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1.  Structural and degradative aspects of ornithine decarboxylase antizyme inhibitor 2 
FEBS Open Bio  2014;4:510-521.
Highlights
•AZIN2, unlike ornithine decarboxylase, exists as a monomer in solution.•Conserved residues among AZIN2 orthologs are critical for the binding to antizymes (AZs).•Substitution of the conserved residues affects the ability of AZIN2 to modulate polyamine levels.•AZIN2 and AZs are extremely labile proteins, which mutually stabilize each other.•Other proteolytic systems, besides the 26S proteasome, might be involved in AZIN2 degradation.
Ornithine decarboxylase (ODC) is the key enzyme in the polyamine biosynthetic pathway. ODC levels are controlled by polyamines through the induction of antizymes (AZs), small proteins that inhibit ODC and target it to proteasomal degradation without ubiquitination. Antizyme inhibitors (AZIN1 and AZIN2) are proteins homologous to ODC that bind to AZs and counteract their negative effect on ODC. Whereas ODC and AZIN1 are well-characterized proteins, little is known on the structure and stability of AZIN2, the lastly discovered member of this regulatory circuit. In this work we first analyzed structural aspects of AZIN2 by combining biochemical and computational approaches. We demonstrated that AZIN2, in contrast to ODC, does not form homodimers, although the predicted tertiary structure of the AZIN2 monomer was similar to that of ODC. Furthermore, we identified conserved residues in the antizyme-binding element, whose substitution drastically affected the capacity of AZIN2 to bind AZ1. On the other hand, we also found that AZIN2 is much more labile than ODC, but it is highly stabilized by its binding to AZs. Interestingly, the administration of the proteasome inhibitor MG132 caused differential effects on the three AZ-binding proteins, having no effect on ODC, preventing the degradation of AZIN1, but unexpectedly increasing the degradation of AZIN2. Inhibitors of the lysosomal function partially prevented the effect of MG132 on AZIN2. These results suggest that the degradation of AZIN2 could be also mediated by an alternative route to that of proteasome. These findings provide new relevant information on this unique regulatory mechanism of polyamine metabolism.
doi:10.1016/j.fob.2014.05.004
PMCID: PMC4066113  PMID: 24967154
AZ, antizyme; AZBE, antizyme-binding element; AZIN, antizyme inhibitor; ERGIC, endoplasmic reticulum-Golgi intermediate compartment; ODC, ornithine decarboxylase; GDT_TS, global distance test total score; HA, hemagglutinin; HEK, human embryonic kidney; PAGE, polyacrylamide gel electrophoresis; RMSD, root-mean-square deviation; TGN, trans-Golgi network; Antizyme; Antizyme-binding element; Homology modeling; Polyamines; Protein degradation; Proteasome inhibitors
2.  Identification and modeling of a phosphatase-like domain in a tRNA 2′-O-ribosyl phosphate transferase Rit1p 
Cell Cycle  2011;10(20):3566-3570.
Cytoplasmic initiator tRNAs from plants and fungi are excluded from participating in translational elongation by the presence of a unique 2′-phosphoribosyl modification of purine 64, introduced posttranscriptionally by the enzyme Rit1p. Members of the Rit1p family show no obvious similarity to other proteins or domains, there is no structural information available to guide experimental analyses, and the mechanism of action of this enzyme remains a mystery. Using protein fold recognition, we identified a phosphatase-like domain in the C-terminal part of Rit1p. A comparative model of the C-terminal domain was constructed and used to predict the function of conserved residues and to propose the mechanism of action of Rit1p. The model will facilitate experimental analyses of Rit1p and its interactions with the initiator tRNA substrate.
doi:10.4161/cc.10.20.17857
PMCID: PMC3266182  PMID: 22030622
fold recognition; homology modeling; tRNA modification; Rit1p; bioinformatics
3.  Identification of a Coding Sequence and Structure Modeling of a Glycine-Rich RNA-Binding Protein (CmGRP1) from Chelidonium majus L. 
