RESUMO
The mammalian high mobility group protein AT-hook 2 (HMGA2) is a multi-functional DNA-binding protein that plays important roles in tumorigenesis and adipogenesis. Previous results showed that HMGA2 is a potential therapeutic target of anticancer and anti-obesity drugs by inhibiting its DNA-binding activities. Here we report the development of a miniaturized, automated AlphaScreen ultra-high-throughput screening assay to identify inhibitors targeting HMGA2-DNA interactions. After screening the LOPAC1280 compound library, we identified several compounds that strongly inhibit HMGA2-DNA interactions including suramin, a century-old, negatively charged antiparasitic drug. Our results show that the inhibition is likely through suramin binding to the "AT-hook" DNA-binding motifs and therefore preventing HMGA2 from binding to the minor groove of AT-rich DNA sequences. Since HMGA1 proteins also carry multiple "AT-hook" DNA-binding motifs, suramin is expected to inhibit HMGA1-DNA interactions as well. Biochemical and biophysical studies show that charge-charge interactions and hydrogen bonding between the suramin sulfonated groups and Arg/Lys residues play critical roles in the binding of suramin to the "AT-hook" DNA-binding motifs. Furthermore, our results suggest that HMGA2 may be one of suramin's cellular targets.
Assuntos
Proteínas de Ligação a DNA/antagonistas & inibidores , Proteína HMGA1a/antagonistas & inibidores , Proteína HMGA2/antagonistas & inibidores , Suramina/química , Adipogenia/efeitos dos fármacos , Motivos de Aminoácidos/efeitos dos fármacos , Sequência de Bases/efeitos dos fármacos , Sítios de Ligação/efeitos dos fármacos , Carcinogênese/efeitos dos fármacos , DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteína HMGA1a/química , Proteína HMGA1a/genética , Proteína HMGA2/química , Proteína HMGA2/genética , Ensaios de Triagem em Larga Escala , Humanos , Suramina/isolamento & purificação , Suramina/farmacologiaRESUMO
Mevalonate pathway inhibitors have been extensively studied for their roles in cholesterol depletion and for inhibiting the prenylation and activation of various proteins. Inhibition of protein prenylation has potential therapeutic uses against neurological disorders, like neural cancers, neurodegeneration, and neurotramatic lesions. Protection against neurodegeneration and promotion of neuronal regeneration is regulated in large part by Ras superfamily small guanosine triphosphatases (GTPases), particularly the Ras, Rho, and Rab subfamilies. These proteins are prenylated to target them to cellular membranes. Prenylation can be specifically inhibited through altering the function of enzymes of the mevalonate pathway necessary for isoprenoid production and attachment to target proteins to elicit a variety of effects on neural cells. However, this approach does not address how prenylation affects a specific protein. This review focuses on the regulation of small GTPase prenylation, the different techniques to inhibit prenylation, and how this inhibition has affected neural cell processes.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Prenilação de Proteína/fisiologia , Acil Coenzima A/metabolismo , Motivos de Aminoácidos/efeitos dos fármacos , Animais , Vias Biossintéticas/efeitos dos fármacos , Membrana Celular/metabolismo , Dimetilaliltranstransferase/metabolismo , Ativação Enzimática , Humanos , Metilação , Ácido Mevalônico/metabolismo , Ligação Proteica , Terpenos/metabolismoRESUMO
A Disintegrin and Metalloproteinase (ADAM) is a family of proteolytic enzymes that possess sheddase function and regulate shedding of membrane-bound proteins, growth factors, cytokines, ligands and receptors. Typically, ADAMs have a pro-domain, and a metalloproteinase, disintegrin, cysteine-rich and a characteristic transmembrane domain. Most ADAMs are activated by proprotein convertases, but can also be regulated by G-protein coupled receptor agonists, Ca2+ ionophores and protein kinase C activators. A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS) is a family of secreted enzymes closely related to ADAMs. Like ADAMs, ADAMTS members have a pro-domain, and a metalloproteinase, disintegrin, and cysteine-rich domain, but they lack a transmembrane domain and instead have characteristic thrombospondin motifs. Activated ADAMs perform several functions and participate in multiple cardiovascular processes including vascular smooth muscle cell proliferation and migration, angiogenesis, vascular cell apoptosis, cell survival, tissue repair, and wound healing. ADAMs may also be involved in pathological conditions and cardiovascular diseases such as atherosclerosis, hypertension, aneurysm, coronary artery disease, myocardial infarction and heart failure. Like ADAMs, ADAMTS have a wide-spectrum role in vascular biology and cardiovascular pathophysiology. ADAMs and ADAMTS activity is naturally controlled by endogenous inhibitors such as tissue inhibitors of metalloproteinases (TIMPs), and their activity can also be suppressed by synthetic small molecule inhibitors. ADAMs and ADAMTS can serve as important diagnostic biomarkers and potential therapeutic targets for cardiovascular disorders. Natural and synthetic inhibitors of ADAMs and ADAMTS could be potential therapeutic tools for the management of cardiovascular diseases.
