RESUMO
UNLABELLED: The aim of this study was to explore the signaling and neuroprotective effect of transactivator of transcription (TAT) protein transduction of the apoptosis repressor with CARD (ARC) in in vitro and in vivo models of cerebral ischemia in mice. In mice, transient focal cerebral ischemia reduced endogenous ARC protein in neurons in the ischemic striatum at early reperfusion time points, and in primary neuronal cultures, RNA interference resulted in greater neuronal susceptibility to oxygen glucose deprivation (OGD). TAT.ARC protein delivery led to a dose-dependent better survival after OGD. Infarct sizes 72 h after 60 min middle cerebral artery occlusion (MCAo) were on average 30 ± 8% (mean ± SD; p = 0.005; T2-weighted MRI) smaller in TAT.ARC-treated mice (1 µg intraventricularly during MCAo) compared with controls. TAT.ARC-treated mice showed better performance in the pole test compared with TAT.ß-Gal-treated controls. Importantly, post-stroke treatment (3 h after MCAo) was still effective in affording reduced lesion volume by 20 ± 7% (mean ± SD; p < 0.05) and better functional outcome compared with controls. Delayed treatment in mice subjected to 30 min MCAo led to sustained neuroprotection and functional behavior benefits for at least 28 d. Functionally, TAT.ARC treatment inhibited DAXX-ASK1-JNK signaling in the ischemic brain. ARC interacts with DAXX in a CARD-dependent manner to block DAXX trafficking and ASK1-JNK activation. Our work identifies for the first time ARC-DAXX binding to block ASK1-JNK activation as an ARC-specific endogenous mechanism that interferes with neuronal cell death and ischemic brain injury. Delayed delivery of TAT.ARC may present a promising target for stroke therapy. SIGNIFICANCE STATEMENT: Up to now, the only successful pharmacological target of human ischemic stroke is thrombolysis. Neuroprotective pharmacological strategies are needed to accompany therapies aiming to achieve reperfusion. We describe that apoptosis repressor with CARD (ARC) interacts and inhibits DAXX and proximal signals of cell death. In a murine stroke model mimicking human malignant infarction in the territory of the middle cerebral artery, TAT.ARC salvages brain tissue when given during occlusion or 3 h delayed with sustained functional benefits (28 d). This is a promising novel therapeutic approach because it appears to be effective in a model producing severe injury by interfering with an array of proximal signals and effectors of the ischemic cascade, upstream of JNK, caspases, and BIM and BAX activation.
Assuntos
Apoptose , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto/metabolismo , Produtos do Gene tat/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Animais , Proteínas Correpressoras , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Chaperonas Moleculares , Ligação Proteica , Mapas de Interação de ProteínasRESUMO
BACKGROUND: Poststroke angiogenesis contributes to long-term recovery after stroke. Signal transducer and activator of transcription-3 (Stat3) is a key regulator for various inflammatory signals and angiogenesis. It was the aim of this study to determine its function in poststroke outcome. METHODS AND RESULTS: We generated a tamoxifen-inducible and endothelial-specific Stat3 knockout mouse model by crossbreeding Stat3(floxed/KO) and Tie2-Cre(ERT2) mice. Cerebral ischemia was induced by 30 minutes of middle cerebral artery occlusion. We demonstrated that endothelial Stat3 ablation did not alter lesion size 2 days after ischemia but did worsen functional outcome at 14 days and increase lesion size at 28 days. At this late time point vascular Stat3 expression and phosphorylation were still increased in wild-type mice. Gene array analysis of a CD31-enriched cell population of the neurovascular niche showed that endothelial Stat3 ablation led to a shift toward an antiangiogenic and axon growth-inhibiting micromilieu after stroke, with an increased expression of Adamts9. Remodeling and glycosylation of the extracellular matrix and microglia proliferation were increased, whereas angiogenesis was reduced. CONCLUSIONS: Endothelial Stat3 regulates angiogenesis, axon growth, and extracellular matrix remodeling and is essential for long-term recovery after stroke. It might serve as a potent target for stroke treatment after the acute phase by fostering angiogenesis and neuroregeneration.
