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1.
Chembiochem ; 21(5): 730-738, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32022370

RESUMO

With the current trajectory of the 2019-nCoV outbreak unknown, public health and medicinal measures will both be needed to contain spreading of the virus and to optimize patient outcomes. Although little is known about the virus, an examination of the genome sequence shows strong homology with its better-studied cousin, SARS-CoV. The spike protein used for host cell infection shows key nonsynonymous mutations that might hamper the efficacy of previously developed therapeutics but remains a viable target for the development of biologics and macrocyclic peptides. Other key drug targets, including RNA-dependent RNA polymerase and coronavirus main proteinase (3CLpro), share a strikingly high (>95 %) homology to SARS-CoV. Herein, we suggest four potential drug candidates (an ACE2-based peptide, remdesivir, 3CLpro-1 and a novel vinylsulfone protease inhibitor) that could be used to treat patients suffering with the 2019-nCoV. We also summarize previous efforts into drugging these targets and hope to help in the development of broad-spectrum anti-coronaviral agents for future epidemics.


Assuntos
Antivirais/uso terapêutico , Betacoronavirus , Infecções por Coronavirus/prevenção & controle , Pneumonia Viral/prevenção & controle , Antivirais/química , Betacoronavirus/enzimologia , Betacoronavirus/genética , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/transmissão , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Desenho de Drogas , Humanos , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/transmissão , RNA Replicase/química , RNA Replicase/genética , RNA Replicase/metabolismo
2.
Phytopathology ; 110(1): 164-173, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31532352

RESUMO

Potato virus Y (PVY; Potyviridae) is a continuing challenge for potato production owing to the increasing popularity of strain-specific resistant cultivars. Hypersensitive resistance (HR) is one type of plant defense responses to restrict virus spread. In many potato cultivars, such as cultivar Premier Russet (PR), local necrosis at the site of infection protects against the most common PVYO strain, but the HR often fails to restrain necrotic strains, which spread systemically. Here, we established the role of callose accumulation in the strain-specific resistance responses to PVY infection. We first uncovered that PVY, independent of the strain, is naturally capable of suppressing pathogenesis-related callose formation in a susceptible host. Such activity can be dissociated from viral replication by the transient expression of the viral-encoded helper component proteinase (HCPro) protein, identifying it as the pathogen elicitor. However, unlike the necrotic strain, PVYO and its corresponding HCPro are unable to block callose accumulation in resistant PR potatoes, in which we observed an abundance of callose deposition and the inability of the virus to spread. The substitution of eight amino acid residues within the HCPro C-terminal region that differ between PVYO and PVYN strains and were previously shown to be responsible for eliciting the HR response, are sufficient to restore the ability of HCProO to suppress callose accumulation, despite the resistant host background, in line with a new viral function in pathogenicity.


Assuntos
Cisteína Endopeptidases , Resistência à Doença , Glucanos , Potyvirus , Solanum tuberosum , Proteínas Virais , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Glucanos/metabolismo , Potyvirus/enzimologia , Potyvirus/genética , Potyvirus/fisiologia , Solanum tuberosum/virologia , Especificidade da Espécie , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral
3.
Chemistry ; 26(9): 2002-2012, 2020 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-31692123

RESUMO

In this work a computational study of the mechanism of inhibition of cruzain, rhodesain, and cathepsin L cysteine proteases by the dipeptidyl nitroalkene Cbz-Phe-Ala-CH=CH-NO2 has been carried out by means of molecular dynamics simulations with hybrid QM/MM potentials. The free-energy surfaces confirmed that the inhibition takes place by the formation of a covalent bond between the protein and the ß-carbon atom of the inhibitor. According to the results, the tested inhibitor should be a much more efficient inhibitor of cruzain than of rhodesain, and little activity would be expected against cathepsin L, in total correspondence with the available experimental data. The origin of these differences may lie in the different stabilizing electrostatic interactions established between the inhibitor and the residues of the active site and S2 pocket of these enzymes. These results may be useful for the rational design of new dipeptidyl nitroalkenes with higher and more selective inhibitory activity against cysteine proteases.


