Cdk1 Restrains NHEJ through Phosphorylation of XRCC4-like Factor Xlf1

Eukaryotic cells use two principal mechanisms for repairing DNA double-strand breaks (DSBs): homologous recombination (HR) and nonhomologous end-joining (NHEJ). DSB repair pathway choice is strongly regulated during the cell cycle. Cyclin-dependent kinase 1 (Cdk1) activates HR by phosphorylation of key recombination factors. However, a mechanism for regulating the NHEJ pathway has not been established. Here, we report that Xlf1, a fission yeast XLF ortholog, is a key regulator of NHEJ activity in the cell cycle. We show that Cdk1 phosphorylates residues in the C terminus of Xlf1 over the course of the cell cycle. Mutation of these residues leads to the loss of Cdk1 phosphorylation, resulting in elevated levels of NHEJ repair in vivo. Together, these data establish that Xlf1 phosphorylation by Cdc2(Cdk1) provides a molecular mechanism for downregulation of NHEJ in fission yeast and indicates that XLF is a key regulator of end-joining processes in eukaryotic organisms.

Zalpha-domains: At the intersection between RNA editing and innate immunity

The involvement of A to I RNA editing in antiviral responses was first indicated by the observation of genomic hyper-mutation for several RNA viruses in the course of persistent infections. However, in only a few cases an antiviral role was ever demonstrated and surprisingly, it turns out that ADARs - the RNA editing enzymes - may have a prominent pro-viral role through the modulation/down-regulation of the interferon response. A key role in this regulatory function of RNA editing is played by ADAR1, an interferon inducible RNA editing enzyme. A distinguishing feature of ADAR1, when compared with other ADARs, is the presence of a Z-DNA binding domain, Zalpha. Since the initial discovery of the specific and high affinity binding of Zalpha to CpG repeats in a left-handed helical conformation, other proteins, all related to the interferon response pathway, were shown to have similar domains throughout the vertebrate lineage. What is the biological function of this domain family remains unclear but a significant body of work provides pieces of a puzzle that points to an important role of Zalpha domains in the recognition of foreign nucleic acids in the cytoplasm by the innate immune system. Here we will provide an overview of our knowledge on ADAR1 function in interferon response with emphasis on Zalpha domains.

A two-step mechanism for epigenetic specification of centromere identity and function

The basic determinant of chromosome inheritance, the centromere, is specified in many eukaryotes by an epigenetic mark. Using gene targeting in human cells and fission yeast, chromatin containing the centromere-specific histone H3 variant CENP-A is demonstrated to be the epigenetic mark that acts through a two-step mechanism to identify, maintain and propagate centromere function indefinitely. Initially, centromere position is replicated and maintained by chromatin assembled with the centromere-targeting domain (CATD) of CENP-A substituted into H3. Subsequently, nucleation of kinetochore assembly onto CATD-containing chromatin is shown to require either the amino- or carboxy-terminal tail of CENP-A for recruitment of inner kinetochore proteins, including stabilizing CENP-B binding to human centromeres or direct recruitment of CENP-C, respectively.

CENP-C reshapes and stabilizes CENP-A nucleosomes at the centromere

Inheritance of each chromosome depends upon its centromere. A histone H3 variant, centromere protein A (CENP-A), is essential for epigenetically marking centromere location. We find that CENP-A is quantitatively retained at the centromere upon which it is initially assembled. CENP-C binds to CENP-A nucleosomes and is a prime candidate to stabilize centromeric chromatin. Using purified components, we find that CENP-C reshapes the octameric histone core of CENP-A nucleosomes, rigidifies both surface and internal nucleosome structure, and modulates terminal DNA to match the loose wrap that is found on native CENP-A nucleosomes at functional human centromeres. Thus, CENP-C affects nucleosome shape and dynamics in a manner analogous to allosteric regulation of enzymes. CENP-C depletion leads to rapid removal of CENP-A from centromeres, indicating their collaboration in maintaining centromere identity.

