RESUMO
The metalloproteinase ADAM10 critically contributes to development, inflammation, and cancer and can be controlled by endogenous or synthetic inhibitors. Here, we demonstrate for the first time that loss of proteolytic activity of ADAM10 by either inhibition or loss of function mutations induces removal of the protease from the cell surface and the whole cell. This process is temperature dependent, restricted to mature ADAM10, and associated with an increased internalization, lysosomal degradation, and release of mature ADAM10 in extracellular vesicles. Recovery from this depletion requires de novo synthesis. Functionally, this is reflected by loss and recovery of ADAM10 substrate shedding. Finally, ADAM10 inhibition in mice reduces systemic ADAM10 levels in different tissues. Thus, ADAM10 activity is critically required for its surface expression in vitro and in vivo. These findings are crucial for development of therapeutic ADAM10 inhibition strategies and may showcase a novel, physiologically relevant mechanism of protease removal due to activity loss.
Assuntos
Proteína ADAM10/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Vesículas Extracelulares/metabolismo , Proteínas de Membrana/metabolismo , Proteína ADAM10/análise , Proteína ADAM10/genética , Secretases da Proteína Precursora do Amiloide/análise , Secretases da Proteína Precursora do Amiloide/genética , Animais , Linhagem Celular , Membrana Celular/genética , Membrana Celular/metabolismo , Vesículas Extracelulares/genética , Humanos , Mutação com Perda de Função , Masculino , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , ProteóliseRESUMO
There is a strong demand for bioanalytical techniques to rapidly detect protease activities with high sensitivity and high specificity. This study reports an activity-based electrochemical method toward this goal. Nanoelectrode arrays (NEAs) fabricated with embedded vertically aligned carbon nanofibers (VACNFs) are functionalized with specific peptide substrates containing a ferrocene (Fc) tag. The kinetic proteolysis curves are measured with continuously repeated ac voltammetry, from which the catalytic activity is derived as the inverse of the exponential decay time constant based on a heterogeneous Michaelis-Menten model. Comparison of three peptide substrates with different lengths reveals that the hexapeptide H2N-(CH2)4-CO-Pro-Leu-Arg-Phe-Gly-Ala-NH-CH2-Fc is the optimal probe for cathepsin B. The activity strongly depends on temperature and is the highest around the body temperature. With the optimized peptide substrate and measuring conditions, the limit of detection of cathepsin B activity and concentration can reach 2.49 × 10-4 s-1 and 0.32 nM, respectively. The peptide substrates show high specificity to the cognate proteases, with negligible cross-reactions among three cancer-related proteases cathepsin B, ADAM10, and ADAM17. This electrochemical method can be developed into multiplex chips for rapid profiling of protease activities in cancer diagnosis and treatment monitoring.
Assuntos
Proteína ADAM10/análise , Proteína ADAM17/análise , Secretases da Proteína Precursora do Amiloide/análise , Carbono/química , Catepsina B/análise , Técnicas Eletroquímicas/métodos , Eletrodos , Proteínas de Membrana/análise , Nanofibras/química , Proteína ADAM10/metabolismo , Proteína ADAM17/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Catepsina B/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Nanotecnologia , ProteóliseRESUMO
ß-Secretase (BACE1) is an important drug target in the treatment of Alzheimer's disease (AD). Therefore, sensitive detection of BACE1 is essential for AD treatment and drug discovery. In this work, a facile and sensitive fluorescence biosensing platform was developed for BACE1 detection. This sensing platform was constituted based on the interaction between a WS2 nanosheet and a peptide sequence. In the absence of BACE1, a FAM-labeled peptide substrate could be adsorbed on the surface of the WS2 nanosheet, thereby quenching its fluorescence. However, in the presence of BACE1, the hydrolysis of the peptide substrate by BACE1 triggers could occur with the subsequent release of short FAM-linked peptide fragments which could not be adsorbed on the surface of the WS2 nanosheet. This resulted in weak fluorescence quenching, thus restoring the fluorescence signal. By measuring the change in the fluorescence of the FAM-labeled peptide substrate, the fluorescence sensing platform based on the WS2 nanosheet could monitor BACE1. The proposed WS2 nanosheet-based platform exhibited excellent specificity and high sensitivity with a detection limit of 66 pM for BACE1. Importantly, we also demonstrated that this platform was suitable for the screening of BACE1 inhibitors. The proposed sensing platform not only provides a novel strategy for the BACE1 assay but also offers a potential tool for screening drugs.
