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1.
Plant Sci ; 308: 110928, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34034876

RESUMO

Cell death is an inevitably cryo-injury in cell and tissue cryopreservation. The research on programmed cell death (PCD) in plant cryopreservation is still in its infancy. In this study, the survival rate of Agapanthus praecox embryogenic callus was significantly improved when the vitrification solution was added with 20 µM E-64, which is an inhibitor of cathepsin B. For further investigating the relation between cathepsin B and cryo-injury, the coding gene of cathepsin B, ApCathB was isolated and characterized. A subcellular localization assay showed that ApCathB was located in cytomembrane. Heterologous overexpression of ApCathB reduced the recovery rate during Arabidopsis seedlings cryopreservation from 29.56 % to 16.46 %. Transgenic seedlings lost most of cell viability in hypocotyl after dehydration and lead to aggravated cryo-injury. The reduced survival rate of ApCathB-overexpressing embryogenic callus of A. praecox further confirmed its negatively function in cryo-injury tolerance. In addition, the survival of ApCathB-overexpressing lines was almost rescued by E-64. TUNEL detection showed intensified signal and ROS was burst, especially for H2O2. Furthermore, VPE, Metacaspase 1, Cyp15a and AIF genes related to cell death regulation were remarkably up-regulated in ApCathB-overexpressing embryogenic callus during cryopreservation. Additionally, the expression level of genes regulating cell degradation was also elevated, indicating accelerated cell death caused by ApCathB-overexpressing. Taken together, this work verified that ApCathB negatively regulated the cryo-injury tolerance and cell viability through mediating the PCD event in plant cryopreservation. Significantly, cathepsin B has potential to be a target to improve survival rate after cryopreservation.


Assuntos
Amaryllidaceae/fisiologia , Arabidopsis/fisiologia , Catepsina B/genética , Resposta ao Choque Frio , Proteínas de Plantas/genética , Amaryllidaceae/genética , Sequência de Aminoácidos , Arabidopsis/genética , Catepsina B/química , Catepsina B/metabolismo , Resposta ao Choque Frio/genética , Congelamento , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/fisiologia , Alinhamento de Sequência
2.
Eur J Med Chem ; 216: 113297, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33677351

RESUMO

Antibody-drug conjugates (ADCs) are currently among the most successful and important strategies for treating patients with solid tumors. ADCs are composed of a monoclonal antibody and warhead, which are conjugated via a linker. Currently, monomethyl auristatin E (MMAE) is the most widely applied warhead in the development of ADCs. However, MMAE-based ADCs are generally constructed using the MC-VC-PABC linker, and this design has limited structural diversity and some disadvantages. Accordingly, in this study, we generated three types of novel linker-MMAE (with alterations in the spacer, catabolizing area, and self-immolative compared with MC-VC-PABC-MMAE) in ADCs, termed SCT200-linker-MMAE conjugates, and then evaluated the linker-drug plasma stability and the rate of drug release by cathepsin B. The binding ability, internalization rates, and efficacy of all SCT200-linker-MMAE ADCs were systematically studied, and the expression of apoptosis-associated proteins and the therapeutic efficacies of SCT200-M-2, -C-2, and -C-4 were evaluated. The results showed that the activities of some of these ADCs were increased for epidermal growth factor receptor-positive tumors. Moreover, the novel linkers designed in this study can be linked with other antibodies to treat other types of cancer. Overall, these findings provide important insights into the application of SCT200-based linkers in ADCs.


Assuntos
Anticorpos Monoclonais/química , Antineoplásicos/síntese química , Imunoconjugados/química , Oligopeptídeos/química , Animais , Antineoplásicos/sangue , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Catepsina B/metabolismo , Linhagem Celular Tumoral , Liberação Controlada de Fármacos , Estabilidade de Medicamentos , Receptores ErbB/genética , Receptores ErbB/imunologia , Receptores ErbB/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imunoconjugados/sangue , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Camundongos , Camundongos Nus , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Oligopeptídeos/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
3.
ACS Chem Neurosci ; 12(5): 930-944, 2021 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-33606519

