RESUMO
Household food insecurity (HFI) during childhood is associated with poor dietary diversity and malnutrition, placing children's growth at risk. Children with growth disorders, such as stunting, are more likely to have poor cognition and educational performance, lower economic status, and an increased risk of nutrition-related chronic diseases in adulthood. Our study aimed to systematically review and conduct a meta-analysis of cohort studies investigating the association between HFI and stunting in children aged 0-59 months. Peer-reviewed and grey literature were systematically searched in electronic databases with no language or date restrictions. Two reviewers independently assessed the studies for pre-established eligibility criteria. Data were extracted using a standard protocol. Random-effects meta-analysis models were used, and I2 > 40% indicated high heterogeneity across studies. We used the Grading of Recommendations Assessment, Development, and Evaluation system to assess the quality of the evidence. Nine cohort studies comprising 46,300 children were included. Approximately 80% (n = 7) of the studies found a positive association between HFI and stunting. Pooled odds ratio was 1.00 (95% confidence interval [CI]: 0.87-1.14; I2: 76.14%). The pooled hazard ratio between moderate and severe HFI and stunting was 1.02 (95% CI: 0.84-1.22; I2: 85.96%). Due to high heterogeneity, the quality of evidence was very low. Individual studies showed an association between HFI and stunting in children aged 0-59 months; however, this association was not sustained in the pooled analysis, possibly because of high heterogeneity across studies.
Assuntos
Características da Família , Insegurança Alimentar , Transtornos do Crescimento , Humanos , Lactente , Transtornos do Crescimento/epidemiologia , Transtornos do Crescimento/etiologia , Pré-Escolar , Recém-Nascido , Estudos de Coortes , Feminino , MasculinoRESUMO
Rab GTPases are compartment-specific molecular switches that regulate intracellular vesicular transport in eukaryotes. GDP/GTP exchange factors (GEFs) control Rab activation, and current models propose that localised and regulated GEF activity is important in targeting Rabs to specific membranes. Here, we investigated the mechanism of GEF function using the Rab27a GEF, Rab3GEP (also known as MADD), in melanocytes as a model. We show that Rab3GEP-deficient melanocytes (melan-R3GKO) manifest partial disruption of melanosome dispersion, a read-out of Rab27a activation and targeting. Using rescue of melanosome dispersion in melan-R3GKO cells and effector pull-down approaches we show that the DENN domain of Rab3GEP (conserved among RabGEFs) is necessary, but insufficient, for its cellular function and GEF activity. Finally, using a mitochondrial re-targeting strategy, we show that Rab3GEP can target Rab27a to specific membranes in a GEF-dependent manner. We conclude that Rab3GEP facilitates the activation and targeting of Rab27a to specific membranes, but that it differs from other DENN-containing RabGEFs in requiring DENN and non-DENN elements for both of these activities and by lacking compartment-specific localisation.
Assuntos
Transporte Biológico/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas rab27 de Ligação ao GTP/metabolismo , Animais , Melanócitos/citologia , Melanócitos/metabolismo , Melanossomas/metabolismo , Camundongos , Deficiência Múltipla de Acil Coenzima A Desidrogenase/metabolismo , Cultura Primária de Células , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab3 de Ligação ao GTP/metabolismoRESUMO
The coordination between late mitotic events such as poleward chromosome motion, spindle elongation, DNA decondensation, and nuclear envelope reformation (NER) is crucial for the completion of chromosome segregation at the anaphase-telophase transition. Mitotic exit is driven by a decrease of Cdk1 kinase activity and an increase of PP1/PP2A phosphatase activities. More recently, Aurora kinases have also emerged as master regulators of late mitotic events and cytokinesis. Aurora A is mainly associated with spindle poles throughout mitosis and midbody during telophase, whereas Aurora B re-localizes from centromeres in early mitosis to the spindle midzone and midbody as cells progress from anaphase to the completion of cytokinesis. Functional studies, together with the identification of a phosphorylation gradient during anaphase, established Aurora B as a major player in the organization of the spindle midzone and in the spatiotemporal coordination between chromosome segregation and NER. Aurora A has been less explored, but a cooperative role in spindle midzone stability has also been proposed, implying that both Aurora A and B contribute to accurate chromosome segregation during mitotic exit. Here, we review the roles of the Aurora kinases in the regulation of late mitotic events and discuss how they work together with other mitotic players to ensure an error-free mitosis.