The family of glycine-rich plant proteins (GRPs) is a large and complex group of proteins that share, as a common feature, the presence of glycine-rich domains arranged in (Gly)n-X repeats that are suggested to be involved in protein–protein interactions, RNA binding, and nucleolar targeting. These proteins are implicated in several independent physiological processes. Some are components of cell walls of many higher plants, while others are involved in molecular responses to environmental stress, and mediated by post-transcriptional regulatory mechanisms. The goals of this study are to identify the coding sequence of a novel glycine-rich RNA-binding protein from Chelidonium majus and to propose its structural model. DNA fragments obtained using degenerate PCR primers showed high sequence identities with glycine-rich RNA-binding protein coding sequences from different plant species. A 439-bp nucleotide sequence is identified coding for a novel polypeptide composed of 146 amino acids, designated as CmGRP1 (C. majus glycine-rich protein 1), with a calculated MW of 14,931 Da (NCBI GenBank accession no. HM173636). Using NCBI CDD and GeneSilico MetaServer, a single conserved domain, the RNA recognition motif (RRM), was detected in CmGRP1. The C-terminal region of CmGRP1 is a glycine-rich motif (GGGGxxGxGGGxxG), and it is predicted to be disordered. Based on a 1fxl crystal structure, a 3D model of CmGRP1 is proposed. CmGRP1 can be classified as a class IVa plant GRP, implicated to play a role in plant defense.
doi:10.1007/s11105-012-0510-y
PMCID: PMC3881573  PMID: 24415842
Glycine-rich protein; RNA-binding; Papaveraceae; Chelidonium majus; Coding sequence; 3D model
4.  Molecular evolution of dihydrouridine synthases 
BMC Bioinformatics  2012;13:153.
Background
Dihydrouridine (D) is a modified base found in conserved positions in the D-loop of tRNA in Bacteria, Eukaryota, and some Archaea. Despite the abundant occurrence of D, little is known about its biochemical roles in mediating tRNA function. It is assumed that D may destabilize the structure of tRNA and thus enhance its conformational flexibility. D is generated post-transcriptionally by the reduction of the 5,6-double bond of a uridine residue in RNA transcripts. The reaction is carried out by dihydrouridine synthases (DUS). DUS constitute a conserved family of enzymes encoded by the orthologous gene family COG0042. In protein sequence databases, members of COG0042 are typically annotated as “predicted TIM-barrel enzymes, possibly dehydrogenases, nifR3 family”.
Results
To elucidate sequence-structure-function relationships in the DUS family, a comprehensive bioinformatic analysis was carried out. We performed extensive database searches to identify all members of the currently known DUS family, followed by clustering analysis to subdivide it into subfamilies of closely related sequences. We analyzed phylogenetic distributions of all members of the DUS family and inferred the evolutionary tree, which suggested a scenario for the evolutionary origin of dihydrouridine-forming enzymes. For a human representative of the DUS family, the hDus2 protein suggested as a potential drug target in cancer, we generated a homology model. While this article was under review, a crystal structure of a DUS representative has been published, giving us an opportunity to validate the model.
Conclusions
We compared sequences and phylogenetic distributions of all members of the DUS family and inferred the phylogenetic tree, which provides a framework to study the functional differences among these proteins and suggests a scenario for the evolutionary origin of dihydrouridine formation. Our evolutionary and structural classification of the DUS family provides a background to study functional differences among these proteins that will guide experimental analyses.
doi:10.1186/1471-2105-13-153
PMCID: PMC3674756  PMID: 22741570
Dihydrouridine synthases; Protein structure prediction; Fold recognition; Remote homology; RNA modification; Molecular evolution; Enzymes acting on RNA
5.  Tridimensional model structure and patterns of molecular evolution of Pepino mosaic virus TGBp3 protein 
Virology Journal  2011;8:318.
