Crystallization, preliminary X-ray analysis and molecular-replacement solution of the carboxy form of haemoglobin I from the fish Brycon cephalus

3 p. : il.Haemoglobin, the `honorary enzyme' [Brunori (1999), Trends Biochem. Sci. 24, 158±161], constitutes a prime prototype for allosteric models. Here, the crystallization and preliminary X-ray analysis of haemoglobin I from the South American ®sh Brycon cephalus are reported. X-ray diffraction data have been collected to 2.5 A Ê resolution using synchrotron radiation (LNLS). Crystals were determined to belong to the space group P6122 and preliminary structural analysis revealed the presence of one dimer ( ) in the asymmetric unit. The structure was determined using standard molecular-replacement techniques

Biophysical and pharmacological characterization of a full-length synthetic analog of the antitumor polypeptide crotamine

Crotamine is a polypeptide isolated from the venom of a South American rattlesnake. Among the properties and biological activities of crotamine, the most extraordinary is its ability to enter cells with unique selective affinity and cytotoxic activity against actively proliferating cells, such as tumor cells. This peptide is also a cargo carrier, and anticipating commercial application of this native polypeptide as a potential theranostic compound against cancer, we performed here a side-by-side characterization of a chemically synthesized full-length crotamine compared with its native counterpart. The structural, biophysical, and pharmacological properties were evaluated. Comparative NMR studies showed structural conservation of synthetic crotamine. Moreover, similarly to native crotamine, the synthetic polypeptide was also capable of inhibiting tumor growth in vivo, increasing the survival of mice bearing subcutaneous tumor. We also confirmed the ability of synthetic crotamine to transfect and transport DNA into eukaryotic cells, in addition to the importance of proteoglycans on cell surface for its internalization. This work opens new opportunities for future evaluation of chimeric and/or point-mutated analogs of this snake polypeptide, aiming for improving crotamine properties and applications, as well as possibly diminishing its potential toxic effects

Protonectin peptides target lipids, act at the interface and selectively kill metastatic breast cancer cells while preserving morphological integrity

© 2021 Elsevier Inc. All rights reserved.Despite the need for innovative compounds as antimicrobial and anticancer agents, natural sources of peptides remain underexplored. Protonectin (PTN), a cationic dodecapeptide of pharmacological interest, presents large hydrophobicity that is associated with the tendency to aggregate and supposedly influences bioactivity. A disaggregating role was assigned to PTN' N-terminal fragment (PTN1-6), which enhances the bioactivity of PTN in a 1:1 mixture (PTN/PTN1-6). Spectroscopic techniques and model membranes (phospholipid bilayers and SDS micelles) revealed that environment-dependent aggregation is reduced for PTN/PTN1-6, but cytotoxicity of PTNs on MDA-MB-231 breast cancer showed the same CC50 values around 16 µM and on MCF-10A epithelial breast cells 6 to 5-fold higher values, revealing a selective interaction. Since PTN1-6 lacks activity on breast cells, its presence should differently affect PTN activity, suggesting that aggregation could modulate activity depending on the membrane characteristics. Indeed, increased partitioning and lytic activity of PTN/PTN1-6 were found in model membranes independently of charge density, but affected by the curvature tendency. PTN and PTN/PTN1-6 do not alter morphology and roughness of cancer cells, indicating a superficial interaction with membranes and consistent with results obtained in NMR experiments. Our results indicate that aggregation of PTNs depends on the membrane characteristics and modulates the activity of the peptides.This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP [Funding Projects Nos. 2012/24259-0, 2014/08372-7, 2016/50178-8] and Fundação para a Ciência e a Tecnologia, Portugal – FCT I. P. [Funding PTDC/BBB-BQB/1693/2014]. DSA has a postdoctoral fellowship (FAPESP grant# 2015/25620-7) and also thanks CAPES for former scholarships. DBM and FDO acknowledge, respectively, CAPES scholarships and FCT I.P. fellowship [PD/BD/135046/2017]. The Brazilian BioscienceNational Laboratory (LNBio) is acknowledged for the NMR timemachine [proposal RMN-23300].info:eu-repo/semantics/publishedVersio

Crystallographic studies on the binding of isonicotinyl-NAD adduct to wild-type and isoniazid resistant 2-trans-enoyl-ACP (CoA) reductase from Mycobacterium tuberculosis

The resumption of tuberculosis led to an increased need to understand the molecular mechanisms of drug action and drug resistance, which should provide significant insight into the development of newer compounds. Isoniazid (INH), the most prescribed drug to treat TB, inhibits an NADH-dependent enoyl-acyl carrier protein reductase (InhA) that provides precursors of mycolic acids, which are components of the mycobacterial cell wall. InhA is the major target of the mode of action of isoniazid. INH is a pro-drug that needs activation to form the inhibitory INH-NAD adduct. Missense mutations in the inhA structural gene have been identified in clinical isolates of Mycobacterium tuberculosis resistant to INH. To understand the mechanism of resistance to INH, we have solved the structure of two InhA mutants (121V and S94A), identified in INH-resistant clinical isolates, and compare them to INH-sensitive WT InhA structure in complex with the INH-NAD adduct. We also solved the structure of unliganded INH-resistant S94A protein, which is the first report on apo form of InhA. The salient features of these structures are discussed and should provide structural information to improve our understanding of the mechanism of action of, and resistance to, INH in M. tuberculosis. The unliganded structure of InhA allows identification of conformational changes upon ligand binding and should help structure-based drug design of more potent antimycobacterial agents. (c) 2007 Elsevier B.V. All rights reserved

