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1.
Arch Insect Biochem Physiol ; 108(3): e21840, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34569086

RESUMO

Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), the cotton bollworm, is a destructive pest which is famous for its resistance to a variety of insecticides. RNA interference is a posttranscriptional gene silencing mechanism that has become a popular tool to control insect pests, triggered by double-stranded RNAs (dsRNAs). The effect of ingestion and injection delivery methods of dsRNA related to some protease genes including Trypsin (Ha-TRY39 and Ha-TRY96), Chymotrypsin (Ha-CHY), and Cathepsin L (Ha-CAT) on growth and development of H. armigera was investigated in this study. All protease genes encoded full ORFs and were expressed in all H. armigera larvae stages and tissues. In both injection and feeding bioassays, Ha-RNAi CHY's performance outperformed that of other protease genes. CHY enzyme activity in the midgut of larvae was significantly reduced after treatment with ds-HaCHY. Oral administration of ds-CHY also resulted in significant mortality of H. armigera larvae. However, because of the high RNase activity in the midgut lumen of lepidoptera, a large amount of dsRNA was needed to effectively kill instars of H. armigera. To reduce dsRNA degradation, bacterial expression and dsRNA formulation were used. After oral administration, it was toxic to H. armigera larvae. Before oral administration, bacterial cells were sonicated to increase dsRNA release. The RNA interference efficiency of sonicated bacteria was significantly increased, resulting in higher larval mortality when administered orally. All of these findings point to Ha-CHY as a new candidate for developing an effective dsRNA-based pesticide for H. armigera control.


Assuntos
Mariposas , Peptídeo Hidrolases , RNA de Cadeia Dupla/farmacologia , Animais , Bactérias/genética , Catepsinas/efeitos dos fármacos , Catepsinas/genética , Quimotripsina/efeitos dos fármacos , Quimotripsina/genética , Proteínas de Insetos/genética , Larva/efeitos dos fármacos , Larva/genética , Larva/crescimento & desenvolvimento , Mortalidade , Mariposas/efeitos dos fármacos , Mariposas/genética , Mariposas/crescimento & desenvolvimento , Organismos Geneticamente Modificados , Peptídeo Hidrolases/efeitos dos fármacos , Peptídeo Hidrolases/genética , Controle de Pragas/métodos , Interferência de RNA , RNA de Cadeia Dupla/biossíntese , RNA de Cadeia Dupla/metabolismo , Tripsina/efeitos dos fármacos , Tripsina/genética
2.
Exp Eye Res ; 211: 108760, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34487726

RESUMO

Little is known about the relationship between stimulation of lacrimal gland (LG) tear protein secretion by parasympathetic versus sympathetic nerves, particularly whether the spectrum of tear proteins evoked through each innervation pathway varies. We have previously shown that activity and abundance of cathepsin S (CTSS), a cysteine protease, is greatly increased in tears of Sjögren's syndrome (SS) patients and in tears from the male NOD mouse of autoimmune dacryoadenitis that recapitulates SS-associated dry eye disease. Beyond the increased synthesis of CTSS detected in the diseased NOD mouse LG, increased tear CTSS secretion in NOD mouse tears was recently linked to increased exocytosis from a novel endolysosomal secretory pathway. Here, we have compared secretion and trafficking of CTSS in healthy mouse LG acinar cells stimulated with either the parasympathetic acetylcholine receptor agonist, carbachol (CCh), or the sympathetic α1-adrenergic agonist, phenylephrine (PE). In situ secretion studies show that PE significantly increases CTSS activity and protein in tears relative to CCh stimulation by 1.2-fold (***, p = 0.0009) and ∼5-fold (*, p-0.0319), respectively. A similar significant increase in CTSS activity with PE relative to CCh is observed when cultured LGAC are stimulated in vitro. CCh stimulation significantly elevates intracellular [Ca2+], an effect associated with increases in the size of Rab3D-enriched vesicles consistent with compound fusion, and subsequently decreases in their intensity of labeling consistent with their exocytosis. PE stimulation induces a lower [Ca2+] response and has minimal effects on Rab3D-enriched SV diameter or the intensity of Rab3D-enriched SV labeling. LG deficient in Rab3D exhibit a higher sensitivity to PE stimulation, and secrete more CTSS activity. Significant increases in the colocalization of endolysosomal vesicle markers (Lamp1, Lamp2, Rab7) with the subapical actin suggestive of fusion of endolysosomal vesicles at the apical membrane occur both with CCh and PE stimulation, but PE demonstrates increased colocalization. In conclusion, the α1-adrenergic agonist, PE, increases CTSS secretion into tears through a pathway independent of the exocytosis of Rab3D-enriched mature SV, possibly representing an alternative endolysosomal secretory pathway.


