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Protein & Peptide Letters
ISSN: 0929-8665
Protein & Peptide Letters
Volume 13, Number 7, 2006
Contents
Mini-Review
Industrial Applications of Hyperthermophilic Enzymes:
A Review Pp. 645-651
T. de M. Bouzas, J. Barros-Velázquez and T.G. Villa
[Abstract]
Regular Papers
A cDNA Sequence Coding for a Glutamic Acid-Rich Protein
Is Differentially Expressed in Cassava Storage Roots Pp.
653-657
C.R. Batista de Souza, L.J.C.B. Carvalho, E.R. Pereira
de Almeida and E.S. Gander
[Abstract]
Efficient In Vitro Refolding and Characterization
of a New Peptide from the Scorpion Buthotus saulcyi
Venom Produced in Escherichia coli Pp. 659-664
M. Nikkhah, H. Naderi-Manesh, M.N. Sarbolouki and B. Ranjbar
[Abstract]
Kinetic and Thermodynamic Properties of an Immobilized
Glucoamylase from a Mesophilic Fungus, Arachniotus citrinus
Pp. 665-671
R. Perveen, M.H. Rashid, M. Saleem, A.M. Khalid and M.I.
Rajoka
[Abstract]
Inhibition of Pancreatic Ribonuclease A Aggregation
by Antibodies Raised Against the Native Enzyme and Its N-Terminal
Dodecapeptide Pp. 673-677
H. Younus, R. Ulbrich-Hofmann and M. Saleemuddin
[Abstract]
The Proline-Rich Domain and the Microtubule Binding
Domain of Protein Tau Acting as RNA Binding Domains Pp.
679-685
X.S. Wang, D.L. Wang, J. Zhao, M.H. Qu, X.H. Zhou, H.J.
He and R.Q. He
[Abstract]
Web-Ware Bioinformatical Analysis and Structure Modelling
of N-Terminus of Hyman Multisynthetase Complex Auxiliary Component
Protein p43 Pp. 687-691
V. Deineko
[Abstract]
Key Residues Involved in Hsp70 Regulatory Activity
and Affect of Co-Chaperones on Mechanism of Action
Pp. 693-698
Y. Tutar
[Abstract]
Heat Shock Proteins, Substrate Specificity and Modulation
of Function Pp. 699-705
Y. Tutar
[Abstract]
Cytotoxic Action in Myoblasts and Myotubes (C2C12)
and Enzymatic Characterization of a New Phospholipase A2
Isoform (Bj-V) from Bothrops jararacussu Venom Pp.
707-713
V.L. Bonfim, L.A. Ponce-Soto, J.C. Novello and S. Marangoni
[Abstract]
Solid-Phase Synthesis of Reactive Peptide Crosslinker
by Selective Deprotection Pp. 715-718
X. He and E. Jabbari
[Abstract]
Improvement of Bacterial Cell Selectivity of Melittin
by a Single Trp Mutation with a Peptoid Residue Pp.
719-725
W.L. Zhu, Y. Park, I.-S. Park, Y.S. Park, Y. Kim, K.-S.
Hahm and S.Y. Shin
[Abstract]
High Level Expression and Characterization of the
Cyclophilin B Gene from the Anaerobic Fungus Orpinomyces
sp. Strain PC-2 Pp. 727-732
H. Chen, X.-L. Li, H. Xu, L.G. Ljungdahl and
C.E. Cerniglia
[Abstract]
Inputting Information About Amino Acid Residues in
Protein Bioinformatics: A Case Study on Predicting Helical
Regions with a Neural Network Pp. 733-736
T. Imoto
[Abstract]
Acidic Extracellular Proteases from Microrganisms
of Fairly Acidic Niche Pp. 737-741
A. Bossi, L. Bonizzato and G. Zapparoli
[Abstract]
A Structural-Energetic Basis for B-Cell Epitope Prediction
Pp. 743-751
S.E.C. Caoili
[Abstract]
Abstracts
[Back to top]
Industrial Applications of Hyperthermophilic Enzymes:
A Review
T. de M. Bouzas, J. Barros-Velázquez and T.G. Villa
Over the past two decades, research scientists have been involved
in the investigation of thermophilic and hyperthermophilic
microorganisms owing to the unique features of their enzymic
systems. Such in-depth investigations are now on their way
to mastering the cloning and industrial exploitation of a
broad variety of genes encoding enzymes involved in starch
hydrolysis, amino acid biosynthesis, protein hydrolysis, etc.
