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There are few biophysical studies or structural characterizations of the type IV pilin system of extremophile bacteria, such as the acidophilic Acidithiobacillus thiooxidans. We set out to analyze their pili-comprising proteins, pilins, because these extracellular proteins are in constant interaction with protons of the acidic medium in which At. thiooxidans grows. We used the web server Operon Mapper to analyze and identify the cluster codified by the minor pilin of At. thiooxidans. In addition, we carried an in-silico characterization of such pilins using the VL-XT algorithm of PONDR® server. Our results showed that structural disorder prevails more in pilins of At. thiooxidans than in non-acidophilic bacteria. Further computational characterization showed that the pilins of At. thiooxidans are significantly enriched in hydroxy (serine and threonine) and amide (glutamine and asparagine) residues, and significantly reduced in charged residues (aspartic acid, glutamic acid, arginine and lysine). Similar results were obtained when comparing pilins from other Acidithiobacillus and other acidophilic bacteria from another genus versus neutrophilic bacteria, suggesting that these properties are intrinsic to pilins from acidic environments, most likely by maintaining solubility and stability in harsh conditions. These results give guidelines for the application of extracellular proteins of acidophiles in protein engineering.
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Acidithiobacillus , Proteínas de Fímbrias , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/química , Proteínas de Fímbrias/metabolismo , Acidithiobacillus thiooxidans/genética , Acidithiobacillus thiooxidans/metabolismo , Aminoácidos/metabolismo , Acidithiobacillus/genética , Acidithiobacillus/metabolismo , ÁcidosRESUMO
Substrate-binding proteins (SBPs) are used by organisms from the three domains of life for transport and signalling. SBPs are composed of two domains that collectively trap ligands with high affinity and selectivity. To explore the role of the domains and the integrity of the hinge region between them in the function and conformation of SBPs, here, we describe the ligand binding, conformational stability and folding kinetics of the Lysine Arginine Ornithine (LAO) binding protein from Salmonella thiphimurium and constructs corresponding to its two independent domains. LAO is a class II SBP formed by a continuous and a discontinuous domain. Contrary to the expected behaviour based on their connectivity, the discontinuous domain shows a stable native-like structure that binds l-arginine with moderate affinity, whereas the continuous domain is barely stable and shows no detectable ligand binding. Regarding folding kinetics, studies of the entire protein revealed the presence of at least two intermediates. While the unfolding and refolding of the continuous domain exhibited only a single intermediate and simpler and faster kinetics than LAO, the folding mechanism of the discontinuous domain was complex and involved multiple intermediates. These findings suggest that in the complete protein the continuous domain nucleates folding and that its presence funnels the folding of the discontinuous domain avoiding nonproductive interactions. The strong dependence of the function, stability and folding pathway of the lobes on their covalent association is most likely the result of the coevolution of both domains as a single unit.
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Proteínas de Transporte , Dobramento de Proteína , Cinética , Lisina , Ligantes , Laos , Desnaturação Proteica , Termodinâmica , Conformação ProteicaRESUMO
Abstract Glutaredoxin (GRXs) protein plays a vital role inside the cell, including redox control of transcription to the cell's antioxidant defense, apoptosis, and cellular differentiation regulation. In this study, we have investigated the energy landscape and characterized the pattern of local frustration in different forms and states of the GRX protein ofE. coli.Analysis was done on the conformational alterations, significant changes in the frustration pattern, and different GRXs such as GRX-II, GRX-III, GRX-II-GSH, and GRX-III-GSH complex. We have found the practice of frustration, and structure was quite similar in the same isoform having different states of protein; however, a significant difference was observed between different isoforms. Moreover, oxidation of GRX-I introduced an extra α-helix increasing the destabilizing interactions within the protein. The study of frustrated contacts on oxidized and reduced GRX and with bound and unbound Glutathione indicates its potential application in activating and regulating the behavior of GRXs.
