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1.
Nat Commun ; 11(1): 229, 2020 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-31932607

RESUMO

Lysosomes are membrane-surrounded cytoplasmic organelles filled with a powerful cocktail of hydrolases. Besides degrading cellular constituents inside the lysosomal lumen, lysosomal hydrolases promote tissue remodeling when delivered to the extracellular space and cell death when released to the cytosol. Here, we show that spatially and temporally controlled lysosomal leakage contributes to the accurate chromosome segregation in normal mammalian cell division. One or more chromatin-proximal lysosomes leak in the majority of prometaphases, after which active cathepsin B (CTSB) localizes to the metaphase chromatin and cleaves a small subset of histone H3. Stabilization of lysosomal membranes or inhibition of CTSB activity during mitotic entry results in a significant increase in telomere-related chromosome segregation defects, whereas cells and tissues lacking CTSB and cells expressing CTSB-resistant histone H3 accumulate micronuclei and other nuclear defects. These data suggest that lysosomal leakage and chromatin-associated CTSB contribute to proper chromosome segregation and maintenance of genomic integrity.


Assuntos
Catepsina B/metabolismo , Cromatina/metabolismo , Segregação de Cromossomos , Lisossomos/metabolismo , Mitose , Animais , Catepsina B/antagonistas & inibidores , Catepsina B/genética , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/patologia , Segregação de Cromossomos/genética , Feminino , Inativação Gênica , Histonas/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Lisossomos/enzimologia , Metáfase , Camundongos , Mitose/genética , Permeabilidade , Telômero/metabolismo
2.
ACS Chem Biol ; 14(12): 2833-2840, 2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31750642

RESUMO

Acquired resistance to apoptotic agents is a long-standing challenge in cancer treatment. Cathepsin B (CTSB) is an enzyme which, among many essential functions, promotes apoptosis during cellular stress through regulation of intracellular proteolytic networks on the minute time scale. Recent data indicate that CTSB inhibition may be a promising method to steer cells away from apoptotic death toward necrosis, a mechanism of cell death that can overcome resistance to apoptotic agents, stimulate an immune response and promote antitumor immunity. Unfortunately, rapid and selective intracellular inactivation of CTSB has not been possible. However, here we report on the synthesis and characterization of photochemical and biological properties of BODIPY-caged inhibitors of CTSB that are cell permeable, highly selective and activated rapidly upon exposure to visible light. Intriguingly, these compounds display tunable photophysical and biological properties based on substituents bound directly to boron. Me2BODIPY-caged compound 8 displays the dual-action capability of light-accelerated CTSB inhibition and singlet oxygen production from a singular molecular entity. The dual-action capacity of 8 leads to a rapid necrotic response in MDA-MB-231 triple negative breast cancer cells with high phototherapeutic indexes (>30) and selectivity vs noncancerous cells that neither CTSB inhibition nor photosensitization gives alone. Our work confirms that singlet oxygen production and CTSB inactivation is highly synergistic and a promising method for killing cancer cells. Furthermore, this ability to trigger intracellular inactivation of CTSB with light provides researchers with a powerful photochemical tool for probing biochemical processes on short time scales.


Assuntos
Apoptose/efeitos dos fármacos , Compostos de Boro/química , Catepsina B/antagonistas & inibidores , Inibidores de Cisteína Proteinase/farmacologia , Luz , Neoplasias/patologia , Linhagem Celular Tumoral , Inibidores de Cisteína Proteinase/química , Humanos , Estresse Oxidativo
3.
Cell Physiol Biochem ; 53(3): 550-572, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31529928

RESUMO

BACKGROUND/AIMS: Atherosclerosis underlies the majority of cardiovascular events, consequent to non-resolving inflammation. Considerable evidence implicates autophagy dysfunction at the core of this inflammatory condition, but the basis of this dysfunction is not fully understood. METHODS: Using an in vitro model of lipid-laden macrophages, activity-based probes and high-throughput techniques, we studied the role of the cysteine proteases cathepsins in autophagy. RESULTS: We showed that cathepsin activity is suppressed by oxidized lipids and that cathepsin has an indispensable role in the autophagy-lysosomal degradation pathway. Accordingly, loss of cathepsin function resulted in autophagy derangement. Shotgun proteomics confirmed autophagy dysfunction and unveiled a pivotal role of cathepsin L in a putative cathepsin degradation network. At the physiological level, cathepsin inhibition resulted in mitochondrial stress, which translated into impaired oxidative metabolism, excessive production of reactive oxygen species and activation of the cellular stress response, driven by ATF4-CHOP transcription factors. In addition, transcriptomic analysis of these cells uncovered some genetic similarities with the inflammatory macrophage phenotype (a.k.a M1 macrophages) and increased expression of inflammatory cytokines. CONCLUSION: Our data highlight the importance of cathepsins for mitochondrial quality control mechanisms and amelioration of vascular inflammation.