Background
Pepino mosaic virus (PepMV) is considered one of the most dangerous pathogens infecting tomatoes worldwide. The virus is highly diverse and four distinct genotypes, as well as inter-strain recombinants, have already been described. The isolates display a wide range on symptoms on infected plant species, ranging from mild mosaic to severe necrosis. However, little is known about the mechanisms and pattern of PepMV molecular evolution and about the role of individual proteins in host-pathogen interactions.
Methods
The nucleotide sequences of the triple gene block 3 (TGB3) from PepMV isolates varying in symptomatology and geographic origin have been analyzed. The modes and patterns of molecular evolution of the TGBp3 protein were investigated by evaluating the selective constraints to which particular amino acid residues have been subjected during the course of diversification. The tridimensional structure of TGBp3 protein has been modeled de novo using the Rosetta algorithm. The correlation between symptoms development and location of specific amino acids residues was analyzed.
Results
The results have shown that TGBp3 has been evolving mainly under the action of purifying selection operating on several amino acid sites, thus highlighting its functional role during PepMV infection. Interestingly, amino acid 67, which has been previously shown to be a necrosis determinant, was found to be under positive selection.
Conclusions
Identification of diverse selection events in TGB3p3 will help unraveling its biological functions and is essential to an understanding of the evolutionary constraints exerted on the Potexvirus genome. The estimated tridimensional structure of TGBp3 will serve as a platform for further sequence, structural and function analysis and will stimulate new experimental advances.
doi:10.1186/1743-422X-8-318
PMCID: PMC3132167  PMID: 21702943
molecular evolution; PepMV; protein modeling; selective constraints; TGBp3; virus evolution
6.  MODOMICS: a database of RNA modification pathways. 2008 update 
Nucleic Acids Research  2008;37(Database issue):D118-D121.
MODOMICS, a database devoted to the systems biology of RNA modification, has been subjected to substantial improvements. It provides comprehensive information on the chemical structure of modified nucleosides, pathways of their biosynthesis, sequences of RNAs containing these modifications and RNA-modifying enzymes. MODOMICS also provides cross-references to other databases and to literature. In addition to the previously available manually curated tRNA sequences from a few model organisms, we have now included additional tRNAs and rRNAs, and all RNAs with 3D structures in the Nucleic Acid Database, in which modified nucleosides are present. In total, 3460 modified bases in RNA sequences of different organisms have been annotated. New RNA-modifying enzymes have been also added. The current collection of enzymes includes mainly proteins for the model organisms Escherichia coli and Saccharomyces cerevisiae, and is currently being expanded to include proteins from other organisms, in particular Archaea and Homo sapiens. For enzymes with known structures, links are provided to the corresponding Protein Data Bank entries, while for many others homology models have been created. Many new options for database searching and querying have been included. MODOMICS can be accessed at http://genesilico.pl/modomics.
doi:10.1093/nar/gkn710
PMCID: PMC2686465  PMID: 18854352
7.  MODOMICS: a database of RNA modification pathways 
Nucleic Acids Research  2005;34(Database issue):D145-D149.
MODOMICS is the first comprehensive database resource for systems biology of RNA modification. It integrates information about the chemical structure of modified nucleosides, their localization in RNA sequences, pathways of their biosynthesis and enzymes that carry out the respective reactions. MODOMICS also provides literature information, and links to other databases, including the available protein sequence and structure data. The current list of modifications and pathways is comprehensive, while the dataset of enzymes is limited to Escherichia coli and Saccharomyces cerevisiae and sequence alignments are presented only for tRNAs from these organisms. RNAs and enzymes from other organisms will be included in the near future. MODOMICS can be queried by the type of nucleoside (e.g. A, G, C, U, I, m1A, nm5s2U, etc.), type of RNA, position of a particular nucleoside, type of reaction (e.g. methylation, thiolation, deamination, etc.) and name or sequence of an enzyme of interest. Options for data presentation include graphs of pathways involving the query nucleoside, multiple sequence alignments of RNA sequences and tabular forms with enzyme and literature data. The contents of MODOMICS can be accessed through the World Wide Web at .
doi:10.1093/nar/gkj084
PMCID: PMC1347447  PMID: 16381833

Results 1-7 (7)