Assuntos
Secretases da Proteína Precursora do Amiloide/análise , Ácido Aspártico Endopeptidases/análise , Técnicas Biossensoriais , Nanoestruturas , Peptídeos/química , Doença de Alzheimer , Animais , Limite de Detecção , Ratos , Espectrometria de FluorescênciaRESUMO
ADAMs (a disintegrin and metalloproteinase) are important mediators of cell signalling events, which play a role in the pathogenesis and progression of cancers. Immunohistochemical method was used to examine the immunoexpression of ADAM10 and microvessel density in 80 cases of oral squamous cell carcinoma (OSCC): without metastases - OSCC M(-) (n = 38), and with metastases - OSCC M(+) (n = 42), in 24 cases of oral leukoplakia (OLK), (15 cases with low-grade dysplasia - OLK-LG, and 9 cases with high-grade dysplasia - OLK-HG), and 19 controls. The immunoexpression of ADAM10 and the mean number of vessels were significantly increased in both groups of OSCC in comparison to both groups of OLK and controls. Moreover, the immunoexpression of ADAM10 and microvessel density were significantly increased in the OSCC M(+) group in comparison to the OSCC M(-) group. No statistically significant differences were found between immunoexpression of ADAM10 and microvessels density in the OLK-LG, OLK-HG, and control cases. In conclusion, the present study revealed overexpression of ADAM10 in OSCCs, especially in OSCC with metastasis. These findings suggest that ADAM10 could potentially contribute to metastases of oral cancer. Although, our findings suggest that ADAM10 may be involved in angiogenesis of OSCC, further studies are required to determine the role of ADAM10 in this process.
Assuntos
Proteína ADAM10/análise , Secretases da Proteína Precursora do Amiloide/análise , Biomarcadores Tumorais/análise , Carcinoma de Células Escamosas/enzimologia , Neoplasias de Cabeça e Pescoço/enzimologia , Proteínas de Membrana/análise , Neoplasias Bucais/enzimologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , Capilares/enzimologia , Capilares/patologia , Carcinoma de Células Escamosas/secundário , Feminino , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Imuno-Histoquímica , Leucoplasia Oral/enzimologia , Leucoplasia Oral/patologia , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/patologia , Neovascularização Patológica , Estudos Retrospectivos , Carcinoma de Células Escamosas de Cabeça e Pescoço , Regulação para CimaRESUMO
Seborrhoeic keratosis (SK) is an age-related skin disease. Amyloid precursor protein (APP) plays an important role in the pathogenesis of age-related Alzheimer's disease. The aim of this study was to elucidate the expression characteristics of APP in SK tissues (n = 50), and explore whether the production of APP is related to the onset of SK and skin ageing, including ultraviolet (UV)-induced ageing, as observed in normal skin (n = 79). The results of immunohistochemistry, Western blotting and quantitative real-time PCR showed that APP and its downstream products (i.e. amyloid-ß42) were more highly expressed in SK than in paired adjacent normal skin tissues. In contrast, the expression of its key secretase (i.e. ß-secretase1) was generally low. Furthermore, APP expression was higher in UV-exposed than non-exposed skin sites, and expression in the older age group (61-85 years) was greater than that in the younger age group (41-60 years) in SK tissues (p<0.05). APP expression correlated positively with age in epidermis (p<0.05), but not in dermis. These findings suggest that overexpression of APP may promote the onset of SK and is a marker of skin ageing and UV damage. Further research will elucidate whether therapeutic mitigation of increased levels of APP in the skin might delay the onset of SK and skin ageing.
