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1.
Regul Toxicol Pharmacol ; 102: 98-107, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30562601

RESUMO

Assessing the safety of genetically engineered crops includes evaluating the risk (hazard and exposure) of consuming their newly expressed proteins. The dicamba monooxygenase (DMO) protein, introduced into soybeans to confer tolerance (DT) to dicamba herbicide, was previously characterized and identified to pose no food or feed safety hazards. Most agricultural commodities (e.g., soybeans, maize) enter the food supply after processing methods that can include exposure to high temperatures, harsh solvents or pH extremes that can adversely impact the structure and function of proteins. To understand the likelihood of exposure to DMO in foods from DT soy, enzymatically active and/or immunodetectable forms of DMO were measured in pilot-scale productions of two soy foods (soymilk and tofu), and eight processed fractions (full fat flour, inactivated full fat flour, defatted flour, toasted meal, protein isolate, protein concentrate, crude lecithin, and refined, bleached and deodorized oil). Western blot analysis detected DMO in tofu and in five of the eight processed fractions. DMO activity was not detected in either soymilk or tofu, nor in six of the eight processed fractions. Therefore, many commercial soy processing methods can denature and/or degrade introduced proteins, like DMO. Although the DMO protein has shown no evidence of hazard, this study demonstrates that processing further reduces any food or feed risk by limiting dietary exposure to intact DMO protein.


Assuntos
Dicamba , Manipulação de Alimentos , Herbicidas , Oxigenases de Função Mista , Plantas Geneticamente Modificadas/enzimologia , Alimentos de Soja/análise , Soja , Exposição Dietética/prevenção & controle , Resistência a Medicamentos , Oxigenases de Função Mista/análise , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Soja/enzimologia , Soja/genética
2.
Regul Toxicol Pharmacol ; 99: 50-60, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30196079

RESUMO

The lepidopteran-active Cry1A.105 protein is a chimeric three-domain insecticidal toxin with distinct structural domains derived from the naturally occurring Cry1Ab, Cry1Ac and Cry1F proteins from the soil bacterium Bacillus thuringiensis (Bt). The X-ray crystal structure of the Cry1A.105 tryptic core at 3.0 Šresolution demonstrates its high structural similarity to the tryptic core of Cry1Ac. Bioinformatics analyses demonstrate that Cry1A.105 has no significant amino acid sequence similarity to known allergens or mammalian toxins, which is the same conclusion reached for its component domains. Like its intact donor proteins, Cry1A.105 was heat labile at temperatures ≥75 °C and degraded upon exposure to gastrointestinal proteases. No adverse effects were observed in mice when Cry1A.105 was dosed orally at 2451 mg/kg body weight. Therefore, the weight of evidence supports that Cry1A.105 is safe for human and animal consumption. These results support the conclusion that the safety of a chimeric protein for human or animal consumption can be evaluated in the context of the safety of its donor proteins.

3.
Regul Toxicol Pharmacol ; 89: 155-164, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28751263

RESUMO

Many insect-protected crops express insecticidal crystal (Cry) proteins derived from the soil bacterium Bacillus thuringiensis (Bt), including both naturally-occurring Cry proteins and chimeric Cry proteins created through biotechnology. The Cry51Aa2 protein is a naturally-occurring Cry protein that was modified to increase its potency and expand its insect activity spectrum through amino acid sequence changes. The improved Cry51Aa2 variant, Cry51Aa2.834_16, and other developmental variants belong to the ETX_MTX2 family of proteins but share a low level of sequence similarity to other members of this family. This similarity is largely localized to the pore-forming and oligomerization protein domains, while sequence divergence is observed within the head domain that confers receptor binding specificity. The intact Cry51Aa2.834_16 protein was heat labile at temperatures ≥55 °C, and was rapidly degraded after exposure to the gastrointestinal protease pepsin. No acute oral toxicity was observed in mice for three protein variants of Cry51Aa2, including Cry51Aa2.834_16, at doses exceeding 1000 mg/kg body weight. The weight-of-evidence therefore supports the conclusion of safety for Cry51Aa2.834_16 and demonstrates that amino acid sequence modifications can be used to substantially increase insecticidal activity of a protein without an increased hazard to mammals.


