RESUMO
Inflammasomes are macromolecular complexes involved in the host response to external and endogenous danger signals. Inflammasome-mediated sterile inflammation plays a central role in several human conditions such as autoimmune diseases, type-2 diabetes, and neurodegenerative disorders, indicating inflammasomes could be appealing therapeutic targets. Previous work has demonstrated that inhibiting the ATPase activity of the nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing protein 3 (NLRP3), disrupts inflammasome assembly and function. However, there is a necessity to find new potent compounds with therapeutic potential. Here we combine computational modeling of the target and virtual screening to discover a group of novel compounds predicted to inhibit NLRP3. We characterized the best compounds and determined their potency, specificity, and ability to inhibit processes downstream from NLRP3 activation. Moreover, we analyzed in mice the competence of a lead candidate to reduce lipopolysaccharide-induced inflammation. We also validated the active pharmacophore shared among all the NLRP3 inhibitors, and through computational docking, we clarify key structural features for compound positioning within the inflammasome ATP-binding site. Our study sets the basis for rational design and optimization of inflammasome-targeting probes and drugs.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Descoberta de Drogas , Inflamassomos/antagonistas & inibidores , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Proteínas Adaptadoras de Sinalização CARD/química , Proteínas de Ligação ao Cálcio/química , Avaliação Pré-Clínica de Medicamentos , Humanos , Inflamassomos/química , Camundongos , Modelos Moleculares , Proteína 3 que Contém Domínio de Pirina da Família NLR/química , Domínios Proteicos , Interface Usuário-ComputadorRESUMO
Systemic TNF neutralization can be used as a therapy for several autoimmune diseases. To evaluate the effects of cell type-restricted TNF blockade, we previously generated bispecific antibodies that can limit TNF secretion by myeloid cells (myeloid cell-specific TNF inhibitors or MYSTIs). In this study several such variable domain (VH) of a camelid heavy-chain only antibody-based TNF inhibitors were compared in relevant experimental models, both in vitro and in vivo. Pretreatment with MYSTI-2, containing the anti-F4/80 module, can restrict the release of human TNF (hTNF) from LPS-activated bone marrow-derived macrophage (BMDM) cultures of humanized TNF knock-in (mice; hTNFKI) more effectively than MYSTI-3, containing the anti-CD11b module. MYSTI-2 was also superior to MYSTI-3 in providing in vivo protection in acute toxicity model. Finally, MYSTI-2 was at least as effective as Infliximab in preventing collagen antibody-induced arthritis. This study demonstrates that a 33 kDa bispecific mini-antibody that specifically restricts TNF secretion by macrophages is efficient for amelioration of experimental arthritis.
Assuntos
Anticorpos Monoclonais/farmacologia , Artrite Experimental/terapia , Antígeno CD11b/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Cadeias Pesadas de Imunoglobulinas/farmacologia , Células Progenitoras Mieloides/efeitos dos fármacos , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Inibidores do Fator de Necrose Tumoral/farmacologia , Animais , Antirreumáticos/farmacologia , Artrite Experimental/genética , Artrite Experimental/imunologia , Artrite Experimental/patologia , Antígeno CD11b/genética , Antígeno CD11b/imunologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/imunologia , Expressão Gênica , Humanos , Infliximab/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Transgênicos , Células Progenitoras Mieloides/imunologia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Uncontrolled activation of NLRP3 inflammasome initiates a series of human inflammatory diseases. Targeting NLRP3 inflammasome has attracted considerable attention in developing potential therapeutic interventions. Here, we reported that dehydrocostus lactone (DCL), a main component of Saussurea lappa from the traditional Chinese medicine, inhibited NLRP3 inflammasome-mediated caspase-1 activation and subsequent interleukin (IL)-1ß production in primary mouse macrophages and human peripheral blood mononuclear cells and exerted an inhibitory effect on NLRP3-driven inflammation. Mechanistically, DCL significantly blocked the ASC oligomerization, which is essential for the assembly of activated inflammasome. Importantly, in vivo experiments showed that DCL reduced IL-1ß secretion and peritoneal neutrophils recruitment in LPS-mediated inflammation mouse model, which is demonstrated to be NLRP3 dependent. These results suggest that DCL is a potent pharmacological inhibitor of NLRP3 inflammasome and may be developed as a therapeutic drug for treating NLRP3-associated diseases.
