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Protein & Peptide Letters
ISSN: 0929-8665
Protein & Peptide Letters
Volume 14, Number 4, 2007
Contents
Copper (II) Complexes with Ac-HXH-NHMe (X=Gly,
Ala and Aib) Peptide Motifs: Influence of Increasing CH3
Groups at Cα
of Residue X on the Coordination in Solution Pp.
305-310
R.K. Singh, V.C. Srivastava and U.P. Singh
[Abstract]
Proteomic Analysis of Jasmonic Acid-Regulated Proteins
in Rice Leaf Blades Pp. 311-319
T. Mahmood, M. Kakishima and S. Komatsu
[Abstract]
Reevaluating the Capability of Taq DNA Polymerase:
Long PCR Amplification Pp. 321-323
H. Lee, K.-N. Kim and Y.K. Chae
[Abstract]
The Blomia tropicalis Allergens
Pp. 325-333
K.Y. Chua, N. Cheong, I.-C. Kuo, B.W. Lee, F.C. Yi, C.-H.
Huang and L.N. Liew
[Abstract]
Probing the Effect of Mutations on Cytochrome c
Stability Pp. 335-339
F. Agueci, F. Polticelli, F Sinibaldi, M.C. Piro, R. Santucci
and L. Fiorucci
[Abstract]
Functional Characterization of Streptomyces coelicolor
FtsY Pp. 341-345
H.-J. Dong, X.-L. Shen, Y.-D. Li and Y.-Q. Li
[Abstract]
A Short C-Terminal Region of Alpha-Synuclein Protects
a (R/S) Non-specific Esterase from Archaeglobus fulgidus
Pp. 347-351
S.B. Kim, M. Kang, Y. Ryu and T.D. Kim
[Abstract]
Conformational Analysis of Ac-NPGQ-NH2
and Ac-VPaH-NH2 by Vibrational Circular Dichroism
Spectroscopy Combined with Molecular Dynamics and Quantum
Chemical Calculations Pp. 353-359
A. Borics, R.F. Murphy and S. Lovas
[Abstract]
The Role of Bax/Bcl-2 and Pro-Caspase Peptides in
Hypoxia/Reperfusion-Dependent Regulation of MAPKERK:
Discordant Proteomic Effect of MAPKp38
Pp. 361-371
J.J. Haddad
[Abstract]
Discordant Tissue-Specific Expression of SAPK/MAPKJNK
Related Cofactors in Hypoxia and Hypoxia/Reoxygenation in
a Model of Anoxia-Tolerance Pp. 373-380
J.J. Haddad
[Abstract]
Molecular Simulations of Rhodopsin Tetrameter
Pp. 381-387
M. Witt, J. Ciarkowski and C. Czaplewski
[Abstract]
New Hope for the Diagnosis and Therapy of Alzheimer’s
Disease Pp. 389-393
B. Kastenholz
[Abstract]
Characterization of a Novel ADPase in Bothrops
jararaca Snake Venom Involved in Nucleotide Hydrolysis
Pp. 395-402
A. Teixeira-Ferreira and E.W. Alves
[Abstract]
Crystallization Reports
Crystallization and Preliminary Crystallographic Study
of DNA Polymerase from Pyrococcus furiosus Pp.
403-405
H. Nishida, M. Tanabe, Y. Ishino, T. Oyama and K. Morikawa
[Abstract]
Crystallization and Preliminary X-Ray Diffraction
Analysis of PD-L1, a Highly Glycosylated Ribosome Inactivating
Protein with DNase Activity Pp. 407-409
A. Ruggiero, A. Chambery, A.D. Maro, A. Mastroianni, A.
Parente and R. Berisio
[Abstract]
Abstracts
[Back to top]
Copper (II) Complexes with Ac-HXH-NHMe (X=Gly, Ala
and Aib) Peptide Motifs: Influence of Increasing CH3
Groups at Cα
of Residue X on the Coordination in Solution
R.K. Singh, V.C. Srivastava and U.P. Singh
The interaction of Cu(II) ion with small peptides has been
an interesting subject to clarify the role of copper in detail.