Assuntos
Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/metabolismo , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Insetos , Controle Biológico de Vetores/métodos , Plantas Geneticamente Modificadas/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Endotoxinas/genética , Proteínas Hemolisinas/genética , Camundongos
4.
PLoS One ; 12(2): e0171926, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28207780

RESUMO

The susceptibility of a dietary protein to proteolytic degradation by digestive enzymes, such as gastric pepsin, provides information on the likelihood of systemic exposure to a structurally intact and biologically active macromolecule, thus informing on the safety of proteins for human and animal consumption. Therefore, the purpose of standardized in vitro degradation studies that are performed during protein safety assessments is to distinguish whether proteins of interest are susceptible or resistant to pepsin degradation via a study design that enables study-to-study comparison. Attempting to assess pepsin degradation under a wide-range of possible physiological conditions poses a problem because of the lack of robust and consistent data collected under a large-range of sub-optimal conditions, which undermines the needs to harmonize in vitro degradation conditions. This report systematically compares the effects of pH, incubation time, and pepsin-to-substrate protein ratio on the relative degradation of five dietary proteins: three pepsin susceptible proteins [ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco), horseradish peroxidase (HRP), hemoglobin (Hb)], and two pepsin resistant proteins [lipid transfer protein (LTP) and soybean trypsin inhibitor (STI)]. The results indicate that proteins susceptible to pepsin degradation are readily distinguishable from pepsin-resistant proteins when the reaction conditions are within the well-characterized optima for pepsin. The current standardized in vitro pepsin resistant assay with low pH and high pepsin-to-substrate ratio fits this purpose. Using non-optimal pH and/or pepsin-to-substrate protein ratios resulted in susceptible proteins no longer being reliably degraded by this stomach enzyme, which compromises the ability of this in vitro assay to distinguish between resistant and susceptible proteins and, therefore, no longer providing useful data to an overall weight-of-evidence approach to assessing safety of proteins.


Assuntos
Proteínas na Dieta/química , Inocuidade dos Alimentos , Pepsina A/química , Proteínas na Dieta/imunologia , Concentração de Íons de Hidrogênio , Fatores de Tempo
5.
J Invertebr Pathol ; 142: 50-59, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27235983

RESUMO

The need for sustainable insect pest control is driving the investigation and discovery of insecticidal proteins outside of the typical 3-domain Cry protein family from Bacillus thuringiensis (Bt). Examples include Cry35 and Cry51 that belong to protein families (Toxin_10, ETX_MTX2) sharing a common ß-pore forming structure and function with known mammalian toxins such as epsilon toxin (ETX). Although ß-pore forming proteins are related to mammalian toxins, there are key differences in sequence and structure that lead to organism specificity that is useful in the weight-of-evidence approach for safety assessment. Despite low overall amino acid sequence identity among ETX_MTX2 proteins, sequence and structural similarities are found in the tail region responsible for the shared oligomerization and pore formation functions (causing the "relatedness"). Conversely, most of the sequence and structural diversity is located in the head region that is likely responsible for differential receptor binding and target species specificity (e.g., insecticidal vs. mammalian). Therefore, inclusion of a domain-based protein characterization approach that includes bioinformatic and functional comparisons of conserved and diverse domains will enhance the overall weight of evidence safety assessment of proteins including recently reported Cry51 protein variants (Cry51Aa1, Cry51Aa2, and Cry51Aa2.834_16).


Assuntos
Biologia Computacional/métodos , Endotoxinas/classificação , Inseticidas/classificação , Modelos Moleculares , Controle Biológico de Vetores/métodos , Sequência de Aminoácidos , Animais , Endotoxinas/química , Endotoxinas/genética , Inseticidas/química , Inseticidas/metabolismo , Relação Estrutura-Atividade
6.
J Biomol NMR ; 61(3-4): 261-74, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25869397