Assuntos
Células Acinares/efeitos dos fármacos , Agonistas de Receptores Adrenérgicos alfa 1/farmacologia , Catepsinas/metabolismo , Aparelho Lacrimal/efeitos dos fármacos , Fenilefrina/farmacologia , Via Secretória/efeitos dos fármacos , Lágrimas/metabolismo , Células Acinares/metabolismo , Animais , Western Blotting , Cálcio/metabolismo , Carbacol/farmacologia , Células Cultivadas , Agonistas Colinérgicos/farmacologia , Modelos Animais de Doenças , Feminino , Inativação Gênica , Aparelho Lacrimal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , beta-N-Acetil-Hexosaminidases/metabolismo , Proteínas rab3 de Ligação ao GTP/genética
3.
Theranostics ; 11(16): 7970-7983, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34335974

RESUMO

The novel ß-coronavirus, SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19), has infected more than 177 million people and resulted in 3.84 million death worldwide. Recent epidemiological studies suggested that some environmental factors, such as air pollution, might be the important contributors to the mortality of COVID-19. However, how environmental exposure enhances the severity of COVID-19 remains to be fully understood. In the present report, we provided evidence showing that mdig, a previously reported environmentally-induced oncogene that antagonizes repressive trimethylation of histone proteins, is an important regulator for SARS-CoV-2 receptors neuropilin-1 (NRP1) and NRP2, cathepsins, glycan metabolism and inflammation, key determinants for viral infection and cytokine storm of the patients. Depletion of mdig in bronchial epithelial cells by CRISPR-Cas-9 gene editing resulted in a decreased expression of NRP1, NRP2, cathepsins, and genes involved in protein glycosylation and inflammation, largely due to a substantial enrichment of lysine 9 and/or lysine 27 trimethylation of histone H3 (H3K9me3/H3K27me3) on these genes as determined by ChIP-seq. Meanwhile, we also validated that environmental factor arsenic is able to induce mdig, NRP1 and NRP2, and genetic disruption of mdig lowered expression of NRP1 and NRP2. Furthermore, mdig may coordinate with the Neanderthal variants linked to an elevated mortality of COVID-19. These data, thus, suggest that mdig is a key mediator for the severity of COVID-19 in response to environmental exposure and targeting mdig may be the one of the effective strategies in ameliorating the symptom and reducing the mortality of COVID-19.


Assuntos
COVID-19/metabolismo , COVID-19/virologia , Dioxigenases/metabolismo , Histona Desmetilases/metabolismo , Neuropilina-1/metabolismo , Proteínas Nucleares/metabolismo , Polissacarídeos/metabolismo , SARS-CoV-2/metabolismo , Células Epiteliais Alveolares/metabolismo , Animais , COVID-19/epidemiologia , Catepsinas/metabolismo , Linhagem Celular , Células Cultivadas , Dioxigenases/biossíntese , Dioxigenases/genética , Exposição Ambiental , Histona Desmetilases/biossíntese , Histona Desmetilases/genética , Histonas/metabolismo , Humanos , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Pandemias , Ratos , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/metabolismo
4.
ACS Chem Biol ; 16(9): 1628-1643, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34416110

