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1.
J Biol Chem ; 289(46): 31736-31750, 2014 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-25288807

RESUMO

Stefin B (cystatin B) is an endogenous cysteine cathepsin inhibitor, and the loss-of-function mutations in the stefin B gene were reported in patients with Unverricht-Lundborg disease (EPM1). In this study we demonstrated that stefin B-deficient (StB KO) mice were significantly more sensitive to the lethal LPS-induced sepsis and secreted higher amounts of pro-inflammatory cytokines IL-1ß and IL-18 in the serum. We further showed that increased caspase-11 gene expression and better pro-inflammatory caspase-1 and -11 activation determined in StB KO bone marrow-derived macrophages resulted in enhanced IL-1ß processing. Pretreatment of macrophages with the cathepsin inhibitor E-64d did not affect secretion of IL-1ß, suggesting that the increased cathepsin activity determined in StB KO bone marrow-derived macrophages is not essential for inflammasome activation. Upon LPS stimulation, stefin B was targeted into the mitochondria, and the lack of stefin B resulted in the increased destabilization of mitochondrial membrane potential and mitochondrial superoxide generation. Collectively, our study demonstrates that the LPS-induced sepsis in StB KO mice is dependent on caspase-11 and mitochondrial reactive oxygen species but is not associated with the lysosomal destabilization and increased cathepsin activity in the cytosol.


Assuntos
Cistatina B/fisiologia , Endotoxemia/metabolismo , Regulação da Expressão Gênica , Inflamação/metabolismo , Animais , Caspases/metabolismo , Caspases Iniciadoras , Escherichia coli/metabolismo , Inflamassomos/metabolismo , Lipopolissacarídeos , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mitocôndrias/metabolismo , NF-kappa B/metabolismo , Espécies Reativas de Oxigênio/metabolismo
2.
Biochim Biophys Acta ; 1843(9): 2089-99, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24909779

RESUMO

EPM1 is a rare progressive myoclonus epilepsy accompanied by apoptosis in the cerebellum of patients. Mutations in the gene of stefin B (cystatin B) are responsible for the primary defect underlying EPM1. Taking stefin B aggregates as a model we asked what comes first, protein aggregation or oxidative stress, and how these two processes correlate with cell death. We studied the aggregation in cells of the stefin B wild type, G4R mutant, and R68X fragment before (Ceru et al., 2010, Biol. Cell). The present study was performed on two more missense mutants of human stefin B, G50E and Q71P, and they similarly showed numerous aggregates upon overexpression. Mutant- and oligomer-dependent increase in oxidative stress and cell death in cells bearing aggregates was shown. On the other hand, there was no correlation between the size and number of the aggregates and cell death. We suggest that differences in toxicity of the aggregates depend on whether they are in oligomeric/protofibrillar or fibrillar form. This in turn likely depends on the mutant's 3D structure where unfolded proteins show lower toxicity. Imaging by transmission electron microscopy showed that the aggregates in cells are of different types: bigger perinuclear, surrounded by membranes and sometimes showing vesicle-like invaginations, or smaller, punctual and dispersed throughout the cytoplasm. All EPM1 mutants studied were inactive as cysteine proteases inhibitors and in this way contribute to loss of stefin B functions. Relevance to EPM1 disease by gain in toxic function is discussed.


Assuntos
Cistatina B/química , Cistatina B/genética , Proteínas Mutantes/química , Estresse Oxidativo , Síndrome de Unverricht-Lundborg/genética , Síndrome de Unverricht-Lundborg/patologia , Amiloide/metabolismo , Animais , Anexina A5/metabolismo , Benzotiazóis , Células CHO , Contagem de Células , Morte Celular , Sobrevivência Celular , Cricetinae , Cricetulus , Cistatina B/ultraestrutura , Células HEK293 , Humanos , Cinética , Proteínas Mutantes/ultraestrutura , Propídio/metabolismo , Estrutura Quaternária de Proteína , Espectrometria de Fluorescência , Tiazóis/metabolismo , Transfecção
3.
Biol Chem ; 394(6): 783-90, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23362198

