RESUMO
BACKGROUND: Decreased cerebral blood flow and systemic inflammation during heart failure (HF) increase the risk for vascular contributions to cognitive impairment and dementia (VCID) and Alzheimer disease-related dementias (ADRD). We previously demonstrated that PNA5, a novel glycosylated angiotensin 1-7 (Ang-(1-7)) Mas receptor (MasR) agonist peptide, is an effective therapy to rescue cognitive impairment in our preclinical model of VCID. Neurofilament light (NfL) protein concentration is correlated with cognitive impairment and elevated in neurodegenerative diseases, hypoxic brain injury, and cardiac disease. The goal of the present study was to determine (1) if treatment with Ang-(1-7)/MasR agonists can rescue cognitive impairment and decrease VCID-induced increases in NfL levels as compared to HF-saline treated mice and, (2) if NfL levels correlate with measures of cognitive function and brain cytokines in our VCID model. METHODS: VCID was induced in C57BL/6 male mice via myocardial infarction (MI). At 5 weeks post-MI, mice were treated with daily subcutaneous injections for 24 days, 5 weeks after MI, with PNA5 or angiotensin 1-7 (500 microg/kg/day or 50 microg/kg/day) or saline (n = 15/group). Following the 24-day treatment protocol, cognitive function was assessed using the Novel Object Recognition (NOR) test. Cardiac function was measured by echocardiography and plasma concentrations of NfL were quantified using a Quanterix Simoa assay. Brain and circulating cytokine levels were determined with a MILLIPLEX MAP Mouse High Sensitivity Multiplex Immunoassay. Treatment groups were compared via ANOVA, significance was set at p < 0.05. RESULTS: Treatment with Ang-(1-7)/MasR agonists reversed VCID-induced cognitive impairment and significantly decreased NfL levels in our mouse model of VCID as compared to HF-saline treated mice. Further, NfL levels were significantly negatively correlated with cognitive scores and the concentrations of multiple pleiotropic cytokines in the brain. CONCLUSIONS: These data show that treatment with Ang-(1-7)/MasR agonists rescues cognitive impairment and decreases plasma NfL relative to HF-saline-treated animals in our VCID mouse model. Further, levels of NfL are significantly negatively correlated with cognitive function and with several brain cytokine concentrations. Based on these preclinical findings, we propose that circulating NfL might be a candidate for a prognostic biomarker for VCID and may also serve as a pharmacodynamic/response biomarker for therapeutic target engagement.
Assuntos
Angiotensina I/agonistas , Angiotensina I/metabolismo , Disfunção Cognitiva/metabolismo , Citocinas/metabolismo , Demência Vascular/metabolismo , Proteínas de Neurofilamentos/metabolismo , Fragmentos de Peptídeos/agonistas , Fragmentos de Peptídeos/metabolismo , Angiotensina I/uso terapêutico , Animais , Biomarcadores/metabolismo , Disfunção Cognitiva/tratamento farmacológico , Disfunção Cognitiva/patologia , Demência Vascular/tratamento farmacológico , Demência Vascular/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos/uso terapêutico , Prognóstico , Volume Sistólico/fisiologiaRESUMO
Nociceptin/orphanin FQ (N/OFQ) controls different biological functions via selective stimulation of the N/OFQ peptide (NOP) receptor. The pleiotropic actions of N/OFQ may limit the development of NOP ligands as innovative drugs in different therapeutic areas. The pharmacological concept of functional selectivity (aka biased agonism) might be useful for amplifying beneficial actions and/or counteracting side effects. Thus, molecules with large bias factors toward G protein or ß arrestin are required for investigating the translational value of NOP biased modulation. Herein, the biased behavior of a heterogeneous library of NOP-targeting peptide derivatives was evaluated in vitro with the aim to provide possible insights into the structural determinants that govern the selective activation of G protein versus ß-arrestin. Our results demonstrate that lipidation of N/OFQ(1-13)-NH2 is a useful strategy for obtaining G protein biased agonists for the NOP receptor.
Assuntos
Peptídeos Opioides/farmacologia , Fragmentos de Peptídeos/agonistas , Receptores Opioides/agonistas , Animais , Proteínas de Ligação ao GTP/química , Fragmentos de Peptídeos/química , Receptores Opioides/química , Relação Estrutura-Atividade , Receptor de Nociceptina , NociceptinaRESUMO
The peptide angiotensin-(1-7) [Ang (1-7)] and its receptor Mas are involved in controlling arterial pressure and display actions on the nervous system. In a previous study, our laboratory showed that A779 [(peptidyl antagonist of the Ang-(1-7)] treatment had a negative effect following a lesion of the sciatic nerve, possibly by delaying the responses of Schwann cells, resulting in a decreased axonal organization along with a slowed functional return. In the present work, we investigated the central cellular changes after sciatic nerve injury in rodents treated with A779 after two weeks. In the lumbar spinal cords, where the neuronal bodies that make up the sciatic are, the treatment with A779 showed reduced reactivity of astrocytes (p = 0.004, Mann-Whitney U test) and less synaptic density (p = 0.004, Mann-Whitney U test) after injury. Also, the treatment upregulated microglia activity in both sides (p = 0.004, Mann-Whitney U test), ipsilateral and contralateral to the lesion, of the spinal cord. In addition, the Mas expression in spine neurons was increased in response to axotomy especially after two weeks (p = 0.03, Mann-Whitney U test) following the nerve lesion in comparison to earlier stages after injury. Therefore, we can conclude that Ang-(1-7)/Mas axis plays a role during spinal cord recovery after peripheral nerve injury.