As various Cu(II)-oligopeptide complexes can also be good
models for the active centers of metalloenzymes, complexes
of tripeptide and tetrapeptides are frequently investigated
instead of the complexes of large peptides. The histidine
side-chains of various metalloproteins frequently take part
in the copper(II) coordination. Accordingly, we studied the
co-ordination of Cu(II) to the N and C terminal protected
tripeptide ligands LA (Ac-HisGlyHis-NHMe), LB
(Ac-HisAlaHis-NHMe) and LC (Ac-HisAibHis-NHMe)
in aqueous solution potentiometrially in order to determine
the effect of Cα
methyl groups at middle residue acid on the ligation of the
backbone NH and also on histidine’s Nim of
coordination. Species distribution curves indicates that in
acidic pH, all three peptides behave as bidentate ligands
and a macrochelate forms on the metal coordination with the
two histidine imidazolyl N. This coordination remains unaffected
with the +I effect of increasing CH3 groups at
Cα
of middle residue. In the pH range 4-8, the tridentate coordination
from the peptide is seen in ligand LA and LB
while it is absent in LC due to +I effect of two
Cα
methyl groups at middle residue as they makes N-terminal NH
deprotonation difficult in this pH range and it takes place
along with C terminal NH and only 4N coordinated species formed
at higher pH. These 4N (Nim, N-, N-,
Nim) coordinated species are formed by all the
three ligands at higher pH values.
[Back to top]
Proteomic Analysis of Jasmonic Acid-Regulated Proteins
in Rice Leaf Blades
T. Mahmood, M. Kakishima and S. Komatsu
Jasmonates play a critical role in plant defense against pathogens
through regulation of the expression of defense-related genes.
To study the role of jasmonic acid (JA) in the rice self-defense
mechanism, a proteomic approach was applied. When 3-week-old
rice cv. Java 14 was treated with 100 µM JA for 3 days,
numerous necrotic brown spots were observed on the leaf blade.
Three-week-old rice was treated with JA and proteins from
cytosolic and membrane fractions of leaf blade were separated
by two-dimensional polyacrylamide gel electrophoresis. A total
of 305 proteins were detected in both cytosolic and membrane
fractions. When rice plant was treated with 100 µM JA
for 2 days, 12 proteins were up-regulated and 2 proteins were
down-regulated. Out of them, 8 proteins were changed in dose
dependence manner, while 4 proteins were changed in a time
course manner. Among them, pathogenesis-related protein 5
(PR5) and probenazole inducible protein 1 (PBZ1) were significantly
induced by 100 µM JA for 2 days. These results suggest
that PR5 and PBZ1 are important proteins expressed down-stream
of JA signals in rice cv. Java 14.
[Back to top]
Reevaluating the Capability of Taq DNA Polymerase:
Long PCR Amplification
H. Lee, K.-N. Kim and Y.K. Chae
We tested the ability of Taq DNA polymerase (Taq)
to amplify long DNA fragments and showed that, if the conditions
were set properly, Taq could successfully perform
the “long PCR” up to 24 kb. The conditions include:
(1) longer primers, (2) a 2-step cycling, and (3) a “long
buffer.” We propose that the most important requirements
are the survival rate of Taq at high temperatures
and that of the primers against the 5’ to 3’ exonuclease
activity of Taq.
[Back to top]
The Blomia tropicalis Allergens
K.Y. Chua, N. Cheong, I.-C. Kuo, B.W. Lee, F.C. Yi, C.-H.
Huang and L.N. Liew
Blomia tropicalis allergens are the most important
mite allergens in tropical regions. Most of them only have
30-40% sequence identity with their Dermatophagoides
counterparts and they share low IgE cross reactivity and exhibit
different immunobiology. Unlike the pyroglyphid counterparts,
Blo t 5 is the major allergen whereas Blo t 1 only has modest
allergenicity.