Assuntos
Alcenos/química , Catepsina L/metabolismo , Inibidores de Cisteína Proteinase/química , Simulação de Dinâmica Molecular , Teoria Quântica , Alcenos/metabolismo , Sítios de Ligação , Domínio Catalítico , Catepsina L/antagonistas & inibidores , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/metabolismo , Dipeptídeos/química , Humanos , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/metabolismo , Termodinâmica
4.
J Enzyme Inhib Med Chem ; 35(1): 145-151, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31724441

RESUMO

There were severe panics caused by Severe Acute Respiratory Syndrome (SARS) and Middle-East Respiratory Syndrome-Coronavirus. Therefore, researches targeting these viruses have been required. Coronaviruses (CoVs) have been rising targets of some flavonoids. The antiviral activity of some flavonoids against CoVs is presumed directly caused by inhibiting 3C-like protease (3CLpro). Here, we applied a flavonoid library to systematically probe inhibitory compounds against SARS-CoV 3CLpro. Herbacetin, rhoifolin and pectolinarin were found to efficiently block the enzymatic activity of SARS-CoV 3CLpro. The interaction of the three flavonoids was confirmed using a tryptophan-based fluorescence method, too. An induced-fit docking analysis indicated that S1, S2 and S3' sites are involved in binding with flavonoids. The comparison with previous studies showed that Triton X-100 played a critical role in objecting false positive or overestimated inhibitory activity of flavonoids. With the systematic analysis, the three flavonoids are suggested to be templates to design functionally improved inhibitors.


Assuntos
Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Vírus da SARS/efeitos dos fármacos , Proteínas Virais/antagonistas & inibidores , Antivirais/síntese química , Antivirais/química , Cisteína Endopeptidases/isolamento & purificação , Cisteína Endopeptidases/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Flavonoides/síntese química , Flavonoides/química , Humanos , Estrutura Molecular , Vírus da SARS/enzimologia , Relação Estrutura-Atividade , Proteínas Virais/isolamento & purificação , Proteínas Virais/metabolismo
5.
PLoS One ; 14(12): e0222055, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31856175

RESUMO

Cruzain, a cysteine protease of Trypanosoma cruzi, is a validated target for the treatment of Chagas disease. Due to its high similarity in three-dimensional structure with human cathepsins and their sequence identity above 70% in the active site regions, identifying potent but selective cruzain inhibitors with low side effects on the host organism represents a significant challenge. Here a panel of nitrile ligands with varying potencies against cathepsin K, cathepsin L and cruzain, are studied by molecular dynamics simulations as both non-covalent and covalent complexes. Principal component analysis (PCA), identifies and quantifies patterns of ligand-induced conformational selection that enable the construction of a decision tree which can predict with high confidence a low-nanomolar inhibitor of each of three proteins, and determine the selectivity for one against others.


Assuntos
Inibidores de Cisteína Proteinase/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Animais , Domínio Catalítico , Catepsina L/metabolismo , Catepsinas/antagonistas & inibidores , Catepsinas/metabolismo , Doença de Chagas , Cisteína Endopeptidases/metabolismo , Cisteína Proteases/metabolismo , Humanos , Ligantes , Simulação de Dinâmica Molecular , Análise de Componente Principal/métodos , Ligação Proteica , Proteínas de Protozoários/metabolismo , Relação Estrutura-Atividade , Trypanosoma cruzi/metabolismo
6.
Parasitol Res ; 118(12): 3377-3386, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31720841

RESUMO

Antibody trapping is a recently described strategy for immune evasion observed in the intestinal trematode Echinostoma caproni, which may aid to avoiding the host humoral response, thus facilitating parasite survival in the presence of high levels of local-specific antibodies. Parasite-derived peptidases carry out the degradation of trapped antibodies, being essential for this mechanism. Herein, we show that cathepsin-like cysteine endopeptidases are active in the excretory/secretory products (ESPs) of E. caproni and play an important role in the context of antibody trapping. Cysteine endopeptidase activity was detected in the ESPs of E. caproni adults. The affinity probe DCG-04 distinguished a cysteine peptidase band in ESPs, which was specifically recognized by an anti-cathepsin L heterologous antibody. The same antibody localized this protein in the gut and syncytial tegument of adult worms. Studies with cultured parasites showed that in vivo-bound antibodies are removed from the parasite surface in the absence of peptidase inhibitors, while addition of cathepsin L inhibitor prevented their degradation. These results indicate that cathepsin L-like peptidases are involved in the degradation of surface-trapped antibodies and suggest that cysteine peptidases are not only crucial for tissue-invading trematodes, but they can be equally relevant at the parasite-host interface in gut-dwelling flukes.