Assuntos
Anti-Inflamatórios/farmacologia , Catepsina B/metabolismo , Catepsina L/metabolismo , Catepsinas/metabolismo , Macrófagos/metabolismo , Animais , Autofagia/efeitos dos fármacos , Células da Medula Óssea/citologia , Catepsina B/antagonistas & inibidores , Catepsina L/antagonistas & inibidores , Células Cultivadas , Colesterol/metabolismo , Humanos , Macrófagos/efeitos dos fármacos , Masculino , Espectrometria de Massas , Camundongos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Estresse Oxidativo/efeitos dos fármacos , Proteômica/métodos , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo
4.
Inorg Chem ; 58(18): 12334-12347, 2019 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-31464130

RESUMO

Lysosomal cysteine peptidase cathepsin B (catB) is an important tumor-promoting factor involved in tumor progression and metastasis representing a relevant target for the development of new antitumor agents. In the present study, we synthesized 11 ruthenium compounds bearing either the clinical agent nitroxoline that was previously identified as potent selective reversible inhibitor of catB activity or its derivatives. We demonstrated that organoruthenation is a viable strategy for obtaining highly effective and specific inhibitors of catB endo- and exopeptidase activity, as shown using enzyme kinetics and microscale thermophoresis. Furthermore, we showed that the novel metallodrugs by catB inhibition significantly impair processes of tumor progression in in vitro cell based functional assays at low noncytotoxic concentrations. Generally, by using metallodrugs we observed an improvement in catB inhibition, a reduction of extracellular matrix degradation and tumor cell invasion in comparison to free ligands, and a correlation with the reactivity of the monodentate halide leaving ligand.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Catepsina B/antagonistas & inibidores , Invasividade Neoplásica/prevenção & controle , Nitroquinolinas/farmacologia , Rutênio/farmacologia , Antineoplásicos/química , Neoplasias da Mama/patologia , Catepsina B/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Modelos Moleculares , Invasividade Neoplásica/patologia , Nitroquinolinas/química , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Rutênio/química
5.
J Pharm Biomed Anal ; 176: 112811, 2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31437748

RESUMO

A simple and valid method for rapid screening of cathepsin B inhibitors from traditional Chinese medicines (TCMs) was established by the combination of immobilized enzyme microreactor (IMER) and capillary electrophoresis. Cathepsin B was immobilized on the inner surface of the capillary by glutaraldehyde method. The separation of substrate and product could be finished by baseline within 3 min. The activity of the immobilized cathepsin B remained approximately 90% after 50 runs. The quantification and statistical analysis of the product peak area was used to evaluate the catalytic activity of cathepsin B. The value of Michaelis-Menten constant of cathepsin B was 0.85 mM. The half-maximal inhibitory concentration (IC50) of L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) was measured as 36.08 nM, which indicated that the cathepsin B reactor was successfully developed and was feasible for inhibitorscreening. The raised method was then applied to discover the inhibitory potential of 17 standard compounds from traditional Chinese medicines. Five natural products, including kaempferol, rutaecarpine, evodiamine, theophylline, lycobetaine showed potential inhibition for cathepsin B. Additionally, molecular docking study was investigated for supporting the interaction between enzyme and inhibitors.


Assuntos
Catepsina B/antagonistas & inibidores , Descoberta de Drogas/métodos , Medicamentos de Ervas Chinesas/análise , Alcaloides de Amaryllidaceae/química , Alcaloides de Amaryllidaceae/isolamento & purificação , Alcaloides de Amaryllidaceae/farmacologia , Catepsina B/química , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/farmacologia , Eletroforese Capilar/métodos , Enzimas Imobilizadas/química , Estudos de Viabilidade , Alcaloides Indólicos/química , Alcaloides Indólicos/isolamento & purificação , Alcaloides Indólicos/farmacologia , Indolizinas/química , Indolizinas/isolamento & purificação , Indolizinas/farmacologia , Quempferóis/química , Quempferóis/isolamento & purificação , Quempferóis/farmacologia , Simulação de Acoplamento Molecular , Quinazolinas/química , Quinazolinas/isolamento & purificação , Quinazolinas/farmacologia , Teofilina/química , Teofilina/isolamento & purificação , Teofilina/farmacologia
6.
Biochimie ; 166: 270-285, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31302164