Assuntos
Precursor de Proteína beta-Amiloide/análise , Ceratose Seborreica/metabolismo , Envelhecimento da Pele , Pele/química , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Secretases da Proteína Precursora do Amiloide/análise , Secretases da Proteína Precursora do Amiloide/genética , Peptídeos beta-Amiloides/análise , Peptídeos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/genética , Ácido Aspártico Endopeptidases/análise , Ácido Aspártico Endopeptidases/genética , Biomarcadores/análise , Estudos de Casos e Controles , Feminino , Humanos , Ceratose Seborreica/genética , Ceratose Seborreica/patologia , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/genética , Fatores de Risco , Pele/patologia , Pele/efeitos da radiação , Envelhecimento da Pele/patologia , Envelhecimento da Pele/efeitos da radiação , Raios Ultravioleta , Regulação para Cima , Adulto JovemRESUMO
We have developed a pair of cell-based reporter gene assays to quantitatively measure γ-secretase cleavage of distinct substrates. This manuscript describes procedures that may be used to monitor γ-secretase-mediated cleavage of either APP-C99 or Notch, using a Gal4 promoter-driven firefly luciferase reporter system. These assays were established by stably co-transfecting HEK293 cells with the Gal4-driven luciferase reporter gene and either the Gal4/VP16-tagged C-terminal fragment of APP (APP-C99; CG cells), or the Gal4/VP16-tagged Notch-ΔE (NΔE; NG cells). Using these reporter assays in parallel, we have demonstrated that an ErbB2 inhibitor, CL-387,785, can preferentially suppress γ-secretase cleavage of APP-C99 in CG cells, but not NΔE in NG cells. The differential responses exhibited by the CG and NG cells, when treated with CL-387,785, represent a preferred characteristic for γ-secretase modulators, and these responses are in stark contrast to the pan-inhibition of γ-secretase induced by DAPT. Our studies provide direct evidence that γ-secretase activities toward different substrates can be differentiated in a cellular context. These new assays may therefore be useful tools in drug discovery for improved AD therapies.
Assuntos
Secretases da Proteína Precursora do Amiloide/análise , Luciferases de Vaga-Lume/química , Receptores Notch/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Diferenciação Celular/fisiologia , Células HEK293 , Humanos , Luciferases de Vaga-Lume/metabolismo , Especificidade por Substrato , TransfecçãoRESUMO
Alzheimer's disease (AD) is a progressive neurodegenerative disorder of elderly and old age people. For intravital diagnosis of the expression of signaling molecules - AD markers, cerebrospinal fluid (CSF) and peripheral tissues are used: lymphocytes and blood platelets, buccal and olfactory epithelium, skin fibroblasts. There are several changes in the production of hyper phosphorylated form of τ-protein, BACE1 and peptide Ðß42 in CSF in case of AD, but CSF taking may have a number of side effects. Less traumatic taking of sampling tissues for the diagnosis of AD is in use of epithelium biopsy and blood portion. An increase in the expression of the hyper phosphorylated form of τ-protein is shown in blood lymphocytes of AD patients. An increase in the content of high molecular weight forms of phosphorylated t-protein and amyloid precursor protein-APP was also revealed in blood platelets of AD patients. Changes in the amount of 2 miRNA families - miR-132 family and miR-134 family were revealed in blood cells 1-5 years before the manifestation of clinical signs of AD. An increase in the concentration of bound calcium, synthesis of peptides Aß40 and Aß42, τ protein was observed in AD skin fibroblasts. In the olfactory and buccal epithelium an increase in the expression of hyper phosphorylated form of τ-protein and Aß peptide was detected in patients with AD. Verification of AD markers in peripheral tissues for biopsy have the important significant for life diagnostics, prevention and and target AD treatment.
Assuntos
Doença de Alzheimer/diagnóstico , Secretases da Proteína Precursora do Amiloide/análise , Peptídeos beta-Amiloides/análise , Ácido Aspártico Endopeptidases/análise , MicroRNAs/análise , Fragmentos de Peptídeos/análise , Proteínas tau/análise , Idoso , Secretases da Proteína Precursora do Amiloide/sangue , Secretases da Proteína Precursora do Amiloide/líquido cefalorraquidiano , Peptídeos beta-Amiloides/sangue , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Ácido Aspártico Endopeptidases/sangue , Ácido Aspártico Endopeptidases/líquido cefalorraquidiano , Biomarcadores/análise , Biomarcadores/sangue , Biomarcadores/líquido cefalorraquidiano , Epitélio/química , Fibroblastos/química , Humanos , MicroRNAs/sangue , MicroRNAs/líquido cefalorraquidiano , Mucosa Olfatória/química , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/líquido cefalorraquidiano , Proteínas tau/sangue , Proteínas tau/líquido cefalorraquidianoRESUMO
