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1.
Ecotoxicol Environ Saf ; 214: 112115, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33691242

RESUMO

Nanoplastics (NPs) are becoming an emerging pollutant of global concern. A potential risk of NPs is that they can serve as carriers and synergistically function with other contaminants to cause diseases. A variety of diseases such as Alzheimer's disease are related to the generation of amyloid fibrils, and insulin is typically used as a model to study the fibrillation process. In this study, we examined the fibrillation of insulin promoted by polystyrene nanoplastics (PSNPs) alone and synergistically with organic contaminants (denoted as X, X = pyrene, bisphenol A, 2,2',4,4'-tetrabromodiphenyl ether, 4,4'-dihydroxydiphenylmethane, or 4-nonylphenol) having different polarities using thioflavin T fluorescence assays, dynamic light scattering, and circular dichroism spectroscopy. The presence of PSNPs and small organic contaminants decreased the lag phase time (tlag) for insulin fibrillation from 54.6 h to 35-51 h and their combination (PS-X) enhanced this process (tlag = 21-30 h). Notably, the lag phase time for insulin fibrillation with PS-nonpolar contaminants, PS-weakly polar contaminants, and PS-polar contaminants is around 20.8, 26.7, and 30.1 h, respectively, indicating the synergistic effect of PS-nonpolar contaminants or PS-weakly polar contaminants was more obvious than that of PS-polar contaminants. Moreover, molecular dynamic simulation reveal the interactions between insulin and PSs or small organic contaminants are primarily driven by van der Waals forces and hydrophobic interactions. Overall, the findings of this study underscore the potentially significant environmental impact of small organic contaminants assisting NPs in promoting insulin fibrillation.


Assuntos
Poluentes Ambientais/química , Insulina/química , Microplásticos/química , Nanopartículas/química , Poliestirenos/química , Compostos Benzidrílicos/química , Éteres Difenil Halogenados/química , Fenóis/química , Pirenos/química
2.
Am J Physiol Endocrinol Metab ; 320(5): E886-E890, 2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33719586

RESUMO

The first therapeutic use of insulin by Frederick Banting and Charles Best in 1921 revolutionized the management of type 1 diabetes and considerably changed the lives of many patients with other types of diabetes. In the past 100 years, significant pharmacological advances took place in the field of insulin therapy, bringing closer the goal of optimal glycemic control along with decreased diabetes-related complications. Despite these developments, several challenges remain, such as increasing treatment flexibility, reducing iatrogenic hypoglycemia, and optimizing patient quality of life. Ongoing innovations in insulin therapy (e.g., new insulin analogs, alternative routes of insulin administration, and closed-loop technology) endeavor to overcome these hurdles and change the landscape of diabetes mellitus management. This report highlights recent advances made in the field of insulin therapy and discusses future perspectives.


Assuntos
Diabetes Mellitus/tratamento farmacológico , Composição de Medicamentos/tendências , Endocrinologia/tendências , Insulina/uso terapêutico , Animais , Preparações de Ação Retardada/farmacocinética , Preparações de Ação Retardada/uso terapêutico , Composição de Medicamentos/história , Composição de Medicamentos/métodos , Endocrinologia/história , Endocrinologia/métodos , História do Século XX , História do Século XXI , Humanos , Insulina/administração & dosagem , Insulina/química , Insulina/farmacocinética , Sistemas de Infusão de Insulina/tendências , Absorção Intestinal/efeitos dos fármacos , Invenções/tendências
3.
J Vis Exp ; (168)2021 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-33616104

RESUMO

These methods describe how to formulate injectable, supramolecular polymer-nanoparticle (PNP) hydrogels for use as biomaterials. PNP hydrogels are composed of two components: hydrophobically modified cellulose as the network polymer and self-assembled core-shell nanoparticles that act as non-covalent cross linkers through dynamic, multivalent interactions. These methods describe both the formation of these self-assembled nanoparticles through nanoprecipitation as well as the formulation and mixing of the two components to form hydrogels with tunable mechanical properties. The use of dynamic light scattering (DLS) and rheology to characterize the quality of the synthesized materials is also detailed. Finally, the utility of these hydrogels for drug delivery, biopharmaceutical stabilization, and cell encapsulation and delivery is demonstrated through in vitro experiments to characterize drug release, thermal stability, and cell settling and viability. Due to its biocompatibility, injectability, and mild gel formation conditions, this hydrogel system is a readily tunable platform suitable for a range of biomedical applications.