Assuntos
Anticorpos Antiprotozoários/imunologia , Cisteína Endopeptidases/metabolismo , Echinostoma/imunologia , Equinostomíase/imunologia , Evasão da Resposta Imune/imunologia , Animais , Catepsina L/antagonistas & inibidores , Echinostoma/metabolismo , Equinostomíase/parasitologia , Proteólise
7.
J Biomed Sci ; 26(1): 79, 2019 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-31629407

RESUMO

BACKGROUND: Neuronal activity-induced changes in gene expression patterns are important mediators of neuronal plasticity. Many neuronal genes can be activated or inactivated in response to neuronal depolarization. Mechanisms that activate gene transcription are well established, but activity-dependent mechanisms that silence transcription are less understood. It is also not clear what is the significance of inhibiting these genes during neuronal activity. METHODS: Quantitative Real Time-PCR, western blot and immunofluorescence staining were performed to examine the expression of Senp1 and GluR1 in mouse cortical neurons. The alterations of Yy1 phosphorylation upon neuronal depolarization and the interaction of Yy1 with Brd4 were studied by protein co-immunoprecipitation. The regulators of Yy1 phosphorylation were identified by phosphatase inhibitors. Chromatin immunoprecipitation, in vitro DNA binding assay, luciferase assay and gene knockdown experiments were used to validate the roles of Yy1 and its phosphorylation as well as Brd4 in regulating Senp1 expression. RESULTS: We report that neuronal depolarization deactivates the transcription of the SUMO protease Senp1, an important component regulating synaptic transmission, scaling, and plasticity, through Yy1. In un-stimulated neurons, Senp1 transcription is activated by a Yy1-Brd4 transcription factor protein complex assembled on the Senp1 promoter. Upon membrane depolarization, however, Yy1 is dephosphorylated and the Yy1-Brd4 complex is evicted from the Senp1 promoter, reducing Senp1 transcription levels. Both Yy1 and Senp1 promote the expression of AMPA receptor subunit GluR1, a pivotal component in learning and memory. CONCLUSIONS: These results reveal an axis of Yy1/Brd4-Senp1 which regulates the expression of GluR1 during neuronal depolarization. This implicates a regulation mechanism in silencing gene expression upon neuronal activity.


Assuntos
Cisteína Endopeptidases/genética , Regulação da Expressão Gênica/genética , Neurônios/fisiologia , Receptores de AMPA/genética , Fator de Transcrição YY1/genética , Animais , Cisteína Endopeptidases/metabolismo , Embrião de Mamíferos/fisiologia , Camundongos Endogâmicos C57BL , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Receptores de AMPA/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Transcrição YY1/metabolismo
8.
Biomed Pharmacother ; 119: 109353, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31521890

RESUMO

OBJECTIVES: The purpose of this study was to investigate the role of legumain in the formation and stability of atherosclerotic plaque, as well as to explore the association between legumain with Smad3 pathway in a rat atherosclerosis model. METHODS: Rat with thoracic aorta atherosclerosis was established and received treatment with statin (n = 15 each) or controls (n = 10). Serum level of legumain was determined by enzyme-linked immunosorbent assay. Legumain and Smad3 aortic expression levels were assessed by immunohistochemistry and fluorescence microscopy. Protein and mRNA levels were analyzed using Western blot analysis and reverse transcriptase coupled polymerase chain reaction, respectively. RESULTS: The atherosclerotic group showed higher serum legumain level than control and statin group. Expression of legumain and Smad3 in macrophages and foam cells was increased in atherosclerotic group compared to control and statin group. The protein and mRNA levels of legumain and Smad3 were significantly attenuated by statin treatment (p < 0.05). For all groups, legumain expression was correlated linearly with Smad3 at mRNA (coefficient: 0.94) and protein (coefficient: 097) level. CONCLUSIONS: Legumain and Smad3 expression is highly expressed in mainly atherosclerotic plaque macrophages and linearly related, which is attenuated by statin therapy, suggesting legumain a potential Smad3 pathway-related marker of atherosclerosis.