RESUMO

The COVID-19 pandemic caused by SARS-CoV-2 represents a global public health emergency. The entry of SARS-CoV-2 into host cells requires the activation of its spike protein by host cell proteases. The serine protease, TMPRSS2, and cysteine proteases, Cathepsins B/L, activate spike protein and enable SARS-CoV-2 entry to the host cell through two completely different and independent pathways. Therefore, inhibiting either TMPRSS2 or cathepsin B/L may not sufficiently block the virus entry. We here hypothesized that simultaneous targeting of both the entry pathways would be more efficient to block the virus entry rather than targeting the entry pathways individually. To this end, we utilized the network-based drug repurposing analyses to identify the possible common drugs that can target both the entry pathways. This study, for the first time, reports the molecules like cyclosporine, calcitriol, and estradiol as candidate drugs with the binding ability to the host proteases, TMPRSS2, and cathepsin B/L. Next, we analyzed drug-gene and gene-gene interaction networks using 332 human targets of SARS-CoV-2 proteins. The network results indicate that, out of 332 human proteins, cyclosporine interacts with 216 (65%) proteins. Furthermore, we performed molecular docking and all-atom molecular dynamics (MD) simulations to explore the binding of drug with TMPRSS2 and cathepsin L. The molecular docking and MD simulation results showed strong and stable binding of cyclosporine A (CsA) with TMPRSS2 and CTSL genes. The above results indicate cyclosporine as a potential drug molecule, as apart from interacting with SARS-CoV-2 entry receptors, it also interacts with most of SARS-CoV-2 target host genes; thus it could potentially interfere with functions of SARS-CoV-2 proteins in human cells. We here also suggest that these antiviral drugs alone or in combination can simultaneously target both the entry pathways and thus can be considered as a potential treatment option for COVID-19.


Assuntos
/virologia , Ciclosporina/farmacologia , Imunossupressores/farmacologia , Internalização do Vírus/efeitos dos fármacos , Antivirais/farmacologia , Catepsina B/metabolismo , Catepsina L/metabolismo , Reposicionamento de Medicamentos , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Pandemias , Serina Endopeptidases/metabolismo
4.
Int J Mol Sci ; 21(24)2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-33333840

RESUMO

Thyroid cancer incidence has increased worldwide; however, investigations of thyroid cancer-related factors as potential prognosis markers remain insufficient. Secreted proteins from the cancer secretome are regulators of several molecular mechanisms and are, thereby, ideal candidates for potential markers. We aimed to identify a specific factor for thyroid cancer by analyzing the secretome from normal thyroid cells, papillary thyroid cancer (PTC) cells, and anaplastic thyroid cancer cells using mass spectrometry (MS). Cathepsin B (CTSB) showed highest expression in PTC cells compared to other cell lines, and CTSB levels in tumor samples were higher than that seen in normal tissue. Further, among thyroid cancer patients, increased CTSB expression was related to higher risk of lymph node metastasis (LNM) and advanced N stage. Overexpression of CTSB in thyroid cancer cell lines activated cell migration by increasing the expression of vimentin and Snail, while its siRNA-mediated silencing inhibited cell migration by decreasing vimentin and Snail expression. Mechanistically, CTSB-associated enhanced cell migration and upregulation of vimentin and Snail occurred via increased phosphorylation of p38. As our results suggest that elevated CTSB in thyroid cancer induces the expression of metastatic proteins and thereby leads to LNM, CTSB may be a good and clinically relevant prognostic marker.


Assuntos
Biomarcadores Tumorais/metabolismo , Catepsina B/metabolismo , Transição Epitelial-Mesenquimal/genética , Câncer Papilífero da Tireoide/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Catepsina B/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Feminino , Humanos , Metástase Linfática , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Fosforilação , Prognóstico , Fatores de Risco , Transdução de Sinais/genética , Fatores de Transcrição da Família Snail/metabolismo , Câncer Papilífero da Tireoide/patologia , Neoplasias da Glândula Tireoide/patologia , Regulação para Cima , Vimentina/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
5.
Ann Surg ; 272(5): 863-870, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32833754