Beta-amyloid (Aß) plaques and oxidative stress are associated with the pathogenesis of Alzheimer's disease (AD). Hydrogen sulfide (H2S) has been recognized as a cytoprotectant, which improves learning memory impairment and exerts antioxidant effects in neurodegenerative disorders, including AD. The experiment was projected to explore the effects of H2S on cognitive deficits, Aß levels and possible antioxidant mechanisms. Here, APP/PS1 transgenic mice were injected sodium hydrosulfide (NaHS, a H2S donor, 2.8mg/kg) once a day for three months. It was found that APP/PS1 transgenic mice exhibited cognitive deficits and a large number of senile plaques, along with neurons decrease and Aß increase. However, intraperitoneal (i.p.) injection of NaHS improved learning memory deficits, decreased the number of senile plaques, Aß1-40 and Aß1-42 levels, suppressed neurons loss, together with up-regulated the levels of cystathionine-ß-synthase (CBS) and 3-mercaptopyruvate-sulfurtransferase (3MST). Furthermore, the protein levels of beta-amyloid precursor (APP) and beta-secretase 1 (BACE1) were dramatically restrained after administration of H2S. In addition, H2S exerted antioxidant effects via up-regulation nuclear factor erythroid-2-related factor 2 (Nrf2), heme oxygenase-1(HO-1) and glutathione S-transferase (GST). Taken together, these findings suggest that H2S ameliorates learning memory impairment, decreases the number of senile plaques in APP/PS1 mice possibly through inhibition of Aß production and activation of Nrf2/antioxidant response element (ARE) pathway.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Precursor de Proteína beta-Amiloide/fisiologia , Sulfeto de Hidrogênio/uso terapêutico , Transtornos da Memória/tratamento farmacológico , Fator 2 Relacionado a NF-E2/fisiologia , Presenilina-1/fisiologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/análise , Precursor de Proteína beta-Amiloide/análise , Animais , Elementos de Resposta Antioxidante/fisiologia , Ácido Aspártico Endopeptidases/análise , Cistationina beta-Sintase/análise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fator 2 Relacionado a NF-E2/análise , Estresse Oxidativo/efeitos dos fármacos , Sulfurtransferases/análiseRESUMO
Due to early onset of local invasion and distant metastasis, pancreatic cancer is the most lethal human malignant tumor, with a 5 year survival rate of less than 5%. As a effective chemotherapy drug for pancreatic cancer patients, gemcitabine is reported to inhibit cell proliferation as a nucleotide analog. In this study, we investigated the role of gemcitabine in immune regulation of pancreatic cancer. Our data showed that the level of soluble ULBP2 (sULBP2), a ligand of NKG2D receptor, decreased in the supernatants of pancreatic cancer cell lines when gemcitabine was added, and sULBP2 level correlated with NK92 cells cytotoxicity to pancreatic cancer cell lines. Importantly, our data showed that gemcitabine promoted PANC-1 cells and MIA PaCa-2 immune evasion by reducing ADAM10 expression, a metalloproteinase involved in sULBP2 shedding from cell membrane. Knockdown of ADAM10 clearly downregulated sULBP2 levels in the culture supernatants and cells became more susceptible to NK92 cytotoxicity. Serum samples and tumor samples were obtained from 45 patients with pancreatic ductal adenocarcinoma (PDAC). Statistical analysis showed a significant correlation between the serum level of sULBP2 with ADAM10 expression in PDAC tissues. In conclusion, our data demostrated that gemcitabine inhibits ULBP2 ectodomain shedding through the suppression of ADAM10 and enhance NK cells cytotoxicity by NKG2D-ULBP2 interaction. The results extends our understanding of gemcitabine in the treatment of pancreatic cancer from cell proliferation inhibition to immune regulation.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/imunologia , Evasão Tumoral/efeitos dos fármacos , Proteína ADAM10/análise , Proteína ADAM10/antagonistas & inibidores , Adulto , Idoso , Secretases da Proteína Precursora do Amiloide/análise , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Desoxicitidina/farmacologia , Regulação para Baixo , Feminino , Proteínas Ligadas por GPI/análise , Proteínas Ligadas por GPI/antagonistas & inibidores , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/análise , Células Matadoras Naturais/imunologia , Masculino , Proteínas de Membrana/análise , Proteínas de Membrana/antagonistas & inibidores , Pessoa de Meia-Idade , Neoplasias Pancreáticas/química , GencitabinaRESUMO
γ-Secretases are a family of intramembrane-cleaving proteases involved in various signaling pathways and diseases, including Alzheimer's disease (AD). Cells co-express differing γ-secretase complexes, including two homologous presenilins (PSENs). We examined the significance of this heterogeneity and identified a unique motif in PSEN2 that directs this γ-secretase to late endosomes/lysosomes via a phosphorylation-dependent interaction with the AP-1 adaptor complex. Accordingly, PSEN2 selectively cleaves late endosomal/lysosomal localized substrates and generates the prominent pool of intracellular Aß that contains longer Aß; familial AD (FAD)-associated mutations in PSEN2 increased the levels of longer Aß further. Moreover, a subset of FAD mutants in PSEN1, normally more broadly distributed in the cell, phenocopies PSEN2 and shifts its localization to late endosomes/lysosomes. Thus, localization of γ-secretases determines substrate specificity, while FAD-causing mutations strongly enhance accumulation of aggregation-prone Aß42 in intracellular acidic compartments. The findings reveal potentially important roles for specific intracellular, localized reactions contributing to AD pathogenesis.