Assuntos
Proteínas ADAM/metabolismo , Desintegrinas/metabolismo , Endotélio Vascular/metabolismo , Trombospondinas/metabolismo , Doenças Vasculares/metabolismo , Proteínas ADAM/antagonistas & inibidores , Motivos de Aminoácidos/efeitos dos fármacos , Motivos de Aminoácidos/fisiologia , Animais , Desintegrinas/antagonistas & inibidores , Endotélio Vascular/efeitos dos fármacos , Humanos , Inibidores de Metaloproteinases de Matriz/administração & dosagem , Trombospondinas/antagonistas & inibidores , Doenças Vasculares/tratamento farmacológicoRESUMO
Background: Neuroblastoma (NB) is one of the most aggressive and common solid tumors in pediatrics. Development of effective new therapeutics for NB is in progress to help reduce mortality and morbidity of the disease, particularly in relapsed patients. The tumor suppressor protein p53 plays a critical role in multiple signaling pathways to maintain cellular hemostasis. Dysregulation of p53 protein and/or molecular aberrations have been associated with multiple human malignancies. p53 stability and protein activity is negatively regulated by the E3 ubiquitin ligase (MDM2). Thus, targeting p53-MDM2 protein-protein interaction is a feasible and promising therapeutic strategy to restore the physiological function of p53 in cancer cells. RG7112 is a highly potent and selective small molecule inhibitor, which target a unique structure located within p53 binding motif of MDM2. Methods: The efficacy of RG7112 in vitro using NB cell lines was examined. Two wild-type (WT)-p53 NB cell lines IMR5 and LAN-5, a mutant p53 cell line SK-N-BE(2), and a WT-p53/p14 deleted cell line SH-EP were employed. Results: Data showed that RG7112 significantly reduced cellular viability of IMR5 (IC50, 562 nM) and LAN-5 (IC50, 430 nM), but not SK-N-BE(2) and SH-EP cells. Further, RG7112 restores p53 and p21 protein levels in IMR5 and LAN-5 in a dose-dependent manner. RG7112 induces cell cycle arresting (60% G1 arresting) in WT-p53 cells (IMR5), but no pronounced effect observed in SK-N-BE(2). In this study, 15 different drugs in combination with RG7112 in IMR5 cell line and identified venetoclax (Bcl-2/Bcl-xL inhibitor) as a promising candidate were evaluated. Conclusions: Taken together, these findings provide initial proof-of-concept data for further investigations of RG7112 in selected subgroups of NB patients.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Imidazolinas/farmacologia , Neuroblastoma/tratamento farmacológico , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Motivos de Aminoácidos/efeitos dos fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Humanos , Imidazolinas/uso terapêutico , Concentração Inibidora 50 , Neuroblastoma/genética , Neuroblastoma/patologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Proteína Supressora de Tumor p53/genéticaRESUMO
Bromodomain and extraterminal (BET) domain containing protein (BRD)-4 modulates the expression of oncogenes such as c-myc, and is a promising therapeutic target in diverse cancer types. We performed pre-clinical studies in myeloma models with bi-functional protein-targeting chimeric molecules (PROTACs) which target BRD4 and other BET family members for ubiquitination and proteasomal degradation. PROTACs potently reduced the viability of myeloma cell lines in a time-dependent and concentration-dependent manner associated with G0/G1 arrest, reduced levels of CDKs 4 and 6, increased p21 levels, and induction of apoptosis. These agents specifically decreased cellular levels of downstream BRD4 targets, including c-MYC and N-MYC, and a Cereblon-targeting PROTAC showed downstream effects similar to those of an immunomodulatory agent. Notably, PROTACs overcame bortezomib, dexamethasone, lenalidomide, and pomalidomide resistance, and their activity was maintained in otherwise isogenic myeloma cells with wild-type or deleted TP53. Combination studies showed synergistic interactions with dexamethasone, BH3 mimetics, and Akt pathway inhibitors. BET-specific PROTACs induced a rapid loss of viability of primary cells from myeloma patients, and delayed growth of MM1.S-based xenografts. Our data demonstrate that BET degraders have promising activity against pre-clinical models of multiple myeloma, and support their translation to the clinic for patients with relapsed and/or refractory disease.