Assuntos
Sistemas de Liberação de Medicamentos , Hidrogéis/química , Injeções , Células-Tronco Mesenquimais/citologia , Nanopartículas/química , Polímeros/química , Animais , Bovinos , Sobrevivência Celular , Precipitação Química , Liberação Controlada de Fármacos , Humanos , Insulina/química , Cinética , Estabilidade Proteica , Reologia , Soroalbumina Bovina/química , Temperatura
4.
Nat Cell Biol ; 23(2): 172-183, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33558728

RESUMO

In patients with advanced-stage cancer, cancer-associated anorexia affects treatment success and patient survival. However, the underlying mechanism is poorly understood. Here, we show that Dilp8, a Drosophila homologue of mammalian insulin-like 3 peptide (INSL3), is secreted from tumour tissues and induces anorexia through the Lgr3 receptor in the brain. Activated Dilp8-Lgr3 signalling upregulated anorexigenic nucleobinding 1 (NUCB1) and downregulated orexigenic short neuropeptide F (sNPF) and NPF expression in the brain. In the cancer condition, the protein expression of Lgr3 and NUCB1 was significantly upregulated in neurons expressing sNPF and NPF. INSL3 levels were increased in tumour-implanted mice and INSL3-treated mouse hypothalamic cells showed Nucb2 upregulation and Npy downregulation. Food consumption was significantly reduced in intracerebrospinal INSL3-injected mice. In patients with pancreatic cancer, higher serum INSL3 levels increased anorexia. These results indicate that tumour-derived Dilp8/INSL3 induces cancer anorexia by regulating feeding hormones through the Lgr3/Lgr8 receptor in Drosophila and mammals.


Assuntos
Anorexia/metabolismo , Encéfalo/metabolismo , Proteínas de Drosophila/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neoplasias/metabolismo , Neuropeptídeos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sequência de Aminoácidos , Animais , Anorexia/etiologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Neoplasias Oculares/patologia , Comportamento Alimentar , Humanos , Hipotálamo/metabolismo , Insulina/sangue , Insulina/química , Insulina/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos Endogâmicos C57BL , Neoplasias/complicações , Neurônios/metabolismo , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/complicações , Proteínas/química , Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais
5.
Int J Nanomedicine ; 16: 297-314, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33488074

RESUMO

The glucose-sensitive self-adjusting drug delivery system simulates the physiological model of the human pancreas-secreting insulin and then precisely regulates the release of hypoglycemic drugs and controls the blood sugar. Thus, it has good application prospects in the treatment of diabetes. Presently, there are three glucose-sensitive drug systems: phenylboronic acid (PBA) and its derivatives, concanavalin A (Con A), and glucose oxidase (GOD). Among these, the glucose-sensitive polymer carrier based on PBA has the advantages of better stability, long-term storage, and reversible glucose response, and the loading of insulin in it can achieve the controlled release of drugs in the human environment. Therefore, it has become a research hotspot in recent years and has been developed very rapidly. In order to further carry out a follow-up study, we focused on the development process, performance, and application of PBA and its derivatives-based glucose-sensitive polymer drug carriers, and the prospects for the development of this field.