RESUMO

Human stefins and cystatins are physiologically important cysteine proteinase inhibitors, acting as a first line of defense against undesirable proteolysis. Mutations in the cystatin B gene cause a rare form of epilepsy EPM1. Its two missense mutants, G50E and Q71P, lack the inhibitory activity and are partially unfolded, which leads to changes in their aggregation behavior, both in vitro and in the cell. SDS-PAGE and MALDI-TOF mass spectrometry were used to follow the hydrolysis of human stefin B wild type, G50E and Q71P, by cathepsins B and S in vitro. Cathepsin S was found to degrade both mutants, with Q71P being degraded faster. This correlates with the openness of the protein structure, Q71P having more exposed hydrophobic surfaces. Cathepsin B acted more selectively, degrading G50E into smaller fragments, while still leaving a portion of the full-length protein intact. Q71P was cleaved only at the exposed N-terminal end. The co-localization of stefin B wild type and EPM1 mutants with cathepsins showed that cathepsins accumulate around the aggregates formed by the EPM1 mutants. We hypothesize that the aggregation of both full-length mutants prevents the cathepsin molecule from accessing the substrate protein's core, whereas the cleaved fragments would be expected to aggregate stronger.


Assuntos
Catepsina B/metabolismo , Catepsinas/metabolismo , Cistatina B/química , Cistatina B/metabolismo , Proteínas Mutantes/metabolismo , Desdobramento de Proteína , Síndrome de Unverricht-Lundborg/metabolismo , Catepsinas/química , Eletroforese em Gel de Poliacrilamida , Imunofluorescência , Humanos , Proteínas Mutantes/química , Estabilidade Proteica , Estrutura Quaternária de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
4.
Cells ; 12(23)2023 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-38067160

RESUMO

Stefin B (cystatin B) is an inhibitor of lysosomal and nuclear cysteine cathepsins. The gene for stefin B is located on human chromosome 21 and its expression is upregulated in the brains of individuals with Down syndrome. Biallelic loss-of-function mutations in the stefin B gene lead to Unverricht-Lundborg disease-progressive myoclonus epilepsy type 1 (EPM1) in humans. In our past study, we demonstrated that mice lacking stefin B were significantly more sensitive to sepsis induced by lipopolysaccharide (LPS) and secreted higher levels of interleukin 1-ß (IL-1ß) due to increased inflammasome activation in bone marrow-derived macrophages. Here, we report lower interleukin 1-ß processing and caspase-11 expression in bone marrow-derived macrophages prepared from mice that have an additional copy of the stefin B gene. Increased expression of stefin B downregulated mitochondrial reactive oxygen species (ROS) generation and lowered the NLR family pyrin domain containing 3 (NLRP3) inflammasome activation in macrophages. We determined higher AMP-activated kinase phosphorylation and downregulation of mTOR activity in stefin B trisomic macrophages-macrophages with increased stefin B expression. Our study showed that increased stefin B expression downregulated mitochondrial ROS generation and increased autophagy. The present work contributes to a better understanding of the role of stefin B in regulation of autophagy and inflammasome activation in macrophages and could help to develop new treatments.


Assuntos
Cistatina B , Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Animais , Humanos , Camundongos , Proteínas Quinases Ativadas por AMP , Cistatina B/fisiologia , Inflamassomos/metabolismo , Interleucina-1 , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Serina-Treonina Quinases TOR , Fatores de Transcrição
5.
J Biol Chem ; 285(13): 10078-10086, 2010 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-20075068