RESUMO

OBJECTIVE: We investigated the activation of pancreatic proenzymes and signs of peripancreatic inflammation in patients with clinically relevant postoperative pancreatic fistulas (POPFs). SUMMARY BACKGROUND DATA: An increase of systemic amylase concentration was associated with POPFs. This suggested parallels in the pathomechanisms between the development of POPFs and pancreatitis. METHODS: Trypsinogen, procathepsin B, and IL-6 concentrations as well as cathepsin B, myeloperoxidase and trypsin activities were determined throughout the first 7 postoperative days in drain fluids of 128 consecutive patients after pancreas resection. Histology and immunohistochemistry were performed in pancreatic specimens after total pancreatectomy due to complications and after placing experimental pancreatic sutures in the pancreatic tail of C57/Bl6 mice. RESULTS: Trypsin activity, cathepsin B activity and myeloperoxidase activity on the first postoperative day were elevated and predictive for clinically relevant pancreatic fistulas. Drain fluid stabilized trypsin activity and prevented the activation of the cascade of digestive enzymes. Leukocytes were the source of cathepsin B in drain fluid. Findings differed between fistulas after distal pancreatectomy and pancreatoduodenectomy. Immunohistochemistry of the pancreatic remnant revealed an inflammatory infiltrate expressing cathepsin B, independent of the presence of pancreatic fistulas. The infiltrate could be reproduced experimentally by sutures placed in the pancreatic tail of C57/Bl6 mice. CONCLUSIONS: Trypsinogen activation, increased cathepsin B activity and inflammation around the pancreato-enteric anastomosis on post operative day 1 are associated with subsequent clinically relevant POPFs after pancreatoduodenectomy. The parenchymal damage seems to be induced by placing sutures in the pancreatic parenchyma during pancreatic surgery.


Assuntos
Pancreatectomia , Fístula Pancreática/enzimologia , Complicações Pós-Operatórias/enzimologia , Amilases/metabolismo , Animais , Catepsina B/metabolismo , Precursores Enzimáticos/metabolismo , Feminino , Humanos , Inflamação/enzimologia , Interleucina-6/metabolismo , Masculino , Camundongos , Peroxidase/metabolismo , Estudos Prospectivos , Tripsina/metabolismo , Tripsinogênio/metabolismo
7.
Viruses ; 12(7)2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32635194

RESUMO

Chikungunya virus (CHIKV) is an enveloped virus that enters host cells and transits within the endosomes before starting its replication cycle, the precise mechanism of which is yet to be elucidated. Endocytosis and endosome acidification inhibitors inhibit infection by CHIKV, murine leukemia virus (MLV), or SARS-coronavirus, indicating that these viral entries into host cells occur through endosomes and require endosome acidification. Although endosomal cathepsin B protease is necessary for MLV, Ebola virus, and SARS-CoV infections, its role in CHIKV infection is unknown. Our results revealed that endocytosis inhibitors attenuated CHIKV-pseudotyped MLV vector infection in 293T cells but not in TE671 cells. In contrast, macropinocytosis inhibitors attenuated CHIKV-pseudotyped MLV vector infection in TE671 cells but not in 293T cells, suggesting that CHIKV host cell entry occurs via endocytosis or macropinocytosis, depending on the cell lines used. Cathepsin B inhibitor and knockdown by an shRNA suppressed CHIKV-pseudotyped MLV vector infection both in 293T and TE671 cells. These results show that cathepsin B facilitates CHIKV infection regardless of the entry pathway.


Assuntos
Catepsina B/metabolismo , Febre de Chikungunya/patologia , Vírus Chikungunya/fisiologia , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus , Catepsina B/antagonistas & inibidores , Linhagem Celular Tumoral , Endocitose/fisiologia , Endossomos/virologia , Células HEK293 , Células HeLa , Humanos , Vírus da Leucemia Murina/fisiologia , Pinocitose/fisiologia , Interferência de RNA , RNA Interferente Pequeno/genética
8.
Signal Transduct Target Ther ; 5(1): 121, 2020 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-32641705
9.
Arch Oral Biol ; 117: 104830, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32673819