Assuntos
Antineoplásicos/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Proteínas/metabolismo , Motivos de Aminoácidos/efeitos dos fármacos , Animais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Proteínas Nucleares/metabolismo , Domínios Proteicos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Ubiquitinação/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
AIMS: The mechanisms underlying numerous biological roles of hydrogen sulfide (H2S) remain largely unknown. We have previously reported an inhibitory role of H2S in the L-type calcium channels in cardiomyocytes. This prompts us to examine the mechanisms underlying the potential regulation of H2S on the ion channels. RESULTS: H2S showed a novel inhibitory effect on Ito potassium channels, and this effect was blocked by mutation at the Cys320 and/or Cys529 residues of the Kv4.2 subunit. H2S broke the disulfide bridge between a pair of oxidized cysteine residues; however, it did not modify single cysteine residues. H2S extended action potential duration in epicardial myocytes and regularized fatal arrhythmia in a rat model of myocardial infarction. H2S treatment significantly increased survival by â¼1.4-fold in the critical 2-h time window after myocardial infarction with a protection against ventricular premature beats and fatal arrhythmia. However, H2S did not change the function of other ion channels, including IK1 and INa. INNOVATION AND CONCLUSION: H2S targets the Cys320/Cys529 motif in Kv4.2 to regulate the Ito potassium channels. H2S also shows a potent regularizing effect against fatal arrhythmia in a rat model of myocardial infarction. The study provides the first piece of evidence for the role of H2S in regulating Ito potassium channels and also the specific motif in an ion channel labile for H2S regulation.
Assuntos
Motivos de Aminoácidos/efeitos dos fármacos , Arritmias Cardíacas/metabolismo , Cisteína/metabolismo , Sulfeto de Hidrogênio/farmacologia , Infarto do Miocárdio/metabolismo , Canais de Potássio Shal/metabolismo , Animais , Arritmias Cardíacas/tratamento farmacológico , Dissulfetos/metabolismo , Células HEK293 , Humanos , Sulfeto de Hidrogênio/uso terapêutico , Masculino , Mutação , Infarto do Miocárdio/tratamento farmacológico , Miócitos Cardíacos/metabolismo , Ratos , Canais de Potássio Shal/antagonistas & inibidores , Canais de Potássio Shal/genéticaRESUMO
In the current study we aimed to show the common YMDD motif mutations in viral polymerase gene in chronic hepatitis B patients during lamivudine and adefovir therapy. Forty-one serum samples obtained from chronic hepatitis B patients (24 male, 17 female; age range: 34-68 years) were included in the study. HBV-DNA was extracted from the peripheral blood of the patients using an extraction kit (Invisorb, Instant Spin DNA/ RNA Virus Mini Kit, Germany). A line probe assay and direct sequencing analyses (INNO-LIPA HBV DR v2; INNOGENETICS N.V, Ghent, Belgium) were applied to determine target mutations of the viral polymerase gene in positive HBV-DNA samples. A total of 41 mutations located in 21 different codons were detected in the current results. In 17 (41.5%) patients various point mutations were detected leading to lamivudin, adefovir and/ or combined drug resistance. Wild polymerase gene profiles were detected in 24 (58.5%) HBV positive patients of the current cohort. Eight of the 17 samples (19.5%) having rtM204V/I/A missense transition and/or transversion point mutations and resistance to lamivudin. Six of the the mutated samples (14.6%) having rtL180M missense transversion mutation and resistance to combined adefovir and lamivudin. Three of the mutated samples (7.5%) having rtG215H by the double base substituation and resistance to adefovir. Three of the mutated samples (7.5%) having codon rtL181W due to the missense transversion point mutations and showed resistance to combined adefovir and lamivudin. Unreported novel point mutations were detected in the different codons of polymerase gene region in the current HBV positive cohort fromTurkish population. The current results provide evidence that rtL180M and rtM204V/I/A mutations of HBV-DNA may be associated with a poor antiviral response and HBV chronicity during conventional therapy in Turkish patients.
Assuntos
Motivos de Aminoácidos/genética , Antivirais/uso terapêutico , DNA Polimerase Dirigida por DNA/genética , Farmacorresistência Viral/genética , Vírus da Hepatite B/genética , Hepatite B Crônica/genética , Mutação/genética , Adenina/análogos & derivados , Adenina/uso terapêutico , Adulto , Idoso , Motivos de Aminoácidos/efeitos dos fármacos , Fármacos Anti-HIV/uso terapêutico , Ácido Aspártico/genética , Estudos de Casos e Controles , DNA Viral/genética , Feminino , Seguimentos , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/enzimologia , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/epidemiologia , Hepatite B Crônica/virologia , Humanos , Lamivudina/uso terapêutico , Masculino , Metionina/genética , Pessoa de Meia-Idade , Organofosfonatos/uso terapêutico , Prognóstico , Estudos Retrospectivos , Turquia/epidemiologia , Tirosina/genéticaRESUMO
Recent epidemiological data have shown that patients suffering from Type 2 Diabetes Mellitus have an increased risk to develop Alzheimer's disease and vice versa. A possible explanation is the cross-sequence interaction between Aß and amylin. Because the resulting amyloid oligomers are difficult to probe in experiments, we investigate stability and conformational changes of Aß-amylin heteroassemblies through molecular dynamics simulations. We find that Aß is a good template for the growth of amylin and vice versa. We see water molecules permeate the ß-strand-turn-ß-strand motif pore of the oligomers, supporting a commonly accepted mechanism for toxicity of ß-rich amyloid oligomers. Aiming for a better understanding of the physical mechanisms of cross-seeding and cell toxicity of amylin and Aß aggregates, our simulations also allow us to identify targets for the rational design of inhibitors against toxic fibril-like oligomers of Aß and amylin oligomers.
