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1.
Mol Cancer ; 22(1): 196, 2023 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-38049829

RESUMO

Pharmacologic targeting of chromatin-associated protein complexes has shown significant responses in KMT2A-rearranged (KMT2A-r) acute myeloid leukemia (AML) but resistance frequently develops to single agents. This points to a need for therapeutic combinations that target multiple mechanisms. To enhance our understanding of functional dependencies in KMT2A-r AML, we have used a proteomic approach to identify the catalytic immunoproteasome subunit PSMB8 as a specific vulnerability. Genetic and pharmacologic inactivation of PSMB8 results in impaired proliferation of murine and human leukemic cells while normal hematopoietic cells remain unaffected. Disruption of immunoproteasome function drives an increase in transcription factor BASP1 which in turn represses KMT2A-fusion protein target genes. Pharmacologic targeting of PSMB8 improves efficacy of Menin-inhibitors, synergistically reduces leukemia in human xenografts and shows preserved activity against Menin-inhibitor resistance mutations. This identifies and validates a cell-intrinsic mechanism whereby selective disruption of proteostasis results in altered transcription factor abundance and repression of oncogene-specific transcriptional networks. These data demonstrate that the immunoproteasome is a relevant therapeutic target in AML and that targeting the immunoproteasome in combination with Menin-inhibition could be a novel approach for treatment of KMT2A-r AML.


Assuntos
Leucemia Mieloide Aguda , Proteômica , Humanos , Camundongos , Animais , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Leucemia Mieloide Aguda/metabolismo , Fatores de Transcrição/genética , Mutação , Expressão Gênica
2.
Haematologica ; 107(4): 947-957, 2022 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-35045692

RESUMO

Vector-based SARS-CoV-2 vaccines have been associated with vaccine- induced thrombosis with thrombocytopenia syndrome (VITT/TTS), but the causative factors are still unresolved. We comprehensively analyzed the ChAdOx1 nCoV-19 (AstraZeneca) and Ad26.COV2.S (Johnson and Johnson) vaccines. ChAdOx1 nCoV-19 contains significant amounts of host cell protein impurities, including functionally active proteasomes, and adenoviral proteins. A much smaller amount of impurities was found in Ad26.COV2.S. Platelet factor 4 formed complexes with ChAdOx1 nCoV-19 constituents, but not with purified virions from ChAdOx1 nCoV-19 or with Ad26.COV2.S. Vascular hyperpermeability was induced by ChAdOx nCoV-19 but not by Ad26.COV2.S. These differences in impurities together with EDTAinduced capillary leakage might contribute to the higher incidence rate of VITT associated with ChAdOx1 nCoV-19 compared to Ad26.COV2.S.


Assuntos
COVID-19 , Vacinas , Ad26COVS1 , Vacinas contra COVID-19/efeitos adversos , ChAdOx1 nCoV-19 , Humanos , SARS-CoV-2
3.
Int J Mol Sci ; 23(7)2022 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-35408787

RESUMO

T cell activation plays a central role in supporting and shaping the immune response. The induction of a functional adaptive immune response requires the control of signaling processes downstream of the T cell receptor (TCR). In this regard, protein phosphorylation and dephosphorylation have been extensively studied. In the past decades, further checkpoints of activation have been identified. These are E3 ligases catalyzing the transfer of ubiquitin or ubiquitin-like proteins to protein substrates, as well as specific peptidases to counteract this reaction, such as deubiquitinating enzymes (DUBs). These posttranslational modifications can critically influence protein interactions by targeting proteins for degradation by proteasomes or mediating the complex formation required for active TCR signaling. Thus, the basic aspects of T cell development and differentiation are controlled by defining, e.g., the threshold of activation in positive and negative selection in the thymus. Furthermore, an emerging role of ubiquitination in peripheral T cell tolerance has been described. Changes in the function and abundance of certain E3 ligases or DUBs involved in T cell homeostasis are associated with the development of autoimmune diseases. This review summarizes the current knowledge of E3 enzymes and their target proteins regulating T cell signaling processes and discusses new approaches for therapeutic intervention.