RESUMO

OBJECTIVE: Cysteine proteases are lysosomal enzymes that, under specific circumstances, may be secreted into the extracellular space and participate in protein turnover. This study investigated the involvement of cathepsin B in the gelatinolytic activity of mature dentin matrices at neutral pH. DESIGN: Human dentin fragments were made into powder and enzymes were extracted using guanidine-HCl/EDTA. Host-derived dentin proteases (cathepsin B, MMP-2 and MMP-9) were identified by immunoblotting, and their activities were evaluated spectrofluorimetrically using fluorogenic substrates. Proteases activities were monitored by measuring the rate of hydrolysis of substrates in the presence/absence of MMP- or cysteine cathepsin inhibitors, at neutral pH (7.4). Mass spectroscopy was used to determine the substrates' cleavage points. Reverse zymography was performed to examine the gelatinolytic activity of cathepsin B. RESULTS: Western-blots of dentin extracts yielded strong bands at 95, 72 and 30 kDa, corresponding respectively to MMP-9, MMP-2 and Cathepsin B. Greater fluorogenic substrates hydrolysis occurred in the absence of MMP and cysteine cathepsin inhibitors than in their presence. Cathepsin B exhibited significant gelatinolytic activity. CONCLUSIONS: Together with MMP-2 and MMP-9, cathepsin B also account for the host-derived gelatinolytic activity and matrix turnover of mature dentin at physiological, neutral pH.


Assuntos
Catepsina B/metabolismo , Dentina/metabolismo , Humanos , Hidrólise , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo
10.
Nat Commun ; 11(1): 3677, 2020 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-32699279

RESUMO

Through the formation of concentration gradients, morphogens drive graded responses to extracellular signals, thereby fine-tuning cell behaviors in complex tissues. Here we show that the chemokine CXCL13 forms both soluble and immobilized gradients. Specifically, CXCL13+ follicular reticular cells form a small-world network of guidance structures, with computer simulations and optimization analysis predicting that immobilized gradients created by this network promote B cell trafficking. Consistent with this prediction, imaging analysis show that CXCL13 binds to extracellular matrix components in situ, constraining its diffusion. CXCL13 solubilization requires the protease cathepsin B that cleaves CXCL13 into a stable product. Mice lacking cathepsin B display aberrant follicular architecture, a phenotype associated with effective B cell homing to but not within lymph nodes. Our data thus suggest that reticular cells of the B cell zone generate microenvironments that shape both immobilized and soluble CXCL13 gradients.


Assuntos
Linfócitos B/imunologia , Microambiente Celular/imunologia , Quimiocina CXCL13/metabolismo , Células Dendríticas Foliculares/imunologia , Imunidade Adaptativa , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Catepsina B/genética , Catepsina B/metabolismo , Linhagem Celular , Quimiocina CXCL13/imunologia , Simulação por Computador , Células Dendríticas Foliculares/citologia , Células Dendríticas Foliculares/metabolismo , Matriz Extracelular/metabolismo , Humanos , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Modelos Biológicos , Tonsila Palatina/citologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Células Estromais/imunologia , Células Estromais/metabolismo
11.
Biochim Biophys Acta Proteins Proteom ; 1868(9): 140465, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32526473

RESUMO

Microglia, the resident mononuclear phagocyte population in the brain, have long been implicated in the pathology of neurodegenerative age-associated disorders. However, activated microglia have now been identified as homeostatic keepers in the brain, because they are involved in the initiation and resolution of neuropathology. The complex roles of activated microglia appear to be linked to change from inflammatory and neurotoxic to anti-inflammatory and neuroprotective phenotypes. Increased expression and secretion of various cathepsins support roles of activated microglia in chronic neuroinflammation, the neurotoxic M1-like polarization and neuronal death. Moreover, changes in expression and localization of microglial cathepsin B play a critical role in the acceleration of the brain aging. Beyond the role as brain-resident macrophages, many lines of evidence have shown that microglia have essential roles in the maturation and maintenance of neuronal circuits in the developing and adult brain. Cathepsin S secreted from microglia induces the diurnal variation of spine density of cortical neurons though proteolytic modification of peri-synaptic extracellular matrix molecules. In this review, I highlight the emerging roles of cathepsins that support the roles of microglia in both normal healthy and pathological brains. In addition, I discuss cathepsin inhibitors as potential therapeutic targets for brain disorders.