In this work, we review the state of the art and future perspectives
of industrial applications of enzymes from hyperthermophilic
and extreme thermophilic microorganisms, special attention
being paid to the biotechnological methods involved in their
industrial exploitation.
[Back to top]
A cDNA Sequence Coding for a Glutamic Acid-Rich Protein
Is Differentially Expressed in Cassava Storage Roots
C.R. Batista de Souza, L.J.C.B. Carvalho, E.R. Pereira
de Almeida and E.S. Gander
We report the isolation and characterization of a cDNA sequence
(Mec1) coding for a glutamic acid-rich protein (Pt2L4) from
cassava storage roots. Comparative sequence analysis showed
a high identity of Pt2L4 with cassava protein C54, which is
expressed in vascular tissues of storage roots. Northern blot
analysis showed that the Mec1 transcript expression pattern
might be related to the maturation of the storage parenchyma
cells.
[Back to top]
Efficient In Vitro Refolding and Characterization
of a New Peptide from the Scorpion Buthotus saulcyi
Venom Produced in Escherichia coli
M. Nikkhah, H. Naderi-Manesh, M.N. Sarbolouki and B. Ranjbar
The selective toxicity of depressant scorpion neurotoxins
to insects is useful in studying the insect sodium channel
gating, as well as being relevant to several other applications.
In order to carry out structure/activity studies, the functional
expression of such polypeptides is required. In the work reported
here, the cDNA of a new peptide from the venom of the scorpion
Buthotus saulcyi was cloned and sequenced. It codes
for a 64 residues peptide (BsaulI) with 8 highly-conserved
cysteines. This peptide shares high sequence similarity with
depressant insect toxins of other scorpion species. Large
amounts of insoluble BsaulI protein were expressed in Escherichia
coli. Purification of this peptide was carried out under
denaturing conditions. Renaturation was performed by pulsed
dilution of the denatured BsaulI in the refolding buffer.
Production of refolded Bsaul1, however, is approximately an
order of magnitude higher than that obtained with similar
scorpion depressant toxins. Intrinsic fluorescence, far-UV
circular dichroism spectra and biological activity assays
indicate that the peptide adopts a folded structure.
[Back to top]
Kinetic and Thermodynamic Properties of an Immobilized
Glucoamylase from a Mesophilic Fungus, Arachniotus citrinus
R. Perveen, M.H. Rashid, M. Saleem, A.M. Khalid and M.I.
Rajoka
Purified glucoamylase from Arachniotus citrinus was
immobilized on polyacrylamide gel with 70% yield of immobilization.
The immobilization improved the pH optima, temperature optima,
values of Km, Vmax, and activation energy.
Irreversible thermal denaturation studies of soluble and immobilized
glucoamylase indicated that immobilization decreased the entropy
and enthalpy of deactivation by magnitudes and made the immobilized
glucoamylase thermodynamically more stable.
[Back to top]
Inhibition of Pancreatic Ribonuclease A Aggregation
by Antibodies Raised Against the Native Enzyme and Its N-Terminal
Dodecapeptide
H. Younus, R. Ulbrich-Hofmann and M. Saleemuddin
Pancreatic ribonuclease A (RNase A) has been shown to aggregate
moderately and gradually at 65oC. Antibodies raised against
the dodecapeptide KETAAAKFERQG corresponding to the N-terminal
1-12 amino acid residues of RNase A (Npep) as well as native
RNase A were effective in lowering RNase A aggregation at
65oC. The antiRNase A antibodies were, however, more protective.
The binding of antiNpep antibodies to the N-terminal region
of RNase A may interfere with initiation of oligomerization
of the enzyme and consequently its aggregation. The antiRNase
A antibodies were presumably more effective in protecting
RNase A against aggregation by binding to multiple epitopes
of the enzyme including the N-terminal region and hence restricting
the interaction of the monomers.
[Back to top]
The Proline-Rich Domain and the Microtubule Binding
Domain of Protein Tau Acting as RNA Binding Domains
X.S. Wang, D.L. Wang, J. Zhao, M.H. Qu, X.H. Zhou, H.J.
He and R.Q. He
Neuronal tau, through its proline-rich domain and the microtubule
binding domain, binds to RNA non-sequence-specifically via
electrostatic interaction. This binding inhibits the activity
of tau. Tau and RNA were also found to co-localize in SH-SY5Y
cells suggesting that RNA has opportunities to interact with
tau in cells.