RESUMO

Cathepsin B is a cysteine protease that normally functions within acidic lysosomes for protein degradation, but in numerous human diseases, cathepsin B translocates to the cytosol having neutral pH where the enzyme activates inflammation and cell death. Cathepsin B is active at both the neutral pH 7.2 of the cytosol and the acidic pH 4.6 within lysosomes. We evaluated the hypothesis that cathepsin B may possess pH-dependent cleavage preferences that can be utilized for design of a selective neutral pH inhibitor by (1) analysis of differential cathepsin B cleavage profiles at neutral pH compared to acidic pH using multiplex substrate profiling by mass spectrometry (MSP-MS), (2) design of pH-selective peptide-7-amino-4-methylcoumarin (AMC) substrates, and (3) design and validation of Z-Arg-Lys-acyloxymethyl ketone (AOMK) as a selective neutral pH inhibitor. Cathepsin B displayed preferences for cleaving peptides with Arg in the P2 position at pH 7.2 and Glu in the P2 position at pH 4.6, represented by its primary dipeptidyl carboxypeptidase and modest endopeptidase activity. These properties led to design of the substrate Z-Arg-Lys-AMC having neutral pH selectivity, and its modification with the AOMK warhead to result in the inhibitor Z-Arg-Lys-AOMK. This irreversible inhibitor displays nanomolar potency with 100-fold selectivity for inhibition of cathepsin B at pH 7.2 compared to pH 4.6, shows specificity for cathepsin B over other cysteine cathepsins, and is cell permeable and inhibits intracellular cathepsin B. These findings demonstrate that cathepsin B possesses pH-dependent cleavage properties that can lead to development of a potent, neutral pH inhibitor of this enzyme.


Assuntos
Catepsina B/antagonistas & inibidores , Inibidores de Cisteína Proteinase/química , Citosol/metabolismo , Lisossomos/metabolismo , Peptídeos/química , Sequência de Aminoácidos , Sítios de Ligação , Catepsinas/metabolismo , Permeabilidade da Membrana Celular , Inibidores de Cisteína Proteinase/metabolismo , Endopeptidases/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Cinética , Espectrometria de Massas , Peptídeos/metabolismo , Ligação Proteica , Especificidade por Substrato
5.
Molecules ; 26(16)2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34443335

RESUMO

The specificity of inhibition by 6,6'-dihydroxythiobinupharidine (DTBN) on cysteine proteases was demonstrated in this work. There were differences in the extent of inhibition, reflecting active site structural-steric and biochemical differences. Cathepsin S (IC50 = 3.2 µM) was most sensitive to inhibition by DTBN compared to Cathepsin B, L and papain (IC50 = 1359.4, 13.2 and 70.4 µM respectively). DTBN is inactive for the inhibition of Mpro of SARS-CoV-2. Docking simulations suggested a mechanism of interaction that was further supported by the biochemical results. In the docking results, it was shown that the cysteine sulphur of Cathepsin S, L and B was in close proximity to the DTBN thiaspirane ring, potentially forming the necessary conditions for a nucleophilic attack to form a disulfide bond. Covalent docking and molecular dynamic simulations were performed to validate disulfide bond formation and to determine the stability of Cathepsins-DTBN complexes, respectively. The lack of reactivity of DTBN against SARS-CoV-2 Mpro was attributed to a mismatch of the binding conformation of DTBN to the catalytic binding site of Mpro. Thus, gradations in reactivity among the tested Cathepsins may be conducive for a mechanism-based search for derivatives of nupharidine against COVID-19. This could be an alternative strategy to the large-scale screening of electrophilic inhibitors.


Assuntos
Alcaloides/farmacologia , Cisteína Proteases/metabolismo , Alcaloides/química , Animais , Antivirais/farmacologia , Sítios de Ligação , COVID-19/tratamento farmacológico , COVID-19/metabolismo , Domínio Catalítico , Catepsinas/farmacologia , Linhagem Celular Tumoral , Cisteína Proteases/química , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/farmacologia , Humanos , Camundongos , Simulação de Acoplamento Molecular/métodos , Nuphar/química , Papaína/farmacologia , Extratos Vegetais/farmacologia , Ligação Proteica , SARS-CoV-2/efeitos dos fármacos
6.
Sci Rep ; 11(1): 16348, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381063

RESUMO

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that affects multiple organs. Recent studies suggest relevance between cysteine protease cathepsin S (CTSS) expression and SLE. To investigate the mechanism of CTSS in SLE, CTSS-overexpressing transgenic (TG) mice were generated, and induced lupus-like symptoms. Eight months later, the TG mice spontaneously developed typical SLE symptoms regardless of the inducement. Furthermore, we observed increased toll-like receptor 7 (TLR7) expression with increased monocyte and neutrophil populations in the TG mice. In conclusion, overexpression of CTSS in mice influences TLR7 expression, autoantibodies and IFN-α, which leads to an autoimmune reaction and exacerbates lupus-like symptoms.


