RESUMO
Alzheimer's disease (AD) is characterized by amyloid beta (Aß) plaques and neurofibrillary tangles. AD drug development has been limited due to the presence of the blood-brain barrier (BBB), which prevents efficient uptake of therapeutics into the brain. To solve this problem, we used trans-activator of transcription (TAT)-transducing domain and added the human serum albumin (HSA) carrier to increase the half-life of the drug within the body. In addition, we included the protein of interest for lowering Aß deposition and/or neurofibrillary tangles. We made HSA fusion protein (designated AL04) which contains Cystatin C (CysC) as core mechanism of action moiety in the construct containing tandem repeat TAT (dTAT). After purification of 80KDa AL04, we investigate the therapeutic potential of AL04 in vitro and AD mouse model Tg2576. We evaluated the permeability of AL04 through the BBB using a cell-basedhuman BBB model and show that dTAT plays a role in facilitating the delivery of 80 kDa protein. We found out that AL04 attenuates Aß-induced neurotoxicity in PC12 cells. In Tg2576 mice brain, Aß plaques were dramatically reduced in AL04 treated mice. These data suggest that BBB-crossing albumin fusion protein AL04 with CysC active moiety can be a disease modifying treatment for AD.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Cistatina C/farmacocinética , Portadores de Fármacos/farmacocinética , Albumina Sérica Humana/farmacocinética , Animais , Barreira Hematoencefálica , Encéfalo/metabolismo , Encéfalo/patologia , Cistatina C/administração & dosagem , Portadores de Fármacos/química , Produtos do Gene tat/farmacocinética , Humanos , Camundongos , Células PC12 , Ratos , Albumina Sérica Humana/químicaRESUMO
Schizophrenia is a chronic debilitating neuropsychiatric disorder that affects about 1 % of the population. Dystrobrevin-binding protein 1 (DTNBP1 or dysbindin) is one of the Research Domain Constructs (RDoC) associated with cognition and is significantly reduced in the brain of schizophrenia patients. To further understand the molecular underpinnings of pathogenesis of schizophrenia, we have performed microarray analyses of the hippocampi from dysbindin knockout mice, and found that genes involved in the lipogenic pathway are suppressed. Moreover, we discovered that maturation of a master transcriptional regulator for lipid synthesis, sterol regulatory element binding protein-1 (SREBP1) is induced by neuronal activity, and is required for induction of the immediate early gene ARC (activity-regulated cytoskeleton-associated protein), necessary for synaptic plasticity and memory. We found that nuclear SREBP1 is dramatically reduced in dysbindin-1 knockout mice and postmortem brain tissues from human patients with schizophrenia. Furthermore, activity-dependent maturation of SREBP1 as well as ARC expression were attenuated in dysbindin-1 knockout mice, and these deficits were restored by an atypical antipsychotic drug, clozapine. Together, results indicate an important role of dysbindin-1 in neuronal activity induced SREBP1 and ARC, which could be related to cognitive deficits in schizophrenia.