Resumo A proteína glutaredoxina (GRXs) desempenha um papel vital dentro da célula, incluindo o controle redox da transcrição para a defesa antioxidante da célula, apoptose e regulação da diferenciação celular. Neste estudo, investigamos a paisagem energética e caracterizamos o padrão de frustração local em diferentes formas e estados da proteína GRX de E. coli. A análise feita foi sobre as alterações conformacionais, mudanças significativas no padrão de frustração e diferentes GRXs, como GRX-II, GRX-III, GRX-II-GSH e complexo GRX-III-GSH. Encontramos a prática da frustração, e a estrutura era bastante semelhante na mesma isoforma com diferentes estados de proteína; no entanto, uma diferença significativa foi observada entre diferentes isoformas. Além disso, a oxidação de GRX-I introduziu uma α-hélice extra, aumentando as interações desestabilizadoras dentro da proteína. O estudo de contatos frustrados em GRX oxidado e reduzido e com glutationa ligada e não ligada indica sua potencial aplicação na ativação e regulação do comportamento de GRXs.
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BACKGROUND: Low-temperature expression of recombinant proteins may be advantageous to support their proper folding and preserve bioactivity. The generation of expression vectors regulated under cold conditions can improve the expression of some target proteins that are difficult to express in different expression systems. The cspA encodes the major cold-shock protein from Escherichia coli (CspA). The promoter of cspA has been widely used to develop cold shock-inducible expression platforms in E. coli. Moreover, it is often necessary to employ expression systems other than bacteria, particularly when recombinant proteins require complex post-translational modifications. Currently, there are no commercial platforms available for expressing target genes by cold shock in eukaryotic cells. Consequently, genetic elements that respond to cold shock offer the possibility of developing novel cold-inducible expression platforms, particularly suitable for yeasts, and mammalian cells. CONCLUSIONS: This review covers the importance of the cellular response to low temperatures and the prospective use of cold-sensitive promoters to direct the expression of recombinant proteins. This concept may contribute to renewing interest in applying white technologies to produce recombinant proteins that are difficult to express.
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BACKGROUND: Newborn screening for glucose-6-phosphate dehydrogenase deficiency (G6PDd) was implemented in Mexico beginning in 2017. In a Mexican population, genotyping analysis of G6PD as a second-tier method identified a previously unreported missense variant, p.(Ser184Cys), which we propose to call "Toluca", and the extremely rare p.(Gln195His) or "Tainan" variant, which was previously described in the Taiwanese population as a Class II allele through in silico evaluations. Here, we sought to perform in vitro biochemical characterizations of the Toluca and Tainan G6PD natural variants and describe their associated phenotypes. METHODS: The "Toluca" and "Tainan" variants were identified in three unrelated G6PDd newborn males, two of whom lacked evidence of acute hemolytic anemia (AHA) or neonatal hyperbilirubinemia (NHB). We constructed wild-type (WT), Tainan, and Toluca G6PD recombinant enzymes and performed in vitro assessments. RESULTS: Both variants had diminished G6PD expression, decreased affinities for glucose-6-phosphate and NADP+ substrates, significant decreases in catalytic efficiency (â¼97 % with respect to WT-G6PD), and diminished thermostabilities that were partially rescued by NADP+. In silico protein modeling predicted that the variants would have destabilizing effects on the protein tertiary structure, potentially reducing the enzyme half-lives and/or catalytic efficiencies. CONCLUSION: Our data suggest that G6PD "Tainan" and "Toluca" are potential Class II natural variants, which agrees with the absence of chronic nonspherocytic hemolytic anemia (CNSHA) in our patients. It remains to be determined whether these variants represent high-risk genetic factors for developing CNSHA, AHA, and/or NHB.