RESUMO

Stefin B (cystatin B) is an endogenous inhibitor of cysteine proteinases localized in the nucleus and the cytosol. Loss-of-function mutations in the stefin B gene (CSTB) gene were reported in patients with Unverricht-Lundborg disease (EPM1). We have identified an interaction between stefin B and nucleosomes, specifically with histones H2A.Z, H2B, and H3. In synchronized T98G cells, stefin B co-immunoprecipitated with histone H3, predominantly in the G(1) phase of the cell cycle. Stefin B-deficient mouse embryonic fibroblasts entered S phase earlier than wild type mouse embryonic fibroblasts. In contrast, increased expression of stefin B in the nucleus delayed cell cycle progression in T98G cells. The delay in cell cycle progression was associated with the inhibition of cathepsin L in the nucleus, as judged from the decreased cleavage of the CUX1 transcription factor. In vitro, inhibition of cathepsin L by stefin B was potentiated in the presence of histones, whereas histones alone did not affect the cathepsin L activity. Interaction of stefin B with the Met-75 truncated form of cathepsin L in the nucleus was confirmed by fluorescence resonance energy transfer experiments in the living cells. Stefin B could thus play an important role in regulating the proteolytic activity of cathepsin L in the nucleus, protecting substrates such as transcription factors from its proteolytic processing.


Assuntos
Catepsina L/metabolismo , Núcleo Celular/metabolismo , Cistatina B/metabolismo , Regulação da Expressão Gênica , Histonas/química , Animais , Ciclo Celular , Linhagem Celular Tumoral , Citosol/metabolismo , Fibroblastos/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Histonas/metabolismo , Humanos , Camundongos , Modelos Biológicos
6.
J Biol Chem ; 285(5): 3201-10, 2010 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-19955183

RESUMO

To contribute to the question of the putative role of cystatins in Alzheimer disease and in neuroprotection in general, we studied the interaction between human stefin B (cystatin B) and amyloid-beta-(1-40) peptide (Abeta). Using surface plasmon resonance and electrospray mass spectrometry we were able to show a direct interaction between the two proteins. As an interesting new fact, we show that stefin B binding to Abeta is oligomer specific. The dimers and tetramers of stefin B, which bind Abeta, are domain-swapped as judged from structural studies. Consistent with the binding results, the same oligomers of stefin B inhibit Abeta fibril formation. When expressed in cultured cells, stefin B co-localizes with Abeta intracellular inclusions. It also co-immunoprecipitates with the APP fragment containing the Abeta epitope. Thus, stefin B is another APP/Abeta-binding protein in vitro and likely in cells.


Assuntos
Peptídeos beta-Amiloides/química , Cistatina B/química , Animais , Benzotiazóis , Células CHO , Cricetinae , Cricetulus , Dimerização , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Humanos , Técnicas In Vitro , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência/métodos , Ligação Proteica , Espectrometria de Massas por Ionização por Electrospray , Ressonância de Plasmônio de Superfície , Tiazóis/química
7.
Biol Cell ; 102(6): 319-34, 2010 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-20078424

RESUMO

BACKGROUND: Protein aggregation is a major contributor to the pathogenic mechanisms of human neurodegenerative diseases. Mutations in the CSTB (cystatin B) gene [StB (stefin B)] cause EPM1 (progressive myoclonus epilepsy of type 1), an epilepsy syndrome with features of neurodegeneration and increased oxidative stress. Oligomerization and aggregation of StB in mammalian cells have recently been reported. It has also been observed that StB is overexpressed after seizures and in certain neurodegenerative conditions, which could potentially lead to its aggregation. Human StB proved to be a good model system to study amyloid fibril formation in vitro and, as we show here, to study protein aggregation in cells. RESULTS: Endogenous human StB formed smaller, occasional cytoplasmic aggregates and chemical inhibition of the UPS (ubiquitin-proteasome system) led to an increase in the amount of the endogenous protein and also increased its aggregation. Further, we characterized both the untagged and T-Sapphire-tagged StB on overexpression in mammalian cells. Compared with wild-type StB, the EPM1 missense mutant (G4R), the aggregate-prone EPM1 mutant (R68X) and the Y31 StB variant (both tagged and untagged) formed larger cytosolic and often perinuclear aggregates accompanied by cytoskeletal reorganization. Non-homogeneous morphology of these large aggregates was revealed using TEM (transmission electron microscopy) with StB detected by immunogold labelling. StB-positive cytoplasmic aggregates were partially co-localized with ubiquitin, proteasome subunits S20 and S26 and components of microfilament and microtubular cytoskeleton using confocal microscopy. StB aggregates also co-localized with LC3 and the protein adaptor p62, markers of autophagy. Flow cytometry showed that protein aggregation was associated with reduced cell viability. CONCLUSIONS: We have shown that endogenous StB aggregates within cells, and that aggregation is increased upon protein overexpression or proteasome inhibition. From confocal and TEM analyses, we conclude that aggregates of StB show some of the molecular characteristics of aggresomes and may be eliminated from the cell by autophagy. Intracellular StB aggregation shows a negative correlation with cell survival.