Assuntos
Disfunção Cognitiva/metabolismo , Disbindina/deficiência , Neurônios/metabolismo , Esquizofrenia/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/biossíntese , Idoso , Idoso de 80 Anos ou mais , Animais , Disfunção Cognitiva/genética , Disfunção Cognitiva/psicologia , Disbindina/genética , Feminino , Redes Reguladoras de Genes/fisiologia , Humanos , Estudos Longitudinais , Masculino , Camundongos , Camundongos Knockout , Técnicas de Cultura de Órgãos , Células PC12 , Distribuição Aleatória , Ratos , Esquizofrenia/genética , Psicologia do Esquizofrênico , Proteína de Ligação a Elemento Regulador de Esterol 1/genéticaRESUMO
NAD(+) plays essential roles in cellular energy homoeostasis and redox state, functioning as a cofactor along the glycolysis and citric acid cycle pathways. Recent discoveries indicated that, through the NAD(+)-consuming enzymes, this molecule may also be involved in many other cellular and biological outcomes such as chromatin remodelling, gene transcription, genomic integrity, cell division, calcium signalling, circadian clock and pluripotency. Poly(ADP-ribose) polymerase 1 (PARP1) is such an enzyme and dysfunctional PARP1 has been linked with the onset and development of various human diseases, including cancer, aging, traumatic brain injury, atherosclerosis, diabetes and inflammation. In the present study, we showed that overexpressed acyl-CoA-binding domain containing 3 (ACBD3), a Golgi-bound protein, significantly reduced cellular NAD(+) content via enhancing PARP1's polymerase activity and enhancing auto-modification of the enzyme in a DNA damage-independent manner. We identified that extracellular signal-regulated kinase (ERK)1/2 as well as de novo fatty acid biosynthesis pathways are involved in ACBD3-mediated activation of PARP1. Importantly, oxidative stress-induced PARP1 activation is greatly attenuated by knocking down the ACBD3 gene. Taken together, these findings suggest that ACBD3 has prominent impacts on cellular NAD(+) metabolism via regulating PARP1 activation-dependent auto-modification and thus cell metabolism and function.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas de Membrana/biossíntese , NAD/metabolismo , NAD/fisiologia , Poli(ADP-Ribose) Polimerases/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Dano ao DNA , Ativação Enzimática/genética , Células HEK293 , Células HeLa , Humanos , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana/genética , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , NAD/genética , Células NIH 3T3 , Estresse Oxidativo/fisiologia , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/genéticaRESUMO
Implantation of self-expanding metal stents (SEMS) is palliation for patients suffering from inoperable malignant obstructions associated with biliary and pancreatic cancers. Chemotherapeutic agent-eluting stents have been developed because SEMS are susceptible to occlusion by tumor in-growth. We reported recently that paclitaxel-eluting SEMS provide enhanced local drug delivery in an animal model. However, little is known about the molecular mechanisms by which paclitaxel-eluting stents attenuate tumor growth. We investigated the signal transduction pathways underlying the antiproliferative effects of a paclitaxel-eluting membrane (PEM) implanted in pancreatic/cholangiocarcinoma tumor bearing nude mice. Molecular and cellular alterations were analyzed in the PEM-implanted pancreatic/cholangiocarcinoma xenograft tumors by Western blot, immunoprecipitation, and immunofluorescence. The quantities of paclitaxel released into the tumor and plasma were determined by liquid chromatography-tandem mass spectroscopy. Paclitaxel from the PEM and its diffusion into the tumor inhibited angiogenesis, which involved suppression of mammalian target of rapamycin (mTOR) through regulation of hypoxia inducible factor (HIF-1) and increased apoptosis. Moreover, implantation of the PEM inhibited tumor-stromal interaction-related expression of proteins such as CD44, SPARC, matrix metalloproteinase-2, and vimentin. Local delivery of paclitaxel from a PEM inhibited growth of pancreatic/cholangiocarcinoma tumors in nude mice by suppressing angiogenesis via the mTOR and inducing apoptosis signal pathway.
