Protein & Peptide Letters, Volume 12, No. 6, 2005
Contents
Meeting
Report
Protein
Misfolding and Misprocessing in Complex Disease Pp.499-506
A.
Fadiel, K.D. Eichenbaum, A. Hamza, O. Tuncalp, J. Luk and F. Naftolin
Mini-Reviews
Pharmacological
Characterisation of Spider Antimicrobial Peptides Pp.507-511
Li
Gao, Jing Zhang, Wei Feng, Na Bao , Daxiang Song and Bao-Cheng Zhu
The
Role of Ab
Peptides in Alzheimer’s Disease Pp.513-519
Anna
K. Tickler, John D. Wade and Frances Separovic
Regular
Papers
Preliminary
Structural Studies of the Hydrophobic Ribosomal P0 Protein from Trypanosoma
cruzi, A Part of the P0/P1/P2 Complex Pp.521-525
Maximiliano
Juri Ayub, Juan A. Barroso, Mariano J. Levin and Carlos F. Aguilar
Expression,
Purification and Partial Characterization of the Kruppel- Associated Box (KRAB)
from the Human ZNF2 Protein Pp.527-532
Vincenzo
Rivieccio, Riccardo Mannini, Luigi Concilio, Sabato D’Auria, Carlo Pedone and
Giovanna Grimaldi
Influence
of Disulfide Bonds on the Conformational Changes and Activities of Refolded
Phytase Pp.533-535
G.Y.
Song, X.Y. Wang and M. Wang
NMR
Assignment and Secondary Structure of the Coxsackievirus and Adenovirus
Receptor Domain 2
Pp.537-539
Shaokai
Jiang and Michael Caffrey
Energy
Barrier for Protein Adhesion and Crystal Nucleation on Flat Alien Substrates Pp.541-546
Daniela
S. Tsekova and Varban Savov
Direct
Comparison of the Crystal and Solution Structure of Glucose/Xylose Isomerase
from Streptomyces rubiginosus Pp.547-550
Maciej
Kozak
Production
of the Human-Beta-Defensin Using Saccharomyces cerevisiae as a Host Pp.551-554
Ingrid
Cipakova and Eva Hostinova
A
General Procedure for the Production of Antibody Reagents Against Eukaryotic
Ribosomal Proteins
Pp.555-560
Giorgio
Dieci, Lorena Bottarelli and Simone Ottonello
Screening
and Purification of a Novel Trypsin Inhibitor from Prosopis juliflora
Seeds with Activity Toward Pest Digestive Enzymes Pp.561-565
S.
Sivakumar, O.L. Franco, P.D. Tagliari, C. Bloch Jr., M. Mohan and B.
Thayumanavan
Characterization
of Expression and Stability of Recombinant Cystein-Rich Protein Human MT1A from
Yeast Pp.567-571
Li
Jie, Shao Kaifeng, Yao Dian, An Lin and Ru Binggen
Global
Changes of Chlorophyll Excitonic Interactions in Photosystem II During Thermal
Denaturation
Pp.573-578
Qiang
Xu, Xiaolan Ding, Yandao Gong, Tingyun Kuang and Nanming Zhao
Further
Studies on the Side Reactions Associated with Use of Np- Benzyloxymethylhistidine Pp.579-582
Kumiko
Yoshizawa-Kumagaye, Takehiro Ishizu, Shuji Isaka, Masaji Tamura, Rumi Okihara,
Yuji Nishiuchi and Terutoshi Kimura
Purification
of a 6.5 kDa Protease Inhibitor from Amazon Inga umbratica Seeds
Effective Against Serine Proteases of the Boll Weevil Anthonomus grandis Pp.583-587
L.A.
Calderon, R.C.L. Teles, J.R.S.A. Leite, O.L. Franco, M.F. Grossi-de-Sa, F.J.
