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Protein & Peptide Letters
ISSN: 0929-8665
Protein & Peptide Letters
Volume 14, Number 2, 2007
Contents
Highly Sequence-Specific Endoribonucleases
Guest Editor: Fakhri Saïda
Editorial Pp. 101
F. Saïda
RNA Recognition and Cleavage by Sequence-Specific
Endoribonucleases Pp. 103-111
F. Saïda and B. Odaert
[Abstract]
Structure and Function of Bacterial Kid-Kis and Related
Toxin-Antitoxin Systems Pp. 113-124
M.B. Kamphuis, M.C. Monti, R.H.H. van den Heuvel, J. López
Villarejo, R. Díaz-Orejas and R. Boelens
[Abstract]
Involvement of Loops L2 and L4 of Ribonucleolytic
Toxin Restrictocin in Its Functional Activity Pp.
125-129
P. Dey, M. Tripathi and J.K. Batra
[Abstract]
An Erythroid-Enriched Endoribonuclease (ErEN) Involved
in α-Globin
mRNA Turnover Pp. 131-136
H. Liu and M. Kiledjian
[Abstract]
tRNase Z Pp. 137-145
M. Ceballos and A. Vioque
[Abstract]
A Sequence-Specific RNase Activity Derived from the
Interface of the Dimeric Immunity Protein of the ColE7 Operon
Pp. 147-150
C.-C. Liao, S.-J. Chang and K.-F. Chak
[Abstract]
Design and Synthesis of Metal-Free Artificial Ribonucleases
Pp. 151-163
L.S. Koroleva, I.Yu. Serpokrylova, V.V. Vlassov and V.N.
Silnikov
[Abstract]
General Articles
Regular Papers
Biochemical and Thermostability Features of Acetyl
Esterase Aes from Escherichia coli Pp. 165-169
T. Farias, L. Mandrich, M. Rossi and G. Manco
[Abstract]
Does Alpha-Helix Folding Necessarily Provide an Energy
Source for the Protein-Lipid Binding? Pp. 171-174
O. Gursky
[Abstract]
The Effect of Disulfide Bond on the Conformational
Stability and Catalytic Activity of Beta-Propeller Phytase
Pp. 175-183
C. Cheng, K.-B. Wong and B.L. Lim
[Abstract]
Prediction of Protein B-Factors Using Multi-Class
Bounded SVM Pp. 185-190
P. Chen, B. Wang, H.-S. Wong and D.-S. Huang
[Abstract]
Improvement of Model for Prediction of Hemagglutinin
Mutations in H5N1 Influenza Viruses with Distinguishing of
Arginine, Leucine and Serine Pp. 191-196
G. Wu and S. Yan
[Abstract]
Inhibitory Effects of Ofloxacin and Cefepime on Enzyme
Activity of 6-Phosphogluconate Dehydrogenase from Chicken
Liver Pp. 197-201
M. Erat and H. Sakiroglu
[Abstract]
Using a New Alignment Kernel Function to Identify
Secretory Proteins Pp. 203-208
H. Liu, J. Yang, D.-Q. Liu, H.-B. Shen and K.-C. Chou
[Abstract]
Crystallization Report
Crystallization and Preliminary Crystallographic Analysis
of Transgelin Pp. 209-211
X. Liao, S. Li, Z. Lou, Z. Chen and M. Bartlam
[Abstract]
Abstracts
[Back to top]
Editorial
Inactivation of RNA molecules by sequence-specific endoribonucleolytic
cleavage is a subtle mechanism by which cells coordinate,
regulate and adapt their complex gene expression patterns.
This special issue entitled "Highly Sequence-Specific
Endoribonucleases" highlights the latest findings in
the field of RNA recognition and cleavage by sequence-specific
endoribonucleases. The aim of this issue is not to provide
an exhaustive collection of sequence-specific endoribonucleases
but rather present an updated, hopefully general, view on
how these particular enzymes work. Our main goal is to attract
the attention of the audience to the exquisite molecular details
that allow these endoribonucleases to recognize and cleave
particular phosphodiester bonds confined within hundreds,
sometimes thousands, of chemically similar bonds.