Assuntos
Aurora Quinases/metabolismo , Mitose , Animais , Aurora Quinases/química , Aurora Quinases/genética , Cromossomos/genética , Cromossomos/metabolismo , Humanos , Fosforilação , Ligação Proteica , Proteólise , Transdução de Sinais , Fuso Acromático/metabolismo , Especificidade por SubstratoRESUMO
The potential of using a synthetic cardosin-based rennet in cheese manufacturing was recently demonstrated with the development and optimization of production of a recombinant form of cardosin B in Kluyveromyces lactis. With the goal of providing a more detailed characterization of this rennet, we herein evaluate the impact of the plant-specific insert (PSI) on cardosin B secretion in this yeast, and provide a thorough analysis of the specificity requirements as well as the biochemical and structural properties of the isolated recombinant protease. We demonstrate that the PSI domain can be substituted by different linker sequences without substantially affecting protein secretion and milk clotting activity. However, the presence of small portions of the PSI results in dramatic reductions of secretion yields in this heterologous system. Kinetic characterization and specificity profiling results clearly suggest that synthetic cardosin B displays lower catalytic efficiency and is more sequence selective than native cardosin B. Elucidation of the structure of synthetic cardosin B confirms the canonical fold of an aspartic protease with the presence of two high mannose-type, N-linked glycan structures; however, there are some differences in the conformation of the flap region when compared to cardosin A. These subtle variations in catalytic properties and the more stringent substrate specificity of synthetic cardosin B help to explain the observed suitability of this rennet for cheese production.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Quimosina/metabolismo , Kluyveromyces/metabolismo , Plantas/enzimologia , Animais , Ácido Aspártico Endopeptidases/genética , Queijo , Quimosina/genética , Glicosilação , Kluyveromyces/genética , Leite/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/genética , Transporte ProteicoRESUMO
Anopheles mosquitoes are vectors of malaria, a potentially fatal blood disease affecting half a billion humans worldwide. These blood-feeding insects include in their antihemostatic arsenal a potent thrombin inhibitor, the flexible and cysteine-less anophelin. Here, we present a thorough structure-and-function analysis of thrombin inhibition by anophelin, including the 2.3-Å crystal structure of the human thrombin·anophelin complex. Anophelin residues 32-61 are well-defined by electron density, completely occupying the long cleft between the active site and exosite I. However, in striking contrast to substrates, the D50-R53 anophelin tetrapeptide occupies the active site cleft of the enzyme, whereas the upstream residues A35-P45 shield the regulatory exosite I, defining a unique reverse-binding mode of an inhibitor to the target proteinase. The extensive interactions established, the disruption of thrombin's active site charge-relay system, and the insertion of residue R53 into the proteinase S(1) pocket in an orientation opposed to productive substrates explain anophelin's remarkable specificity and resistance to proteolysis by thrombin. Complementary biophysical and functional characterization of point mutants and truncated versions of anophelin unambiguously establish the molecular mechanism of action of this family of serine proteinase inhibitors (I77). These findings have implications for the design of novel antithrombotics.
