Mechanical Strength of 17 134 Model Proteins and Cysteine Slipknots

A new theoretical survey of proteins' resistance to constant speed stretching is performed for a set of 17 134 proteins as described by a structure-based model. The proteins selected have no gaps in their structure determination and consist of no more than 250 amino acids. Our previous studies have dealt with 7510 proteins of no more than 150 amino acids. The proteins are ranked according to the strength of the resistance. Most of the predicted top-strength proteins have not yet been studied experimentally. Architectures and folds which are likely to yield large forces are identified. New types of potent force clamps are discovered. They involve disulphide bridges and, in particular, cysteine slipknots. An effective energy parameter of the model is estimated by comparing the theoretical data on characteristic forces to the corresponding experimental values combined with an extrapolation of the theoretical data to the experimental pulling speeds. These studies provide guidance for future experiments on single molecule manipulation and should lead to selection of proteins for applications. A new class of proteins, involving cystein slipknots, is identified as one that is expected to lead to the strongest force clamps known. This class is characterized through molecular dynamics simulations.Comment: 40 pages, 13 PostScript figure

Helical Chirality: a Link between Local Interactions and Global Topology in DNA

DNA supercoiling plays a major role in many cellular functions. The global DNA conformation is however intimately linked to local DNA-DNA interactions influencing both the physical properties and the biological functions of the supercoiled molecule. Juxtaposition of DNA double helices in ubiquitous crossover arrangements participates in multiple functions such as recombination, gene regulation and DNA packaging. However, little is currently known about how the structure and stability of direct DNA-DNA interactions influence the topological state of DNA. Here, a crystallographic analysis shows that due to the intrinsic helical chirality of DNA, crossovers of opposite handedness exhibit markedly different geometries. While right-handed crossovers are self-fitted by sequence-specific groove-backbone interaction and bridging Mg2+ sites, left-handed crossovers are juxtaposed by groove-groove interaction. Our previous calculations have shown that the different geometries result in differential stabilisation in solution, in the presence of divalent cations. The present study reveals that the various topological states of the cell are associated with different inter-segmental interactions. While the unstable left-handed crossovers are exclusively formed in negatively supercoiled DNA, stable right-handed crossovers constitute the local signature of an unusual topological state in the cell, such as the positively supercoiled or relaxed DNA. These findings not only provide a simple mechanism for locally sensing the DNA topology but also lead to the prediction that, due to their different tertiary intra-molecular interactions, supercoiled molecules of opposite signs must display markedly different physical properties. Sticky inter-segmental interactions in positively supercoiled or relaxed DNA are expected to greatly slow down the slithering dynamics of DNA. We therefore suggest that the intrinsic helical chirality of DNA may have oriented the early evolutionary choices for DNA topology

Using Entropy Maximization to Understand the Determinants of Structural Dynamics beyond Native Contact Topology

Comparison of elastic network model predictions with experimental data has provided important insights on the dominant role of the network of inter-residue contacts in defining the global dynamics of proteins. Most of these studies have focused on interpreting the mean-square fluctuations of residues, or deriving the most collective, or softest, modes of motions that are known to be insensitive to structural and energetic details. However, with increasing structural data, we are in a position to perform a more critical assessment of the structure-dynamics relations in proteins, and gain a deeper understanding of the major determinants of not only the mean-square fluctuations and lowest frequency modes, but the covariance or the cross-correlations between residue fluctuations and the shapes of higher modes. A systematic study of a large set of NMR-determined proteins is analyzed using a novel method based on entropy maximization to demonstrate that the next level of refinement in the elastic network model description of proteins ought to take into consideration properties such as contact order (or sequential separation between contacting residues) and the secondary structure types of the interacting residues, whereas the types of amino acids do not play a critical role. Most importantly, an optimal description of observed cross-correlations requires the inclusion of destabilizing, as opposed to exclusively stabilizing, interactions, stipulating the functional significance of local frustration in imparting native-like dynamics. This study provides us with a deeper understanding of the structural basis of experimentally observed behavior, and opens the way to the development of more accurate models for exploring protein dynamics

Impact of carbohydrate restriction with and without fatty acid loading on myocardial 18F-FDG uptake during PET: A randomized controlled trial

