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1.
Altern Lab Anim ; 52(1): 60-68, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-38061994

RESUMEN

The Brazilian National Network of Alternative Methods (RENAMA), which is linked to the Ministry of Science, Technology and Innovation, is currently comprised of 51 laboratories from CROs, academia, industry and government. RENAMA's aim is to develop and validate new approach methodologies (NAMs), as well as train researchers and disseminate information on their use - thus reducing Brazilian, and consequently Latin American, dependence on external technology. Moreover, it promotes the adoption of NAMs by educators and trained researchers, as well as the implementation of good laboratory practice (GLP) and the use of certified products. The RENAMA network started its activities in 2012, and was originally comprised of three central laboratories - the National Institute of Metrology, Quality and Technology (INMETRO); the National Institute of Quality Control in Health (INCQS); and the National Brazilian Biosciences Laboratory (LNBio) - and ten associated laboratories. In 2022, RENAMA celebrated its 10th anniversary, a milestone commemorated by the organisation of a meeting attended by different stakeholders, including the RENAMA-associated laboratories, academia, non-governmental organisations and industry. Ninety-six participants attended the meeting, held on 26 May 2022 in Balneário Camboriú, SC, Brazil, as part of the programme of the XXIII Brazilian Congress of Toxicology 2022. Significant moments of the RENAMA were remembered, and new goals and discussion themes were established. The lectures highlighted recent innovations in the toxicological sciences that have translated into the assessment of consumer product safety through the use of human-relevant NAMs instead of the use of existing animal-based approaches. The challenges and opportunities in accepting such practices for regulatory purposes were also presented and discussed.


Asunto(s)
Aniversarios y Eventos Especiales , Laboratorios , Animales , Humanos , Brasil
2.
J Biol Chem ; 296: 100385, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33556371

RESUMEN

Glycoside hydrolases (GHs) are involved in the degradation of a wide diversity of carbohydrates and present several biotechnological applications. Many GH families are composed of enzymes with a single well-defined specificity. In contrast, enzymes from the GH16 family can act on a range of different polysaccharides, including ß-glucans and galactans. SCLam, a GH16 member derived from a soil metagenome, an endo-ß-1,3(4)-glucanase (EC 3.2.1.6), can cleave both ß-1,3 and ß-1,4 glycosidic bonds in glucans, such as laminarin, barley ß-glucan, and cello-oligosaccharides. A similar cleavage pattern was previously reported for other GH16 family members. However, the molecular mechanisms for this dual cleavage activity on (1,3)- and (1,4)-ß-D-glycosidic bonds by laminarinases have not been elucidated. In this sense, we determined the X-ray structure of a presumably inactive form of SCLam cocrystallized with different oligosaccharides. The solved structures revealed general bound products that are formed owing to residual activities of hydrolysis and transglycosylation. Biochemical and biophysical analyses and molecular dynamics simulations help to rationalize differences in activity toward different substrates. Our results depicted a bulky aromatic residue near the catalytic site critical to select the preferable configuration of glycosidic bonds in the binding cleft. Altogether, these data contribute to understanding the structural basis of recognition and hydrolysis of ß-1,3 and ß-1,4 glycosidic linkages of the laminarinase enzyme class, which is valuable for future studies on the GH16 family members and applications related to biomass conversion into feedstocks and bioproducts.


Asunto(s)
Proteínas Bacterianas/metabolismo , Celulasas/metabolismo , Glucanos/metabolismo , Proteínas Bacterianas/química , Secuencia de Carbohidratos , Dominio Catalítico , Celulasas/química , Cristalografía por Rayos X/métodos , Glucanos/clasificación , Glicósidos/química , Glicósidos/metabolismo , Hidrólisis , Simulación de Dinámica Molecular , Microbiología del Suelo , Especificidad por Sustrato
3.
J Mol Evol ; 89(6): 370-383, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-33956179