Assuntos
Catepsinas/metabolismo , Microglia/metabolismo , Animais , Encéfalo/metabolismo , Catepsina B/metabolismo , Catepsina H/metabolismo , Dor Crônica/metabolismo , Humanos , Inflamação , Microglia/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Fenótipo
12.
Transl Res ; 222: 28-40, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32434697

RESUMO

Nonalcoholic steatohepatitis (NASH) is a severe form of nonalcoholic fatty liver disease characterized by fat accumulation and inflammation in liver. Yet, the mechanistic insight and diagnostic and therapeutic options of NASH remain incompletely understood. This study tested the roles of cysteine protease cathepsin B (CatB) in mouse NASH development. Immunoblot revealed increased liver CatB expression in NASH mice. Fructose-palmitate-cholesterol diet increased body weight gain, liver to body weight ratio, blood fasting glucose, plasma total cholesterol and alanine transaminase levels, and liver triglyceride, but decreased plasma high-density lipoprotein in wild-type mice. All these changes were blunted in CatB-deficient (Ctsb-/-) mice. In parallel to reduced expression of genes involved in liver lipid transport and lipogenesis, liver CD36, FABP4, and PPARγ protein levels were also significantly decreased in Ctsb-/- mice, although CatB deficiency did not affect liver gluconeogenesis and fatty acid beta-oxidation-associated gene expression. Mechanistic studies showed that CatB deficiency decreased liver expression of adhesion molecules, inflammatory cytokine, and chemokine, along with reduced liver inflammatory cell infiltration. CatB deficiency also promoted M2 macrophage polarization and reduced liver TGF-ß1 signaling and fibrosis. Together, CatB deficiency improves liver function in NASH mice by suppressing de novo lipogenesis and liver inflammation and fibrosis.


Assuntos
Catepsina B/deficiência , Dieta Hiperlipídica , Inflamação/patologia , Metabolismo dos Lipídeos , Cirrose Hepática/complicações , Cirrose Hepática/enzimologia , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/complicações , Animais , Caderinas , Catepsina B/metabolismo , Polaridade Celular , Fígado/patologia , Fígado/fisiopatologia , Cirrose Hepática/fisiopatologia , Macrófagos/patologia , Masculino , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/enzimologia , Hepatopatia Gordurosa não Alcoólica/fisiopatologia , Transdução de Sinais , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Ganho de Peso
13.
Int J Antimicrob Agents ; 55(6): 106004, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32361028

RESUMO

SARS-coronavirus 2 is the causal agent of the COVID-19 outbreak. SARS-Cov-2 entry into a cell is dependent upon binding of the viral spike (S) protein to cellular receptor and on cleavage of the spike protein by the host cell proteases such as Cathepsin L and Cathepsin B. CTSL/B are crucial elements of lysosomal pathway and both enzymes are almost exclusively located in the lysosomes. CTSL disruption offers potential for CoVID-19 therapies. The mechanisms of disruption include: decreasing expression of CTSL, direct inhibition of CTSL activity and affecting the conditions of CTSL environment (increase pH in the lysosomes). We have conducted a high throughput drug screen gene expression analysis to identify compounds that would downregulate the expression of CTSL/CTSB. One of the top significant results shown to downregulate the expression of the CTSL gene is amantadine (10uM). Amantadine was approved by the US Food and Drug Administration in 1968 as a prophylactic agent for influenza and later for Parkinson's disease. It is available as a generic drug. Amantadine in addition to downregulating CTSL appears to further disrupt lysosomal pathway, hence, interfering with the capacity of the virus to replicate. It acts as a lysosomotropic agent altering the CTSL functional environment.  We hypothesize that amantadine could decrease the viral load in SARS-CoV-2 positive patients and as such it may serve as a potent therapeutic decreasing the replication and infectivity of the virus likely leading to better clinical outcomes. Clinical studies will be needed to examine the therapeutic utility of amantadine in COVID-19 infection.


Assuntos
Amantadina/uso terapêutico , Antivirais/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Catepsina L/metabolismo , Infecções por Coronavirus/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Catepsina B/metabolismo , Catepsina L/genética , Linhagem Celular , Regulação para Baixo/efeitos dos fármacos , Humanos , Lisossomos/metabolismo , Pandemias
14.
Biochim Biophys Acta Proteins Proteom ; 1868(8): 140428, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32305689