Assuntos
Quimiotaxia de Leucócito/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Inflamassomos/efeitos dos fármacos , Inflamação/prevenção & controle , Lactonas/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Sesquiterpenos/farmacologia , Adulto , Animais , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/fisiologia , Proteínas Adaptadoras de Sinalização CARD/antagonistas & inibidores , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/fisiologia , Caspase 1/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/fisiologia , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Interleucina-1beta/biossíntese , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/toxicidade , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/efeitos dos fármacos , Nigericina/farmacologia , Poli I-C/farmacologia , Polimerização/efeitos dos fármacos , Organismos Livres de Patógenos Específicos , Ácido Úrico/farmacologiaRESUMO
N(6Aminohexyl)5chloro1naphthalenesulfonamide (W-7), a kind of adjuvant chemotherapy, can bind to calmodulin and inhibit Ca2+/calmodulin-regulated enzyme activities and cell proliferation. Similar to calmodulin, euplotes octocarinatus centrin (EoCen) belongs to EF-hand superfamily of calcium-binding proteins. It is associated with nucleotide excision repair (NER), cell division cycle and ciliogenesis. In the present study, the comparative interaction of W-7 with EoCen was first examined by using various spectroscopic, calorimetric methods and molecular docking. The obtain results recommend that only one W-7 molecule is identified binding to the C-terminal hydrophobic pocket of centrin that normally plays a role in anchoring targets. Methyl groups of Ala126, Met141, Ile161 and M162 of C-terminal may react with W-7 chloronaphthalene ring, other aliphatic or aromatic side-chains in a deep hydrophobic pocket of protein. Circular dichroism (CD) and fluorescence lifetime experiments reveal that W-7 triggers a conformational change of centrin. As a result, W-7 is identified to be an antagonist of centrin. It appears to inhibit the centrin-mediated activation of target proteins by blocking the hydrophobic pocket. Moreover, the complex formation leads to affinity decrease of Tb3+ binding to C-terminal of protein and self-assembly affected. Our present study provides the first view of centrin recognizing a naphthalene-sulfonamide derivative. It is proposed that W-7 and its analogues can serve as a useful tool for research on the participation of centrin in biological processes and cell biology-related studies.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Sulfonamidas/metabolismo , Térbio/metabolismo , Sítios de Ligação , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/química , Euplotes/química , Simulação de Acoplamento Molecular , Ligação Proteica , Sulfonamidas/química , Térbio/químicaRESUMO
OBJECTIVE: To address whether matrix Gla protein (MGP) can inhibit mineralization in normal rat kidney tubular cells (NRK-52E) under high concentration of calcium. MATERIALS AND METHODS: NRK-52E cells were treated with high concentration of calcium. The viability and apoptosis of cells were detected by cell counting kit-8 and flow cytology, respectively. Real-time-polymerase chain, Western blotting, and immunofluorescence analysis were conducted to detect the expression of MGP. Cells were transfected with plasmid-MGP or siRNA-MGP for up- or down-regulation of the expression of MGP, respectively. Rat recombinant MGP was also used as supplementation of exogenous MGP. Alizarin red staining was conducted to detect the adherent and deposition of calcium salt. RESULTS: High concentration of calcium suppressed MGP expression in NRK-52E cells. There was significant mineralization when NRK-52E cells were treated with high concentration of calcium. Supplementation with exogenous rat recombinant MGP and overexpression of endogenous MGP both decreased the adherent and deposition of calcium salt to NRK-52E cells, while silence of MGP showed reverse results. CONCLUSION: MGP plays an inhibitory role in the stone formation. However, high concentration of calcium significantly inhibits the expression of MGP and then promotes mineralization in NRK-52E cells.
Assuntos
Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/biossíntese , Cálcio/metabolismo , Proteínas da Matriz Extracelular/antagonistas & inibidores , Proteínas da Matriz Extracelular/biossíntese , Nefropatias/etiologia , Animais , Calcinose/etiologia , Células Cultivadas , Ratos , Proteína de Matriz GlaRESUMO
Postmenopausal osteoporosis is a common disorder accompanied with estrogen deficiency in women. Plants containing phytoestrogens and amino acids have been used in the osteoporosis treatment. The present study aims to evaluate the estrogen-like activity of the Cicer arietinum extract (CAE) and its ability to inhibit osteoclastogenesis process. These achieved by investigating the binding of its active phytoestrogens (genistein, daidzein, formononetin and biochanin A) to the estrogen receptors (ER) α and ß of rats and human in silico. In addition, in vivo study on ovariectomized (OVX) rats is performed. For in vivo study, twenty four rats were divided into four groups (n= 6). Group I is the sham control rats which administered distilled water. Groups II, III, and IV are OVX groups which administered distilled water, CAE (500 mg/kg), and alendronate; respectively. The docking study revealed that the phytoestrogens docked into the protein active site with binding energies comparable with that of estrogens (estriol and ß-estradiol) which means the similarity between the estrogenic contents of CAE and the ensogenous ones. Additionally, in vivo study revealed that CAE reverse TRAP5b and RANKL levels that drastically increased in the untreated OVX group. But, it trigger upregulation of OPG, enhance the OPG/RANKL ratio and modulate the bone and uterus alterations of OVX group. Phytoestrogens and the bone-protective amino acids contents of CAE could be responsible for their estrogen-like effect and antiosteoporotic activity. These results concluded that CAE is an attractive candidate for developing a potential therapeutic cheap agent used as an alternative to the synthetic estrogen replacement therapy. Further, in vivo validation is required for its clinical application.