RESUMEN

Transthyretin was discovered in the 1940s, named after its ability to bind thyroid hormones and retinol. In the genomic era, transthyretins were found to be part of a larger family with homologs of no obvious function, then called transthyretin-related proteins. Thus, it was proposed that the transthyretin gene could be the result of gene duplication of an ancestral of this newly identified homolog, later found out to be an enzyme involved in uric acid degradation, then named HIUase (5-hydroxy-isourate hydrolase). Here, we sought to re-enact the evolutionary history of this protein family by reconstructing, from a phylogeny inferred from 123 vertebrate sequences, three ancestors corresponding to key moments in their evolution-before duplication; the common transthyretin ancestor after gene duplication and the common ancestor of Eutheria transthyretins. Experimental and computational characterization showed the reconstructed ancestor before duplication was unable to bind thyroxine and likely presented the modern HIUase reaction mechanism, while the substitutions after duplication prevented that activity and were enough to provide stable thyroxine binding, as confirmed by calorimetry and x-ray diffraction. The Eutheria transthyretin ancestor was less prone to characterization, but limited data suggested thyroxine binding as expected. Sequence/structure analysis suggests an early ability to bind the Retinol Binding Protein. We solved the X-ray structures from the two first ancestors, the first at 1.46 resolution, the second at 1.55 resolution with well-defined electron density for thyroxine, providing a useful tool for the understanding of structural adaptation from enzyme to hormone distributor.


Asunto(s)
Evolución Molecular , Prealbúmina , Amidohidrolasas/metabolismo , Secuencia de Aminoácidos , Filogenia , Prealbúmina/genética
4.
Redox Biol ; 37: 101735, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-33011677

RESUMEN

The activity of Thioredoxin-1 (Trx-1) is adjusted by the balance of its monomeric, active and its dimeric, inactive state. The regulation of this balance is not completely understood. We have previously shown that the cytoplasmic domain of the transmembrane protein A Disintegrin And Metalloprotease 17 (ADAM17cyto) binds to Thioredoxin-1 (Trx-1) and the destabilization of this interaction favors the dimeric state of Trx-1. Here, we investigate whether ADAM17 plays a role in the conformation and activation of Trx-1. We found that disrupting the interacting interface with Trx-1 by a site-directed mutagenesis in ADAM17 (ADAM17cytoF730A) caused a decrease of Trx-1 reductive capacity and activity. Moreover, we observed that ADAM17 overexpressing cells favor the monomeric state of Trx-1 while knockdown cells do not. As a result, there is a decrease of cell oxidant levels and ADAM17 sheddase activity and an increase in the reduced cysteine-containing peptides in intracellular proteins in ADAM17cyto overexpressing cells. A mechanistic explanation that ADAM17cyto favors the monomeric, active state of Trx-1 is the formation of a disulfide bond between Cys824 at the C-terminal of ADAM17cyto with the Cys73 of Trx-1, which is involved in the dimerization site of Trx-1. In summary, we propose that ADAM17 is able to modulate Trx-1 conformation affecting its activity and intracellular redox state, bringing up a novel possibility for positive regulation of thiol isomerase activity in the cell by mammalian metalloproteinases.


Asunto(s)
Proteína ADAM17 , Cisteína , Tiorredoxinas , Cisteína/metabolismo , Células HEK293 , Humanos , Conformación Molecular , Oxidación-Reducción , Compuestos de Sulfhidrilo , Tiorredoxinas/genética , Tiorredoxinas/metabolismo
5.
Mol Cell Endocrinol ; 484: 1-14, 2019 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-30703486

RESUMEN

Nuclear receptors (NRs) are a superfamily of ligand-dependent transcription factors that modulate several biological processes. Traditionally, modulation of NRs has been focused on the development of ligands that recognize and bind to the ligand binding domain (LBD), resulting in activation or repression of transcription through the recruitment of coregulators. However, for more severe diseases, such as breast and prostate cancer, the conventional treatment addressing LBD modulation is not always successful, due to tumor resistance. To overcome these challenges and aiming to modulate NR activity by inhibiting the NR-DNA interaction, new studies focus on the development of molecules targeting alternative sites and domains on NRs. Here, we discuss two different approaches for this alternative NR modulation: one targeting the NR DNA binding domain (DBD); and the other targeting the DNA sites recognized by NRs. Our aim is to present the challenges and perspectives for developing specific inhibitors for each purpose, alongside with already reported examples.


Asunto(s)
ADN/química , ADN/metabolismo , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/metabolismo , Sitios de Unión/efectos de los fármacos , Humanos , Inactivación Metabólica , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Relación Estructura-Actividad
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