Terpyridine platinum(II) complexes inhibit cysteine proteases by binding to active-site cysteine

Platinum(II) complexes have been demonstrated to form covalent bonds with sulfur-donating ligands (in glutathione, metallothionein and other sulfur-containing biomolecules) or coordination bonds with nitrogen-donating ligands (such as histidine and guanine). To investigate how these compounds interact with cysteine proteases, we chose terpyridine platinum(II) (TP-Pt(II)) complexes as a model system. By using X-ray crystallography, we demonstrated that TP-Pt(II) formed a covalent bond with the catalytic cysteine residue in pyroglutamyl peptidase I. Moreover, by using MALDI (matrix-assisted laser desorption/ionization) and TOF-TOF (time of flight) mass spectrometry, we elucidated that the TP-Pt(II) complex formed a covalent bond with the active-site cysteine residue in two other types of cysteine protease. Taken together, the results unequivocally showed that TP-Pt(II) complexes can selectively bind to the active site of most cysteine proteases. Our findings here can be useful in the design of new anti-cancer, anti-parasite or anti-virus platinum(II) compounds.     

Crystal structure and substrate-binding mode of cellulase 12A from Thermotoga maritima

Cellulases have been used in many applications to treat various carbohydrate-containing materials. Thermotoga maritima cellulase 12A (TmCel12A) belongs to the GH12 family of glycoside hydrolases. It is a β-1,4-endoglucanase that degrades cellulose molecules into smaller fragments, facilitating further utilization of the carbohydrate. Because of its hyperthermophilic nature, the enzyme is especially suitable for industrial applications. Here the crystal structure of TmCel12A was determined by using an active-site mutant E134C and its mercury-containing derivatives. It adopts a β-jellyroll protein fold typical of the GH12-family enzymes, with two curved β-sheets A and B and a central active-site cleft. Structural comparison with other GH12 enzymes shows significant differences, as found in two longer and highly twisted β-strands B8 and B9 and several loops. A unique Loop A3-B3 that contains Arg60 and Tyr61 stabilizes the substrate by hydrogen bonding and stacking, as observed in the complex crystals with cellotetraose and cellobiose. The high-resolution structures allow clear elucidation of the network of interactions between the enzyme and its substrate. The sugar residues bound to the enzyme appear to be more ordered in the -2 and -1 subsites than in the +1, +2 and -3 subsites. In the E134C crystals the bound -1 sugar at the cleavage site consistently show the α-anomeric configuration, implicating an intermediate-like structure.     

Structure and mechanism of Escherichia coli glutathionylspermidine amidase belonging to the family of cysteine; histidine-dependent amidohydrolases/peptidases

The bifunctional Escherichia coli glutathionylspermidine synthetase/amidase (GspSA) catalyzes both the synthesis and hydrolysis of Gsp. Its amidase domain (GspA), which catalyzes the hydrolysis of Gsp into glutathione and spermidine, plays an important role in redox sensing and protein S-thiolation. To gain insight of the regulation and catalytic mechanism of and further understand the recycling of the Gsp dimer and Gsp-S-protein adducts, we solved two crystal structures of GspA and GspSA both with the C59A mutation and bound with the substrate, Gsp. In both structures, Cys59, His131, and Glu147 form the catalytic triad, which is similar to other cysteine proteases. Comparison of the GspA_Gsp complex and apo GspSA structures indicates that on binding with Gsp, the side chains of Asn149 and Gln58 of the amidase domain are induced to move closer to the carbonyl oxygen of the cleaved amide bond of Gsp, thereby participating in catalysis. In addition, the helix-loop region of GspA, corresponding to the sequence (30)YSSLDPQEYEDDA(42), involves in regulating the substrate binding. Our previous study indicated that the thiol of Cys59 of GspA is only oxidized to sulfenic acid by H(2)O(2). When comparing the active site of GspA with those of other cysteine proteases, we found that limited space and hydrophobicity of the environment around Cys59 play an important role to inhibit its further oxidation. The structural results presented here not only elucidate the catalytic mechanism and regulation of GspA but also help us to design small molecules to inhibit or probe for the activity of GspA.     

The many blades of the β-propeller proteins: conserved but versatile

The β-propeller is a highly symmetrical structure with 4-10 repeats of a four-stranded antiparallel β-sheet motif. Although β-propeller proteins with different blade numbers all adopt disc-like shapes, they are involved in a diverse set of functions, and defects in this family of proteins have been associated with human diseases. However, it has remained ambiguous how variations in blade number could alter the function of β-propellers. In addition to the regularly arranged β-propeller topology, a recently discovered β-pinwheel propeller has been found. Here, we review the structural and functional diversity of β-propeller proteins, including β-pinwheels, as well as recent advances in the typical and atypical propeller structures.     

DAPK activates MARK1/2 to regulate microtubule assembly, neuronal differentiation, and tau toxicity

Death-associated protein kinase (DAPK) is a key player in several modes of neuronal death/injury and has been implicated in the late-onset Alzheimer's disease (AD). DAPK promotes cell death partly through its effect on regulating actin cytoskeletons. In this study, we report that DAPK inhibits microtubule (MT) assembly by activating MARK/PAR-1 family kinases MARK1/2, which destabilize MT by phosphorylating tau and related MAP2/4. DAPK death domain, but not catalytic activity, is responsible for this activation by binding to MARK1/2 spacer region, thereby disrupting an intramolecular interaction that inhibits MARK1/2. Accordingly, DAPK(-/-) mice brain displays a reduction of tau phosphorylation and DAPK enhances the effect of MARK2 on regulating polarized neurite outgrowth. Using a well-characterized Drosophila model of tauopathy, we show that DAPK exerts an effect in part through MARK Drosophila ortholog PAR-1 to induce rough eye and loss of photoreceptor neurons. Furthermore, DAPK enhances tau toxicity through a PAR-1 phosphorylation-dependent mechanism. Together, our study reveals a novel mechanism of MARK activation, uncovers DAPK functions in modulating MT assembly and neuronal differentiation, and provides a molecular link of DAPK to tau phosphorylation, an event associated with AD pathology.     