This special issue opens with a manuscript analyzing and comparing
structural and biochemical data from nine highly sequence-specific
endoribonucleases that target messenger, ribosomal and transfer
RNA molecules. A special emphasis is given to phage T4 endoribonuclease
RegB and is based on our recently published solution structure
of the enzyme. This manuscript is aimed to show that despite
the absence of any relevant sequence similarities, even between
functionally related ribonucleases, and the wide variety of
biological sources; the analyzed sequence-specific endoribonucleases
adopt limited structural folds, catalyze their cleavage reaction
using the same chemistry and use a small set of amino acids
for both RNA recognition and cleavage.
Following this analysis is a manuscript that reviews structural
and functional data, obtained by the R. Boelens group, for
sequence-specific endoribonuclease Kid and its specific inhibitor
Kis. The authors compare the properties of their Kid-Kis system
to other known bacterial toxin-antitoxin systems.
A third manuscript is dedicated to ribotoxin restrictocin.
This manuscript presents original research results dealing
with the role of particular residues in loops L2 and L4 in
the activity of restrictocin.
The fourth manuscript treats the case of endoribonuclease
ErEN. This enzyme can destabilize α-globin
mRNA through a sequence-specific cleavage in its 3' untranslated
region. Interestingly, a multi-protein complex is reported
to regulate the activity of this endoribonuclease.
The fifth manuscript deals with tRNase Z. The authors describe
the diversity of tRNase Z activities, the structural basis
for substrate specificity and the potential application of
the enzyme for sequence-specific cleavage of target RNA molecules.
The manuscript written by K. Chak describes an unusual sequence-specific
ribonuclease activity derived from the dimeric interface of
the ImE7 protein (a protein encoded in the ColE7 operon).
Although the exact identity of this ribonuclease activity
is still elusive, the authors show strong evidences for the
involvement of ImE7 in the regulation of the stability of
its own mRNA through a sequence-specific cleavage.
The manuscript by L. Koroleva presents a detailed and comprehensive
view of the recent advances in the design of metal-free ribonuclease
mimics as well as sequence-specific oligonucleotide-based
artificial ribonucleases.
I hope that the audience will find in this selection of papers
not only a summary of the present understanding on how sequence-specific
endoribonucleases work but also an invitation for future investigations
in this exciting field.
Finally, I express my gratitude to all contributing authors
and reviewers and to Protein & Peptide Letters for giving
me the chance to compose this special issue.
Dr. Fakhri Saïda
Guest Editor
Protein & Peptide Letters
Center for Molecular Genetics,
University of California San Diego,
USA
E-mail: fsaida@biomail.ucsd.edu
[Back to top]
RNA Recognition and Cleavage by Sequence-Specific
Endoribonucleases
F. Saïda and B. Odaert
Inactivation of RNA molecules by sequence-specific endoribonucleolytic
cleavage is a subtle mechanism by which cells regulate gene
expression. Sequence-specific endoribonucleases can recognize
and cleave particular phosphodiester bonds confined within
hundreds/thousands of chemically similar bonds. Here, we present
a comparative analysis of the mechanisms used by endoribonucleases
to select and cleave their target RNA molecules. This analysis
is based on the very recent molecular details obtained from
the structural and/or biochemical studies of nine sequence-specific
ribonucleases that target messenger, ribosomal, and transfer
RNA molecules. This analysis shows that despite the absence
of sequence homologies and the wide diversity of biological
sources (prokaryotes, archaea and eukaryotes), the sequence-specific
ribonucleases studied here adopt limited structural folds,
catalyze their cleavage reactions using a common chemistry
and involve a very limited set of amino acids for both RNA
binding and processing.
[Back to top]
Structure and Function of Bacterial Kid-Kis and Related
Toxin-Antitoxin Systems
M.B. Kamphuis, M.C. Monti, R.H.H. van den Heuvel, J. López
Villarejo, R. Díaz-Orejas and R. Boelens
Toxin-antitoxin systems were discovered as plasmid auxiliary
maintenance cassettes. In recent years, an increasing amount
of structural and functional information has become available
about the proteins involved, allowing the understanding of
bacterial cell growth inhibition by the toxins on a molecular
level. A well-studied TA system is formed by the proteins
Kid and Kis, encoded by the parD operon of the Escherichia
coli plasmid R1. The toxicity of Kid has been related
to its endoribonuclease activity, which is counteracted by
binding of the antitoxin Kis at the proposed active site.