Assuntos
Deficiência de Glucosefosfato Desidrogenase , Humanos , Masculino , Recém-Nascido , Deficiência de Glucosefosfato Desidrogenase/diagnóstico , Deficiência de Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/química , Triagem Neonatal , NADP , MéxicoRESUMO
Oryzalin (ORY) is a dinitroaniline derivative that inhibits the microtubule polymerization in plants and parasitic protozoa by selectively binding to the α-tubulin subunit. This herbicidal agent exhibits good antiprotozoal activity against major human parasites, such as Toxoplasma gondii (toxoplasmosis), Leishmania mexicana (leishmaniasis), and Plasmodium falciparum (malaria). Previous chemical mutagenesis assays on T. gondii α-tubulin (TgAT) have identified key mutations that lead to ORY resistance. Herein, we employed alchemical free energy methods and molecular dynamics simulations to determine if the ORY resistance mutations either decrease the TgAT's affinity of the compound or increase the protein stability. Our results here suggest that L136F and V202F mutations significantly decrease the affinity of ORY to TgAT, while T239I and V252L mutations diminish TgAT's flexibility. On the other hand, protein stability predictors determined that R243S mutation reduces TgAT stability due to the loss of its salt bridge interaction with E27. Interestingly, molecular dynamics simulations confirm that the loss of this key interaction leads to ORY binding site closure. Our study provides a better insight into the TgAT-ORY interaction, further supporting our recently proposed ORY-binding site.
Assuntos
Toxoplasma , Humanos , Toxoplasma/genética , Toxoplasma/metabolismo , Tubulina (Proteína)/química , Dinitrobenzenos/química , Dinitrobenzenos/metabolismo , Dinitrobenzenos/farmacologia , Sítios de LigaçãoRESUMO
Leukocyte immunoglobulin (Ig)-like receptors (LILR) LILRB1 and LILRB2 may play a pivotal role in maintaining self-tolerance and modulating the immune response through interaction with classical and nonclassical HLA molecules. Although both diversity and natural selection patterns over HLA genes have been extensively evaluated, little information is available concerning the genetic diversity and selection signatures on the LILRB1/2 regions. Therefore, we identified the LILRB1/2 genetic diversity using next-generation sequencing in a population sample from São Paulo State, Brazil. We identified 58 LILRB1 Single Nucleotide Variants (SNVs), which gave rise to 13 haplotypes, and 41 LILRB2 SNVs arranged into 11 haplotypes. Although we may not exclude as a possible effect of population structure, we found evidence of either positive or purifying selection on LILRB1/2 coding regions. Some residues in both proteins showed to be under the effect of positive selection, suggesting that amino acid replacements in these proteins resulted in beneficial functional changes. Finally, we have revealed that allelic variation (six and five amino acid exchanges in LILRB1 and LILRB2, respectively) affects the structure and/or stability of both molecules. Nonetheless, LILRB2 has shown higher average stability, with no D1/D2 residue affecting protein structure. Overall, our findings demonstrate that LILRB1 and LILRB2 are as polymorphic as HLA class Ib genes and provide strong evidence supporting the directional selection regime hypothesis.
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Antígenos CD , Receptor B1 de Leucócitos Semelhante a Imunoglobulina , Glicoproteínas de Membrana , Receptores Imunológicos , Alelos , Aminoácidos , Antígenos CD/genética , Brasil , Variação Genética , Humanos , Receptor B1 de Leucócitos Semelhante a Imunoglobulina/genética , Glicoproteínas de Membrana/genética , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismoRESUMO
All biological processes rely on the formation of protein-ligand, protein-peptide and protein-protein complexes. Studying the affinity, kinetics and thermodynamics of binding between these pairs is critical for understanding basic cellular mechanisms. Many different technologies have been designed for probing interactions between biomolecules, each based on measuring different signals (fluorescence, heat, thermophoresis, scattering and interference, among others). Evaluation of the data from binding experiments and their fitting is an essential step towards the quantification of binding affinities. Here, user-friendly online tools to analyze biophysical data from steady-state fluorescence spectroscopy, microscale thermophoresis and differential scanning fluorimetry experiments are presented. The modules of the data-analysis platform (https://spc.embl-hamburg.de/) contain classical thermodynamic models and clear user guidelines for the determination of equilibrium dissociation constants (Kd) and thermal unfolding parameters such as melting temperatures (Tm).
