of the hexamers in the presence of DNA have been studied here. One model is assumed that, of the six nucleotide-binding sites, three are noncatalytic and three are catalytic. The other model is assumed that all the six nucleotide-binding sites are catalytic. In terms of the sequential NTPase activity around the ring and the previous determined crystal structure of bacteriophage T7 helicase it is shown that the obtained mechanical behaviors such as the ssDNA-translocation size and DNA-unwinding size per dTTPase cycle using the former model are in good quantitative agreement with the previous experimental results for T7 helicase. Moreover, the acceleration of DNA unwinding rate with the stimulation of DNA synthesis by DNA polymerase can also be well explained by using the former model. In contrast, the ssDNA-translocation size and DNA-unwinding size per dTTPase cycle obtained by using the latter model are not consistent with the experimental results for T7 helicase. Thus it is preferred that the former model is the appropriate one for the T7 helicase. Furthermore, using the former model some dynamic behaviors such as the rotational speeds of DNA relative to the T7 helicase when translocation along ssDNA and when unwinding dsDNA have been predicted, which are expected to test in order to further verify the model.

Keywords: Helicase; Translocation mechanism; DNA unwinding; Hexamer; Molecular motor

Effect of selected Ser/Ala and Xaa/Pro mutations on the stability and catalytic properties of a cold adapted subtilisin-like serine proteinase by Jóhanna Arnórsdóttir; Sunna Helgadóttir; Sigrídur H. Thorbjarnardóttir; Gudmundur Eggertsson; Magnús M. Kristjánsson (pp. 749-755).

A subtilisin-like serine proteinase from a psychrotrophic Vibrio species (VPR) shows distinct cold adapted traits regarding stability and catalytic properties, while sharing high sequence homology with enzymes adapted to higher temperatures. Based on comparisons of sequences and examination of 3D structural models of VPR and related enzymes of higher temperature origin, five sites were chosen to be subject to site directed mutagenesis. Three serine residues were substituted with alanine and two residues in loops were substituted with proline. The single mutations were combined to make double and triple mutants. The single Ser/Ala mutations had a moderately stabilizing effect and concomitantly decreased catalytic efficiency. Introducing a second Ser/Ala mutation did not have additive effect on stability; on the contrary a double Ser/Ala mutant had reduced stability with regard to both wild type and single mutants. The Xaa/Pro mutations stabilized the enzyme and did also tend to decrease the catalytic efficiency more than the Ser/Ala mutations.

Keywords: Cold adaptation; Stability; Kinetic properties; Site directed mutagenesis; Subtilsin-like; Proteinase K; Vibrio; -proteinase

Pseudomonas aeruginosa contains multiple glyoxalase I-encoding genes from both metal activation classes by Nicole Sukdeo; John F. Honek (pp. 756-763).

The glyoxalase (Glx) system is a critical detoxification enzyme system that is widely distributed in prokaryotic and eukaryotic organisms. Glyoxalase I (GlxI), the first enzyme in the system, is a divalent metal-ion dependent lyase (isomerizing), and its homologs have recently been categorized into two metal activation classes which are either Zn²⁺-dependent or non-Zn²⁺ dependent (Ni²⁺-/Co²⁺-activated). The latter class encompasses enzymes of predominantly bacterial origin. We have identified two genes in Pseudomonas aeruginosa PAO1 encoding glyoxalase I enzymes in addition to the gloA1 sequence recently reported and characterized. The gloA1 and gloA2 genes encode non-Zn²⁺ dependent glyoxalase I enzymes and the gloA3 gene remarkably encodes a Zn²⁺-dependent homolog. To our knowledge this is the first report of a eubacterial species with several GlxI encoding genes, and also of an organism possessing GlxI enzymes from both metal activation classes.