Low-carbohydrate (LC) and high-fat, low-carbohydrate (HFLC) dietary preparations may enhance 18F-FDG-PET-based imaging of small, inflamed structures near the heart by suppressing myocardial FDG signal. We compared myocardial 18F-FDG uptake in patients randomized to LC, HFLC, and unrestricted (UR) preparations prior to 18F-FDG-PET. We randomized 63 outpatients referred for oncologic 18F-FDG-PET to LC, HFLC, or UR dietary preparations (1:1:1 allocation) starting the evening before PET. After eating dinner according to instructions, UR and LC patients fasted until FDG injection (mean time 745 minutes for UR, 899 minutes for LC), and HFLC patients drank a fatty drink 60-70 minutes prior to FDG injection. Attenuation-corrected PET imaging was performed 60 minutes after FDG administration. Maximal myocardial standard uptake values (MyoSUVmax) were systematically measured in axial view and compared between the three groups. Using UR patients as reference, mean MyoSUVmax was lower in LC patients (3.3 ± 2.7 vs 6.2 ± 5.2, P = .03) but not in HFLC patients (5.5 ± 4.2, P = .63). Ratios of MyoSUVmax to liver SUVmax, calculated to control for background uptake, were not significantly different amongst the groups (1.9 ± 2.1 LC, 2.6 ± 2.3 HFLC, 3.6 ± 3.5 UR). In this small randomized controlled trial using UR diet as reference, LC dietary preparation followed by extended fasting resulted in significant myocardial uptake suppression

Big Genomes Facilitate the Comparative Identification of Regulatory Elements

The identification of regulatory sequences in animal genomes remains a significant challenge. Comparative genomic methods that use patterns of evolutionary conservation to identify non-coding sequences with regulatory function have yielded many new vertebrate enhancers. However, these methods have not contributed significantly to the identification of regulatory sequences in sequenced invertebrate taxa. We demonstrate here that this differential success, which is often attributed to fundamental differences in the nature of vertebrate and invertebrate regulatory sequences, is instead primarily a product of the relatively small size of sequenced invertebrate genomes. We sequenced and compared loci involved in early embryonic patterning from four species of true fruit flies (family Tephritidae) that have genomes four to six times larger than those of Drosophila melanogaster. Unlike in Drosophila, where virtually all non-coding DNA is highly conserved, blocks of conserved non-coding sequence in tephritids are flanked by large stretches of poorly conserved sequence, similar to what is observed in vertebrate genomes. We tested the activities of nine conserved non-coding sequences flanking the even-skipped gene of the teprhitid Ceratis capitata in transgenic D. melanogaster embryos, six of which drove patterns that recapitulate those of known D. melanogaster enhancers. In contrast, none of the three non-conserved tephritid non-coding sequences that we tested drove expression in D. melanogaster embryos. Based on the landscape of non-coding conservation in tephritids, and our initial success in using conservation in tephritids to identify D. melanogaster regulatory sequences, we suggest that comparison of tephritid genomes may provide a systematic means to annotate the non-coding portion of the D. melanogaster genome. We also propose that large genomes be given more consideration in the selection of species for comparative genomics projects, to provide increased power to detect functional non-coding DNAs and to provide a less biased view of the evolution and function of animal genomes

The Pioneer Anomaly

Radio-metric Doppler tracking data received from the Pioneer 10 and 11 spacecraft from heliocentric distances of 20-70 AU has consistently indicated the presence of a small, anomalous, blue-shifted frequency drift uniformly changing with a rate of ~6 x 10^{-9} Hz/s. Ultimately, the drift was interpreted as a constant sunward deceleration of each particular spacecraft at the level of a_P = (8.74 +/- 1.33) x 10^{-10} m/s^2. This apparent violation of the Newton's gravitational inverse-square law has become known as the Pioneer anomaly; the nature of this anomaly remains unexplained. In this review, we summarize the current knowledge of the physical properties of the anomaly and the conditions that led to its detection and characterization. We review various mechanisms proposed to explain the anomaly and discuss the current state of efforts to determine its nature. A comprehensive new investigation of the anomalous behavior of the two Pioneers has begun recently. The new efforts rely on the much-extended set of radio-metric Doppler data for both spacecraft in conjunction with the newly available complete record of their telemetry files and a large archive of original project documentation. As the new study is yet to report its findings, this review provides the necessary background for the new results to appear in the near future. In particular, we provide a significant amount of information on the design, operations and behavior of the two Pioneers during their entire missions, including descriptions of various data formats and techniques used for their navigation and radio-science data analysis. As most of this information was recovered relatively recently, it was not used in the previous studies of the Pioneer anomaly, but it is critical for the new investigation.Comment: 165 pages, 40 figures, 16 tables; accepted for publication in Living Reviews in Relativit

