1,721,039 research outputs found
Structural bioinformatic analyses of (macro)molecular interactions of biomedical relevance: an experimental validation
No full textStructural bioinformatics, like many other subdisciplines within bioinformatics, is characterized by the establishment of general purpose methods for manipulating information about biological macromolecules, and the application of these methods to solving problems in biology and creating new knowledge. Among its capabilities, structural bioinformatics can analyse the feasible (macro)molecular interactions and assists, or sometimes anticipates, the experimental approaches in biological research, even starting from a prediction analysis of the three-dimensional structures of the partners. This thesis reports on the in silico and in vitro characterization of a selection on physiologically relevant processes involving binding between proteins and endogenous and exogenous ligands, with results confirming the well-founded capability of the bioinformatic methods to clarify these issues. The general approach consisted in: (i) the in silico derivation of the predictive structural and equilibrium parameters for the ligand-receptor complexes starting from either deposited crystallographic (where available) or homology modeled structures; (ii) the experimental validation of the computational data according to both "in solution" and "on surface" in vitro studies; (iii) the final evaluation of the effects of the interactions on cell based models. Specifically, during the PhD period my interest was mainly focused on the characterization of the molecular basis of the systemic sclerosis (SSc), a rare human auto-immune disease, with particular emphasis on the interaction between platelet-derived growth factor receptor and a selection of human autoantibodies expressed in SSc patients (the revised version of this manuscript is currently under evaluation in Nature Communications). This project was paralleled by several other studies, among which the modulation by natural polyphenols of two human enzymes, HMG-CoA reductase and plasmin, involved in cholesterol biosynthetic pathway and in cellular adhesion and mobility, respectively. The results of these studies were published in impacted scientific journals
Rapid reverse phase-HPLC assay of HMG-CoA reductase activity.
Full text linkRadioisotope-based and mass spectrometry coupled to chromatographic techniques are the conventional methods for monitoring HMG-CoA reductase (HMGR) activity. Irrespective of offering adequate sensitivity, these methods are often cumbersome and time-consuming, requiring the handling of radiolabeled chemicals or elaborate ad-hoc derivatizing procedures. We propose a rapid and versatile reverse phase-HPLC method for assaying HMGR activity capable of monitoring the levels of both substrates (HMG-CoA and NADPH) and products (CoA, mevalonate, and NADP(+)) in a single 20 min run with no pretreatment required. The linear dynamic range was 10-26 pmol for HMG-CoA, 7-27 nmol for NADPH, 0.5-40 pmol for CoA and mevalonate, and 2-27 nmol for NADP(+), and limit of detection values were 2.67 pmol, 2.77 nmol, 0.27 pmol, and 1.3 nmol, respectively
Understanding the function of bacterial and eukaryotic thiolases II by integrating evolutionary and functional approaches
Full text linkAcetoacetyl-CoA thiolase (EC 2.3.1.9), commonly named thiolase II, condenses two molecules of acetyl-CoA to give acetoacetyl-CoA and CoA. This enzyme acts in anabolic processes as the first step in the biosynthesis of isoprenoids and polyhydroxybutyrate in eukaryotes and bacteria, respectively. We have recently reported the evolutionary and functional equivalence of these enzymes, suggesting that thiolase II could be the rate limiting enzyme in these pathways and presented evidence indicating that this enzyme modulates the availability of reducing equivalents during abiotic stress adaptation in bacteria and plants. However, these results are not sufficient to clarify why thiolase II was evolutionary selected as a critical enzyme in the production of antioxidant compounds. Regarding this intriguing topic, we propose that thiolase II could sense changes in the acetyl-CoA/CoA ratio induced by the inhibition of the tricarboxylic acid cycle under abiotic stress. Thus, the high level of evolutionary and functional constraint of thiolase II may be due to the connection of this enzyme with an ancient and conserved metabolic route.Fil: Fox, Ana Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Soto, Gabriela Cynthia. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Mozzicafreddo, Matteo. University of Camerino. School of Biosciences and Biotechnology; ItaliaFil: García, Araceli Nora. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronómicas. Instituto de Genética; ArgentinaFil: Cuccioloni, Massimiliano. University of Camerino. School of Biosciences and Biotechnology; ItaliaFil: Angeletti, Mauro. University of Camerino. School of Biosciences and Biotechnology; ItaliaFil: Salerno, Juan Carlos. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronómicas. Instituto de Genética; ArgentinaFil: Ayub, Nicolas Daniel. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin
An In-Silico Comparative Study of Lipases from the Antarctic Psychrophilic Ciliate Euplotes focardii and the Mesophilic Congeneric Species Euplotes crassus: Insight into Molecular Cold-Adaptation
Full text linkCold-adapted enzymes produced by psychrophilic organisms have elevated catalytic activities at low temperatures compared to their mesophilic counterparts. This is largely due to amino acids changes in the protein sequence that often confer increased molecular flexibility in the cold. Comparison of structural changes between psychrophilic and mesophilic enzymes often reveal molecular cold adaptation. In the present study, we performed an in-silico comparative analysis of 104 hydrolytic enzymes belonging to the family of lipases from two evolutionary close marine ciliate species: The Antarctic psychrophilic Euplotes focardii and the mesophilic Euplotes crassus. By applying bioinformatics approaches, we compared amino acid composition and predicted secondary and tertiary structures of these lipases to extract relevant information relative to cold adaptation. Our results not only confirm the importance of several previous recognized amino acid substitutions for cold adaptation, as the preference for small amino acid, but also identify some new factors correlated with the secondary structure possibly responsible for enhanced enzyme activity at low temperatures. This study emphasizes the subtle sequence and structural modifications that may help to transform mesophilic into psychrophilic enzymes for industrial applications by protein engineering