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Proteínas Quinases Dependentes de GMP Cíclico/química , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Fluorescência , Mycobacterium tuberculosis/metabolismo , Sistemas On-Line , Temperatura , Termodinâmica , Cinética , Ligantes , Ligação Proteica , Espectrometria de FluorescênciaRESUMO
Trypanosoma cruzi is a flagellate protozoa being the etiological agent of Chagas disease, a neglected tropical disease, which still poses a public health problem worldwide. The intricate molecular changes during T. cruzi-host interaction have been explored using different largescale omics techniques. However, protein stability is largely unknown. Thermal proteome profiling (TPP) methodology has the potential to characterize proteome-wide stability highlighting key proteins during T. cruzi infection and life stage transition from the invertebrate to the mammalian host. In the present work, T. cruzi epimastigotes and trypomastigotes cell lysates were subjected to TPP workflow and analyzed by quantitative large-scale mass spectrometry-based proteomics to fit a melting profile for each protein. A total of 2884 proteins were identified and associated to 1741 melting curves being 1370 in trypomastigotes (TmAVG 53.53 °C) and 1279 in epimastigotes (TmAVG 50.89 °C). A total of 453 proteins were identified with statistically different melting profiles between the two life stages. Proteins associated to pathogenesis and intracellular transport had regulated melting temperatures. Membrane and glycosylated proteins had a higher average Tm in trypomastigotes compared to epimastigotes. This study represents the first large-scale comparison of parasite protein stability between life stages. SIGNIFICANCE: Trypanosoma cruzi, a unicellular flagellate parasite, is the etiological agent of Chagas disease, endemic in South America and affecting more that 7 million people worldwide. There is an intense research to identify novel chemotherapeutic and diagnostic targets of Chagas disease. Proteomic approaches have helped in elucidating the quantitative proteome and PTMs changes of T. cruzi during life cycle transition and upon different biotic and abiotic stimuli. However, a comprehensive knowledge of the protein-protein interaction and protein conformation is still missing. In order to fill this gap, this manuscript elucidates the T. cruzi Y strain proteome-wide thermal stability map in the epimastigote and trypomastigote life stages. Comparison between life stages showed a higher average melting temperature stability for trypomastigotes than epimastigotes indicating a host temperature adaptation. Both presented a selective thermal stability shift for cellular compartments, molecular functions and biological processes based on the T. cruzi life stage. Membrane and glycosylated proteins presented a higher thermal stability in trypomastigotes when compared to the epimastigotes.
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Doença de Chagas , Trypanosoma cruzi , Animais , Humanos , Estágios do Ciclo de Vida , Proteoma , Proteômica , Proteínas de ProtozoáriosRESUMO
Centralities determined from Residue Interaction Networks (RIN) in proteins have been used to predict aspects of their structure and dynamics. Here, we correlate the Eigenvector Centrality (Ec) with the rate constant for thermal denaturation (kden) of the HisF protein from Thermotoga maritima based on 12 single alanine substitution mutants. The molecular basis for this correlation was further explored by studying a mutant containing a replacement of a high Ec residue, Y182A, which displayed increased kden at 80 °C. The crystallographic structure of this mutant showed few changes, mostly in two flexible loops. The 1H-15N -HSQC showed only subtle changes of cross peak positions for residues located near the mutation site and scattered throughout the structure. However, the comparison of the RIN showed that Y182 is the vertex of a set of high centrality residues that spreads throughout the HisF structure, which is lacking in the mutant. Cross-correlation displacements of Cα calculated from a molecular dynamics simulation at different temperatures showed that the Y182A mutation reduced the correlated movements in the HisF structure above 70 °C. 1H-15N NMR chemical shift covariance using temperature as perturbation were consistent with these results. In conclusion the increase in temperature drives the structure of the mutant HisF-Y182A into a less connected state, richer in non-concerted motions, located predominantly in the C-terminal half of the protein where Y182 is placed. Conversely, wild-type HisF responds to increased temperature as a single unit. Hence the replacement of a high Ec residue alters the distribution of thermal energy through HisF structure.