Assuntos
Enzimas Desubiquitinantes , Receptores de Antígenos de Linfócitos T , Transdução de Sinais , Ubiquitina-Proteína Ligases , Enzimas Desubiquitinantes/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
4.
Eur J Immunol ; 50(2): 270-283, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31729751

RESUMO

Dissecting the different steps of the processing and presentation of tumor-associated antigens is a key aspect of immunotherapies enabling to tackle the immune response evasion attempts of cancer cells. The immunodominant glycoprotein gp100209-217 epitope, which is liberated from the melanoma differentiation antigen gp100PMEL17 , is part of immunotherapy trials. By analyzing different human melanoma cell lines, we here demonstrate that a pool of N-terminal extended peptides sharing the common minimal epitope is generated by melanoma proteasome subtypes. In vitro and in cellulo experiments indicate that ER-resident aminopeptidase 1 (ERAP1)-but not ERAP2-defines the processing of this peptide pool thereby modulating the T-cell recognition of melanoma cells. By combining the outcomes of our studies and others, we can sketch the complex processing and endogenous presentation pathway of the gp100209-217 -containing epitope/peptides, which are produced by proteasomes and are translocated to the vesicular compartment through different pathways, where the precursor peptides that reach the endoplasmic reticulum are further processed by ERAP1. The latter step enhances the activation of epitope-specific T lymphocytes, which might be a target to improve the efficiency of anti-melanoma immunotherapy.


Assuntos
Aminopeptidases/imunologia , Apresentação de Antígeno/imunologia , Retículo Endoplasmático/imunologia , Epitopos de Linfócito T/imunologia , Melanoma/imunologia , Melanoma/terapia , Antígenos de Histocompatibilidade Menor/imunologia , Antígenos de Neoplasias , Linhagem Celular Tumoral , Células HeLa , Humanos , Fatores Imunológicos/imunologia , Imunoterapia/métodos , Peptídeos/imunologia , Complexo de Endopeptidases do Proteassoma/imunologia , Linfócitos T/imunologia
5.
Transfusion ; 61(9): 2746-2755, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34331776

RESUMO

OBJECTIVES: Characterization of the proteasome and its stability in buffy-coat derived platelet concentrates (PCs) during storage. BACKGROUND: The proteasome plays a key role in cell homeostasis by processing misfolded or abnormal proteins and regulating the levels and activities of a high number of proteins contributing to cell cycle, survival, and proliferation. Controversial data exist, whether inhibition of the proteasome affects platelet function. Little is known about function, expression, and stability of the proteasome in PCs during storage, and the potential role of the platelet proteasome in storage lesions. STUDY DESIGN AND METHODS: PCs were produced by the buffy-coat method in additive solution and stored at room temperature under agitation. Platelet aggregation was monitored by light transmission aggregometry. Proteasome complexes were assessed by immunoprecipitation and immunoblotting, and proteasome activity was measured using fluorogenic substrates specific for the three different proteolytic activities over 7 days of storage. RESULTS: Proteasome inhibition led to a decreased platelet aggregation response after activation with collagen, ADP, TRAP-6, and thrombin. There were no changes in the expression of the catalytic active subunits as well as the proteasome activity during storage of PCs, comparing baseline and day 7. DISCUSSION: Platelet proteasome function is relevant for platelet aggregation in response to various agonists. The constitutive and stable expression of the active standard- and immunoproteasome in platelets makes it unlikely that loss of proteasome function is a relevant cause of storage lesions.


Assuntos
Plaquetas/citologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Buffy Coat/citologia , Plaquetas/metabolismo , Preservação de Sangue , Humanos , Ativação Plaquetária , Agregação Plaquetária , Testes de Função Plaquetária
6.
Brain Behav Immun ; 80: 35-43, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30797047

RESUMO

Proteolysis as mediated by one of the major cellular protein degradation pathways, the ubiquitin-proteasome system (UPS), plays an essential role in learning and memory formation. However, the functional relevance of immunoproteasomes in the healthy brain and especially their impact on normal brain function including processes of learning and memory has not been investigated so far. In the present study, we analyzed the phenotypic effects of an impaired immunoproteasome formation using a ß5i/LMP7-deficient mouse model in different behavioral paradigms focusing on locomotor activity, exploratory behavior, innate anxiety, startle response, prepulse inhibition, as well as fear and safety conditioning. Overall, our results demonstrate no strong effects of constitutive ß5i/LMP7-deficiency on gross locomotor abilities and anxiety-related behavior in general. However, ß5i/LMP7-deficient mice expressed more anxiety after mild stress and increased cued fear after fear conditioning. These findings indicate that the basal proper formation of immunoproteasomes and/or at least the expression of ß5i/LMP7 in healthy mice seem to be involved in the regulation of anxiety and cued fear levels.


Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Estresse Psicológico/metabolismo , Animais , Ansiedade/metabolismo , Sinais (Psicologia) , Modelos Animais de Doenças , Medo/fisiologia , Feminino , Masculino , Memória/fisiologia , Camundongos , Camundongos Knockout , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/imunologia , Complexo de Endopeptidases do Proteassoma/fisiologia , Proteólise , Reflexo de Sobressalto/fisiologia , Estresse Psicológico/imunologia
7.
Cell Commun Signal ; 17(1): 5, 2019 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-30651113

RESUMO

BACKGROUND: Treatment of acute leukemia is challenging and long-lasting remissions are difficult to induce. Innovative therapy approaches aim to complement standard chemotherapy to improve drug efficacy and decrease toxicity. Promising new therapeutic targets in cancer therapy include voltage-gated Kv1.3 potassium channels, but their role in acute leukemia is unclear. We reported that Kv1.3 channels of lymphocytes are blocked by memantine, which is known as an antagonist of neuronal N-methyl-D-aspartate type glutamate receptors and clinically applied in therapy of advanced Alzheimer disease. Here we evaluated whether pharmacological targeting of Kv1.3 channels by memantine promotes cell death of acute leukemia cells induced by chemotherapeutic cytarabine. METHODS: We analyzed acute lymphoid (Jurkat, CEM) and myeloid (HL-60, Molm-13, OCI-AML-3) leukemia cell lines and patients' acute leukemic blasts after treatment with either drug alone or the combination of cytarabine and memantine. Patch-clamp analysis was performed to evaluate inhibition of Kv1.3 channels and membrane depolarization by memantine. Cell death was determined with propidium iodide, Annexin V and SYTOX staining and cytochrome C release assay. Molecular effects of memantine co-treatment on activation of Caspases, AKT, ERK1/2, and JNK signaling were analysed by Western blot. Kv1.3 channel expression in Jurkat cells was downregulated by shRNA. RESULTS: Our study demonstrates that memantine inhibits Kv1.3 channels of acute leukemia cells and in combination with cytarabine potentiates cell death of acute lymphoid and myeloid leukemia cell lines as well as primary leukemic blasts from acute leukemia patients. At molecular level, memantine co-application fosters concurrent inhibition of AKT, S6 and ERK1/2 and reinforces nuclear down-regulation of MYC, a common target of AKT and ERK1/2 signaling. In addition, it augments mitochondrial dysfunction resulting in enhanced cytochrome C release and activation of Caspase-9 and Caspase-3 leading to amplified apoptosis. CONCLUSIONS: Our study underlines inhibition of Kv1.3 channels as a therapeutic strategy in acute leukemia and proposes co-treatment with memantine, a licensed and safe drug, as a potential approach to promote cytarabine-based cell death of various subtypes of acute leukemia.


Assuntos
Apoptose/efeitos dos fármacos , Citarabina/farmacologia , Canal de Potássio Kv1.3/antagonistas & inibidores , Leucemia Mieloide Aguda/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Memantina/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Trifosfato de Adenosina/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citocromos c/metabolismo , Humanos , Canal de Potássio Kv1.3/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo
8.
BMC Cell Biol ; 17(1): 28, 2016 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-27387758