In this review, the structural studies on the Kid-Kis system
are compared to those of three related toxin-antitoxin systems:
MazF-MazE, CcdB-CcdA and RelE-RelB.
[Back to top]
Involvement of Loops L2 and L4 of Ribonucleolytic
Toxin Restrictocin in Its Functional Activity
P. Dey, M. Tripathi and J.K. Batra
Restrictocin, a member of the fungal ribotoxin family, specifically
cleaves a single phosphodiester bond in the 28S rRNA and potently
inhibits eukaryotic protein synthesis. The long loops in restrictocin
molecule have been shown structurally to be involved in target
RNA recognition. In this study we have investigated the role
of some putative substrate-interacting residues in loops L2
and L4, spanning residues 36-48 and 99-117, respectively in
restrictocin catalysis. The residues Lys42, Ser46, Pro48 and
Lys111 were individually mutated to alanine to probe their
role in restrictocin function. The mutation of Lys111 to alanine,
although did not affect the ribonucleolytic activity, rendered
the toxin completely inactive in inhibiting translation in
HeLa cells as well in an in vitro cell free system.
The loop L4 in restrictocin appears to be more critical compared
to loop L2 for its interaction with the specific substrate.
[Back to top]
An Erythroid-Enriched Endoribonuclease (ErEN) Involved
in α-Globin
mRNA Turnover
H. Liu and M. Kiledjian
Messenger RNA (mRNA) decay utilizes both exoribonucleolytic
and endoribonucleolytic enzymes where the latter are generally
more prone to be transcript-specific. An erythroid-enriched
endoribonuclease, ErEN, can destabilize the α-globin
mRNA through directing a site-specific cleavage within the
3' untranslated region (3' UTR) both in vitro and
in vivo. ErEN activity is sequence- and/or local
structure-specific as the minimal recognition/cleavage sequence
can be conferred onto a heterologous RNA and mutations at
the cleavage site immunize the mRNA from ErEN hydrolysis.
Interestingly, the ErEN cleavage activity is regulated by
an mRNA stability complex (α-complex).
An interaction between the α-complex
and the poly(A)-binding protein (PABP) accentuates α-complex
binding to a region overlapping the ErEN cleavage site and
further prevents premature ErEN-mediated decay. At present
the identity of ErEN remains elusive, yet its identification
will provide mechanistic and functional insights into the
general processes of endoribonuclease-mediated mRNA turnover
and erythropoiesis.
[Back to top]
tRNase Z
M. Ceballos and A. Vioque
Endonuclease tRNase Z catalyzes the generation of the mature
3’ end of tRNA precursors through specific endonucleolytic
cleavage. The enzyme has been characterized from organisms
representative of all domains of life as well as from organelles,
and the crystal structure of three bacterial enzymes has been
determined. This review presents an overview of its properties
and what is known about its structure, substrate recognition,
cleavage site definition, and potential practical applications.
[Back to top]
A Sequence-Specific RNase Activity Derived from the
Interface of the Dimeric Immunity Protein of the ColE7 Operon
C.-C. Liao, S.-J. Chang and K.-F. Chak
Recently, two sequence-specific cleavage sites were found
in the ceiE7 gene of the cea-cei-cel polycistronic
transcript from the ColE7 operon. The crystal structure of
the ColE7 immunity protein (ImE7) suggested that a novel ribonuclease
active site is created at the interface of the dimeric structure
of the protein. Frame shift mutation of the ceiE7
gene and mutation of histidine residues at the putative active
site of the dimeric ImE7 protein respectively abolished and
significantly reduced the observed ribonucleolytic cleavage
indicating that the dimeric ImE7 protein is indeed involved
in this sequence-specific cleavage at the ceiE7 mRNA.