Assuntos
Cicer/química , Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio/genética , Osteogênese/efeitos dos fármacos , Osteoporose/tratamento farmacológico , Fitoestrógenos/farmacologia , Fitoterapia , Alendronato/química , Alendronato/farmacologia , Animais , Conservadores da Densidade Óssea/química , Conservadores da Densidade Óssea/isolamento & purificação , Conservadores da Densidade Óssea/farmacologia , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Modelos Animais de Doenças , Estradiol/química , Estradiol/farmacologia , Receptor alfa de Estrogênio/agonistas , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/agonistas , Receptor beta de Estrogênio/metabolismo , Feminino , Regulação da Expressão Gênica , Genisteína/química , Genisteína/isolamento & purificação , Genisteína/farmacologia , Humanos , Isoflavonas/química , Isoflavonas/isolamento & purificação , Isoflavonas/farmacologia , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Simulação de Acoplamento Molecular , Osteogênese/genética , Osteoporose/genética , Osteoporose/metabolismo , Osteoporose/patologia , Osteoprotegerina/agonistas , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Ovariectomia , Fitoestrógenos/química , Fitoestrógenos/isolamento & purificação , Estrutura Secundária de Proteína , Ligante RANK/agonistas , Ligante RANK/genética , Ligante RANK/metabolismo , Ratos , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Peptídeos/antagonistas & inibidores , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismoRESUMO
Mitochondrial Ca2+ overload is a main contributor to mitochondrial damage hence cardiomyocyte death in myocardial ischemia/reperfusion (MI/R) injury. MICU1 has been recently identified as an important regulator of mitochondrial Ca2+ homeostasis. Here we try to identify the role of MICU1 in MI/R, and to investigate whether the mitochondrial importer receptor Tom70 possesses critical roles in the mitochondrial translocation of MICU1 and MI/R. Specific small interfering RNA (20 µg) against MICU1 and Tom70, and lentivirus vectors carrying the Tom70a sequences (3.3 × 107 TU) were delivered through intramyocardial injection. Seventy-two hours after injection, mice were subjected to 30 min of MI followed by 3 h (for cell apoptosis and mitochondrial damage assessment) or 24 h (for cardiac function and infarct size determination) of reperfusion. MI/R had no significant effect on total MICU1 expression, but caused significant reduction of MICU1 in mitochondria. Knockdown of MICU1 significantly aggravated MI/R injury, as evidenced by enlarged infarct size, depressed cardiac function and increased myocardial apoptosis. Moreover, MICU1 deficiency resulted in markedly aggravated mitochondrial Ca2+ overload, consequently destructed mitochondrial morphology and suppressed mitochondrial function (evidenced by decreased ATP production). Interestingly, mitochondrial Tom70 was also decreased in MI/R. Genetic loss-function study revealed that mitochondrial MICU1 expression was depressed by Tom70 ablation. Furthermore, Tom70 deficiency significantly aggravated MI/R injury and worsened mitochondrial Ca2+ overload. However, supplementation of Tom70 significantly attenuated MI/R injury, preserved mitochondrial morphology and function, and inhibited mitochondrial Ca2+ overload, all of which were abolished by MICU1 suppression. Mitochondrial Tom70/MICU1 pathway protects against MI/R injury, in which mitochondrial localization of MICU1 is governed by Tom70, and MICU1 serves as an indispensable factor in Tom70's cardioprotection.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Animais , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/genética , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Masculino , Potencial da Membrana Mitocondrial , Camundongos , Microscopia Eletrônica de Transmissão , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas de Transporte da Membrana Mitocondrial/antagonistas & inibidores , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Miocárdio/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Troponina I/análiseRESUMO