RESUMO

BACKGROUND: Antigenic stimulation of the T cell receptor (TCR) initiates a change from a resting state into an activated one, which ultimately results in proliferation and the acquisition of effector functions. To accomplish this task, T cells require dramatic changes in metabolism. Therefore, we investigated changes of metabolic intermediates indicating for crucial metabolic pathways reflecting the status of T cells. Moreover we analyzed possible regulatory molecules required for the initiation of the metabolic changes. RESULTS: We found that proliferation inducing conditions result in an increase in key glycolytic metabolites, whereas the citric acid cycle remains unaffected. The upregulation of glycolysis led to a strong lactate production, which depends upon AKT/PKB, but not mTOR. The observed upregulation of lactate dehydrogenase results in increased lactate production, which we found to be dependent on IL-2 and to be required for proliferation. Additionally we observed upregulation of Glucose-transporter 1 (GLUT1) and glucose uptake upon stimulation, which were surprisingly not influenced by AKT inhibition. CONCLUSIONS: Our findings suggest that AKT plays a central role in upregulating glycolysis via induction of lactate dehydrogenase expression, but has no impact on glucose uptake of T cells. Furthermore, under apoptosis inducing conditions, T cells are not able to upregulate glycolysis and induce lactate production. In addition maintaining high glycolytic rates strongly depends on IL-2 production.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Ativação Linfocitária , Metabolômica , Trifosfato de Adenosina/metabolismo , Animais , Anticorpos/farmacologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Antígeno CTLA-4/metabolismo , Proliferação de Células/efeitos dos fármacos , Transporte de Elétrons/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Humanos , Interleucina-2/metabolismo , L-Lactato Desidrogenase/metabolismo , Lactatos/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo
9.
Cell Commun Signal ; 11(1): 28, 2013 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-23601194

RESUMO

BACKGROUND: PAG/Cbp represents a ubiquitous mechanism for regulating Src family kinases by recruiting Csk to the plasma membrane, thereby controlling cellular activation. Since Src kinases are known oncogenes, we used RNA interference in primary human T cells to test whether the loss of PAG resulted in lymphocyte transformation. RESULTS: PAG-depletion enhanced Src kinase activity and augmented proximal T-cell receptor signaling; exactly the phenotype expected for loss of this negative regulator. Surprisingly, rather than becoming hyper-proliferative, PAG-suppressed T cells became unresponsive. This was mediated by a Fyn-dependent hyper-phosphorylation of the inhibitory receptor CTLA-4, which recruited the protein tyrosine phosphatase Shp-1 to lipid rafts. Co-suppression of CTLA-4 abrogates this inhibition and restores proliferation to T cells. CONCLUSION: We have identified a fail-safe mechanism as well as a novel contribution of CTLA-4 to setting the activation threshold in T cells.

10.
Biomed Pharmacother ; 168: 115635, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37816303

RESUMO

Primary and acquired therapy resistance is a major problem in patients with BRAF-mutant melanomas being treated with BRAF and MEK inhibitors (BRAFI, MEKi). Therefore, development of alternative therapy regimes is still required. In this regard, new drug combinations targeting different pathways to induce apoptosis could offer promising alternative approaches. Here, we investigated the combination of proteasome and Kv1.3 potassium channel inhibition on chemo-resistant, BRAF inhibitor-resistant as well as sensitive human melanoma cells. Our experiments demonstrated that all analyzed melanoma cell lines were sensitive to proteasome inhibitor treatment at concentrations that are not toxic to primary human fibroblasts. To further reduce proteasome inhibitor-associated side effects, and to foster apoptosis, potassium channels, which are other targets to induce pro-apoptotic effects in cancer cells, were blocked. In support, combined exposure of melanoma cells to proteasome and Kv1.3 channel inhibitor resulted in synergistic effects and significantly reduced cell viability. On the molecular level, enhanced apoptosis correlated with an increase of intracellular Kv1.3 channels and pro-apoptotic proteins such as Noxa and Bak and a reduction of anti-apoptotic proteins. Thus, use of combined therapeutic strategies triggering different apoptotic pathways may efficiently prevent the outgrowth of drug-resistant and -sensitive BRAF-mutant melanoma cells. In addition, this could be the basis for an alternative approach to treat other tumors expressing mutated BRAF such as non-small-cell lung cancer.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Melanoma , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Canal de Potássio Kv1.3/genética , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Linhagem Celular Tumoral , Neoplasias Pulmonares/tratamento farmacológico , Melanoma/patologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Reguladoras de Apoptose/metabolismo , Mutação
11.
Front Immunol ; 14: 1229562, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37731490