Assuntos
Anticoagulantes/farmacologia , Antitrombinas/farmacologia , Proteínas de Insetos/farmacologia , Insetos Vetores/química , Malária/parasitologia , Proteínas e Peptídeos Salivares/farmacologia , Trombina/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Anopheles/química , Anticoagulantes/química , Antitrombinas/química , Arginina/metabolismo , Coagulação Sanguínea/efeitos dos fármacos , Domínio Catalítico , Sequência Conservada , Cristalografia por Raios X , Humanos , Proteínas Imobilizadas/metabolismo , Proteínas de Insetos/química , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Proteínas e Peptídeos Salivares/química , Alinhamento de Sequência , Relação Estrutura-Atividade , Especificidade por Substrato/efeitos dos fármacos , Ressonância de Plasmônio de Superfície , Trombina/metabolismo , Tempo de TrombinaRESUMO
Rab GTPases are important determinants of organelle identity and regulators of vesicular transport pathways. Consequently, each Rab occupies a highly specific subcellular localization. However, the precise mechanisms governing Rab targeting remain unclear. Guanine nucleotide exchange factors (GEFs), putative membrane-resident targeting factors and effector binding have all been implicated as critical regulators of Rab targeting. Here, we address these issues using Rab27a targeting to melanosomes as a model system. Rab27a regulates motility of lysosome-related organelles and secretory granules. Its effectors have been characterized extensively, and we have identified Rab3GEP as the non-redundant Rab27a GEF in melanocytes (Figueiredo AC et al. Rab3GEP is the non-redundant guanine nucleotide exchange factor for Rab27a in melanocytes. J Biol Chem 2008;283:23209-23216). Using Rab27a mutants that show impaired binding to representatives of all four Rab27a effector subgroups, we present evidence that effector binding is not essential for targeting of Rab27a to melanosomes. In contrast, we observed that knockdown of Rab3GEP resulted in mis-targeting of Rab27a, suggesting that Rab3GEP activity is required for correct targeting of Rab27a. However, the identification of Rab27a mutants that undergo efficient GDP/GTP exchange in the presence of Rab3GEP in vitro but are mis-targeted in a cellular context indicates that nucleotide loading is not the sole determinant of subcellular targeting of Rab27a. Our data support a model in which exchange activity, but not effector binding, represents one essential factor that contributes to membrane targeting of Rab proteins.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Melanócitos/metabolismo , Melanossomas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Células Cultivadas , Lisossomos/metabolismo , Melanócitos/ultraestrutura , Melanossomas/ultraestrutura , Camundongos , Mutagênese Sítio-Dirigida , Ligação Proteica , Vesículas Secretórias/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab27 de Ligação ao GTP , Proteínas rab3 de Ligação ao GTP/metabolismoRESUMO
Boophilin is a tight-binding thrombin inhibitor composed of two canonical Kunitz-type domains in a tandem arrangement. Thrombin-bound boophilin can inhibit a second trypsin-like serine proteinase, most likely through the reactive loop of its N-terminal Kunitz domain. Here, the crystallization and preliminary crystallographic analysis of the isolated N-terminal domain of boophilin is reported. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1) and diffracted to beyond 1.8 Å resolution using a sealed-tube home source and to 0.87 Å resolution at a synchrotron source.
Assuntos
Inibidores Enzimáticos/química , Proteínas Fúngicas/química , Pichia/química , Sequência de Aminoácidos , Cristalização , Cristalografia por Raios X , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Trombina/antagonistas & inibidoresRESUMO
Micronuclei are a hallmark of cancer and several other human disorders. Recently, micronuclei were implicated in chromothripsis, a series of massive genomic rearrangements that may drive tumor evolution and progression. Here, we show that Aurora B kinase mediates a surveillance mechanism that integrates error correction during anaphase with spatial control of nuclear envelope reassembly to prevent micronuclei formation. Using high-resolution live-cell imaging of human cancer and non-cancer cells, we uncover that anaphase lagging chromosomes are more frequent than previously anticipated, yet they rarely form micronuclei. Micronuclei formation from anaphase lagging chromosomes is prevented by a midzone-based Aurora B phosphorylation gradient that stabilizes kinetochore-microtubule attachments and assists spindle forces required for anaphase error correction while delaying nuclear envelope reassembly on lagging chromosomes, independently of microtubule density. We propose that a midzone-based Aurora B phosphorylation gradient actively monitors and corrects frequent chromosome segregation errors to prevent micronuclei formation during human cell division.
