Structure of the bifunctional dCTP deaminase-dUTPase from Methanocaldococcus jannaschii and its relation to other homotrimeric dUTPases

The bifunctional dCTP deaminase-dUTPase (DCD-DUT) from Methanocaldococcus jannaschii catalyzes the deamination of the cytosine moiety in dCTP and the hydrolysis of the triphosphate moiety forming dUMP, thereby preventing uracil from being incorporated into DNA. The crystal structure of DCD-DUT has been determined to 1.88-A resolution and represents the first known structure of an enzyme catalyzing dCTP deamination. The functional form of DCD-DUT is a homotrimer wherein the subunits are composed of a central distorted beta-barrel surrounded by two beta-sheets and four helices. The trimeric DCD-DUT shows structural similarity to trimeric dUTPases at the tertiary and quaternary levels. There are also additional structural elements in DCD-DUT compared with dUTPase because of a longer primary structure. Four of the five conserved sequence motifs that create the active sites in dUTPase are found in structurally equivalent positions in DCD-DUT. The last 25 C-terminal residues of the 204-residue-long DCD-DUT are not visible in the electron density map, but, analogous to dUTPases, the C terminus is probably ordered, closing the active site upon catalysis. Unlike other enzymes catalyzing the deamination of cytosine compounds, DCD-DUT is not exploiting an enzyme-bound metal ion such as zinc or iron for nucleophile generation. The active site contains two water molecules that are engaged in hydrogen bonds to the invariant residues Ser118, Arg122, Thr130, and Glu145. These water molecules are potential nucleophile candidates in the deamination reaction.     

Domain organization and structure-function relationship of the HET-s prion protein of Podospora anserina

The [Het-s] infectious element of the fungus Podospora anserina is a prion protein involved in a genetically controlled cell death reaction termed heterokaryon incompatibility. Previous analyses indicate that [Het-s] propagates as a self-perpetuating amyloid aggregate. The HET-s protein is 289 amino acids in length. Herein, we identify the region of the HET-s protein that is responsible for amyloid formation and prion propagation. The region of HET-s spanning residues 218-289 forms amyloid fibers in vitro and allows prion propagation in vivo. Conversely, a C-terminal deletion in HET-s prevents amyloid aggregation in vitro and prion propagation in vivo, and abolishes the incompatibility function. In the soluble form of HET-s, the region from residue 1 to 227 forms a well-folded domain while the C-terminal region is highly flexible. Together, our data establish a domain structure-function relationship for HET-s amyloid formation, prion propagation and incompatibility activity.     

'Cyclic alopecia' in Msx2 mutants: defects in hair cycling and hair shaft differentiation

Msx2-deficient mice exhibit progressive hair loss, starting at P14 and followed by successive cycles of wavelike regrowth and loss. During the hair cycle, Msx2 deficiency shortens anagen phase, but prolongs catagen and telogen. Msx2-deficient hair shafts are structurally abnormal. Molecular analyses suggest a Bmp4/Bmp2/Msx2/Foxn1 acidic hair keratin pathway is involved. These structurally abnormal hairs are easily dislodged in catagen implying a precocious exogen. Deficiency in Msx2 helps to reveal the distinctive skin domains on the same mouse. Each domain cycles asynchronously - although hairs within each skin domain cycle in synchronized waves. Thus, the combinatorial defects in hair cycling and differentiation, together with concealed skin domains, account for the cyclic alopecia phenotype.     

Evolutionary transformations of myoseptal tendons in gnathostomes

Axial undulations in fishes are powered by a series of three-dimensionally folded myomeres separated by sheets of connective tissue, the myosepta. Myosepta have been hypothesized to function as transmitters of muscular forces to axial structures during swimming, but the difficulty of studying these delicate complex structures has precluded a more complete understanding of myoseptal mechanics. We have developed a new combination of techniques for visualizing the three-dimensional morphology of myosepta, and here we present their collagen-fibre architecture based on examination of 62 species representing all of the major clades of notochordates. In all gnathostome fishes, each myoseptum bears a set of six specifically arranged tendons. Because these tendons are not present outside the gnathostomes (i.e. they are absent from lampreys, hagfishes and lancelets), they represent evolutionary novelties of the gnathostome ancestor. This arrangement has remained unchanged throughout 400 Myr of gnathostome evolution, changing only on the transition to land. The high uniformity of myoseptal architecture in gnathostome fishes indicates functional significance and may be a key to understanding general principles of fish swimming mechanics. In the design of future experiments or biomechanical models, myosepta have to be regarded as tendons that can distribute forces in specific directions.     

Enhanced pro-inflammatory cytokine production in Galphai2-deficient mice on colitis prone and colitis resistant 129Sv genetic backgrounds

Mice deficient in G-protein subunit alphai2 develop colitis closely resembling human ulcerative colitis when raised on 129SvEv background. When backcrossing the Galphai2-deficiency into a 129SvJBom genetic background, surprisingly, mice did not develop colitis. In vitro stimulation of splenocytes with formalin-killed Staphylococcus aureus resulted in significantly increased production of interleukin-1beta, tumor necrosis factor, and interleukin-12p40 in Galphai2(-/-) as compared to control mice. The enhanced production of pro-inflammatory cytokines was seen in colitis prone as well as in colitis resistant genetic background. A similar outcome was seen upon stimulation with toxic shock syndrome toxin-1, a T cell superantigen, except that Galphai2(-/-) colitis resistant 129SvJBom splenocytes did not show increased production of IL-12p40 as compared to their controls.     

