Protein and Peptide Letters - Volume 14, Issue 2, 2007

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 metalfree 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.

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 sequencespecific 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.

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.

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.

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.

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. Z.

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 stemloop structure might participate in mediating the formation of a degradosome-like complex required for this specific ribonucleolytic activity.

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.

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 (t1/2 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 t1/2 of inactivation of 30 min at 65 °C.

Lipid-induced α -helix folding, which occurs in many lipid surface-binding proteins and peptides such as apo- lipoproteins 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.

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.

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.

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.

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 noncompetitive 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.

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.

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 .