We have previously shown that the antiallergic drug cromolyn blocks S100P interaction with its receptor receptor for advanced glycation end product (RAGE) and improves gemcitabine effectiveness in pancreatic ductal adenocarcinoma (PDAC). However, the concentration required to achieve its effectiveness was high (100 µmol/L). In this study, we designed and synthesized analogs of cromolyn and analyzed their effectiveness compared with the parent molecule. An ELISA was used to confirm the binding of S100P with RAGE and to test the effectiveness of the different analogs. Analog 5-methyl cromolyn (C5OH) blocked S100P binding as well as the increases in NF-κB activity, cell growth, and apoptosis normally caused by S100P. In vivo C5OH systemic delivery reduced NF-κB activity to a greater extent than cromolyn and at 10 times lesser dose (50 mg vs. 5 mg). Treatment of mice-bearing syngeneic PDAC tumors showed that C5OH treatment reduced both tumor growth and metastasis. C5OH treatment of nude mice bearing orthotopic highly aggressive pancreatic Mpanc96 cells increased the overall animal survival. Therefore, the cromolyn analog, C5OH, was found to be more efficient and potent than cromolyn as a therapeutic for PDAC.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Carcinoma Ductal Pancreático/metabolismo , Cromolina Sódica/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias Pancreáticas/metabolismo , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Proteínas de Ligação ao Cálcio/metabolismo , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/mortalidade , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromolina Sódica/análogos & derivados , Cromolina Sódica/química , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Masculino , Camundongos , Camundongos Nus , NF-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Ligação Proteica/efeitos dos fármacos , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The interplay between cardiac sarcoplasmic Ca(2+)ATPase and phospholamban is a key regulating factor of contraction and relaxation in the cardiac muscle. In heart failure, aberrations in the inhibition of sarcoplasmic Ca(2+)ATPase by phospholamban are associated with anomalies in cardiac functions. In experimental heart failure models, modulation of the interaction between these two proteins has been shown to be a potential therapeutic approach. The aim of our research was to find molecules able to interfere with the inhibitory activity of phospholamban on sarcoplasmic Ca(2+)ATPase. For this purpose, a portion of phospholamban was synthesized and used as target for a phage-display peptide library screening. The cyclic peptide C-Y-W-E-L-E-W-L-P-C-A was found to bind to phospholamban (1-36) with high specificity. Its functional activity was tested in Ca(2+)uptake assays utilizing preparations from cardiac sarcoplasmic reticulum. By synthesizing and testing a series of alanine point-mutated cyclic peptides, we identified which amino acid was important for the inhibition of the phospholamban function. The structures of active and inactive alanine-mutated cyclic peptides, and of phospholamban (1-36), were determined by NMR. This structure-activity analysis allowed building a model of phospholamban -cyclic peptide complex. Thereafter, a simple pharmacophore was defined and used for the design of small molecules. Finally, examples of such molecules were synthesized and characterized as phospholamban inhibitors.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Peptídeos Cíclicos/química , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/síntese química , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Cobaias , Coração/efeitos dos fármacos , Humanos , Modelos Moleculares , Miocárdio/metabolismo , Biblioteca de Peptídeos , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/farmacologia , Ligação Proteica , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-AtividadeRESUMO
BACKGROUND: We conducted an in vivo experiment to determine whether vitamin D3 acts as a fat synthesizer and/or meat tenderizer in mice. At 6 weeks of age, 20 male C57BL/6 wild-type mice were randomly divided into two groups (10 mice per group) and fed a modified AIN93G diet with (vitamin D3 diet) or without (basal diet) 10 IU 25-OH-cholecalciferol kg⻳ for 3 weeks. RESULTS: When vitamin D3 was fed to mice for 3 weeks, body fat was significantly increased compared to mice fed a basal diet. There was, however, no difference in body weight between the two groups. Vitamin D3 increased the gene expressions of pro-inflammatory cytokines and peroxisome proliferator-activated receptor gamma, but decreased interleukin-15 in adipose tissue through nuclear vitamin D receptor and uncoupling protein-2 signals. The muscle inducible nitrate oxide synthase content of mice fed vitamin D3 was higher than those fed a basal diet, while muscle arginase l showed a reverse phenomenon. longissimuss dorsi muscle of vitamin D3-fed mice showed more severe fat deposition than those fed a basal diet. Vitamin D3 amplified muscle u- and m-calpain protein content and suppressed muscle calpastatin protein content. CONCLUSION: These findings suggest that vitamin D3 can be used as a fat synthesizer and meat tenderizer in meat-producing animals.