Assuntos
Ácidos Borônicos/química , Diabetes Mellitus/tratamento farmacológico , Portadores de Fármacos/química , Hipoglicemiantes/farmacologia , Ácidos Borônicos/metabolismo , Diabetes Mellitus/metabolismo , Portadores de Fármacos/metabolismo , Seguimentos , Glucose/metabolismo , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/uso terapêutico , Insulina/química , Insulina/farmacologia , Insulina/uso terapêutico
6.
Anal Chem ; 93(4): 2215-2225, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33433190

RESUMO

Infrared (IR) absorption spectroscopy is a powerful tool that can quantify complex biomolecules and their structural conformations. However, conventional approaches to protein analysis in aqueous solutions have been significantly challenged because the strong IR absorption of water overwhelms the limited dynamic range of the detection system and thus allows only a very short path length and a limited concentration sensitivity. Here, we demonstrate a solvent absorption compensation (SAC) approach that can improve the concentration sensitivity and extend the available path length by distinguishing the analyte signal over the full dynamic range at each wavelength. Absorption spectra without any postprocessing show good linearity from 100 to 0.1 mg/mL protein concentration, allowing a >100 times enhanced signal-to-noise ratio in the amide I band compared to the non-SAC results. We apply this method to in situ investigate the isothermal kinetics of insulin fibrillation at two clinical concentrations at 74 °C for 18 h. Simultaneous monitoring of both reactants (native forms) and products (fibrils) allows quantitative discussion of the detailed fibrillation mechanisms, which are not accessible with other single modality measurements. This simple optical technique can be applied to other absorption spectroscopies of analytes in strongly absorbing solvents, allowing for enhanced sensitivity without changing the detection system.


Assuntos
Anticorpos Monoclonais/química , Proteínas/química , Soroalbumina Bovina/química , Espectrofotometria Infravermelho/métodos , Água/química , Insulina/química , Cinética , Modelos Químicos , Estrutura Secundária de Proteína
7.
Carbohydr Polym ; 256: 117414, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33483009

RESUMO

The major role of biomolecules in treatment of different diseases has been proven by several studies. However, the main drawback in successful treatment by these molecules is designing of efficient delivery systems to fulfill all of the delivery purposes. In this regard, many polymeric vehicles have been introduced for protecting and delivery of biomolecules to the target site. Chitosan as a unique biopolymer with special properties has been widely used for biomolecule delivery. Several research groups have focused on developing and applying of chitosan as a versatile machine in biomolecule delivery. In this review the unique properties of chitosan have been discussed at first and then its application as a delivery machine for different types of biomolecules include protein and peptides, nucleic acids and vaccines has been considered. Furthermore, the targeting approach by conjugation of various ligands to the chitosan and also the current challenges for development of chitosan vehicles will be discussed for biomolecule delivery.


Assuntos
Biopolímeros/química , Quitosana/química , Portadores de Fármacos , Polímeros/química , Vacinas/química , Animais , Carboidratos/química , Humanos , Insulina/química , Ligantes , Camundongos , Nanopartículas/química , Ácido Oleico/química , Peptídeos/química , Poliésteres/química , Polietilenoglicóis/química , Ratos , Zinco/química
8.
J Med Chem ; 64(1): 616-628, 2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33356257

RESUMO

Recently, the first basal oral insulin (OI338) was shown to provide similar treatment outcomes to insulin glargine in a phase 2a clinical trial. Here, we report the engineering of a novel class of basal oral insulin analogues of which OI338, 10, in this publication, was successfully tested in the phase 2a clinical trial. We found that the introduction of two insulin substitutions, A14E and B25H, was needed to provide increased stability toward proteolysis. Ultralong pharmacokinetic profiles were obtained by attaching an albumin-binding side chain derived from octadecanedioic (C18) or icosanedioic acid (C20) to the lysine in position B29. Crucial for obtaining the ultralong PK profile was also a significant reduction of insulin receptor affinity. Oral bioavailability in dogs indicated that C18-based analogues were superior to C20-based analogues. These studies led to the identification of the two clinical candidates OI338 and OI320 (10 and 24, respectively).