Medrano, C. Bloch Jr. and S.M. Freitas
Two
Novel Lectins from Parkia biglandulosa and Parkia roxburghii:
Isolation, Physicochemical Characterization, Mitogenicity and Anti-Proliferative
Activity
Pp.589-595
Navjot
Kaur, Jatinder Singh, Sukhdev Singh Kamboj, Javed N. Agrewala and Manpreet Kaur
Effects
of Trehalose on Pressure-Induced Inactivation of Yeast Alcohol Dehydrogenase Pp.597-599
Hyun Park, Gene Kidman and Dexter B. Northrop
Innovative,
Multifunctional Sequential Oligopeptide Carriers Socn-I and SOCn-II:
Functions-Technology-Perspectives Pp.601-607
Charalampos Alexopoulos, Dimitrios Krikorian, Eugenia Panou-Pomonis, Maria Sakarellos- Daitsiotis and Constantinos Sakarellos
Crystallization
Report
Preliminary
X-Ray Crystallographic Analysis of Centrin from Ciliate Euplotes
octocarinatus
Pp.609-611
Xiao-jing He, Liu Li, Hai Hou, Yaqin Zhao, Aihua Liang and Zihe Rao
Abstracts
[Back to top] Protein
Misfolding and Misprocessing in Complex Disease
A.
Fadiel, K.D. Eichenbaum, A. Hamza, O. Tuncalp, J. Luk and F. Naftolin
Scientists from over 20 major research centers recently convened to discuss advances and new discoveries in “Protein MisFolding and MisProcessing in Disease.” Understanding protein mechanisms the underlying etiology of complex diseases lies in analyzing the associated biochemical mechanisms, which include folding patterns, processing patterns, chaperone regulators, stress pathways, and signal transduction.
[Back to top]
Pharmacological
Characterisation of Spider Antimicrobial Peptides
Li
Gao, Jing Zhang, Wei Feng, Na Bao , Daxiang Song and Bao-Cheng Zhu
Most spider antimicrobial peptides share a common mechanism of membrane permeabilisation and the innate immune systems of the pathogen. In this review, we present recent accounts of the application at the preclinical level that should be tried and the range of bioactivities and their particular structure can be harnessed for molecular engineering applications and in drug design. Structural analyses such as amino acid sequence and circular dichroism are described. Conductance measurements and pharmacological studies of the action on the inner or outer membranes of anti-microbe will reveal more about the mode of action of the antimicrobial peptides of spider.
[Back to top]
The Role of Ab Peptides in Alzheimer’s Disease
Anna
K. Tickler, John D. Wade and Frances Separovic
The Ab peptide has been identified as central to the onset and development of Alzheimer’s disease (AD) and several hypotheses about toxicity involving Ab peptides have been proposed including mechanisms of oxidative stress and disruption of calcium homeostasis. The biology, structure and physical properties of Ab peptides are discussed, as well as existing therapeutics and future strategies for the treatment of AD.
[Back to top] Preliminary Structural Studies of the Hydrophobic Ribosomal
P0 Protein from Trypanosoma cruzi, A Part of the P0/P1/P2 Complex
Maximiliano
Juri Ayub, Juan A. Barroso, Mariano J. Levin and Carlos F. Aguilar
The Trypanosoma cruzi ribosomal P0 protein (TcP0) is part of the ribosomal stalk, which is an elongated lateral protuberance of the large ribosomal subunit involved in the translocation step of protein synthesis. The TcP0 C-terminal peptide is highly antigenic and a major target of the antibody response in patients with systemic lupus erythematosus and patients suffering chronic heart disease produced by Trypanosoma cruzi infection. The structural properties of TcP0 have been explored by circular dichroism, tryptophan fluorescence and limited proteolysis experiments. These studies were complemented by secondary structure consensus prediction analysis. The results suggest that the tertiary structure of TcP0 could be described as a compact, stable, trypsin-resistant, 200 residues long N–terminal domain belonging to the a/b class and a more flexible, degradable, helical, 123 residues long C-terminal domain which could be involved in the formation of an unusual hydrophobic zipper with the ribosomal P1/P2 proteins to form the P0/P1/P2 complex.