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Proteínas , Thermotoga maritima , Modelos Moleculares , Conformação Proteica , Thermotoga maritima/genéticaRESUMO
Resumen Los microacarreadores basados en microcápsulas y microesferas han sido ampliamente estudiados y ensayados para controlar la liberación de medicamentos biotecnológicos (MB), disminuyendo la dosificación o modificando la vía de administración. Los métodos para la obtención de microacarreadores, son complejos y variados, por lo que es necesario determinar los requisitos mínimos que debe cumplir el sistema. El objetivo de este trabajo fue establecer las principales características que deben ser evaluadas en los microacarreadores para garantizar que la actividad biológica de los medicamentos biotecnológicos permanezca intacta a través del proceso de microencapsulación y, por lo tanto, que la seguridad del MB (desarrollo de reacciones inmunes) se mantenga inalterada. Las características a evaluar de un microacarreador deben describir las propiedades del material, tamaño y forma del sistema, carga de la partícula, funcionalidad, eficiencia de la microencapsulación y la cinética de liberación. Mientras que la integridad de los MB puede ser evaluada a partir de parámetros críticos de calidad: estructura y función biológica del MB, pureza del producto, presencia de agregados de alto peso molecular, estructura de orden superior y ensayos de actividad biológica. La caracterización de los microacarreadores debe enfocarse en la seguridad del biopolímero y proteínas ensayadas.
Abstract Microcarriers based on microcapsules and microspheres have been widely studied and tested to control the release of biotechnological drugs (BD), diminishing the dosage or modifying the route of administration. The methods for obtaining microcarriers are complex and varied, so it is necessary to determine the minimum characteristics with which the system must comply. The aim of this work was to establish the main characteristics that should be evaluated in the microcarriers in order to guarantee that the biological activity of biotechnological drugs remains intact through the microencapsulation process, and therefore the safety of the BD (development of immune reactions) remains unaltered. The characteristics of a microcarrier to be evaluated must describe the properties of the material, the size and shape of the system, the particle load, functionalization, the microencapsulation efficiency, and the kinetics of liberation. Whereas the integrity of BD can be evaluated by critical quality parameter such as: structure and biological function of the BD, product purity, presence of high molecular weight aggregations, higher order structure and biological activity tests. The characterization of the microcarriers must focus on the safety of the biopolymer and proteins tested.
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L-asparaginase (ASNase) from Escherichia coli (EcAII) is used in the treatment of acute lymphoblastic leukaemia (ALL). EcAII activity in vivo has been described to be influenced by the human lysosomal proteases asparaginyl endopeptidase (AEP) and cathepsin B (CTSB); these hydrolases cleave and could expose epitopes associated with the immune response against EcAII. In this work, we show that ASNase resistance to CTSB and/or AEP influences the formation of anti-ASNase antibodies, one of the main causes of hypersensitivity reactions in patients. Error-prone polymerase chain reaction was used to produce variants of EcAII more resistant to proteolytic cleavage by AEP and CTSB. The variants with enzymatic activity and cytotoxicity levels equivalent to or better than EcAII WT were submitted to in vivo assays. Only one of the mutants presented increased serum half-life, so resistance to these proteases is not the only feature involved in EcAII stability in vivo. Our results showed alteration of the phenotypic profile of B cells isolated after animal treatment with different protease-resistant proteoforms. Furthermore, mice that were exposed to the protease-resistant proteoforms presented lower anti-asparaginase antibodies production in vivo. Our data suggest that modulating resistance to lysosomal proteases can result in less immunogenic protein drugs.