Assuntos
Hipoglicemiantes/administração & dosagem , Insulina/administração & dosagem , Acilação , Administração Oral , Sequência de Aminoácidos , Animais , Disponibilidade Biológica , Preparações de Ação Retardada , Cães , Meia-Vida , Humanos , Hipoglicemiantes/farmacocinética , Insulina/química , Insulina/farmacocinética , Ratos
9.
PLoS One ; 15(12): e0244296, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33362209

RESUMO

There are many reports demonstrating that various derivatives of carbon nanoparticles are effective inhibitors of protein aggregation. As surface structural features of nanoparticles play a key role on modulating amyloid fibrillation process, in the present in vitro study, bovine insulin and hen egg white lysozyme (HEWL) were selected as two model proteins to investigate the reducing effect of graphene oxide quantum dots (GOQDs) on their assembly under amyloidogenic conditions. GOQDs were prepared through direct pyrolysis of citric acid, and the reduction step was carried out using ascorbic acid. The prepared nanoparticles were characterized by UV-Vis, X-ray photoelectron, and FT-IR spectroscopies, transmission electron and atomic force microscopies, zeta potential measurement, and Nile red fluorescence assay. They showed the tendencies to modulate the assembly of the proteins through different mechanisms. While GOQDs appeared to have the capacity to inhibit fibrillation, the presence of reduced GOQDs (rGOQDs) was found to promote protein assembly via shortening the nucleation phase, as suggested by ThT fluorescence data. Moreover, the structures produced in the presence of GOQDs or rGOQDs were totally nontoxic. We suggest that surface properties of these particles may be part of the differences in their mechanism(s) of action.


Assuntos
Grafite/química , Grafite/metabolismo , Oxigênio/metabolismo , Amiloide/química , Amiloide/metabolismo , Proteínas Amiloidogênicas/química , Amiloidose/metabolismo , Animais , Bovinos , Insulina/química , Modelos Biológicos , Muramidase/química , Nanopartículas/química , Oxigênio/fisiologia , Agregados Proteicos/efeitos dos fármacos , Agregados Proteicos/fisiologia , Pontos Quânticos/química , Propriedades de Superfície/efeitos dos fármacos
10.
Int J Vitam Nutr Res ; 90(5-6): 425-429, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32729784

RESUMO

Objective: The objective of the present study is to investigate the effects of glutamine administration on postprandial glycemia, insulin, and C-peptide concentration in patients with type 2 diabetes. Methods: A randomized, double-blind, placebo-controlled trial was conducted on patients with type 2 diabetes so that 33 subjects were recruited in each group. The patients were randomly allocated to receive either 30 g/d glutamine or placebo (with instructions to take in half glass of ice-cold water 5 to 10 min before each main meal) for 6 weeks. Postprandial C-peptide, insulin, and glucose were measured at the baseline and at the end of the study at 30 and 90 min after consuming a meal comprising wheat-cake and reduced fat milk. Results: The repeated measures ANOVA revealed no significant difference between the groups for glucose and insulin after 6 weeks of intervention (p > 0.05). However, C-peptide was reduced in both intervention groups at all measurement points. Between-group differences remained significant by the end of the study (p = 0.02). Conclusions: Glutamine supplementation before each main meal does not represent an effective nutritional strategy to improve postprandial glycemic control or postprandial insulin secretion in type 2 diabetes patients.


Assuntos
Diabetes Mellitus Tipo 2 , Secreção de Insulina , Glicemia/metabolismo , Método Duplo-Cego , Glutamina/metabolismo , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/metabolismo , Insulina/química
11.
Nat Commun ; 11(1): 3746, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32719315

RESUMO

Recently, the clinical proof of concept for the first ultra-long oral insulin was reported, showing efficacy and safety similar to subcutaneously administered insulin glargine. Here, we report the molecular engineering as well as biological and pharmacological properties of these insulin analogues. Molecules were designed to have ultra-long pharmacokinetic profile to minimize variability in plasma exposure. Elimination plasma half-life of ~20 h in dogs and ~70 h in man is achieved by a strong albumin binding, and by lowering the insulin receptor affinity 500-fold to slow down receptor mediated clearance. These insulin analogues still stimulate efficient glucose disposal in rats, pigs and dogs during constant intravenous infusion and euglycemic clamp conditions. The albumin binding facilitates initial high plasma exposure with a concomitant delay in distribution to peripheral tissues. This slow appearance in the periphery mediates an early transient hepato-centric insulin action and blunts hypoglycaemia in dogs in response to overdosing.