[Back to top]
Web-Ware Bioinformatical Analysis and Structure Modelling
of N-Terminus of Hyman Multisynthetase Complex Auxiliary Component
Protein p43
V. Deineko
Human multisynthetase complex auxiliary component, protein
p43 is an endothelial monocyte-activating polypeptide II precursor.
In this study, comprehensive sequence analysis of N-terminus
has been performed to identify structural domains, motifs,
sites of post-translation modification and other functionally
important parameters. The spatial structure model of full-chain
protein p43 is obtained.
[Back to top]
Key Residues Involved in Hsp70 Regulatory Activity
and Affect of Co-Chaperones on Mechanism of Action
Y. Tutar
Hsp70 proteins assist refolding of polypeptides in an ATP
dependent manner. Crystal structure of intact Hsp70 protein
has not been determined yet however, structures of its two
domains were solved separately. Allostery between ATPase domain
and peptide-binding domain facilitates unfolded substrate
processing. To elucidate function of key residues and affect
of other factors involved in this allosteric mechanism, a
biochemical study was undertaken.
[Back to top]
Heat Shock Proteins, Substrate Specificity and Modulation
of Function
Y. Tutar
Hsp70 is a universally conserved essential protein chaperone.
In addition to its roles in many cellular process, Hsp70 protects
cells from stress by binding partially unfolded proteins.
Therefore, Hsp70 prevents protein aggregation and prion formation.
Prions are infectious agents and are responsible for several
fatal neurodegenerative diseases. Eukaryotic cells have several
cytosolic Hsp70 isoforms, some constitutively expressed (Hsc70s),
and others expressed only when cells are exposed to stress
(Hsp70s). To determine which factors conferred functional
specificity, we constructed hybrid Hsc/Hsp chaperones. All
hybrids supported growth except those that contained the ATPase
domain derived from inducible Hsp70. Thus, regulation of peptide
binding by ATP hydrolysis must differ significantly between
Hsc- and Hsp70 isoforms. In this work, nucleotide and peptide
binding domain communication of Hsp70 proteins during their
interaction with nucleotides and peptide substrates were investigated
in vitro by using hybrid constructs.
[Back to top]
Cytotoxic Action in Myoblasts and Myotubes (C2C12)
and Enzymatic Characterization of a New Phospholipase A2
Isoform (Bj-V) from Bothrops jararacussu Venom
V.L. Bonfim, L.A. Ponce-Soto, J.C. Novello and S. Marangoni
A new PLA2 Bj-V from Bothrops jararacussu
(14039.49 Da determined by MALDI-TOF mass spectrometry) was
isolated in only one chromatographic step by HPLC ion-exchange
and its purity was confirmed by reverse phase. Amino acid
analysis showed a high content of hydrophobic and basic amino
acids as well as 14 half-cysteine residues. The N-terminal
sequence (DLWQFGQMIL KETGKIPFPY YGAYGCYCGW GGRGGKPKDG TDRCCYVHD
. . .) showed a high degree of homology with basic D49 PLA2
myotoxins from other Bothrops venoms. Bj V showed
discrete sigmoidal enzymatic behavior, with maximal activity
at pH 8.4 and 35–40°C. Full PLA2 activity
required Ca2+ (10 mM) and there was little catalytic
activity in the presence of 1 mM Ca2+.The addition
of Mn2+ or Mg2+ (10 mM) in the presence
of low (1 mM) Ca2+ slightly increased the enzyme
activity, whereas Zn2+ and Cu2+ (10
mM) diminished the activity. The substitution of Ca2+
for Mg2+ or Cu2+ also reduced the enzymatic
activity. Bj V had PLA2 activity and produced cytotoxicity
in murine C2C12 skeletal muscle myoblasts and myotubes. The
isolation of these isoforms Bj-IV [1] and Bj-V (described
herein) found in a fraction previously described as homogeneous
shows us the importance of optimization in purification techniques
in order to better understand their biological behavior.
[Back to top]
Solid-Phase Synthesis of Reactive Peptide Crosslinker
by Selective Deprotection
X. He and E. Jabbari
An effective and simple strategy for preparing peptide crosslinkers
is described. An MMP-13 degradable peptide QPQGLAK-NH2 was
prepared on the solid-phase using Fmoc chemistry. The peptide
crosslinker was synthesized on-bead by the coupling reaction
between acrylic acid and the amine groups of glutamine and
lysine residues. The synthetic procedure employed the acid-labile
Fmoc-Lys (Mtt)-OH and base-labile Fmoc-AA-OH derivatives.
