Cyclic AMP-induced Conformational Changes in Mycobacterial Protein Acetyltransferases

The activities of a number of proteins are regulated by the binding of cAMP and cGMP to cyclic nucleotide binding (CNB) domains that are found associated with one or more effector domains with diverse functions. Although the conserved architecture of CNB domains has been extensively studied by x-ray crystallography, the key to unraveling the mechanisms of cAMP action has been protein dynamics analyses. Recently, we have identified a novel cAMP-binding protein from mycobacteria, where cAMP regulates the activity of an associated protein acetyltransferase domain. In the current study, we have monitored the conformational changes that occur upon cAMP binding to the CNB domain in these proteins, using a combination of bioluminescence resonance energy transfer and amide hydrogen/deuterium exchange mass spectrometry. Coupled with mutational analyses, our studies reveal the critical role of the linker region (positioned between the CNB domain and the acetyltransferase domain) in allosteric coupling of cAMP binding to activation of acetyltransferase catalysis. Importantly, major differences in conformational change upon cAMP binding were accompanied by stabilization of the CNB and linker domain alone. This is in contrast to other cAMP-binding proteins, where cyclic nucleotide binding has been shown to involve intricate and parallel allosteric relays. Finally, this powerful convergence of results from bioluminescence resonance energy transfer and hydrogen/deuterium exchange mass spectrometry reaffirms the power of solution biophysical tools in unraveling mechanistic bases of regulation of proteins in the absence of high resolution structural information.

Damage limit states of reinforced concrete beams subjected to incremental cyclic loading using relaxation ratio analysis of AE parameters

This paper presents an experimental study on damage assessment of reinforced concrete (RC) beams subjected to incremental cyclic loading. During testing acoustic emissions (AEs) were recorded. The analysis of the AE released was carried out by using parameters relaxation ratio, load ratio and calm ratio. Digital image correlation (DIC) technique and tracking with available MATLAB program were used to measure the displacement and surface strains in concrete. Earlier researchers classified the damage in RC beams using Kaiser effect, crack mouth opening displacement and proposed a standard. In general (or in practical situations), multiple cracks occur in reinforced concrete beams. In the present study damage assessment in RC beams was studied according to different limit states specified by the code of practice IS-456:2000 and AE technique. Based on the two ratios namely load ratio and calm ratio and when the deflection reached approximately 85% of the maximum allowable deflection it was observed that the RC beams were heavily damaged. The combination of AE and DIC techniques has the potential to provide the state of damage in RC structures.

A cyclic peptide mimic of an RNA recognition motif of human La protein is a potent inhibitor of hepatitis C virus

Due to limited available therapeutic options, developing new lead compounds against hepatitis C virus is an urgent need. Human La protein stimulates hepatitis C virus translation through interaction with the hepatitis C viral RNA. A cyclic peptide mimicking the beta-turn of the human La protein that interacts with the viral RNA was synthesized. It inhibits hepatitis C viral RNA translation significantly better than the corresponding linear peptide at longer post-treatment times. The cyclic peptide also inhibited replication as measured by replicon RNA levels using real time RT-PCR. The cyclic peptide emerges as a promising lead compound against hepatitis C.

Processing of DNA double-stranded breaks and intermediates of recombination and repair by saccharomyces cerevisiae Mre11 and its stimulation by Rad50, Xrs2, and Sae2 proteins

Saccharomyces cerevisiae RAD50, MRE11, and XRS2 genes are essential for telomere length maintenance, cell cycle checkpoint signaling, meiotic recombination, and DNA double-stranded break (DSB) repair via nonhomologous end joining and homologous recombination. The DSB repair pathways that draw upon Mre11-Rad50-Xrs2 subunits are complex, so their mechanistic features remain poorly understood. Moreover, the molecular basis of DSB end resection in yeast mre11-nuclease deficient mutants and Mre11 nuclease-independent activation of ATM in mammals remains unknown and adds a new dimension to many unanswered questions about the mechanism of DSB repair. Here, we demonstrate that S. cerevisiae Mre11 (ScMre11) exhibits higher binding affinity for single-over double-stranded DNA and intermediates of recombination and repair and catalyzes robust unwinding of substrates possessing a 3' single-stranded DNA overhang but not of 5' overhangs or blunt-ended DNA fragments. Additional evidence disclosed that ScMre11 nuclease activity is dispensable for its DNA binding and unwinding activity, thus uncovering the molecular basis underlying DSB end processing in mre11 nuclease deficient mutants. Significantly, Rad50, Xrs2, and Sae2 potentiate the DNA unwinding activity of Mre11, thus underscoring functional interaction among the components of DSB end repair machinery. Our results also show that ScMre11 by itself binds to DSB ends, then promotes end bridging of duplex DNA, and directly interacts with Sae2. We discuss the implications of these results in the context of an alternative mechanism for DSB end processing and the generation of single-stranded DNA for DNA repair and homologous recombination.