Assuntos
Catepsinas/metabolismo , Interferon-alfa/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptor 7 Toll-Like/metabolismo , Regulação para Cima/fisiologia , Animais , Autoanticorpos , Feminino , Humanos , Lúpus Eritematoso Sistêmico/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Monócitos/metabolismo , Neutrófilos/metabolismo
7.
Int J Mol Sci ; 22(12)2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34208434

RESUMO

Skullcapflavone II (SFII), a flavonoid derived from Scutellaria baicalensis, has been reported to have anti-inflammatory properties. However, its therapeutic potential for skin inflammatory diseases and its mechanism are unknown. Therefore, this study aimed to investigate the effect of SFII on TNF-α/IFN-γ-induced atopic dermatitis (AD)-associated cytokines, such as thymus- and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC). Co-stimulation with TNF-α/IFN-γ in HaCaT cells is a well-established model for induction of pro-inflammatory cytokines. We treated cells with SFII prior to TNF-α/IFN-γ-stimulation and confirmed that it significantly inhibited TARC and MDC expression at the mRNA and protein levels. Additionally, SFII also inhibited the expression of cathepsin S (CTSS), which is associated with itching in patients with AD. Using specific inhibitors, we demonstrated that STAT1, NF-κB, and p38 MAPK mediate TNF-α/IFN-γ-induced TARC and MDC, as well as CTSS expression. Finally, we confirmed that SFII significantly suppressed TNF-α/IFN-γ-induced phosphorylation of STAT1, NF-κB, and p38 MAPK. Taken together, our study indicates that SFII inhibits TNF-α/IFN-γ-induced TARC, MDC, and CTSS expression by regulating STAT1, NF-κB, and p38 MAPK signaling pathways.


Assuntos
Catepsinas/biossíntese , Quimiocina CCL17/biossíntese , Quimiocina CCL22/biossíntese , Flavonoides/farmacologia , Interferon gama/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Catepsinas/genética , Sobrevivência Celular/efeitos dos fármacos , Quimiocina CCL17/genética , Quimiocina CCL22/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Células HaCaT , Humanos , Queratinócitos/metabolismo , NF-kappa B/metabolismo , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
8.
Virol J ; 18(1): 154, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301275

RESUMO

The COVID-19 pandemic has put healthcare infrastructures and our social and economic lives under unprecedented strain. Effective solutions are needed to end the pandemic while significantly lessening its further impact on mortality and social and economic life. Effective and widely-available vaccines have appropriately long been seen as the best way to end the pandemic. Indeed, the current availability of several effective vaccines are already making a significant progress towards achieving that goal. Nevertheless, concerns have risen due to new SARS-CoV-2 variants that harbor mutations against which current vaccines are less effective. Furthermore, some individuals are unwilling or unable to take the vaccine. As health officials across the globe scramble to vaccinate their populations to reach herd immunity, the challenges noted above indicate that COVID-19 therapeutics are still needed to work alongside the vaccines. Here we describe the impact that neutralizing antibodies have had on those with early or mild COVID-19, and what their approval for early management of COVID-19 means for other viral entry inhibitors that have a similar mechanism of action. Importantly, we also highlight studies that show that therapeutic strategies involving various viral entry inhibitors such as multivalent antibodies, recombinant ACE2 and miniproteins can be effective not only for pre-exposure prophylaxis, but also in protecting against SARS-CoV-2 antigenic drift and future zoonotic sarbecoviruses.