[Back to top]
Probing the Effect of Mutations on Cytochrome c
Stability
F. Agueci, F. Polticelli, F Sinibaldi, M.C. Piro, R. Santucci
and L. Fiorucci
Although the tertiary structures of mitochondrial cytochromes
c (cyts c) seem to be remarkably similar,
there are variations in their amino acid sequences, stability
and functional properties. GdnHCl-induced unfolding experiments
on engineered yeast and horse cyt c were carried
out with the aim to to clarify, at molecular level, some aspects
concerning the stability of this class of proteins. The results
obtained are discussed in the light of the three-dimensional
structures of the two proteins.
[Back to top]
Functional Characterization of Streptomyces coelicolor
FtsY
H.-J. Dong, X.-L. Shen, Y.-D. Li and Y.-Q. Li
This study indicated that the N-terminus was dispensable for
FtsY GTPase activity, and that the N-domain plays an essential
role in the GTPase activity of the NG domain. In addition,
the S.scoelicolor FtsY was able to restore function
in an E. coli mutant. However, its NG domain was
unable to play any roles.
[Back to top]
A Short C-Terminal Region of Alpha-Synuclein Protects
a (R/S) Non-specific Esterase from Archaeglobus fulgidus
S.B. Kim, M. Kang, Y. Ryu and T.D. Kim
We show that α-synuclein
could assist the molecular activity of a ketoprofen-(R/S)
nonspecific esterase from Archaeglobus Fulgidus.
Specifically, several synthetic peptides from α-synuclein,
each having random coil conformation in far-UV spectra, could
protect the enzyme activity against stress conditions such
as heat and organic solvents.
[Back to top]
Conformational Analysis of Ac-NPGQ-NH2
and Ac-VPaH-NH2 by Vibrational Circular Dichroism
Spectroscopy Combined with Molecular Dynamics and Quantum
Chemical Calculations
A. Borics, R.F. Murphy and S. Lovas
Conformational properties of two potentially β-turn
forming peptides were determined using a strategy which combines
MD simulations, IR and VCD spectroscopy and quantum chemical
calculations. This strategy could be a useful alternative
for solution conformational analysis of short flexible peptides
and could help to identify VCD features which are as yet unknown.
[Back to top]
The Role of Bax/Bcl-2 and Pro-Caspase Peptides in
Hypoxia/Reperfusion-Dependent Regulation of MAPKERK:
Discordant Proteomic Effect of MAPKp38
J.J. Haddad
Background: The molecular regulation of MAPKs and
apoptosis was investigated in a model of hypoxia-tolerance.
Survival of neurons in Chrysemys picta bellii, an
anoxia-tolerant turtle, involves a reduction in energy metabolism.
The biochemical/physiological mechanisms of anoxia tolerance
have been examined at the level of ion transport and ATP turnover.
However, changes in the phosphorylation state of key enzymes
and kinases, mainly, MAPKs, may occur during anoxia, thereby
reversible protein phosphorylation could be a critical factor
and major mechanism of metabolic re-organization for enduring
anaerobiosis.
Methods: If a turtle were to undergo hypoxia akin
to that experienced in its native habitat, it was placed in
a glass aquarium filled with water to within a half inch of
the top. After the turtle was anesthetized, through extended
hypoxia or anesthesia, the animal was sacrificed by decapitation.
The brain was then excised and placed in anoxic artificial
cerebrospinal fluid. Total protein extraction was performed
by homogenizing brain in a buffer, followed by threonine and
tyrosine phos-phorylation determination of MAPKs, and caspase
activity.
Results: MAPKp38
was decreased after reoxygenation following 1 day and 1 week
hypoxia. The effect of hypoxia on the phosphorylation of MAPKERK
was biphasic: Enhancement at 5h and inhibition at 6 weeks.