Assuntos
Adiposidade , Calcifediol/metabolismo , Calpaína/metabolismo , Citocinas/metabolismo , Regulação da Expressão Gênica , Músculo Esquelético/metabolismo , Animais , Arginase/antagonistas & inibidores , Arginase/metabolismo , Calcifediol/deficiência , Calcifediol/uso terapêutico , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Calpaína/química , Calpaína/genética , Citocinas/sangue , Citocinas/genética , Suplementos Nutricionais , Canais Iônicos/agonistas , Canais Iônicos/metabolismo , Metabolismo dos Lipídeos , Masculino , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais/agonistas , Proteínas Mitocondriais/metabolismo , Músculo Esquelético/enzimologia , Músculo Esquelético/imunologia , Óxido Nítrico Sintase Tipo II/química , Óxido Nítrico Sintase Tipo II/metabolismo , PPAR gama/agonistas , PPAR gama/genética , PPAR gama/metabolismo , Distribuição Aleatória , Receptores de Calcitriol/agonistas , Receptores de Calcitriol/metabolismo , Transdução de Sinais , Proteína Desacopladora 2RESUMO
Classical serine proteases use the conserved Ser/His/Asp catalytic triad to hydrolyze substrates. Here, we show that longistatin, a salivary gland protein with two EF-hand domains from the vector tick Haemaphysalis longicornis, does not have the conserved catalytic triad, but still functions as a serine protease. Longistatin was synthesized in and secreted from the salivary glands of ticks, and is injected into host tissues during the acquisition of blood-meals. Longistatin hydrolyzed fibrinogen, an essential plasma protein in the coagulation cascade, and activated plasminogen, into its active form plasmin, a serine protease that dissolves fibrin clots. Longistatin efficiently hydrolyzed several serine protease-specific substrates showing its specificity to the amide bond of Arg. Longistatin did not hydrolyze synthetic substrates specific for other groups of proteases. The enzyme was active at a wide range of temperatures and pHs, with the optimum at 37°C and pH 7. Its activity was efficiently inhibited by various serine protease inhibitors such as phenylmethanesulfonyl fluoride (PMSF), aprotinin, antipain, and leupeptin with the estimated IC(50) of 278.57 µM, 0.35 µM, 41.56 µM and 198.86 µM, respectively. In addition, longistatin was also potently inhibited by Zinc (Zn(2+)) in a concentration-dependent manner with an IC(50) value of 275 µM, and the inhibitory effect of Zn(2+) was revived by ethylenediaminetetra acetic acid (EDTA). Immunization studies revealed that longistatin sharply induced high levels of protective IgG antibodies against ticks. Immunization with longistatin reduced repletion of ticks by about 54%, post engorgement body weight by >11% and molting of nymphs by approximately 34%; thus, the vaccination trial was approximately 73% effective against tick infestation. Taken together, our results suggest that longistatin is a new potent atypical serine protease, and may be an interesting candidate for the development of anti-tick vaccines.
Assuntos
Proteínas de Ligação ao Cálcio/imunologia , Ixodidae/enzimologia , Ixodidae/imunologia , Proteínas e Peptídeos Salivares/imunologia , Infestações por Carrapato/imunologia , Animais , Anticorpos/imunologia , Antipaína/farmacologia , Aprotinina/farmacologia , Arginina/metabolismo , Peso Corporal , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Ácido Edético/farmacologia , Ativação Enzimática , Fibrinogênio/metabolismo , Hidrólise , Concentração Inibidora 50 , Ixodidae/patogenicidade , Leupeptinas/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Ativadores de Plasminogênio/metabolismo , Coelhos , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Glândulas Salivares/enzimologia , Glândulas Salivares/imunologia , Proteínas e Peptídeos Salivares/antagonistas & inibidores , Serina Proteases/imunologia , Inibidores de Serina Proteinase/farmacologia , Especificidade por Substrato , Temperatura , Infestações por Carrapato/parasitologia , Infestações por Carrapato/terapia , Compostos de Tosil/farmacologia , Vacinação , Zinco/farmacologiaRESUMO
Derangements in calcium cycling have been described in failing hearts, and preclinical studies have suggested that therapies aimed at correcting this defect can lead to improvements in cardiac function and survival. One strategy to improve calcium cycling would be to inhibit phospholamban (PLB), the negative regulator of SERCA2a that is upregulated in failing hearts. The goal of this study was to evaluate the safety and efficacy of using adeno-associated virus (AAV)-mediated cardiac gene transfer of short hairpin RNA (shRNA) to knock down expression of PLB. Six dogs were treated with self-complementary AAV serotype 6 (scAAV6) expressing shRNA against PLB. Three control dogs were treated with empty AAV6 capsid, and two control dogs were treated with scAAV6 expressing dominant negative PLB. Vector was delivered via a percutaneously inserted cardiac injection catheter. PLB mRNA and protein expression were analyzed in three of six shRNA dogs between days 16 and 26. The other three shRNA dogs and five control dogs were monitored long-term to assess cardiac safety. PLB mRNA was reduced 16-fold, and PLB protein was reduced 5-fold, with treatment. Serum troponin elevation and depressed cardiac function were observed in the shRNA group only at 4 weeks. An enzyme-linked immunospot assay failed to detect any T cells reactive to AAV6 capsid in peripheral blood mononuclear cells, heart, or spleen. Microarray analysis revealed alterations in cardiac expression of several microRNAs with shRNA treatment. AAV6-mediated cardiac gene transfer of shRNA effectively knocks down PLB expression but is associated with severe cardiac toxicity. Toxicity may result from dysregulation of endogenous microRNA pathways.