Assuntos
Angiotensina II/análogos & derivados , Angiotensina I/agonistas , Axotomia , Gliose/tratamento farmacológico , Gliose/patologia , Fragmentos de Peptídeos/agonistas , Fragmentos de Peptídeos/uso terapêutico , Receptores Acoplados a Proteínas G/agonistas , Sinapses/efeitos dos fármacos , Angiotensina II/uso terapêutico , Animais , Astrócitos/efeitos dos fármacos , Imunofluorescência , Masculino , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Medula Espinal/efeitos dos fármacosRESUMO
The neurotensin receptors are attractive targets for the development of new analgesic compounds. They represent potential alternatives or adjuvants to opioids. Herein, we report the structural optimization of our recently reported macrocyclic peptide analogues of NT(8-13). The macrocycle was formed via ring-closing metathesis (RCM) between an ortho allylated tyrosine residue in position 11 and the side chain of alkene-functionalized amino acid in position 8 of NT(8-13). Minute modifications led to significant binding affinity improvement ( Ki improved from 5600 to 15 nM) with greatly improved plasma stability compared to NT(8-13). This study also delineates the structural features influencing these parameters. The signaling profiles of the new macrocycles were determined on the NTS1 receptor, and the physiological effects of the two most potent and stable analogues were assessed in vivo using rodent models. Both compounds displayed strong analgesic effects.
Assuntos
Analgésicos não Narcóticos/química , Analgésicos não Narcóticos/farmacologia , Neurotensina/farmacologia , Fragmentos de Peptídeos/farmacologia , Peptídeos Cíclicos/química , Receptores de Neurotensina/metabolismo , Animais , Ligação Competitiva , Pressão Sanguínea/efeitos dos fármacos , Temperatura Corporal/efeitos dos fármacos , Células CHO , Cricetulus , Ciclização , Avaliação Pré-Clínica de Medicamentos/métodos , Estabilidade de Medicamentos , Masculino , Simulação de Acoplamento Molecular , Neurotensina/agonistas , Neurotensina/química , Fragmentos de Peptídeos/agonistas , Fragmentos de Peptídeos/química , Peptídeos Cíclicos/sangue , Peptídeos Cíclicos/farmacologia , Ratos Sprague-Dawley , Receptores de Neurotensina/química , Relação Estrutura-Atividade , Tirosina/químicaRESUMO
AIMS: Ovarian cancer (OC) has the highest mortality rate of all gynecological cancers. Currently, the first-line OC treatment consists of cytoreductive surgery and platinum-based chemotherapy. However, most patients develop chemoresistance after the first-line treatment limits the success of treatment. Therefore, there is an urgent need to identify effective therapeutic agents. MAIN METHODS: Cell viabilities were detected by MTS assay; Annexin V-FITC/PI assay and western blotting assay were performed to analyze the apoptotic cells in vitro; An immunofluorescence assay was performed to analyze the TUNEL+ apoptotic cells in vivo; Patient-derived xenografts were established to test the in vivo antitumor effects; The key proteins of p53, caspase-mediated apoptotic pathway and Akt/mTOR pathway were detected by Western blotting. KEY FINDINGS: Icaritin, a prenylflavonoid derivative from Epimedium Genus, inhibited the proliferation of drug-sensitive OC cells (OV2008 and C13*) and cisplatin resistant OC cells A2780cp. Icaritin induced OC cell apoptosis in vitro, as indicated by the increase of Annexin V+/PI+ apoptotic cells analyzed with flow cytometry, and the cleavage of caspase 9, caspase 3 and poly-ADP-ribose polymerase (PARP) detected with western blotting. Icaritin also inhibited tumor growth and induced OC cells apoptosis in patient-derived xenografts, as indicated by the tumor growth delay and increase of TUNEL-positive cells in tumor tissues. The icaritin-induced OC cell apoptosis may be associated with the activation of p53 and the suppression of Akt/mTOR pathway. SIGNIFICANCE: This study sheds light on the underlying mechanisms of antitumor effect of icaritin, and warrants clinical trial for treatment of OC.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Flavonoides/farmacologia , Proteína Oncogênica v-akt/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Fragmentos de Peptídeos/agonistas , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Proteína Supressora de Tumor p53/agonistas , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Nus , Camundongos SCID , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Glucagon-like peptide-1 (GLP-1) is a 30 amino acid long peptide hormone derived from the proglucagon gene and secreted in the distal small intestine when food enters the duodenum. GLP-1 is also produced in the central nervous system (CNS), predominantly in the brainstem, and subsequently transported to a large number of regions in the CNS. Neuronal cells in nucleus tractus solitarius (NTS) can synthesize GLP-1 and extends to hypothalamus, some thalamic and cortical areas. A G protein coupled receptor (GPCR) provides the majority of GLP-1 actions. GLP-1 receptor activation triggers some in vivo signaling pathways. GLP-1 receptor agonists (GLP-1 RA) are used in the treatment diabetes and obesity. GLP-1 stimulates insulin secretion, inhibits glucagon secretion, decreases food intake, reduces appetite, delays gastric emptying, provides weight reduction, and protects ß cells from apoptosis. Alzheimer's disease (AD) is the most prevalent form of dementia. It is characterized by cognitive insufficiencies and behavioral changes that impact memory and learning abilities, daily functioning and quality of life. Hyperinsulinemia and insulin resistance, which are known as pathophysiological features of the T2DM, have also been demonstrated to have significant impact on cognitive impairment. It is thought that GLP-1 affects neurological and cognitive functions, as well as its regulatory effect on glucose metabolism. The pathophysiological relationship between GLP-1 and AD is discussed in this review.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Diabetes Mellitus Tipo 2/complicações , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Fragmentos de Peptídeos/agonistas , Fragmentos de Peptídeos/metabolismo , Doença de Alzheimer/etiologia , Humanos , Qualidade de VidaRESUMO