Assuntos
Cistatina B/metabolismo , Cistatina B/ultraestrutura , Autofagia/fisiologia , Western Blotting , Linhagem Celular Tumoral , Separação Celular , Sobrevivência Celular , Citoplasma/metabolismo , Citometria de Fluxo , Humanos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Complexo de Endopeptidases do Proteassoma/metabolismo , Transfecção
8.
Cells ; 10(2)2021 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-33572835

RESUMO

Glioblastoma is the most common brain malignant tumor in the adult population, and immunotherapy is playing an increasingly central role in the treatment of many cancers. Nevertheless, the search for effective immunotherapeutic approaches for glioblastoma patients continues. The goal of immunotherapy is to promote tumor eradication, boost the patient's innate and adaptive immune responses, and overcome tumor immune resistance. A range of new, promising immunotherapeutic strategies has been applied for glioblastoma, including vaccines, oncolytic viruses, immune checkpoint inhibitors, and adoptive cell transfer. However, the main challenges of immunotherapy for glioblastoma are the intracranial location and heterogeneity of the tumor as well as the unique, immunosuppressive tumor microenvironment. Owing to the lack of appropriate tumor models, there are discrepancies in the efficiency of various immunotherapeutic strategies between preclinical studies (with in vitro and animal models) on the one hand and clinical studies (on humans) on the other hand. In this review, we summarize the glioblastoma characteristics that drive tolerance to immunotherapy, the currently used immunotherapeutic approaches against glioblastoma, and the most suitable tumor models to mimic conditions in glioblastoma patients. These models are improving and can more precisely predict patients' responses to immunotherapeutic treatments, either alone or in combination with standard treatment.


Assuntos
Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/terapia , Glioblastoma/imunologia , Glioblastoma/terapia , Imunoterapia , Modelos Biológicos , Animais , Modelos Animais de Doenças , Humanos , Terapia de Imunossupressão
9.
Antioxidants (Basel) ; 10(3)2021 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-33673502

RESUMO

Stefin B (cystatin B) is an inhibitor of endo-lysosomal cysteine cathepsin, and the loss-of-function mutations in the stefin B gene were reported in patients with Unverricht-Lundborg disease (EPM1), a form of progressive myoclonus epilepsy. Stefin B-deficient mice, a mouse model of the disease, display key features of EPM1, including myoclonic seizures. Although the underlying mechanism is not yet completely clear, it was reported that the impaired redox homeostasis and inflammation in the brain contribute to the progression of the disease. In the present study, we investigated if lipopolysaccharide (LPS)-triggered neuroinflammation affected the protein levels of redox-sensitive proteins: thioredoxin (Trx1), thioredoxin reductase (TrxR), peroxiredoxins (Prxs) in brain and cerebella of stefin B-deficient mice. LPS challenge was found to result in a marked elevation of Trx1 and TrxR in the brain and cerebella of stefin B deficient mice, while Prx1 was upregulated only in cerebella after LPS challenge. Mitochondrial peroxiredoxin 3 (Prx3), was upregulated also in the cerebellar tissue lysates prepared from unchallenged stefin B deficient mice, while after LPS challenge Prx3 was upregulated in stefin B deficient brain and cerebella. Our results imply the role of oxidative stress in the progression of the disease.