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Técnicas de Transferência de Genes , RNA Interferente Pequeno/genética , Animais , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/metabolismo , Dependovirus/genética , Cães , Expressão Gênica , Técnicas de Silenciamento de Genes , Técnicas de Transferência de Genes/efeitos adversos , Vetores Genéticos , Monócitos/citologia , Monócitos/metabolismo , Miócitos Cardíacos/metabolismo , RNA Mensageiro/metabolismo , Troponina/sangueRESUMO
UNLABELLED: Calciphylaxis is a rare, usually fatal vasculopathic disorder characterized by cutaneous ischemia and necrosis due to calcification of arterioles. Although calciphylaxis is most frequently associated with end-stage renal disease (ESRD) and secondary hyperparathyroidism, it has been reported infrequently among patients on warfarin. No standard treatment has been established for atypical calciphylaxis; however, a potentially beneficial treatment is hyperbaric oxygen therapy (HBOT). A high degree of clinical suspicion, early diagnosis, and understanding the pathophysiology of this disease promotes the optimal management of this extremely morbid and often fatal condition. CASE REPORT: We present a 63-year-old Polynesian woman with biopsy-proven calciphylaxis in the absence of ESRD or elevated serum calcium levels while taking warfarin. Therapeutic dose enoxaparin was substituted for warfarin and she received 40 sessions of HBOT during which lower extremity ulcers resolved. DISCUSSION: Warfarin has been implicated when calciphylaxis presents in an atypical fashion. No guidelines exist for treatment of atypical calciphylaxis in the setting of concomitant warfarin therapy. Up to 80% of calciphylaxis patients die within 1 year of diagnosis. Our patient was changed to low-molecular-weight heparin and received HBOT. CONCLUSION: We present what we believe is the first case of atypical calciphylaxis thought to be attributable to warfarin treated with a therapeutic substitution of anticoagulant and HBOT leading to resolution of cutaneous lesions.
Assuntos
Anticoagulantes/efeitos adversos , Calciofilaxia/induzido quimicamente , Oxigenoterapia Hiperbárica , Úlcera da Perna/etiologia , Varfarina/efeitos adversos , Antibacterianos/uso terapêutico , Anticoagulantes/uso terapêutico , Calciofilaxia/diagnóstico , Calciofilaxia/cirurgia , Calciofilaxia/terapia , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Celulite (Flegmão)/diagnóstico , Celulite (Flegmão)/tratamento farmacológico , Terapia Combinada , Desbridamento , Diabetes Mellitus Tipo 2/complicações , Proteínas da Matriz Extracelular/antagonistas & inibidores , Feminino , Humanos , Úlcera da Perna/cirurgia , Úlcera da Perna/terapia , Pessoa de Meia-Idade , Obesidade Mórbida/complicações , Fatores de Risco , Trombofilia/tratamento farmacológico , Varfarina/uso terapêutico , Proteína de Matriz GlaRESUMO
Notch1 is an evolutionarily conserved receptor that regulates cell fate, including such events as differentiation, proliferation, and apoptosis. Myofibroblast differentiation is a key feature of lung fibrosis. Found in inflammatory zone 1 (FIZZ1) has direct fibrogenic properties because of its ability to induce myofibroblast differentiation. However, the downstream signaling pathway that mediates FIZZ1 induction of myofibroblast differentiation remains unknown. The objective of this study was to investigate the involvement of Notch signaling in FIZZ1 induction of lung myofibroblast differentiation and thus explore the potential role of Notch1 in pulmonary fibrosis. The results showed that FIZZ1 increased the expression levels of activated intracellular domain of Notch1 (NIC), its ligand Jagged1, and its target gene Hes1, which were associated with elevated alpha-smooth muscle actin expression levels. Fibroblast alpha-smooth muscle actin expression is induced by the overexpression of NIC but is suppressed by the inhibition of NIC. Moreover, lung fibroblasts that were isolated from mice lacking the GDP-4-keto-6-deoxymannose3,5-epimerase-4-reductase enzyme (FX knockout) exhibited significantly reduced responsiveness to FIZZ1, which was reversed by fucose supplementation. In the absence of exogenous fucose, these FX-deficient cells exhibited defective fucosylation, which is required for Notch signaling. These knockout mice also showed impaired lung fibrosis. These findings suggest that Notch1 signaling in response to FIZZ1 may play a significant role in myofibroblast differentiation during lung fibrosis.