Assuntos
Peptídeos beta-Amiloides/efeitos adversos , Diabetes Mellitus Tipo 2/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/efeitos adversos , Emaranhados Neurofibrilares/química , Motivos de Aminoácidos/efeitos dos fármacos , Peptídeos beta-Amiloides/biossíntese , Peptídeos beta-Amiloides/química , Permeabilidade da Membrana Celular/efeitos dos fármacos , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/patologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Polipeptídeo Amiloide das Ilhotas Pancreáticas/biossíntese , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Modelos Químicos , Simulação de Dinâmica Molecular , Emaranhados Neurofibrilares/efeitos dos fármacos , Emaranhados Neurofibrilares/patologia , Estabilidade Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos dos fármacos , Proteínas tau/química , Proteínas tau/metabolismoRESUMO
Selective control of receptor trafficking provides a mechanism for remodeling the receptor composition of excitatory synapses, and thus supports synaptic transmission, plasticity, and development. GluN3A (formerly NR3A) is a nonconventional member of the NMDA receptor (NMDAR) subunit family, which endows NMDAR channels with low calcium permeability and reduced magnesium sensitivity compared with NMDARs comprising only GluN1 and GluN2 subunits. Because of these special properties, GluN3A subunits act as a molecular brake to limit the plasticity and maturation of excitatory synapses, pointing toward GluN3A removal as a critical step in the development of neuronal circuitry. However, the molecular signals mediating GluN3A endocytic removal remain unclear. Here we define a novel endocytic motif (YWL), which is located within the cytoplasmic C-terminal tail of GluN3A and mediates its binding to the clathrin adaptor AP2. Alanine mutations within the GluN3A endocytic motif inhibited clathrin-dependent internalization and led to accumulation of GluN3A-containing NMDARs at the cell surface, whereas mimicking phosphorylation of the tyrosine residue promoted internalization and reduced cell-surface expression as shown by immunocytochemical and electrophysiological approaches in recombinant systems and rat neurons in primary culture. We further demonstrate that the tyrosine residue is phosphorylated by Src family kinases, and that Src-activation limits surface GluN3A expression in neurons. Together, our results identify a new molecular signal for GluN3A internalization that couples the functional surface expression of GluN3A-containing receptors to the phosphorylation state of GluN3A subunits, and provides a molecular framework for the regulation of NMDAR subunit composition with implications for synaptic plasticity and neurodevelopment.
Assuntos
Endocitose/fisiologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Neurônios/metabolismo , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismo , Tirosina/metabolismo , Trifosfato de Adenosina/farmacocinética , Motivos de Aminoácidos/efeitos dos fármacos , Motivos de Aminoácidos/genética , Análise de Variância , Animais , Biofísica , Biotinilação , Células Cultivadas , Córtex Cerebral/citologia , Chlorocebus aethiops , Clatrina/farmacologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Estimulação Elétrica , Embrião de Mamíferos , Endocitose/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Ácido Glutâmico/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/citologia , Humanos , Imunoprecipitação , Mutagênese/fisiologia , Mutação/fisiologia , Neurônios/efeitos dos fármacos , Neurotransmissores/farmacologia , Técnicas de Patch-Clamp , Isótopos de Fósforo/farmacocinética , Fosforilação/efeitos dos fármacos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Conformação Proteica , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Receptores de N-Metil-D-Aspartato/genética , Transfecção , Transferrina/metabolismoRESUMO
OBJECTIVE: This study aimed to determine the natural prevalence of variants of tyrosine-methionine-aspartic acid-aspartic acid (YMDD) motif in patients with chronic hepatitis B (CHB), and to explore its relation with demographic and clinical features, hepatitis B virus (HBV) genotypes, and HBV DNA levels. METHODS: A total of 1,042 antiviral treatment naïve CHB patients (including with lamivudine [LAM]) in the past year were recruited from outpatient and inpatient departments of six centers from December 2008 to June 2010. YMDD variants were analyzed using the HBV drug resistance line probe assay (Inno-Lipa HBV-DR). HBV genotypes were detected with polymerase chain reaction (PCR) microcosmic nucleic acid cross-ELISA, and HBV deoxyribonucleic acid (DNA) was quantitated with real-time PCR. All serum samples underwent tests for HBV, HCV, and HDV with ELISA. RESULTS: YMDD variants were detected in 23.3% (243/1042) of CHB patients. YMDD mutation was accompanied by L180M mutation in 154 (76.9%) patients. Both wild-type HBV and YMDD variant HBV were present in 231 of 243 patients. Interestingly, 12 patients had only YIDD and/or YVDD variants without wild YMDD motif. In addition, 27.2% (98/359) of HbeAg-positive patients had YMDD mutations, which was higher than that in HbeAg-negative patients (21.2%, 145/683). The incidence of YMDD varied among patients with different HBV genotypes, but the difference was not significant. Moreover, the incidence of YMDD in patients with high HBV DNA level was significantly higher than that in those with low HBV DNA level. CONCLUSION: Mutation of YMDD motif was detectable at a high rate in CHB patients in this study. The incidence of YMDD may be correlated with HBeAg and HBV DNA level.