Assuntos
Insulina/administração & dosagem , Engenharia de Proteínas , Administração Oral , Sequência de Aminoácidos , Animais , Glicemia/metabolismo , Simulação por Computador , Cães , Relação Dose-Resposta a Droga , Overdose de Drogas/sangue , Técnica Clamp de Glucose , Meia-Vida , Humanos , Hiperinsulinismo/tratamento farmacológico , Hipoglicemia/diagnóstico , Insulina/análogos & derivados , Insulina/química , Insulina/farmacocinética , Masculino , Estabilidade Proteica , Proteólise , Ratos Sprague-Dawley , Suínos , Resultado do Tratamento
12.
Life Sci ; 256: 117910, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32504753

RESUMO

AIMS: Insulin (Ins) covalently modified by catecholestrogens (CEs) was commonly found in diabetic patients who have developed insulin resistance. Estrogenization of insulin altered its molecular function and effect carbohydrates metabolisms in these patients. Insulin resistance is a common phenomenon in diabetes but the exact mechanism remains unknown. In this study, binding specificity and affinity of autoantibodies against estrogenized insulin (4-hydroxyestradiol-insulin; 4-OHE2-Ins) were assayed in the serum of type 1 diabetes (T1D) patients in order to explain the phenomena behind insulin resistance. MATERIALS AND METHODS: Specificity and affinity of autoantibodies from the sera of 66 T1D patients and 41 controls were analyzed by direct binding, competition ELISA and quantitative precipitin titration. Insulin was also estimated in the serum of T1D patients by ELISA. KEY FINDING: Estrogenized insulin (4-OHE2-Ins) exhibited high affinity and specificity to T1D autoantibodies in comparison to Ins (p < .05) or 4-OHE2 (p < .001). Estrogenization of insulin alters its interaction with the insulin receptor (IR). The affinity constant of 4-OHE2-Ins with the T1D autoantibodies was found to be 1.41 × 10-7 M. SIGNIFICANCE: Estrogenization of insulin by catecholestrogen makes these molecules highly antigenic and produced high-affinity autoantibodies in T1D patients. As a result, patients develop insulin resistance and presented this molecule as a potential biomarker for T1D.


Assuntos
Autoanticorpos/imunologia , Diabetes Mellitus Tipo 1/tratamento farmacológico , Estrogênios de Catecol/química , Hipoglicemiantes/química , Insulina/química , Adulto , Autoanticorpos/metabolismo , Biomarcadores/metabolismo , Glicemia/análise , Coleta de Amostras Sanguíneas , Proposta de Concorrência , Ensaio de Imunoadsorção Enzimática , Estrogênios de Catecol/uso terapêutico , Feminino , Humanos , Hipoglicemiantes/uso terapêutico , Insulina/uso terapêutico , Resistência à Insulina , Masculino , Pessoa de Meia-Idade , Receptor de Insulina/imunologia , Receptor de Insulina/metabolismo , Sensibilidade e Especificidade
13.
Nat Struct Mol Biol ; 27(7): 615-624, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32483339

RESUMO

Human insulin and its current therapeutic analogs all show propensity, albeit varyingly, to self-associate into dimers and hexamers, which delays their onset of action and makes blood glucose management difficult for people with diabetes. Recently, we described a monomeric, insulin-like peptide in cone-snail venom with moderate human insulin-like bioactivity. Here, with insights from structural biology studies, we report the development of mini-Ins-a human des-octapeptide insulin analog-as a structurally minimal, full-potency insulin. Mini-Ins is monomeric and, despite the lack of the canonical B-chain C-terminal octapeptide, has similar receptor binding affinity to human insulin. Four mutations compensate for the lack of contacts normally made by the octapeptide. Mini-Ins also has similar in vitro insulin signaling and in vivo bioactivities to human insulin. The full bioactivity of mini-Ins demonstrates the dispensability of the PheB24-PheB25-TyrB26 aromatic triplet and opens a new direction for therapeutic insulin development.