Assuntos
Anáfase , Aurora Quinase B/metabolismo , Segregação de Cromossomos , Cinetocoros/enzimologia , Micronúcleos com Defeito Cromossômico , Membrana Nuclear/enzimologia , Fuso Acromático/enzimologia , Células HeLa , Humanos , Mecanotransdução Celular , Membrana Nuclear/genética , Fosforilação , Fuso Acromático/genética , Fatores de TempoRESUMO
AIM: The present study systematically reviewed the characteristics of the food environment in schools and their immediate vicinities associated with excess weight in adolescents. METHODS: We searched for relevant articles in seven databases. No restrictions were applied on language, publication date, and status of publication. The study selection process and data extraction were conducted by two authors independently. For meta-analyses, the random-effects model and the maximum-likelihood method were applied. RESULTS: A total of 9327 publications were identified from the initial search. Of these, 20 studies met the inclusion criteria and were included in the systematic review. The sale of food in school or immediate vicinity was associated with high BMI [Odds ratio (OR)= 1.14, 95% confidence interval (CI) 1.01, 2.06]. The availability of healthy food provided by school significantly decreased the odds of obesity [OR= 0.89, 95%CI 0.82, 0.96]. However, the presence of nutrition policies or programs at school was not associated with obesity (OR= 0.81, 95%CI 0.57, 1.16). CONCLUSIONS: This study highlights the need for improvement in school food environment, including restricting students' exposure to unhealthy foods. The quality of food (healthy vs unhealthy) provided or made available to adolescents in the school food environment influences their weight status. Therefore, promotion of healthy food programs and availability of healthy food at school would limit the prevalence of excess weight in adolescents.
Assuntos
Instituições Acadêmicas , Estudantes , Adolescente , Humanos , Política Nutricional , Obesidade , Aumento de PesoRESUMO
BACKGROUND: Periodontitis and the Triglyceride/High Density Lipoprotein Cholesterol (TG/HDL-C) ratio have both been associated with cardiovascular disease, metabolic syndrome, and obesity. Additionally, the ratio is a possible substitute for predicting insulin resistance. This study investigated the association between periodontitis, its severity levels (exposures), and the TG/HDL-C ratio (outcome). METHODS: A cross-sectional study of public health service users in Brazil considered socioeconomic-demographic characteristics, lifestyle behavior, and general and oral health conditions. Anthropometric measurements and blood pressure were also measured. Systemic biomarker data were obtained, as well as assessment of periodontal diagnosis and its severity. The TG/HDL-C ratio was calculated using the serum triglyceride level over HDL cholesterol and the cut-off point, TG/HDL-C ≥2.3 serving as the cutoff indicting dyslipidemia. Logistic and linear regressions were used to statistically analyze the data. RESULTS: A total of 1011 participants were included, with 84.17% having periodontitis and 49.85% having a TG/HDL-C ratio ≥2.3. For individuals with periodontitis, the odds of TG/HDL-C ratio ≥2.3 were 1.47 times greater than in those without periodontitis (ORAdjusted = 1.47, 95% CI: 1.02-2.14). Similar results were found for those with moderate and severe periodontitis, with a slight increase in the measurement magnitude with disease severity. CONCLUSION: A positive relationship between periodontitis and the TG/HDL-C ratio ≥2.3 was found, suggesting a possible association with periodontal disease severity.