10.
Cells ; 10(8)2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34440840

RESUMO

Cystatin C is a potent cysteine protease inhibitor that plays an important role in various biological processes including cancer, cardiovascular diseases and neurodegenerative diseases. However, the role of CstC in inflammation is still unclear. In this study we demonstrated that cystatin C-deficient mice were significantly more sensitive to the lethal LPS-induced sepsis. We further showed increased caspase-11 gene expression and enhanced processing of pro-inflammatory cytokines IL-1ß and IL-18 in CstC KO bone marrow-derived macrophages (BMDM) upon LPS and ATP stimulation. Pre-treatment of BMDMs with the cysteine cathepsin inhibitor E-64d did not reverse the effect of CstC deficiency on IL-1ß processing and secretion, suggesting that the increased cysteine cathepsin activity determined in CstC KO BMDMs is not essential for NLRP3 inflammasome activation. The CstC deficiency had no effect on (mitochondrial) reactive oxygen species (ROS) generation, the MAPK signaling pathway or the secretion of anti-inflammatory cytokine IL-10. However, CstC-deficient BMDMs showed dysfunctional autophagy, as autophagy induction via mTOR and AMPK signaling pathways was suppressed and accumulation of SQSTM1/p62 indicated a reduced autophagic flux. Collectively, our study demonstrates that the excessive inflammatory response to the LPS-induced sepsis in CstC KO mice is dependent on increased caspase-11 expression and impaired autophagy, but is not associated with increased cysteine cathepsin activity.


Assuntos
Cistatina C/genética , Lipopolissacarídeos/efeitos adversos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Sepse/etiologia , Animais , Autofagia/genética , Caspases Iniciadoras/genética , Caspases Iniciadoras/metabolismo , Cistatina C/deficiência , Inflamassomos/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Leucina/análogos & derivados , Leucina/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Espécies Reativas de Oxigênio/metabolismo , Sepse/mortalidade , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais , Taxa de Sobrevida , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Regulação para Cima
11.
Immunology ; 131(2): 257-67, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20497254

RESUMO

The pore-forming protein perforin is synthesized as an inactive precursor in natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), and becomes active when a short C-terminal peptide is cleaved within acidic lysosome-like cytotoxic granules. Although it was shown more than a decade ago that this cleavage is pH dependent and can be inhibited by the generic cysteine cathepsin inhibitor E-64d, no protease capable of processing the perforin C terminus has been identified. Neither is it known whether a single protease is responsible or the processing has inbuilt redundancy. Here, we show that incubation of human NK cells and primary antigen-restricted mouse CTLs with the cathepsin L (CatL) inhibitor L1 resulted in a marked inhibition of perforin-dependent target cell death and reduced perforin processing. In vitro, CatL preferentially cleaved a site on full-length recombinant perforin close to its C terminus. The NK cells of mice deficient in CatL showed a reduction but not a complete absence of processed perforin, indicating that cysteine proteases other than CatL are also able to process perforin. We conclude that granule-bound cathepsins are essential for processing perforin to its active form, and that CatL is an important, but not exclusive, participant in this process.


Assuntos
Catepsina L/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Animais , Biocatálise/efeitos dos fármacos , Catepsina L/antagonistas & inibidores , Catepsina L/genética , Linhagem Celular Tumoral , Inibidores de Cisteína Proteinase/farmacologia , Citotoxicidade Imunológica/efeitos dos fármacos , Citotoxicidade Imunológica/genética , Citotoxicidade Imunológica/imunologia , Ácido Egtázico/farmacologia , Granzimas/metabolismo , Humanos , Células K562 , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Leucina/análogos & derivados , Leucina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Ovalbumina/imunologia , Fragmentos de Peptídeos/imunologia , Peptídeo Hidrolases/metabolismo , Perforina , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia
12.
Biol Chem ; 391(8): 923-35, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20536394

RESUMO

The cysteine peptidase cathepsin B is important in thyroid physiology by being involved in thyroid prohormone processing initiated in the follicular lumen and completed in endo-lysosomal compartments. However, cathepsin B has also been localized to the extrafollicular space and is therefore suggested to promote invasiveness and metastasis in thyroid carcinomas through, e.g., ECM degradation. In this study, immunofluorescence and biochemical data from subcellular fractionation revealed that cathepsin B, in its single- and two-chain forms, is localized to endo-lysosomes in the papillary thyroid carcinoma cell line KTC-1 and in the anaplastic thyroid carcinoma cell lines HTh7 and HTh74. This distribution is not affected by thyroid stimulating hormone (TSH) incubation of HTh74, the only cell line that expresses a functional TSH-receptor. Immunofluorescence data disclosed an additional nuclear localization of cathepsin B immunoreactivity. This was supported by biochemical data showing a proteolytically active variant slightly smaller than the cathepsin B proform in nuclear fractions. We also demonstrate that immunoreactions specific for cathepsin V, but not cathepsin L, are localized to the nucleus in HTh74 in peri-nucleolar patterns. As deduced from co-localization studies and in vitro degradation assays, we suggest that nuclear variants of cathepsins are involved in the development of thyroid malignancies through modification of DNA-associated proteins.