The precision with which single cells of Dictyostelium discoideum can locate a source of cyclic AMP

When stimulated by a point source of cyclic AMP, a starved amoeba of Dictyostelium discoideum responds by putting out a hollow balloon-like membrane extension followed by a pseudopod. The effect of the stimulus is to influence the position where either of these protrusions is made on the cell rather than to cause them to be made. Because the pseudopod forms perpendicular to the cell surface, its location is a measure of the precision with which the cell can locate the cAMP source. Cells beyond 1 h of starvation respond non-randomly with a precision that improves steadily thereafter. A cell that is starved for 1-2 h can locate the source accurately 43% of the time; and if starved for 6-7 h, 87% of the time. The response always has a high scatter; population-level heterogeneity reflects stochasticity in single cell behaviour. From the angular distribution of the response its maximum information content is estimated to be 2-3 bits. In summary, we quantitatively demonstrate the stochastic nature of the directional response and the increase in its accuracy over time.

Cyclic AMP-dependent protein lysine acylation in mycobacteria regulates fatty acid and propionate metabolism

Acetylation of lysine residues is a posttranslational modification that is used by both eukaryotes and prokaryotes to regulate a variety of biological processes. Here we identify multiple substrates for the cAMP-dependent protein lysine acetyltransferase from Mycobacterium tuberculosis (KATmt). We demonstrate that a catalytically important lysine residue in a number of FadD (fatty acyl CoA synthetase) enzymes is acetylated by KATmt in a cAMP-dependent manner and that acetylation inhibits the activity of FadD enzymes. A sirtuin-like enzyme can deacetylate multiple FadDs, thus completing the regulatory cycle. Using a strain deleted for the KATmt ortholog in Mycobacterium bovis Bacillus Calmette-Guerin (BCG), we show for the first time that acetylation is dependent on intracellular cAMP levels. KATmt can utilize propionyl CoA as a substrate and, therefore, plays a critical role in alleviating propionyl CoA toxicity in mycobacteria by inactivating acyl CoA synthetase (ACS). The precision by which mycobacteria can regulate the metabolism of fatty acids in a cAMP-dependent manner appears to be unparalleled in other biological organisms and is ideally suited to adapt to the complex environment that pathogenic mycobacteria experience in the host.

Cyclic nucleotide signaling in intestinal epithelia: getting to the gut of the matter

The intestine is the primary site of nutrient absorption, fluid-ion secretion, and home to trillions of symbiotic microbiota. The high turnover of the intestinal epithelia also renders it susceptible to neoplastic growth. These diverse processes are carefully regulated by an intricate signaling network. Among the myriad molecules involved in intestinal epithelial cell homeostasis are the second messengers, cyclic AMP (cAMP) and cyclic GMP (cGMP). These cyclic nucleotides are synthesized by nucleotidyl cyclases whose activities are regulated by extrinsic and intrinsic cues. Downstream effectors of cAMP and cGMP include protein kinases, cyclic nucleotide gated ion channels, and transcription factors, which modulate key processes such as ion-balance, immune response, and cell proliferation. The web of interaction involving the major signaling pathways of cAMP and cGMP in the intestinal epithelial cell, and possible cross-talk among the pathways, are highlighted in this review. Deregulation of these pathways occurs during infection by pathogens, intestinal inflammation, and cancer. Thus, an appreciation of the importance of cyclic nucleotide signaling in the intestine furthers our understanding of bowel disease, thereby aiding in the development of therapeutic approaches.

Cyclic AMP in Mycobacteria: the second messenger comes first

Cyclic AMP (cAMP) has emerged as a pivotal molecule for signalling in all life forms. Mycobacterial genomes have been found to encode for numerous proteins that are involved in cAMP generation, degradation and utilization. Many of these proteins have domain organizations unique to mycobacteria. This review summarizes recent advances in mechanisms of cAMP synthesis and degradation, focusing on the processes by which cAMP modulates mycobacterial signalling. We explore its impact on the physiology of the organism and on the discourse between M. tuberculosis and its host.

Generalization of deviated linear cyclic pursuit

Earlier work on cyclic pursuit systems has shown that using heterogeneous gains for agents in linear cyclic pursuit, the point of convergence (rendezvous point) can be chosen arbitrarily. But there are some restrictions on this set of reachable points. The use of deviated cyclic pursuit, as discussed in this paper, expands this set of reachable points to include points which are not reachable by any known linear cyclic pursuit scheme. The limits on the deviations are determined by stability considerations. Such limits have been analytically obtained in this paper along with results on the expansion in reachable set and the latter has also been verified through simulations.

Elastic-plastic contact conditions for frictionally constrained bodies under cyclic tangential loading

A Frictionally constrained condition implies dependence of friction force on tangential displacement amplitude. The condition may occur due to chemical, physical, and/or mechanical interaction between the contacting surfaces. The condition, sometimes also referred to as the presliding condition or partial slip condition, is characterized under fretting. Under such conditions, various experimental studies indicate the existence of two distinguishable regions, that is, stick region and slip region. In the present study, frictionally constrained conditions are identified and the evolutions of stick-slip regions are investigated in detail. Investigations have been performed on self-mated stainless steel and chromium carbide coated surfaces mated against stainless steel, under both vacuum and ambient conditions. Contact conditions prevailing at the contact interface were identified based on the mechanical responses and were correlated with the surface damage observed. Surface degradation has been observed in the form of microcracks and material transfer. Detailed numerical analysis has also been performed in order to understand the energy dissipation and the damage mode involved in the surface or subsurface damage. It has been observed that under frictionally constrained conditions, the occurrence of annular slip features are mainly due to the junction growth, resulting from elastic-plastic deformation at the contact interface. Ratcheting has been observed as the governing damage mode under cyclic tangential loading condition.