Assuntos
Antígenos CD/química , Insulina/química , Venenos de Moluscos/química , Venenos de Moluscos/metabolismo , Receptor de Insulina/química , Substituição de Aminoácidos , Animais , Antígenos CD/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Insulina/análogos & derivados , Insulina/metabolismo , Insulina/farmacologia , Camundongos Endogâmicos C57BL , Modelos Moleculares , Simulação de Dinâmica Molecular , Venenos de Moluscos/genética , Venenos de Moluscos/farmacologia , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Conformação Proteica , Ratos Sprague-Dawley , Receptor de Insulina/metabolismo , Relação Estrutura-Atividade , Tirosina
14.
Sci Rep ; 10(1): 7862, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32398693

RESUMO

Deposits of protein misfolding and/or aggregates are a pathological hallmark of amyloid-related diseases. For instance, insulin amyloid fibril deposits have been observed in patients with insulin-dependent diabetes mellitus after insulin administration. Here, we report on the use of AuNPs functionalized with linear- (i.e. dextrin and chitosan) and branched- (i.e. dextran-40 and dextran-10) biopolymers as potential agents to inhibit insulin fibril formation. Our dynamic light scattering analyses showed a size decrease of the amyloid fibrils in the presence of functionalized AuNPs. Circular dichroism spectroscopy as well as enzyme-linked immunosorbent assay data demonstrated that the secondary structural transition from α-helix to ß-sheet (which is characteristic for insulin amyloid fibril formation) was significantly suppressed by all biopolymer-coated AuNPs, and in particular, by those functionalized with linear biopolymers. Both transmission electron microscopy and atomic force microscopy analyses showed that the long thick amyloid fibrils formed by insulin alone become shorter, thinner or cluster when incubated with biopolymer-coated AuNPs. Dextrin- and chitosan-coated AuNPs were found to be the best inhibitors of the fibril formation. Based on these results, we propose a mechanism for the inhibition of insulin amyloid fibrils: biopolymer-coated AuNPsstrongly interact with the insulin monomers and inhibit the oligomer formation as well as elongation of the protofibrils.Moreover, cytotoxicity experiments showed that AuNP-insulin amyloid fibrils are less toxic compared to insulin amyloid fibrils alone. Our results suggest that both dextrin- and chitosan-AuNPs could be used as therapeutic agents for the treatment of amyloid-related disorders.


Assuntos
Amiloide/química , Amiloidose/prevenção & controle , Biopolímeros/química , Materiais Revestidos Biocompatíveis/farmacologia , Ouro/química , Insulina/química , Nanopartículas Metálicas/administração & dosagem , Quitosana/química , Dicroísmo Circular/métodos , Materiais Revestidos Biocompatíveis/química , Dextrinas/química , Difusão Dinâmica da Luz , Humanos , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Microscopia de Força Atômica/métodos , Microscopia Eletrônica de Transmissão/métodos , Espectrofotometria
15.
J Phys Chem Lett ; 11(11): 4353-4358, 2020 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-32401513

RESUMO

Solvent-protein interactions are important for protein biological functions, especially for a coupled folding and binding system such as insulin. By monitoring the change in the conformation of insulin dimers during dissociation with temperature-jump infrared spectroscopy, we show that co-solvents can significantly destabilize the dimers by perturbing their hydrophobic center. The transition from the native to intermediate dimer state is observed as the buried residues are exposed to solvents in the presence of 10% dimethyl sulfoxide and with α-helices unfolding when ethanol is present, which reduces the dissociation time dramatically to 50% and 20% of the value in a D2O solution, respectively. We propose a self-consistent analysis using complementary methods to resolve this coupled folding and binding process and obtain a much higher rate of monomer association than of intermediate folding. Our results demonstrate that the conformational changes are critical in dimer formation and strongly affected by co-solvents.