Assuntos
Carcinoma/enzimologia , Catepsina B/metabolismo , Núcleo Celular/enzimologia , Variação Genética , Neoplasias da Glândula Tireoide/enzimologia , Carcinoma/metabolismo , Carcinoma/patologia , Catepsina B/química , Catepsinas/química , Catepsinas/metabolismo , Fracionamento Celular , Linhagem Celular Tumoral , Núcleo Celular/patologia , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Lisossomos/enzimologia , Microscopia Confocal , Microscopia de Fluorescência , Microscopia de Contraste de Fase , Peso Molecular , Proteínas Nucleares/metabolismo , Subunidades Proteicas/metabolismo , RNA Mensageiro/metabolismo , Receptores da Tireotropina/genética , Receptores da Tireotropina/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Tireotropina/metabolismo
13.
Front Immunol ; 11: 591803, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33163006

RESUMO

The NLRP3 inflammasome is cytosolic multi-protein complex that induces inflammation and pyroptotic cell death in response to both pathogen (PAMPs) and endogenous activators (DAMPs). Recognition of PAMPs or DAMPs leads to formation of the inflammasome complex, which results in activation of caspase-1, followed by cleavage and release of pro-inflammatory cytokines. Excessive activation of NLRP3 inflammasome can contribute to development of inflammatory diseases and cancer. Autophagy is vital intracellular process for recycling and removal of damaged proteins and organelles, as well as destruction of intracellular pathogens. Cytosolic components are sequestered in a double-membrane vesicle-autophagosome, which then fuses with lysosome resulting in degradation of the cargo. The autophagy dysfunction can lead to diseases with hyperinflammation and excessive activation of NLRP3 inflammasome and thus acts as a major regulator of inflammasomes. Autophagic removal of NLRP3 inflammasome activators, such as intracellular DAMPs, NLRP3 inflammasome components, and cytokines can reduce inflammasome activation and inflammatory response. Likewise, inflammasome signaling pathways can regulate autophagic process necessary for balance between required host defense inflammatory response and prevention of excessive and detrimental inflammation. Autophagy has a protective role in some inflammatory diseases associated with NLRP3 inflammasome, including gouty arthritis, familial Mediterranean fever (FMF), and sepsis. Understanding the interregulation between these two essential biological processes is necessary to comprehend the biological mechanisms and designing possible treatments for multiple inflammatory diseases.


Assuntos
Autofagia , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Biomarcadores , Citocinas/metabolismo , Suscetibilidade a Doenças , Interações Hospedeiro-Patógeno , Humanos , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitofagia , Especificidade de Órgãos , Transdução de Sinais
15.
Nat Microbiol ; 5(9): 1119-1133, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32514074

RESUMO

The interplay between host and pathogen relies heavily on rapid protein synthesis and accurate protein targeting to ensure pathogen destruction. To gain insight into this dynamic interface, we combined Click chemistry with pulsed stable isotope labelling of amino acids in cell culture to quantify the host proteome response during macrophage infection with the intracellular bacterial pathogen Salmonella enterica Typhimurium. We monitored newly synthesized proteins across different host cell compartments and infection stages. Within this rich resource, we detected aberrant trafficking of lysosomal proteases to the extracellular space and the nucleus. We verified that active cathepsins re-traffic to the nucleus and that these are linked to cell death. Pharmacological cathepsin inhibition and nuclear targeting of a cellular cathepsin inhibitor (stefin B) suppressed S. enterica Typhimurium-induced cell death. We demonstrate that cathepsin activity is required for pyroptotic cell death via the non-canonical inflammasome, and that lipopolysaccharide transfection into the host cytoplasm is sufficient to trigger active cathepsin accumulation in the host nucleus and cathepsin-dependent cell death. Finally, cathepsin inhibition reduced gasdermin D expression, thus revealing an unexpected role for cathepsin activity in non-canonical inflammasome regulation. Overall, our study illustrates how resolution of host proteome dynamics during infection can drive the discovery of biological mechanisms at the host-microbe interface.