RESUMO
Metformin is a biguanide drug that is widely prescribed for type 2 diabetes. Metformin suppresses hepatic gluconeogenesis and increases fatty acid oxidation. Although studies have suggested that metformin acts, at least in part, via activation of the liver kinase B1 (LKB1)/AMP-activated protein kinase (AMPK) pathway, the specific molecular mechanisms underlying metformin's regulation of glucose and lipid metabolism have not been well delineated. Recently, we have shown that inositol polyphosphate multikinase (IPMK) plays an important role in cellular energy metabolism and glucose-mediated AMPK regulation. Here we investigated the role of IPMK in metformin-induced AMPK activation. We observed that metformin-mediated activation of AMPK was impaired in the absence of IPMK. Overexpression of wild-type IPMK was sufficient to restore LKB1-AMPK activation by either metformin or AICAR in IPMK(-/-) murine embryonic fibroblast cells, suggesting that IPMK may act as an upstream regulator of LKB1-AMPK signaling in response to metformin. Moreover, this regulation was mediated by protein-protein interaction between IPMK and LKB1 as a dominant-negative peptide, which abrogates this interaction, attenuated metformin's ability to activate AMPK. Our data demonstrate that IPMK plays an important role in LKB1/AMPK signaling and may be targeted for treatment of metabolic diseases.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Hipoglicemiantes/farmacologia , Metformina/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Linhagem Celular Tumoral , Diabetes Mellitus Tipo 2/tratamento farmacológico , Metabolismo Energético , Ácidos Graxos/metabolismo , Técnicas de Inativação de Genes , Gluconeogênese/efeitos dos fármacos , Glucose/metabolismo , Células HEK293 , Células HeLa , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Oxirredução/efeitos dos fármacos , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/biossíntese , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Ribonucleotídeos/farmacologiaRESUMO
Insulin resistance and other features of the metabolic syndrome are increasingly recognized for their effects on cognitive health. To ascertain mechanisms by which this occurs, we fed mice a very high fat diet (60% kcal by fat) for 17days or a moderate high fat diet (HFD, 45% kcal by fat) for 8weeks and examined changes in brain insulin signaling responses, hippocampal synaptodendritic protein expression, and spatial working memory. Compared to normal control diet mice, cerebral cortex tissues of HFD mice were insulin-resistant as evidenced by failed activation of Akt, S6 and GSK3ß with ex-vivo insulin stimulation. Importantly, we found that expression of brain IPMK, which is necessary for mTOR/Akt signaling, remained decreased in HFD mice upon activation of AMPK. HFD mouse hippocampus exhibited increased expression of serine-phosphorylated insulin receptor substrate 1 (IRS1-pS(616)), a marker of insulin resistance, as well as decreased expression of PSD-95, a scaffolding protein enriched in post-synaptic densities, and synaptopodin, an actin-associated protein enriched in spine apparatuses. Spatial working memory was impaired as assessed by decreased spontaneous alternation in a T-maze. These findings indicate that HFD is associated with telencephalic insulin resistance and deleterious effects on synaptic integrity and cognitive behaviors.
Assuntos
Encéfalo/metabolismo , Dendritos/metabolismo , Dieta Hiperlipídica/efeitos adversos , Resistência à Insulina , Memória Espacial/fisiologia , Sinapses/metabolismo , Animais , Hiperglicemia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células PC12 , Ratos , Transdução de SinaisRESUMO
Ras homolog enriched in striatum (Rhes), is a highly conserved small guanosine-5'-triphosphate (GTP) binding protein belonging to the Ras superfamily. Rhes is involved in the dopamine receptor-mediated signaling and behavior though adenylyl cyclase. The striatum-specific GTPase share a close homology with Dexras1, which regulates iron trafficking in the neurons when activated though the post-translational modification called s-nitrosylation by nitric oxide (NO). We report that Rhes physiologically interacted with Peripheral benzodiazepine receptor-associated protein7 and participated in iron uptake via divalent metal transporter 1 similar to Dexras1. Interestingly, Rhes is not S-nitrosylated by NO-treatment, however phosphorylated by protein kinase A at the site of serine-239. Two Rhes mutants - the phosphomimetic form (serine 239 to aspartic acid) and constitutively active form (alanine 173 to valine) - displayed an increase in iron uptake compared to the wild-type Rhes. These findings suggest that Rhes may play a crucial role in striatal iron homeostasis.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Ferro/metabolismo , Mutação/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/genética , Proteínas de Transporte de Cátions/metabolismo , Desferroxamina/farmacologia , Compostos Férricos/farmacologia , Proteínas de Ligação ao GTP/genética , Glutationa Transferase/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Imunoprecipitação , Proteínas Reguladoras de Ferro/genética , Proteínas Reguladoras de Ferro/metabolismo , Proteínas de Membrana/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Compostos de Amônio Quaternário/farmacologia , Serina/metabolismo , Sideróforos/farmacologia , TransfecçãoRESUMO
Dexras1, a small G-protein localized predominantly to the brain, is transcriptionally upregulated by the synthetic glucocorticoid dexamethasone. It has close homology to the Ras subfamily but differs in that Dexras1 contains an extended 7 kDa C-terminal tail. Previous studies in our laboratory showed that NMDA receptor activation, via NO and Dexras1, physiologically stimulates DMT1, the major iron importer. A membrane-permeable iron chelator substantially reduces NMDA excitotoxicity, suggesting that Dexras1-mediated iron influx plays a crucial role in NMDA/NO-mediated cell death. We here report that iron influx is elicited by nitric oxide but not by other proapoptotic stimuli, such as H2O2 or staurosporine. Deletion of Dexras1 in mice attenuates NO-mediated cell death in dissociated primary cortical neurons and retinal ganglion cells in vivo. Thus, Dexras1 appears to mediate NMDA-elicited neurotoxicity via NO and iron influx.