Shallow genetic divergence and species delineations in the endemic Labeobarbus species flock of Lake Tana,Ethiopia

To assess whether the species distinctions of Lake Tana's Labeobarbus spp. are supported by genetic information, microsatellite markers were used. A total of 376 Labeobarbus spp., belonging to 24 populations of 11 species from three regions of the lake (north, south and east), were sampled. Eight microsatellite markers were analysed. In general, differences between conspecific populations were smaller than differences between populations of different species. For six species, conspecific populations from different regions in the lake were consistently more similar than populations of other species from the same region. For four species this was not the case, while for one species two populations were similar, but different from the third population. River‐spawning species appeared to be more distinct than presumed lake spawners. On the species level, there was a significant correlation between genetic and morphological differentiation, especially in morphological aspects associated with ecological functioning. This suggests that genetic differentiation arose together with adaptive radiation, although the overall genetic differentiation among the Lake Tana Labeobarbus spp. is small.     

Gene Expression in Chicken Reveals Correlation with Structural Genomic Features and Conserved Patterns of Transcription in the Terrestrial Vertebrates

Background

The chicken is an important agricultural and avian-model species. A survey of gene expression in a range of different tissues will provide a benchmark for understanding expression levels under normal physiological conditions in birds. With expression data for birds being very scant, this benchmark is of particular interest for comparative expression analysis among various terrestrial vertebrates.

Methodology/Principal Findings

We carried out a gene expression survey in eight major chicken tissues using whole genome microarrays. A global picture of gene expression is presented for the eight tissues, and tissue specific as well as common gene expression were identified. A Gene Ontology (GO) term enrichment analysis showed that tissue-specific genes are enriched with GO terms reflecting the physiological functions of the specific tissue, and housekeeping genes are enriched with GO terms related to essential biological functions. Comparisons of structural genomic features between tissue-specific genes and housekeeping genes show that housekeeping genes are more compact. Specifically, coding sequence and particularly introns are shorter than genes that display more variation in expression between tissues, and in addition intergenic space was also shorter. Meanwhile, housekeeping genes are more likely to co-localize with other abundantly or highly expressed genes on the same chromosomal regions. Furthermore, comparisons of gene expression in a panel of five common tissues between birds, mammals and amphibians showed that the expression patterns across tissues are highly similar for orthologuous genes compared to random gene pairs within each pair-wise comparison, indicating a high degree of functional conservation in gene expression among terrestrial vertebrates.

Conclusions

The housekeeping genes identified in this study have shorter gene length, shorter coding sequence length, shorter introns, and shorter intergenic regions, there seems to be selection pressure on economy in genes with a wide tissue distribution, i.e. these genes are more compact. A comparative analysis showed that the expression patterns of orthologous genes are conserved in the terrestrial vertebrates during evolution.     

Persistence of bovine viral diarrhea virus is determined by a cellular cofactor of a viral autoprotease

Polyprotein processing control is a crucial step in the life cycle of positive-strand RNA viruses. Recently, a vital autoprotease generating an essential viral replication factor was identified in such a virus, namely, the pestivirus bovine viral diarrhea virus. Surprisingly, the activity of this protease, which resides in nonstructural protein 2 (NS2), diminishes early after infection, resulting in the limitation of viral RNA replication. Here, we describe that a cellular chaperone termed Jiv (J-domain protein interacting with viral protein) acts as a cofactor of the NS2 protease. Consumption of the intracellular Jiv pool is responsible for temporal regulation of protease activity: overexpression of Jiv interfered with regulation and correlated with increased accumulation of viral RNA; downregulation of the cellular Jiv level accelerated the decline of protease activity and reduced intracellular viral RNA levels and virion production. Accordingly, the amount of a cellular protein controls pestiviral replication by limiting the generation of active viral protease molecules and replication complexes. Importantly, this unique mechanism of replication control is essential for maintenance of the noncytopathogenic phenotype of the virus and thereby for its ability to establish persistent infections. These results add an entirely novel aspect to the understanding of the molecular basis of viral persistence.     

Rapid evolution of the MH class I locus results in different allelic compositions in recently diverged populations of Atlantic salmon

We compared major histocompatibility class I allelic diversity in two currently reproductively isolated Atlantic salmon (Salmo salar) populations (Irish and Norwegian) with a common postglacial origin in order to test for among-population differences in allelic composition and patterns of recombination and point mutation. We also examined the evidence for adaptive molecular divergence at this locus by analyzing the rate of amino acid replacement in relation to a neutral expectation. Contrary to our prediction, and in contrast to the situation for other genetic markers, the two populations have almost nonoverlapping sets of major histocompatibility class I alleles. Although there is a strong signal of point mutation that predates population divergence, recent recombination, acting in similar, but not identical, ways in both populations appears to be a significant force in creating new alleles. Moreover, selection acting on peptide-binding residues seems to favor new recombinant alleles and is likely to be responsible for the rapid divergence between populations.