Assuntos
Síndrome Metabólica , Periodontite , Biomarcadores , HDL-Colesterol , Estudos Transversais , Humanos , Periodontite/complicações , Periodontite/epidemiologia , Fatores de Risco , TriglicerídeosRESUMO
CLASPs are conserved microtubule plus-end-tracking proteins that suppress microtubule catastrophes and independently localize to kinetochores during mitosis. Thus, CLASPs are ideally positioned to regulate kinetochore-microtubule dynamics required for chromosome segregation fidelity, but the underlying mechanism remains unknown. Here, we found that human CLASP2 exists predominantly as a monomer in solution, but it can self-associate through its C-terminal kinetochore-binding domain. Kinetochore localization was independent of self-association, and driving monomeric CLASP2 to kinetochores fully rescued normal kinetochore-microtubule dynamics, while partially sustaining mitosis. CLASP2 kinetochore localization, recognition of growing microtubule plus-ends through EB-protein interaction, and the ability to associate with curved microtubule protofilaments through TOG2 and TOG3 domains independently sustained normal spindle length, timely spindle assembly checkpoint satisfaction, chromosome congression, and faithful segregation. Measurements of kinetochore-microtubule half-life and poleward flux revealed that CLASP2 regulates kinetochore-microtubule dynamics by integrating distinctive microtubule-binding properties at the kinetochore-microtubule interface. We propose that kinetochore CLASP2 suppresses microtubule depolymerization and detachment by binding to curved protofilaments at microtubule plus-ends.
Assuntos
Cinetocoros/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular/genética , Proteínas Associadas aos Microtúbulos/genética , Segregação de Cromossomos/genética , Células HeLa , Humanos , Microtúbulos/genética , Mitose/genética , Ligação Proteica/genética , Domínios Proteicos , Fuso Acromático/genéticaRESUMO
Accurate chromosome segregation relies on microtubule end conversion, the ill-understood ability of kinetochores to transit from lateral microtubule attachment to durable association with dynamic microtubule plus-ends. The molecular requirements for this conversion and the underlying biophysical mechanisms are elusive. We reconstituted end conversion in vitro using two kinetochore components: the plus end-directed kinesin CENP-E and microtubule-binding Ndc80 complex, combined on the surface of a microbead. The primary role of CENP-E is to ensure close proximity between Ndc80 complexes and the microtubule plus-end, whereas Ndc80 complexes provide lasting microtubule association by diffusing on the microtubule wall near its tip. Together, these proteins mediate robust plus-end coupling during several rounds of microtubule dynamics, in the absence of any specialized tip-binding or regulatory proteins. Using a Brownian dynamics model, we show that end conversion is an emergent property of multimolecular ensembles of microtubule wall-binding proteins with finely tuned force-dependent motility characteristics.
Assuntos
Segregação de Cromossomos , Cinesinas/metabolismo , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Animais , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/isolamento & purificação , Proteínas Cromossômicas não Histona/metabolismo , Proteínas do Citoesqueleto , Microscopia de Fluorescência , Modelos Biológicos , Dinâmica não Linear , Proteínas Nucleares/genética , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Células Sf9 , Imagem Individual de Molécula , Processos Estocásticos , Proteínas de Xenopus/genética , Proteínas de Xenopus/isolamento & purificação , Proteínas de Xenopus/metabolismoRESUMO
Mitosis is an essential process that takes place in all eukaryotes and involves the equal division of genetic material from a parental cell into two identical daughter cells. During mitosis, chromosome movement and segregation are orchestrated by a specialized structure known as the mitotic spindle, composed of a bipolar array of microtubules. The fundamental structure of microtubules comprises of α/ß-tubulin heterodimers that associate head-to-tail and laterally to form hollow filaments. In vivo, microtubules are modified by abundant and evolutionarily conserved tubulin posttranslational modifications (PTMs), giving these filaments the potential for a wide chemical diversity. In recent years, the concept of a "tubulin code" has emerged as an extralayer of regulation governing microtubule function. A range of tubulin isoforms, each with a diverse set of PTMs, provides a readable code for microtubule motors and other microtubule-associated proteins. This chapter focuses on the complexity of tubulin PTMs with an emphasis on detyrosination and summarizes the methods currently used in our laboratory to experimentally manipulate these modifications and study their impact in mitosis.