Pro-caspases 8/9 were unchanged by hypoxia until increasing
at 6 weeks. Both pro-caspases were upregulated by reoxygenation
at 1 day or 6 weeks hypoxia. Neither hypoxia nor reoxygenation
induced the cleavage of pro-caspases 8/9 into p20 and p10,
respectively. Furthermore, hypoxia induced Bax at 3 days and
1 week, and reoxygenation increased Bax ≈ 4-fold at
1 day. Although the expression of Bcl-2 was slightly increased
by hypoxia, [Bcl-2] was 3-4-fold smaller in comparison with
Bax.
Conclusion: These results indicate that hypoxia up-regulates
MAPKERK but not MAPKp38;
hypoxia/reperfusion increases the expression of caspases and
pro-apoptotic cofactors. The patterns of MAPK regulation suggest
the significance of these kinases in cellular adaptation to
oxygen deprivation with biomedical correlations, and thereby
identify novel natural responsive signaling cofactors in Chrysemys
picta bellii with potential pharmacologic and clinical
applications.
[Back to top]
Discordant Tissue-Specific Expression of SAPK/MAPKJNK
Related Cofactors in Hypoxia and Hypoxia/Reoxygenation in
a Model of Anoxia-Tolerance
J.J. Haddad
Background: Pursuant to establishing the proteomic
distribution of MAPKERK/MAPKp38
in the brain in a model of hypoxia-tolerance [Haddad, Protein
Pept Lett, In press, 2007], I therein exclusively report the
differential expression of MAPKJNK
and related upstream and downstream kinases in various organs
of the anoxia-tolerant turtle. Despite the fact that the aforementioned
mechanisms involved dual expression of MAPKERK,
the mechanistic distribution of MAPKJNK
has not been previously unraveled. Changes in the phosphorylation
state of MAPKs may occur during anoxia, thereby reversible
protein phosphorylation could be a critical factor and major
mechanism of metabolic reorganization for enduring anaerobiosis.
Methods: If a turtle were to undergo hypoxia akin
to that experienced in its native habitat, it was placed in
a glass aquarium filled with water to within a half inch of
the top. After the turtle was anesthetized, through extended
hypoxia or anes-thesia, the animal was sacrificed by decapitation.
The brain and other organs were then excised and placed in
anoxic artificial cerebrospinal fluid. Total protein extraction
was performed by homogenizing various organs in a suitable
buffer, followed by determination of the phosphorylation states
of SEK-1/MKK-4, SAPK/MAPKJNK
and c-Jun activating protein (AP)-1.
Results: SEK-1/MKK-4 expression was mild in the cortex
as compared with the manifold hypoxic (2h) induction in the
liver. Continuous imposition of hypoxia (1 day – 1 week)
increased the expression of SEK-1/MKK-4, thereafter declined
at 3 weeks hypoxia. Hypoxia/reoxygenation weakly induced SEK-1/MKK-4
expression in cortex, in contrast with a strong induction
in the liver, but not in other organs. Hypoxia (2h –
3 weeks) did not induce SAPK/MAPKJNK
expression in cortex, despite prominent increase in liver,
with mild reoxygenation effect. The normoxic induction of
c-Jun AP-1 in cortex and rest of brain (ROB) was reduced with
imposition of hypoxia (2h – 1 week). Furthermore, hypoxia
(2h – 3 weeks) upregulated expression of c-Jun AP-1
in liver, heart and spleen, an effect abrogated with hypoxia/reoxygenation.
Conclusion: These results indicate that hypoxia differentially
up-regulates the expression of MAPKJNK-related
cofactors with organ-specific distribution. Since these modules
are involved with neuroprotection in Chrysemys picta bellii,
the expression of MAPKs bears relative mechanisms of specific
responses to hypoxia tolerance.