Assuntos
Antineoplásicos/farmacologia , Asparaginase/farmacologia , Produtos Biológicos/farmacologia , Fenômenos Imunogenéticos/efeitos dos fármacos , Lisossomos/imunologia , Peptídeo Hidrolases/farmacologia , Sequência de Aminoácidos , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Asparaginase/química , Asparaginase/uso terapêutico , Produtos Biológicos/química , Produtos Biológicos/uso terapêutico , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Galinhas , Relação Dose-Resposta a Droga , Escherichia coli , Feminino , Cavalos , Humanos , Fenômenos Imunogenéticos/fisiologia , Células Jurkat , Lisossomos/química , Camundongos , Camundongos Endogâmicos BALB C , Peptídeo Hidrolases/química , Peptídeo Hidrolases/uso terapêutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Estrutura Secundária de ProteínaRESUMO
Endothelin-converting enzyme-1 (ECE1) activates the endothelin-1 peptide, which upregulates pathways that are related to diverse hallmarks of cancer. ECE1 is expressed as four isoforms differing in their N-terminal domains. Protein kinase CK2 phosphorylates the N-terminus of isoform ECE1c, enhancing its stability and promoting invasiveness of colorectal cancer cells. However, the specific residues in ECE1c that are phosphorylated by CK2 and how this phosphorylation promotes invasiveness was unknown. Here we demonstrate that Ser-18 and Ser-20 are the bona fide residues phosphorylated by CK2 in ECE1c. Thus, biphospho-mimetic ECE1cDD and biphospho-resistant ECE1cAA mutants were constructed and stably expressed in different colorectal cancer cells through lentiviral transduction. Biphospho-mimetic ECE1cDD displayed the highest stability in cells, even in the presence of the specific CK2 inhibitor silmitasertib. Concordantly, ECE1cDD-expressing cells showed enhanced hallmarks of cancer, such as proliferation, migration, invasiveness, and self-renewal capacities. Conversely, cells expressing the less-stable biphospho-resistant ECE1cAA showed a reduction in these features, but also displayed an important sensitization to 5-fluorouracil, an antineoplastic agent traditionally used as therapy in colorectal cancer patients. Altogether, these findings suggest that phosphorylation of ECE1c at Ser-18 and Ser-20 by CK2 promotes aggressiveness in colorectal cancer cells. Therefore, phospho-ECE1c may constitute a novel biomarker of poor prognosis and CK2 inhibition may be envisioned as a potential therapy for colorectal cancer patients.
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Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most frequent human enzymopathy, affecting over 400 million people globally. Worldwide, 217 mutations have been reported at the genetic level, and only 19 have been found in Mexico. The objective of this work was to contribute to the knowledge of the function and structure of three single natural variants (G6PD A+, G6PD San Luis Potosi, and G6PD Guadalajara) and a double mutant (G6PD Mount Sinai), each localized in a different region of the three-dimensional (3D) structure. In the functional characterization of the mutants, we observed a decrease in specific activity, protein expression and purification, catalytic efficiency, and substrate affinity in comparison with wild-type (WT) G6PD. Moreover, the analysis of the effect of all mutations on the structural stability showed that its presence increases denaturation and lability with temperature and it is more sensible to trypsin digestion protease and guanidine hydrochloride compared with WT G6PD. This could be explained by accelerated degradation of the variant enzymes due to reduced stability of the protein, as is shown in patients with G6PD deficiency.
Assuntos
Deficiência de Glucosefosfato Desidrogenase/enzimologia , Deficiência de Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/química , Glucosefosfato Desidrogenase/metabolismo , Naftalenossulfonato de Anilina/química , Catálise , Dicroísmo Circular , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/isolamento & purificação , Deficiência de Glucosefosfato Desidrogenase/metabolismo , Guanidina , Humanos , Cinética , México , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Estabilidade Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Software , Temperatura , Tripsina/químicaRESUMO
Protein drugs present specific challenges to the maintenance of long-term stability, which can be accomplished by altering parameters of obtention, purification, molecule structure and formulation. As we believe, commercial formulations are undervalued; therefore, this review focuses on screening, categorising and discussing all formulations of protein drugs approved and not withdrawn by regulatory agencies from United States, Canada and Europe until mid-2018. Peptides (<50 amino acids) were not included to allow a more precise evaluation of choices for larger molecules. We extracted data from the DrugBank database, cross-checked it with the FDA purple book and supplemented it with patient information leaflets and papers. We further classified and discussed the entries according to protein function, drug delivery, route of administration and types of excipient (freeze-dried forms). In addition, alternative choices of excipients were discussed. Experimental work included here relates to targeting strategies with verified pharmacokinetics or in vivo effectiveness to identify physiologically relevant options. Although no single rule can be set for efficient protein formulation, our data help to better understand and optimise the choice for excipients and pharmaceutical dosage forms. For more information, see the Supplemental Data.