RESUMO

Investigations of Alzheimer's disease (AD), traumatic brain injury (TBI), and related brain disorders have provided extensive evidence for involvement of cathepsin B, a lysosomal cysteine protease, in mediating the behavioral deficits and neuropathology of these neurodegenerative diseases. This review integrates findings of cathepsin B regulation in clinical biomarker studies, animal model genetic and inhibitor evaluations, structural studies, and lysosomal cell biological mechanisms in AD, TBI, and related brain disorders. The results together indicate the role of cathepsin B in the behavioral deficits and neuropathology of these disorders. Lysosomal leakage occurs in AD and TBI, and related neurodegeneration, which leads to the hypothesis that cathepsin B is redistributed from the lysosome to the cytosol where it initiates cell death and inflammation processes associated with neurodegeneration. These results together implicate cathepsin B as a major contributor to these neuropathological changes and behavioral deficits. These findings support the investigation of cathepsin B as a potential drug target for therapeutic discovery and treatment of AD, TBI, and TBI-related brain disorders.


Assuntos
Doença de Alzheimer/enzimologia , Lesões Encefálicas Traumáticas/enzimologia , Encéfalo/enzimologia , Catepsina B/genética , Transtornos Neurocognitivos/enzimologia , Neurônios/enzimologia , Adulto , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Lesões Encefálicas Traumáticas/tratamento farmacológico , Lesões Encefálicas Traumáticas/genética , Lesões Encefálicas Traumáticas/patologia , Catepsina B/antagonistas & inibidores , Catepsina B/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Criança , Citosol/efeitos dos fármacos , Citosol/enzimologia , Modelos Animais de Doenças , Feto , Regulação da Expressão Gênica , Humanos , Lactente , Lisossomos/efeitos dos fármacos , Lisossomos/enzimologia , Terapia de Alvo Molecular , Transtornos Neurocognitivos/tratamento farmacológico , Transtornos Neurocognitivos/genética , Transtornos Neurocognitivos/patologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Fármacos Neuroprotetores/uso terapêutico , Transdução de Sinais
15.
Biochim Biophys Acta Proteins Proteom ; 1868(7): 140423, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32247787

RESUMO

The human genome encodes for 11 papain-like endolysosomal cysteine peptidases, collectively known as the cysteine cathepsins. Based on their biochemical properties and with the help of experiments in cell culture, the cysteine cathepsins have acquired a reputation as promotors of progression and metastasis of various cancer entities. However, tumors are known to be complex tissues in which non-cancerous cells are also critical for tumorigenesis. Here we discuss the results of the intense investigation of cathepsins in mouse models of human cancers. We focus on models in immunocompetent mice, because only such models allow for analysis of cathepsins in a fully functional tumor microenvironment. An important outcome of those studies was the identification of cancer-promoting cathepsins in tumor-associated macrophages. Another interesting outcome of these animal studies was the identification of a homeostatic tumor-suppressive role for cathepsin L in skin and intestinal cancers. Taken together, these in vivo findings provide a basis for the use of cysteine cathepsins as therapeutic targets, prodrug activators, or as proteases for imaging tumors.


Assuntos
Catepsinas/metabolismo , Cisteína/metabolismo , Neoplasias/metabolismo , Microambiente Tumoral/fisiologia , Aloenxertos , Animais , Neoplasias da Mama/metabolismo , Carcinogênese , Catepsina B/genética , Catepsina B/metabolismo , Catepsina L , Catepsinas/genética , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Intestinais/metabolismo , Camundongos , Neoplasias/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Cutâneas/metabolismo , Microambiente Tumoral/genética
16.
Biochemistry ; 59(14): 1420-1427, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32212642

RESUMO

Cathepsin B (CTSB) is an abundant cysteine protease that functions in both endolysosomal compartments and extracellular regions. A considerable number of preclinical and clinical studies indicate that CTSB is implicated in many human diseases. Expression levels and activity of CTSB significantly correlate with disease progression and severity. Current inhibitors of CTSB are lack of adequate specificity and pharmacological activities. Through structure-guided rational design, we hereby designed and generated a humanized antibody inhibitor targeting human CTSB. This was achieved by genetically fusing the propeptide of procathepsin B, a naturally occurring inhibitor of CTSB, into heavy chain complementarity-determining region 3 (CDR3H) of Herceptin that is used in the clinic for the treatment of breast cancer. The resulting antibody-propeptide fusion displayed high specificity for inhibiting CTSB proteolytic activity at nanomolar levels. Pharmacokinetic studies in mice revealed a plasma half-life of approximately 42 h for this anti-CTSB antibody inhibitor, comparable to that of the parental Herceptin scaffold. This study demonstrates a new approach for the efficient generation of humanized antibody inhibitors with high potency and specificity for human CTSB, which may be extended to develop antibody inhibitors against other disease relevant cathepsin proteases.