Assuntos
Aorta Torácica/metabolismo , Aorta Torácica/patologia , Cisteína Endopeptidases/metabolismo , Placa Aterosclerótica/metabolismo , Proteína Smad3/metabolismo , Animais , Peso Corporal , Cisteína Endopeptidases/sangue , Cisteína Endopeptidases/genética , Lipídeos/sangue , Masculino , Placa Aterosclerótica/sangue , Placa Aterosclerótica/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Wistar , Proteína Smad3/genética
9.
J Microbiol Biotechnol ; 29(10): 1603-1606, 2019 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-31474099

RESUMO

Sortase A (SrtA), a type of transpeptidase responsible for anchoring surface proteins to the peptidoglycan cell wall, is important in the virulence of gram-positive bacteria. Three compounds were isolated from marine-derived Streptomyces sp. MBTH32 using various chromatography techniques. The structures of these compounds were determined based on spectroscopic data and comparisons with previously reported data. Among the metabolites tested, lumichrome showed strong inhibitory activity against Staphylococcus aureus SrtA without affecting cell viability. The results of cell clumping activity assessment suggest the potential for using this compound to treat S. aureus infection by inhibiting SrtA activity.


Assuntos
Aminoaciltransferases/antagonistas & inibidores , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Fibrinogênio/metabolismo , Staphylococcus aureus/patogenicidade , Streptomyces/química , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismo , Aderência Bacteriana/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Flavinas/química , Flavinas/isolamento & purificação , Flavinas/farmacologia , Concentração Inibidora 50 , Estrutura Molecular , Mutação , Staphylococcus aureus/enzimologia , Streptomyces/metabolismo , Virulência/efeitos dos fármacos
10.
Nat Commun ; 10(1): 3987, 2019 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-31485003

RESUMO

In contrast to our extensive knowledge on ubiquitin polymer signaling, we are severely limited in our understanding of poly-SUMO signaling. We set out to identify substrates conjugated to SUMO polymers, using knockdown of the poly-SUMO2/3 protease SENP6. We identify over 180 SENP6 regulated proteins that represent highly interconnected functional groups of proteins including the constitutive centromere-associated network (CCAN), the CENP-A loading factors Mis18BP1 and Mis18A and DNA damage response factors. Our results indicate a striking protein group de-modification by SENP6. SENP6 deficient cells are severely compromised for proliferation, accumulate in G2/M and frequently form micronuclei. Accumulation of CENP-T, CENP-W and CENP-A to centromeres is impaired in the absence of SENP6. Surprisingly, the increase of SUMO chains does not lead to ubiquitin-dependent proteasomal degradation of the CCAN subunits. Our results indicate that SUMO polymers can act in a proteolysis-independent manner and consequently, have a more diverse signaling function than previously expected.


Assuntos
Centrômero/metabolismo , Cisteína Endopeptidases/metabolismo , Mapas de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Linhagem Celular Tumoral , Proliferação de Células/genética , Proteína Centromérica A/genética , Proteína Centromérica A/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cisteína Endopeptidases/genética , Células HEK293 , Células HeLa , Humanos , Interferência de RNA , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Ubiquitinas/genética , Ubiquitinas/metabolismo
11.
Nat Commun ; 10(1): 3812, 2019 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-31444354

RESUMO

Acute myeloid leukemia (AML) is a genetically heterogeneous malignant disorder of the hematopoietic system, characterized by the accumulation of DNA-damaged immature myeloid precursors. Here, we find that hCINAP is involved in the repair of double-stranded DNA breaks (DSB) and that its expression correlates with AML prognosis. Following DSB, hCINAP is recruited to damage sites where it promotes SENP3-dependent deSUMOylation of NPM1. This in turn results in the dissociation of RAP80 from the damage site and CTIP-dependent DNA resection and homologous recombination. NPM1 SUMOylation is required for recruitment of DNA repair proteins at the early stage of DNA-damage response (DDR), and SUMOylated NPM1 impacts the assembly of the BRCA1 complex. Knockdown of hCINAP also sensitizes a patient-derived xenograft (PDX) mouse model to chemotherapy. In clinical AML samples, low hCINAP expression is associated with a higher overall survival rate in patients. These results provide mechanistic insight into the function of hCINAP during the DNA-damage response and its role in AML resistance to therapy.