Assuntos
Insulina/química , Dimerização , Dimetil Sulfóxido/química , Etanol/química , Conformação Proteica , Solventes/química
16.
J Chromatogr A ; 1622: 461093, 2020 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-32340726

RESUMO

The Peptide RPC Column Characterisation Protocol was applied to 38 stationary phases, varying in ligand chemistry, base silica, end capping and pore size, which are suitable for the analysis of peptides. The protocol at low and intermediate pH is based on measuring retention time differences between peptides of different functionality to calculate selectivity delta values. The characterisation was designed to explore increases / decreases in positive or negative charge (deamidation), steric effect (i.e. racemisation / switch in amino acid order), oxidation and addition / removal of aromatic moieties. The necessity of developing a characterisation protocol specifically for peptide analysis was highlighted by the fact that the small molecule databases (Snyder's Hydrophobic Subtraction Model and the extended Tanaka protocol) failed to correlate with the Peptide RPC Column Characterisation Protocol. Principal Component Analysis was used to demonstrate that the protocol could be used to identify columns with similar or dissimilar chromatographic selectivity for the purpose of selectivity back-up or method development columns respectively. This was validated using peptide fragments derived from the tryptic digest of bovine insulin and carbonic anhydrase. It was also demonstrated that the presence of positively charged functional groups on the stationary phase was advantageous as it yielded very different chromatographic selectivity and improved peak shape.


Assuntos
Técnicas de Química Analítica/métodos , Cromatografia de Fase Reversa , Bases de Dados Factuais , Peptídeos/isolamento & purificação , Animais , Bovinos , Interações Hidrofóbicas e Hidrofílicas , Insulina/química , Peptídeos/química , Análise de Componente Principal , Dióxido de Silício/química
17.
Phys Chem Chem Phys ; 22(17): 9573-9586, 2020 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-32322851

RESUMO

Synthesizing and understanding phase transition behavior of novel block copolymers is very crucial for fabricating next generation of smart materials with foreseeable applications. In this regard, we synthesized three random (r) copolymers of poly(N-vinyl-caprolactam) (PVCL) and poly(2-dimethyl amino ethyl methacrylate) (PDMAEMA) with varying percentages of each block and characterized them using nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD) patterns, time-resolved fluorescence spectroscopy, and atomic force microscopy (AFM). Synthesized copolymers i.e. PVCL30-PDMAEMA70, PVCL50-PDMAEMA50 and PVCL70-PDMAEMA30 have fluorescence properties, which were confirmed by time-resolved fluorescence spectra and emission spectra, and emission bands were observed at ∼310, ∼435 and ∼424 nm, respectively. The fluorescence lifetime for PVCL50-PDMAEMA50 is larger than those of the other two copolymers suggesting a slow decay of the excited state. The copolymers have spherical geometry as micelles, which were confirmed by TEM. We observed patterned arrangement of micelles and the arranged micelles appear to be pentagon in shape, creating space in between the arranged micelles; however, for PVCL50-PDMAEMA50, the arranged micelles do not form any particular shape. The thermal phase transition of PVCL-r-PDMAEMA in aqueous solution was studied by differential scanning calorimetry and thermal fluorescence spectroscopy. In order to design a biomimetic polymer for bio-specific applications and to understand novel concepts towards polymer-protein interactions, we studied the effect of insulin on lower critical solution temperature (LCST) of PVCL-r-PDMAEMA using multiple sophisticated techniques. The LCST is finely tuned by incorporation of two blocks with various block compositions and the value falls within the range of human body temperature, making PVCL50-PDMAEMA50 a highly compatible material for bio-medical and bio-material applications. Insulin forms a self-assembly with the monomers of PVCL-r-PDMAEMA, which leads to enhancing the micellar aggregates and the eventual decrease in the LCST of the diblock copolymer aqueous solution. The present study provides new insights into insulin-copolymer interactions and can be used for self-assembling nanocarriers and designing protein resistance surfaces.