Assuntos
Diferenciação Celular , Fibroblastos/citologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Pulmão/citologia , Receptor Notch1/metabolismo , Actinas/metabolismo , Animais , Antibióticos Antineoplásicos/toxicidade , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Bleomicina/toxicidade , Western Blotting , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Fibroblastos/metabolismo , Imunofluorescência , Fucose/administração & dosagem , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Hidroliases/fisiologia , Hidroxiprolina/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteína Jagged-1 , Pulmão/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso/citologia , Músculo Liso/metabolismo , Regiões Promotoras Genéticas , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor Notch1/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Serrate-Jagged , Fatores de Transcrição HES-1RESUMO
RNA interference (RNAi) by means of short hairpin RNA (shRNA) has developed into a powerful tool for loss-of-function analysis in mammalian cells. The principal problem in RNAi experiments is off-target effects, and the most vigorous demonstration of the specificity of shRNA is the rescue of the RNAi effects with a shRNA-resistant target gene. This presents its own problems, including the unpredictable relative expression of shRNA and rescue cDNA in individual cells, and the difficulty in generating stable cell lines. In this report, we evaluated the plausibility of combining the expression of shRNA and rescue cDNA in the same vector. In addition to facilitate the validation of shRNA specificity, this system also considerably simplifies the generation of shRNA-expressing cell lines. Since the compensatory cDNA is under the control of an inducible promoter, stable shRNA-expressing cells can be generated before the knockdown phenotypes are studied by conditionally turning off the rescue protein. Conversely, the rescue protein can be activated after the endogenous protein is completely repressed. This approach is particularly suitable when prolonged expression of either the shRNA or the compensatory cDNA is detrimental to cell growth. This system allows a convenient one-step validation of shRNA and generation of stable shRNA-expressing cells.
Assuntos
Interferência de RNA , RNA não Traduzido/biossíntese , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Ciclina A/antagonistas & inibidores , Ciclina A/genética , DNA Complementar/biossíntese , DNA Complementar/genética , Engenharia Genética/métodos , Vetores Genéticos , Células HeLa , Humanos , Proteínas Mad2 , Fenótipo , Plasmídeos/genética , RNA não Traduzido/química , RNA não Traduzido/genética , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genéticaRESUMO
Signals from the hypothalamus govern food intake and energy balance. A new study describes nesfatin-1, a hypothalamic and brainstem peptide whose expression decreases during fasting. Although central treatment with nesfatin-1 inhibited food intake and nesfatin-1 blockade increased food intake, the role and mechanism of nesfatin in energy balance remains unclear.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ingestão de Alimentos , Metabolismo Energético , Hipotálamo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais , Animais , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ligação a DNA/antagonistas & inibidores , Metabolismo Energético/efeitos dos fármacos , Jejum/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/farmacologia , Nucleobindinas , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
Kv4.3 channels conduct transient outward K(+) currents in the human heart and brain where they mediate the early phase of action potential repolarization. KChIP2 proteins are members of a new class of calcium sensors that modulate the surface expression and biophysical properties of Kv4 K(+) channels. Here we describe three novel isoforms of KChIP2 with an alternatively spliced C-terminus (KChIP2e, KChIP2f) or N-terminus (KChIP2g). KChIP2e and KChIP2f are expressed in the human atrium, whereas KChIP2g is predominantly expressed in the brain. The KChIP2 isoforms were coexpressed with Kv4.3 channels in Xenopus oocytes and currents recorded with two-microelectrode voltage-clamp techniques. KChIP2e caused a reduction in current amplitude, an acceleration of inactivation and a slowing of the recovery from inactivation of Kv4.3 currents. KChIP2f increased the current amplitude and slowed the rate of inactivation, but did not alter the recovery from inactivation or the voltage of half-maximal inactivation of Kv4.3 channels. KChIP2g increased current amplitudes, slowed the rate of inactivation and shifted the voltage of half-maximal inactivation to more negative potentials. The biophysical changes induced by these alternatively spliced KChIP2 proteins differ markedly from previously described KChIP2 proteins and would be expected to increase the diversity of native transient outward K(+) currents.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Canais de Potássio/metabolismo , Sequência de Aminoácidos , Animais , Química Encefálica , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Clonagem Molecular , DNA Complementar/biossíntese , DNA Complementar/genética , Estimulação Elétrica , Eletrofisiologia , Humanos , Ativação do Canal Iônico , Isomerismo , Proteínas Interatuantes com Canais de Kv , Medições Luminescentes , Potenciais da Membrana/fisiologia , Dados de Sequência Molecular , Miocárdio/metabolismo , Oócitos/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/efeitos dos fármacos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canais de Potássio Shal , Xenopus laevisRESUMO