It is noteworthy that E. coli S-30 cell extracts
must be added to the in vitro reactions in order
to detect this ribonucleolytic cleavage. In addition, mutation
of the T1 stem-loop structure located between the ceiE7
and the celE7 genes completely turned off the ribonuclease
activity in vivo, implying that the T1 stem-loop
structure might participate in mediating the formation of
a degradosome-like complex required for this specific ribonucleolytic
activity.
[Back to top]
Design and Synthesis of Metal-Free Artificial Ribonucleases
L.S. Koroleva, I.Yu. Serpokrylova, V.V. Vlassov and V.N.
Silnikov
The present review is aimed at giving a general overview of
our results in the field of designing and synthesizing simple
peptide-like molecules that mimic structural and functional
aspects of natural ribonucleases, as well as designing oligonucleotide-based
artificial ribonucleases.
[Back to top]
Biochemical and Thermostability Features of Acetyl
Esterase Aes from Escherichia coli
T. Farias, L. Mandrich, M. Rossi and G. Manco
Previously we characterized an acetyl-esterase from Escherichia
coli, formally Aes, from a thermodynamic point of view
in comparative studies with thermophilic homologs. Since the
enzyme appeared unusually resistant to the thermal denaturation
we analysed the kinetic behaviour with respect to the temperature.
The enzyme displays a surprising optimal temperature at 65°C,
showing a specific activity of 250 U/mg using pNP-butanoate
as substrate, but a low kinetic stability at the same temperature
(t½
of inactivation=5 min). By a random mutagenesis approach we
searched for mutated versions of Aes with increased thermostability.
We found the mutant T74A, which shows the same specific activity
of wild type but a t½
of inactivation of 30 min at 65 °C.
[Back to top]
Does Alpha-Helix Folding Necessarily Provide an Energy
Source for the Protein-Lipid Binding?
O. Gursky
Lipid-induced α-helix
folding, which occurs in many lipid surface-binding proteins
and peptides such as apolipoproteins and synucleins, has been
proposed to provide an energy source for protein-lipid interactions.
We propose that in a system comprised of a phospholipid surface
and a small polypeptide that is unfolded in solution and binds
reversibly to lipid surface, helical folding involves expenditure
of free energy as compared to a similar polypeptide that is
α-helical
in solution. This is a consequence of the entropic cost of
helix folding that is illustrated in a simple thermodynamic
model and exemplifies the general ”key-into-lock”
paradigm of protein-ligand binding. Even though this simple
model does not explicitly address the protein-induced lipid
re-arrangement and may not directly apply to large proteins
that undergo significant tertiary structural changes upon
lipid binding, it suggests that the notion of helix folding
as an energy source for lipid binding should be treated with
caution.
[Back to top]
The Effect of Disulfide Bond on the Conformational
Stability and Catalytic Activity of Beta-Propeller Phytase
C. Cheng, K.-B. Wong and B.L. Lim
While beta-propeller phytases (BPPs) from Gram-positive bacteria
do not carry disulfide bonding, their counterparts from Gram-negative
bacteria contain cysteine residues that may form disulfide
bonds. By molecular modeling, two amino acid residues of B.
subtilis 168 phytase (168PhyA), Ser-161 and Leu-212,
were mutated to cysteine residues. Although the double cysteine
mutant was secreted from B. subtilis at an expression
level that was 3.5 times higher than that of the wild type,
the biochemical and enzymatic properties were unaltered. In
CD spectrometric analysis, both enzymes exhibited similar
apparent melting temperatures and mid-points of transition
under thermal and guanidine hydrochloride induced denaturation,
respectively. In enzyme assays, the mutant phytase exhibited
a poor refolding ability after thermal denaturation. We postulate
that the disulfide bond in BPP sequences from Gram-negative
bacteria is beneficial to their stability in the periplasmic
compartment. In contrast, the lack of periplasmic space in
Bacillus species and the fact that Bacillus
BPPs are released extracellularly may render disulfide bonds
unnecessary. This may explain why in evolution, BPPs in Bacillus
species do not carry disulfide bonds.
