The role of carbohydrates on protein function : the case of PGHSs

Estima-se que em torno de metade das proteínas existentes na natureza sejam glicosiladas. Ao ligarem-se às proteínas, os carboidratos podem alterar diversas de suas propriedades, tanto físico-químicas quanto biológicas, tornando-se fundamentais no entendimento de suas funções e, como consequência, na sua modulação com finalidades terapêuticas. Uma das formas de obtermos informações acerca do papel da porção sacarídica de uma glicoproteína envolve o estudo da estrutura tridimensional destas biomacromoléculas, por exemplo, através de cálculos de modelagem molecular, particularmente simulações de dinâmica molecular. Nosso grupo de pesquisas vem se dedicando ao desenvolvimento e a validação de abordagens de baixo custo computacional e baseadas em ferramentas gratuitas capazes de contribuírem para o entendimento de fenômenos biológicos. Estes procedimentos serão apresentados a seguir, tomando como exemplo as enzimas denominadas Prostaglandinas Endoperóxido Sintases, em suas isoformas 1 e 2.It is estimated that about half of all existing proteins are glycosylated. When bound to proteins, carbohydrates are able to affect several properties of such molecules, including physicochemical and biological properties, and therefore they became essential for understanding its function and its theraupeutical modulation. One of many strategies to obtain information on the role of a glycoprotein`s saccharidic moiety involves the tridimensional study of such macromolecules. This can be achieved using molecular modeling, specifically, molecular dynamics simulations. Therefore, our research group has been dedicated to the development and validation of low computational cost approaches, based on free of charge tools, capable of contribute to the understanding of biological phenomena. These procedures are presented next, using Prostaglandin Endoperoxide Synthase 1 and 2 as example

The calcium goes meow : effects of ions and glycosylation on Fel d 1, the major cat allergen

The major cat allergen, Fel d 1, is a structurally complex protein with two N-glycosylation sites that may be filled by different glycoforms. In addition, the protein contains three putative Ca2+ binding sites. Since the impact of these Fel d 1 structure modifications on the protein dynamics, physiology and pathology are not well established, the present work employed computational biology techniques to tackle these issues. While conformational effects brought upon by glycosylation were identified, potentially involved in cavity volume regulation, our results indicate that only the central Ca2+ion remains coordinated to Fel d 1 in biological solutions, impairing its proposed role in modulating phospholipase A2 activity. As these results increase our understanding of Fel d 1 structural biology, they may offer new support for understanding its physiological role and impact into cat-promoted allergy

Glycyrrhizin and glycyrrhetic acid: scaffolds to promising new pharmacologically active compounds

Glycyrrhizinic acid (GL), also known as glycyrrhizin, is a triterpene saponin, a natural product found on the root of Glycyrrhyza glabra L. (“licquorice”), used worldwide as sweetener and in the traditional eastern medicines. This review is focused on a series of new derivatives synthesized using GL and its aglycon, glycyrrhetinic acid (GLA), as starting materials, the pharmacological activities described for those compounds, as well as new activities reported for GL and GLA themselves.O ácido glicirrizínico (GL), também conhecido como glicirrizina, é uma saponina triterpênica, um produto natural encontrado na raiz de Glycyrrhyza glabra L. (“licquorice” ou “alcaçuz”), utilizada mundialmente como edulcorante e também na medicina tradicional do Oriente. Este artigo de revisão enfoca os novos compostos sintetizados usando GL ou sua aglicona, o ácido glicirretínico (GLA), como materiais de partida e as atividades farmacológicas descritas para os mesmos e seus derivados

Insights into the N-Sulfation Mechanism: Molecular Dynamics Simulations of the N-Sulfotransferase Domain of Ndst1 and Mutants