RESUMO

Taar1 is a G protein-coupled receptor (GPCR) confined to primary cilia of rodent thyroid epithelial cells. Taar1-deficient mouse thyroid follicles feature luminal accumulation of thyroglobulin suggesting that Taar1 acts as a regulator of extra- and pericellular thyroglobulin processing, which is mediated by cysteine cathepsin proteases present at the apical plasma membrane of rodent thyrocytes. Here, by immunostaining and confocal laser scanning microscopy, we demonstrated co-localization of cathepsin L, but only little cathepsin B, with Taar1 at primary cilia of rat thyrocytes, the FRT cells. Because proteases were shown to affect half-lives of certain receptors, we determined the effect of cathepsin activity inhibition on sub-cellular localization of Taar1 in FRT cells, whereupon Taar1 localization altered such that it was retained in compartments of the secretory pathway. Since the same effect on Taar1 localization was observed in both cathepsin B and L inhibitor-treated cells, the interaction of cathepsin activities and sub-cellular localization of Taar1 was thought to be indirect. Indeed, we observed that cathepsin inhibition resulted in a lack of primary cilia from FRT cells. Next, we proved that primary cilia are a necessity for Taar1 trafficking to reach the plasma membrane of FRT cells, since the disruption of primary cilia by treatment with ß-cyclodextrin resulted in Taar1 retention in compartments of the secretory pathway. Furthermore, in less well-polarized rat thyrocytes, namely in FRTL-5 cells lacking primary cilia, Taar1 was mainly confined to the compartments of the secretory pathway. We conclude that Taar1 localization in polarized thyroid epithelial cells requires the presence of primary cilia, which is dependent on the proteolytic activity of cysteine cathepsins B and L.


Assuntos
Catepsina B/metabolismo , Catepsina L/metabolismo , Cílios/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Células Epiteliais da Tireoide/metabolismo , Animais , Catepsina B/antagonistas & inibidores , Catepsina L/antagonistas & inibidores , Linhagem Celular , Transporte Proteico/efeitos dos fármacos , Células Epiteliais da Tireoide/citologia
7.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao ; 41(2): 208-215, 2019 Apr 28.
Artigo em Chinês | MEDLINE | ID: mdl-31060676

RESUMO

Objective To explore the effects of cathepsin B(CTSB)on the activation of nucleotide-binding domain and leucine-rich-repeat-containing family and pyrin domain-containing 3(NLRP3)inflammasome via transient receptor potential mucolipin-1(TRPML1)in cell oxidative stress model and specific gene silencing cell model. Methods BV2 cells cultured in vivo were treated separately or simultaneously with hydrogen peroxide(H2O2),calcium-sensitive receptor agonist gadolinium trichloride(GdCl3),and CTSB inhibitor CA-074Me,and interleukin-1(IL-1)beta and caspase-1 protein were detected by enzyme-linked immunosorbent assay.The growth activity of BV2 cells in each group was measured by MTT.BV2 cells were treated with different concentrations of H2O2.Cystatin C mRNA and TRPML1 mRNA in BV2 cells were detected by real-time quantitative polymerase chain reaction and the proteins of TRPML1,CTSB,cathepsin D(CTSD),cathepsin L(CTSL)and cathepsin V(CTSV)were detected by Western blot.Specific small interfering RNA was designed for TRPML1 gene target sequence.TRPML1 gene silencing cell lines(named Tr-si-Bv2 cells)were established in BV2 cells and treated with or without H2O2.TRPML1,CTSB and transcription factor EB(TFEB)proteins in Tr-si-Bv2 cells or control cells were detected by Western blot. Results After treatment with H2O2,the expression of caspase-1 protein and NLRP3 mRNA in BV2 cells was increased,and IL-1beta protein in BV2 cells was significantly increased after treatment with GdCl3(P=0.0036).After treatment with CA-074Me,the doses of NLRP3 mRNA(P=0.037),caspase-1(P=0.021),and IL-1ß(P= 0.036)were significantly reduced.Cells in the H2O2 group and H2O2+GdCl3 group grew more slowly.The expressions of CTSB mRNA and TRPML1 mRNA,or CTSB and TRPML1 proteins in BV2 cells in the treatment group with 200 µmol/L of H2O2 concentration were similar.H2O2-induced CTSB protein expression was inhibited after silencing TRPML1 gene.The changes of other cathepsins were not affected for the different concentration of H2O2.In the BV2 cells treated with TRPML1 gene silencing,the expression of CTSB protein was significantly reduced and the difference was statistically significant(P=0.021)between the H2O2 +siRNA treatment group and the H2O2 treatment group.Conclusion CTSB regulates the activation of NLRP3 inflammasome in the oxidative stress model of microglia cells,probably mediated by calcium channel protein TRPML1.


Assuntos
Catepsina B/metabolismo , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Estresse Oxidativo , Canais de Receptores Transientes de Potencial/metabolismo , Animais , Catepsina B/antagonistas & inibidores , Linhagem Celular , Inativação Gênica , Peróxido de Hidrogênio , Interleucina-1beta , Camundongos , Microglia , Domínio Pirina
8.
PLoS One ; 14(4): e0214968, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30973897