Assuntos
Endotélio Vascular/metabolismo , Infarto da Artéria Cerebral Média/fisiopatologia , Neovascularização Fisiológica/fisiologia , Plasticidade Neuronal/fisiologia , Fator de Transcrição STAT3/fisiologia , Proteínas ADAM/biossíntese , Proteínas ADAM/genética , Proteína ADAMTS9 , Animais , Axônios/fisiologia , Encéfalo/patologia , Microambiente Celular , Circulação Cerebrovascular , Convalescença , Proteínas da Matriz Extracelular/metabolismo , Perfilação da Expressão Gênica , Infarto da Artéria Cerebral Média/patologia , Camundongos , Camundongos Knockout , Microglia/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Processamento de Proteína Pós-Traducional , Recuperação de Função Fisiológica , Fator de Transcrição STAT3/deficiência , Fator de Transcrição STAT3/genética , Transdução de Sinais/fisiologiaRESUMO
Hemorrhagic shock (HS), a leading cause of preventable death, is characterized by severe blood loss and inadequate tissue perfusion. Reoxygenation of ischemic tissues exacerbates organ damage through ischemia-reperfusion injury. SUMOylation has been shown to protect neurons after stroke and is upregulated in response to cellular stress. However, the role of SUMOylation in organ protection after HS is unknown. This study aimed to investigate SUMOylation-mediated organ protection following HS. Male Wistar rats were subjected to HS (blood pressure of 40 ± 2 mmHg, for 90 min) followed by reperfusion. Blood, kidney, and liver samples were collected at various time points after reperfusion to assess organ damage and investigate the profile of SUMO1 and SUMO2/3 conjugation. In addition, human kidney cells (HK-2), treated with the SUMOylation inhibitor TAK-981 or overexpressing SUMO proteins, were subjected to oxygen and glucose deprivation to investigate the role of SUMOylation in hypoxia/reoxygenation injury. The animals presented progressive multiorgan dysfunction, except for the renal system, which showed improvement over time. Compared to the liver, the kidneys displayed distinct patterns in terms of oxidative stress, apoptosis activation, and tissue damage. The global level of SUMO2/3 in renal tissue was also distinct, suggesting a differential role. Pharmacological inhibition of SUMOylation reduced cell viability after hypoxia-reoxygenation damage, while overexpression of SUMO1 or SUMO2 protected the cells. These findings suggest that SUMOylation might play a critical role in cellular protection during ischemia-reperfusion injury in the kidneys, a role not observed in the liver. This difference potentially explains the renal resilience observed in HS animals when compared to other systems.
Assuntos
Ratos Wistar , Choque Hemorrágico , Sumoilação , Animais , Masculino , Choque Hemorrágico/metabolismo , Sumoilação/efeitos dos fármacos , Sumoilação/fisiologia , Ratos , Humanos , Rim/metabolismo , Rim/patologia , Rim/efeitos dos fármacos , Traumatismo por Reperfusão/metabolismo , Linhagem CelularRESUMO
BACKGROUND & AIMS: Acetaminophen (AAP) overdose is the most frequent cause of drug-induced liver failure. c-Jun N-terminal kinase (JNK) is thought to play a central role in AAP-induced hepatocellular necrosis. The apoptosis repressor with caspase recruitment domain (ARC) is a death repressor that inhibits death receptor and mitochondrial apoptotic signaling. Here, we investigated ARC's therapeutic effect and molecular mechanisms on AAP-induced hepatocellular necrosis. METHODS: We tested the in vivo and in vitro effects of ARC fused with the transduction domain of HIV-1 (TAT-ARC) on murine AAP hepatotoxicity. RESULTS: Treatment with TAT-ARC protein completely abrogated otherwise lethal liver failure induced by AAP overdose in C57BL/6 mice. AAP triggered caspase-independent necrosis, as evidenced by liver histology, elevated serum transaminases, and secreted HMGB1 that was inhibited by ARC. ARC-mediated hepatoprotection was not caused by an alteration of AAP metabolism, but resulted in reduced oxidative stress. AAP overdose led to induction of RIP-dependent signaling with subsequent JNK activation. Ectopic ARC inhibited JNK activation by specific interactions between ARC and JNK1 and JNK2. Importantly, survival of mice was even preserved when ARC therapy was initiated in a delayed manner after AAP administration. CONCLUSIONS: This work identifies for the first time ARC-JNK-binding with subsequent inhibition of JNK signaling as a specific mechanism of ARC to interfere with AAP-dependent necrosis. Our data suggests that AAP-mediated induction of RIP signaling serves as a critical switch for hepatocellular necrosis. The efficacy of TAT-ARC protein transduction in murine AAP hepatotoxicity suggests its therapeutic potential for reversing AAP intoxication also in humans.