[Back
to top] Expression, Purification and
Partial Characterization of the Kruppel- Associated Box (KRAB) from the Human
ZNF2 Protein
Vincenzo
Rivieccio, Riccardo Mannini, Luigi Concilio, Sabato D’Auria, Carlo Pedone and
Giovanna Grimaldi
The Krüppel-associated box (KRAB) domain is a potent transcription repression bipartite domain, shared by over 400 zinc finger proteins in humans, involved in the regulation of many functions. KRAB domains are both physically and functionally bipartite (A and B modules). The lone KRAB-A and composite KRAB-AB domains from the human ZNF2 protein were over-expressed as recombinant proteins in E. coli, isolated and purified to homogeneity to investigate their structure to function relationship.
[Back to top] Influence of Disulfide Bonds on the
Conformational Changes and Activities of Refolded Phytase
G.Y.
Song, X.Y. Wang and M. Wang
Aspergillus sp. phytase contains five disulfide bonds. In order to elucidate their role, the reactivation and refolding of phytase in the absence and presence of dithiothreitol (DTT) was investigated. The results indicated that the disulfide bonds play an important role in the catalytic activity and conformational stability of the enzyme.
[Back to top] NMR Assignment and Secondary
Structure of the Coxsackievirus and Adenovirus Receptor Domain 2
Shaokai
Jiang and Michael Caffrey
The Coxsackievirus and Adenovirus receptor (CAR) mediates entry of coxsackievirus and adenovirus. CAR possesses an extracellular region that is comprised of 2 Ig domains termed CAR-D1 and CAR-D2. The 1H, 13C and 15N resonances of CAR-D2 have been assigned and the secondary structure has been deduced.
[Back to top] Energy Barrier for Protein
Adhesion and Crystal Nucleation on Flat Alien Substrates
Daniela
S. Tsekova and Varban Savov
From the experimentally defined rate of heterogeneous formation of protein crystals and number of collisions between protein molecules with substrate, the energy barrier for adhesion of protein molecule to alien materials is estimated. According to the Boltzmann equation for energy distribution this barrier is of the order of 10-12 erg per molecule.
[Back to top] Direct Comparison of the Crystal
and Solution Structure of Glucose/Xylose Isomerase from Streptomyces
rubiginosus
Maciej
Kozak
Glucose isomerase (D-xylose ketol-isomerase, EC 5.3.1.5.) catalyses the isomerization reaction of glucose and xylose. The small angle X-ray scattering (SAXS) data of glucose/xylose isomerase from Streptomyces rubiginosus were recorded for protein solution using synchrotron radiation. The experimental data were compared with theoretical scattering calculated on the basis of the known crystal structure (PDB code: 1OAD). The radius of gyration measured by SAXS (RG=3.30 nm) was almost identical and the maximum dimension in the distance distribution function was by about 2.5 % lower than the corresponding values calculated on the basis of the crystal structure.
[Back to top] Production
of the Human-Beta-Defensin Using Saccharomyces cerevisiae as a Host
Ingrid
Cipakova and Eva Hostinova
Studies of the human defensins have been hampered by the lack of a simple expression system allowing for rapid production of functional peptide forms. Here, we describe a Saccharomyces cerevisiae AH22 expression system that meets that condition. The 42 amino acid form of human b-defensin-1 was expressed under the control of the ADH1 promoter. The optimum conditions for expression were determined and the stable maintenance of the pVT103L-hBD-1 chimeric vector in the yeast population was confirmed. Expressed hBD-1 was secreted into the medium (~55 mg l-1) and purified using cation-exchange chromatography. Isolated defensin exhibited strong bactericidal effect on Escherichia coli ML-35p. We conclude that the expression system described here will be a useful tool where readily prepared and active forms of the human defensins are needed.
[Back to top] A
General Procedure for the Production of Antibody Reagents Against Eukaryotic
Ribosomal Proteins
Giorgio
Dieci, Lorena Bottarelli and Simone Ottonello
Despite recent progress in the structural and functional analysis of bacterial and archaeal ribosomes, the structure and biogenesis of eukaryotic ribosomes still awaits a detailed characterization. Ribosomal protein-specific antibodies would be valuable tools for such studies, but their production is commonly hindered by the poor expression and solubility of eukaryotic ribosomal proteins in E. coli. We report here an improved general procedure for the over-production of recombinant eukaryotic ribosomal proteins and for the generation of the corresponding polyclonal antibodies. The specificity and sensitivity of detection of the antibodies produced by this procedure are documented.