Assuntos
Técnicas Citológicas/métodos , Mitose , Tubulina (Proteína)/metabolismo , Linhagem Celular Tumoral , Humanos , Lentivirus/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas Motores Moleculares/metabolismo , Processamento de Proteína Pós-Traducional , RNA Interferente Pequeno/metabolismo , Transdução Genética , Tirosina/metabolismoRESUMO
BACKGROUND: The comparison between long acting insulin analogues (LAIA) and human insulin (NPH) has been investigated for decades, with many randomized controlled trials (RCTs) and systematic reviews giving mixed results. This overlapping and contradictory evidence has increased uncertainty on coverage decisions at health systems level. AIM: To conduct an overview of systematic reviews and update existing reviews, preparing new meta-analysis to determine whether LAIA are effective for T1D patients compared to NPH. METHODS: We identified systematic reviews of RCTs that evaluated the efficacy of LAIA glargine or detemir, compared to NPH insulin for T1D, assessing glycated hemoglobin (A1C) and hypoglycemia. Data sources included Pubmed, Cochrane Library, EMBASE and hand-searching. The methodological quality of studies was independently assessed by two reviewers, using AMSTAR and Jadad scale. We found 11 eligible systematic reviews that contained a total of 25 relevant clinical trials. Two reviewers independently abstracted data. RESULTS: We found evidence that LAIA are efficacious compared to NPH, with estimates showing a reduction in nocturnal hypoglycemia episodes (RR 0.66; 95% CI 0.57; 0.76) and A1C (95% CI 0.23; 0.12). No significance was found related to severe hypoglycemia (RR 0.94; 95% CI 0.71; 1.24). CONCLUSION: This study design has allowed us to carry out the most comprehensive assessment of RCTs on this subject, filling a gap in diabetes research. Our paper addresses a question that is important not only for decision makers but also for clinicians.
Assuntos
Diabetes Mellitus Tipo 1/tratamento farmacológico , Insulina de Ação Prolongada/uso terapêutico , Hemoglobinas Glicadas/química , Humanos , Hipoglicemia/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Insulina Detemir/uso terapêutico , Insulina Glargina/uso terapêutico , Modelos Estatísticos , Ensaios Clínicos Controlados Aleatórios como Assunto , Análise de RegressãoRESUMO
OBJECTIVE: To systematically analyze the relationship between maternal anemia and low birth weight. METHODS: A search of studies was conducted in the main databases (Medline, Embase, Scopus, Web of Science, SciELO, and Lilacs), the gray literature, and the reference lists of selected articles. Cohort and case-control studies that met the eligibility criteria were included in the review. There was no limitation on the language or date of publication. Article selection and data extraction were performed by two independent reviewers. Meta-analyses with random effects, subgroup analyses and meta-regressions were performed. Publication bias was measured using Egger regression and visual funnel plot inspection. RESULTS: A total of 7243 articles were found, of which 71 comprised the systematic review and 68 were included in the meta-analyses. Maternal anemia was associated with low birth weight with an adjusted OR: 1.23 (95% CI: 1.06â»1.43) and I²: 58%. The meta-regressions confirmed that the sample size and the methodological quality may partially explain the statistical heterogeneity. CONCLUSIONS: Maternal anemia was considered a risk factor for low birth weight.