Assuntos
Insulina/química , Micelas , Conformação Molecular , Polímeros/química , Etilaminas/química , Metacrilatos/química , Multimerização Proteica
18.
Gen Comp Endocrinol ; 293: 113478, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32243957

RESUMO

This study identified an insulin-like peptide (ILP) in Macrobrachium rosenbergii termed Mr-ILP and further investigated its function through glucose injection and RNAi. With the analysis of five other glucose metabolism related genes, this study shed light on the molecular mechanism of carbohydrate metabolism in crustaceans. Mr-ILP shared the typical skeleton with six conserved cysteine and mainly expressed in neuroendocrine system. In M. rosenbergii, the elevated hemolymph glucose concentration after glucose injection returned to basal levels in short time, implying an efficient regulatory system in carbohydrate metabolism. Hyperglycemic related genes answered the elevated hemolymph glucose concentration quickly with significant decreased expression level, while Mr-ILP showed delayed response. Instead, glycolysis increased after glucose injection, which indicated glycolysis might play an important role in lowering the abnormally high glucose level. In vivo silencing of Mr-ILP, by injecting the prawns with double-stranded RNA (dsRNA) for 21 days reduced its expression by approximately 75%. Accordingly, glycogen synthase decreased and the trehalose and glycogen level in the hepatopancreas were significantly reduced, indicating the function of Mr-ILP in oligosaccharide and polysaccharide accumulation. When Mr-ILP was silenced, the expression of hyperglycemic related genes were enhanced, but the hemolymph glucose level was not elevated significantly, which might attribute to the increased glycolysis to keep a balanced glucose level in hemolymph.


Assuntos
Metabolismo dos Carboidratos , Insulina/metabolismo , Palaemonidae/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Metabolismo dos Carboidratos/genética , DNA Complementar/genética , Feminino , Regulação da Expressão Gênica , Glucose/administração & dosagem , Hemolinfa/metabolismo , Insulina/química , Insulina/genética , Masculino , Palaemonidae/genética , Filogenia
19.
Acta Crystallogr D Struct Biol ; 76(Pt 4): 366-374, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32254061

RESUMO

In this study, the first crystal structure of a novel crystal form of human insulin bound to meta-cresol in an acidic environment is reported. The combination of single-crystal and powder X-ray diffraction crystallography led to the detection of a previously unknown monoclinic phase (P21). The structure was identified from the powder patterns and was solved using single-crystal diffraction data at 2.2 Šresolution. The unit-cell parameters at pH 6.1 are a = 47.66, b = 70.36, c = 84.75 Å, ß = 105.21°. The structure consists of two insulin hexamers per asymmetric unit. The potential use of this insulin form in microcrystalline drugs is discussed.


Assuntos
Cresóis/química , Insulina/química , Cristalografia por Raios X , Humanos , Modelos Moleculares , Multimerização Proteica , Estrutura Quaternária de Proteína , Difração de Raios X
20.
Chemistry ; 26(38): 8341-8357, 2020 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-32196765

RESUMO

Insulin is a small protein crucial for regulating the blood glucose level in all animals. Since 1922 it has been used for the treatment of patients with diabetes. Despite consisting of just 51 amino acids, insulin contains 17 of the proteinogenic amino acids, A- and B-chains, three disulfide bridges, and it folds with 3 α-helices and a short ß-sheet segment. Insulin associates into dimers and further into hexamers with stabilization by Zn2+ and phenolic ligands. Selective chemical modification of proteins is at the forefront of developments in chemical biology and biopharmaceuticals. Insulin's structure has made it amenable to organic and inorganic chemical reactions. This Review provides a synthetic organic chemistry perspective on this small protein. It gives an overview of key chemical and physico-chemical aspects of the insulin molecule, with a focus on chemoselective reactions. This includes N-acylations at the N-termini or at LysB29 by pH control, introduction of protecting groups on insulin, binding of metal ions, ligands to control the nano-scale assembly of insulin, and more.


Assuntos
Aminoácidos/química , Dissulfetos/química , Insulina/química , Acilação , Aminoácidos/metabolismo , Animais , Química Orgânica , Humanos , Modelos Moleculares
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