Assuntos
Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/análise , Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/metabolismo , Presenilina-2/análise , Complexo 1 de Proteínas Adaptadoras/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Motivos de Aminoácidos , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Linhagem Celular Tumoral , Endossomos/química , Humanos , Lisossomos/química , Camundongos , Presenilina-1/análise , Presenilina-1/química , Presenilina-1/genética , Presenilina-1/metabolismo , Presenilina-2/química , Presenilina-2/genética , Presenilina-2/metabolismo , Ratos , Especificidade por SubstratoRESUMO
The present study aimed to determine the effect and mechanism of fuzhisan (FZS) and donepezil on the SIRT1 signaling pathway and the metabolism of the amyloid precursor protein (APP) in PC12 cells. An experimental cell model of PC12 cells with Aß2535induced neurotoxicity was established and cell proliferation was determined by the MTT assay following treatment with donepezil and FZS. In addition, cell apoptosis was determined using DAPI staining and light microscopy. Furthermore, western blot analysis and ELISA were utilized to evaluate the expression levels of associated APP, Aß40, Aß42, sAPPα, sAPPß, ADAM10, sirtuin 1 (SIRT1) and forkhead box O (FoxO) protein. The results indicated that the cell model was successfully established and FZS protected the PC12 cells from the neurotoxic effects of Aß2535, in a similar effect to donepezil, in a dosedependent manner. The expression of APP remained at the same level during the experimental period. The levels of Aß40, Aß42 and sAPPß were downregulated, where as sAPPα, ADAM10, SIRT1 and FoxO expression levels were upregulated. In conclusion, FZS treatment attenuated the Aß2535induced neurotoxicity in vitro. The neuroprotective mechanism of FZS was determined, including the induction of ADAM10 and SIRT1FoxO pathway, which participated in the process of neuroprotection. The present study identified the neuroprotective function of FZS, which may protect against Aßinduced toxicity. Therefore, FZS may be used clinically as a beneficial therapeutic drug for the development or progression of Alzheimer's disease.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Indanos/farmacologia , Piperidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sirtuína 1/metabolismo , Proteína ADAM10/metabolismo , Secretases da Proteína Precursora do Amiloide/análise , Animais , Western Blotting , Donepezila , Ensaio de Imunoadsorção Enzimática , Fatores de Transcrição Forkhead/metabolismo , Células PC12 , Ratos , Sirtuína 1/análiseRESUMO
Membrane-type 5-matrix metalloproteinase (MT5-MMP) is a proteinase mainly expressed in the nervous system with emerging roles in brain pathophysiology. The implication of MT5-MMP in Alzheimer's disease (AD), notably its interplay with the amyloidogenic process, remains elusive. Accordingly, we crossed the genetically engineered 5xFAD mouse model of AD with MT5-MMP-deficient mice and examined the impact of MT5-MMP deficiency in bigenic 5xFAD/MT5-MMP(-/-) mice. At early stages (4 months) of the pathology, the levels of amyloid beta peptide (Aß) and its amyloid precursor protein (APP) C-terminal fragment C99 were largely reduced in the cortex and hippocampus of 5xFAD/MT5-MMP(-/-), compared to 5xFAD mice. Reduced amyloidosis in bigenic mice was concomitant with decreased glial reactivity and interleukin-1ß (IL-1ß) levels, and the preservation of long-term potentiation (LTP) and spatial learning, without changes in the activity of α-, ß- and γ-secretases. The positive impact of MT5-MMP deficiency was still noticeable at 16 months of age, as illustrated by reduced amyloid burden and gliosis, and a better preservation of the cortical neuronal network and synaptophysin levels in bigenic mice. MT5-MMP expressed in HEKswe cells colocalized and co-immunoprecipitated with APP and significantly increased the levels of Aß and C99. MT5-MMP also promoted the release of a soluble APP fragment of 95 kDa (sAPP95) in HEKswe cells. sAPP95 levels were significantly reduced in brain homogenates of 5xFAD/MT5-MMP(-/-) mice, supporting altogether the idea that MT5-MMP influences APP processing. MT5-MMP emerges as a new pro-amyloidogenic regulator of APP metabolism, whose deficiency alleviates amyloid pathology, neuroinflammation and cognitive decline.
