Cyclic adenosine monophosphate

Cyclic adenosine monophosphate (cAMP, cyclic AMP, or 3',5'-cyclic adenosine monophosphate) is a second messenger, or cellular signal occurring within cells, that is important in many biological processes. cAMP is a derivative of adenosine triphosphate (ATP) and used for intracellular signal transduction in many different organisms, conveying the cAMP-dependent pathway. History Earl Sutherland of Vanderbilt University won a Nobel Prize in Physiology or Medicine in 1971 "for his discoveries concerning the mechanisms of the action of hormones", especially epinephrine, via second messengers (such as cyclic adenosine monophosphate, cyclic AMP). Synthesis Cyclic AMP is synthesized from ATP by adenylate cyclase located on the inner side of the plasma membrane and anchored at various locations in the interior of the cell. Adenylate cyclase is activated by a range of signaling molecules through the activation of adenylate cyclase stimulatory G (Gs)-protein-coupled receptors

Metabolic modeling and experimental studies on carbon and energy metabolism of Mycobacterium tuberculosis

Muhittin Emre Özdemir

Tuberculosis is an ancient human disease affecting millions of people every year. The common causative agent, Mycobacterium tuberculosis, is a peculiar pathogen that can persist in the host tissues for decades. Many of the key attributes of M. tuberculosis can be traced to its metabolism. Therefore, novel insights into the metabolism of this bacterium could lead to the identification of new drug targets as well as better methods to understand the mechanism of action of anti-tuberculosis drugs. Analysis of the metabolic network of this bacterium using a genome-scale model offers an unconventional approach to understanding utilization of metabolic pathways of this pathogen during growth and non-replicating persistence. Metabolic adaptation of a pathogen is central to its life style in vivo. To understand the capacity and limitations of M. tuberculosis, we developed an updated and thermodynamically curated genome scale model – iOS783. This model has been verified with respect to the observed phenotypes for targeted gene deletion studies reported previously. Evaluation of iOS783 with thermodynamics based flux balance analysis (TFBA) improved overall reliability of the model results and led to identification of possible bottleneck reactions. In addition, several new hypotheses were generated with iOS783, involving the role of isocitrate lyase enzymes, propionate toxicity, and energy metabolism of this pathogen. Analysis of iOS783 for conditional gene essentiality emphasizes the importance of the carbon source available to the pathogen. Several genes show a conditional impact on biomass production rate of the model, depending on the availability of carbohydrates or fatty acids as the carbon substrate. Conditional essentiality is even more pronounced when ATP replenishment capacity is used as a measure of a pathogen’s ability to survive under stress in a non-growing state. As expected, ATP synthase plays a major role in estimations of ATP replenishment capacity based on iOS783. Curiously, computational analysis of the metabolic network with ATP synthase activity blocked indicates that the damaged network can use substrate-level phosphorylation reactions to sustain ATP production to fulfill non-growth-associated ATP requirements when glucose is available. In order to establish the dependence of M. tuberculosis on ATP synthase, we attempted to construct a deletion of the chromosomal atp operon. Our results show that the native copy of these genes can be deleted only when a second set of genes are introduced elsewhere in the genome, indicating essentiality of the ATP synthase for growth. Although our results indicate that ATP synthase is essential for growth, we observed carbon source specific efficacy of an ATP synthase inhibitor, bedaquiline, on M. tuberculosis. These experimental results are consistent with ATP turnover estimations based on modeling results. Furthermore, analysis of iOS783 suggests a novel role in energy metabolism for recently described glycine dehydrogenase (GDH) on energy metabolism. Deletion of ald gene responsible for GDH activity reduces the ATP replenishment capacity of iOS783, especially during metabolism of fatty acid derivative carbon sources. Consistent with these modeling results, wetlab experiments designed to test this hypothesis show that bedaquiline has a stronger bactericidal effect on an M. tuberculosis ∆ald mutant, compared to the wild-type parental strain, when cells are cultured in medium containing acetate as the carbon source. Metabolic modelling is already well established as an invaluable tool for engineering of strains with desired characteristics. For pathogenic species, however, the application of this approach has so far been quite limited. The work presented in this thesis exemplifies an interdisciplinary approach combining computational modeling and experimental studies of M. tuberculosis metabolism. First, iOS783 was utilized to interpret existing experimental results and to generate novel hypotheses, thereby providing the impetus for new wetlab experiments. Conversely, results from iOS783-inspired wetlab experiments were used to further refine and expand the iOS783 model. This cyclic exchange between computational modeling and wetlab experimentation illustrates how a close interdisciplinary partnership can create a positive feedback loop that enriches both disciplines.

EPFL2013

Regulatory elements involved in the expression of competence genes in naturally transformable Vibrio cholerae

Melanie Blokesch, Mirella Lo Scrudato

BackgroundThe human pathogen Vibrio cholerae normally enters the developmental program of natural competence for transformation after colonizing chitinous surfaces. Natural competence is regulated by at least three pathways in this organism: chitin sensing/degradation, quorum sensing and carbon catabolite repression (CCR). The cyclic adenosine monophosphate (cAMP) receptor protein CRP, which is the global regulator of CCR, binds to regulatory DNA elements called CRP sites when in complex with cAMP. Previous studies in Haemophilus influenzae suggested that the CRP protein binds competence-specific CRP-S sites under competence-inducing conditions, most likely in concert with the master regulator of transformation Sxy/TfoX.ResultsIn this study, we investigated the regulation of the competence genes qstR and comEA as an example of the complex process that controls competence gene activation in V. cholerae. We identified previously unrecognized putative CRP-S sites upstream of both genes. Deletion of these motifs significantly impaired natural transformability. Moreover, site-directed mutagenesis of these sites resulted in altered gene expression. This altered gene expression also correlated directly with protein levels, bacterial capacity for DNA uptake, and natural transformability.ConclusionsBased on the data provided in this study we suggest that the identified sites are important for the expression of the competence genes qstR and comEA and therefore for natural transformability of V. cholerae even though the motifs might not reflect bona fide CRP-S sites.

BioMed Central2014

Astrocytic release of ATP through type 2 inositol 1,4,5‐trisphosphate receptor calcium signaling and social dominance behavior in mice

Jocelin Grosse, Marie-Isabelle Guillot de Suduiraut, Fiona Hollis, Eva Ramos Fernandez, Maria del Carmen Sandi Perez

Brain mitochondrial function is critical for numerous neuronal processes. We recently identified a link between brain energy and social dominance, where higher levels of mitochondrial function resulted in increased social competitive ability. The underlying mechanism of this link, however, remains unclear. Here, we investigated the contribution of astrocytic release of adenosine triphosphate (ATP) through the type 2 inositol 1,4,5‐triphosphate receptor to social dominance behavior. Mice lacking the type 2 inositol 1,4,5‐triphosphate receptor were characterized for their social dominance behavior, as well as their performance on a nonsocial task, the Morris Water Maze. In parallel, we also examined mitochondrial function in the medial prefrontal cortex, nucleus accumbens, and hippocampus to investigate how deficiencies in astrocytic ATP could modulate overall mitochondrial function. While knockout mice showed similar competitive ability compared with their wild‐type littermates, dominant knockout mice exhibited a significant delay in exerting their dominance during the initial encounter. Otherwise, there were no differences in anxiety and exploratory traits, spatial learning and memory, or brain mitochondrial function in either light or dark circadian phases. Our findings point to a marginal role of astrocytic ATP through IP3R2 in social competition, suggesting that, under basal conditions, the neuronal compartment is predominant for social dominance exertion.

2020