Assuntos
Adenilato Quinase/metabolismo , Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Leucemia Mieloide Aguda/genética , Reparo de DNA por Recombinação , Adenilato Quinase/genética , Adenilato Quinase/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antineoplásicos/uso terapêutico , Proteína BRCA1/metabolismo , Cisteína Endopeptidases/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Feminino , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Células HEK293 , Células HeLa , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Masculino , Camundongos , Pessoa de Meia-Idade , Proteínas Nucleares/metabolismo , Sumoilação , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto Jovem
12.
Oxid Med Cell Longev ; 2019: 1232146, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31428220

RESUMO

Background: Zinc plays a role in mitophagy and protects cardiomyocytes from ischemia/reperfusion injury. This study is aimed at investigating whether SUMOylation of Drp1 is involved in the protection of zinc ion on cardiac I/R injury. Methods: Mouse hearts were subjected to 30 minutes of regional ischemia followed by 2 hours of reperfusion (ischemia/reoxygenation (I/R)). Infarct size and apoptosis were assessed. HL-1 cells were subjected to 24 hours of hypoxia and 6 hours of reoxygenation (hypoxia/reoxygenation (H/R)). Zinc was given 5 min before reperfusion for 30 min. SENP2 overexpression plasmid (Flag-SENP2), Drp1 mutation plasmid (Myc-Drp1 4KR), and SUMO1 siRNA were transfected into HL-1 cells for 48 h before hypoxia. Effects of zinc on SUMO family members were analyzed by Western blotting. SUMOylation of Drp1, apoptosis and the collapse of mitochondrial membrane potential (ΔΨm), and mitophagy were evaluated. Results: Compared with the control, SUMO1 modification level of proteins in the H/R decreased, while this effect was reversed by zinc. In the setting of H/R, zinc attenuated myocardial apoptosis, which was reversed by SUMO1 siRNA. Similar effects were observed in SUMO1 KO mice exposed to H/R. In addition, the dynamin-related protein 1 (Drp1) is a target protein of SUMO1. The SUMOylation of Drp1 induced by zinc regulated mitophagy and contributed to the protective effect of zinc on H/R injury. Conclusions: SUMOylation of Drp1 played an essential role in zinc-induced cardio protection against I/R injury. Our findings provide a promising therapeutic approach for acute myocardial I/R injury.


Assuntos
Dinaminas/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Substâncias Protetoras/farmacologia , Zinco/farmacologia , Animais , Apoptose/efeitos dos fármacos , Hipóxia Celular , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Dinaminas/genética , Coração/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutagênese , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Substâncias Protetoras/uso terapêutico , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína SUMO-1/antagonistas & inibidores , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , Sumoilação/efeitos dos fármacos
13.
Redox Biol ; 26: 101265, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31299612

RESUMO

Protein:protein interactions are the basis of molecular communication and are usually of transient non-covalent nature, while covalent interactions other than ubiquitination are rare. For cellular adaptations, the cellular oxygen and peroxide sensor factor inhibiting HIF (FIH) confers oxygen and oxidant stress sensitivity to the hypoxia inducible factor (HIF) by asparagine hydroxylation. We investigated whether FIH contributes to hypoxia adaptation also through other mechanisms and identified a hypoxia sensitive, likely covalent, bond formation by FIH with several client proteins, including the deubiquitinase ovarian tumor domain containing ubiquitin aldehyde binding protein 1 (OTUB1). Biochemical analyses were consistent with a co-translational amide bond formation between FIH and OTUB1, occurring within mammalian and bacterial cells but not between separately purified proteins. Bond formation is catalysed by FIH and highly dependent on oxygen availability in the cellular microenvironment. Within cells, a heterotrimeric complex is formed, consisting of two FIH and one covalently linked OTUB1. Complexation of OTUB1 by FIH regulates OTUB1 deubiquitinase activity. Our findings reveal an alternative mechanism for hypoxia adaptation with remarkably high oxygen sensitivity, mediated through covalent protein-protein interactions catalysed by an asparagine modifying dioxygenase.