Hemorheological alterations in sickle cell anemia and their clinical consequences: the role of genetic modulators

Sickle cell anemia (SCA) is an autosomal recessive chronic hemolytic anemia, caused by homozygosity for the HBB:c.20A>T mutation. The disease presents with high clinical heterogeneity, stroke being the most devastating manifestation. This study aimed to identify genetic modulators of severe hemolysis and stroke risk in children with SCA, as well as understand their consequences at the hemorheological level. Sixty-six children with SCA were categorised according to their degree of cerebral vasculopathy (Stroke/Risk/Control). Relevant data were collected from patients’ medical records. Several polymorphic regions in genes related to vascular cell adhesion and tonus were characterized by molecular methodologies. Data analyses were performed using R software. Several in silico tools (e.g. TFBind, MatInspector) were applied to investigate the main variant consequences. Some genetic variants in vascular adhesion molecule-1 gene promoter and endothelial nitric oxide synthase gene were associated with higher levels of hemolysis and stroke events. They modify important transcription factor binding sites or disturb the corresponding protein structure/function. Our findings emphasize the relevance of the genetic variants in modulating the degree of hemolysis and development of cerebral vasculopathy due to their effect on gene expression, modification of protein biological activities related with erythrocyte/endothelial interactions and consequent hemorheological abnormalities in SCA.

CemaT1 is an active transposon within the Caenorhabditis elegans genome

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and T-C transposons, with a distribution thus far limited to a few invertebrate species. In the nematode Caenorhabditis elegans, there are eight copies of CemaT1 that are predicted to encode a functional transposase, with five copies being >99% identical. We present evidence, based on searches of publicly available databases and on PCR-based mobility assays, that the CemaT1 transposase is expressed in C. elegans and that the CemaT transposons are capable of excising in both somatic and germline tissues. We also show that the frequency of CemaT1 excisions within the genome of the N2 strain of C. elegans is comparable to that of the Tc1 transposon. However, unlike T-C transposons in mutator strains of C elegans, maT transposons do not exhibit increased frequencies of mobility, suggesting that maT is not regulated by the same factors that control T-C activity in these strains. Finally, we show that CemaT1 transposons are capable of precise transpositions as well as orientation inversions at some loci, and thereby become members of an increasing number of identified active transposons within the C. elegans genome. (C) 2004 Elsevier B.V. All rights reserved.

SVMtm: Support vector machines to predict transmembrane segments

A new method has been developed for prediction of transmembrane helices using support vector machines. Different coding schemes of protein sequences were explored, and their performances were assessed by crossvalidation tests. The best performance method can predict the transmembrane helices with sensitivity of 93.4% and precision of 92.0%. For each predicted transmembrane segment, a score is given to show the strength of transmembrane signal and the prediction reliability. In particular, this method can distinguish transmembrane proteins from soluble proteins with an accuracy of similar to99%. This method can be used to complement current transmembrane helix prediction methods and can be Used for consensus analysis of entire proteomes . The predictor is located at http://genet.imb.uq.edu.au/predictors/ SVMtm. (C) 2004 Wiley Periodicals, Inc.

The Caenorhabditis briggsae genome contains active CbmaT1 and Tcb1 transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.

Capsule and fimbria interaction in Klebsiella pneumoniae

The capsular polysaccharide and type I fimbriae are two of the major surface-located virulence properties associated with the pathogenesis of Klebsiella pneumoniae. The capsule is an elaborate polysaccharide matrix that encases the entire cell surface and provides resistance against many host defense mechanisms. In contrast, type 1 fimbriae are thin adhesive thread-like surface organelles that can extend beyond the capsular matrix and mediate D-mannose-sensitive adhesion to host epithelial cells. These fimbriae are archetypical and consist of a major building block protein (FimA) that comprises the bulk of the organelle and a tip-located adhesin (FimH). It is assumed that the extended major-subunit protein structure permits the FimH adhesin to function independently of the presence of a capsule. In this study, we have employed a defined set of K. pneumoniae capsulated and noncapsulated strains to show that the function of type I fimbriae is actually impeded by the concomitant expression of a polysaccharide capsule. Capsule expression had significant effects on two parameters commonly used to define FimH function, namely, yeast cell agglutination and biofilm formation. Our data suggest that this effect is not due to transcriptional/translational changes in fimbrial gene/protein expression but rather the result of direct physical interference. This was further demonstrated by the fact that we could restore fimbrial function by inhibiting capsule synthesis. It remains to be determined whether the expression of these very different surface components occurs simply via random events of phase variation or in a coordinated manner in response to specific environmental cues.