Assuntos
Anticorpos/química , Catepsina B/antagonistas & inibidores , Inibidores Enzimáticos/química , Animais , Anticorpos/administração & dosagem , Anticorpos/genética , Anticorpos/metabolismo , Catepsina B/química , Catepsina B/genética , Catepsina B/metabolismo , Desenho de Fármacos , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/metabolismo , Feminino , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Domínios Proteicos
17.
Res Vet Sci ; 130: 110-117, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32171999

RESUMO

Cathepsin B is one member of cysteine protease family and widely distributed in organisms, it plays an important function in parasite penetrating, migrating, molting and immune escaping. The aim of this work was to investigate whether exist interaction between a Trichinella spiralis cathepsin B (TsCB) and mouse intestinal epithelium cells (IECs), and its influence in the process of larva cell invasion. The results of ELISA, indirect immunofluorescence assay (IIFA), confocal microscopy and Far western blotting showed that there was a strong specific binding of rTsCB and IEC proteins, and the binding positions were located in cytoplasm and nuclei of IECs. The results of the in vitro larva penetration test revealed that rTsCB facilitated the larva invasion of IECs, whereas anti-rTsCB antibodies impeded partially the larva intrusion of enterocytes, this promotive or inhibitory roles were dose-dependent of rTsCB or anti-rTsCB antibodies. Silencing TsCB by siRNA mediated RNA interference reduced the TsCB expression in T. spiralis larvae, and markedly inhibited the larva penetration of enterocytes. The results indicated that TsCB binding to IECs promoted larva penetration of host's enteral epithelia, and it is a promising molecular target against intestinal invasive stages of T. spiralis.


Assuntos
Catepsina B/genética , Enterócitos/parasitologia , Células Epiteliais/parasitologia , Proteínas de Helminto/genética , Mucosa Intestinal/parasitologia , Trichinella spiralis/fisiologia , Animais , Catepsina B/metabolismo , Feminino , Proteínas de Helminto/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Larva/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Análise de Sequência de DNA/veterinária , Trichinella spiralis/genética , Trichinella spiralis/crescimento & desenvolvimento , Triquinelose/parasitologia
18.
Food Chem Toxicol ; 138: 111246, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32156567

RESUMO

The presence of acrylamide (ACR) in food results in evident cognitive decline, accumulation of misfolded proteins, neurotoxicity, neuroinflammation, and neuronal apoptosis leading to progressive neurodegeneration. Here, we used 4 dpf zebrafish larvae exposed to ACR (1mM/3days) as our model, and neuronal proteins were analyzed. Next, we tested the effect of garcinol (GAR), a natural histone-acetylation inhibitor, whose neuroprotection mechanism of action remains to be fully elucidated. Our result revealed that ACR exposure significantly impaired cognitive behavior, downregulated oxidative repair machinery, and enhanced microglia-induced neuronal apoptosis. Moreover, ACR mediated cathepsin-B (CAT-B) translocation acted as the intracellular secretase for the processing of amyloid precursor protein (APP) and served as an additional risk factor for tau hyper-phosphorylation. Here, GAR suppresses ACR mediated CATB translocation as similar with standard inhibitor CA-074. And, this pharmacological repression helped in inhibiting amyloidogenic APP processing and downstream tau hyper-phosphorylation. GAR neuroprotection was accompanied by CREB, ATF1, and BDNF activation promoting neuronal survival. At the same time, GAR subdued cdk5 and GSK3ß, the link between APP processing and tau hyper-phosphorylation. Taken together, our findings indicate that GAR rescued from ACR mediated behavioral defects, oxidative injury, neuroinflammation, undesirable APP processing, tau hyper-phosphorylation which in turn found to be CATB dependent.