Assuntos
Cisteína Endopeptidases/genética , Fator 1 Induzível por Hipóxia/metabolismo , Oxigênio/metabolismo , Linhagem Celular Tumoral , Cisteína Endopeptidases/metabolismo , Humanos , Espectrometria de Massas , Oxirredução , Oxigênio/química
14.
Oxid Med Cell Longev ; 2019: 2140427, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31281568

RESUMO

Background: Neurotoxicity induced by the amyloid-ß (Aß) peptide is one of the most important pathological mechanisms of Alzheimer's disease (AD). Based on accumulating evidence in AD research, both endoplasmic reticulum stress (ER stress) and alterations in the microRNA (miRNA) network contribute to the pathogenesis of the disease, making them potential therapeutic targets for AD. The present study was performed to investigate whether miR-34a and the inositol-requiring enzyme 1 (IRE1) are involved in the regulation of Aß-induced cytotoxicity. Methods: Human neuroblastoma SH-SY5Y cells were treated with Aß1-40. Cell viability was assessed by the MTT assay. The integrity of the plasma membrane was assessed by LDH release. The expression levels of XBP1s, IRE1α, p-IRE1α, and Caspase-2 were detected by Western blot analysis. Spliced-XBP1 mRNA and miR-34a were detected by reverse transcription- (RT-) PCR and quantitative real-time PCR, respectively. Caspase-2 activity was measured using the Caspase-2 cellular activity assay kit. The IRE1 inhibitor (STF-083010) was used to determine the role of IRE1α on miR-34a expression. SH-SY5Y cells were transfected with miR-34a mimics to assess the role of miR-34a on the activation of Caspase-2 and the viability of Aß-exposed SH-SY5Y cells. Results: We showed that Aß caused concentration- and duration-dependent death of SH-SY5Y cells. The expression levels of XBP1s, p-IRE1α, and Caspase-2 were increased, along with a corresponding decrease in the miR-34a levels in Aß-exposed SH-SY5Y cells. The IRE1 inhibitor (STF-083010) upregulated the expression of miR-34a and suppressed the activation of Caspase-2, effectively alleviating the Aß-induced death of SH-SY5Y cells. Transfection studies show that miR-34a mimics inhibit the expression of Caspase-2 and restore the viability of Aß-exposed SH-SY5Y cells. Conclusion: Aß peptide induced downregulation of miR-34a through the activation of IRE1α, which may induce cytotoxicity by targeting Caspase-2. Upregulation of miR-34a by inhibition of IRE1α has protective effects against Aß-induced injury in SH-SY5Y cells.


Assuntos
Peptídeos beta-Amiloides/toxicidade , Caspase 2/metabolismo , Cisteína Endopeptidases/metabolismo , Endorribonucleases/antagonistas & inibidores , MicroRNAs/metabolismo , Fragmentos de Peptídeos/toxicidade , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Caspase 2/biossíntese , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisteína Endopeptidases/biossíntese , Endorribonucleases/metabolismo , Humanos , MicroRNAs/administração & dosagem , MicroRNAs/biossíntese , MicroRNAs/genética , Neuroblastoma , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Transfecção , Regulação para Cima
15.
Mol Cell ; 75(4): 823-834.e5, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31302001

RESUMO

Sirt3, as a major mitochondrial nicotinamide adenine dinucleotide (NAD)-dependent deacetylase, is required for mitochondrial metabolic adaption to various stresses. However, how to regulate Sirt3 activity responding to metabolic stress remains largely unknown. Here, we report Sirt3 as a SUMOylated protein in mitochondria. SUMOylation suppresses Sirt3 catalytic activity. SUMOylation-deficient Sirt3 shows elevated deacetylation on mitochondrial proteins and increased fatty acid oxidation. During fasting, SUMO-specific protease SENP1 is accumulated in mitochondria and quickly de-SUMOylates and activates Sirt3. SENP1 deficiency results in hyper-SUMOylation of Sirt3 and hyper-acetylation of mitochondrial proteins, which reduces mitochondrial metabolic adaption responding to fasting. Furthermore, we find that fasting induces SENP1 translocation into mitochondria to activate Sirt3. The studies on mice show that Sirt3 SUMOylation mutation reduces fat mass and antagonizes high-fat diet (HFD)-induced obesity via increasing oxidative phosphorylation and energy expenditure. Our results reveal that SENP1-Sirt3 signaling modulates Sirt3 activation and mitochondrial metabolism during metabolic stress.