Assuntos
Anemia Ferropriva/epidemiologia , Recém-Nascido de Baixo Peso , Feminino , Hemoglobinas/análise , Humanos , Recém-Nascido , Estudos Observacionais como Assunto , Gravidez , Fatores de RiscoRESUMO
Blood-contacting medical devices have been associated with severe clinical complications, such as thrombus formation, triggered by the activation of the coagulation cascade due to the adsorption of certain plasma proteins on the surface of biomaterials. Hence, the coating of such surfaces with antithrombotic agents has been used to increase biomaterial haemocompatibility. Biomaterial-induced clotting may also be decreased by albumin adsorption from blood plasma in a selective and reversible way, since this protein is not involved in the coagulation cascade. In this context, this paper reports that the immobilization of the thrombin inhibitor D-Phe-Pro-D-Arg-D-Thr-CONH2 (fPrt) onto nanostructured surfaces induces selective and reversible adsorption of albumin, delaying the clotting time when compared to peptide-free surfaces. fPrt, synthesized with two glycine residues attached to the N-terminus (GGfPrt), was covalently immobilized onto self-assembled monolayers (SAMs) having different ratios of carboxylate-hexa(ethylene glycol)- and tri(ethylene glycol)-terminated thiols (EG6-COOH/EG3) that were specifically designed to control GGfPrt orientation, exposure and density at the molecular level. In solution, GGfPrt was able to inactivate the enzymatic activity of thrombin and to delay plasma clotting time in a concentration-dependent way. After surface immobilization, and independently of its concentration, GGfPrt lost its selectivity to thrombin and its capacity to inhibit thrombin enzymatic activity against the chromogenic substrate n-p-tosyl-Gly-Pro-Arg-p-nitroanilide. Nevertheless, surfaces with low concentrations of GGfPrt could delay the capacity of adsorbed thrombin to cleave fibrinogen. In contrast, GGfPrt immobilized in high concentrations was found to induce the procoagulant activity of the adsorbed thrombin. However, all surfaces containing GGfPrt have a plasma clotting time similar to the negative control (empty polystyrene wells), showing resistance to coagulation, which is explained by its capacity to adsorb albumin in a selective and reversible way. This work opens new perspectives to the improvement of the haemocompatibility of blood-contacting medical devices.
Assuntos
Albuminas/metabolismo , Antitrombinas/farmacologia , Peptídeos/farmacologia , Trombina/farmacologia , Adsorção , Fibrinogênio/metabolismo , Ouro/farmacologia , Humanos , Hidrólise/efeitos dos fármacos , Proteínas Imobilizadas/farmacologia , Radioisótopos do Iodo , Nitrogênio/farmacologia , Peptídeos/química , Ligação Proteica/efeitos dos fármacos , Espectroscopia de Infravermelho com Transformada de Fourier , Propriedades de SuperfícieRESUMO
The cysteine-less peptidic anticoagulants madanin-1 and madanin-2 from the bush tick Haemaphysalis longicornis are the founding members of the MEROPS inhibitor family I53. It has been previously suggested that madanins exert their functional activity by competing with physiological substrates for binding to the positively charged exosite I (fibrinogen-binding exosite) of α-thrombin. We hereby demonstrate that competitive inhibition of α-thrombin by madanin-1 or madanin-2 involves binding to the enzyme's active site. Moreover, the blood coagulation factors IIa and Xa are shown to hydrolyze both inhibitors at different, although partially overlapping cleavage sites. Finally, the three-dimensional structure of the complex formed between human α-thrombin and a proteolytic fragment of madanin-1, determined by X-ray crystallography, elucidates the molecular details of madanin-1 recognition and processing by the proteinase. Taken together, the current findings establish the mechanism of action of madanins, natural anticoagulants that behave as cleavable competitive inhibitors of thrombin.