RESUMO

Emerging viruses such as severe fever and thrombocytopenia syndrome virus (SFTSV) and Ebola virus (EBOV) are responsible for significant morbidity and mortality. Host cell proteases that process the glycoproteins of these viruses are potential targets for antiviral intervention. The aspartyl protease signal peptide peptidase (SPP) has recently been shown to be required for processing of the glycoprotein precursor, Gn/Gc, of Bunyamwera virus and for viral infectivity. Here, we investigated whether SPP is also required for infectivity of particles bearing SFTSV-Gn/Gc. Entry driven by the EBOV glycoprotein (GP) and the Lassa virus glycoprotein (LASV-GPC) depends on the cysteine proteases cathepsin B and L (CatB/CatL) and the serine protease subtilisin/kexin-isozyme 1 (SKI-1), respectively, and was examined in parallel for control purposes. We found that inhibition of SPP and SKI-1 did not interfere with SFTSV Gn + Gc-driven entry but, unexpectedly, blocked entry mediated by EBOV-GP. The inhibition occurred at the stage of proteolytic activation and the SPP inhibitor was found to block CatL/CatB activity. In contrast, the SKI-1 inhibitor did not interfere with CatB/CatL activity but disrupted CatB localization in endo/lysosomes, the site of EBOV-GP processing. These results underline the potential of protease inhibitors for antiviral therapy but also show that previously characterized compounds might exert broader specificity than initially appreciated and might block viral entry via diverse mechanisms.


Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Catepsina B/metabolismo , Catepsina L/metabolismo , Endossomos , Glicoproteínas/metabolismo , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus , Animais , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/genética , Células COS , Catepsina B/antagonistas & inibidores , Catepsina B/genética , Catepsina L/antagonistas & inibidores , Catepsina L/genética , Chlorocebus aethiops , Ebolavirus/genética , Endossomos/enzimologia , Endossomos/genética , Endossomos/virologia , Glicoproteínas/genética , Células HEK293 , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Células Vero , Proteínas do Envelope Viral/genética
10.
Basic Clin Pharmacol Toxicol ; 125(2): 89-99, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30916878

RESUMO

Proton pump inhibitors (PPIs) are prodrugs used in the treatment of peptic ulcer diseases. Once activated by acidic pH, the PPIs subsequently inhibit the secretion of gastric acid by covalently forming disulphide bonds with the SH groups of the parietal proton pump, that is the H+ /K+ -ATPase. Long-term use of PPIs has been associated with numerous adverse effects, including bone fractures. Considering the mechanism of activation, PPIs could also be active in acidic micro-environments such as in lysosomes, tumours and bone resorption sites. We suggested that the SH group in the active site of cysteine proteases could be susceptible for inhibition by PPIs. In this study, the inhibition by lansoprazole was shown on the cysteine proteases legumain and cathepsin B by incubating purified proteases or cell lysates with lansoprazole at different concentrations and pH conditions. The mechanism of legumain inhibition was shown to be a direct interaction of lansoprazole with the SH group in the active site, and thus blocking binding of the legumain-selective activity-based probe MP-L01. Lansoprazole was also shown to inhibit both legumain and cathepsin B in various cell models like HEK293, monoclonal legumain over-expressing HEK293 cells (M38L) and RAW264.7 macrophages, but not in human bone marrow-derived skeletal (mesenchymal) stem cells (hBMSC-TERT). During hBMSC-TERT differentiation to osteoblasts, lansoprazole inhibited legumain secretion, alkaline phosphatase activity, but had no effects on in vitro mineralization capacity. In conclusion, lansoprazole acts as a direct covalent inhibitor of cysteine proteases via disulphide bonds with the SH group in the protease active site. Such inhibition of cysteine proteases could explain some of the off-target effects of PPIs.


Assuntos
Inibidores de Cisteína Proteinase/efeitos adversos , Lansoprazol/efeitos adversos , Inibidores da Bomba de Prótons/efeitos adversos , Animais , Calcificação Fisiológica/efeitos dos fármacos , Domínio Catalítico/efeitos dos fármacos , Catepsina B/antagonistas & inibidores , Diferenciação Celular/efeitos dos fármacos , Cisteína Endopeptidases/metabolismo , Fraturas Ósseas/induzido quimicamente , Células HEK293 , Humanos , Células-Tronco Mesenquimais , Camundongos , Osteoblastos/efeitos dos fármacos , Osteoblastos/fisiologia , Úlcera Péptica/tratamento farmacológico , Ligação Proteica , Células RAW 264.7
11.
Cancer Lett ; 449: 207-214, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30796968

RESUMO

Clinical, biochemical and molecular biology studies have identified lysosome-encapsulated cellular proteases as critical risk factors for cancer progression. Cathepsins represent a group of such proteases aimed at maintenance of cellular homeostasis. Nevertheless, recent reports suggest that Cathepsin B executes other cellular programs such as controlling tumor growth, migration, invasion, angiogenesis, and metastases development. In fact, elevated levels of Cathepsins are found under different pathological conditions including inflammation, infection, neurodegenerative disease, and cancer. Furthermore, the discovery of Cathepsin B secretion and function as an extracellular matrix protein has broadened our appreciation for the impact of Cathepsin B on cancer progression. Underneath a façade of an intracellular protease with limited therapeutic potential hides a central role of cathepsins in extracellular functions. Moreover, this role is incredibly diverse from one condition to the next - from driving caspase-dependent apoptosis to facilitating tumor neovascularization and metastasis. Here we discuss the role of Cathepsin B in the oncogenic process and perspective the use of Cathepsin B for diagnostic and therapeutic applications.