Assuntos
Doença de Alzheimer/enzimologia , Doença de Alzheimer/fisiopatologia , Hipocampo/enzimologia , Hipocampo/fisiopatologia , Metaloproteinases da Matriz Associadas à Membrana/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/análise , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/análise , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/análise , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Cognição , Feminino , Deleção de Genes , Células HEK293 , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Potenciação de Longa Duração , Masculino , Metaloproteinases da Matriz Associadas à Membrana/análise , Metaloproteinases da Matriz Associadas à Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Aprendizagem EspacialAssuntos
Envelhecimento , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/análise , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/análise , Precursor de Proteína beta-Amiloide/análise , Amiloidose/metabolismo , Animais , Modelos Animais de Doenças , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
The metalloprotease ADAM10 mediates the shedding of the ectodomain of various cell membrane proteins, including APP, the precursor of the amyloid peptide Aß, and Notch receptors following ligand binding. ADAM10 associates with the members of an evolutionary conserved subgroup of tetraspanins, referred to as TspanC8, which regulate its exit from the endoplasmic reticulum. Here we show that 4 of these TspanC8 (Tspan5, Tspan14, Tspan15 and Tspan33) which positively regulate ADAM10 surface expression levels differentially impact ADAM10-dependent Notch activation and the cleavage of several ADAM10 substrates, including APP, N-cadherin and CD44. Sucrose gradient fractionation, single molecule tracking and quantitative mass-spectrometry analysis of the repertoire of molecules co-immunoprecipitated with Tspan5, Tspan15 and ADAM10 show that these two tetraspanins differentially regulate ADAM10 membrane compartmentalization. These data represent a unique example where several tetraspanins differentially regulate the function of a common partner protein through a distinct membrane compartmentalization.
Assuntos
Proteínas ADAM/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Proteínas de Membrana/metabolismo , Receptor Notch1/metabolismo , Tetraspaninas/metabolismo , Proteínas ADAM/análise , Proteínas ADAM/genética , Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide/análise , Secretases da Proteína Precursora do Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Caderinas/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Humanos , Receptores de Hialuronatos/metabolismo , Imunoprecipitação , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Microscopia Confocal , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptor Notch1/genética , Especificidade por Substrato , Espectrometria de Massas em Tandem , Tetraspaninas/antagonistas & inibidores , Tetraspaninas/genéticaRESUMO
A Disintegrin and Metalloproteinase 10 (ADAM10) and ADAM17 catalyze ectodomain shedding of a number of cell surface proteins important for embryonic development and tissue homeostasis. Changes in the expression levels or dysregulated proteolytic activity of ADAM10 and ADAM17 have been shown to play important roles in multiple diseases such as inflammation, cancer, and neurodegenerative disorders. Despite the well documented substrate repertoire of ADAM10 and ADAM17, little is known about their cleavage site specificity. We optimized Q-PICS (Quantitative Proteomics for the Identification of Cleavage Sites) to elucidate the cleavage site specificity of recombinant murine ADAM10 and ADAM17. Two different yeast proteome-derived peptide libraries were used and samples were analyzed by LC-MALDI and LC-ESI MS in parallel. We show that the largest difference in the cleavage site specificities of ADAM10 and ADAM17 is at the P1' site: while both enzymes cleave N-terminal of leucine, only ADAM10 shows additional preference toward aromatic amino acids, whereas ADAM17 exhibits the highest preference for valine. Together with further amino acid preferences more adjacent to the scissile bond, our data is in good agreement with ADAM10/17 cleavage sites previously identified in native substrates. Overall, the precise identification of ADAM10 and ADAM17 cleavage site specificity provides the basis for better substrate identification in vivo and the generation of specific inhibitors or activity based probes.