CD16a/Fc γ receptor IIIa is the most abundant antibody Fc receptor expressed on human natural killer (NK) cells and activates a protective cytotoxic response following engagement with antibody clustered on the surface of a pathogen or diseased tissue. Therapeutic monoclonal antibodies (mAbs) with greater Fc-mediated affinity for CD16a show superior therapeutic outcome; however, one significant factor that promotes antibody-CD16a interactions, the asparagine-linked carbohydrates (N-glycans), remains undefined. Here, we purified CD16a from the primary NK cells of three donors and identified a large proportion of hybrid (22%) and oligomannose N-glycans (23%). These proportions indicated restricted N-glycan processing and were unlike those of the recombinant CD16a forms, which have predominantly complex-type N-glycans (82%). Tethering recombinant CD16a to the membrane by including the transmembrane and intracellular domains and via coexpression with the Fc ϵ receptor γ-chain in HEK293F cells was expected to produce N-glycoforms similar to NK cell-derived CD16a but yielded N-glycoforms different from NK cell-derived CD16a and recombinant soluble CD16a. Of note, these differences in CD16a N-glycan composition affected antibody binding: CD16a with oligomannose N-glycans bound IgG1 Fc with 12-fold greater affinity than did CD16a having primarily complex-type and highly branched N-glycans. The changes in binding activity mirrored changes in NMR spectra of the two CD16a glycoforms, indicating that CD16a glycan composition also affects the glycoprotein's structure. These results indicated that CD16a from primary human NK cells is compositionally, and likely also functionally, distinct from commonly used recombinant forms. Furthermore, our study provides critical evidence that cell lineage determines CD16a N-glycan composition and antibody-binding affinity.
Assuntos
Fragmentos Fc das Imunoglobulinas/metabolismo , Células Matadoras Naturais/metabolismo , Modelos Moleculares , Polissacarídeos/metabolismo , Processamento de Proteína Pós-Traducional , Receptores de IgG/metabolismo , Idoso , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacologia , Sequência de Carboidratos , Linhagem da Célula , Células Cultivadas , Glicosilação , Células HEK293 , Humanos , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/genética , Células Matadoras Naturais/citologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Ligantes , Masculino , Fragmentos de Peptídeos/agonistas , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Polissacarídeos/química , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores de IgG/agonistas , Receptores de IgG/química , Receptores de IgG/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , SolubilidadeRESUMO
CD300 molecules (CD300s) belong to paired activating and inhibitory receptor families, which mediate immune responses. Human CD300e (hCD300e) is expressed in monocytes and myeloid dendritic cells and transmits an immune-activating signal by interacting with DNAX-activating protein 12 (DAP12). However, the CD300e ortholog in mice (mCD300e) is poorly characterized. Here, we found that mCD300e is also an immune-activating receptor. We found that mCD300e engagement triggers cytokine production in mCD300e-transduced bone marrow-derived mast cells (BMMCs). Loss of DAP12 and another signaling protein, FcRγ, did not affect surface expression of transduced mCD300e, but abrogated mCD300e-mediated cytokine production in the BMMCs. Co-immunoprecipitation experiments revealed that mCD300e physically interacts with both FcRγ and DAP12, suggesting that mCD300e delivers an activating signal via these two proteins. Binding and reporter assays with the mCD300e extracellular domain identified sphingomyelin as a ligand of both mCD300e and hCD300e. Notably, the binding of sphingomyelin to mCD300e stimulated cytokine production in the transduced BMMCs in an FcRγ- and DAP12-dependent manner. Flow cytometric analysis with an mCD300e-specific Ab disclosed that mCD300e expression is highly restricted to CD115+Ly-6Clow/int peripheral blood monocytes, corresponding to CD14dim/+CD16+ human nonclassical and intermediate monocytes. Loss of FcRγ or DAP12 lowered the surface expression of endogenous mCD300e in the CD115+Ly-6Clow/int monocytes. Stimulation with sphingomyelin failed to activate the CD115+Ly-6Clow/int mouse monocytes, but induced hCD300e-mediated cytokine production in the CD14dimCD16+ human monocytes. Taken together, these observations indicate that mCD300e recognizes sphingomyelin and thereby regulates nonclassical and intermediate monocyte functions through FcRγ and DAP12.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Mastócitos/metabolismo , Monócitos/metabolismo , Processamento de Proteína Pós-Traducional , Receptores de IgG/metabolismo , Receptores Imunológicos/agonistas , Esfingomielinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Substituição de Aminoácidos , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Linhagem Celular , Citocinas/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Ligantes , Mastócitos/citologia , Mastócitos/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/citologia , Monócitos/imunologia , Mutação , Fragmentos de Peptídeos/agonistas , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Receptores de IgG/química , Receptores de IgG/genética , Receptores Imunológicos/química , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
In the yeast Saccharomyces cerevisiae, the exposure to mating pheromone activates a prototypic mitogen-activated protein kinase (MAPK) cascade and triggers a dose-dependent differentiation response. Whereas a high pheromone dose induces growth arrest and formation of a shmoo-like morphology in yeast cells, lower pheromone doses elicit elongated cell growth. Previous population-level analysis has revealed that the MAPK Fus3 plays an important role in mediating this differentiation switch. To further investigate how Fus3 controls the fate decision process at the single-cell level, we developed a specific translocation-based reporter for monitoring Fus3 activity in individual live cells. Using this reporter, we observed strikingly different dynamic patterns of Fus3 activation in single cells differentiated into distinct fates. Cells committed to growth arrest and shmoo formation exhibited sustained Fus3 activation. In contrast, most cells undergoing elongated growth showed either a delayed gradual increase or pulsatile dynamics of Fus3 activity. Furthermore, we found that chemically perturbing Fus3 dynamics with a specific inhibitor could effectively redirect the mating differentiation, confirming the causative role of Fus3 dynamics in driving cell fate decisions. MAPKs mediate proliferation and differentiation signals in mammals and are therapeutic targets in many cancers. Our results highlight the importance of MAPK dynamics in regulating single-cell responses and open up the possibility that MAPK signaling dynamics could be a pharmacological target in therapeutic interventions.