Assuntos
Biomarcadores Tumorais/metabolismo , Catepsina B/metabolismo , Neoplasias/enzimologia , Animais , Antineoplásicos/uso terapêutico , Apoptose , Autofagia , Catepsina B/antagonistas & inibidores , Catepsina B/genética , Movimento Celular , Humanos , Invasividade Neoplásica , Neoplasias/genética , Neoplasias/patologia , Neoplasias/terapia , Inibidores de Proteases/uso terapêutico , Terapêutica com RNAi , Transdução de Sinais
12.
Cell Death Dis ; 10(2): 121, 2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30741926

RESUMO

In obesity, adipocytes exhibit high metabolic activity accompanied by an increase in lipid mobilization. Recent findings indicate that autophagy plays an important role in metabolic homeostasis. However, the role of this process in adipocytes remains controversial. Therefore, we performed an overall analysis of the expression profiles of 322 lysosomal/autophagic genes in the omental adipose tissue of lean and obese individuals, and found that among 35 significantly differentially expressed genes, 34 genes were upregulated. A large number of lysosomal/autophagic genes also were upregulated in murine 3T3-L1 adipocytes challenged with tumor necrosis factor α (TNFα) (within 24 h), which is in accordance with increased autophagy flux in adipocytes. SQSTM1/p62, a selective autophagy receptor that recognizes and binds specifically to ubiquitinated proteins, is transcriptionally upregulated upon TNFα stimulation as well. Perilipin 1 (PLIN1), a crucial lipid droplet protein, can be ubiquitinated and interacts with SQSTM1 directly. Thus, TNFα-induced autophagy is a more selective process that signals through SQSTM1 and can selectively degrade PLIN1. Our study indicates that local proinflammatory cytokines in obese adipose tissue impair triglyceride storage via autophagy induction.


Assuntos
Autofagia , Lisossomos/metabolismo , Obesidade/patologia , Perilipina-1/metabolismo , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Autofagia/efeitos dos fármacos , Catepsina B/antagonistas & inibidores , Catepsina B/metabolismo , Meios de Cultivo Condicionados/farmacologia , Humanos , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/complicações , Palmitatos/farmacologia , Perilipina-1/genética , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Ubiquitinação/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
13.
Fitoterapia ; 132: 26-29, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30114470

RESUMO

A new flavone, 4'-hydroxy-6,7-methylenedioxy-3-methoxyflavone 1, and two other nucleosides, ribavirin 2 and adenosine 3, were isolated from the leaves of Dulacia egleri. The nucleosides were identified by spectroscopic techniques (1D, 2D-NMR) while the structure of the flavonoid was established by 1D, 2D-NMR analysis, including HRESIMS data. The results obtained in the biological assays showed that the compound 1 was able to inhibit cathepsins B and L with IC50 of 14.88 ±â€¯0.18 µM and 3.19 ±â€¯0.07 µM, respectively. The mechanism of inhibition for both enzymes were determined showing to be competitive at cathepsin B with Ki = 12.8 ±â€¯0.6 µM and non-linear non-competitive with positive cooperativity inhibition at cathepsin L with Ki = 322 ±â€¯33 µM, αKi = 133 ±â€¯15 µM, ßKi = 5.14 ±â€¯0.41 µM and γKi = 13.2 ±â€¯13 µM.


Assuntos
Catepsina B/antagonistas & inibidores , Catepsina L/antagonistas & inibidores , Inibidores Enzimáticos/química , Flavonoides/química , Olacaceae/química , Brasil , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Flavonoides/isolamento & purificação , Flavonoides/farmacologia , Estrutura Molecular , Folhas de Planta/química
14.
J Alzheimers Dis ; 66(4): 1397-1408, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30400084

RESUMO

Amyloid-ß (Aß), a major component of senile plaques, is generated via the proteolysis of amyloid-ß protein precursor (AßPP). This cleavage also produces AßPP fragment-derived oligomers which can be highly neurotoxic. AßPP metabolism/processing is affected by many factors, one of which is oxidative stress (OS). Associated with aging, OS is an important risk factor for Alzheimer's disease. In addition, the protein degradation systems, especially those involving cathepsins, are impaired in aging brains. Moreover, cathepsin B (CTSB) is a cysteine protease with potentially specific roles in AßPP proteolysis (ß-secretase activity) and Aß clearance (Aß degradative activity). The present work examines the effect of OS and the involvement of CTSB in amyloid oligomer formation. The xanthine/xanthine oxidase (X-XOD) free radical generating system induced the partial inhibition of CTSB activity, which was accompanied by an increase in large amyloid oligomers. These were located throughout the cytosol and in endo-lysosomal vesicles. Cells treated with the CTSB inhibitor CA-074Me also showed increased amyloid oligomer levels, whereas those subjected to OS in the presence of the inhibitor showed no such increase. However, CTSB inhibition clearly modulated the AßPP metabolism/processing induced by X-XOD, as revealed by the increase in intracellular AßPP and secreted α-secretase-cleaved soluble AßPP. The present results suggest that CTSB participates in the changes of amyloid oligomer induced by mild OS.


Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Catepsina B/metabolismo , Radicais Livres/metabolismo , Estresse Oxidativo/fisiologia , Envelhecimento/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Catepsina B/antagonistas & inibidores , Linhagem Celular Tumoral , Dipeptídeos/farmacologia , Humanos , Lisossomos/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Estresse Oxidativo/efeitos dos fármacos
15.
J. physiol. biochem ; 74(4): 503-510, nov. 2018. ilus, graf
Artigo em Inglês | IBECS | ID: ibc-179028

RESUMO

Non-alcoholic fatty liver disease (NAFLD) has emerged as the most common chronic liver disease. NLRP3 inflammasome activation has been widely studied in the pathogenesis of NAFLD. Cathepsin B (CTSB) is a ubiquitous cysteine cathepsin, and the role of CTSB in the progression and development of NAFLD has received extensive concern. However, the exact roles of CTSB in the NAFLD development and NLRP3 inflammasome activation are yet to be evaluated. In the present study, we used methionine choline-deficient (MCD) diet to establish mice NASH model. CTSB inhibitor (CA-074) was used to suppress the expression of CSTB. Expressions of CTSB and caspase-1 were evaluated by immunohistochemical staining. Serum IL-1Beta and IL-18 levels were also determined. Palmitic acid was used to stimulate Kupffer cells (KCs), and protein expressions of CTSB, NLRP3, ASC (apoptosis-associated speck-like protein containing CARD), and caspase-1 in KCs were detected. The levels of IL-1Beta and IL-18 in the supernatant of KCs were evaluated by enzyme-linked immunosorbent assay (ELISA). Our results showed that CTSB inhibition improved the liver function and reduced hepatic inflammation and ballooning, and the levels of pro-inflammatory cytokines IL-1Beta and IL-18 were decreased. The expressions of CTSB and caspase-1 in liver tissues were increased in the NASH group. In in vitro experiments, PA stimulation could increase the expressions of CTSB and NLRP3 inflammasome in KCs, and CTSB inhibition downregulated the expression of NLRP3 inflammasome in KCs, when challenged by PA. Moreover, CTSB inhibition effectively suppressed the expression and activity of caspase-1 and subsequently secretions of IL-1Beta and IL-18. Collectively, these results suggest that CTSB inhibition limits NLRP3 inflammasome-dependent NASH formation through regulating the expression and activity of caspase-1, thus providing a novel anti-inflammatory signal pathway for the therapy of NAFLD


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Assuntos
Animais , Masculino , Camundongos , Caspase 1/metabolismo , Catepsina B/antagonistas & inibidores , Inibidores de Cisteína Proteinase/uso terapêutico , Dipeptídeos/uso terapêutico , Modelos Animais de Doenças , Fígado , Anti-Inflamatórios não Esteroides/administração & dosagem , Anti-Inflamatórios não Esteroides/uso terapêutico , Caspase 1/química , Catepsina B/metabolismo , Inibidores de Cisteína Proteinase/administração & dosagem , Dipeptídeos/administração & dosagem , Ativação Enzimática , Técnica Indireta de Fluorescência para Anticorpo , Imuno-Histoquímica , Interleucina-18/antagonistas & inibidores
16.
Cell Physiol Biochem ; 50(4): 1585-1600, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30359991

RESUMO

BACKGROUND/AIMS: Angiotensin II (Ang II) is an octapeptide hormone that plays a significant role in mediating hypertension. Although hypertension is considered a chronic inflammatory disease, the molecular basis of the sterile inflammatory response involved in hypertension remains unclear. METHODS: We investigated the role of macrophage NLRP3 inflammasomes in engulfing and digesting microbes, a key macrophage function, and in early onset of hypertension-associated macrophage injury using biochemical analyses, gene silencing, molecular biotechnology, immunofluorescence, and microbiology. RESULTS: Ang II stimulation decreased nitric oxide (NO) release and macrophage digestion in cultured THP-1 cells and markedly increased NLRP3 inflammasome formation and activation. NO release and macrophage digestion were restored by NLRP3 inflammasome inhibition with isoliquiritigenin and gene silencing. This Ang II-induced upregulation of NLRP3 inflammasomes in macrophages was attributed to lysosomal damage and release of cathepsin B. Mechanistically, losartan, a nonpeptide Ang II receptor antagonist, decreased Ang II-induced NLRP3 inflammasome activation, lysosomal membrane permeability, lysosomal cathepsin B release, and macrophage digestion dysfunction. Similarly, Ang II-induced macrophage microbe digestion and NO production, which were blocked by ATI gene silencing. In addition, in vivo experiments showed that the bacteria scavenging function was clearly decreased in macrophages from Ang II-induced hypertensive mice. CONCLUSION: Angiotensin II enhances lysosomal membrane permeabilization and the consequent release of lysosomal cathepsin B, resulting in activation of the macrophage NLRP3 inflammasome. This may contribute to NO mediation of dysfunction in digesting microbes.