Assuntos
Adulto , Feminino , Humanos , Masculino , Antivirais/uso terapêutico , Ácido Aspártico/genética , Vírus da Hepatite B/genética , Hepatite B Crônica/tratamento farmacológico , Metionina/genética , Mutação/genética , Tirosina/genética , Motivos de Aminoácidos/efeitos dos fármacos , Motivos de Aminoácidos/genética , DNA Viral/análise , Genótipo , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B Crônica/virologia , Reação em Cadeia da PolimeraseRESUMO
In the paramyxovirus cell entry process, receptor binding triggers conformational changes in the fusion protein (F) leading to viral and cellular membrane fusion. Peptides derived from C-terminal heptad repeat (HRC) regions in F have been shown to inhibit fusion by preventing formation of the fusogenic six-helix bundle. We recently showed that the addition of a cholesterol group to HRC peptides active against Nipah virus targets these peptides to the membrane where fusion occurs, dramatically increasing their antiviral effect. In this work, we report that unlike the untagged HRC peptides, which bind to the postulated extended intermediate state bridging the viral and cell membranes, the cholesterol tagged HRC-derived peptides interact with F before the fusion peptide inserts into the target cell membrane, thus capturing an earlier stage in the F-activation process. Furthermore, we show that cholesterol tagging renders these peptides active in vivo: the cholesterol-tagged peptides cross the blood brain barrier, and effectively prevent and treat in an established animal model what would otherwise be fatal Nipah virus encephalitis. The in vivo efficacy of cholesterol-tagged peptides, and in particular their ability to penetrate the CNS, suggests that they are promising candidates for the prevention or therapy of infection by Nipah and other lethal paramyxoviruses.
Assuntos
Colesterol/uso terapêutico , Infecções por Henipavirus/prevenção & controle , Vírus Nipah/fisiologia , Paramyxovirinae/fisiologia , Proteínas Virais de Fusão/antagonistas & inibidores , Internalização do Vírus , Motivos de Aminoácidos/efeitos dos fármacos , Motivos de Aminoácidos/fisiologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Chlorocebus aethiops , Colesterol/química , Colesterol/farmacologia , Regulação para Baixo , Infecções por Henipavirus/imunologia , Infecções por Henipavirus/terapia , Humanos , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Terapia de Alvo Molecular , Vírus Nipah/efeitos dos fármacos , Vírus Nipah/imunologia , Vírus Nipah/patogenicidade , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/uso terapêutico , Células Vero , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/metabolismo , Proteínas Virais de Fusão/fisiologiaRESUMO
Thioredoxin reductase (TrxR) is a selenocysteine-containing flavoprotein that catalyzes the NADPH-dependent reduction of oxidized thioredoxin and plays a key role in regulating cellular redox homeostasis. In the present studies, we examined the effects of 2-chloroethyl ethyl sulfide (CEES), a model sulfur mustard vesicant, on TrxR in lung epithelial cells. We speculated that vesicant-induced alterations in TrxR contribute to oxidative stress and toxicity. The treatment of human lung A549 epithelial cells with CEES resulted in a time- and concentration-dependent inhibition of TrxR. Using purified rat liver TrxR, we demonstrated that only the reduced enzyme was inhibited and that this inhibition was irreversible. The reaction of TrxR with iodoacetamide, which selectively modifies free thiol or selenol on proteins, was also markedly reduced by CEES, suggesting that CEES induces covalent modification of the reduced selenocysteine-containing active site in the enzyme. This was supported by our findings that recombinant mutant TrxR, in which selenocysteine was replaced by cysteine, was markedly less sensitive to inhibition by CEES and that the vesicant preferentially alkylated selenocysteine in the C-terminal redox motif of TrxR. TrxR also catalyzes quinone redox cycling, a process that generates reactive oxygen species. In contrast to its inhibitory effects on TrxR activity, CEES was found to stimulate redox cycling. Taken together, these data suggest that sulfur mustard vesicants target TrxR and that this may be an important mechanism mediating oxidative stress and tissue injury.