Assuntos
Catepsinas/metabolismo , Morte Celular/fisiologia , Macrófagos/metabolismo , Proteômica , Infecções por Salmonella/metabolismo , Salmonella typhimurium/metabolismo , Animais , Catepsinas/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Cistatina B/antagonistas & inibidores , Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipopolissacarídeos/metabolismo , Lisossomos/metabolismo , Macrófagos/microbiologia , Camundongos , Peptídeo Hidrolases/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Proteoma , Células RAW 264.7 , Infecções por Salmonella/microbiologia
16.
J Neurochem ; 110(2): 486-95, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19457110

RESUMO

Neuroimage analysis in alcoholic corpus callosum (CC) suggests that microstructural abnormalities are higher in the genu followed by the body and the splenium. Molecular mechanisms underlying these dysmorphologys are still unclear. Protein expression was performed using the CC body samples [(nine controls, seven uncomplicated, and six complicated (with liver cirrhosis) alcoholics] through proteomics approach. Thirty-nine protein spots in uncomplicated and 60 in complicated alcoholics were differentially altered compared with the control (p < 0.05). Comparison between alcoholic groups revealed that 40% more protein showed altered expression in complicated compared with uncomplicated. This result suggests that alcohol-related liver dysfunction has synergetic effects on brain protein expression. Subregional expression profiles indicate that the highest numbers of region-specific proteins were in the genus followed by the CC body and the splenium. Interestingly, abnormal thiamine cascade was strongly suggested in the genu, and to a lesser extent in the CC body, but no such cascade was observed in the splenium. Therefore, alcohol-induced microstructural damage detected by image analysis in the CC, possibly involves multiple biochemical mechanisms.


Assuntos
Alcoolismo/metabolismo , Corpo Caloso/fisiologia , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/fisiologia , Biossíntese de Proteínas , Alcoolismo/genética , Alcoolismo/patologia , Corpo Caloso/patologia , Proteína Glial Fibrilar Ácida/biossíntese , Proteína Glial Fibrilar Ácida/genética , Humanos , Estresse Oxidativo/fisiologia , Biossíntese de Proteínas/genética , Proteômica/métodos , Proteínas de Ligação ao Retinol/biossíntese , Proteínas de Ligação ao Retinol/genética , Transdução de Sinais/fisiologia
17.
Cells ; 8(12)2019 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-31766320

RESUMO

Stefin B (cystatin B) is an intracellular inhibitor of cysteine cathepsins and mutations in the stefin B gene, resulting in the development of Unverricht-Lundborg disease, which is a form of myoclonic epilepsy. It was suggested that a key mechanism behind stefin B-mediated disease progression was impaired redox homeostasis. Stefin B-deficient mice were found more sensitive to lipopolysaccharide (LPS)-induced sepsis as a consequence of increased expression of caspase-11 and Nucleotide-binding oligomerization domain, Leucine rich Repeat and Pyrin domain containing (NLRP nflammasome activation and higher levels of mitochondrial reactive oxygen species (ROS). In the present study, we investigated if LPS-triggered oxidative stress affected the protein levels and redox status of redox sensitive proteins-thioredoxin, peroxiredoxins, and superoxide dismutases in macrophages and spleens of LPS-injected mice. LPS challenge was found to result in a marked elevation in mitochondrial peroxiredoxin 3 (Prx3), sulfiredoxin, and superoxide dismutase 2 (Sod2) in stefin B-deficient macrophages and spleens. We determined that sulfiredoxin is targeted to mitochondria after LPS challenge. In conclusion, the upregulation of mitochondrial redox-sensitive proteins Prx3 and Sod2 in stefin B-deficient cells implies a protective role of stefin B in mitochondrial function.