RESUMO

Life-threatening toxic shock syndrome is often caused by the superantigen toxic shock syndrome toxin-1 (TSST-1) produced by Staphylococcus aureus. A well-known risk factor is the lack of neutralizing antibodies. To identify determinants of the anti-TSST-1 antibody response, we examined 976 participants of the German population-based epidemiological Study of Health in Pomerania (SHIP-TREND-0). We measured anti-TSST-1 antibody levels, analyzed the colonization with TSST-1-encoding S. aureus strains, and performed a genome-wide association analysis of genetic risk factors. TSST-1-specific serum IgG levels varied over a range of 4.2 logs and were elevated by a factor of 12.3 upon nasal colonization with TSST-1-encoding S. aureus. Moreover, the anti-TSST-1 antibody levels were strongly associated with HLA class II gene loci. HLA-DRB1*03:01 and HLA-DQB1*02:01 were positively, and HLA-DRB1*01:01 as well as HLA-DQB1*05:01 negatively associated with the anti-TSST-1 antibody levels. Thus, both toxin exposure and HLA alleles affect the human antibody response to TSST-1.


Assuntos
Choque Séptico , Infecções Estafilocócicas , Humanos , Staphylococcus aureus , Alelos , Estudo de Associação Genômica Ampla , Choque Séptico/genética , Superantígenos/genética , Infecções Estafilocócicas/genética
12.
Front Cell Infect Microbiol ; 12: 817532, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35281454

RESUMO

Epithelial cells are an important line of defense within the lung. Disruption of the epithelial barrier by pathogens enables the systemic dissemination of bacteria or viruses within the host leading to severe diseases with fatal outcomes. Thus, the lung epithelium can be damaged by seasonal and pandemic influenza A viruses. Influenza A virus infection induced dysregulation of the immune system is beneficial for the dissemination of bacteria to the lower respiratory tract, causing bacterial and viral co-infection. Host cells regulate protein homeostasis and the response to different perturbances, for instance provoked by infections, by post translational modification of proteins. Aside from protein phosphorylation, ubiquitination of proteins is an essential regulatory tool in virtually every cellular process such as protein homeostasis, host immune response, cell morphology, and in clearing of cytosolic pathogens. Here, we analyzed the proteome and ubiquitinome of A549 alveolar lung epithelial cells in response to infection by either Streptococcus pneumoniae D39Δcps or influenza A virus H1N1 as well as bacterial and viral co-infection. Pneumococcal infection induced alterations in the ubiquitination of proteins involved in the organization of the actin cytoskeleton and Rho GTPases, but had minor effects on the abundance of host proteins. H1N1 infection results in an anti-viral state of A549 cells. Finally, co-infection resembled the imprints of both infecting pathogens with a minor increase in the observed alterations in protein and ubiquitination abundance.


Assuntos
Coinfecção , Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A , Influenza Humana , Células A549 , Coinfecção/microbiologia , Humanos , Vírus da Influenza A Subtipo H1N1/fisiologia , Streptococcus pneumoniae , Ubiquitinação
13.
Front Immunol ; 13: 958616, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35983034

RESUMO

Upon antigen recognition by the T cell receptor (TCR), a complex signaling network orchestrated by protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs) regulates the transmission of the extracellular signal to the nucleus. The role of the PTPs Src-homology 2 (SH2) domain-containing phosphatase 1 (SHP1, Ptpn6) and Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2, Ptpn11) have been studied in various cell types including T cells. Whereas SHP1 acts as an essential negative regulator of the proximal steps in T cell signalling, the role of SHP2 in T cell activation is still a matter of debate. Here, we analyzed the role of the constitutively active SHP2-D61Y-mutant in T cell activation using knock-in mice expressing the mutant form Ptpn11D61Y in T cells. We observed reduced numbers of CD8+ and increased numbers of CD4+ T cells in the bone marrow and spleen of young and aged SHP2-D61Y-mutant mice as well as in Influenza A Virus (IAV)-infected mice compared to controls. In addition, we found elevated frequencies of effector memory CD8+ T cells and an upregulation of the programmed cell death protein 1 (PD-1)-receptor on both CD4+ and CD8+ T cells. Functional analysis of SHP2-D61Y-mutated T cells revealed an induction of late apoptosis/necrosis, a reduced proliferation and altered signaling upon TCR stimulation. However, the ability of D61Y-mutant mice to clear viral infection was not affected. In conclusion, our data indicate an important regulatory role of SHP2 in T cell function, where the effect is determined by the kinetics of SHP2 phosphatase activity and differs in the presence of the permanently active and the temporally regulated phosphatase. Due to interaction of SHP2 with the PD-1-receptor targeting the protein-tyrosine phosphatase might be a valuable tool to enhance T cell activities in immunotherapy.