Assuntos
Proteínas ADAM/análise , Secretases da Proteína Precursora do Amiloide/análise , Proteínas de Membrana/análise , Fragmentos de Peptídeos/análise , Proteoma/análise , Proteômica/métodos , Proteínas ADAM/química , Proteínas ADAM/metabolismo , Proteína ADAM10 , Proteína ADAM17 , Secretases da Proteína Precursora do Amiloide/química , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Cromatografia Líquida , Humanos , Espectrometria de Massas , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Camundongos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Biblioteca de Peptídeos , Proteoma/química , Proteoma/metabolismoRESUMO
BACKGROUND: Osteosarcoma is the most common type of bone cancer and is notorious for its rapid progression. The Notch signaling pathway has recently been shown to be involved in osteosarcoma. As a major sheddase of Notch receptors, ADAM10 has been implicated in many types of cancers, but its role in osteosarcoma has not been investigated. Previous studies have shown that the expression of CD31 was significantly elevated in metastatic osteosarcoma; however, its expression in nonmetastatic groups is not known. In addition, the mysterious multinucleated giant cell in giant cell-rich osteosarcoma was previously regarded as an osteoclast-like cell, but its exact identity is unclear. METHOD: Tissue chip samples from 40 cases of nonmetastatic osteosarcoma were stained for cytoplasmic ADAM10, activated Notch1 and CD31. Osteoclasts in tumor sections were also stained for tartrate-resistant acid phosphatase (TRAP). RESULTS: Immunofluorescence staining revealed that ADAM10 expression significantly increased with the progression of osteosarcoma as well as in osteoblastic osteosarcoma, whereas the expression of the Notch intracellular domain (NICD) and CD31 was not significantly altered between different pathological stages. In addition, multinucleated giant cells in giant cell-rich osteosarcoma were also found to coexpress CD31, ADAM10 and NICD, but were negative for TRAP staining. CONCLUSIONS: Our results highlight the importance of ADAM10 in the progression of osteosarcoma and suggest that the protein might be a potential therapeutic target in osteosarcoma treatment. This study also demonstrates that the multinucleated giant cell is an angiogenic tumor cell, rather than an osteoclast, and involves ADAM10/Notch1 signaling activation.
Assuntos
Proteínas ADAM/biossíntese , Secretases da Proteína Precursora do Amiloide/biossíntese , Biomarcadores Tumorais/análise , Neoplasias Ósseas/patologia , Proteínas de Membrana/biossíntese , Osteossarcoma/patologia , Proteínas ADAM/análise , Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide/análise , Neoplasias Ósseas/metabolismo , Progressão da Doença , Imunofluorescência , Células Gigantes/metabolismo , Células Gigantes/patologia , Humanos , Proteínas de Membrana/análise , Osteossarcoma/metabolismo , PrognósticoRESUMO
The zinc metallopeptidase, neprilysin (NEP), is an endothelin-1 degrading enzyme whose expression is extensively downregulated in prostate cancer. The expression of NEP in neuronal cells is regulated by intramembrane proteolysis of the amyloid precursor protein (APP) through its intracellular domain (AICD) facilitating histone acetylation of the NEP promoter and gene transcription. The present study has examined whether similar mechanisms operate in prostate cell lines. The expression of APP and its processing enzymes (ß- and γ-secretases) was examined in a number of prostate cell lines, and the effect of γ-secretase inhibition was explored on NEP expression and activity. The potential interaction of AICD with the NEP promoter was examined by chromatin immunoprecipitation. Our results indicated that all key components involved in APP processing were expressed in prostate cancer cell lines but suppression of AICD production using a γ-secretase inhibitor did not decrease NEP expression and activity, and no direct AICD-NEP promoter interaction could be detected. However, histone deacetylase inhibitors (valproate and trichostatin A) caused a 2- to 3-fold increase in NEP expression in PC-3 cells, and combinatorial treatment with the DNA demethylating agent, AzaC, further increased NEP expression levels. Although AICD is detectable in prostate cell lines, it does not appear to regulate NEP by AICD-mediated signalling. Apart from promoter de-methylation, the data suggest that histone acetylation may facilitate partial re-activation of NEP expression in advanced prostate cancer cells. Upregulation of this tumour-suppressing protein may provide a novel therapeutic strategy in prostate cancer.