Assuntos
COVID-19/tratamento farmacológico , COVID-19/virologia , SARS-CoV-2/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/epidemiologia , Vacinas contra COVID-19/farmacologia , Catepsinas/metabolismo , Humanos , Mutação , Pandemias , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Serina Endopeptidases/efeitos dos fármacos , Serina Endopeptidases/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo
9.
Immunology ; 164(3): 494-506, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34110622

RESUMO

An exclusive feature of dendritic cells (DCs) is their capacity to present exogenous antigens by MHC class I molecules, called cross-presentation. Here, we show that protein antigen can be conserved in mature murine DCs for several days in a lysosome-like storage compartment, distinct from MHC class II and early endosomal compartments, as an internal source for the supply of MHC class I ligands. Using two different uptake routes via Fcγ receptors and C-type lectin receptors, we could show that antigens were routed towards the same endolysosomal compartments after 48 h. The antigen-containing compartments lacked co-expression of molecules involved in MHC class I processing and presentation including TAP and proteasome subunits as shown by single-cell imaging flow cytometry. Moreover, we observed the absence of cathepsin S but selective co-localization of active cathepsin X with protein antigen in the storage compartments. This indicates cathepsin S-independent antigen degradation and a novel but yet undefined role for cathepsin X in antigen processing and cross-presentation by DCs. In summary, our data suggest that these antigen-containing compartments in DCs can conserve protein antigens from different uptake routes and contribute to long-lasting antigen cross-presentation.


Assuntos
Antígenos/metabolismo , Apresentação Cruzada , Células Dendríticas/imunologia , Lectinas Tipo C/metabolismo , Receptores de IgG/metabolismo , Animais , Apresentação do Antígeno , Antígenos/imunologia , Catepsinas/metabolismo , Células Dendríticas/metabolismo , Células Dendríticas/ultraestrutura , Endossomos/imunologia , Endossomos/metabolismo , Endossomos/ultraestrutura , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Lisossomos/imunologia , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Camundongos , Microscopia Eletrônica de Transmissão , Modelos Animais , Células NIH 3T3 , Cultura Primária de Células
10.
Aging (Albany NY) ; 13(11): 15638-15658, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34077394

RESUMO

Dendritic cell-derived exosomes have been proven to be efficient adjuvant options for anti-tumor vaccines in cancer immunotherapy. However, their potency in atherosclerosis remains unclear. Here we summarize the association of microRNA-203-3p (miR-203-3p) with dendritic cell-derived exosomes and atherosclerosis. Firstly, dendritic cell-derived exosomes and bone marrow-derived macrophages were isolated, after which expression of miR-203-3p and cathepsin S was determined. After the establishment of atherosclerosis mouse models, gain- and loss-of-function experiments were conducted for the analysis of effects of miR-203-3p and cathepsin S on foam-cell formation, lipid accumulation, collagen deposition and serum total cholesterol. The results found high expression of cathepsin S in atherosclerosis mice and downregulation of miR-203-3p in the serum of atherosclerosis patients and ox-LDL-simulated bone marrow-derived macrophages. Cathepsin S was the target gene of miR-203-3p. miR-203-3p transporting from exosomes to bone marrow-derived macrophages resulted in inhibition of cathepsin S expression and atherosclerosis-related phenotypes in bone marrow-derived macrophages, thus alleviating atherosclerosis in mice, and this process was found to involve the p38/MAPK signaling pathway. These findings provided evidence that the transfer of miR-203-3p by dendritic cell-derived exosomes targeted cathepsin S in bone marrow-derived macrophages to attenuate atherosclerosis progression in mice, serving as a promising clinical target for atherosclerosis.


Assuntos
Aterosclerose/genética , Catepsinas/genética , Células Dendríticas/metabolismo , Regulação para Baixo/genética , Exossomos/genética , Macrófagos/metabolismo , MicroRNAs/metabolismo , Animais , Sequência de Bases , Catepsinas/metabolismo , Movimento Celular/efeitos dos fármacos , Progressão da Doença , Feminino , Inativação Gênica , Humanos , Lipoproteínas LDL/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Pessoa de Meia-Idade , Modelos Biológicos , Fenótipo , Transporte de RNA/efeitos dos fármacos , Reprodutibilidade dos Testes
11.
Methods Mol Biol ; 2322: 63-72, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34043193

RESUMO

Leucine-rich repeat kinase 2 (LRRK2) is a causative gene product of autosomal-dominant Parkinson's disease and has been shown to play a role in lysosomal regulation. We have previously shown that endogenous LRRK2 recruited its substrates Rab8a and Rab10 onto overloaded lysosomes depending on their phosphorylation, which functioned in the suppression of lysosomal enlargement as well as the promotion of the exocytic release of lysosomal cathepsins. In this chapter, we introduce two methods to analyze cellular functions of LRRK2 upon exposure to lysosomal overload stress in RAW264.7 cells.