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Preparações Farmacêuticas/química , Proteínas/química , Animais , Química Farmacêutica/métodos , Sistemas de Liberação de Medicamentos/métodos , Estabilidade de Medicamentos , Excipientes/química , HumanosRESUMO
BACKGROUND: The 11S globulin from amaranth is the most abundant storage protein in mature seeds and is well recognized for its nutritional value. We used this globulin to engineer a new protein by adding a four valinetyrosine antihypertensive peptide at its C-terminal end to improve its functionality. The new protein was named AMR5 and expressed in the Escherichia coli BL21-CodonPlus(DE3)-RIL strain using a custom medium (F8PW) designed for this work. RESULTS: The alternative medium allowed for the production of 652 mg/L expressed protein at the flask level, mostly in an insoluble form, and this protein was subjected to in vitro refolding. The spectrometric analysis suggests that the protein adopts a ß/α structure with a small increment of α-helix conformation relative to the native amaranth 11S globulin. Thermal and urea denaturation experiments determined apparent Tm and C1/2 values of 50.4°C and 3.04 M, respectively, thus indicating that the antihypertensive peptide insertion destabilized the modified protein relative to the native one. AMR5 hydrolyzed by trypsin and chymotrypsin showed 14- and 1.3-fold stronger inhibitory activity against angiotensin I-converting enzyme (IC50 of 0.034 mg/mL) than the unmodified protein and the previously reported amaranth acidic subunit modified with antihypertensive peptides, respectively. CONCLUSION: The inserted peptide decreases the structural stability of amaranth 11S globulin and improves its antihypertensive activity.
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Peptídeos/metabolismo , Proteínas/metabolismo , Globulinas/metabolismo , Anti-Hipertensivos/metabolismo , Sementes , Temperatura , Meios de Cultura , Amaranthus , Estabilidade Proteica , Compostos FitoquímicosRESUMO
Deep Eutectic Solvents (DES) were investigated as new reaction media for the synthesis of alkyl glycosides catalyzed by the thermostable α-amylase from Thermotoga maritima Amy A. The enzyme was almost completely deactivated when assayed in a series of pure DES, but as cosolvents, DES containing alcohols, sugars, and amides as hydrogen-bond donors (HBD) performed best. A choline chloride:urea based DES was further characterized for the alcoholysis reaction using methanol as a nucleophile. As a cosolvent, this DES increased the hydrolytic and alcoholytic activity of the enzyme at low methanol concentrations, even when both activities drastically dropped when methanol concentration was increased. To explain this phenomenon, variable-temperature, circular dichroism characterization of the protein was conducted, finding that above 60 °C, Amy A underwent large conformational changes not observed in aqueous medium. Thus, 60 °C was set as the temperature limit to carry out alcoholysis reactions. Higher DES contents at this temperature had a detrimental but differential effect on hydrolysis and alcoholysis reactions, thus increasing the alcoholyisis/hydrolysis ratio. To the best of our knowledge, this is the first report on the effect of DES and temperature on an enzyme in which structural studies made it possible to establish the temperature limit for a thermostable enzyme in DES.
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Proteínas de Bactérias/metabolismo , Glicosídeos/metabolismo , Solventes/química , Thermotoga maritima/enzimologia , alfa-Amilases/metabolismo , Proteínas de Bactérias/química , Biocatálise , Colina/química , Dicroísmo Circular , Estabilidade Enzimática , Temperatura Alta , Ligação de Hidrogênio , Hidrólise , Metanol/química , Conformação Proteica , Ureia/química , alfa-Amilases/químicaRESUMO
Hemocyanins are widely used as carriers, adjuvants, and nonspecific immunostimulants in cancer because they promote Th1 immunity in mammals. Hemocyanins also interact with glycan-recognizing innate immune receptors on antigen-presenting cells, such as the C-type lectin immune receptors mannose receptor (MR), macrophage galactose lectin (MGL), and the Toll-like receptors (TLRs), stimulating proinflammatory cytokine secretion. However, the role of N-linked oligosaccharides on the structural and immunological properties of hemocyanin is unclear. Mollusk hemocyanins, such as Concholepas concholepas (CCH), Fissurella latimarginata (FLH), and Megathura crenulata (KLH), are oligomeric glycoproteins with complex dodecameric quaternary structures and heterogeneous glycosylation patterns, primarily consisting of mannose-rich N-glycans. Here, we report that enzyme-catalyzed N-deglycosylation of CCH, FLH, and KLH disrupts their quaternary structure and impairs their immunogenic effects. Biochemical analyses revealed that the deglycosylation does not change hemocyanin secondary structure but alters their refolding mechanism and dodecameric structure. Immunochemical analyses indicated decreased binding of N-deglycosylated hemocyanins to the MR and MGL receptors and TLR4 and reduced endocytosis concomitant with an impaired production of tumor necrosis factor α, and interleukins 6 and 12 (IL-6 and IL-12p40, respectively) in macrophages. Evaluating the function of N-deglycosylated hemocyanins in the humoral immune response and their nonspecific antitumor effects in the B16F10 melanoma model, we found that compared with native hemocyanins N-deglycosylated hemocyanins elicited reduced antibody titers, as well as partially diminished antitumor effects and altered carrier activities. In conclusion, the glycan content of hemocyanins is, among other structural characteristics, critically required for their immunological activities and should be considered in biomedical applications.