Assuntos
Acetaminofen/efeitos adversos , Proteínas Reguladoras de Apoptose/uso terapêutico , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/tratamento farmacológico , Proteínas Musculares/uso terapêutico , Proteínas Recombinantes de Fusão/uso terapêutico , Animais , Proteínas Reguladoras de Apoptose/farmacologia , Carcinoma Hepatocelular/patologia , Modelos Animais de Doenças , Glutationa/metabolismo , HIV-1 , Neoplasias Hepáticas/patologia , MAP Quinase Quinase 4/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Musculares/farmacologia , Necrose/induzido quimicamente , Necrose/prevenção & controle , Espécies Reativas de Oxigênio/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Transdução de Sinais/efeitos dos fármacos , Resultado do TratamentoRESUMO
Magnetic resonance imaging (MRI) with contrast agents that target specific inflammatory components of atherosclerotic lesions has the potential to emerge as promising diagnostic modality for detecting unstable plaques. Since a high content of macrophages and alterations of the extracellular matrix are hallmarks of plaque instability, these structures represent attractive targets for new imaging modalities. In this study, we compared in vitro uptake and binding of electrostatically stabilized citrate-coated very small superparamagnetic iron oxide particles (VSOP) to THP-1 cells with sterically stabilized carboxydextran-coated Resovist(®). Uptake of VSOP in both THP-1 monocytic cells and THP-derived macrophages (THP-MΦ) was more efficient compared to Resovist(®) without inducing cytotoxicity or modifying normal cellular functions (no changes in levels of reactive oxygen species, caspase-3 activity, proliferation, cytokine production). Importantly, VSOP bound with high affinity to the cell surface and to apoptotic membrane vesicles. Inhibition of glycosaminoglycan (GAG) synthesis by glucose deprivation in THP-MΦ was associated with a significant reduction of VSOP attachment suggesting that the strong interaction of VSOP with the membranes of cells and apoptotic vesicles occurs via binding to negatively charged GAGs. These in vitro experiments show that VSOP-enhanced MRI may represent a new imaging approach for visualizing high-risk plaques on the basis of targeting pathologically increased GAGs or apoptotic membrane vesicles in atherosclerotic lesions. VSOP should be investigated further in appropriate in vivo experiments to characterize accumulation in unstable plaque.
Assuntos
Meios de Contraste/metabolismo , Dextranos/metabolismo , Monócitos/metabolismo , Linhagem Celular , Glicosaminoglicanos/metabolismo , Humanos , Técnicas In Vitro , Macrófagos/metabolismo , Imageamento por Ressonância Magnética , Nanopartículas de Magnetita/química , Microscopia Eletrônica , Placa Aterosclerótica/diagnósticoRESUMO
UNLABELLED: Acute liver failure (ALF) is associated with massive hepatocyte cell death and high mortality rates. Therapeutic approaches targeting hepatocyte injury in ALF are hampered by the activation of distinct stimulus-dependent pathways, mechanism of cell death, and a limited therapeutic window. The apoptosis repressor with caspase recruitment domain (ARC) is a recently discovered death repressor that inhibits both death receptor and mitochondrial apoptotic signaling. Here, we investigated the in vivo effects of ARC fused with the transduction domain of human immunodeficiency virus 1 (HIV-1) (TAT-ARC) on Fas- and tumor necrosis factor (TNF)-mediated murine models of fulminant liver failure. Treatment with TAT-ARC protein completely abrogated otherwise lethal liver failure induced by Fas-agonistic antibody (Jo2), concanavalin A (ConA), or D-galactosamine/lipopolysaccharide (GalN/LPS) administration. Importantly, survival of mice was even preserved when TAT-ARC therapy was initiated in a delayed manner after stimulation with Jo2, ConA, or GalN/LPS. ARC blocked hepatocyte apoptosis by directly interacting with members of the death-inducing signaling complex. TNF-mediated liver damage was inhibited by two independent mechanisms: inhibition of jun kinase (JNK)-mediated TNF-α expression and prevention of hepatocyte apoptosis by inhibition of both death receptor and mitochondrial death signaling. We identified JNK as a novel target of ARC. ARC's caspase recruitment domain (CARD) directly interacts with JNK1 and JNK2, which correlates with decreased JNK activation and JNK-dependent TNF-α production. CONCLUSION: This work suggests that ARC confers hepatoprotection upstream and at the hepatocyte level. The efficacy of TAT-ARC protein transduction in multiple murine models of ALF demonstrates its therapeutic potential for reversing liver failure.