RESUMO

Solution NMR spectroscopy has become a robust method to determine structures and explore the dynamics of integral membrane proteins. The vast majority of previous studies on membrane proteins by solution NMR have been conducted in lipid micelles. Contrary to the lipids that form a lipid bilayer in biological membranes, micellar lipids typically contain only a single hydrocarbon chain or two chains that are too short to form a bilayer. Therefore, there is a need to explore alternative more bilayer-like media to mimic the natural environment of membrane proteins. Lipid bicelles and lipid nanodiscs have emerged as two alternative membrane mimetics that are compatible with solution NMR spectroscopy. Here, we have conducted a comprehensive comparison of the physical and spectroscopic behavior of two outer membrane proteins from Pseudomonas aeruginosa, OprG and OprH, in lipid micelles, bicelles, and nanodiscs of five different sizes. Bicelles stabilized with a fraction of negatively charged lipids yielded spectra of almost comparable quality as in the best micellar solutions and the secondary structures were found to be almost indistinguishable in the two environments. Of the five nanodiscs tested, nanodiscs assembled from MSP1D1ΔH5 performed the best with both proteins in terms of sample stability and spectral resolution. Even in these optimal nanodiscs some broad signals from the membrane embedded barrel were severely overlapped with sharp signals from the flexible loops making their assignments difficult. A mutant OprH that had two of the flexible loops truncated yielded very promising spectra for further structural and dynamical analysis in MSP1D1ΔH5 nanodiscs.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Micelas , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Clonagem Molecular , Bicamadas Lipídicas/química , Modelos Moleculares , Fosfolipídeos/química , Estrutura Terciária de Proteína , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo
7.
J Biol Chem ; 286(45): 39211-23, 2011 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-21865172

RESUMO

Pseudomonas aeruginosa is a major nosocomial pathogen that infects cystic fibrosis and immunocompromised patients. The impermeability of the P. aeruginosa outer membrane contributes substantially to the notorious antibiotic resistance of this human pathogen. This impermeability is partially imparted by the outer membrane protein H (OprH). Here we have solved the structure of OprH in a lipid environment by solution NMR. The structure reveals an eight-stranded ß-barrel protein with four extracellular loops of unequal size. Fast time-scale dynamics measurements show that the extracellular loops are disordered and unstructured. It was previously suggested that the function of OprH is to provide increased stability to the outer membranes of P. aeruginosa by directly interacting with lipopolysaccharide (LPS) molecules. Using in vivo and in vitro biochemical assays, we show that OprH indeed interacts with LPS in P. aeruginosa outer membranes. Based upon NMR chemical shift perturbations observed upon the addition of LPS to OprH in lipid micelles, we conclude that the interaction is predominantly electrostatic and localized to charged regions near both rims of the barrel, but also through two conspicuous tyrosines in the middle of the bilayer. These results provide the first molecular structure of OprH and offer evidence for multiple interactions between OprH and LPS that likely contribute to the antibiotic resistance of P. aeruginosa.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Lipopolissacarídeos/química , Pseudomonas aeruginosa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Lipopolissacarídeos/metabolismo , Micelas , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Pseudomonas aeruginosa/metabolismo , Relação Estrutura-Atividade
9.
Biophys J ; 95(6): 2859-66, 2008 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-18586850

RESUMO

The photoreceptor rhodopsin is a G-protein coupled receptor that has recently been proposed to exist as a dimer or higher order oligomer, in contrast to the previously described monomer, in retinal rod outer segment disk membranes. Rhodopsin exhibits considerably greater thermal stability than opsin (the bleached form of the receptor), which is reflected in an approximately 15 degrees C difference in the thermal denaturation temperatures (T(m)) of rhodopsin and opsin as measured by differential scanning calorimetry. Here we use differential scanning calorimetry to investigate the effect of partial bleaching of disk membranes on the T(m) of rhodopsin and of opsin in native disk membranes, as well as in cross-linked disk membranes in which rhodopsin dimers are known to be present. The T(m)s of rhodopsin and opsin are expected to be perturbed if mixed oligomers are present. The T(m) remained constant for rhodopsin and opsin in native disks regardless of the level of bleaching. In contrast, the T(m) of cross-linked rhodopsin in disk membranes was dependent on the extent of bleaching. The energy of activation for denaturation of rhodopsin and cross-linked rhodopsin was calculated. Cross-linking rhodopsin significantly decreased the energy of activation. We conclude that in native disk membranes, rhodopsin behaves predominantly as a monomer.


Assuntos
Membrana Celular/metabolismo , Rodopsina/metabolismo , Segmento Externo da Célula Bastonete/citologia , Opsinas de Bastonetes/metabolismo , Animais , Varredura Diferencial de Calorimetria , Bovinos , Reagentes para Ligações Cruzadas/metabolismo , Dimerização , Cinética , Desnaturação Proteica , Estrutura Terciária de Proteína , Rodopsina/química , Opsinas de Bastonetes/química , Temperatura de Transição
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