[Back to top]
Prediction of Protein B-Factors Using Multi-Class
Bounded SVM
P. Chen, B. Wang, H.-S. Wong and D.-S. Huang
In this paper, we propose the adoption of the bounded support
vector machine (BSVM) to predict the B-factors of residues
based on a number of distinctive properties of residues. Due
to the ability of multi-class classification of the BSVM,
we can elaborately distinguish our targets and obtain relatively
higher accuracy.
[Back to top]
Improvement of Model for Prediction of Hemagglutinin
Mutations in H5N1 Influenza Viruses with Distinguishing of
Arginine, Leucine and Serine
G. Wu and S. Yan
In a continuation of our attempt to predict mutations in proteins
from influenza A virus, this study attempted to answer the
question of whether distinguishing between arginine, leucine
and serine can improve the predictability as these residues
are governed by different probabilistic mechanism translating
from RNA codons to amino acids. In this study, we made the
prediction based on the mutation relation among 299 H5N1 hemagglutinins
of influenza A virus. Then, we compared the results based
on the distinguishing of arginine, leucine and serine with
the results without distinguishing of arginine, leucine and
serine. The results show that the prediction together with
distinguishing between arginine, leucine and serine is better
than prediction without distinguishing between these residues.
[Back to top]
Inhibitory Effects of Ofloxacin and Cefepime on Enzyme
Activity of 6-Phosphogluconate Dehydrogenase from Chicken
Liver
M. Erat and H. Sakiroglu
In this study, effects of some antibiotics, namely, ofloxacin,
cefepime, cefazolin, and ampicillin on the in vitro
enzyme activity of 6-phosphogluconate dehydrogenase have been
investigated. For this purpose, 6-phosphogluconate dehydrogenase
was purified from chicken liver 535-fold with a yield of 18%
by using ammonium sulphate precipitation, 2',5'-ADP Sepharose
4B affinity chromatography, and Sephadex G-200 gel filtration
chromatography. In order to check the purity of the enzyme,
SDS polyacylamide gel electrophoresis (SDS-PAGE) was performed.
This analysis revealed a highly pure enzyme band on the gel.
Among the antibiotics, ofloxacin and cefepime exhibited inhibitory
effects, but cefazolin and ampicillin showed neither important
inhibitory nor activatory effects on the enzyme activity.
The measured I50 values by plotting activity percent
vs. inhibitor concentration, [I50] were
0.1713 mM for ofloxacin and 6.0028 mM for cefepime. Inhibition
constants, Ki, for ofloxacin and cefepime were
also calculated as 0.2740 ± 0.1080 mM and 12.869 ±
16.6540 mM by means of Lineweaver-Burk graphs, and inhibition
types of the antibiotics were found out to be non-competitive
and competitive, respectively. It has been understood from
the calculated inhibitory parameters that the purified chicken
enzyme has been quite inhibited by these two antimicrobials.
[Back to top]
Using a New Alignment Kernel Function to Identify
Secretory Proteins
H. Liu, J. Yang, D.-Q. Liu, H.-B. Shen and K.-C. Chou
As the knowledge of protein signal peptides can be used to
reprogram cells in a desired way for gene therapy, signal
peptides have become a crucial tool for researchers to design
new drugs for targeting a particular organelle to correct
a specific defect. To effectively use such a technique, however,
we have to develop an automated method for fast and accurately
predicting signal peptides and their cleavage sites, particularly
in the post-genomic era when the number of protein sequences
is being explosively increased. To realize this, the first
important thing is to discriminate secretory proteins from
non-secretory proteins. On the basis of the Needleman-Wunsch
algorithm, we proposed a new alignment kernel function. The
novel approach can be effectively used to extract the statistical
properties of protein sequences for machine learning, leading
to a higher prediction success rate.
[Back to top]
Crystallization and Preliminary Crystallographic Analysis
of Transgelin
X. Liao, S. Li, Z. Lou, Z. Chen and M. Bartlam
Transgelin was solubly expressed in E. coli. Crystals
of transgelin have been grown at 291K using sodium formate
or PEG-4000 as precipitants. X-ray diffraction by the crystal
extends to 2.3 Å resolution. The crystal belongs to
the space group P21, with the unit cell parameters
a=39.3, b=61.9, c=56.0 Å and β=90.2°.
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