Assuntos
Sistema de Sinalização das MAP Quinases , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Substituição de Aminoácidos , Proteínas do Citoesqueleto/agonistas , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Ativação Enzimática/efeitos dos fármacos , Deleção de Genes , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Reporter/efeitos dos fármacos , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Cinética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fator de Acasalamento/agonistas , Fator de Acasalamento/metabolismo , Proteínas de Membrana/agonistas , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Quinases Ativadas por Mitógeno/química , Proteínas Quinases Ativadas por Mitógeno/genética , Mutação , Fragmentos de Peptídeos/agonistas , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Feromônios/farmacologia , Transporte Proteico/efeitos dos fármacos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/agonistas , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Análise de Célula ÚnicaRESUMO
Unlike age-matched men, premenopausal women benefit from cardiovascular protection. Estrogens protect against apoptosis of endothelial cells (ECs), one of the hallmarks of endothelial dysfunction leading to cardiovascular disorders, but the underlying molecular mechanisms remain poorly understood. The inflammatory cytokine TNFα causes EC apoptosis while dysregulating the Notch pathway, a major contributor to EC survival. We have previously reported that 17ß-estradiol (E2) treatment activates Notch signaling in ECs. Here, we sought to assess whether in TNFα-induced inflammation Notch is involved in E2-mediated protection of the endothelium. We treated human umbilical vein endothelial cells (HUVECs) with E2, TNFα, or both and found that E2 counteracts TNFα-induced apoptosis. When Notch1 was inhibited, this E2-mediated protection was not observed, whereas ectopic overexpression of Notch1 diminished TNFα-induced apoptosis. Moreover, TNFα reduced the levels of active Notch1 protein, which were partially restored by E2 treatment. Moreover, siRNA-mediated knockdown of estrogen receptor ß (ERß), but not ERα, abolished the effect of E2 on apoptosis. Additionally, the E2-mediated regulation of the levels of active Notch1 was abrogated after silencing ERß. In summary, our results indicate that E2 requires active Notch1 through a mechanism involving ERß to protect the endothelium in TNFα-induced inflammation. These findings could be relevant for assessing the efficacy and applicability of menopausal hormone treatment, because they may indicate that in women with impaired Notch signaling, hormone therapy might not effectively protect the endothelium.
Assuntos
Apoptose , Endotélio Vascular/metabolismo , Estradiol/metabolismo , Receptor beta de Estrogênio/agonistas , Receptor Notch1/agonistas , Transdução de Sinais , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Apoptose/efeitos dos fármacos , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/imunologia , Receptor alfa de Estrogênio/agonistas , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/antagonistas & inibidores , Receptor beta de Estrogênio/genética , Receptor beta de Estrogênio/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Fragmentos de Peptídeos/agonistas , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Domínios e Motivos de Interação entre Proteínas , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/agonistas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Receptor Notch1/química , Receptor Notch1/genética , Receptor Notch1/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Blood coagulation is a finely regulated physiological process culminating with the factor Xa (FXa)-mediated conversion of the prothrombin (ProT) zymogen to active α-thrombin (αT). In the prothrombinase complex on the platelet surface, FXa cleaves ProT at Arg-271, generating the inactive precursor prethrombin-2 (Pre2), which is further attacked at Arg-320-Ile-321 to yield mature αT. Whereas the mechanism of physiological ProT activation has been elucidated in great detail, little is known about the role of bacterial proteases, possibly released in the bloodstream during infection, in inducing blood coagulation by direct proteolytic ProT activation. This knowledge gap is particularly concerning, as bacterial infections are frequently complicated by severe coagulopathies. Here, we show that addition of subtilisin (50 nm to 2 µm), a serine protease secreted by the non-pathogenic bacterium Bacillus subtilis, induces plasma clotting by proteolytically converting ProT into active σPre2, a nicked Pre2 derivative with a single cleaved Ala-470-Asn-471 bond. Notably, we found that this non-canonical cleavage at Ala-470-Asn-471 is instrumental for the onset of catalysis in σPre2, which was, however, reduced about 100-200-fold compared with αT. Of note, σPre2 could generate fibrin clots from fibrinogen, either in solution or in blood plasma, and could aggregate human platelets, either isolated or in whole blood. Our findings demonstrate that alternative cleavage of ProT by proteases, even by those secreted by non-virulent bacteria such as B. subtilis, can shift the delicate procoagulant-anticoagulant equilibrium toward thrombosis.
Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Coagulação Sanguínea , Modelos Moleculares , Agregação Plaquetária , Protrombina/agonistas , Subtilisina/metabolismo , Adulto , Proteínas de Bactérias/antagonistas & inibidores , Coagulação Sanguínea/efeitos dos fármacos , Domínio Catalítico , Ativação Enzimática/efeitos dos fármacos , Estabilidade Enzimática/efeitos dos fármacos , Feminino , Humanos , Masculino , Fragmentos de Peptídeos/agonistas , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteólise/efeitos dos fármacos , Protrombina/química , Protrombina/genética , Protrombina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Inibidores de Serina Proteinase/farmacologia , Especificidade por Substrato , Subtilisina/antagonistas & inibidores , Trombose/etiologia , Trombose/metabolismoRESUMO
Apolipoprotein B mRNA-editing enzyme catalytic subunit 3 (APOBEC-3) enzymes are cytidine deaminases that are broadly and constitutively expressed. They are often up-regulated during carcinogenesis and candidate genes for causing the major single-base substitution in cancer-associated DNA mutations. Moreover, APOBEC-3s are involved in host innate immunity against many viruses. However, how APOBEC-3 mutational activity is regulated in normal and pathological conditions remains largely unknown. Heat shock protein levels are often elevated in both carcinogenesis and viral infection and are associated with DNA mutations. Here, using mutational analyses of hepatitis B virus (HBV), we found that Hsp90 stimulates deamination activity of APOBEC-3G (A3G), A3B, and A3C during co-expression in human liver HepG2 cells. Hsp90 directly stimulated A3G deamination activity when the purified proteins were used in in vitro reactions. Hsp40, -60, and -70 also had variable stimulatory effects in the cellular assay, but not in vitro Sequencing analyses further demonstrated that Hsp90 increased both A3G cytosine mutation efficiency on HBV DNA and total HBV mutation frequency. In addition, Hsp90 shifted A3G's cytosine region selection in HBV DNA and increased A3G's 5' nucleoside preference for deoxycytidine (5'-CC). Furthermore, the Hsp90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin dose dependently inhibited A3G and A3B mutational activity on HBV viral DNA. Hsp90 knockdown by siRNA or by Hsp90 active-site mutation also decreased A3G activity. These results indicate that heat shock proteins, in particular Hsp90, stimulate APOBEC-3-mediated DNA deamination activity, suggesting a potential physiological role in carcinogenesis and viral innate immunity.
Assuntos
Desaminase APOBEC-3G/metabolismo , Citidina Desaminase/metabolismo , DNA Viral/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Vírus da Hepatite B/metabolismo , Hepatócitos/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Desaminase APOBEC-3G/química , Desaminase APOBEC-3G/genética , Carcinogênese , Citidina/metabolismo , Citidina Desaminase/química , Citidina Desaminase/genética , Análise Mutacional de DNA , DNA Recombinante/química , DNA Recombinante/metabolismo , DNA Viral/química , Desaminação , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/genética , Células Hep G2 , Vírus da Hepatite B/genética , Vírus da Hepatite B/imunologia , Hepatócitos/imunologia , Hepatócitos/virologia , Humanos , Imunidade Inata , Antígenos de Histocompatibilidade Menor/química , Antígenos de Histocompatibilidade Menor/genética , Mutagênese , Taxa de Mutação , Fragmentos de Peptídeos/agonistas , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Mutação Puntual , Interferência de RNA , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
The type 1 ryanodine receptor (RyR1) mediates Ca2+ release from the sarcoplasmic reticulum to initiate skeletal muscle contraction and is associated with muscle diseases, malignant hyperthermia, and central core disease. To better understand RyR1 channel function, we investigated the molecular mechanisms of channel gating and ion permeation. An adequate model of channel gating requires accurate, high-resolution models of both open and closed states of the channel. To this end, we generated an open-channel RyR1 model using molecular simulations to pull Ca2+ through the pore constriction site of a closed-channel RyR1 structure determined at 3.8-Å resolution. Importantly, we find that our open-channel model is consistent with the RyR1 and cardiac RyR (RyR2) open-channel structures reported while this paper was in preparation. Both our model and the published structures show similar rotation of the upper portion of the pore-lining S6 helix away from the 4-fold channel axis and twisting of Ile-4937 at the channel constriction site out of the channel pore. These motions result in a minimum open-channel pore radius of â¼3 Å formed by Gln-4933, rather than Ile-4937 in the closed-channel structure. We also present functional support for our model by mutations around the closed- and open-channel constriction sites (Gln-4933 and Ile-4937). Our results indicate that use of ion-pulling simulations produces a RyR1 open-channel model, which can provide insights into the mechanisms of channel opening complementing those from the structural data.
