Conserved amino acids in each subunit of the heteroligomeric tRNA m1A58 Mtase from Saccharomyces cerevisiae contribute to tRNA binding

In Saccharomyces cerevisiae, a two-subunit methyltransferase (Mtase) encoded by the essential genes TRM6 and TRM61 is responsible for the formation of 1-methyladenosine, a modified nucleoside found at position 58 in tRNA that is critical for the stability of tRNA(Met)i The crystal structure of the homotetrameric m1A58 tRNA Mtase from Mycobacterium tuberculosis, TrmI, has been solved and was used as a template to build a model of the yeast m1A58 tRNA Mtase heterotetramer. We altered amino acids in TRM6 and TRM61 that were predicted to be important for the stability of the heteroligomer based on this model. Yeast strains expressing trm6 and trm61 mutants exhibited growth phenotypes indicative of reduced m1A formation. In addition, recombinant mutant enzymes had reduced in vitro Mtase activity. We demonstrate that the mutations introduced do not prevent heteroligomer formation and do not disrupt binding of the cofactor S-adenosyl-L-methionine. Instead, amino acid substitutions in either Trm6p or Trm61p destroy the ability of the yeast m1A58 tRNA Mtase to bind tRNA(Met)i, indicating that each subunit contributes to tRNA binding and suggesting a structural alteration of the substrate-binding pocket occurs when these mutations are present.     

The development of 2-benzimidazole substituted pyrimidine based inhibitors of lymphocyte specific kinase (Lck)

This communication details the synthesis, biological activity, and binding mode of a novel class of 2-benzimidazole substituted pyrimidines. The most potent analogs disclosed showed low nanomolar activity for the inhibition of Lck kinase and a representative analog was co-crystallized with Hck (a structurally related member of the Src family kinases).     

A homology model of restriction endonuclease SfiI in complex with DNA

Background

Restriction enzymes (REases) are commercial reagents commonly used in recombinant DNA technologies. They are attractive models for studying protein-DNA interactions and valuable targets for protein engineering. They are, however, extremely divergent: the amino acid sequence of a typical REase usually shows no detectable similarities to any other proteins, with rare exceptions of other REases that recognize identical or very similar sequences. From structural analyses and bioinformatics studies it has been learned that some REases belong to at least four unrelated and structurally distinct superfamilies of nucleases, PD-DxK, PLD, HNH, and GIY-YIG. Hence, they are extremely hard targets for structure prediction and homology-based inference of sequence-function relationships and the great majority of REases remain structurally and evolutionarily unclassified.

Results

SfiI is a REase which recognizes the interrupted palindromic sequence 5'GGCCNNNN^NGGCC3' and generates 3 nt long 3' overhangs upon cleavage. SfiI is an archetypal Type IIF enzyme, which functions as a tetramer and cleaves two copies of the recognition site in a concerted manner. Its sequence shows no similarity to other proteins and nothing is known about the localization of its active site or residues important for oligomerization. Using the threading approach for protein fold-recognition, we identified a remote relationship between SfiI and BglI, a dimeric Type IIP restriction enzyme from the PD-DxK superfamily of nucleases, which recognizes the 5'GCCNNNN^NGGC3' sequence and whose structure in complex with the substrate DNA is available. We constructed a homology model of SfiI in complex with its target sequence and used it to predict residues important for dimerization, tetramerization, DNA binding and catalysis.

Conclusions

The bioinformatics analysis suggest that SfiI, a Type IIF enzyme, is more closely related to BglI, an "orthodox" Type IIP restriction enzyme, than to any other REase, including other Type IIF REases with known structures, such as NgoMIV. NgoMIV and BglI belong to two different, very remotely related branches of the PD-DxK superfamily: the α-class (EcoRI-like), and the β-class (EcoRV-like), respectively. Thus, our analysis provides evidence that the ability to tetramerize and cut the two DNA sequences in a concerted manner was developed independently at least two times in the evolution of the PD-DxK superfamily of REases. The model of SfiI will also serve as a convenient platform for further experimental analyses.     

