RESUMO
The PKC-related kinases (PRKs, also termed PKNs) are important in cell migration, cancer, hepatitis C infection, and nutrient sensing. They belong to a group of protein kinases called AGC kinases that share common features like a C-terminal extension to the catalytic domain comprising a hydrophobic motif. PRKs are regulated by N-terminal domains, a pseudosubstrate sequence, Rho-binding domains, and a C2 domain involved in inhibition and dimerization, while Rho and lipids are activators. We investigated the allosteric regulation of PRK2 and its interaction with its upstream kinase PDK1 using a chemical biology approach. We confirmed the phosphoinositide-dependent protein kinase 1 (PDK1)-interacting fragment (PIF)-mediated docking interaction of PRK2 with PDK1 and showed that this interaction can be modulated allosterically. We showed that the polypeptide PIFtide and a small compound binding to the PIF-pocket of PRK2 were allosteric activators, by displacing the pseudosubstrate PKL region from the active site. In addition, a small compound binding to the PIF-pocket allosterically inhibited the catalytic activity of PRK2. Together, we confirmed the docking interaction and allostery between PRK2 and PDK1 and described an allosteric communication between the PIF-pocket and the active site of PRK2, both modulating the conformation of the ATP-binding site and the pseudosubstrate PKL-binding site. Our study highlights the allosteric modulation of the activity and the conformation of PRK2 in addition to the existence of at least two different complexes between PRK2 and its upstream kinase PDK1. Finally, the study highlights the potential for developing allosteric drugs to modulate PRK2 kinase conformations and catalytic activity.
Assuntos
Proteína Quinase C , Piruvato Desidrogenase Quinase de Transferência de Acetil , Humanos , Regulação Alostérica , Proteína Quinase C/metabolismo , Proteína Quinase C/genética , Proteína Quinase C/química , Piruvato Desidrogenase Quinase de Transferência de Acetil/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil/genética , Domínio Catalítico , Simulação de Acoplamento Molecular , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/química , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/genética , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/química , Ligação ProteicaRESUMO
The selective, rapid detection of low levels of hormones in drinking water and foodstuffs requires materials suitable for inexpensive sensing platforms. We report on core-shell Ag@C nanocables (NCs) decorated with carbon spherical shells (CSSs) and silver nanoparticles (AgNPs) by using a hydrothermal green approach. Sensors were fabricated with homogeneous, porous films on screen-printed electrodes, which comprised a 115 nm silver core covered by a 122 nm thick carbon layer and CSSs with 168 nm in diameter. NCs and CSSs were also decorated with 10-25 nm AgNPs. The NC/CSS/AgNP sensor was used to detect ethinylestradiol using square wave voltammetry in 0.1 M phosphate buffer (pH 7.0) over the 1.0-10.0 µM linear range with a detection limit of 0.76 µM. The sensor was then applied to detect ethinylestradiol in tap water samples and a contraceptive pill with recovery percentages between 93 and 101%. The high performance in terms of sensitivity and selectivity for hormones is attributed to the synergy between the carbon nanomaterials and AgNPs, which not only increased the sensor surface area and provided sites for electron exchange but also imparted an increased surface area.
Assuntos
Carbono , Nanopartículas Metálicas , Prata , Etinilestradiol , Água , Hormônios , Eletrodos , Técnicas EletroquímicasRESUMO
The protein kinase PDK1 phosphorylates at least 24 distinct substrates, all of which belong to the AGC protein kinase group. Some substrates, such as conventional PKCs, undergo phosphorylation by PDK1 during their synthesis and subsequently get activated by DAG and Calcium. On the other hand, other substrates, including members of the Akt/PKB, S6K, SGK, and RSK families, undergo phosphorylation and activation downstream of PI3-kinase signaling. This review presents two accepted molecular mechanisms that determine the precise and timely phosphorylation of different substrates by PDK1. The first mechanism involves the colocalization of PDK1 with Akt/PKB in the presence of PIP3. The second mechanism involves the regulated docking interaction between the hydrophobic motif (HM) of substrates and the PIF-pocket of PDK1. This interaction, in trans, is equivalent to the molecular mechanism that governs the activity of AGC kinases through their HMs intramolecularly. PDK1 has been instrumental in illustrating the bi-directional allosteric communication between the PIF-pocket and the ATP-binding site and the potential of the system for drug discovery. PDK1's interaction with substrates is not solely regulated by the substrates themselves. Recent research indicates that full-length PDK1 can adopt various conformations based on the positioning of the PH domain relative to the catalytic domain. These distinct conformations of full-length PDK1 can influence the interaction and phosphorylation of substrates. Finally, we critically discuss recent findings proposing that PIP3 can directly regulate the activity of PDK1, which contradicts extensive in vitro and in vivo studies conducted over the years.