Assuntos
Catepsinas/farmacologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Lisossomos/metabolismo , Doença de Parkinson/metabolismo , Animais , Linhagem Celular , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Camundongos , Mutação/efeitos dos fármacos , Mutação/genética , Doença de Parkinson/genética , Fosforilação/efeitos dos fármacos , Células RAW 264.7
13.
Front Immunol ; 12: 647728, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33841429

RESUMO

Despite the available antibiotics, tuberculosis (TB) has made its return since the 90's of the last century as a global threat mostly due to co-infection with HIV, to the emergence of drug resistant strains and the lack of an effective vaccine. Host-directed strategies could be exploited to improve treatment efficacy, contain drug-resistant strains, improve immune responses and reduce disease severity. Macrophages in the lungs are often found infected with Mycobacterium tuberculosis (Mtb) and/or with HIV. The long-term survival of lung macrophages infected with Mtb or with HIV, together with their ability to produce viral particles, especially during TB, makes these niches major contributors to the pathogenicity of the infection. Among the available drugs to control HIV infection, protease inhibitors (PIs), acting at post-integrational stages of virus replication cycle, are the only drugs able to interfere with virus production and release from macrophages during chronic infection. For Mtb we recently found that the pathogen induces a general down-regulation of lysosomal proteases, helping bacteria to establish an intracellular niche in macrophages. Here we found that the PI saquinavir, contrary to ritonavir, is able to induce an increase of endolysosomal proteases activity especially of cathepsin S in Mtb infected macrophages and during co-infection with HIV. Our results indicate that saquinavir treatment of infected macrophages led not only to a significant intracellular killing of Mtb but also: (i) to an improved expression of the HLA class II antigen presentation machinery at the cell surface; (ii) to increased T-lymphocyte priming and proliferation; and (iii) to increased secretion of IFN-γ. All together the results indicate saquinavir as a potential host directed therapy for tuberculosis.


Assuntos
Coinfecção/imunologia , Reposicionamento de Medicamentos/métodos , Infecções por HIV/imunologia , Inibidores da Protease de HIV/farmacologia , HIV-1/genética , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/virologia , Mycobacterium tuberculosis/efeitos dos fármacos , Saquinavir/farmacologia , Tuberculose/imunologia , Doadores de Sangue , Linfócitos T CD4-Positivos/imunologia , Catepsinas/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Coinfecção/virologia , Infecções por HIV/virologia , HIV-1/isolamento & purificação , Humanos , Interferon gama/metabolismo , Macrófagos Alveolares/enzimologia , Transdução de Sinais/efeitos dos fármacos , Tuberculose/microbiologia
14.
Nat Microbiol ; 6(7): 899-909, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33907312

RESUMO

SARS-CoV-2 entry requires sequential cleavage of the spike glycoprotein at the S1/S2 and the S2' cleavage sites to mediate membrane fusion. SARS-CoV-2 has a polybasic insertion (PRRAR) at the S1/S2 cleavage site that can be cleaved by furin. Using lentiviral pseudotypes and a cell-culture-adapted SARS-CoV-2 virus with an S1/S2 deletion, we show that the polybasic insertion endows SARS-CoV-2 with a selective advantage in lung cells and primary human airway epithelial cells, but impairs replication in Vero E6, a cell line used for passaging SARS-CoV-2. Using engineered spike variants and live virus competition assays and by measuring growth kinetics, we find that the selective advantage in lung and primary human airway epithelial cells depends on the expression of the cell surface protease TMPRSS2, which enables endosome-independent virus entry by a route that avoids antiviral IFITM proteins. SARS-CoV-2 virus lacking the S1/S2 furin cleavage site was shed to lower titres from infected ferrets and was not transmitted to cohoused sentinel animals, unlike wild-type virus. Analysis of 100,000 SARS-CoV-2 sequences derived from patients and 24 human postmortem tissues showed low frequencies of naturally occurring mutants that harbour deletions at the polybasic site. Taken together, our findings reveal that the furin cleavage site is an important determinant of SARS-CoV-2 transmission.