Assuntos
Angiotensina II/farmacologia , Catepsina B/metabolismo , Inflamassomos/metabolismo , Macrófagos/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Catepsina B/antagonistas & inibidores , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Chalconas/farmacologia , Escherichia coli/fisiologia , Hipertensão/metabolismo , Hipertensão/patologia , Losartan/farmacologia , Lisossomos/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Óxido Nítrico/metabolismo , Fagocitose/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/metabolismo
17.
J Am Chem Soc ; 140(38): 12010-12020, 2018 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-30148621

RESUMO

X-ray CT instruments are among the most available, efficient, and cost-effective imaging modalities in hospitals. The field of CT molecular imaging is emerging which relies mainly on the detection of gold nanoparticles and iodine-containing compounds directed to tagging a variety of abundant biomolecules. Here for the first time we attempted to detect enzymatic activity, while the low sensitivity of CT scanners to contrast reagents made this a challenging task. Therefore, we developed a new class of nanosized cathepsin-targeted activity-based probes (ABPs) for functional CT imaging of cancer. ABPs are small molecules designed to covalently modify enzyme targets in an activity-dependent manner. Using a CT instrument, these novel probes enable detection of the elevated cathepsin activity within cancerous tissue, thus creating a direct link between biological processes and imaging signals. We present the generation and biochemical evaluation of a library of ABPs tagged with different sized gold nanoparticles (GNPs), with various ratios of cathepsin-targeting moiety and a combination of different polyethylene glycol (PEG) protective layers. The most potent and stable GNP-ABPs were applied for noninvasive cancer imaging in mice. Surprisingly, detection of CT contrast from the tumor had reverse correlation to GNP size and the amount of targeting moiety. Interestingly, TEM images of tumor sections show intercellular lysosomal subcellular localization of the GNP-ABPs. In conclusion, we demonstrate that the covalent linkage is key for detection using low sensitive imaging modalities and the utility of GNP-ABPs as a promising tool for enzymatic-based CT imaging.


Assuntos
Catepsina B/metabolismo , Dipeptídeos/farmacologia , Inibidores Enzimáticos/farmacologia , Nanopartículas Metálicas/química , Neoplasias/metabolismo , Animais , Catepsina B/antagonistas & inibidores , Linhagem Celular Tumoral , Dipeptídeos/síntese química , Dipeptídeos/química , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Ouro/química , Humanos , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Células NIH 3T3 , Neoplasias/diagnóstico por imagem , Neoplasias/patologia , Polietilenoglicóis/química , Tomografia Computadorizada por Raios X/métodos
18.
Bioorg Med Chem ; 26(16): 4624-4634, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-30037754

RESUMO

A family of dipeptidyl enoates has been prepared and tested against the parasitic cysteine proteases rhodesain, cruzain and falcipain-2 related to sleeping sickness, Chagas disease and malaria, respectively. They have also been tested against human cathepsins B and L1 for selectivity. Dipeptidyl enoates resulted to be irreversible inhibitors of these enzymes. Some of the members of the family are very potent inhibitors of parasitic cysteine proteases displaying k2nd (M-1s-1) values of seven orders of magnitude. In vivo antiprotozoal testing was also performed. Inhibitors exhibited IC50 values in the micromolar range against Plasmodium falciparum, Trypanosoma brucei, Trypanosoma cruzi and even more promising lower values against Leishmania donovanii.


Assuntos
Antiprotozoários/química , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/química , Dipeptídeos/química , Antiprotozoários/síntese química , Antiprotozoários/farmacologia , Sítios de Ligação , Catepsina B/antagonistas & inibidores , Catepsina B/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Cisteína Endopeptidases/química , Inibidores de Cisteína Proteinase/síntese química , Inibidores de Cisteína Proteinase/farmacologia , Dipeptídeos/síntese química , Dipeptídeos/farmacologia , Células Hep G2 , Humanos , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/enzimologia
19.
J Physiol Biochem ; 74(4): 503-510, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30019185