Assuntos
Sinalização do Cálcio , Bicamadas Lipídicas/química , Modelos Moleculares , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Substituição de Aminoácidos , Animais , Cafeína/química , Cafeína/metabolismo , Cafeína/farmacologia , Agonistas dos Canais de Cálcio/química , Agonistas dos Canais de Cálcio/metabolismo , Agonistas dos Canais de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Glutamina/química , Células HEK293 , Humanos , Isoleucina/química , Ligantes , Simulação de Dinâmica Molecular , Mutação , Fragmentos de Peptídeos/agonistas , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Rianodina/química , Rianodina/metabolismo , Rianodina/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismoRESUMO
The inositol 1,4,5 trisphosphate receptor (IP3R) is an intracellular Ca2+ release channel expressed predominately on the membranes of the endoplasmic reticulum. IP3R1 can be cleaved by caspase or calpain into at least two receptor fragments. However, the functional consequences of receptor fragmentation are poorly understood. Our previous work has demonstrated that IP3R1 channels, formed following either enzymatic fragmentation or expression of the corresponding complementary polypeptide chains, retain tetrameric architecture and are still activated by IP3 binding despite the loss of peptide continuity. In this study, we demonstrate that region-specific receptor fragmentation modifies channel regulation. Specifically, the agonist-evoked temporal Ca2+ release profile and protein kinase A modulation of Ca2+ release are markedly altered. Moreover, we also demonstrate that activation of fragmented IP3R1 can result in a distinct functional outcome. Our work suggests that proteolysis of IP3R1 may represent a novel form of modulation of IP3R1 channel function and increases the repertoire of Ca2+ signals achievable through this channel.
Assuntos
Sinalização do Cálcio , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Processamento de Proteína Pós-Traducional , Substituição de Aminoácidos , Animais , Linhagem Celular , Galinhas , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Técnicas de Inativação de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Receptores de Inositol 1,4,5-Trifosfato/agonistas , Receptores de Inositol 1,4,5-Trifosfato/química , Receptores de Inositol 1,4,5-Trifosfato/genética , Cinética , Mutação , Técnicas de Patch-Clamp , Fragmentos de Peptídeos/agonistas , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosforilação , Proteólise , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Regulação para CimaRESUMO
STUDY QUESTION: Do angiotensin (Ang)-(1-7) levels in human ovarian follicular fluid (FF) correlate with the number and proportion of mature oocytes obtained for IVF? SUMMARY ANSWER: The present study shows for the first time that Ang-(1-7) levels in human FF correlate with the proportion of mature oocytes collected upon ovarian stimulation for IVF. WHAT IS KNOWN ALREADY: Ang-(1-7) is an active peptide of the renin-angiotensin system that stimulates oocyte maturation in isolated rabbit and rat ovaries. However, its role in human ovulation remains unexplored. STUDY DESIGN, SIZE, DURATION: This was a prospective cohort study including 64 participants from a single IVF center. Sample size was calculated to achieve a statistical power of 80% in detecting 20% differences in the proportion of mature oocytes between groups. The participants were enrolled in the study during six consecutive months. PARTICIPANTS/MATERIALS, SETTING, METHODS: Plasma samples were obtained from all subjects at Day 21 of the last menstrual cycle before starting pituitary blockade and controlled ovarian stimulation (COS). Plasma and FF samples were quickly mixed with a protease inhibitor cocktail and stored at -80°C. Ang-(1-7) was quantified in plasma and FF samples by a highly sensitive and specific radioimmunoassay, which was preceded by solid phase extraction, speed vacuum concentration and sample reconstitution in assay buffer. FF Ang-(1-7) levels were stratified into tertiles and the patients of each tertile were compared for COS/IVF outcomes using Kruskal-Wallis ANOVA. Multiple regression analysis was used to adjust correlations for potential confounders. The mRNA encoding for Mas, a receptor for Ang-(1-7), was investigated by real-time PCR in luteinized granulosa cells purified from the FF. MAIN RESULTS AND THE ROLE OF CHANCE: There was a four-fold increase in plasma Ang-(1-7) after ovulation induction (median 160.9 vs 41.4 pg/ml, P < 0.0001). FF Ang-(1-7) levels were similar to (169.9 pg/ml) but did not correlate with plasma Ang-(1-7) levels (r = -0.05, P = 0.665). Patients at the highest FF Ang-(1-7) tertile had a higher proportion of mature oocytes compared to patients at the lower FF Ang-(1-7) tertile (median 100% vs 70%, P < 0.01). There was a linear correlation between FF Ang-(1-7) and the proportion of mature oocytes (r = 0.380, P < 0.01), which remained significant after adjustment for age and duration of infertility (r = 0.447, P < 0.001). The luteinized granulosa cells expressed Mas receptor mRNA, which was positively correlated to the number of mature oocytes in women with more than three mature oocytes retrieved (r = 0.42, P < 0.01). LIMITATIONS, REASONS FOR CAUTION: This is an observational study, therefore, no causal relationship can be established between Ang-(1-7) and human oocyte maturation. Mas protein expression was not quantified due to limited availability of granulosa cells. WIDER IMPLICATIONS OF THE FINDINGS: Since this peptide promotes oocyte maturation in other species, it deserves further investigation as a potential maturation factor to human oocytes. STUDY FUNDING AND COMPETING INTEREST(S): Research supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG). The authors have nothing to disclose.