Assuntos
Células Epiteliais/efeitos dos fármacos , Pulmão/citologia , Gás de Mostarda/análogos & derivados , Selenocisteína/metabolismo , Tiorredoxina Dissulfeto Redutase/metabolismo , Motivos de Aminoácidos/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Células Epiteliais/enzimologia , Humanos , Gás de Mostarda/efeitos adversos , Gás de Mostarda/farmacologia , Proteínas Mutantes/antagonistas & inibidores , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Oxirredução/efeitos dos fármacos , Ratos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tiorredoxina Dissulfeto Redutase/antagonistas & inibidores , Tiorredoxina Dissulfeto Redutase/genéticaRESUMO
There is experimental evidence that the transmembrane fragment spanning amino acids 70-86 of glycophorin A, GpA70-86, forms amyloid fibrils and the inhibitor RGTFEGKF prevents GpA70-86 fibril formation at an equimolar ratio. Both the GpA70-86 and inhibitor peptides contain a GxxxG motif as found in many amyloid proteins such as the Alzheimer's amyloid beta-peptide and prion protein. To explore the intrinsic, early interaction and inhibition mechanism, we have determined the structures of GpA70-86 in the absence and presence of the inhibitor by means of extensive molecular dynamics simulations in explicit solvent. Consistent with experiments on the fibrils, our simulations show that the two GxxxG motifs interact significantly at the monomer level. They go, however, one step beyond by indicating that the inhibitor has a significant impact on the global structure of GpA70-86, but a limited influence on the conformations of the GxxxG motif. Implications of our simulations on amyloid propagation of proteins containing GxxxG motifs are discussed.
Assuntos
Glicoforinas/antagonistas & inibidores , Glicoforinas/química , Peptídeos/farmacologia , Conformação Proteica/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Motivos de Aminoácidos/efeitos dos fármacos , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Peptídeos/químicaRESUMO
Viruses need to deliver their genomic information into the host cell lumen to establish productive infection. Enveloped viruses accomplish this task by fusing their membrane with a host cell membrane. Membrane fusion is facilitated by specialized viral membrane proteins, which mediate binding and entry into host cells. The architecture of the fusion machinery of envelope proteins can differ between viruses, and class I, II and III fusion systems have been described. However, the conformational rearrangements associated with membrane fusion are comparable and constitute attractive targets for intervention. The fusion apparatus of the human immunodeficiency virus (HIV) envelope protein (Env), a class I fusion protein, is located in the transmembrane unit gP41 of Env. The fusion machinery is activated by Env binding to CD4 and a chemokine coreceptor, and the structural rearrangements in gp41 associated with membrane fusion comprise the insertion of a fusion peptide into the target cell membrane and the formation of a stable six-helix bundle structure. These processes can be efficiently inhibited by peptides mimicking conserved functional elements in gp41. A prominent example for such peptides, termed fusion inhibitors, is the peptide T-20 (enfuvirtide, Fuzeon) which is used as salvage therapy of HIV/AIDS. Here, we will discuss how HIV mediates fusion with host cell membranes and how this process can be blocked by peptides targeting gp41. In addition, we will discuss peptide inhibitors of other class I viral fusion proteins.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Proteína gp41 do Envelope de HIV/antagonistas & inibidores , Inibidores da Fusão de HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , HIV/efeitos dos fármacos , Peptídeos/uso terapêutico , Motivos de Aminoácidos/efeitos dos fármacos , Sequência de Aminoácidos , Fármacos Anti-HIV/farmacologia , Farmacorresistência Viral/genética , HIV/patogenicidade , Proteína gp41 do Envelope de HIV/biossíntese , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/metabolismo , Inibidores da Fusão de HIV/classificação , Inibidores da Fusão de HIV/farmacologia , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Peptídeos/farmacologiaRESUMO
Expression of the MHC class I chain related molecules A and B (MICA/B) on tumor cell surface can signal the immune receptor NKG2D for tumor immune destruction. However, MIC was found to be shed by tumors in cancer patients, which negatively regulates host immunity and promotes tumor immune evasion and progression. The mechanisms by which tumors shed MIC are not well understood although diverse groups of enzymes are suggested to be involved. The functional complexity of these enzymes makes them unfeasible therapeutic targets for inhibiting MIC shedding. Here we identified an six-amino acid (6-aa) motif in the alpha3 domain of MIC that is critical for the interaction of MIC with ERp5 to enable shedding. Mutations in this motif prevented MIC shedding but did not interfere with NKG2D-mediated recognition of MIC. Our study suggests that the 6-aa motif is a feasible target to inhibit MIC shedding for cancer therapy.