Assuntos
Proteínas do Citoesqueleto/genética , Terapia Genética/métodos , Falência Hepática Aguda/genética , Falência Hepática Aguda/terapia , Proteínas do Tecido Nervoso/genética , Proteínas Recombinantes de Fusão/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Animais , Apoptose/fisiologia , Caspases/química , Caspases/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Feminino , Hepatócitos/citologia , Hepatócitos/fisiologia , Falência Hepática Aguda/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Estrutura Terciária de Proteína , Transdução Genética/métodos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Paracrine cerebral Interleukin 6 (Il6) is relevant for stroke recovery, but systemic Il6 elevation may worsen outcome. Hence, paracrine Il6 response modulation within the neurovascular unit has emerged as an attractive therapeutic approach. Lithium modulates Il6 responses and improves stroke outcome. However, lithium may cause serious adverse effects. Here, we report that Zincfinger protein 580 (Zfp580) mediates the effects of lithium on Il6 signaling. In contrast to lithium, Zfp580 inactivation had no neurotoxic effects, and Zfp580 knock out mice showed no phenotypic changes in cognitive and motor function behavioral tests. We discovered that lithium and hypoxia disinhibited Il6 via Zfp580 suppression and post-translational modification by small ubiquitin-like modifier (SUMO). After transient middle cerebral artery occlusion, loss of Zfp580 reduced paracrine Il6 and increased Il6 trans-signaling. Aside from modulating Il6 signaling, Zfp580 loss improved endothelial resilience to ischemia, was highly neuroprotective resulting in smaller infarcts and enhanced use-dependent neuroplasticity, all of which led to improved functional outcome. In conclusion, inactivation of Zfp580 exerts positive effects on multiple key mechanisms without exhibiting relevant adverse side effects, making it potentially a more specific and effective treatment target for stroke recovery than lithium. To fully assess its potential, Zfp580 inhibitors must be developed.
Assuntos
Isquemia Encefálica , Acidente Vascular Cerebral , Camundongos , Animais , Interleucina-6 , Lítio , Fatores de Transcrição/metabolismo , Acidente Vascular Cerebral/tratamento farmacológico , Transdução de SinaisRESUMO
Cell-penetrating peptides like the cationic HIV1 TAT peptide are able to translocate across cell membranes and to carry molecular cargoes into the cellular interior. For most of these peptides, the biophysical mechanism of the membrane translocation is still quite unknown. We analyzed HIV1 TAT peptide binding and mobility within biological model membranes. To this end, we generated neutral and anionic giant unilamellar vesicles (GUVs) containing DPPC, DOPC, and cholesterol and containing DPPC, DOPC, cholesterol, and DPPS (DOPS), respectively. First, we characterized the mobility of fluorescently labeled lipids (TR-DHPE) within liquid-ordered and liquid-disordered lipid phases by single-molecule tracking, yielding a D(LO) of 0.6 +/- 0.05 microm(2)/s and a D(LD) of 2.5 +/- 0.05 microm(2)/s, respectively, as a reference. Fluorescently labeled TAT peptides accumulated on neutral GUVs but bound very efficiently to anionic GUVs. Single-molecule tracking revealed that HIV1 TAT peptides move on neutral and anionic GUV surfaces with a D(N,TAT) of 5.3 +/- 0.2 microm(2)/s and a D(A,TAT) of 3.3 +/- 0.2 mum(2)/s, respectively. TAT peptide diffusion was faster than fluorescent lipid diffusion, and also independent of the phase state of the membrane. We concluded that TAT peptides are not incorporated into but rather floating on lipid bilayers, but they immerged deeper into the headgroup domain of anionic lipids. The diffusion constants were not dependent on the TAT concentration ranging from 150 pM to 2 microM, indicating that the peptides were not aggregated on the membrane and not forming any "carpet".