Keywords: Abbreviations; Glx; Glyoxalase; GlxI; Glyoxalase I; GlxII; Glyoxalase II; MG; methylglyoxal; GSH; glutathione; TSH; N; ¹; ,; N; ⁸; -bis(glutathionyl)spermidine; PCR; polymerase chain reaction; TB; terrific broth; DPA; 2,6-pyridinedicarboxylic acid; IPTG; isopropyl β-; d; -thiogalactoside; DTT; dithiothreitol; KPB; potassium phosphate buffer; PMSF; phenylmethylsulphonyl fluoride; SDS/PAGE; sodium dodecylsulphate polyacrylamide gel electrophoresis; ESMS; electrospray ionization mass spectrometry; ICP-MS; inductively coupled plasma mass spectrometry; PAR; 4-(2-pyridylazo)resorcinol; EDTA; ethylenediaminetetraacetic acidGlyoxalase I; Pseudomonas aeruginosa; Homologues; Metal activation; Nickel; Zinc

Serum proteomic-based analysis of pancreatic carcinoma for the identification of potential cancer biomarkers by Zhi-Ling Sun; Yi Zhu; Fu-Qiang Wang; Rui Chen; Tao Peng; Zhi-Ning Fan; Ze-Kuan Xu; Yi Miao (pp. 764-771).

To identify new biomarkers that improve the early diagnosis and lead to possible therapeutic targets in pancreatic carcinoma, we performed a proteomic approach to compare serum protein expression patterns of pancreatic carcinoma patients with that of gastric cancer patients, other pancreatic disease patients, and healthy volunteers. By two-dimensional gel electrophoresis (2-DE) analyses and mass spectroscopic identification, 10 protein spots were found significantly changed in pancreatic carcinoma and 5 proteins including cyclin I, Rab GDP dissociation inhibitor β (GDI2), α-1 antitrypsin precursor, Haptoglobin precursor, and Serotransferrin precursor were successfully identified. The increased levels of cyclin I and GDI2 found to be associated with pancreatic carcinoma were further confirmed by Western blot analyses in an independent series of serum samples and/or pancreatic juice samples. Applying immunohistochemistry, we further validated expression of cyclin I and GDI2 in additional pancreatic carcinomas. These results indicate that cyclin I and GDI2 may be potential molecular targets for pancreatic cancer diagnostics and therapeutics.

Keywords: Biomarkers; Pancreatic carcinoma; Serum proteomics; Two-dimensional electrophoresis

Thermal unfolding of human BRCA1 BRCT-domain variants by George Nikolopoulos; Serapion Pyrpassopoulos; Angelos Thanassoulas; Persefoni Klimentzou; Christos Zikos; Metaxia Vlassi; Constantinos E. Vorgias; Drakoulis Yannoukakos; George Nounesis (pp. 772-780).

Missense mutations at the BRCT domain of human BRCA1 protein have been associated with an elevated risk for hereditary breast/ovarian cancer. They have been shown to affect the binding site and they have also been proposed to affect domain stability, severely hampering the protein's tumor suppressor function. In order to assess the impact of various such mutations upon the stability and the function of the BRCT domain, heat-induced denaturation has been employed to study the thermal unfolding of variants M1775R and R1699W, which have been linked with the disease, as well as of V1833M, which has been reported for patients with a family history. Calorimetric and circular dichroism results reveal that in pH 9.0, 5 mM borate buffer, 200 mM NaCl, analogously to wild type BRCT, all three variants undergo partial thermal unfolding to a denatured state, which retains most of the native's structural characteristics. With respect to wild-type BRCT, the mutation M1775R induces the most severe effects especially upon the thermostability, while R1699W also has a strong impact. On the other hand, the thermal unfolding of variant V1833M is only moderately affected relative to wild-type BRCT. Moreover, isothermal titration calorimetric measurements reveal that contrary to M1775R and R1699W variants, V1833M binds to BACH1 and CtIP phosphopeptides.

Keywords: BRCA1-BRCT domain; Thermal unfolding; DSC; CD; ITC; V1833M; M1775R; R1699W

Sections

Personal tools

BBA - Proteins and Proteomics (v.1774, #6)

Sections

Personal tools

Local resources

BBA - Proteins and Proteomics (v.1774, #6)