[Back to top]
Molecular Simulations of Rhodopsin Tetrameter
M. Witt, J. Ciarkowski and C. Czaplewski
Di/oligomerization of G-protein coupled receptors (GPCRs)
is well established, however very little is known regarding
the interaction details. Current paper presents results of
molecular dynamics simulations of theoretical model of rhodopsin
tetramer with transducine (Gt) in lipid bilayer. Ligand-protein
and receptor-receptor interactions have been proposed.
[Back to top]
New Hope for the Diagnosis and Therapy of Alzheimer’s
Disease
B. Kastenholz
Improperly folded metal cofactor-containing proteins (e.g.,
copper chaperone for superoxide dismutase, CCS) are believed
to play a key role in several protein-misfolding diseases
(e.g., Alzheimer’s disease or Amyotrophic Lateral Sclerosis)
because under regular physiological conditions, metallochaperones
activate or stabilize the native conformation of important
metalloproteins (e.g., superoxide dismutase) in certain cellular
processes. For an improved diagnosis and therapy of neurodegenerative
diseases, new methodologies have to be developed that enable
a well-defined differentiation between properly folded and
inactive metalloproteins in clinical samples. In the literature
it is reported that different high molecular mass metal-containing
proteins were isolated in brain samples from Alzheimer’s
patients and in vegetables by using a 2-dimensional polyacrylamide
gel electrophoresis (2-DE) procedure. In the present article,
selected results of these studies are scrutinized and compared
with some results obtained by a standardized method termed
‘quantitative preparative native continuous polyacrylamide
gel electrophoresis (QPNC-PAGE)’. Conclusively, QPNC-PAGE
is a highly efficient approach used by biochemists to resolve
native and denatured metalloproteins (MW 6 - ≥ 200 kDa)
in complex protein mixtures.
[Back to top]
Characterization of a Novel ADPase in Bothrops
jararaca Snake Venom Involved in Nucleotide Hydrolysis
A. Teixeira-Ferreira and E.W. Alves
Snake venoms hydrolyze several phosphorylated substrates.
However, not is clearly understood which enzyme(s) is (are)
involved in these process. Here, we propose the existence
of an independent ADPase activity. In addition, we studied
the reactions mechanism of nucleotide hydrolysis. This system
resembles membrane ecto-nucleotidases and acts with a multi
enzymatic complex transforming ATP in adenosine without the
accumulation of intermediates.
[Back to top]
Crystallization and Preliminary Crystallographic Study
of DNA Polymerase from Pyrococcus furiosus
H. Nishida, M. Tanabe, Y. Ishino, T. Oyama and K. Morikawa
A new member of archaeal DNA polymerase from Pyrococcus
furiosus was crystallized. Diffraction data to 3.1 Å
of the selenomethionine-derivatized crystal were collected,
and preliminary crystallographic study has been completed.
The crystal belongs to the space group C2 with unit
cell parameters of α
= 93.2 Å, b = 124.9 Å, c = 87.7
Å, α
= 90°, β
= 109.7°, and γ
= 90°. Assuming the presence of one molecule in the asymmetric
unit, the solvent content of the crystal is estimated to be
54%, corresponding to a Matthews coefficient VM
of 2.7Å3 Da-1.
[Back to top]
Crystallization and Preliminary X-Ray Diffraction
Analysis of PD-L1, a Highly Glycosylated Ribosome Inactivating
Protein with DNase Activity
A. Ruggiero, A. Chambery, A.D. Maro, A. Mastroianni, A.
Parente and R. Berisio
PD-L1 is a highly glycosylated type 1 ribosome inactivating
protein, from Phytolacca dioica leaves, with the
peculiarity to act also as a DNase. PD-L1 has been successfully
crystallized using vapour diffusion and seeding techniques.
Crystals belong to the monoclinic C2 space group, with unit
cell dimensions a=161.01, b=34.73, c=120.63 Å, β=127.99°.
Two molecules are present in the asymmetric unit. Phase determination
has been achieved using molecular replacement.
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