Assuntos
Córtex Cerebral/fisiologia , Ácido Glutâmico/toxicidade , N-Metilaspartato/toxicidade , Células Ganglionares da Retina/fisiologia , Proteínas ras/fisiologia , Animais , Córtex Cerebral/efeitos dos fármacos , Ácido Glutâmico/fisiologia , Células HEK293 , Humanos , Ferro/metabolismo , Ferro/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Monoméricas de Ligação ao GTP/deficiência , Proteínas Monoméricas de Ligação ao GTP/fisiologia , N-Metilaspartato/fisiologia , Óxido Nítrico/fisiologia , Óxido Nítrico/toxicidade , Células PC12 , Ratos , Proteínas ras/deficiênciaRESUMO
Imbalance of lipid metabolism has been linked with pathogenesis of a variety of human pathological conditions such as diabetes, obesity, cancer and neurodegeneration. Sterol regulatory element binding proteins (SREBPs) are the master transcription factors controlling the homeostasis of fatty acids and cholesterol in the body. Transcription, expression, and activity of SREBPs are regulated by various nutritional, hormonal or stressful stimuli, yet the molecular and cellular mechanisms involved in these adaptative responses remains elusive. In the present study, we found that overexpressed acyl-CoA binding domain containing 3 (ACBD3), a Golgi-associated protein, dramatically inhibited SREBP1-sensitive promoter activity of fatty acid synthase (FASN). Moreover, lipid deprivation-stimulated SREBP1 maturation was significantly attenuated by ACBD3. With cell fractionation, gene knockdown and immunoprecipitation assays, it was showed that ACBD3 blocked intracellular maturation of SREBP1 probably through directly binding with the lipid regulator rather than disrupted SREBP1-SCAP-Insig1 interaction. Further investigation revealed that acyl-CoA domain-containing N-terminal sequence of ACBD3 contributed to its inhibitory effects on the production of nuclear SREBP1. In addition, mRNA and protein levels of FASN and de novo palmitate biosynthesis were remarkably reduced in cells overexpressed with ACBD3. These findings suggest that ACBD3 plays an essential role in maintaining lipid homeostasis via regulating SREBP1's processing pathway and thus impacting cellular lipogenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Ácido Graxo Sintases/metabolismo , Regulação da Expressão Gênica/fisiologia , Homeostase/fisiologia , Metabolismo dos Lipídeos/fisiologia , Proteínas de Membrana/metabolismo , Conformação Proteica , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Fracionamento Celular , Ácido Graxo Sintases/genética , Técnicas de Silenciamento de Genes , Humanos , Imunoprecipitação , Luciferases , Proteínas de Membrana/químicaRESUMO
The Rhes/RASD2 GTPase complex is involved in dopamine D1/D2 receptor-mediated signaling and behavior. This GTP binding protein belongs to the RAS superfamily, along with Dexras1/RASD1, and is primarily expressed in the striatum. RASDs differ from typical small GTPases as they have an extended C-terminal tail of roughly 7 kDa. Previously, it has been shown that dopamine depletion reduces Rhes mRNA expression in the brain. Here we show that Rhes interacts with p85, the regulatory subunit of PI3K. Specifically, the C-terminal unique tail region of Rhes is responsible for this interaction. The interaction between p85 and the C-terminal region of Rhes is enhanced upon growth factor treatment in vitro, while AKT translocation to the membrane is facilitated in the presence of Rhes or the Rhes-p85 complex. These findings suggest that Rhes is a novel striatal regulator of the AKT-mediated pathway in the striatum.
Assuntos
Corpo Estriado/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas Proto-Oncogênicas c-akt/fisiologia , Animais , Membrana Celular/metabolismo , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Fatores de Crescimento de Fibroblastos/farmacologia , Proteínas de Ligação ao GTP/genética , Células HEK293 , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , Células PC12 , Fosforilação , Fator de Crescimento Derivado de Plaquetas/farmacologia , Transporte Proteico , Ratos , Transdução de Sinais , TransfecçãoRESUMO
Fenretinide (4-HPR) cytotoxicity relative to glutathione levels in pediatric acute lymphoblastic leukemia cell lines cultured at bone marrow level hypoxia (5% O2) is evaluated. 4-HPR cytotoxicity correlated with reactive oxygen species generation (P < 0.001),but not with levels of intracellular glutathione, g-glutamylcysteine synthase, or glutathione peroxidase. Buthionine sulfoximine (BSO)reduced glutathione levels in 10 cell lines (P < 0.001), but 4-HPR þ BSO was markedly synergistic in only 1 of 10 lines. Pretreatment with N-acetylcysteine increased glutathione (P < 0.02)but did not alter 4-HPR cytotoxicity. Our data suggest that 4-HPR cytotoxicity is independent of glutathione under physiologic oxygen tension.
Assuntos
Antineoplásicos/farmacologia , Fenretinida/farmacologia , Glutationa/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The AMP-activated kinase (AMPK) senses the energy status of cells and regulates fuel availability, whereas hypothalamic AMPK regulates food intake. We report that inositol polyphosphate multikinase (IPMK) regulates glucose signaling to AMPK in a pathway whereby glucose activates phosphorylation of IPMK at tyrosine 174 enabling the enzyme to bind to AMPK and regulate its activation. Thus, refeeding fasted mice rapidly and markedly stimulates transcriptional enhancement of IPMK expression while down-regulating AMPK. Also, AMPK is up-regulated in mice with genetic depletion of hypothalamic IPMK. IPMK physiologically binds AMPK, with binding enhanced by glucose treatment. Regulation by glucose of phospho-AMPK in hypothalamic cell lines is prevented by blocking AMPK-IPMK binding. These findings imply that IPMK inhibitors will be beneficial in treating obesity and diabetes.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Regulação da Expressão Gênica/fisiologia , Glucose/metabolismo , Hipotálamo/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Transdução de Sinais/fisiologia , Animais , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Imunoprecipitação , Camundongos , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Tirosina/metabolismoRESUMO
PURPOSE: RFB4 (dsFv)-PE38 (BL22) is a recombinant immunotoxin containing an anti-CD22 (Fv) fused to truncated Pseudomonas exotoxin A, which induces a high complete remission rate in patients with purine analogue-resistant hairy cell leukemia. HA22 is a mutant of BL22 with mutations in heavy-chain CDR3 resulting in increased cytotoxic activity. Our goal was to improve the activity of HA22. EXPERIMENTAL DESIGN: Arg(490), which is located in the catalytic domain (III) of the immunotoxin HA22, was mutated to alanine. Purified immunotoxins were produced and tested for cytotoxic activity in cell culture and for antitumor activity and nonspecific toxicity in mice. ADP-ribosylation activity was also measured. RESULTS: HA22 (R490A) is approximately 2-fold more cytotoxic than HA22 on several CD22-positive cell lines. When injected i.v., HA22 (R490A) has more potent antitumor activity than HA22 against CA46 tumors in mice. HA22 and HA22 (R490A) have similar LD(50)s (approximately 1.3 mg/kg) and similar plasma half-lives. The R490A mutation also improved the cytotoxicity of the antimesothelin recombinant immunotoxin SS1 (dsFv)-PE38 (SS1P). In vitro ADP-ribosylation assays show that HA22 R490A has increased activity. Increased cytotoxic activity is probably related to this increase in ADP-ribosylation activity. CONCLUSION: Protein engineering can be used to increase the efficacy of recombinant immunotoxins. Because HA22 (R490A) has increased antitumor activity without increased animal toxicity, immunotoxins with this mutation are candidates for clinical development.