Interaction between wheat alpha-amylase/trypsin bi-functional inhibitor and mammalian digestive enzymes: Kinetic, equilibrium and structural characterization of binding
No full textAlpha-amylase/trypsin bi-functional inhibitors (ATIs) are non-gluten protein components of wheat and other cereals that can hypersensitise the human gastrointestinal tract, eventually causing enteropathies in predisposed individuals. These inhibitory proteins can act both directly by targeting specific pro-inflammatory receptors, and indirectly by impairing the activity of digestive enzymes, the latter event causing the accumulation of undigested peptides with potential immunogenic properties. Herein, according to a concerted approach based on in vitro and in silico methods we characterized kinetics, equilibrium parameters and modes of binding of the complexes formed between wheat ATI and two representative mammalian digestive enzymes, namely trypsin and alpha-amylase. Interestingly, we demonstrated ATI to target both enzymes with independent binding sites and with moderately high affinity
Targeting phosphoglycerate kinases by tatridin A, a natural sesquiterpenoid endowed with anti-cancer activity, using a proteomic platform
Full text linkTatridin A (TatA) is a germacrane sesquiterpenoid containing one E-double bond and one Z-double bond in its 10-membered ring, which is fused to a 3- methylene-dihydrofuran-2-one moiety. Tatridin A bioactivity has been poorly investigated despite its interesting chemical structure. Here, a functional proteomic platform was adapted to disclose its most reliable targets in leukemia monocytic cells, and phosphoglycerate kinases were recognized as the most affine enzymes. Through a combination of limited proteolysis and molecular docking, it has been discovered that tatridin A interacts with the active domains of phosphoglycerate kinase 1, altering its hinge region, and it can be accountable for tatridin A inhibition potency on enzyme activity. A more detailed tatridin A biological profile showed that it is also fully active against gastric cancer cells, downregulating the mRNA levels of chemokine receptor 4 and β-catenin and inhibiting the invasiveness of living KATO III cells as a direct consequence of phosphoglycerate kinase 1 antagonism
Draft Genome Sequence of a New Pseudomonas sp. Strain, ef1, Associated with the Psychrophilic Antarctic Ciliate Euplotes focardii
Full text linkWe announce here the draft genome sequence of a new Pseudomonas strain, named Pseudomonas sp. strain ef1, associated with the cold-adapted Antarctic ciliate Euplotes focardii The genome sequence is 6,228,167 bp long with a G+C content of 59.7%
The relationship between the 20S proteasomes and prion-mediated neurodegenerations: potential therapeutic opportunities.
No full textThe dysfunction of cellular degradation pathways of aberrant and misfolded proteins is a critical event in the onset of neurodegenerative disorders. Among these pathologies, prion diseases are a unique class of transmissible fatal disorders affecting mammals, characterized by the presence of an abnormal isoform of a membrane-bound protein, namely the prion protein. The proteasome is the main proteolytic machinery in charge of removing damaged, oxidized and misfolded proteins and numerous authors have approached the involvement of this complex in the prion protein cellular processing. Herein, we described the general features of prion disorders focusing our attention on the available data on the interplay between the infectious agent and the proteasome system, exploring its implications in prion-mediated toxicity. Finally, considering the proteasome as a potential drug target, we reviewed possible therapeutic opportunities in the treatment of such pathologies. © 2010 Springer Science+Business Media, LLC
Molecular Evolution and Adaptation Strategies in Marine Ciliates: An Inspiration for Cold-Adapted Enzyme Engineering and Drug Binding Analysis
Full text linkIn the present review, we summarize genome mining of genomic data obtained from the psychrophilic Antarctic marine ciliate Euplotes focardii and its evolutionary-close mesophilic cosmopolitan counterpart E. crassus. This analysis highlights adaptation strategies that are unique to the Antarctic ciliate, including antioxidant gene duplication and distinctive substitutions that may play roles in increased drug binding affinity and enzyme reaction rate in cold environments. Enzymes from psychrophiles are usually characterized by high activities and reaction rates at low temperatures compared with their counterparts from mesophiles and thermophiles. As a rule, catalyst cold activity derives from an increased structural flexibility that may lead to protein denaturation in response to temperature fluctuation. Molecular thermolability has been a major drawback of using macromolecules from psychrophiles in industrial applications. Here, we report a case study in which the role of peculiar amino acid substitution in cold adaptation is demonstrated by site-directed mutagenesis. Combined with a rational design approach, these substitutions can be used for site-directed mutagenesis to obtain cold-active catalysts that are structurally stable. Furthermore, molecular docking analysis of β-tubulin isotypes extrapolated from E. focardii and E. crassus genomes allowed us to obtain additional insight on the taxol binding site and drug affinity. E. focardii genome mining and the comparison with the mesophilic sibling counterpart can be used as an inspiration for molecular engineering for medical and industrial applications
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