Assuntos
Angiotensina I/agonistas , Fármacos para a Fertilidade Feminina/uso terapêutico , Líquido Folicular/efeitos dos fármacos , Infertilidade Feminina/terapia , Oogênese/efeitos dos fármacos , Indução da Ovulação , Fragmentos de Peptídeos/agonistas , Regulação para Cima/efeitos dos fármacos , Adulto , Angiotensina I/sangue , Angiotensina I/metabolismo , Blastocisto/citologia , Blastocisto/patologia , Estudos de Coortes , Características da Família , Feminino , Fertilização in vitro , Líquido Folicular/metabolismo , Humanos , Infertilidade Feminina/sangue , Infertilidade Feminina/metabolismo , Infertilidade Feminina/patologia , Infertilidade Masculina , Masculino , Recuperação de Oócitos , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/metabolismo , Estudos Prospectivos , Radioimunoensaio , Extração em Fase SólidaRESUMO
The glucagon receptor (GCGR) belongs to the secretin-like (class B) family of G protein-coupled receptors (GPCRs) and is activated by the peptide hormone glucagon. The structures of an activated class B GPCR have remained unsolved, preventing a mechanistic understanding of how these receptors are activated. Using a combination of structural modeling and mutagenesis studies, we present here two modes of ligand-independent activation of GCGR. First, we identified a GCGR-specific hydrophobic lock comprising Met-338 and Phe-345 within the IC3 loop and transmembrane helix 6 (TM6) and found that this lock stabilizes the TM6 helix in the inactive conformation. Disruption of this hydrophobic lock led to constitutive G protein and arrestin signaling. Second, we discovered a polar core comprising conserved residues in TM2, TM3, TM6, and TM7, and mutations that disrupt this polar core led to constitutive GCGR activity. On the basis of these results, we propose a mechanistic model of GCGR activation in which TM6 is held in an inactive conformation by the conserved polar core and the hydrophobic lock. Mutations that disrupt these inhibitory elements allow TM6 to swing outward to adopt an active TM6 conformation similar to that of the canonical ß2-adrenergic receptor complexed with G protein and to that of rhodopsin complexed with arrestin. Importantly, mutations in the corresponding polar core of several other members of class B GPCRs, including PTH1R, PAC1R, VIP1R, and CRFR1, also induce constitutive G protein signaling, suggesting that the rearrangement of the polar core is a conserved mechanism for class B GPCR activation.
Assuntos
Modelos Moleculares , Receptor Tipo 1 de Hormônio Paratireóideo/agonistas , Receptores de Hormônio Liberador da Corticotropina/agonistas , Receptores de Glucagon/agonistas , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/agonistas , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/agonistas , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Linhagem Celular , Sequência Conservada , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Mutagênese Sítio-Dirigida , Mutação , Fragmentos de Peptídeos/agonistas , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Receptor Tipo 1 de Hormônio Paratireóideo/química , Receptor Tipo 1 de Hormônio Paratireóideo/genética , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Receptores de Hormônio Liberador da Corticotropina/química , Receptores de Hormônio Liberador da Corticotropina/genética , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Receptores de Glucagon/química , Receptores de Glucagon/genética , Receptores de Glucagon/metabolismo , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/química , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/química , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/genética , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sistemas do Segundo Mensageiro , Homologia Estrutural de ProteínaRESUMO
Food contamination by the trichothecene mycotoxin deoxynivalenol (DON, vomitoxin) has the potential to adversely affect animal and human health by suppressing food intake and impairing growth. In mice, the DON-induced anorectic response results from aberrant satiety hormone secretion by enteroendocrine cells (EECs) of the gastrointestinal tract. Recent in vitro studies in the murine STC-1 EEC model have linked DON-induced satiety hormone secretion to activation of calcium-sensing receptor (CaSR), a G-coupled protein receptor, and transient receptor potential ankyrin-1 (TRPA1), a TRP channel. However, it is unknown whether similar mechanisms mediate DON's anorectic effects in vivo. Here, we tested the hypothesis that DON-induced food refusal and satiety hormone release in the mouse are linked to activation of CaSR and TRPA1. Oral treatment with selective agonists for CaSR (R-568) or TRPA1 (allyl isothiocyanate (AITC)) suppressed food intake in mice, and the agonist's effects were suppressed by pretreatment with corresponding antagonists NPS-2143 or ruthenium red (RR), respectively. Importantly, NPS-2143 or RR inhibited both DON-induced food refusal and plasma elevations of the satiety hormones cholecystokinin (CCK) and peptide YY3-36 (PYY3-36); cotreatment with both antagonists additively suppressed both anorectic and hormone responses to DON. Taken together, these in vivo data along with prior in vitro findings support the contention that activation of CaSR and TRPA1 contributes to DON-induced food refusal by mediating satiety hormone exocytosis from EEC.