Sulfation patterns along glycosaminoglycan (GAG) chains dictate their functional role. the N-deacetylase N-sulfotransferase family (NDST) catalyzes the initial downstream modification of heparan sulfate and heparin chains by removing acetyl groups from subsets of N-acetylglucosamine units and, subsequently, sulfating the residual free amino groups. These enzymes transfer the sulfuryl group from 3'-phosphoadenosine-5'-phosphosulfate (PAPS), yielding sulfated sugar chains and 3'-phosphoadenosine-5'-phosphate (PAP). for the N-sulfotransferase domain of NDST1, Lys833 has been implicated to play a role in holding the substrate glycan moiety close to the PAPS cofactor. Additionally, Lys833 together with His716 interact with the sulfonate group, stabilizing the transition state. Such a role seems to be shared by Lys614 through donation of a proton to the bridging oxygen of the cofactor, thereby acting as a catalytic acid. However, the relevance of these boundary residues at the hydrophobic cleft is still unclear. Moreover, whether Lys833, His716 and Lys614 play a role in both glycan recognition and glycan sulfation remains elusive. in this study we evaluate the contribution of NDST mutants (Lys833, His716 and Lys614) to dynamical effects during sulfate transfer using comprehensive combined docking and essential dynamics. in addition, the binding location of the glycan moiety, PAPS and PAP within the active site of NDST1 throughout the sulfate transfer were determined by intermediate state analysis. Furthermore, NDST1 mutants unveiled Lys833 as vital for both the glycan binding and subsequent N-sulfotransferase activity of NDST1.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional do Desenvolvimento Cientifico e TecnologicoCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo, Dept Bioquim, São Paulo, BrazilUniv Fed Rio Grande do Sul, Ctr Biotecnol, Porto Alegre, RS, BrazilUniversidade Federal de São Paulo, Dept Bioquim, São Paulo, BrazilFAPESP: 2010/52426-3Web of Scienc

Molecular Insight into Silk Fibroin Based Delivery Vehicle for Amphiphilic Drugs: Synthesis, Characterization and Molecular Dynamics Studies

©2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ This document is the Accepted Manuscript version of a Published Work that appeared in final form in Journal of Molecular Liquids. To access the final edited and published work see DOI: 10.1016/j.molliq.2019.112156Recent emergence of natural biopolymers as drug delivery vehicles is attributed to their biodegradability and less systemic toxicity. Here, we have synthesized curcumin, indomethacin and emodin-loaded silk fibroin nanoparticles (SFNs) and characterized several pharmacokinetic parameters (Drug Loading and Encapsulation Efficiency). Silk fibroin is a highly promising bio-material with impressive mechanical properties, high bio-compatibility and it does not exert any immunological responses in vivo. Our results show that emodin almost released completely within 144 hr, however a steady release profile has been observed for indomethacin which is attributed to its moderate loading and encapsulation efficiency by SFNs. On the other hand, complete release of curcumin is not observed even in 168 hr. Curcumin also shows very promising drug loading and encapsulation efficiency when loaded within the SFNs matrix. Molecular level characterization with the aid of blind docking and molecular dynamics simulation reveals that the encapsulation efficiency of the drugs exactly follows the interaction energy patterns obtained from MM/PBSA calculation, i.e., curcumin > indomethacin > emodin. Strong binding energy of curcumin with the fibroin protein is attributed to the formation of more number of hydrogen bonds compared to the other two drugs and involvement in additional π-π stacking interactions. Indomethacin interacts moderately with the SFN primarily mediated through several van der Waals interactions which accounts for its sustained release from the SFN matrix. Emodin interacts with the fibroin protein very weakly which is responsible for its low encapsulation and observed diffusion controlled release behavior within the fibroin matrix

Anticoagulant Activity of a Unique Sulfated Pyranosic (133) - ß - L - Arabinan through Direct Interaction with Thrombin