RESUMO

Non-alcoholic fatty liver disease (NAFLD) has emerged as the most common chronic liver disease. NLRP3 inflammasome activation has been widely studied in the pathogenesis of NAFLD. Cathepsin B (CTSB) is a ubiquitous cysteine cathepsin, and the role of CTSB in the progression and development of NAFLD has received extensive concern. However, the exact roles of CTSB in the NAFLD development and NLRP3 inflammasome activation are yet to be evaluated. In the present study, we used methionine choline-deficient (MCD) diet to establish mice NASH model. CTSB inhibitor (CA-074) was used to suppress the expression of CSTB. Expressions of CTSB and caspase-1 were evaluated by immunohistochemical staining. Serum IL-1ß and IL-18 levels were also determined. Palmitic acid was used to stimulate Kupffer cells (KCs), and protein expressions of CTSB, NLRP3, ASC (apoptosis-associated speck-like protein containing CARD), and caspase-1 in KCs were detected. The levels of IL-1ß and IL-18 in the supernatant of KCs were evaluated by enzyme-linked immunosorbent assay (ELISA). Our results showed that CTSB inhibition improved the liver function and reduced hepatic inflammation and ballooning, and the levels of pro-inflammatory cytokines IL-1ß and IL-18 were decreased. The expressions of CTSB and caspase-1 in liver tissues were increased in the NASH group. In in vitro experiments, PA stimulation could increase the expressions of CTSB and NLRP3 inflammasome in KCs, and CTSB inhibition downregulated the expression of NLRP3 inflammasome in KCs, when challenged by PA. Moreover, CTSB inhibition effectively suppressed the expression and activity of caspase-1 and subsequently secretions of IL-1ß and IL-18. Collectively, these results suggest that CTSB inhibition limits NLRP3 inflammasome-dependent NASH formation through regulating the expression and activity of caspase-1, thus providing a novel anti-inflammatory signal pathway for the therapy of NAFLD.


Assuntos
Caspase 1/metabolismo , Catepsina B/antagonistas & inibidores , Inibidores de Cisteína Proteinase/uso terapêutico , Dipeptídeos/uso terapêutico , Modelos Animais de Doenças , Fígado/efeitos dos fármacos , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Animais , Anti-Inflamatórios não Esteroides/administração & dosagem , Anti-Inflamatórios não Esteroides/uso terapêutico , Caspase 1/química , Catepsina B/metabolismo , Células Cultivadas , Inibidores de Cisteína Proteinase/administração & dosagem , Dipeptídeos/administração & dosagem , Ativação Enzimática/efeitos dos fármacos , Técnica Indireta de Fluorescência para Anticorpo , Imuno-Histoquímica , Inflamassomos/efeitos dos fármacos , Inflamassomos/imunologia , Inflamassomos/metabolismo , Injeções Intraperitoneais , Interleucina-18/antagonistas & inibidores , Interleucina-18/sangue , Interleucina-1beta/sangue , Interleucina-1beta/metabolismo , Macrófagos do Fígado/efeitos dos fármacos , Macrófagos do Fígado/imunologia , Macrófagos do Fígado/metabolismo , Macrófagos do Fígado/patologia , Fígado/imunologia , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/agonistas , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Hepatopatia Gordurosa não Alcoólica/imunologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Distribuição Aleatória
20.
J Virol ; 92(19)2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30021905

RESUMO

Middle East respiratory syndrome coronavirus (MERS-CoV) utilizes host cellular proteases to enter cells. A previous report shows that furin, which is distributed mainly in the Golgi apparatus and cycled to the cell surface and endosomes, proteolytically activates the MERS-CoV spike (S) protein following receptor binding to mediate fusion between the viral and cellular membranes. In this study, we reexamined furin usage by MERS-CoV using a real-time PCR-based virus cell entry assay after inhibition of cellular proteases. We found that the furin inhibitor dec-RVKR-CMK blocked entry of MERS-CoV harboring an S protein lacking furin cleavage sites; it even blocked entry into furin-deficient LoVo cells. In addition, dec-RVKR-CMK inhibited not only the enzymatic activity of furin but also those of cathepsin L, cathepsin B, trypsin, papain, and TMPRSS2. Furthermore, a virus cell entry assay and a cell-cell fusion assay provided no evidence that the S protein was activated by exogenous furin. Therefore, we conclude that furin does not play a role in entry of MERS-CoV into cells and that the inhibitory effect of dec-RVKR-CMK is specific for TMPRSS2 and cathepsin L rather than furin.IMPORTANCE Previous studies using the furin inhibitor dec-RVKR-CMK suggest that MERS-CoV utilizes a cellular protease, furin, to activate viral glycoproteins during cell entry. However, we found that dec-RVKR-CMK inhibits not only furin but also other proteases. Furthermore, we found no evidence that MERS-CoV uses furin. These findings suggest that previous studies in the virology field based on dec-RVKR-CMK should be reexamined carefully. Here we describe appropriate experiments that can be used to assess the effect of protease inhibitors on virus cell entry.


Assuntos
Furina/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Proteólise , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do Vírus , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Catepsina B/antagonistas & inibidores , Catepsina B/genética , Catepsina B/metabolismo , Catepsina L/antagonistas & inibidores , Catepsina L/genética , Catepsina L/metabolismo , Chlorocebus aethiops , Furina/antagonistas & inibidores , Furina/genética , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Papaína/antagonistas & inibidores , Papaína/genética , Papaína/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Células Vero
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