Assuntos
Produtos do Gene tat/química , Produtos do Gene tat/metabolismo , Bicamadas Lipídicas/metabolismo , Modelos Biológicos , Peptídeos/química , Peptídeos/metabolismo , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismo , Ânions/metabolismo , Cátions/metabolismo , Corantes Fluorescentes/metabolismo , Produtos do Gene tat/fisiologia , HIV-1/química , HIV-1/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/química , Peptídeos/fisiologia , Permeabilidade , Fosfatidiletanolaminas/metabolismo , Transporte Proteico/fisiologia , Eletricidade Estática , ViscosidadeRESUMO
Phosphatase and tensin homolog (PTEN) signalling might influence neuronal survival after brain ischemia. However, the influence of the less studied longer variant termed PTEN-L (or PTENα) has not been studied to date. Therefore, we examined the translational variant PTEN-L in the context of neuronal survival. We identified PTEN-L by proteomics in murine neuronal cultures and brain lysates and established a novel model to analyse PTEN or PTEN-L variants independently in vitro while avoiding overexpression. We found that PTEN-L, unlike PTEN, localises predominantly in the cytosol and translocates to the nucleus 10-20 minutes after glutamate stress. Genomic ablation of PTEN and PTEN-L increased neuronal susceptibility to oxygen-glucose deprivation. This effect was rescued by expression of either PTEN-L indicating that both PTEN isoforms might contribute to a neuroprotective response. However, in direct comparison, PTEN-L replaced neurons were protected against ischemic-like stress compared to neurons expressing PTEN. Neurons expressing strictly nuclear PTEN-L NLS showed increased vulnerability, indicating that nuclear PTEN-L alone is not sufficient in protecting against stress. We identified mutually exclusive binding partners of PTEN-L or PTEN in cytosolic or nuclear fractions, which were regulated after ischemic-like stress. GRB2-associated-binding protein 2, which is known to interact with phosphoinositol-3-kinase, was enriched specifically with PTEN-L in the cytosol in proximity to the plasma membrane and their interaction was lost after glutamate exposure. The present study revealed that PTEN and PTEN-L have distinct functions in response to stress and might be involved in different mechanisms of neuroprotection.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Isquemia Encefálica/genética , Encéfalo/metabolismo , PTEN Fosfo-Hidrolase/genética , Acidente Vascular Cerebral/genética , Animais , Encéfalo/patologia , Isquemia Encefálica/patologia , Núcleo Celular/genética , Modelos Animais de Doenças , Proteína Adaptadora GRB2/genética , Regulação da Expressão Gênica/genética , Glucose/metabolismo , Humanos , Camundongos , Neurônios/metabolismo , Neurônios/patologia , Neuroproteção/genética , Oxigênio/metabolismo , Isoformas de Proteínas/genética , Proteômica/métodos , Transdução de Sinais/genética , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologiaRESUMO
In contrast to immortal cell lines, primary cells are hardly susceptible to intracellular delivery methods such as transfection. In this study, we evaluated the direct delivery of several cell-permeable peptides under noninvasive conditions into living primary adult rat cardiomyocytes. We specifically monitored the functional effects of a cell-permeable peptide containing the 15 amino acid N-terminal peptide from human ventricular light chain-1 (VLC-1) on contraction and intracellular Ca2+ signals after electrical stimulation in primary adult cardiomyocytes. The transducible VLC-1 variant was taken up by cardiomyocytes within 5 min with more than 95% efficiency and localized to sarcomeric structures. Analysis of the functional effects of the cell-permeable VLC-1 revealed an enhancement of the intrinsic contractility of cardiomyocytes without affecting the intracellular Ca2+. Therefore, peptide transduction mediated by cell-penetrating peptides represents not only a unique strategy to enhance heart muscle function with no secondary effect on intracellular Ca2+ but also an invaluable tool for the modulation and manipulation of protein interactions in general and in primary cells.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Cardiotônicos/farmacologia , Permeabilidade da Membrana Celular , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Cadeias Leves de Miosina/farmacologia , Fragmentos de Peptídeos/farmacologia , Miosinas Ventriculares/farmacologia , Animais , Cardiotônicos/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Estimulação Elétrica , Humanos , Microscopia Confocal , Miócitos Cardíacos/metabolismo , Cadeias Leves de Miosina/metabolismo , Fragmentos de Peptídeos/metabolismo , Ratos , Ratos Endogâmicos WKY , Sarcômeros/efeitos dos fármacos , Sarcômeros/metabolismo , Miosinas Ventriculares/metabolismoRESUMO
Cell-penetrating peptides (CPPs) are capable of introducing a wide range of cargoes into living cells. Descriptions of the internalization process vary from energy-independent cell penetration of membranes to endocytic uptake. To elucidate whether the mechanism of entry of CPP constructs might be influenced by the properties of the cargo, we used time lapse confocal microscopy analysis of living mammalian cells to directly compare the uptake of the well-studied CPP TAT fused to a protein (>50 amino acids) or peptide (<50 amino acids) cargo. We also analyzed various constructs for their subcellular distribution and mobility after the internalization event. TAT fusion proteins were taken up largely into cytoplasmic vesicles whereas peptides fused to TAT entered the cell in a rapid manner that was dependent on membrane potential. Despite their accumulation in the nucleolus, photobleaching of TAT fusion peptides revealed their mobility. The bioavailability of internalized TAT peptides was tested and confirmed by the strong inhibitory effect on cell cycle progression of two TAT fusion peptides derived from the tumor suppressor p21(WAF/Cip) and DNA Ligase I measured in living cells.