Assuntos
Cisteína Endopeptidases/metabolismo , Mitocôndrias/metabolismo , Mutação , Obesidade/metabolismo , Transdução de Sinais , Sirtuína 3/metabolismo , Sumoilação , Acetilação , Animais , Cisteína Endopeptidases/genética , Gorduras na Dieta/efeitos adversos , Gorduras na Dieta/farmacologia , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Mutantes , Mitocôndrias/genética , Mitocôndrias/patologia , Obesidade/induzido quimicamente , Obesidade/genética , Obesidade/patologia , Sirtuína 3/genética
16.
Int J Biol Macromol ; 137: 703-711, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31279878

RESUMO

The vacuolar processing enzyme (VPE) plays an important role in PCD and was originally identified as the proteinase responsible for the maturation and activation of vacuolar proteins in plants. We found that γVPE is involved in PCD of xylem fiber cells through the activation of CEP1 proproteases into mature protease in Arabidopsis. The γVPE protein was expressed specifically in cambium cells cambium, the primary phloem and the primary xylem during stem development. The recombinant γVPE appearing as a proenzyme at pH 7.0, and then transforming into a 40-kD mature enzyme at pH 5.5 in vitro by self-cleaving. The mature γVPE protein activated CEP1 maturation in vitro, whereas this activity was inhibited in the γvpe mutant. Transmission electron microscopy showed delayed PCD in fiber cells and thickening of secondary fiber cell walls in the γvpe mutant. Transcriptome analysis showed that the expression of 2001 genes was significantly altered expression in the γvpe mutants, and most of them are important for secondary cell wall formation and PCD. Our results demonstrate that γVPE is a crucial processing enzyme for xylem fiber cells PCD during stem development.


Assuntos
Apoptose , Arabidopsis/citologia , Arabidopsis/enzimologia , Cisteína Endopeptidases/metabolismo , Xilema/citologia , Arabidopsis/genética , Arabidopsis/metabolismo , Cisteína Endopeptidases/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Concentração de Íons de Hidrogênio , Mutação , Biossíntese de Proteínas
17.
ACS Chem Biol ; 14(8): 1836-1844, 2019 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-31348637

RESUMO

Commonly used methods to monitor internalization of cell surface structures involve application of fluorescently or otherwise labeled antibodies against the target of interest. Genetic modification of the protein of interest, for example through creation of fusions with fluorescent or enzymatically active protein domains, is another approach to follow trafficking behavior. The former approach requires indirect methods, such as multiple rounds of cell staining, to distinguish between a target that remains surface-disposed and an internalized and/or recycled species. The latter approach necessitates the creation of fusions whose behavior may not accurately reflect that of their unmodified counterparts. Here, we report a method for the characterization of protein internalization in real time through sortase-mediated, site-specific labeling of single-domain antibodies or viral proteins with a newly developed, cathepsin-sensitive quenched-fluorophore probe. Quenched probes of this type have been used to measure enzyme activity in complex environments and for different cell types, but not as a sensor of protein movement into living cells. This approach allows a quantitative assessment of the movement of proteins into protease-containing endosomes in real time in living cells. We demonstrate considerable variation in the rate of endosomal delivery for different cell surface receptors. We were also able to characterize the kinetics of influenza virus delivery to cathepsin-positive compartments, showing highly coordinated arrival in endosomal compartments. This approach should be useful for identifying proteins expressed on cells of interest for targeted endosomal delivery of payloads, such as antibody-drug conjugates or antigens that require processing.