Assuntos
Piruvato Desidrogenase Quinase de Transferência de Acetil , Humanos , Sítios de Ligação , Fosfatidilinositol 3-Quinase , Fosforilação , Proteínas Proto-Oncogênicas c-akt , Piruvato Desidrogenase Quinase de Transferência de Acetil/metabolismoRESUMO
Abstract Objective To determine the prevalence of the atypical glandular cells (AGCs) cytology and to analyze its clinical significance in different age ranges. Methods Retrospective observational study using computerized data from the Brazilian National Cancer Institute, including women screened between January 2002 and December 2008. The women included were those with an AGC result who were properly followed-up with colposcopy and a second cytology. Results A total of 132,147 cytopathological exams were performed during the study period. Five-hundred and thirty-three (0.4%) women with AGC cytology were identified and, of these, 69.41% (370/533) were properly referred for colposcopy and a new cytology. Most of the women (79.2%) with a 1st or 2nd AGC cytology were between the ages of 25 and 54 years. The 2nd cytology demonstrated 67.6% (250/370) of normality, 24.5% (91/370) of squamous atypia, and 6.2% (23/370) of AGC, 0.8% (3/370) adenocarcinoma in situ and 0.8% (3/370) adenocarcinoma invasor. On biopsy of the women with a second AGC cytology, 43.4% (10/23) had normal histology, 43.4% (10/23) had squamous lesions, 8.7% (2/23) had invasive adenocarcinoma, and 1.2% (1/23) had an inconclusive report. All of the women with high-grade squamous intraepithelial lesion (HSIL) or invasive adenocarcinoma (respectively 5 and 2 patients), after a 2nd AGC cytology were 25 years old or older. Conclusion The prevalence of the AGC cytology was low in the studied population. Most of the AGC cytology cases occurred in adult women between the ages of 25 and 54. Although most of the patients had normal histology after follow-up, several of them presented with squamous intraepithelial lesions or invasive adenocarcinoma.
Resumo Objetivo Determinar a prevalência de citologia com laudo de células glandulares atípicas (AGCs, na sigla em inglês) e analisar a significância clínica nas diferentes faixas etárias Métodos Estudo observacional retrospectivo, usando os dados arquivados no sistema do Instituto Nacional de Câncer no Brasil, que incluiu mulheres rastreadas entre janeiro de 2002 a dezembro de 2008. As mulheres incluídas tinham citologia com resultado de AGCs, que foram acompanhadas com colposcopia e nova citologia Resultados Um total de132,147 exames citopatológicos foram incluídos durante o período de estudo. Quinhentas e trinta e três mulheres com citologia de AGC foram identificadas e destas, 69.41% (370) foram encaminhadas para colposcopia e nova citologia. A prevalência de citologia de AGC na população estudada foi 0.4%. A maioria das mulheres (79.22%) com resultado citológico de AGC tinham idade entre 25 e 54 anos. A segunda citologia demonstrou 67.56% (250/370) de normalidade, 24.5% (91/370) de atipias escamosas, e 6.2% (23/370) de AGC. Na biopsia das mulheres com a 2ª citologia de AGC, 43.4% (10/23) tinham histologia normal, 43.4% (10/23) tinha lesões escamosas, 8.7% (2/23) tinha adenocarcinoma invasor e 1.2% (1/23) tinha laudo inconclusivo. Todas as mulheres com lesões intraepiteliais escamosas de alto grau (HSIL, na sigla em inglês) ou adenocarcinoma invasor (respectivamente 5 e 2pacientes), após a 2ª citologia com AGC, tinham 25 anos de idade ou mais. Conclusão A prevalência de citologia com AGC foi baixa na população estudada. Muitos casos de citologia com AGC apareceram em mulheres adultas, entre 25 e 54 anos de idade. Embora a maioria das pacientes tiveram histologia normal após seguimento, várias apresentaram lesões intraepiteliais escamosas ou glandulares invasoras.