Assuntos
Anticoagulantes/metabolismo , Fator Xa/metabolismo , Proteínas de Insetos/metabolismo , Proteínas e Peptídeos Salivares/metabolismo , Trombina/metabolismo , Sequência de Aminoácidos , Anticoagulantes/química , Antitrombinas/química , Antitrombinas/isolamento & purificação , Antitrombinas/metabolismo , Ligação Competitiva , Coagulação Sanguínea/efeitos dos fármacos , Domínio Catalítico , Fator Xa/química , Humanos , Proteínas de Insetos/química , Proteínas de Insetos/genética , Proteínas de Insetos/isolamento & purificação , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas e Peptídeos Salivares/química , Proteínas e Peptídeos Salivares/genética , Proteínas e Peptídeos Salivares/isolamento & purificação , Alinhamento de Sequência , Trombina/químicaRESUMO
The tremendous social and economic impact of thrombotic disorders, together with the considerable risks associated to the currently available therapies, prompt for the development of more efficient and safer anticoagulants. Novel peptide-based thrombin inhibitors were identified using in silico structure-based design and further validated in vitro. The best candidate compounds contained both L- and D-amino acids, with the general sequence D-Phe(P3)-Pro(P2)-D-Arg(P1)-P1'-CONH2. The P1' position was scanned with L- and D-isomers of natural or unnatural amino acids, covering the major chemical classes. The most potent non-covalent and proteolysis-resistant inhibitors contain small hydrophobic or polar amino acids (Gly, Ala, Ser, Cys, Thr) at the P1' position. The lead tetrapeptide, D-Phe-Pro-D-Arg-D-Thr-CONH2, competitively inhibits α-thrombin's cleavage of the S2238 chromogenic substrate with a K(i) of 0.92 µM. In order to understand the molecular details of their inhibitory action, the three-dimensional structure of three peptides (with P1' L-isoleucine (fPrI), L-cysteine (fPrC) or D-threonine (fPrt)) in complex with human α-thrombin were determined by X-ray crystallography. All the inhibitors bind in a substrate-like orientation to the active site of the enzyme. The contacts established between the D-Arg residue in position P1 and thrombin are similar to those observed for the L-isomer in other substrates and inhibitors. However, fPrC and fPrt disrupt the active site His57-Ser195 hydrogen bond, while the combination of a P1 D-Arg and a bulkier P1' residue in fPrI induce an unfavorable geometry for the nucleophilic attack of the scissile bond by the catalytic serine. The experimental models explain the observed relative potency of the inhibitors, as well as their stability to proteolysis. Moreover, the newly identified direct thrombin inhibitors provide a novel pharmacophore platform for developing antithrombotic agents by exploring the conformational constrains imposed by the D-stereochemistry of the residues at positions P1 and P1'.
Assuntos
Antitrombinas/farmacologia , Desenho de Fármacos , Oligopeptídeos/farmacologia , Antitrombinas/química , Humanos , Oligopeptídeos/químicaRESUMO
Rab geranylgeranyl transferase (RGGT) catalyzes the post-translational geranylgeranyl (GG) modification of (usually) two C-terminal cysteines in Rab GTPases. Here we studied the mechanism of the Rab geranylgeranylation reaction by bisphosphonate analogs in which one phosphonate group is replaced by a carboxylate (phosphonocarboxylate, PC). The phosphonocarboxylates used were 3-PEHPC, which was previously reported, and 2-hydroxy-3-imidazo[1,2-a]pyridin-3-yl-2-phosphonopropionic acid ((+)-3-IPEHPC), a >25-fold more potent related compound as measured by both IC50 and Ki.(+)-3-IPEHPC behaves as a mixed-type inhibitor with respect to GG pyrophosphate (GGPP) and an uncompetitive inhibitor with respect to Rab substrates. We propose that phosphonocarboxylates prevent only the second GG transfer onto Rabs based on the following evidence. First, geranylgeranylation of Rab proteins ending with a single cysteine motif such as CAAX, is not affected by the inhibitors, either in vitro or in vivo. Second, the addition of an -AAX sequence onto Rab-CC proteins protects the substrate from inhibition by the inhibitors. Third, we demonstrate directly that in the presence of (+)-3-IPEHPC, Rab-CC and Rab-CXC proteins are modified by only a single GG addition. The presence of (+)-3-IPEHPC resulted in a preference for the Rab N-terminal cysteine to be modified first, suggesting an order of cysteine geranylgeranylation in RGGT catalysis. Our results further suggest that the inhibitor binds to a site distinct from the GGPP-binding site on RGGT. We suggest that phosphonocarboxylate inhibitors bind to a GG-cysteine binding site adjacent to the active site, which is necessary to align the mono-GG-Rab for the second GG addition. These inhibitors may represent a novel therapeutic approach in Rab-mediated diseases.