Fernández, Paula Virginia. Universidad de Buenos Aires. Facultad de Agronomía. Departamento de Biología Aplicada y Alimentos. Cátedra de Química de Biomoléculas. Buenos Aires, Argentina.Quintana, Irene. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Hemostasia y Trombosis. Buenos Aires, Argentina.Cerezo, Alberto S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica. Subsede del Centro de Investigación de Hidratos de Carbono (CIHIDECAR). Ciudad Universitaria, Buenos Aires, Argentina.Caramelo, Julio J. CONICET - Instituto de Investigaciones Bioquímicas de Buenos Aires (IIBBA). Buenos Aires, Argentina.Pol Fachin, Laercio. Universidade Federal do Rio Grande do Sul. Faculdade de Farmácia. Programa de Pos-Graduação em Biologia Celular e Molecular. Centro de Biotecnologia. Rio Grande do Sul, Brasil.Verli, Hugo. Universidade Federal do Rio Grande do Sul.Centro de Biotecnologia. Programa de Pos-Graduação em Biologia Celular e Molecular. Rio Grande do Sul, Brasil.Estevez, José Manuel. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias (IFIByNE). Buenos Aires, Argentina.Ciancia, Marina. Universidad de Buenos Aires. Facultad de Agronomía. Departamento de Biología Aplicada y Alimentos. Cátedra de Química de Biomoléculas. Buenos Aires, Argentina.223–233Background: Many seaweed polysaccharides have anticoagulant activity, but the mechanism of action was elucidated in a few cases. Results: A highly sulfated pyranosic ß-arabinan exerts its activity through direct and indirect inhibition of thrombin. Conclusion: The structure and mechanism of action of the arabinan are different from those found for other polysaccharides. Significance: This arabinan could be an alternative anticoagulant in certain specific cases

Homology modeling and molecular dynamics provide structural insights into tospovirus nucleoprotein

Background: Tospovirus is a plant-infecting genus within the family Bunyaviridae, which also includes four animalinfecting genera: Hantavirus, Nairovirus, Phlebovirus and Orthobunyavirus. Compared to these members, the structures of Tospovirus proteins still are poorly understood. Despite multiple studies have attempted to identify candidate N protein regions involved in RNA binding and protein multimerization for tospovirus using yeast two-hybrid systems (Y2HS) and site-directed mutagenesis, the tospovirus ribonucleocapsids (RNPs) remains largely uncharacterized at the molecular level and the lack of structural information prevents detailed insight into these interactions. Results: Here we used the nucleoprotein structure of LACV (La Crosse virus-Orthobunyavirus) and molecular dynamics simulations to access the structure and dynamics of the nucleoprotein from tospovirus GRSV (Groundnut ringspot virus). The resulting model is a monomer composed by a flexible N-terminal and C-terminal arms and a globular domain with a positively charged groove in which RNA is deeply encompassed. This model allowed identifying the candidate amino acids residues involved in RNA interaction and N-N multimerization. Moreover, most residues predicted to be involved in these interactions are highly conserved among tospoviruses. Conclusions: Crucially, the interaction model proposed here for GRSV N is further corroborated by the all available mutational studies on TSWV (Tomato spotted wilt virus) N, so far. Our data will help designing further and more accurate mutational and functional studies of tospovirus N proteins. In addition, the proposed model may shed light on the mechanisms of RNP shaping and could allow the identification of essential amino acid residues as potential targets for tospovirus control strategies

Thermal-induced conformational changes in the product release area drive the enzymatic activity of xylanases 10B: Crystal structure, conformational stability and functional characterization of the xylanase 10B from Thermotoga petrophila RKU-1

AbstractEndo-xylanases play a key role in the depolymerization of xylan and recently, they have attracted much attention owing to their potential applications on biofuels and paper industries. In this work, we have investigated the molecular basis for the action mode of xylanases 10B at high temperatures using biochemical, biophysical and crystallographic methods. The crystal structure of xylanase 10B from hyperthermophilic bacterium Thermotoga petrophila RKU-1 (TpXyl10B) has been solved in the native state and in complex with xylobiose. The complex crystal structure showed a classical binding mode shared among other xylanases, which encompasses the −1 and −2 subsites. Interestingly, TpXyl10B displayed a temperature-dependent action mode producing xylobiose and xylotriose at 20°C, and exclusively xylobiose at 90°C as assessed by capillary zone electrophoresis. Moreover, circular dichroism spectroscopy suggested a coupling effect of temperature-induced structural changes with this particular enzymatic behavior. Molecular dynamics simulations supported the CD analysis suggesting that an open conformational state adopted by the catalytic loop (Trp297-Lys326) provokes significant modifications in the product release area (+1,+2 and +3 subsites), which drives the enzymatic activity to the specific release of xylobiose at high temperatures