Structural Basis for Type VI Secretion Effector Recognition by a Cognate Immunity Protein

The type VI secretion system (T6SS) has emerged as an important mediator of interbacterial interactions. A T6SS from Pseudomonas aeruginosa targets at least three effector proteins, type VI secretion exported 1–3 (Tse1–3), to recipient Gram-negative cells. The Tse2 protein is a cytoplasmic effector that acts as a potent inhibitor of target cell proliferation, thus providing a pronounced fitness advantage for P. aeruginosa donor cells. P. aeruginosa utilizes a dedicated immunity protein, type VI secretion immunity 2 (Tsi2), to protect against endogenous and intercellularly-transferred Tse2. Here we show that Tse2 delivered by the T6SS efficiently induces quiescence, not death, within recipient cells. We demonstrate that despite direct interaction of Tsi2 and Tse2 in the cytoplasm, Tsi2 is dispensable for targeting the toxin to the secretory apparatus. To gain insights into the molecular basis of Tse2 immunity, we solved the 1.00 Å X-ray crystal structure of Tsi2. The structure shows that Tsi2 assembles as a dimer that does not resemble previously characterized immunity or antitoxin proteins. A genetic screen for Tsi2 mutants deficient in Tse2 interaction revealed an acidic patch distal to the Tsi2 homodimer interface that mediates toxin interaction and immunity. Consistent with this finding, we observed that destabilization of the Tsi2 dimer does not impact Tse2 interaction. The molecular insights into Tsi2 structure and function garnered from this study shed light on the mechanisms of T6 effector secretion, and indicate that the Tse2–Tsi2 effector–immunity pair has features distinguishing it from previously characterized toxin–immunity and toxin–antitoxin systems

Silencing α-Synuclein Gene Expression Enhances Tyrosine Hydroxylase Activity in MN9D Cells

α-Synuclein has been implicated in the pathogenesis of Parkinson’s disease (PD). Previous studies have shown that α-synuclein is involved in the regulation of dopamine (DA) metabolism, possibly by down-regulating the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in DA biosynthesis. In this study, we constructed α-synuclein stably silenced MN9D/α-SYN− cells by vector mediated RNA interference and examined its effects on DA metabolism. We found that there were no significant differences in TH protein and mRNA levels between MN9D, MN9D/α-SYN− and MN9D/CON cells, suggesting that silencing α-synuclein expression does not affect TH gene expression. However, significant increases in phosphorylated TH, cytosolic 3, 4-dihydroxyphenylalanine (l-DOPA) and DA levels were observed in MN9D/α-SYN− cells. Our data show that TH activity and DA biosynthesis were enhanced by down-regulation of α-synuclein, suggesting that α-synuclein may act as a negative regulator of cytosolic DA. With respect to PD pathology, a loss of functional α-synuclein may result in increased DA levels in neurons that may lead to cell injury or even death

Drug Discovery Using Chemical Systems Biology: Identification of the Protein-Ligand Binding Network To Explain the Side Effects of CETP Inhibitors

Systematic identification of protein-drug interaction networks is crucial to correlate complex modes of drug action to clinical indications. We introduce a novel computational strategy to identify protein-ligand binding profiles on a genome-wide scale and apply it to elucidating the molecular mechanisms associated with the adverse drug effects of Cholesteryl Ester Transfer Protein (CETP) inhibitors. CETP inhibitors are a new class of preventive therapies for the treatment of cardiovascular disease. However, clinical studies indicated that one CETP inhibitor, Torcetrapib, has deadly off-target effects as a result of hypertension, and hence it has been withdrawn from phase III clinical trials. We have identified a panel of off-targets for Torcetrapib and other CETP inhibitors from the human structural genome and map those targets to biological pathways via the literature. The predicted protein-ligand network is consistent with experimental results from multiple sources and reveals that the side-effect of CETP inhibitors is modulated through the combinatorial control of multiple interconnected pathways. Given that combinatorial control is a common phenomenon observed in many biological processes, our findings suggest that adverse drug effects might be minimized by fine-tuning multiple off-target interactions using single or multiple therapies. This work extends the scope of chemogenomics approaches and exemplifies the role that systems biology has in the future of drug discovery