Assuntos
Anorexia/induzido quimicamente , Depressores do Apetite/toxicidade , Poluentes Ambientais/toxicidade , Modelos Biológicos , Receptores Acoplados a Proteínas G/agonistas , Canais de Potencial de Receptor Transitório/agonistas , Tricotecenos/toxicidade , Animais , Anorexia/metabolismo , Anorexia/prevenção & controle , Depressores do Apetite/química , Estimulantes do Apetite/uso terapêutico , Comportamento Animal/efeitos dos fármacos , Colecistocinina/agonistas , Colecistocinina/antagonistas & inibidores , Colecistocinina/sangue , Quimioterapia Combinada , Ingestão de Energia/efeitos dos fármacos , Poluentes Ambientais/antagonistas & inibidores , Feminino , Fragmentos de Peptídeos/agonistas , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/sangue , Peptídeo YY/agonistas , Peptídeo YY/antagonistas & inibidores , Peptídeo YY/sangue , Distribuição Aleatória , Receptores de Detecção de Cálcio , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/metabolismo , Resposta de Saciedade/efeitos dos fármacos , Canal de Cátion TRPA1 , Canais de Potencial de Receptor Transitório/antagonistas & inibidores , Canais de Potencial de Receptor Transitório/metabolismo , Tricotecenos/antagonistas & inibidoresRESUMO
γ-secretase mediates the intramembranous proteolysis of amyloid precursor protein (APP) and determines the generation of Aß which is associated with Alzheimer's disease (AD). Here we identified that an anti-Parkinson's disease drug, Istradefylline, could enhance Aß generation in various cell lines and primary neuronal cells of APP/PS1 mouse. Moreover, the increased generation of Aß42 was detected in the cortex of APP/PS1 mouse after chronic treatment with Istradefylline. Istradefylline promoted the activity of γ-secretase which could lead to increased Aß production. These effects of Istradefylline were reduced by the knockdown of A2AR but independent of A2AR-mediated G protein- or ß-arrestin-dependent signal pathway. We further observed that A2AR colocalized with γ-secretase in endosomes and physically interacted with the catalytic subunit presenilin-1 (PS1). Interestingly, Istradefylline attenuated the interaction in time- and dosage-dependent manners. Moreover the knockdown of A2AR which in theory would release PS1 potentiated both Aß generation and γ-secretase activity. Thus, our study implies that the association of A2AR could modulate γ-secretase activity. Istradefylline enhance Aß generation and γ-secretase activity possibly via modulating the interaction between A2AR and γ-secretase, which may bring some undesired effects in the central nervous system (CNS).
Assuntos
Antagonistas do Receptor A2 de Adenosina/efeitos adversos , Secretases da Proteína Precursora do Amiloide/genética , Precursor de Proteína beta-Amiloide/genética , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/efeitos adversos , Purinas/efeitos adversos , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/agonistas , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Linhagem Celular , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Neurônios/citologia , Neurônios/metabolismo , Fragmentos de Peptídeos/agonistas , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Presenilina-1/genética , Presenilina-1/metabolismo , Cultura Primária de Células , Receptor A2A de Adenosina/deficiência , Receptor A2A de Adenosina/genética , Transdução de Sinais , beta-Arrestinas/genética , beta-Arrestinas/metabolismoRESUMO
Proteolytic cleavage of the amyloid-ß protein precursor (AßPP) by secretases leads to extracellular release of amyloid-ß (Aß) peptides. Increased production of Aß42 over Aß40 and aggregation into oligomers and plaques constitute an Alzheimer's disease (AD) hallmark. Identifying products of the 'human chemical exposome' (HCE) able to induce Aß42 production may be a key to understanding some of the initiating causes of AD and to generate non-genetic, chemically-induced AD animal models. A cell model was used to screen HCE libraries for Aß42 inducers. Out of 3500+ compounds, six triazine herbicides were found that induced a ß- and γ-secretases-dependent, 2-10 fold increase in the production of extracellular Aß42 in various cell lines, primary neuronal cells, and neurons differentiated from human-induced pluripotent stem cells (iPSCs). Immunoprecipitation/mass spectrometry analyses show enhanced production of Aß peptides cleaved at positions 42/43, and reduced production of peptides cleaved at positions 38 and lower, a characteristic of AD. Neurons derived from iPSCs obtained from a familial AD (FAD) patient (AßPP K724N) produced more Aß42 versus Aß40 than neurons derived from healthy controls iPSCs (AßPP WT). Triazines enhanced Aß42 production in both control and AD iPSCs-derived neurons. Triazines also shifted the cleavage pattern of alcadeinα, another γ-secretase substrate, suggesting a direct effect of triazines on γ-secretase activity. In conclusion, several widely used triazines enhance the production of toxic, aggregation prone Aß42/Aß43 amyloids, suggesting the possible existence of environmental "Alzheimerogens" which may contribute to the initiation and propagation of the amyloidogenic process in late-onset AD.
Assuntos
Peptídeos beta-Amiloides/biossíntese , Herbicidas/química , Herbicidas/farmacologia , Fragmentos de Peptídeos/biossíntese , Triazinas/química , Triazinas/farmacologia , Adulto , Peptídeos beta-Amiloides/agonistas , Animais , Células CHO , Linhagem Celular Tumoral , Células Cultivadas , Cricetinae , Cricetulus , Feminino , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Fragmentos de Peptídeos/agonistas , RatosRESUMO
Polyphosphate (polyP), a several billion-year-old biopolymer, is produced in every cell, tissue, and organism studied. Structurally extremely simple, polyP consists of long chains of covalently linked inorganic phosphate groups. We report here the surprising discovery that polyP shows a remarkable efficacy in accelerating amyloid fibril formation. We found that polyP serves as an effective nucleation source for various different amyloid proteins, ranging from bacterial CsgA to human α-synuclein, Aß1-40/42, and Tau. polyP-associated α-synuclein fibrils show distinct differences in seeding behavior, morphology, and fibril stability compared with fibrils formed in the absence of polyP. In vivo, the amyloid-stimulating and fibril-stabilizing effects of polyP have wide-reaching consequences, increasing the rate of biofilm formation in pathogenic bacteria and mitigating amyloid toxicity in differentiated neuroblastoma cells and C. elegans strains that serve as models for human folding diseases. These results suggest that we have discovered a conserved cytoprotective modifier of amyloidogenic processes.