Assuntos
Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Receptor de Morte Celular Programada 1 , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células de Memória Imunológica , Ativação Linfocitária , Camundongos , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Domínios de Homologia de src
14.
Anticancer Res ; 41(7): 3271-3279, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34230121

RESUMO

BACKGROUND/AIM: Androgen receptor (AR) degradation is the primary regulator of androgen receptor activity. This study was designed to investigate the influence of the proteasome on AR protein stability after enzalutamide (Enz) treatment. MATERIALS AND METHODS: Cell counting after treatment was utilized to assess the effect of Enz on cell proliferation. Changes in mRNA levels were evaluated using reverse transcription-polymerase chain reaction (RT-PCR). Proteasome activity was assessed by measurement of the chymotrypsin-like activity of the beta-5 subunit of the proteasome. Changes in protein levels after treatment with Enz, MG132 (MG), bortezomib (Bor), or their combination were assessed using western blot analysis. RESULTS: Treatment with Enz led to a significant reduction of cell proliferation and AR protein levels. However, AR mRNA levels were unchanged. Inhibition of proteasome activity by MG counteracts the Enz-mediated AR degradation transiently, whereas Bor showed no inhibition of the Enz-mediated AR degradation. CONCLUSION: Enz-mediated change in AR stability as an early and essential event after treatment was shown. However, investigations of the ubiquitin/proteasome system indicate involvement of several proteases in the Enz-mediated AR degradation process.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Benzamidas/farmacologia , Nitrilas/farmacologia , Feniltioidantoína/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise/efeitos dos fármacos , Receptores Androgênicos/metabolismo , Bortezomib/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Leupeptinas/farmacologia , Masculino , Próstata/efeitos dos fármacos , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , RNA Mensageiro/metabolismo
15.
Front Immunol ; 12: 619465, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33968021

RESUMO

Cell survival and function critically relies on the fine-tuned balance of protein synthesis and degradation. In the steady state, the standard proteasome is sufficient to maintain this proteostasis. However, upon inflammation, the sharp increase in protein production requires additional mechanisms to limit protein-associated cellular stress. Under inflammatory conditions and the release of interferons, the immunoproteasome (IP) is induced to support protein processing and recycling. In antigen-presenting cells constitutively expressing IPs, inflammation-related mechanisms contribute to the formation of MHC class I/II-peptide complexes, which are required for the induction of T cell responses. The control of Toxoplasma gondii infection relies on Interferon-γ (IFNγ)-related T cell responses. Whether and how the IP affects the course of anti-parasitic T cell responses along the infection as well as inflammation of the central nervous system is still unknown. To answer this question we used triple knockout (TKO) mice lacking the 3 catalytic subunits of the immunoproteasome (ß1i/LMP2, ß2i/MECL-1 and ß5i/LMP7). Here we show that the numbers of dendritic cells, monocytes and CD8+ T cells were reduced in Toxoplasma gondii-infected TKO mice. Furthermore, impaired IFNγ, TNF and iNOS production was accompanied by dysregulated chemokine expression and altered immune cell recruitment to the brain. T cell differentiation was altered, apoptosis rates of microglia and monocytes were elevated and STAT3 downstream signaling was diminished. Consequently, anti-parasitic immune responses were impaired in TKO mice leading to elevated T. gondii burden and prolonged neuroinflammation. In summary we provide evidence for a critical role of the IP subunits ß1i/LMP2, ß2i/MECL-1 and ß5i/LMP7 for the control of cerebral Toxoplasma gondii infection and subsequent neuroinflammation.