Acetohydroxyacid synthase and its role in the biosynthetic pathway for branched-chain amino acids

The branched-chain amino acids are synthesized by plants, fungi and microorganisms, but not by animals. Therefore, the enzymes of this pathway are potential target sites for the development of antifungal agents, antimicrobials and herbicides. Most research has focused upon the first enzyme in this biosynthetic pathway, acetohydroxyacid synthase (AHAS) largely because it is the target site for many commercial herbicides. In this review we provide a brief overview of the important properties of each enzyme within the pathway and a detailed summary of the most recent AHAS research, against the perspective of work that has been carried out over the past 50 years.

Identification and molecular modeling of a novel, plant-like, human purple acid phosphatase

Purple acid phosphatases are a family of binuclear metallohydrolases that have been identified in plants, animals and fungi. Only one isoform of similar to 35 kDa has been isolated from animals, where it is associated with bone resorption and microbial killing through its phosphatase activity, and hydroxyl radical production, respectively. Using the sensitive PSI-BLAST search method, sequences representing new purple acid phosphatase-like proteins have been identified in mammals, insects and nematodes. These new putative isoforms are closely related to the similar to 55 kDa purple acid phosphatase characterized from plants. Secondary structure prediction of the new human isoform further confirms its similarity to a purple acid phosphatase from the red kidney bean. A structural model for the human enzyme was constructed based on the red kidney bean purple acid phosphatase structure. This model shows that the catalytic centre observed in other purple acid phosphatases is also present in this new isoform. These observations suggest that the sequences identified in this study represent a novel subfamily of plant-like purple acid phosphatases in animals and humans. (c) 2006 Elsevier B.V. All rights reserved.

Molecular dynamics study of MscL interactions with a curved lipid bilayer

Mechanosensitivity is a ubiquitous sensory mechanism found in living organisms. The simplest known mechanotransducing mechanism is found in bacteria in the form of the mechanosensitive membrane channel of large conductance, MscL. This channel has been studied extensively using a variety of methods at a functional and structural level. The channel is gated by membrane tension in the lipid bilayer alone. It serves as a safety valve protecting bacterial cells against hypoosmotic shock. MscL of Escherichia coli embedded in bilayers composed of asymmetric amounts of single-tailed and double-tailed lipids has been shown to gate spontaneously, even in the absence of membrane tension. To gain insight into the effect of the lipid membrane composition and geometry on MscL structure, a fully solvated, all-atom model of MscL in a stress-free curved bilayer composed of double- and single-tailed lipids was studied using a 9.5-ns molecular dynamics simulation. The bilayer was modeled as a domed structure accommodating the asymmetric composition of the monolayers. During the course of the simulation a spontaneous restructuring of the periplasmic loops occurred, leading to interactions between one of the loops and phospholipid headgroups. Previous experimental studies of the role of the loops agree with the observation that opening starts with a restructuring of the periplasmic loop, suggesting an effect of the curved bilayer. Because of limited resources, only one simulation of the large system was performed. However, the results obtained suggest that through the geometry and composition of the bilayer the protein structure can be affected even on short timescales.