Assuntos
Células/metabolismo , Produtos do Gene tat/metabolismo , Fragmentos de Peptídeos/metabolismo , Sequência de Aminoácidos , Animais , Disponibilidade Biológica , Transporte Biológico , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/química , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Humanos , Integrases/metabolismo , Cinética , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
In this study we investigated whether the expression of N-terminal myosin light chain-1 (MLC-1) peptides could improve the intrinsic contractility of the whole heart. We generated transgenic rats (TGR) that overexpressed minigenes encoding the N-terminal 15 amino acids of human atrial MLC-1 (TGR/hALC-1/1-15, lines 7475 and 3966) or human ventricular MLC-1 (TGR/hVLC-1/1-15, lines 6113 and 6114) isoforms in cardiomyocytes. Synthetic N-terminal peptides revealed specific actin binding, with a significantly (P<0.01) lower dissociation constant (K(D)) for the hVLC-1/1-15-actin complex compared with the K(D) value of the hALC-1/1-15-actin complex. Using synthetic hVLC-1/1-15 as a TAT fusion peptide labeled with the fluorochrome TAMRA, we observed specific accumulation of the N-terminal MLC-1 peptide at the sarcomere predominantly within the actin-containing I-band, but also within the actin-myosin overlap zone (A-band) in intact adult cardiomyocytes. For the first time we show that the expression of N-terminal human MLC-1 peptides in TGR (range: 3-6 muM) correlated positively with significant (P<0.001) improvements of the intrinsic contractile state of the isolated perfused heart (Langendorff mode): systolic force generation, as well as the rates of both force generation and relaxation, rose in TGR lines that expressed the transgenic human MLC-1 peptide, but not in a TGR line with undetectable transgene expression levels. The positive inotropic effect of MLC-1 peptides occurred in the absence of a hypertrophic response. Thus, expression of N-terminal domains of MLC-1 represent a valuable tool for the treatment of the failing heart.
Assuntos
Coração/fisiologia , Contração Miocárdica/genética , Contração Miocárdica/fisiologia , Cadeias Leves de Miosina/química , Cadeias Leves de Miosina/metabolismo , Actinas , Animais , Animais Geneticamente Modificados , Regulação da Expressão Gênica , Terapia Genética , Humanos , Masculino , Miócitos Cardíacos/metabolismo , Cadeias Leves de Miosina/genética , Ligação Proteica , Ratos , Ratos Endogâmicos WKYRESUMO
The nucleolus is the hallmark of nuclear compartmentalization and has been shown to exert multiple roles in cellular metabolism besides its main function as the place of rRNA synthesis and assembly of ribosomes. Nucleolar proteins dynamically localize and accumulate in this nuclear compartment relative to the surrounding nucleoplasm. In this study, we have assessed the molecular requirements that are necessary and sufficient for the localization and accumulation of peptides and proteins inside the nucleoli of living cells. The data showed that positively charged peptide entities composed of arginines alone and with an isoelectric point at and above 12.6 are necessary and sufficient for mediating significant nucleolar accumulation. A threshold of 6 arginines is necessary for peptides to accumulate in nucleoli, but already 4 arginines are sufficient when fused within 15 amino acid residues of a nuclear localization signal of a protein. Using a pH sensitive dye, we found that the nucleolar compartment is particularly acidic when compared to the surrounding nucleoplasm and, hence, provides the ideal electrochemical environment to bind poly-arginine containing proteins. In fact, we found that oligo-arginine peptides and GFP fusions bind RNA in vitro. Consistent with RNA being the main binding partner for arginines in the nucleolus, we found that the same principles apply to cells from insects to man, indicating that this mechanism is highly conserved throughout evolution.
Assuntos
Nucléolo Celular/metabolismo , Transporte Proteico , Animais , Arginina/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Células HEK293 , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Sinais de Localização Nuclear/metabolismo , Proteínas Nucleares/metabolismo , TransfecçãoRESUMO
Proliferating Cell Nuclear Antigen (PCNA) is a key protein in DNA replication and repair. The dynamics of replication and repair in live cells is usually studied introducing translational fusions of PCNA. To obviate the need for transfection and bypass the problem of difficult to transfect and/or short lived cells, we have now developed a cell permeable replication and/or repair marker. The design of this marker has three essential molecular components: (1) an optimized artificial PCNA binding peptide; (2) a cell-penetrating peptide, derived from the HIV-1 Trans Activator of Transcription (TAT); (3) an in vivo cleavable linker, linking the two peptides. The resulting construct was taken up by human, hamster and mouse cells within minutes of addition to the media. Inside the cells, the cargo separated from the vector peptide and bound PCNA effectively. Both replication and repair sites could be directly labeled in live cells making it the first in vivo cell permeable peptide marker for these two fundamental cellular processes. Concurrently, we also introduced a quick peptide based PCNA staining method as an alternative to PCNA antibodies for immunofluorescence applications. In summary, we present here a versatile tool to instantaneously label repair and replication processes in fixed and live cells.