Detection of bacterial DNA in blood samples from febrile patients: underestimated infection or emerging contamination?

We applied real-time broad-range polymerase chain reaction (PCR) to detect bacteraemia in blood from febrile patients. Interpretation of amplification results in relation to clinical data and blood culture outcome was complex, although the reproducibility of the PCR results was good. Sequencing analysis of the PCR products revealed the presence of Burkholderia species DNA while no Burkholderia species grew in culture. The source of this contamination was shown to be the commercial DNA isolation kit used in the automated MagNA Pure Isolation Robot. A high degree of suspicion is required when uncommon or unexpected pathogens are diagnosed by molecular methods as clinical consequences can be serious.     

Analysis of curated and predicted plastid subproteomes of Arabidopsis. Subcellular compartmentalization leads to distinctive proteome properties

Carefully curated proteomes of the inner envelope membrane, the thylakoid membrane, and the thylakoid lumen of chloroplasts from Arabidopsis were assembled based on published, well-documented localizations. These curated proteomes were evaluated for distribution of physical-chemical parameters, with the goal of extracting parameters for improved subcellular prediction and subsequent identification of additional (low abundant) components of each membrane system. The assembly of rigorously curated subcellular proteomes is in itself also important as a parts list for plant and systems biology. Transmembrane and subcellular prediction strategies were evaluated using the curated data sets. The three curated proteomes differ strongly in average isoelectric point and protein size, as well as transmembrane distribution. Removal of the cleavable, N-terminal transit peptide sequences greatly affected isoelectric point and size distribution. Unexpectedly, the Cys content was much lower for the thylakoid proteomes than for the inner envelope. This likely relates to the role of the thylakoid membrane in light-driven electron transport and helps to avoid unwanted oxidation-reduction reactions. A rule of thumb for discriminating between the predicted integral inner envelope membrane and integral thylakoid membrane proteins is suggested. Using a combination of predictors and experimentally derived parameters, four plastid subproteomes were predicted from the fully annotated Arabidopsis genome. These predicted subproteomes were analyzed for their properties and compared to the curated proteomes. The sensitivity and accuracy of the prediction strategies are discussed. Data can be extracted from the new plastid proteome database (http://ppdb.tc.cornell.edu).     

Silanized surfaces for in vitro studies of actomyosin function and nanotechnology applications

We have previously shown that selective heavy meromyosin (HMM) adsorption to predefined regions of nanostructured polymer resist surfaces may be used to produce a nanostructured in vitro motility assay. However, actomyosin function was of lower quality than on conventional nitrocellulose films. We have therefore studied actomyosin function on differently derivatized glass surfaces with the aim to find a substitute for the polymer resists. We have found that surfaces derivatized with trimethylchlorosilane (TMCS) were superior to all other surfaces tested, including nitrocellulose. High-quality actin filament motility was observed up to 6 days after incubation with HMM and the fraction of motile actin filaments and the velocity of smooth sliding were generally higher on TMCS than on nitrocellulose. The actomyosin function on TMCS-derivatized glass and nitrocellulose is considered in relation to roughness and hydrophobicity of these surfaces. The results suggest that TMCS is an ideal substitute for polymer resists in the nanostructured in vitro motility assay. Furthermore, TMCS derivatized glass also seems to offer several advantages over nitrocellulose for HMM adsorption in the ordinary in vitro motility assay.     

A bifunctional dCTP deaminase-dUTP nucleotidohydrolase from the hyperthermophilic archaeon Methanocaldococcus jannaschii

By the sequential action of dCTP deaminase and dUTPase, dCTP is converted to dUMP, the precursor of thymidine nucleotides. In addition, dUTPase has an essential role as a safeguard against uracil incorporation in DNA. The putative dCTP deaminase (MJ0430) and dUTPase (MJ1102) from the hyperthermophilic archaeon Methanocaldococcus jannaschii were overproduced in Escherichia coli. Unexpectedly, we found the MJ0430 protein capable of both reactions, i.e. hydrolytic deamination of the cytosine ring and hydrolytic cleavage of the phosphoanhydride bond between the alpha- and beta-phosphates. When the reaction was followed by thin layer chromatography using [3H]dCTP as substrate, dUMP and not dUTP was identified as a reaction product. In the presence of unlabeled dUTP, which acted as an inhibitor, no label was transferred from [3H]dCTP to the pool of dUTP. This finding strongly suggests that the two consecutive steps of the reaction are tightly coupled within the enzyme. The hitherto unknown bifunctionality of the MJ0430 protein appears beneficial for the cells because the toxic intermediate dUTP is never released. The MJ0430 protein also catalyzed the hydrolysis of dUTP to dUMP but with a low affinity for the substrate (Km >100 micro m). According to limited proteolysis, the C-terminal residues constitute a flexible region. The other protein investigated, MJ1102, is a specific dUTPase with a Km for dUTP (0.4 micro m) comparable in magnitude with that found for previously characterized dUTPases. Its physiological function is probably to degrade dUTP derived from other reactions in nucleotide metabolism.