Synthesis and characterization of peptide-chitosan conjugates (PepChis) with lipid bilayer affinity and antibacterial activity

© 2019 American Chemical SocietyAntimicrobial peptides appear among innovative biopolymers with potential therapeutic interest. Nevertheless, issues concerning efficiency, production costs, and toxicity persist. Herein, we show that conjugation of peptides with chitosans can represent an alternative in the search for these needs. To increase solubility, deacetylated and degraded chitosans were prepared. Then, they were functionalized via N-succinimidyl-S-acetylthiopropionate or via glutathione (GSH), an endogenous peptide linker. To the best of our knowledge, it is the first time that GSH is used as a thiolating agent for the conjugation of peptides. Next, thiolated chitosans were conjugated through a disulfide bond with designed shortchain peptides, one of them derived from the antimicrobial peptide Jelleine-I. Conjugates and respective reaction intermediates were characterized by absorciometry, attenuated total reflectance−Fourier transform infrared, and 1H NMR. Zeta potential measurements showed the cationic nature of these biomacromolecules and their preferential partitioning to Gram-positive bacterial-like model membranes. In vitro investigation using representative Gram-positive and -negative bacteria (Staphylococcus aureus and Escherichia coli, respectively) showed that the conjugation strategies lead to enhanced activity in relation to the unconjugated peptide and to the unconjugated chitosan. The obtained products showed selectivity toward S. aureus at low cytotoxicity as determined in NIH/3T3 cells. Overall, our study demonstrates that an appropriate choice of antimicrobial peptide and chitosan characteristics leads to increased antimicrobial activity of the conjugated product and represents a strategy to modulate the activity and selectivity of antimicrobials resorting to low-cost chemicals. The present proposal starts from less expensive raw materials (chitosan and short-chain peptide), is based on aqueous solvents, and minimizes the use of reactants with a higher environmental impact. The final biopolymer contains the backbone of chitosan, just 3−6% peptide derived from royal jelly and GSH, all of them considered safe for human use or as a physiological molecule.This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo—FAPESP nos. 2012/24259-0, 2012/02065-0, 2014/08372-7, 2015/07548-7, 2016/13368-4, 2016/50178-8, FCT (Fundação para a Ciência e a Tecnologia, Portugal) grant PTDC/QEQ-MED/4412/2014, and European Commission, Marie S. Curie action RISE, H2020-MSCA-RISE-2014, grant 644167. DBM was a recipient of the CAPES scholarships, LMS is a recipient of the CNPq scholarship, and AC and SAD are recipients of scholarships PD/BD/136866/2018 and PD/BD/114425/2016 from FCT, respectively.info:eu-repo/semantics/publishedVersio

Crystallographic studies of fish hemoglobins

O presente trabalho relata nossa experiência relacionada à cristalização de quatro hemoglobinas de peixe de três espécies brasileiras de Teleostes: Liposarcus anisitsi, Brycon cephalus e Piaractus mesopotamicus. Os dados, aqui apresentados, são parte de um estudo funcional e estrutural sistemático de hemoglobinas de peixe com o objetivo de melhor entender a ampla faixa de propriedades funcionais e estruturais exibidas por estas proteínas. Nós também apresentamos um método otimizado para cristalização de hemoglobinas de peixes, que pode reduzir a quantidade de hemoglobina inicialmente usada nos experimentos de cristalização.The present work reports our succesfull experience concerning crystallization of four fish hemoglobins from three Brazilian species of Teleosts: Liposarcus anisitsi, Brycon cephalus and Piaractus mesopotamicus. The data shown here is part of a systematic functional and structural study of fish hemoglobins with the aim of better understanding the outstanding range of functional and structural properties exhibited by these proteins. We also present a reduced sparse-matrix method for crystallization of fish hemoglobins, which can reduce the amount of hemoglobin initially used in the crystallization experiments.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Ineraction Model Between HRSV G-Protein and Flavonoids

Background: Acute respiratory infections (ARI) are the leading cause of infant mortality in the world, and human respiratory syncytial virus (HRSV) is one of the main agents of ARI. One of the key targets of the adaptive host immune response is the RSV G-protein, which is responsible for attachment to the host cell. There is evidence that compounds such as flavonoids can inhibit viral infection in vitro. With this in mind, the main purpose of this study was to determine, using computational tools, the potential sites for interactions between G-protein and flavonoids. Results: Our study allowed the recognition of an hRSV G-protein model, as well as a model of the interaction with flavonoids. These models were composed, mainly, of -helix and random coil proteins. The docking process showed that molecular interactions are likely to occur. The flavonoid kaempferol-3-O-α-L-arabinopyranosil-(2 → 1)-α-L-apiofuranoside-7-O-α-L-rhamnopyranoside was selected as a candidate inhibitor. The main forces of the interaction were hydrophobic, hydrogen and electrostatic. Conclusions: The model of G-protein is consistent with literature expectations, since it was mostly composed of random coils (highly glycosylated sites) and -helices (lipid regions), which are common in transmembrane proteins. The docking analysis showed that flavonoids interact with G-protein in an important ectodomain region, addressing experimental studies to these sites. The determination of the G-protein structure is of great importance to elucidate the mechanism of viral infectivity, and the results obtained in this study will allow us to propose mechanisms of cellular recognition and to coordinate further experimental studies in order to discover effective inhibitors of attachment proteins