Assuntos
Reparo do DNA/genética , Replicação do DNA/genética , Antígeno Nuclear de Célula em Proliferação/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Células 3T3 , Animais , Ciclo Celular/genética , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/genética , Peptídeos Penetradores de Células/metabolismo , Cricetinae , HIV-1/química , Células HeLa , Humanos , Camundongos , Antígeno Nuclear de Célula em Proliferação/química , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ligação Proteica , Conformação Proteica , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Small ubiquitin-like modifier (SUMO)2/3 but not SUMO1 conjugation is activated after transient cerebral ischemia. To investigate its function, we blocked neuronal SUMO2/3 translation through lentiviral microRNA delivery in primary cortical neurons. Viability was unaffected by SUMO2/3 silencing unless neurons were stressed by transient oxygen-glucose deprivation (OGD). Both 15 and 45 minutes of OGD were tolerated by control microRNA-expressing neurons but damaged >60% of neurons expressing SUMO2/3 microRNA. Damaging OGD (75 minutes) increased neuronal loss to 54% (control microRNA) and to 99% (SUMO2/3 microRNA). This suggests that activation of SUMO2/3 conjugation is an endogenous neuroprotective stress response.
Assuntos
Ataque Isquêmico Transitório/prevenção & controle , Neurônios/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Técnicas de Cultura de Células , Hipóxia Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Vetores Genéticos , Glucose/metabolismo , Imuno-Histoquímica , Ataque Isquêmico Transitório/metabolismo , Ataque Isquêmico Transitório/patologia , Lentivirus/genética , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/administração & dosagem , MicroRNAs/genética , MicroRNAs/farmacologia , Neurônios/patologia , Oxigênio/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Sumoilação/efeitos dos fármacos , Sumoilação/genética , Ubiquitinas/genéticaRESUMO
In addition to endocytosis-mediated cellular uptake, hydrophilic cell-penetrating peptides are able to traverse biological membranes in a non-endocytic mode termed transduction, resulting in immediate bioavailability. Here we analysed structural requirements for the non-endocytic uptake mode of arginine-rich cell-penetrating peptides, by a combination of live-cell microscopy, molecular dynamics simulations and analytical ultracentrifugation. We demonstrate that the transduction efficiency of arginine-rich peptides increases with higher peptide structural rigidity. Consequently, cyclic arginine-rich cell-penetrating peptides showed enhanced cellular uptake kinetics relative to their linear and more flexible counterpart. We propose that guanidinium groups are forced into maximally distant positions by cyclization. This orientation increases membrane contacts leading to enhanced cell penetration.
Assuntos
Arginina/química , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/metabolismo , Células/metabolismo , Guanidina/química , Animais , Arginina/metabolismo , Transporte Biológico , Linhagem Celular , Membrana Celular/química , Membrana Celular/metabolismo , Células/química , Guanidina/metabolismo , Cinética , Camundongos , Estrutura MolecularRESUMO
Arginine-rich peptides are a subclass of cell-penetrating peptides that are taken up by living cells and can be detected freely diffusing inside the cytoplasm and nucleoplasm. This phenomenon has been attributed to either an endocytic mode of uptake and a subsequent release from vesicles or to direct membrane penetration (transduction). To distinguish between both possibilities, we have blocked endocytic pathways suggested to be involved in uptake of cell-penetrating peptides. We have then monitored by confocal microscopy the uptake and distribution of the cell-penetrating transactivator of transcription (TAT) peptide into living mammalian cells over time. To prevent side effects of chemical inhibitors, we used genetically engineered cells as well as different temperature. We found that a knockdown of clathrin-mediated endocytosis and a knock-out of caveolin-mediated endocytosis did not affect the ability of TAT to enter cells. In addition, the TAT peptide showed the same intracellular distribution throughout the cytoplasm and nucleus as in control cells. Even incubation of cells at 4 degrees C did not abrogate TAT uptake nor change its intracellular distribution. We therefore conclude that this distribution results from TAT peptide that directly penetrated (transduced) the plasma membrane. The formation of nonselective pores is unlikely, because simultaneously added fluorophores were not taken up together with the TAT peptide. In summary, although the frequency and kinetics of TAT transduction varied between cell types, it was independent of endocytosis.