Tetrabromobenzotriazole (TBBt) and tetrabromobenzimidazole (TBBz) as selective inhibitors of protein kinase CK2: evaluation of their effects on cells and different molecular forms of human CK2

The development of selective cell-permeable inhibitors of protein kinase CK2 has represented an important advance in the field. However, it is important to not overlook the existence of discrete molecular forms of CK2 that arise from the presence of distinct isozymic forms, and the existence of the catalytic CK2 subunits as free subunits and in complexes with the regulatory CK2beta subunits and, possibly, other proteins. This review examines two recently developed, and presently widely applied, CK2 inhibitors, 4,5,6,7-tetrabromobenzotriazole (TBBt) and the related 4,5,6,7-tetrabromobenzimidazole (TBBz), the latter of which was previously shown to discriminate between different molecular forms of CK2 in yeast. We have shown, by spectrophotometric titration, that TBBt, with a pK(a) approximately 5, exists in solution at physiological pH almost exclusively (>99%) as the monoanion; whereas TBBz, with a pKa approximately 9, is predominantly (>95%) in the neutral form, both of obvious relevance to their modes of binding. In vitro, TBBt inhibits different forms of CK2 with Ki values ranging from 80 to 210 nM. TBBz better discriminates between CK2 forms, with Ki values ranging from 70 to 510 nM. Despite their general similar in vitro activities, TBBz is more effective than TBBt in inducing apoptosis and, to a lesser degree, necrosis, in transformed human cell lines. Finally, development of shRNA strategies for the selective knockdown of the CK2alpha and CK2alpha' isoforms reinforces the foregoing results, indicating that inhibition of CK2 leads to attenuation of proliferation.     

Polish Glomales

Spores of Acaulospora dilatata and Scutellospora dipurpurascens found in Poland are described and illustrated and their occurrence and distribution are characterized and mapped. Spores of Acaulospora dilatata from Poland do not differ from those originally described in the United States of America. The germination shield found in a number of spores is described and illustrated, and compared with that occurring in members of the genus Scutellospora. Acaulospora dilatata was found in five of the 303 soil samples taken from around the roots of Ammophila arenaria colonizing maritime sand dunes of the Sowinski National Park. Polish specimens of S. dipurpurascens are similar in size, wall structure, and reaction in Melzer's reagent to those described from the type localized in the United States of America. However, some spores from Poland have a thicker wall, greater sporogenous cells, and are somewhat darker coloured. They were recovered from 34 soils sampled from forests, gardens, sand dunes, and both cultivated and uncultivated soils. S. dipurpurascens was commonly associated with different plants of the Hel Peninsula and occurred regularly among the roots of Ammophila arenaria growing in the Slowinski National Park. Both species were found for the first time in Poland and are probably new to Europe.     

RIBER/DIBER: a software suite for crystal content analysis in the studies of protein-nucleic acid complexes

Co-crystallization experiments of proteins with nucleic acids do not guarantee that both components are present in the crystal. We have previously developed DIBER to predict crystal content when protein and DNA are present in the crystallization mix. Here, we present RIBER, which should be used when protein and RNA are in the crystallization drop. The combined RIBER/DIBER suite builds on machine learning techniques to make reliable, quantitative predictions of crystal content for non-expert users and high-throughput crystallography.     

Analogues of cyclolinopeptide A containing alpha-hydroxymethyl amino acid residues

Linear and cyclic cyclolinopeptide A (CLA) analogues containing alpha-hydroxymethylleucine (HmL) in positions 1, 4, and 1&4, and alpha-hydroxymethylvaline (HmV) in position 5, were synthesized by the solid-phase peptide strategy and cyclized with the 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide/1-hydroxy-7-azabenzotriazole (EDC/HOAt) reagent. The peptides were examined for their immunosuppressive activity in the lymphocyte proliferation assays (LPA). Only HmL-containing peptides demonstrated at about 25% lower immunosuppressive activity, but they are four times more soluble in water solutions than the native CLA. It seems from the LPA results that peptide [(HmL4)CLA] is the most promising for further studies. This peptide was characterized in solution, at room temperature in CDCl3, and the conformation compared with that observed for CLA in the solid state.     

Mutational analysis in podocin-associated hereditary nephrotic syndrome in Polish patients: founder effect in the Kashubian population

Hereditary nephrotic syndrome is caused by mutations in a number of different genes, the most common being NPHS2. The aim of the study was to identify the spectrum of NPHS2 mutations in Polish patients with the disease. A total of 141 children with steroid-resistant nephrotic syndrome (SRNS) were enrolled in the study. Mutational analysis included the entire coding sequence and intron boundaries of the NPHS2 gene. Restriction fragment length polymorphism (RFLP) and TaqMan genotyping assay were applied to detect selected NPHS2 sequence variants in 575 population-matched controls. Twenty patients (14 %) had homozygous or compound heterozygous NPHS2 mutations, the most frequent being c.1032delT found in 11 children and p.R138Q found in four patients. Carriers of the c.1032delT allele were exclusively found in the Pomeranian (Kashubian) region, suggesting a founder effect origin. The 14 % NPHS2 gene mutation detection rate is similar to that observed in other populations. The heterogeneity of mutations detected in the studied group confirms the requirement of genetic testing the entire NPHS2 coding sequence in Polish patients, with the exception of Kashubs, who should be initially screened for the c.1032delT deletion.