Role of melt convection on optimization of PCM-based heat sink under cyclic heat load

In this article, we study the thermal performance of phase-change material (PCM)-based heat sinks under cyclic heat load and subjected to melt convection. Plate fin type heat sinks made of aluminum and filled with PCM are considered in this study. The heat sink is heated from the bottom. For a prescribed value of heat flux, design of such a heat sink can be optimized with respect to its geometry, with the objective of minimizing the temperature rise during heating and ensuring complete solidification of PCM at the end of the cooling period for a given cycle. For given length and base plate thickness of a heat sink, a genetic algorithm (GA)-based optimization is carried out with respect to geometrical variables such as fin thickness, fin height, and the number of fins. The thermal performance of the heat sink for a given set of parameters is evaluated using an enthalpy-based heat transfer model, which provides the necessary data for the optimization algorithm. The effect of melt convection is studied by taking two cases, one without melt convection (conduction regime) and the other with convection. The results show that melt convection alters the results of geometrical optimization.

Pulsed Electrical Stimulation and Surface Charge Induced Cell Growth on Multistage Spark Plasma Sintered Hydroxyapatite-Barium Titanate Piezobiocomposite

The primary purpose of the present work was to illustrate whether cell proliferation can be enhanced on electroactive bioceramic composite, when the cells are cultured in the presence of external electrical stimulation. The two different aspects of the influence of electric field (E-field) application toward stimulating the growth/proliferation of bone/connective tissue cells in vitro, (a) intermittent delivery of extremely low strength pulsed electrical stimulation (0.5-4V/cm, 400s DC pulse) and (b) surface charge generated by electrical poling (10kV/cm) of hydroxyapatite (HA)-BaTiO3 piezobiocomposite have been demonstrated. The experimental results establish that the cell growth can be enhanced using the new culture protocol of the intermittent delivery of electrical pulses within a narrow range of stimulation parameters. The optimal E-field strength for enhanced cellular response for mouse fibroblast L929 and osteogenic cells is in the range of 0.5-1V/cm. The MTT 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] assay results suggested the increased viability of E-field treated cells over 7d in culture, implicating the positive impact of electrical pulses on proliferation behavior. The alizarin red assay results showed noticeable increase in Ca-deposition on the E-field treated samples in comparison to their untreated counterparts. The negatively charged surfaces of developed piezocomposite stimulated the cell growth in a statistically noticeable manner as compared with the uncharged or positively charged surfaces of similar composition.

The influence of mesoporous silica in low T-g cyclic olefin copolymer nanocomposite films: Mechanical and moisture barrier studies

In this study, mesoporous silica-cyclic olefin copolymer nanocomposite films were fabricated by solution casting. With an increase in silica loading, the stiffness of the matrix increased. The nanocomposite film shows increased strain to failure with moisture after aging by matrix plasticization. The storage modulus and loss factor for samples with silica content show better results compared with pristine polymer, as indicated by dynamic mechanical analysis. The interaction between filler-polymer chain exhibit hydrophobicity compared to the neat polymer. Water absorption studies at room temperature and near the T-g of the polymer (similar to 64 degrees C) were carried out. The nanocomposites up to 4 wt% filler reduces the water diffusion by forming hydrogen and chemical bonding. The result by calcium degradation test method for moisture permeability and Schottky structured organic device encapsulation under weathering condition confirms the effective reinforcement effect of silica particles in the matrix. (C) 2014 Elsevier Ltd. All rights reserved.

Conformational Analysis of a 20-Membered Cyclic Peptide Disulfide from Conus virgo with a WPW Segment: Evidence for an Aromatic-Proline Sandwich

A novel peptide containing a single disulfide bond, CIWPWC (Vi804), has been isolated and characterised from the venom of the marine cone snail, Conus virgo. A precursor polypeptide sequence derived from complementary DNA, corresponding to the M-superfamily conotoxins, has been identified. The identity of the synthetic and natural peptide sequence has been established. A detailed analysis of the conformation in solution is reported for Vi804 and a synthetic analogue, (CIWPWC)-W-D ((D)W3-Vi804), in order to establish the structure of the novel WPW motif, which occurs in the context of a 20-membered macrocyclic disulfide. Vi804 exists exclusively in the cis W3P4 conformer in water and methanol, whereas (D)W3-Vi804 occurs exclusively as the trans conformer. NMR spectra revealed a W3P4 typeVI turn in Vi804 and a typeII turn in the analogue peptide, (D)W3-Vi804. The extremely high-field chemical shifts of the proline ring protons, together with specific nuclear Overhauser effects, are used to establish a conformation in which the proline ring is sandwiched between the flanking Trp residues, which emphasises a stabilising role for the aromatic-proline interactions, mediated predominantly by dispersion forces.