Predicting residue-wise contact orders in proteins by support vector regression

Background: The residue-wise contact order (RWCO) describes the sequence separations between the residues of interest and its contacting residues in a protein sequence. It is a new kind of one-dimensional protein structure that represents the extent of long-range contacts and is considered as a generalization of contact order. Together with secondary structure, accessible surface area, the B factor, and contact number, RWCO provides comprehensive and indispensable important information to reconstructing the protein three-dimensional structure from a set of one-dimensional structural properties. Accurately predicting RWCO values could have many important applications in protein three-dimensional structure prediction and protein folding rate prediction, and give deep insights into protein sequence-structure relationships. Results: We developed a novel approach to predict residue-wise contact order values in proteins based on support vector regression (SVR), starting from primary amino acid sequences. We explored seven different sequence encoding schemes to examine their effects on the prediction performance, including local sequence in the form of PSI-BLAST profiles, local sequence plus amino acid composition, local sequence plus molecular weight, local sequence plus secondary structure predicted by PSIPRED, local sequence plus molecular weight and amino acid composition, local sequence plus molecular weight and predicted secondary structure, and local sequence plus molecular weight, amino acid composition and predicted secondary structure. When using local sequences with multiple sequence alignments in the form of PSI-BLAST profiles, we could predict the RWCO distribution with a Pearson correlation coefficient (CC) between the predicted and observed RWCO values of 0.55, and root mean square error (RMSE) of 0.82, based on a well-defined dataset with 680 protein sequences. Moreover, by incorporating global features such as molecular weight and amino acid composition we could further improve the prediction performance with the CC to 0.57 and an RMSE of 0.79. In addition, combining the predicted secondary structure by PSIPRED was found to significantly improve the prediction performance and could yield the best prediction accuracy with a CC of 0.60 and RMSE of 0.78, which provided at least comparable performance compared with the other existing methods. Conclusion: The SVR method shows a prediction performance competitive with or at least comparable to the previously developed linear regression-based methods for predicting RWCO values. In contrast to support vector classification (SVC), SVR is very good at estimating the raw value profiles of the samples. The successful application of the SVR approach in this study reinforces the fact that support vector regression is a powerful tool in extracting the protein sequence-structure relationship and in estimating the protein structural profiles from amino acid sequences.

Toxin insights into nicotinic acetylcholine receptors

Venomous species have evolved cocktails of bioactive peptides to facilitate prey capture. Given their often exquisite potency and target selectivity, venom peptides provide unique biochemical tools for probing the function of membrane proteins at the molecular level. in the field of the nicotinic acetylcholine receptors (nAChRs), the subtype specific snake alpha-neurotoxins and cone snail alpha-conotoxins have been widely used to probe receptor structure and function in native tissues and recombinant systems. However, only recently has it been possible to generate an accurate molecular view of these nAChR-toxin interactions. Crystal structures of AChBP, a homologue of the nAChR ligand binding domain, have now been solved in complex with alpha-cobratoxin, alpha-conotoxin PnIA and alpha-conotoxin Iml. The orientation of all three toxins in the ACh binding site confirms many of the predictions obtained from mutagenesis and docking simulations on homology models of mammalian nAChR. The precise understanding of the molecular determinants of these complexes is expected to contribute to the development of more selective nAChR modulators. In this commentary, we review the structural data on nAChR-toxin interactions and discuss their implications for the design of novel ligands acting at the nAChR. (c) 2006 Elsevier Inc. All rights reserved.

Focusing in on structural genomics: The University of Queensland structural biology pipeline

The flood of new genomic sequence information together with technological innovations in protein structure determination have led to worldwide structural genomics (SG) initiatives. The goals of SG initiatives are to accelerate the process of protein structure determination, to fill in protein fold space and to provide information about the function of uncharacterized proteins. In the long-term, these outcomes are likely to impact on medical biotechnology and drug discovery, leading to a better understanding of disease as well as the development of new therapeutics. Here we describe the high throughput pipeline established at the University of Queensland in Australia. In this focused pipeline, the targets for structure determination are proteins that are expressed in mouse macrophage cells and that are inferred to have a role in innate immunity. The aim is to characterize the molecular structure and the biochemical and cellular function of these targets by using a parallel processing pipeline. The pipeline is designed to work with tens to hundreds of target gene products and comprises target selection, cloning, expression, purification, crystallization and structure determination. The structures from this pipeline will provide insights into the function of previously uncharacterized macrophage proteins and could lead to the validation of new drug targets for chronic obstructive pulmonary disease and arthritis. (c) 2006 Elsevier B.V. All rights reserved.