Assuntos
Hemocianinas/química , Hemocianinas/imunologia , Imunidade Humoral , Moluscos/química , Adjuvantes Imunológicos , Animais , Linhagem Celular , Citocinas/imunologia , Galactose/química , Glicosilação , Lectinas/química , Lectinas Tipo C/química , Macrófagos/imunologia , Receptor de Manose , Lectinas de Ligação a Manose/química , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/química , Polissacarídeos/química , Dobramento de Proteína , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Receptores de Superfície Celular/químicaRESUMO
Glutamate exerts its actions through the activation of membrane receptors expressed in neurons and glia cells. The signaling properties of glutamate transporters have been characterized recently, suggesting a complex array of signaling transactions triggered by presynaptic released glutamate. In the cerebellar molecular layer, glutamatergic synapses are surrounded by Bergmann glia cells, compulsory participants of glutamate turnover and supply to neurons. Since a glutamate-dependent increase in cGMP levels has been described in these cells and the nitric oxide-cGMP signaling cascade increases their glutamate uptake activity, we describe here the Bergmann glia expression of neuronal nitric oxide synthetase. An augmentation of neuronal nitric oxide synthase was found upon glutamate exposure. This effect is mediated by glutamate transporters and is related to an increase in the stability of the enzyme. These results strengthen the notion of a complex regulation of glial glutamate uptake that supports neuronal glutamate signaling.
Assuntos
Cerebelo/metabolismo , Ácido Glutâmico/metabolismo , Neuroglia/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Sistema X-AG de Transporte de Aminoácidos/metabolismo , Animais , Células Cultivadas , Embrião de Galinha , Transdução de Sinais/fisiologiaRESUMO
Snake venom L-amino acid oxidases (svLAAOs) are an interesting class of enzymes with important biological activities. Their participation in key metabolic processes, including pathological disorders, suggest that svLAAOs are potential lead compounds in drug discovery. However, their short-term stability defies their applications. This paper describes the stability studies together with functional and structural characterization of the LAAO bordonein-L. It has 498â¯amino acid residues, one N-glycosylation site and two disulfide bonds, revealed by high-resolution MS/MS. Molecular modeling approach showed its monomer folds into three conserved domains: FAD, substrate and helical domains. Differential scanning fluorimetry showed the enzyme tends to destabilize from neutral to basic pHs and in presence of mono/bivalent ions and it is highly stabilized by acid pHs and its substrates. However, high concentrations of L-amino acids decrease bordonein-L enzyme activity. Dynamic light scattering revealed bordonein-L remains in the dimeric and monodisperse form, so aggregation does not cause the rapidly decrease of enzyme activity. In vitro, the enzyme exhibited cytotoxicity against fibroblast cell line and killed Leishmania amazonensis promastigotes, intensified by substrate addition. Concluding, our results provide biochemistry and biophysical insights to improve LAAOs stability and better approaches to long-term storage. Moreover, our study emphasizes the importance of proper buffers choice mainly in cell-based assays.