Assuntos
Corantes Fluorescentes/química , Vírus da Influenza A/fisiologia , Proteínas de Membrana/metabolismo , Peptídeos/química , Rodaminas/química , Aminoaciltransferases/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Linhagem Celular Tumoral , Cisteína Endopeptidases/metabolismo , Cães , Endossomos/metabolismo , Células Madin Darby de Rim Canino , Camundongos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Transporte Proteico , Internalização do Vírus
18.
Nat Immunol ; 20(7): 879-889, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31182807

RESUMO

CD8+ T cells and natural killer (NK) cells are central cellular components of immune responses against pathogens and cancer, which rely on interleukin (IL)-15 for homeostasis. Here we show that IL-15 also mediates homeostatic priming of CD8+ T cells for antigen-stimulated activation, which is controlled by a deubiquitinase, Otub1. IL-15 mediates membrane recruitment of Otub1, which inhibits ubiquitin-dependent activation of AKT, a kinase that is pivotal for T cell activation and metabolism. Otub1 deficiency in mice causes aberrant responses of CD8+ T cells to IL-15, rendering naive CD8+ T cells hypersensitive to antigen stimulation characterized by enhanced metabolic reprograming and effector functions. Otub1 also controls the maturation and activation of NK cells. Deletion of Otub1 profoundly enhances anticancer immunity by unleashing the activity of CD8+ T cells and NK cells. These findings suggest that Otub1 controls the activation of CD8+ T cells and NK cells by functioning as a checkpoint of IL-15-mediated priming.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Cisteína Endopeptidases/metabolismo , Interleucina-15/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Cisteína Endopeptidases/deficiência , Enzimas Desubiquitinantes/metabolismo , Modelos Animais de Doenças , Metabolismo Energético , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Interleucina-15/genética , Melanoma Experimental , Camundongos , Camundongos Transgênicos , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Interleucina-15/metabolismo , Tolerância a Antígenos Próprios/genética , Tolerância a Antígenos Próprios/imunologia , Transdução de Sinais , Especificidade do Receptor de Antígeno de Linfócitos T , Ubiquitinação
19.
Nat Chem ; 11(7): 644-652, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31182821

RESUMO

A promising approach in cancer therapy is to find ligands that directly bind ubiquitin (Ub) chains. However, finding molecules capable of tightly and specifically binding Ub chains is challenging given the range of Ub polymer lengths and linkages and their subtle structural differences. Here, we use total chemical synthesis of proteins to generate highly homogeneous Ub chains for screening against trillion-member macrocyclic peptide libraries (RaPID system). De novo cyclic peptides were found that can bind tightly and specifically to K48-linked Ub chains, confirmed by NMR studies. These cyclic peptides protected K48-linked Ub chains from deubiquitinating enzymes and prevented proteasomal degradation of Ub-tagged proteins. The cyclic peptides could enter cells, inhibit growth and induce programmed cell death, opening new opportunities for therapeutic intervention. This highly synthetic approach, with both protein target generation and cyclic peptide discovery performed in vitro, will make other elaborate post-translationally modified targets accessible for drug discovery.


Assuntos
Lisina/química , Peptídeos Cíclicos/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Ubiquitinas/metabolismo , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Células HeLa , Humanos , Estrutura Molecular , Peptídeos Cíclicos/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Ligação Proteica , Bibliotecas de Moléculas Pequenas/farmacologia , Ubiquitinas/síntese química , Ubiquitinas/química
20.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 6): 419-427, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31204688

RESUMO

Chagas disease, which is caused by Trypanosoma cruzi, affects more than six million people worldwide. Cruzain is the major cysteine protease involved in the survival of this parasite. Here, the expression, purification and crystallization of this enzyme are reported. The cruzain crystals diffracted to 1.2 Šresolution, yielding two novel cruzain structures: apocruzain and cruzain bound to the reversible covalent inhibitor S-methyl thiomethanesulfonate. Mass-spectrometric experiments confirmed the presence of a methylthiol group attached to the catalytic cysteine. Comparison of these structures with previously published structures indicates the rigidity of the cruzain structure. These results provide further structural information about the enzyme and may help in new in silico studies to identify or optimize novel prototypes of cruzain inhibitors.


Assuntos
Apoproteínas/química , Apoproteínas/metabolismo , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/metabolismo , Desenho de Drogas , Metanossulfonato de Metila/análogos & derivados , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Cristalografia por Raios X , Inibidores de Cisteína Proteinase/química , Metanossulfonato de Metila/química , Metanossulfonato de Metila/metabolismo , Modelos Moleculares , Conformação Proteica
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