Assuntos
Humanos , Feminino , Displasia do Colo do Útero , Células Epiteliais , Detecção Precoce de CâncerRESUMO
Abstract Leptospirosis is a wide spread bacterial zoonosis that is common worldwide. The disease symptoms are mild or acute. Leptospira has pathogenic and non-pathogenic species; it has a lot of surface antigens. Adenylate Guanylate Cyclase (AGC) is a membrane protein that is found only in pathogenic species. In this study, the complete coding sequences of AGC protein of 242 pathogen serovars were investigated by bioinformatics tools. A Pattern was selected as a target sequence based on high prevalence pathogenic serovars in Iran Antigen sites; moreover, B-cell and T-cell epitopes were predicted by IEDB web server. An antigen site amino acid (D259-R462) in complete coding sequence of AGC protein was selected. This nucleotide related sequence was cloned into the pET32a+ expression vector. Expression of recombinant protein was optimized in E. coli strain Bl21-DE3 by 0.2mM IPTG after 16-hour incubation at 37 ͦ C and confirmed by 10% SDS-PAGE and western blotting. Antigenic peptide D259-R462 was highly expressed as Trx tag fusion protein. Recombinant peptide (rAcB) was purified by 6M urea from inclusion body with high extent yield 514.2 mg per 1000ml culture of E. coli. 20µg rAcB protein with montanide adjuvant was injected subcutaneously in BALB/c mice. Results showed that the recombinant peptide D259-R462 was produced significant antibody compared to adjuvant and PBS groups. The induced antibody in sera of immunized animal with Leptospira vaccine was detected by 250 ng of rAcB coated in ELISA microplate. This study demonstrated that antigenic region (D259-R462) of AGC protein might be useful for evaluation of antibody level in vaccinated animal.
Assuntos
Guanilato Ciclase , Proteínas Recombinantes , Ensaio de Imunoadsorção Enzimática , Adenilil Ciclases , LeptospiroseRESUMO
Allostery is a basic principle that enables proteins to process and transmit cellular information. Protein kinases evolved allosteric mechanisms to transduce cellular signals to downstream signalling components or effector molecules. Protein kinases catalyse the transfer of the terminal phosphate from ATP to protein substrates upon specific stimuli. Protein kinases are targets for the development of small molecule inhibitors for the treatment of human diseases. Drug development has focussed on ATP-binding site, while there is increase interest in the development of drugs targeting alternative sites, i.e. allosteric sites. Here, we review the mechanism of regulation of protein kinases, which often involve the allosteric modulation of the ATP-binding site, enhancing or inhibiting activity. We exemplify the molecular mechanism of allostery in protein kinases downstream of PI3-kinase signalling with a focus on phosphoinositide-dependent protein kinase 1 (PDK1), a model kinase where small compounds can allosterically modulate the conformation of the kinase bidirectionally.
Assuntos
Desenvolvimento de Medicamentos , Fosfatidilinositol 3-Quinases , Proteínas Quinases , Transdução de Sinais , Regulação Alostérica , Sítio Alostérico , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Abstract Introduction Long-term electrocardiogram (ECG) recordings are widely employed to assist the diagnosis of cardiac and sleep disorders. However, variability of ECG amplitude during the recordings hampers the detection of QRS complexes by algorithms. This work presents a simple electronic circuit to automatically normalize the ECG amplitude, improving its sampling by analog to digital converters (ADCs). Methods The proposed circuit consists of an analog divider that normalizes the ECG amplitude using its absolute peak value as reference. The reference value is obtained by means of a full-wave rectifier and a peak voltage detector. The circuit and tasks of its different stages are described. Results Example of the circuit performance for a bradycardia ECG signal (40bpm) is presented; the signal has its amplitude suddenly halved, and later, restored. The signal is automatically normalized after 5 heart beats for the amplitude drop. For the amplitude increase, the signal is promptly normalized. Conclusion The proposed circuit adjusts the ECG amplitude to the input voltage range of ADC, avoiding signal to noise ratio degradation of the sampled waveform in order to allow a better performance of processing algorithms.
RESUMO
In this review we discuss the structure and functions of the aspartate/glutamate carriers (AGC1-aralar and AGC2-citrin). Those proteins supply the aspartate synthesized within mitochondrial matrix to the cytosol in exchange for glutamate and a proton. A structure of an AGC carrier is not available yet but comparative 3D models were proposed. Moreover, transport assays performed by using the recombinant AGC1 and AGC2, reconstituted into liposome vesicles, allowed to explore the kinetics of those carriers and to reveal their specific transport properties. AGCs participate to a wide range of cellular functions, as the control of mitochondrial respiration, calcium signaling and antioxydant defenses. AGC1 might also play peculiar tissue-specific functions, as it was found to participate to cell-to-cell metabolic symbiosis in the retina. On the other hand, AGC1 is involved in the glutamate-mediated excitotoxicity in neurons and AGC gene or protein alterations were discovered in rare human diseases. Accordingly, a mice model of AGC1 gene knock-out presented with growth delay and generalized tremor, with myelinisation defects. More recently, AGC was proposed to play a crucial role in tumor metabolism as observed from metabolomic studies showing that the asparate exported from the mitochondrion by AGC1 is employed in the regeneration of cytosolic glutathione. Therefore, given the central role of AGCs in cell metabolism and human pathology, drug screening are now being developed to identify pharmacological modulators of those carriers. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou.