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1.
Mol Biol (Mosk) ; 53(6): 1049-1056, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31876283

RESUMO

Zinc ions and glycosaminoglycans (GAGs) are found in amyloid deposits and are known to modulate the ß-amyloid peptide (Aß) aggregation, which is thought to be a key event in the pathogenesis of Alzheimer's disease (AD). Correlation spectroscopy was used to study how the H6R and D7H mutations of the metal-binding domain (MBD) of Aß42 affect the modulation of its zinc-induced aggregation by the model GAG heparin. The H6R mutation was shown to decrease and the D7H mutation to increase the Aß42 propensity to aggregate in the presence of zinc ions. In addition, H6R diminished and D7H enhanced the modulating effect of heparin. The difference in the heparin-dependent modulation was associated with coordination of zinc ions within the MBDs of the mutant peptides. The findings indicate that anion-binding sites formed by complexes of zinc ions with the Aß MBD play an essential role in the interaction of zinc-induced Aß aggregates with heparin.


Assuntos
Peptídeos beta-Amiloides/efeitos dos fármacos , Peptídeos beta-Amiloides/genética , Heparina/farmacologia , Mutação , Fragmentos de Peptídeos/efeitos dos fármacos , Fragmentos de Peptídeos/genética , Agregação Patológica de Proteínas/genética , Zinco/farmacologia , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/química , Humanos , Fragmentos de Peptídeos/química
2.
Mol Cell Biol ; 39(22)2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31501275

RESUMO

The MYC oncogene is upregulated in human cancers by translocation, amplification, and mutation of cellular pathways that regulate Myc. Myc/Max heterodimers bind to E box sequences in the promoter regions of genes and activate transcription. The MYC inhibitor Omomyc can reduce the ability of MYC to bind specific box sequences in promoters of MYC target genes by binding directly to E box sequences as demonstrated by chromatin immunoprecipitation (CHIP). Here, we demonstrate by both a proximity ligation assay (PLA) and double chromatin immunoprecipitation (ReCHIP) that Omomyc preferentially binds to Max, not Myc, to mediate inhibition of MYC-mediated transcription by replacing MYC/MAX heterodimers with Omomyc/MAX heterodimers. The formation of Myc/Max and Omomyc/Max heterodimers occurs cotranslationally; Myc, Max, and Omomyc can interact with ribosomes and Max RNA under conditions in which ribosomes are intact. Taken together, our data suggest that the mechanism of action of Omomyc is to bind DNA as either a homodimer or a heterodimer with Max that is formed cotranslationally, revealing a novel mechanism to inhibit the MYC oncogene. We find that in vivo, Omomyc distributes quickly to kidneys and liver and has a short effective half-life in plasma, which could limit its use in vivo.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Genes myc , Fragmentos de Peptídeos/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Sequência de Aminoácidos , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Linhagem Celular , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina/métodos , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Feminino , Células HCT116 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/farmacologia , Proteínas Recombinantes/farmacologia , Transcrição Genética , Ativação Transcricional
3.
Int J Mol Sci ; 20(18)2019 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-31505809

RESUMO

Many neurodegenerative disorders have lysosomal impediments, and the list of proposed treatments targeting lysosomes is growing. We investigated the role of lysosomes in Alzheimer's disease (AD) and other age-related disorders, as well as in a strategy to compensate for lysosomal disturbances. Comprehensive immunostaining was used to analyze brains from wild-type mice vs. amyloid precursor protein/presenilin-1 (APP/PS1) mice that express mutant proteins linked to familial AD. Also, lysosomal modulation was evaluated for inducing synaptic and behavioral improvements in transgenic models of AD and Parkinson's disease, and in models of mild cognitive impairment (MCI). Amyloid plaques were surrounded by swollen organelles positive for the lysosome-associated membrane protein 1 (LAMP1) in the APP/PS1 cortex and hippocampus, regions with robust synaptic deterioration. Within neurons, lysosomes contain the amyloid ß 42 (Aß42) degradation product Aß38, and this indicator of Aß42 detoxification was augmented by Z-Phe-Ala-diazomethylketone (PADK; also known as ZFAD) as it enhanced the lysosomal hydrolase cathepsin B (CatB). PADK promoted Aß42 colocalization with CatB in lysosomes that formed clusters in neurons, while reducing Aß deposits as well. PADK also reduced amyloidogenic peptides and α-synuclein in correspondence with restored synaptic markers, and both synaptic and cognitive measures were improved in the APP/PS1 and MCI models. These findings indicate that lysosomal perturbation contributes to synaptic and cognitive decay, whereas safely enhancing protein clearance through modulated CatB ameliorates the compromised synapses and cognition, thus supporting early CatB upregulation as a disease-modifying therapy that may also slow the MCI to dementia continuum.


Assuntos
Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Disfunção Cognitiva/metabolismo , Lisossomos/metabolismo , Doença de Parkinson/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Encéfalo/patologia , Disfunção Cognitiva/genética , Disfunção Cognitiva/patologia , Humanos , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Lisossomos/genética , Lisossomos/patologia , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo , Neurônios/patologia , Doença de Parkinson/genética , Doença de Parkinson/patologia , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Sinapses/metabolismo , Sinapses/patologia
4.
PLoS Pathog ; 15(9): e1008036, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31525249

RESUMO

Cytomegalovirus (CMV) is a ubiquitous ß-herpesvirus that establishes life-long latent infection in a high percentage of the population worldwide. CMV induces the strongest and most durable CD8+ T cell response known in human clinical medicine. Due to its unique properties, the virus represents a promising candidate vaccine vector for the induction of persistent cellular immunity. To take advantage of this, we constructed a recombinant murine CMV (MCMV) expressing an MHC-I restricted epitope from influenza A virus (IAV) H1N1 within the immediate early 2 (ie2) gene. Only mice that were immunized intranasally (i.n.) were capable of controlling IAV infection, despite the greater potency of the intraperitoneally (i.p.) vaccination in inducing a systemic IAV-specific CD8+ T cell response. The protective capacity of the i.n. immunization was associated with its ability to induce IAV-specific tissue-resident memory CD8+ T (CD8TRM) cells in the lungs. Our data demonstrate that the protective effect exerted by the i.n. immunization was critically mediated by antigen-specific CD8+ T cells. CD8TRM cells promoted the induction of IFNγ and chemokines that facilitate the recruitment of antigen-specific CD8+ T cells to the lungs. Overall, our results showed that locally applied MCMV vectors could induce mucosal immunity at sites of entry, providing superior immune protection against respiratory infections.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Imunidade nas Mucosas , Vacinas contra Influenza/imunologia , Muromegalovirus/imunologia , Administração Intranasal , Sequência de Aminoácidos , Animais , Linhagem Celular , Quimiocinas/biossíntese , Epitopos de Linfócito T/administração & dosagem , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Feminino , Produtos do Gene env/administração & dosagem , Produtos do Gene env/genética , Produtos do Gene env/imunologia , Vetores Genéticos , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/genética , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Pulmão/imunologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , Muromegalovirus/genética , Células NIH 3T3 , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/virologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia
5.
Molecules ; 24(15)2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31370315

RESUMO

Conflicting values, obtained by different techniques and often under different experimental conditions have been reported on the affinity of Zn2+ for amyloid-ß, that is recognized as the major interaction responsible for Alzheimer's disease. Here, we compare the approaches employed so far, i.e., the evaluation of Kd and the determination of the stability constants to quantitatively express the affinity of Zn2+ for the amyloid-ß peptide, evidencing the pros and cons of the two approaches. We also comment on the different techniques and conditions employed that may lead to divergent data. Through the analysis of the species distribution obtained for two selected examples, we show the implications that the speciation, based on stoichiometric constants rather than on Kd, may have on data interpretation. The paper also demonstrates that the problem is further complicated by the occurrence of multiple equilibria over a relatively narrow pH range.


Assuntos
Doença de Alzheimer/genética , Peptídeos beta-Amiloides/química , Fragmentos de Peptídeos/química , Zinco/química , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/genética , Humanos , Concentração de Íons de Hidrogênio , Cinética , Fragmentos de Peptídeos/genética , Ligação Proteica/genética
6.
Pathologica ; 111(2): 58-61, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31388196

RESUMO

Introduction: The 2011 WHO Classification for lung adenocarcinoma enlightened the need for a wise use of immunohistochemistry to preserve tissue for both diagnosis and molecular studies. The current recommendation is to use a panel comprising TTF1 and p40 to classify tumors with no clear squamous or glandular differentiation as many studies have showed the higher specificity of p40 over p63 as marker of squamous differentiation. However, the co-expression of both markers opens a new scenario with subsequent classification and potentially treatment issues. Materials and methods: We report a case of a non-small lung cell carcinoma (NSCLC) with coexistent expression of TTF1 and p40 in the same tumour cells. To our knowledge, this peculiar immunohistochemical profile is very rare, and thus a review of the clinical and molecular features including molecular variances of the tumour was performed. Review of the pertinent literature was also carried out. Results: Two additional articles describing unusual cases of NSCLC with coexistent expression of TTF1 and p40 were found and compared to our case. Interestingly, they all carried out aberrant mutation in TP53 oncogene and were of advance stage. Conclusion: The positivity for both "squamous" and "adenocarcinomatous" markers and mutations of TP53 could be the expression of a not fully recognized variant of NSCLC with possible implications for classification, diagnosis and therapy.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Epitopos Imunodominantes/genética , Neoplasias Pulmonares/genética , Mutação/genética , Fragmentos de Peptídeos/genética , Fatores de Transcrição/genética , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proteínas de Ligação a DNA/biossíntese , Humanos , Epitopos Imunodominantes/biossíntese , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/metabolismo , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/biossíntese , Fatores de Transcrição/biossíntese
7.
Cancer Immunol Immunother ; 68(9): 1401-1415, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31414180

RESUMO

Although CAR T-cell therapy has demonstrated tremendous clinical efficacy especially in hematological malignancies, severe treatment-associated toxicities still compromise the widespread application of this innovative technology. Therefore, developing novel approaches to abrogate CAR T-cell-mediated side effects is of great relevance. Several promising strategies pursue the selective antibody-based depletion of adoptively transferred T cells via elimination markers. However, given the limited half-life and tissue penetration, dependence on the patients' immune system and on-target/off-side effects of proposed monoclonal antibodies, we sought to exploit αCAR-engineered T cells to efficiently eliminate CAR T cells. For comprehensive and specific recognition, a small peptide epitope (E-tag) was incorporated into the extracellular spacer region of CAR constructs. We provide first proof-of-concept for targeting this epitope by αE-tag CAR T cells, allowing an effective killing of autologous E-tagged CAR T cells both in vitro and in vivo whilst sparing cells lacking the E-tag. In addition to CAR T-cell cytotoxicity, the αE-tag-specific T cells can be empowered with cancer-fighting ability in case of relapse, hence, have versatile utility. Our proposed methodology can most probably be implemented in CAR T-cell therapies regardless of the targeted tumor antigen aiding in improving overall safety and survival control of highly potent gene-modified cells.


Assuntos
Epitopos de Linfócito T/genética , Imunoterapia Adotiva/métodos , Fragmentos de Peptídeos/genética , Neoplasias da Próstata/terapia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos Quiméricos/genética , Linfócitos T Citotóxicos/imunologia , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Autoantígenos/imunologia , Citotoxicidade Imunológica , Epitopos de Linfócito T/imunologia , Engenharia Genética , Humanos , Masculino , Camundongos , Recidiva Local de Neoplasia , Células PC-3 , Neoplasias da Próstata/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Immunol Med ; 42(2): 53-64, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31449478

RESUMO

Autoimmune disease is induced by the breakdown of immune tolerance to self-antigens. This is brought about by an imbalance between the activation and the repression of immune responses. Dysregulation of the immune response is driven by the excess of proinflammatory cytokines such as IL-6 and TNF, which play a central role in the pathogenesis of a set of autoimmune diseases. The expression of proinflammatory mediator genes is tightly controlled by post-transcriptional regulation, which is mediated by a set of immune-related RNA binding proteins, such as tristetraprolin, Roquin, and Regnase-1. These proteins coordinately control the stability of proinflammatory mRNAs to regulate aberrant immune reactions. In this review, we discuss the roles of RNA binding proteins which are associated with the immune regulation and autoimmune pathogenesis.


Assuntos
Doenças Autoimunes/etiologia , Doenças Autoimunes/imunologia , Proteínas de Ligação a RNA/fisiologia , Humanos , Mediadores da Inflamação/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , RNA Mensageiro/metabolismo , Ribonucleases/fisiologia , Fatores de Transcrição/fisiologia , Tristetraprolina/fisiologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitina-Proteína Ligases/fisiologia
9.
PLoS One ; 14(7): e0219465, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31291354

RESUMO

Accumulation of amyloid ß (Aß) peptides, the major component of amyloid fibrils in senile plaques, is one of the main causes of Alzheimer's disease. Docosahexaenoic acid (DHA) is a fatty acid abundant in the brain, and is reported to have protective effects against Alzheimer's disease, although the mechanistic effects of DHA against Alzheimer's pathophysiology remain unclear. Because dietary supplementation of DHA in Aß precursor protein transgenic mice ameliorates Aß pathology and behavioral deficits, we hypothesize that DHA may affect the fibrillization and deposition of Aß. Here we studied the effect of different types of fatty acids on Aß fibril formation by in vitro Aß fibrillization assay. Formation of amyloid fibrils consists of two steps, i.e., the initial nucleation phase and the following elongation phase. We found that unsaturated fatty acids, especially DHA, accelerated the formation of Aß fibrils with a unique short and curved morphology in its nucleation phase, which did not elongate further into the long and straight, mature Aß fibrils. Addition of DHA afterwards did not modify the morphology of the mature Aß(1-40) fibrils. The short and curved Aß fibrils formed in the presence of DHA did not facilitate the elongation phase of Aß fibril formation, suggesting that DHA promotes the formation of "off-pathway" conformers of Aß. Our study unravels a possible mechanism of how DHA acts protectively against the pathophysiology of Alzheimer's disease.


Assuntos
Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Ácidos Graxos Insaturados/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Amiloide/genética , Amiloide/metabolismo , Peptídeos beta-Amiloides/química , Precursor de Proteína beta-Amiloide/metabolismo , Amiloidose/genética , Amiloidose/metabolismo , Amiloidose/patologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Suplementos Nutricionais , Modelos Animais de Doenças , Ácidos Docosa-Hexaenoicos/metabolismo , Ácidos Docosa-Hexaenoicos/farmacologia , Ácidos Graxos Insaturados/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo
10.
Nature ; 572(7768): 270-274, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31291642

RESUMO

Receptor kinases of the Catharanthus roseus RLK1-like (CrRLK1L) family have emerged as important regulators of plant reproduction, growth and responses to the environment1. Endogenous RAPID ALKALINIZATION FACTOR (RALF) peptides2 have previously been proposed as ligands for several members of the CrRLK1L family1. However, the mechanistic basis of this perception is unknown. Here we report that RALF23 induces a complex between the CrRLK1L FERONIA (FER) and LORELEI (LRE)-LIKE GLYCOSYLPHOSPHATIDYLINOSITOL (GPI)-ANCHORED PROTEIN 1 (LLG1) to regulate immune signalling. Structural and biochemical data indicate that LLG1 (which is genetically important for RALF23 responses) and the related LLG2 directly bind RALF23 to nucleate the assembly of RALF23-LLG1-FER and RALF23-LLG2-FER heterocomplexes, respectively. A conserved N-terminal region of RALF23 is sufficient for the biochemical recognition of RALF23 by LLG1, LLG2 or LLG3, and binding assays suggest that other RALF peptides that share this conserved N-terminal region may be perceived by LLG proteins in a similar manner. Structural data also show that RALF23 recognition is governed by the conformationally flexible C-terminal sides of LLG1, LLG2 and LLG3. Our work reveals a mechanism of peptide perception in plants by GPI-anchored proteins that act together with a phylogenetically unrelated receptor kinase. This provides a molecular framework for understanding how diverse RALF peptides may regulate multiple processes, through perception by distinct heterocomplexes of CrRLK1L receptor kinases and GPI-anchored proteins of the LRE and LLG family.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Arabidopsis/metabolismo , Proteínas Ligadas por GPI/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Fragmentos de Peptídeos/metabolismo , Fosfotransferases/metabolismo , Proteínas de Arabidopsis/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Modelos Moleculares , Mutagênese , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fosfotransferases/genética , Maleabilidade , Ligação Proteica/genética , Conformação Proteica , Multimerização Proteica
13.
Int J Med Sci ; 16(7): 1032-1041, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31341417

RESUMO

AP25 is an anti-tumor peptide with a high affinity for integrins. It exerts its anti-tumor activity by inhibiting angiogenesis and by directly inhibiting the growth of tumor cells. Its half-life time in vivo is only about 50 minutes, which limits its clinical application. In order to prolong the half-life time of AP25 while preserving its anti-tumor activity, several fusion proteins of AP25 and IgG4 Fc were designed and expressed; their anti-tumor activity and pharmacokinetics properties were evaluated. Firstly, four AP25-Fc fusion protein sequences were designed, and the corresponding proteins were expressed and purified. Based on the results of HUVEC migration inhibition assay, HUVEC and tumor cell proliferation inhibition assay and yields of expression by HEK293 cells, the fusion protein designated PSG4R was selected for further evaluation. The anti-tumor effect of PSG4R was then evaluated in vivo on HCT-116 nude mice xenograft model. And the pharmacokinetics properties of PSG4R were investigated in rats. The results showed that PSG4R could inhibit the growth of xenografts of human colon cancer cell line HCT-116 in nude mice by intravenous administration of 40 mg/kg once every two days. The half-life time of PSG4R was 56.270 ± 15.398 h. This study showed that the construction of AP25-Fc fusion protein could significantly prolong the half-life of AP25 while retaining its anti-tumor activity, which provides a new direction for new drug development of AP25.


Assuntos
Endostatinas/farmacologia , Imunoconjugados/farmacologia , Imunoglobulina G/farmacologia , Neoplasias/tratamento farmacológico , Fragmentos de Peptídeos/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Administração Intravenosa , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Endostatinas/genética , Endostatinas/uso terapêutico , Feminino , Células HCT116 , Células HEK293 , Meia-Vida , Células Endoteliais da Veia Umbilical Humana , Humanos , Imunoconjugados/genética , Imunoconjugados/uso terapêutico , Imunoglobulina G/genética , Imunoglobulina G/uso terapêutico , Masculino , Camundongos , Modelos Animais , Neoplasias/patologia , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/uso terapêutico , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/uso terapêutico , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Int J Oncol ; 55(1): 309-319, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31180549

RESUMO

Fusion toxins consisting of an affinity protein fused to toxic polypeptides derived from Pseudomonas exotoxin A (ETA) are promising agents for targeted cancer therapy. In this study, we examined whether fusion toxins consisting of an albumin binding domain­derived affinity protein (ADAPT) interacting with human epidermal growth factor receptor 2 (HER2), coupled to the ETA­derived polypeptides PE38X8 or PE25, with or without an albumin binding domain (ABD) for half­life extension, can be used for specific killing of HER2­expressing cells. The fusion toxins could easily be expressed in a soluble form in Escherichia coli and purified to homogeneity. All constructs had strong affinity for HER2 (KD 10 to 26 nM) and no tendency for aggregation could be detected. The fusion toxins including the ABD showed strong interaction with human and mouse serum albumin [equilibrium dissociation constant (KD) 1 to 3 nM and 2 to 10 nM, respectively]. The in vitro investigation of the cytotoxic potential revealed IC50­values in the picomolar range for cells expressing high levels of HER2. The specificity was also demonstrated, by showing that free HER2 receptors on the target cells are required for fusion toxin activity. In mice, the fusion toxins containing the ABD exhibited an appreciably longer time in circulation. The uptake was highest in liver and kidney. Fusion with PE25 was associated with the highest hepatic uptake. Collectively, the results suggest that fusion toxins consisting of ADAPTs and ETA­derivatives are promising agents for targeted cancer therapy.


Assuntos
ADP Ribose Transferases/administração & dosagem , Toxinas Bacterianas/administração & dosagem , Exotoxinas/administração & dosagem , Neoplasias/tratamento farmacológico , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusão/administração & dosagem , Fatores de Virulência/administração & dosagem , ADP Ribose Transferases/química , ADP Ribose Transferases/genética , ADP Ribose Transferases/farmacocinética , Albuminas/administração & dosagem , Albuminas/química , Animais , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/farmacocinética , Linhagem Celular Tumoral , Exotoxinas/química , Exotoxinas/genética , Exotoxinas/farmacocinética , Feminino , Humanos , Camundongos , Neoplasias/metabolismo , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/farmacocinética , Ligação Proteica , Domínios Proteicos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacocinética , Ressonância de Plasmônio de Superfície , Distribuição Tecidual , Fatores de Virulência/química , Fatores de Virulência/genética , Fatores de Virulência/farmacocinética
15.
Proc Jpn Acad Ser B Phys Biol Sci ; 95(6): 295-302, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31189782

RESUMO

The herb Ruta chalepensis L. exhibits medical effects, such as anti-inflammatory, central nervous system depressant, and antipyretic activities. However, a genetic transformation method has not yet been developed for this species. In this paper, a simple and efficient tissue culture and genetic transformation system for R. chalepensis is reported. An amyloid ß-peptide (Aß) gene, which is considered to be a causative agent of Alzheimer's disease (AD), fused with green-fluorescent protein (GFP), was introduced into R. chalepensis. When the leaves of R. chalepensis expressing Aß-GFP were administered orally to C57BL/6J mice, serum anti-Aß antibody titers of several mice were elevated without the use of an adjuvant. These results indicated that an oral vaccine against AD using R. chalepensis may be feasible. R. chalepensis is rich in bioactive compounds that may have synergistic effects with the vaccine for AD. Plant-derived vaccines are safer and cheaper than those produced from animal cells or microbes, because plants can serve as biofactories at low cost and with high biosynthetic capacity.


Assuntos
Peptídeos beta-Amiloides/genética , Engenharia Genética/métodos , Proteínas de Fluorescência Verde/genética , Fragmentos de Peptídeos/genética , Proteínas Recombinantes de Fusão/genética , Ruta/genética , Doença de Alzheimer/imunologia , Peptídeos beta-Amiloides/imunologia , Animais , Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos/imunologia , Proteínas Recombinantes de Fusão/imunologia , Transformação Genética , Vacinas/genética , Vacinas/imunologia
16.
Nat Rev Cardiol ; 16(10): 612-622, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31186538

RESUMO

G protein-coupled receptors (GPCRs) are critical cellular sensors that mediate numerous physiological processes. In the heart, multiple GPCRs are expressed on various cell types, where they coordinate to regulate cardiac function by modulating critical processes such as contractility and blood flow. Under pathological settings, these receptors undergo aberrant changes in expression levels, localization and capacity to couple to downstream signalling pathways. Conventional therapies for heart failure work by targeting GPCRs, such as ß-adrenergic receptor and angiotensin II receptor antagonists. Although these treatments have improved patient survival, heart failure remains one of the leading causes of mortality worldwide. GPCR kinases (GRKs) are responsible for GPCR phosphorylation and, therefore, desensitization and downregulation of GPCRs. In this Review, we discuss the GPCR signalling pathways and the GRKs involved in the pathophysiology of heart disease. Given that increased expression and activity of GRK2 and GRK5 contribute to the loss of contractile reserve in the stressed and failing heart, inhibition of overactive GRKs has been proposed as a novel therapeutic approach to treat heart failure.


Assuntos
Quinases de Receptores Acoplados a Proteína G/antagonistas & inibidores , Quinases de Receptores Acoplados a Proteína G/metabolismo , Cardiopatias/tratamento farmacológico , Cardiopatias/fisiopatologia , Antagonistas Adrenérgicos beta/uso terapêutico , Animais , Catecolaminas/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Quinase 2 de Receptor Acoplado a Proteína G/antagonistas & inibidores , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Quinase 5 de Receptor Acoplado a Proteína G/antagonistas & inibidores , Quinase 5 de Receptor Acoplado a Proteína G/metabolismo , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/fisiopatologia , Humanos , Contração Muscular , Miócitos Cardíacos , Fragmentos de Peptídeos/genética , Receptores Adrenérgicos/metabolismo , Proteínas Recombinantes/genética , Transdução de Sinais/genética , beta-Arrestinas/metabolismo
17.
J Chem Phys ; 150(22): 225101, 2019 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-31202253

RESUMO

Understanding the key factors that govern the rate of protein aggregation is of immense interest since protein aggregation is associated with a number of neurodegenerative diseases. Previous experimental and theoretical studies have revealed that the hydrophobicity, charge, and population of the fibril-prone monomeric state control the fibril formation rate. Because the fibril structures consist of cross beta sheets, it is widely believed that those sequences that have a high beta content (ß) in the monomeric state should have high aggregation rates as the monomer can serve as a template for fibril growth. However, this important fact has never been explicitly proven, motivating us to carry out this study. Using replica exchange molecular dynamics simulation with implicit water, we have computed ß of 19 mutations of amyloid beta peptide of 42 residues (Aß42) for which the aggregation rate κ has been measured experimentally. We have found that κ depends on ß in such a way that the higher the propensity to aggregation, the higher the beta content in the monomeric state. Thus, we have solved a long-standing problem of the dependence of fibril formation time of the ß-structure on a quantitative level.


Assuntos
Peptídeos beta-Amiloides/química , Fragmentos de Peptídeos/química , Multimerização Proteica , Peptídeos beta-Amiloides/genética , Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular , Mutação , Fragmentos de Peptídeos/genética , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Termodinâmica
18.
Oxid Med Cell Longev ; 2019: 5123565, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31198491

RESUMO

Despite the fact that harboring the apolipoprotein E4 (APOE4) allele represents the single greatest risk factor for late-onset Alzheimer's disease (AD), the exact mechanism by which apoE4 contributes to disease progression remains unknown. Recently, we demonstrated that a 151 amino-terminal fragment of apoE4 (nApoE41-151) localizes within the nucleus of microglia in the human AD brain, suggesting a potential role in gene expression. In the present study, we investigated this possibility utilizing BV2 microglia cells treated exogenously with nApoE41-151. The results indicated that nApoE41-151 leads to morphological activation of microglia cells through, at least in part, the downregulation of a novel ER-associated protein, CXorf56. Moreover, treatment of BV2 cells with nApoE41-151 resulted in a 68-fold increase in the expression of the inflammatory cytokine, TNFα, a key trigger of microglia activation. In this regard, we also observed a specific binding interaction of nApoE41-151 with the TNFα promoter region. Collectively, these data identify a novel gene-regulatory pathway involving CXorf56 that may link apoE4 to microglia activation and inflammation associated with AD.


Assuntos
Apolipoproteína E4/metabolismo , Regulação da Expressão Gênica , Microglia/fisiologia , Fragmentos de Peptídeos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Apolipoproteína E4/genética , Astrócitos/citologia , Astrócitos/fisiologia , Células Cultivadas , Citocinas/metabolismo , Humanos , Camundongos , Microglia/citologia , Fragmentos de Peptídeos/genética , Fatores de Transcrição/genética
19.
PLoS One ; 14(5): e0217746, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31150491

RESUMO

Although the aggregation of the amyloid-ß peptide (Aß) into amyloid fibrils is a well-established hallmark of Alzheimer's disease, the complex mechanisms linking this process to neurodegeneration are still incompletely understood. The nematode worm C. elegans is a valuable model organism through which to study these mechanisms because of its simple nervous system and its relatively short lifespan. Standard Aß-based C. elegans models of Alzheimer's disease are designed to study the toxic effects of the overexpression of Aß in the muscle or nervous systems. However, the wide variety of effects associated with the tissue-level overexpression of Aß makes it difficult to single out and study specific cellular mechanisms related to the onset of Alzheimer's disease. Here, to better understand how to investigate the early events affecting neuronal signalling, we created a C. elegans model expressing Aß42, the 42-residue form of Aß, from a single-copy gene insertion in just one pair of glutamatergic sensory neurons, the BAG neurons. In behavioural assays, we found that the Aß42-expressing animals displayed a subtle modulation of the response to CO2, compared to controls. Ca2+ imaging revealed that the BAG neurons in young Aß42-expressing nematodes were activated more strongly than in control animals, and that neuronal activation remained intact until old age. Taken together, our results suggest that Aß42-expression in this very subtle model of AD is sufficient to modulate the behavioural response but not strong enough to generate significant neurotoxicity, suggesting that slightly more aggressive perturbations will enable effectively studies of the links between the modulation of a physiological response and its associated neurotoxicity.


Assuntos
Doença de Alzheimer/genética , Peptídeos beta-Amiloides/genética , Fragmentos de Peptídeos/genética , Células Receptoras Sensoriais/metabolismo , Doença de Alzheimer/patologia , Amiloide , Animais , Comportamento Animal/fisiologia , Caenorhabditis elegans/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Humanos , Células Receptoras Sensoriais/patologia
20.
Artigo em Inglês | MEDLINE | ID: mdl-31165052

RESUMO

Porcine circovirus 2 (PCV2) is a major etiological agent for porcine circovirus-associated diseases and causes enormous economic losses in domestic and overseas swine production. However, there are currently no suitable cell models to study the cytopathic effects (CPE) of PCV2 in vitro, which severely restricts the study of PCV2 pathogenesis. In the present study, we established an immortalized porcine oral mucosal epithelial cell line (hTERT-POMEC) by introducing the hTERT gene into primary porcine oral mucosal epithelial cells (POMECs) derived from a neonatal, unsuckled piglet. The hTERT-POMEC cells have a homogeneous cobblestone-like morphology and retain the basic physiological properties of primary POMECs. No chromosome abnormality and tumorigenicity transformation was observed in immortalized hTERT-POMECs. Viral infection assays demonstrated that PCV2 propagated and caused CPE in hTERT-POMECs. We conclude that the immortalized cell line hTERT-POMEC is a crucial tool for further research into the pathogenesis of PCV2.


Assuntos
Infecções por Circoviridae/virologia , Circovirus/patogenicidade , Células Epiteliais/virologia , Animais , Linhagem Celular , Proliferação de Células , Infecções por Circoviridae/genética , Circovirus/genética , Células HeLa , Humanos , Mucosa Bucal , Fragmentos de Peptídeos/genética , Cultura Primária de Células/métodos , Suínos , Doenças dos Suínos/virologia , Telomerase/genética , Replicação Viral
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