Assuntos
Cisteína Endopeptidases/metabolismo , Imunomodulação , Complexo de Endopeptidases do Proteassoma/metabolismo , Toxoplasmose Cerebral/etiologia , Toxoplasmose Cerebral/metabolismo , Animais , Apoptose , Biomarcadores , Citocinas/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Camundongos , Transdução de Sinais , Toxoplasma
16.
J Thromb Haemost ; 18(4): 771-780, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31898400

RESUMO

Platelets are small anucleate blood cells with a life span of 7 to 10 days. They are main regulators of hemostasis. Balanced platelet activity is crucial to prevent bleeding or occlusive thrombus formation. Growing evidence supports that platelets also participate in immune reactions, and interaction between platelets and leukocytes contributes to both thrombosis and inflammation. The ubiquitin-proteasome system (UPS) plays a key role in maintaining cellular protein homeostasis by its ability to degrade non-functional self-, foreign, or short-lived regulatory proteins. Platelets express standard and immunoproteasomes. Inhibition of the proteasome impairs platelet production and platelet function. Platelets also express major histocompatibility complex (MHC) class I molecules. Peptide fragments released by proteasomes can bind to MHC class I, which makes it also likely that platelets can activate epitope specific cytotoxic T lymphocytes (CTLs). In this review, we focus on current knowledge on the significance of the proteasome for the functions of platelets as critical regulators of hemostasis as well as modulators of the immune response.


Assuntos
Complexo de Endopeptidases do Proteassoma , Ubiquitina , Plaquetas , Antígenos de Histocompatibilidade Classe I , Humanos , Testes de Função Plaquetária
17.
Eur J Microbiol Immunol (Bp) ; 7(3): 234-245, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29034113

RESUMO

Application of the proteasome inhibitor Bortezomib for the treatment of haematopoietic malignancies such as multiple myeloma significantly improves the average overall survival of patients. However, one of the most severe side effects is the development of peripheral neuropathies caused by neurotoxic effects of Bortezomib limiting its therapeutic efficacy. With ONX-0914 a specific inhibitor of the ß5i (LMP7)-immunosubunit containing proteasomes was developed that targets exclusively the proteasome subtypes mainly expressed in immune cells including B lymphocytes as the origin of multiple myeloma. Furthermore, immunosubunitspecific inhibitors have been shown to be promising tools for the therapy of autoimmune disorders. In the presented study, we analysed the concentration-dependent impact of both inhibitors on primary neurons regarding survival rate, morphological changes, and overall viability. Our results clearly demonstrate that ONX-0914, compared to Bortezomib, is less neurotoxic suggesting its potential as a putative antineoplastic drug and as a candidate for the treatment of autoimmune disorders affecting the peripheral and/or central nervous system.

18.
Exp Mol Med ; 48(11): e270, 2016 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-27833096

RESUMO

By changing the relative abundance of generated antigenic peptides through alterations in the proteolytic activity, interferon (IFN)-γ-induced immunoproteasomes influence the outcome of CD8+ cytotoxic T lymphocyte responses. In the present study, we investigated the effects of hepatitis C virus (HCV) infection on IFN-γ-induced immunoproteasome expression using a HCV infection cell culture system. We found that, although IFN-γ induced the transcriptional expression of mRNAs encoding the ß1i/LMP2, ß2i/MECL-1 and ß5i/LMP7 immunoproteasome subunits, the formation of immunoproteasomes was significantly suppressed in HCV-infected cells. This finding indicated that immunoproteasome induction was impaired at the translational or posttranslational level by HCV infection. Gene silencing studies showed that the suppression of immunoproteasome induction is essentially dependent on protein kinase R (PKR). Indeed, the generation of a strictly immunoproteasome-dependent cytotoxic T lymphocyte epitope was impaired in in vitro processing experiments using isolated 20S proteasomes from HCV-infected cells and was restored by the silencing of PKR expression. In conclusion, our data point to a novel mechanism of immune regulation by HCV that affects the antigen-processing machinery through the PKR-mediated suppression of immunoproteasome induction in infected cells.


Assuntos
Hepacivirus/imunologia , Hepatite C/imunologia , Complexo de Endopeptidases do Proteassoma/imunologia , eIF-2 Quinase/imunologia , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Hepacivirus/genética , Humanos , Interferon gama/imunologia , Complexo de Endopeptidases do Proteassoma/genética , RNA Mensageiro/genética
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