Assuntos
Cistatina B/metabolismo , Macrófagos/metabolismo , Mitocôndrias/metabolismo , Animais , Caspases/metabolismo , Catepsinas/metabolismo , Cistatina B/fisiologia , Cistatinas/metabolismo , Feminino , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos , Mitocôndrias/fisiologia , Proteínas Mitocondriais/metabolismo , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Peroxirredoxinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Tiorredoxinas/metabolismo , Fatores de Transcrição/metabolismo
18.
Front Immunol ; 8: 873, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28791024

RESUMO

Inflammation is an essential physiological process, which enables survival during infection and maintains tissue homeostasis. Interferons (IFNs) and pro- and anti-inflammatory cytokines are crucial for appropriate response to pathogens, damaged cells, or irritants in inflammatory response. The inflammasom is multiprotein complex, which initiates cleavage of pro-inflammatory cytokines IL-1ß and IL-18 into active forms. In addition, inflammasomes initiate pyroptotic cell death. In the present review, I summarize and analyze recent findings regarding the cross talk of IFNs and inflammasomes.

19.
FEBS Lett ; 580(27): 6295-301, 2006 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-17098233

RESUMO

The cystatins constitute a large group of evolutionary related proteins with diverse biological activities. Initially, they were characterized as inhibitors of lysosomal cysteine proteases - cathepsins. Cathepsins are involved in processing and presentation of antigens, as well as several pathological conditions such as inflammation and cancer. Recently, alternative functions of cystatins have been proposed: they also induce tumour necrosis factor and interleukin 10 synthesis and stimulate nitric oxide production. The aim of the present review was the analysis of data on cystatins from NCBI GEO database and the literature, and obtained in microarray and serial analysis of gene expression (SAGE) experiments. The expression of cystatins A, B, C, and F in macrophages, dendritic cells and natural killer cells of the immune system, during differentiation and activation is discussed.


Assuntos
Apresentação de Antígeno , Diferenciação Celular/imunologia , Cistatinas/imunologia , Neoplasias/imunologia , Animais , Catepsinas/antagonistas & inibidores , Catepsinas/imunologia , Células Dendríticas/imunologia , Humanos , Inflamação/imunologia , Interleucina-10/imunologia , Células Matadoras Naturais/imunologia , Lisossomos/imunologia , Macrófagos/imunologia , Óxido Nítrico/imunologia , Fator de Necrose Tumoral alfa
20.
FEBS Lett ; 579(10): 2149-55, 2005 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-15811333

RESUMO

Stefin B (cystatin B) is an inhibitor of lysosomal cysteine cathepsins and does not inhibit cathepsin D, E (aspartic) or cathepsin G (serine) proteinases. In this study, we have investigated apoptosis triggered by camptothecin, staurosporin (STS), and anti-CD95 monoclonal antibody in the thymocytes from the stefin B-deficient mice and wild-type mice. We have observed increased sensibility to STS-induced apoptosis in the thymocytes of stefin B-deficient mice. Pretreatment of cells with pan-caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethylketone completely inhibited phosphatidylserine externalization and caspase activation, while treatment with inhibitor of calpains- and papain-like cathepsins (2S,3S)-trans-epoxysuccinyl-leucylamido-3-methyl-butane ethyl ester did not prevent caspase activation nor phosphatidylserine exposure. We conclude that sensitization to apoptosis induced by STS in thymocytes of stefin B-deficient and wild-type mice is not dependent on cathepsin inhibition by stefin B.


Assuntos
Apoptose/efeitos dos fármacos , Catepsinas/metabolismo , Cistatinas/metabolismo , Estaurosporina/farmacologia , Timo/efeitos dos fármacos , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Catepsinas/antagonistas & inibidores , Células Cultivadas , Cistatina B , Inibidores de Cisteína Proteinase/farmacologia , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos BALB C , Timo/citologia , Timo/enzimologia , Timo/metabolismo
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