Assuntos
Acrilamida/efeitos adversos , Apoptose/efeitos dos fármacos , Encéfalo/metabolismo , Catepsina B/metabolismo , Disfunção Cognitiva/induzido quimicamente , Inflamação/induzido quimicamente , Neurônios/efeitos dos fármacos , Terpenos/efeitos adversos , Animais , Comportamento Animal , Modelos Animais de Doenças , Larva/efeitos dos fármacos , Larva/metabolismo , Mitocôndrias/efeitos dos fármacos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteínas do Tecido Nervoso/metabolismo , Peixe-Zebra
19.
Parasite Immunol ; 42(6): e12710, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32145079

RESUMO

Trichobilharzia regenti (Schistosomatidae) percutaneously infects birds and mammals and invades their central nervous system (CNS). Here, we characterized the peripheral immune response of infected mice and showed how it was influenced by the parasite-induced inflammation in the skin and the CNS. As revealed by flow cytometry, T cells expanded in the spleen and the CNS-draining lymph nodes 7-14 days post-infection. Both T-bet+ and GATA-3+ T cells were markedly elevated suggesting a mixed type 1/2 immune response. However, it dropped after 7 dpi most likely being unaffected by the neuroinflammation. Splenocytes from infected mice produced a high amount of IFN-γ and, to a lesser extent, IL-10, IL-4 and IL-17 after in vitro stimulation by cercarial homogenate. Nevertheless, it had only a limited capacity to alter the maturation status of bone marrow-derived dendritic cells (BMDCs), contrary to the recombinant T. regenti cathepsin B2, which also strongly augmented expression of Ccl5, Cxcl10, Il12a, Il33 and Il10 by BMDCs. Taken together, mice infected with T. regenti developed the mixed type 1/2 immune response, which was driven by the early skin inflammation rather than the late neuroinflammation. Parasite peptidases might play an active role in triggering the host immune response.


Assuntos
Cercárias/imunologia , Dermatite/parasitologia , Schistosomatidae/imunologia , Linfócitos T/imunologia , Infecções por Trematódeos/imunologia , Animais , Catepsina B/metabolismo , Citocinas/imunologia , Células Dendríticas/imunologia , Dermatite/imunologia , Dermatite/patologia , Feminino , Inflamação/parasitologia , Inflamação/patologia , Camundongos , Camundongos Endogâmicos C57BL , Pele/imunologia , Pele/parasitologia , Pele/patologia , Infecções por Trematódeos/parasitologia
20.
Arch Gynecol Obstet ; 301(4): 955-962, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32140809

RESUMO

PURPOSE: Preeclampsia is associated with abnormal invasion of the trophoblast through decidua and subsequently altered remodeling of the maternal spiral arteries and endothelial dysfunction. This phenomenon is explained by the dysregulation of various kinds of vascular factors and proteases. The purpose of this study was to compare the circulating levels of sFlt-1, cathepsin B, and cystatin C in preeclamptic and normotensive pregnancies. STUDY DESIGN: Sixty-two pregnant women were enrolled in this prospective study. Twenty women were preeclamptic and 42 were normotensive. Serum levels of sFlt-1, cathepsin B, and cystatin C were measured using an enzyme-linked immunosorbent assay kit. RESULTS: Circulating levels of sFlt-1, cathepsin B, and cystatin C were significantly higher in preeclamptic than in normotensive pregnant women (p < 0.001; p = 0.017; p = 0.003). Preeclamptic women with severe features demonstrated significantly higher levels of cathepsin B (p = 0.05). Serum sFlt-1 and cystatin C levels were positively correlated with elevated systolic and diastolic blood pressure. The levels of cathepsin B were positively correlated with alanine and aspartate aminotransferase. The amount of 24 h proteinuria was positively, but non-significantly correlated with sFlt-1 and cystatin C. CONCLUSIONS: In addition to sFlt-1 levels, the serum levels of cathepsin B and cystatin C significantly change when preeclampsia develops. These markers are associated with severity markers of elevated blood pressure and liver injury in preeclampsia.


Assuntos
Biomarcadores/sangue , Catepsina B/metabolismo , Cistatina C/metabolismo , Pré-Eclâmpsia/sangue , Pré-Eclâmpsia/diagnóstico , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Adulto , Feminino , Humanos , Projetos Piloto , Pré-Eclâmpsia/patologia , Gravidez , Estudos Prospectivos
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