[Back to top] Screening
and Purification of a Novel Trypsin Inhibitor from Prosopis juliflora
Seeds with Activity Toward Pest Digestive Enzymes
S.
Sivakumar, O.L. Franco, P.D. Tagliari, C. Bloch Jr., M. Mohan and B.
Thayumanavan
Several pests are capable of decreasing crop production causing severe economical and social losses. Aiming to find novel molecules that could impede the digestion process of different pests, a screening of a-amylase and trypsin-like proteinase inhibitors was carried out in Prosopis juliflora, showing the presence of both in dry seeds. Furthermore, a novel trypsin inhibitor, with molecular mass of 13,292 Da, was purified showing remarkable in vitro activity against T. castaneum and C. maculatus.
[Back to top] Characterization
of Expression and Stability of Recombinant Cystein-Rich Protein Human MT1A from
Yeast
Li
Jie, Shao Kaifeng, Yao Dian, An Lin and Ru Binggen
Metallothionein (MT) is the protein that has been shown to bind heavy metals, scavenge free radicals, protect DNA from radiation damage, and alleviate disease symptoms. However, only very limited success has been achieved in expression and production of active recombinant metallothionein. In this study, human metallothionein 1A (hMT1A) was transformed into yeast Pichia pastoris for expression with secretion of the protein into the medium. The expression system was optimized to obtain the targeted protein in active form at 335mg per litre culture. hMT1A showed the character of extreme instability in the experiment. High concentration, aeration and heavy metal ions are the main factors affecting hMT1A stability.
[Back to top] Global
Changes of Chlorophyll Excitonic Interactions in Photosystem II During Thermal
Denaturation
Qiang
Xu, Xiaolan Ding, Yandao Gong, Tingyun Kuang and Nanming Zhao
Photosystem II (PSII) is a multisubunit chlorophyll-binding enzyme that absorbs light to catalyze water oxidation and plastoquinone reduction. Chlorophyll excitonic interaction changes in PSII were studied by absorption and circular dichroism spectra from 25°C to 80°C, and protein subunit denaturation was monitored by differential scanning calorimetry. A four-stage process of chlorophyll excitonic interaction change was observed being correlated with the denaturation of protein subunits.
[Back to top] Further
Studies on the Side Reactions Associated with Use of Np- Benzyloxymethylhistidine
Kumiko
Yoshizawa-Kumagaye, Takehiro Ishizu, Shuji Isaka, Masaji Tamura, Rumi Okihara,
Yuji Nishiuchi and Terutoshi Kimura
The use of Na-tert.-butyloxycarbonyl-Np-benzyloxymethylhistidine [Boc-His(Bom)] in peptide synthesis results in a serious level of side products arising from the generation of formaldehyde during the HF cleavage reaction. In particular, when treating a His(Bom)-containing peptide having Cys at the N-terminus by HF, this leads to almost complete conversion of the Cys-peptide to thiazolidyl (Thz)-peptide unless precautions are taken. Also, the reaction of formaldehyde with the N-terminal Trp and the N-methylanthranyl (Nma) group was found to produce tetrahydro-b-carboline and dihydroquinazolin derivatives, respectively, upon isolation from HF mixtures. The addition of cysteine as a scavenger in HF proved to be effective for suppressing modification arising from the generation of formaldehyde.
[Back to top] Purification
of a 6.5 kDa Protease Inhibitor from Amazon Inga umbratica Seeds
Effective Against Serine Proteases of the Boll Weevil Anthonomus grandis
L.A.
Calderon, R.C.L. Teles, J.R.S.A. Leite, O.L. Franco, M.F. Grossi-de-Sa, F.J.