Assuntos
Caveolinas/metabolismo , Núcleo Celular/metabolismo , Clatrina/metabolismo , Endocitose/fisiologia , Produtos do Gene tat/metabolismo , Peptídeos/metabolismo , Células 3T3 , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Arginina/genética , Arginina/metabolismo , Caveolinas/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Núcleo Celular/genética , Clatrina/genética , Cricetinae , Citoplasma/genética , Citoplasma/metabolismo , Produtos do Gene tat/genética , Camundongos , Camundongos Knockout , Peptídeos/genéticaRESUMO
Cell penetrating peptides (CPPs) are useful tools to deliver low-molecular-weight cargoes into cells; however, their mode of uptake is still controversial. The most efficient CPPs belong to the group of arginine-rich peptides, but a systematic assessment of their potential toxicity is lacking. In this study we combined data on the membrane translocation abilities of oligo-arginines in living cells as a function of their chain length, concentration, stability and toxicity. Using confocal microscopy analysis of living cells we evaluated the transduction frequency of the L-isoforms of oligo-arginines and lysines and then monitored their associated toxicity by concomitant addition of propidium iodide. Whereas lysines showed virtually no transduction, the transduction ability of arginines increased with the number of consecutive residues and the peptide concentration, with L-R9 and L-R10 performing overall best. We further compared the L- and D-R9 isomers and found that the D-isoform always showed a higher transduction as compared to the L-counterpart in all cell types. Notably, the transduction difference between D- and L-forms was highly variable between cell types, emphasizing the need for protease-resistant peptides as vectors for drug delivery. Real-time kinetic analysis of the D- and L-isomers applied simultaneously to the cells revealed a much faster transduction for the D-variant. The latter underlies the fact that the isomers do not mix, and penetration of one peptide does not perturb the membrane in a way that gives access to the other peptide. Finally, we performed short- and long-term cell viability and cell cycle progression analyses with the protease-resistant D-R9. Altogether, our results identified concentration windows with low toxicity and high transduction efficiency, resulting in fully bioavailable intracellular peptides.
Assuntos
Arginina/metabolismo , Permeabilidade da Membrana Celular , Membrana Celular/metabolismo , Oligonucleotídeos Antissenso/metabolismo , Sequência de Aminoácidos , Animais , Arginina/química , Arginina/genética , Transporte Biológico , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultura , Cães , Relação Dose-Resposta a Droga , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Células HeLa , Humanos , Indicadores e Reagentes , Lisina/química , Lisina/genética , Lisina/metabolismo , Masculino , Microscopia Confocal , Mioblastos/metabolismo , Miócitos Cardíacos/metabolismo , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/genética , Propídio , Isoformas de Proteínas/metabolismo , Ratos , Ratos Endogâmicos WKY , Ratos Wistar , Fatores de Tempo , Transdução GenéticaRESUMO
Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae. Infection by EAV requires the release of the viral genome by fusion with the respective target membrane of the host cell. We have investigated the entry pathway of EAV into Baby Hamster Kidney cells (BHK). Infection of cells assessed by the plaque reduction assay was strongly inhibited by substances which interfere with clathrin-dependent endocytosis and by lysosomotropic compounds. Furthermore, infection of BHK cells was suppressed when clathrin-dependent endocytosis was inhibited by expression of antisense RNA of the clathrin-heavy chain before infection. These results strongly suggest that EAV is taken up via clathrin-dependent endocytosis and is delivered to acidic endosomal compartments.
Assuntos
Clatrina/metabolismo , Endocitose/fisiologia , Endossomos/virologia , Equartevirus/fisiologia , Animais , Infecções por Arterivirus/metabolismo , Linhagem Celular , Cricetinae , Endocitose/efeitos dos fármacos , Endossomos/metabolismo , Equartevirus/genética , Equartevirus/metabolismoRESUMO
In the recent molecular and cell biological research, there is an increasing need for labeling of subcellular structures in living cells. Here, we present the use of a fluorescently labeled cell penetrating peptide for fast labeling of nucleoli in living cells of different species and origin. We show that the short peptide with ten amino acids was able to cross cellular membranes and reach the nucleolar target sites, thereby marking this subnuclear structure in living cells. The treatment of cells with actinomycin D and labeling of B23 protein and fibrillarin provided evidence for a localization to the granular component of the nucleolus. The fluorescently conjugated nucleolar marker could be used in combination with different fluorophores like fluorescent proteins or DNA dyes, and nucleolar labeling was also preserved during fixation and staining of the cells. Furthermore, we observed a high stability of the label in long-term studies over 24 h as well as no effect on the cellular viability and proliferation and on rDNA transcription. The transducible nucleolar marker is therefore a valuable molecular tool for cell biology that allows a fast and easy labeling of this structure in living cells.