Selective deprotection, of –Mtt and -Fmoc groups on-bead,
freed the amine end-groups on glutamine and lysine residues
for coupling reaction with acrylic acid while maintaining
the peptide attached to the resin. Subsequent cleavage from
the resin yielded a peptide crosslinker with two unsaturated
acrylate end-groups with high yield and purity. This method
can be generally employed for the synthesis of a wide range
of peptides with one or more reactive groups for grafting
in the fabrication of biomimetic scaffolds in tissue engineering
applications.
[Back to top]
Improvement of Bacterial Cell Selectivity of Melittin
by a Single Trp Mutation with a Peptoid Residue
W.L. Zhu, Y. Park, I.-S. Park, Y.S. Park, Y. Kim, K.-S.
Hahm and S.Y. Shin
To design melittin (ME) analogues that are not cytotoxic against
mammalian cells but which possessing potent antimicrobial
activity, we synthesized a ME analogue (ME-w) in which the
Trp-19 residue of ME was replaced by a Trp-peptoid residue
(Nhtrp). ME-w exhibited similar antimicrobial activity compared
to ME against the tested six bacteria and C. albicans.
However, it was much less cytotoxic against the hRBCs and
HeLa and NIH-3T3 cells than ME. Tryptophan fluorescence and
CD spectra revealed that the Trp-19 →
Nhtrp substitution in ME contributed to a much lower helical
assembly in an aqueous environment and structural flexibility
and exterior localization to zwitterionic membrane which modulates
its selectivity toward bacterial cells.
[Back to top]
High Level Expression and Characterization of the
Cyclophilin B Gene from the Anaerobic Fungus Orpinomyces
sp. Strain PC-2
H. Chen, X.-L. Li, H. Xu, L.G. Ljungdahl and
C.E. Cerniglia
Cyclophilins are an evolutionarily conserved family of peptidyl-prolyl
cis-trans isomerases (PPIases). A cyclophilin B (cypB)
gene from the anaerobic fungus Orpinomyces sp. strain
PC-2 was cloned and overexpressed in Escherichia coli.
It was expressed as an amino-terminal 6 x His-tagged recombinant
protein to facilitate purification. Highly purified protein
(26.5 kDa) was isolated by two chromatographic steps involving
affinity and gel filtration for biochemical studies of the
enzyme. The recombinant CypB displayed PPIase activity with
a kcat/Km of 8.9 x
10 6
M -1
s-1 at 10°C and pH 7.8. It was inhibited by
cyclosporin A (CsA) with an IC50 of 23.5 nM, similar
to those of the native protein and other cyclophilin B enzymes
from animals. Genomic DNA analysis of cypB revealed
that it was present as a single copy in Orpinomyces
PC-2 and contained two introns, indicating it has a eukaryotic
origin. It is one of the most heavily interrupted genes with
intron sequences found in anaerobic fungi. The three-dimensional
model of Orpinomyces PC-2 CypB was predicted with
a homology modeling approach using the Swiss-Model Protein
Modeling Server and three dimensional structure of human CypB
as a template. The overall architecture of the CypB molecule
is very similar to that of human CypB.
[Back to top]
Inputting Information About Amino Acid Residues in
Protein Bioinformatics: A Case Study on Predicting Helical
Regions with a Neural Network
T. Imoto
The best way to introduce information about amino acid residues
into calculations of protein bioinformatics was examined.
That was done for predicting helical regions with a neural
network. Several fundamental and instructive ways for information
processing were developed and are described.
[Back to top]
Acidic Extracellular Proteases from Microrganisms
of Fairly Acidic Niche
A. Bossi, L. Bonizzato and G. Zapparoli
The protein population secreted from three wine-contaminant
microorganisms was studied by two-dimensional electrophoresis
and screened for proteases. Proteolytic enzymes were electrophoretically
purified and their activity, optimum pH and temperature determined.
The protease of Acetobacter aceti maintained its
activity over the range of pHs 3.0-5.0, thus being of potential
biotechnological interest.
[Back to top]
A Structural-Energetic Basis for B-Cell Epitope Prediction
S.E.C. Caoili
Structural-energetic analysis of peptide and protein antigens
in the context of binding to antibody reveals fundamental
differences between the cross-reactions of antipeptide antibody
with protein and antiprotein antibody with peptide, providing
a physicochemical basis for B-cell epitope prediction as applied
to the development of peptide-based vaccines and immunodiagnostics.
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