Medrano, C. Bloch Jr. and S.M. Freitas
A 6.5 kDa serine protease inhibitor was purified by anion-exchange chromatography from the crude extract of the Inga umbratica seeds, containing inhibitor isoforms ranging from 6.3 to 6.7 kDa and protease inhibitors of ~19 kDa. The purified protein was characterized as a potent inhibitor against trypsin and chymotrypsin and it was named I. umbratica trypsin and chymotrypsin inhibitor (IUTCI). MALDI-TOF spectra of the IUTCI, in the presence of DTT, showed six disulfide bonds content, suggesting that this inhibitor belongs to Bowman-Birk family. The circular dichroism spectroscopy indicates that IUTCI is predominantly formed by unordered and b-sheet secondary structure. It was also characterized, by fluorescence spectroscopy, as a stable protein at range of pH from 5.0 to 7.0. Moreover, this inhibitor at concentration of 75 mM presented a remarkable inhibitory activity (60%) against digestive serine proteases from boll weevil Anthonomus grandis, an important economical cotton pest.
[Back to top] Two
Novel Lectins from Parkia biglandulosa and Parkia roxburghii:
Isolation, Physicochemical Characterization, Mitogenicity and Anti-
Proliferative Activity
Navjot
Kaur, Jatinder Singh, Sukhdev Singh Kamboj, Javed N. Agrewala and Manpreet Kaur
Two mannose/glucose specific seed lectins were isolated from Parkia biglandulosa and Parkia roxburghii and were characterized w.r.t various physicochemical properties. Unlike other Parkia lectins a comparison of native and subunit molecular mass showed that both Parkia lectins were heterotetramers. Parkia biglandulosa lectin was found to be T-cell mitogen as revealed by IL-2 bioassay. These lectins showed anti-proliferative effect on two murine macrophage cancer cell lines i.e. P 388DI (50%) and J774 (70%). In addition Parkia roxburghii also inhibited proliferation of HB98 (65.47%), a B-cell hybridoma cell line.
[Back to top] Effects of Trehalose on
Pressure-Induced Inactivation of Yeast Alcohol Dehydrogenase
Hyun Park, Gene Kidman and Dexter B. Northrop
Isozymes of yeast alcohol dehydrogenase are slowly denatured at moderate hydrostatic pressures (<3 kbar). The time courses for inactivation are biphasic and both phases of both isozymes are protected by trehalose. ADH-I is slightly more barostable than ADH-II which is opposite to their thermostabilities. Trehalose at 1M extends their half-lives about 6-fold at 2 kbar, pH 7.5 and 25 °C. In contrast, 1M sucrose provides only 4.4-fold protection under identical conditions, a finding consistent with the superior protein stabilization of trehalose to other denaturants.
[Back to top] Innovative, Multifunctional
Sequential Oligopeptide Carriers Socn-I and SOCn-II:
Functions-Technology-Perspectives
Charalampos Alexopoulos, Dimitrios Krikorian, Eugenia Panou-Pomonis, Maria Sakarellos- Daitsiotis and Constantinos Sakarellos
An innovative type of multifunctional helicoid artificial carriers, formed by the repetitive Lys-Aib-Gly (SOCn-I) or by the Aib-Lys-Aib-Gly (SOCn-II), with structural rigidity and regularity were successfully applied in our laboratory for anchoring antigenic/immunogenic peptides. The carriers, designed to display a predetermined 3D structure, adopt the 310 helical conformation, while the attached peptides preserve their original “active” conformation. The constructed conjugates were used as substrates in solid phase immunoassays, as well as for generating potent and specific immune responses.
[Back to top] Preliminary X-Ray
Crystallographic Analysis of Centrin from Ciliate Euplotes octocarinatus
Xiao-jing He, Liu Li, Hai Hou, Yaqin Zhao, Aihua Liang and Zihe Rao
Centrins are four-EF-hand Ca2+-binding proteins, which belong to the CaM super family. The centrin from ciliate Euplotes Octocarinatus has been expressed in Escherichia coli, purified and crystallized using the hanging-drop method. Rod-like crystals were grown and diffracted to 2.0 Å. The crystals belong to space group P212121 and the unit-cell parameters are a=34.442 Å, b=48.954 Å, c=72.583 Å.