Assuntos
Antineoplásicos/farmacologia , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/efeitos dos fármacos , Neoplasias/metabolismo , Motivos de Aminoácidos/efeitos dos fármacos , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Dados de Sequência Molecular , Mutação , Subfamília K de Receptores Semelhantes a Lectina de Células NK/química , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Neoplasias/química , Neoplasias/tratamento farmacológico , Isomerases de Dissulfetos de Proteínas/química , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Estrutura Terciária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/genéticaRESUMO
BACKGROUND: All aerobically grown living cells are exposed to oxidative damage by reactive oxygen species (ROS). A major damage by ROS to proteins is caused by covalent modifications of methionine residues giving methionine sulfoxide (Met-SO). Methionine sulfoxide reductases are enzymes able to regenerate methionine and restore protein function after oxidative damage. RESULTS: We characterized the methionine sulfoxide reductase genes msrA and msrB in Bacillus subtilis, forming an operon transcribed from a single sigma A-dependent promoter. The msrAB operon was specifically induced by oxidative stress caused by paraquat (PQ) but not by H2O2. Spx, a global oxidative stress regulator in B. subtilis, is primarily responsible for this PQ-specific induction of msrAB expression. In support of this finding, an spx deletion mutant is extremely sensitive to PQ, and increased expression of msrA was identified in a clpX mutant in which Spx accumulated. However, the Spx effect was also visible under conditions where the protein did not accumulate (PQ treatment), suggesting a specific molecular effect at the level of the Spx protein. Indeed, the CXXC motif of Spx was found essential for its function in the PQ-specific induction of msrAB expression. PQ caused a modification of Spx requiring at least one of the cysteines of the CXXC motif of Spx. The PQ modified form of Spx showed a dynamic change in vivo. CONCLUSION: The Spx mediated PQ-specific regulation pathway of the msrAB operon in B. subtilis is reported. Our results suggest that PQ induced the expression of msrAB partially through an oxidation on Spx via modification of its CXXC motif.
Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Genes Reguladores , Óperon , Estresse Oxidativo , Oxirredutases/metabolismo , Motivos de Aminoácidos/efeitos dos fármacos , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , Regulação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Reguladores/efeitos dos fármacos , Metionina/metabolismo , Metionina Sulfóxido Redutases , Óperon/efeitos dos fármacos , Oxirredutases/química , Oxirredutases/genética , Paraquat/farmacologia , Regiões Promotoras GenéticasRESUMO
Coiled coils are bundles of intertwined alpha-helices that provide protein-protein interaction sites for the dynamic assembly and disassembly of protein complexes. The coiled-coil motif combines structural versatility and adaptability with mechanical strength and specificity. Multimeric proteins that rely on coiled-coil interactions are structurally and functionally very diverse, ranging from simple homodimeric transcription factors to elaborate heteromultimeric scaffolding clusters. Several coiled-coil-bearing proteins are of outstanding pharmacological importance, most notably SNARE proteins involved in vesicular trafficking of neurotransmitters and viral fusion proteins. Together with their crucial roles in many physiological and pathological processes, the structural simplicity and reversible nature of coiled-coil associations render them a promising target for pharmacological interference, as successfully exemplified by botulinum toxins and viral fusion inhibitors. The alpha-helical coiled coil is a ubiquitous protein domain that mediates highly specific homo- and heteromeric protein-protein interactions among a wide range of proteins. The coiled-coil motif was first proposed by Crick on the basis of X-ray diffraction data on alpha-keratin more than 50 years ago (Crick 1952, 1953) and nowadays belongs to the best-characterized protein interaction modules. By definition, a coiled coil is an oligomeric protein assembly consisting of several right-handed amphipathic alpha-helices that wind around each other into a superhelix (or a supercoil) in which the hydrophobic surfaces of the constituent helices are in continuous contact, forming a hydrophobic core. Both homomeric and heteromeric coiled coils with different stoichiometries are possible, and the helices can be aligned in either a parallel or an antiparallel topology (Harbury et al. 1993, 1994). Stoichiometry and topology are governed by the primary structure, that is, the sequence of the polypeptide chains, and a given protein can participate in multiple assembly-disassembly equilibria among several coiled coils differing in stoichiometry and topology (Portwich et al. 2007). Protein complexes whose oligomeric quaternary structures - and, hence, biological activities - depend on coiled-coil interactions include transcription factors, tRNA synthetases (Biou et al. 1994; Cusack et al. 1990), cytoskeletal and signal-transduction proteins, enzyme complexes, proteins involved in vesicular trafficking, viral coat proteins, and membrane proteins (Langosch and Heringa 1998). It is thus not surprising that coiled-coil motifs have gained great attention as potential targets for modulating protein-protein interactions implicated in a large number of diseases. In this review, we will first discuss some fundamental functional and structural aspects of a simple and well-characterized representative of coiled-coil transcription factors (Sect. 1) before considering two more complex coiled coils found in scaffolding proteins involved in mitosis and meiosis and vesicular trafficking Sect. 2). This will set the stage for addressing the role of coiled coils in viral infection (Sect. 3) as well as strategies of interfering with such protein-protein interactions therapeutically (Sect. 4 and 5).