Assuntos
Precursor de Proteína beta-Amiloide/fisiologia , Regulação Neoplásica da Expressão Gênica , Inibidores de Histona Desacetilases/farmacologia , Neprilisina/genética , Neoplasias da Próstata/metabolismo , Transdução de Sinais/fisiologia , Secretases da Proteína Precursora do Amiloide/análise , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Precursor de Proteína beta-Amiloide/análise , Precursor de Proteína beta-Amiloide/química , Linhagem Celular Tumoral , Humanos , Insulisina/metabolismo , Masculino , Regiões Promotoras Genéticas , Neoplasias da Próstata/patologia , Estrutura Terciária de ProteínaRESUMO
BACKGROUND: The metalloproteinase domain-containing protein 10 (ADAM 10) has been implicated in the development and progression of gastric cancer. METHODS: Expression of ADAM 10 and C-erbB-2 were examined immunochemically in 436 clinicopathologically characterized gastric cancer cases. RESULTS: Protein levels of ADAM 10 and C-erbB-2 were up-regulated in gastric cancer lesions compared with adjacent non-cancerous tissues. Positive expression of ADAM 10 correlated with age, size of tumor, location of tumor, depth of invasion, vessel invasion, lymph node, and distant metastasis and TNM stage, and also with expression of C-erbB-2. In stages I, II, and III, the 5-year survival rate of patients with high ADAM 10 expression was significantly lower than in patients with low expression. However, in stage IV, ADAM 10 expression did not correlate with the 5-year survival rate. Further multivariate analysis suggests that up-regulation of ADAM 10 and C-erbB-2 were independent prognostic indicators for the disease, along with depth of invasion, lymph node and distant metastasis and TNM stage. CONCLUSION: Expression of ADAM 10 in gastric cancer is significantly associated with lymph node and distant metastasis, high C-erbB-2 expression, and poor prognosis. ADAM 10 and C-erbB-2 proteins could be useful markers to predict tumor progression and prognosis.
Assuntos
Proteínas ADAM/análise , Adenocarcinoma/química , Adenocarcinoma/secundário , Secretases da Proteína Precursora do Amiloide/análise , Biomarcadores Tumorais/análise , Carcinoma de Células em Anel de Sinete/química , Proteínas de Membrana/análise , Receptor ErbB-2/análise , Neoplasias Gástricas/química , Neoplasias Gástricas/patologia , Proteína ADAM10 , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células em Anel de Sinete/mortalidade , Carcinoma de Células em Anel de Sinete/patologia , Carcinoma de Células em Anel de Sinete/secundário , Progressão da Doença , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Neoplasias Gástricas/mortalidadeRESUMO
gamma-Secretase is an aspartyl protease that cleaves multiple substrates including the amyloid precursor protein (APP) and the Notch proteins. Abnormal proteolysis of APP is involved in the pathogenesis of Alzheimer's disease (AD) and overactive Notch signaling plays an oncogenic role in a variety of cancers. gamma-Secretase has emerged as a promising target for drug development in the treatment of AD and cancer. Assays with increased capacity for high-throughput screening would allow for quicker screening of chemical libraries and facilitate inhibitor development. We have developed a homogeneous time-resolved fluorescence (HTRF)-based assay that makes use of a novel biotinylated recombinant APP substrate and solubilized membrane preparation as the source of the gamma-secretase enzyme. The assay was miniaturized to a 1536-well format and validated in a pilot screen against a library of approximately 3,000 compounds. The overall assay performance was robust due to a calculated Z' factor of 0.74 and its demonstrated ability to identify known gamma-secretase inhibitors such as pepstatin A. This validated assay can readily be used for primary screening against large chemical libraries searching for novel inhibitors of gamma-secretase activity that may represent potential therapeutics for AD and a variety of neoplasms.
Assuntos
Secretases da Proteína Precursora do Amiloide/análise , Avaliação Pré-Clínica de Medicamentos/instrumentação , Secretases da Proteína Precursora do Amiloide/metabolismo , Automação , Análise Custo-Benefício , Relação Dose-Resposta a Droga , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos/métodos , Células HeLa , Humanos , Indicadores e Reagentes , Luminescência , MiniaturizaçãoRESUMO
Cleavage of APP by BACE is the first proteolytic step in the production of Amyloid beta (Abeta, which accumulates in senile plaques in Alzheimer's disease. BACE-cleavage of APP is thought to happen in endosomes. However, there are controversial data whether APP and BACE can already interact on the cell surface dependent on the cholesterol level. To examine whether APP and BACE come into close proximity on the cell surface in living cells, we employed a novel technique by combining time-resolved Förster resonance energy transfer (FRET) measurements with total internal reflection microscopy (TIRET microscopy). Our data indicate that BACE and APP come into close proximity within the cell, but probably not on the cell surface. To analyze the impact of alterations in cholesterol level upon BACE-cleavage, we measured sAPP secretion. Alteration of APP processing and BACE proximity by cholesterol might be explained by alterations in cell membrane fluidity.