The mitochondrial carriers are a family of transport proteins that, with a few exceptions, are found in the inner membranes of mitochondria. They shuttle metabolites and cofactors through this membrane, and connect cytoplasmic functions with others in the matrix. SAM (S-adenosylmethionine) has to be transported into the mitochondria where it is converted into S-adenosylhomocysteine in methylation reactions of DNA, RNA and proteins. The transport of SAM has been investigated in rat liver mitochondria, but no protein has ever been associated with this activity. By using information derived from the phylogenetically distant yeast mitochondrial carrier for SAM and from related human expressed sequence tags, a human cDNA sequence was completed. This sequence was overexpressed in bacteria, and its product was purified, reconstituted into phospholipid vesicles and identified from its transport properties as the human mitochondrial SAM carrier (SAMC). Unlike the yeast orthologue, SAMC catalysed virtually only countertransport, exhibited a higher transport affinity for SAM and was strongly inhibited by tannic acid and Bromocresol Purple. SAMC was found to be expressed in all human tissues examined and was localized to the mitochondria. The physiological role of SAMC is probably to exchange cytosolic SAM for mitochondrial S-adenosylhomocysteine. This is the first report describing the identification and characterization of the human SAMC and its gene.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Genes , Proteínas de Membrana Transportadoras/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/metabolismo , Sequência de Aminoácidos , Sistemas de Transporte de Aminoácidos , Animais , Transporte Biológico/efeitos dos fármacos , Química Encefálica , Púrpura de Bromocresol/farmacologia , Células CHO , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/isolamento & purificação , Proteínas de Ligação ao Cálcio/fisiologia , Clonagem Molecular , Cricetinae , Citosol/metabolismo , DNA Complementar/genética , Escherichia coli , Etiquetas de Sequências Expressas , Humanos , Taninos Hidrolisáveis/farmacologia , Moduladores de Transporte de Membrana , Proteínas de Membrana Transportadoras/antagonistas & inibidores , Proteínas de Membrana Transportadoras/isolamento & purificação , Proteínas de Membrana Transportadoras/fisiologia , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/isolamento & purificação , Proteínas Mitocondriais/fisiologia , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/isolamento & purificação , Especificidade de Órgãos , Filogenia , RNA Mensageiro/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Synaptotagmin I (Syt I), a low-affinity Ca(2+)-binding protein, is thought to serve as the Ca(2+) sensor in the release of neurotransmitter. However, functional studies on the calyx of Held synapse revealed that the rapid release of neurotransmitter requires only approximately micromolar [Ca(2+)], suggesting that Syt I may play a more complex role in determining the high-affinity Ca(2+) dependence of exocytosis. Here we tested this hypothesis by studying pituitary cells, which possess high- and low-affinity Ca(2+)-dependent exocytic pathways and express Syt I. Using patch-clamp capacitance measurements to monitor secretion and the acute antisense deletion of Syt I from differentiated cells, we have shown that the rapid and the most Ca(2+)-sensitive pathway of exocytosis in rat melanotrophs requires Syt I. Furthermore, stimulation of the Ca(2+)-dependent exocytosis by cytosol dialysis with solutions containing 1 microM [Ca(2+)] was completely abolished in the absence of Syt I. Similar results were obtained by the preinjection of antibodies against the CAPS (Ca(2+)-dependent activator protein for secretion) protein. These results indicate that synaptotagmin I and CAPS proteins increase the probability of vesicle fusion at low cytosolic [Ca(2+)].
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/deficiência , Células Epiteliais/metabolismo , Exocitose/genética , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Hipófise/metabolismo , Vesículas Secretórias/metabolismo , Proteínas de Transporte Vesicular , Animais , Sinalização do Cálcio/efeitos dos fármacos , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/metabolismo , Células Cultivadas , DNA Complementar/genética , Endocitose/efeitos dos fármacos , Endocitose/genética , Células Epiteliais/efeitos dos fármacos , Exocitose/efeitos dos fármacos , Masculino , Glicoproteínas de Membrana/genética , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Oligonucleotídeos Antissenso , Hipófise/efeitos dos fármacos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , Ratos , Proteínas SNARE , Vesículas Secretórias/efeitos dos fármacos , Sinaptotagmina I , SinaptotagminasRESUMO
1. The present study was undertaken to determine whether calponin (CaP) participates in the regulation of vascular smooth muscle contraction and, if so, to investigate the mechanism. 2. By PCR homology cloning, the cDNA sequence of ferret basic (h1) CaP was determined and phosphorothioate antisense and random oligonucleotides were synthesized and introduced into strips of ferret aorta by a chemical loading procedure. 3. Treatment of ferret aorta with CaP antisense oligonucleotides resulted in a decrease in protein levels of CaP to 54% of that in random sequence-loaded muscles, but no change in the protein levels of caldesmon (CaD), actin, desmin or extracellular regulated protein kinase (ERK). 4. Contraction in response to phenylephrine or a phorbol ester was significantly decreased in antisense-treated muscles compared to random sequence-loaded controls. Neither basal intrinsic tone nor the contraction in response to 51 mM KCl was significantly affected by antisense treatment. 5. During phenylephrine contractions, phospho-ERK levels increased, as did myosin light chain (LC20) phosphorylation. Phenylephrine-induced ERK phosphorylation and CaD phosphorylation at an ERK site were significantly decreased by CaP antisense. Increases in myosin light chain phosphorylation were unaffected. 6. The data indicate that CaP plays a significant role in the regulation of contraction and suggest that in a tonically active smooth muscle CaP may function as a signalling protein to facilitate ERK-dependent signalling, but not as a direct regulator of actomyosin interactions at the myofilament level.