Assuntos
Motivos de Aminoácidos/fisiologia , Sistemas de Liberação de Medicamentos , Proteínas/metabolismo , Motivos de Aminoácidos/efeitos dos fármacos , Fatores de Transcrição de Zíper de Leucina Básica , Sítios de Ligação , Proteínas de Ligação a DNA/metabolismo , Humanos , Zíper de Leucina/fisiologia , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Viroses/metabolismoRESUMO
We have shown earlier that platelet-activating factor (PAF) causes apoptosis in enterocytes via a mechanism that involves Bax translocation to mitochondria, followed by caspase activation and DNA fragmentation. Herein we report that, in rat small intestinal epithelial cells (IEC-6), these downstream apoptotic effects are mediated by a PAF-induced inhibition of the phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (Akt) signaling pathway. Treatment with PAF results in rapid dephosphorylation of Akt, phosphoinositide-dependent kinase-1, and the YXXM p85 binding motif of several proteins and redistribution of Akt-pleckstrin homology domain-green fluorescent protein, i.e., an in vivo phosphatidylinositol (3,4,5)-trisphosphate sensor, from membrane to cytosol. The proapoptotic effects of PAF were inhibited by both n-3 and n-6 polyunsaturated fatty acids but not by a saturated fatty acid palmitate. Indomethacin, an inhibitor of prostaglandin biosynthesis, did not influence the baseline or PAF-induced apoptosis, but 2-bromopalmitate, an inhibitor of protein palmitoylation, inhibited all of the proapoptotic effects of PAF. Our data strongly suggest that an inhibition of the PI 3-kinase/Akt signaling pathway is the main mechanism of PAF-induced apoptosis in enterocytes and that polyunsaturated fatty acids block this mechanism very early in the signaling cascade independently of any effect on prostaglandin synthesis, and probably directly via an effect on protein palmitoylation.
Assuntos
Apoptose/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Ácidos Graxos Insaturados/farmacologia , Proteína Oncogênica v-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fator de Ativação de Plaquetas/farmacologia , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Motivos de Aminoácidos/efeitos dos fármacos , Animais , Ácido Araquidônico/farmacologia , Caspase 3/metabolismo , Linhagem Celular , Cromonas/farmacologia , Ácidos Docosa-Hexaenoicos/farmacologia , Inibidores Enzimáticos/farmacologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Expressão Gênica/efeitos dos fármacos , Indometacina/farmacologia , Intestinos/citologia , Lipoilação/efeitos dos fármacos , Modelos Biológicos , Morfolinas/farmacologia , Palmitatos/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RatosRESUMO
OBJECTIVES: A few reports exist on hepatitis B virus (HBV) genotype distribution in Iran; however the sample sizes of these studies are insufficient. The first objective of this study was to determine the HBV genotype distribution with a large sample size (147 specimens). The second objective was to determine the incidence of the lamivudine-resistant YMDD mutant profile among HBV-infected patients not treated with lamivudine; some studies have reported that YMDD mutants are detectable even before antiviral treatment. METHODS: We used two cost-effective PCR-based methods that have been developed in-house: gap-PCR and artificially created restriction site-PCR (ACRS-PCR). Also, 11 samples were randomly selected and bi-directionally sequenced and subjected to phylogenetic analysis. RESULTS: Gap-PCR results revealed genotype D of HBV in all patients, and ACRS-PCR results disclosed the absence of mutation within the YMDD motif before antiviral therapy in the study population. Phylogenetic analysis supported the former genotyping results with the segregation of all Iranian HBV isolates in the genotype D branch with a high bootstrap value (99%, 1000 replicates). CONCLUSIONS: The present study using two cost-effective methods showed that genotype D of HBV is dominant among Iranian HBV-infected subjects, and HBV lamivudine-resistant strains do not exist naturally among Iranian patients not treated with lamivudine.
Assuntos
Motivos de Aminoácidos/genética , Produtos do Gene pol/genética , Vírus da Hepatite B/genética , Hepatite B Crônica/genética , Lamivudina/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Adulto , Idoso , Motivos de Aminoácidos/efeitos dos fármacos , Farmacorresistência Viral/genética , Feminino , Genótipo , Vírus da Hepatite B/classificação , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B Crônica/epidemiologia , Humanos , Irã (Geográfico)/epidemiologia , Lamivudina/administração & dosagem , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Mutação/genética , Filogenia , Inibidores da Transcriptase Reversa/administração & dosagem , Análise de Sequência de DNARESUMO
The microphthalmia-associated transcription factor Mitf plays a critical role in regulating many aspects of melanocyte biology. It is required for melanoblast and postnatal melanocyte survival, regulates proliferation, and activates genes associated with differentiation such as tyrosinase and related genes involved in melanogenesis. Identifying the signals that regulate Mitf expression is crucial if we are to understand how cells of the melanocyte lineage respond to environmental cues. Here we show that the Mitf promoter is induced by lipid signalling via the p38 stress-activated kinase pathway that is also activated by a wide range of receptors as well as UV irradiation. Signalling via p38 leads to increased phosphorylation and activation of cyclic adenosine monophosphate response element-binding (CREB) that binds and activates the Mitf promoter via the cyclic adenosine monophosphate (cAMP) response element. Moreover, we also show that activation of p38 mediated by lipids is potentiated by inhibition of the PI3kinase pathway but not by inhibition of protein kinase A (PKA). The results identify a mechanism in which stress signalling via p38 leads to activation of CREB, enhanced Mitf expression and consequently increased tyrosinase expression. The results are relevant for the regulation of melanocytes by Mitf, but also raise the possibility that lipid mediated activation of p38 signalling may represent a potential therapy for vitiligo.