Assuntos
COVID-19/transmissão , Furina/metabolismo , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Animais , COVID-19/virologia , Catepsinas/metabolismo , Chlorocebus aethiops , Endossomos/metabolismo , Células Epiteliais , Furões , Humanos , Evasão da Resposta Imune , Proteínas de Membrana/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sistema Respiratório/citologia , Sistema Respiratório/virologia , Serina Endopeptidases/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Células Vero , Empacotamento do Genoma Viral , Internalização do Vírus , Replicação Viral , Eliminação de Partículas Virais
15.
Genes (Basel) ; 12(5)2021 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-33919403

RESUMO

A mutant of the ridgetail white prawn, which exhibited rare orange-red body color with a higher level of free astaxanthin (ASTX) concentration than that in the wild-type prawn, was obtained in our lab. In order to understand the underlying mechanism for the existence of a high level of free astaxanthin, transcriptome analysis was performed to identify the differentially expressed genes (DEGs) between the mutant and wild-type prawns. A total of 78,224 unigenes were obtained, and 1863 were identified as DEGs, in which 902 unigenes showed higher expression levels, while 961 unigenes presented lower expression levels in the mutant in comparison with the wild-type prawns. Based on Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes analysis, as well as further investigation of annotated DEGs, we found that the biological processes related to astaxanthin binding, transport, and metabolism presented significant differences between the mutant and the wild-type prawns. Some genes related to these processes, including crustacyanin, apolipoprotein D (ApoD), cathepsin, and cuticle proteins, were identified as DEGs between the two types of prawns. These data may provide important information for us to understand the molecular mechanism of the existence of a high level of free astaxanthin in the prawn.


Assuntos
Palaemonidae/genética , Transcriptoma , Animais , Apolipoproteínas D/genética , Apolipoproteínas D/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Catepsinas/genética , Catepsinas/metabolismo , Mutação , Palaemonidae/metabolismo , Xantofilas/metabolismo
16.
Virol Sin ; 36(5): 1036-1051, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33830433

RESUMO

3h-31 of Heliothis virescens ascovirus 3h (HvAV-3h) is a highly conserved gene of ascoviruses. As an early gene of HvAV-3h, 3h-31 codes for a non-structural protein (3H-31) of HvAV-3h. In the study, 3h-31 was initially transcribed and expressed at 3 h post-infection (hpi) in the infected Spodoptera exigua fat body cells (SeFB). 3h-31 was further inserted into the bacmid of Autographa californica nucleopolyhedrovirus (AcMNPV) to generate an infectious baculovirus (AcMNPV-31). In vivo experiments showed that budded virus production and viral DNA replication decreased with the expression of 3H-31, and lucent tubular structures were found around the virogenic stroma in the AcMNPV-31-infected SeFB cells. In vivo, both LD50 and LD90 values of AcMNPV-31 were significantly higher than those of the wild-type AcMNPV (AcMNPV-wt) in third instar S. exigua larvae. An interesting finding was that the liquefaction of the larvae killed by the infection of AcMNPV-31 was delayed. Chitinase and cathepsin activities of AcMNPV-31-infected larvae were significantly lower than those of AcMNPV-wt-infected larvae. The possible regulatory function of the chitinase and cathepsin for 3H-31 was further confirmed by RNAi, which showed that larval cathepsin activity was significantly upregulated, but chitinase activity was not significantly changed due to the RNAi of 3h-31. Based on the obtained results, we assumed that the function of 3H-31 was associated with the inhibition of host larval chitinase and cathepsin activities, so as to restrain the hosts in their larval stages.


Assuntos
Ascoviridae , Quitinases , Animais , Ascoviridae/genética , Catepsinas/genética , Quitinases/genética , Replicação do DNA , DNA Viral , Larva , Spodoptera , Replicação Viral
17.
Int J Mol Sci ; 22(9)2021 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-33925117

RESUMO

Taken with the growing importance of cathepsin-mediated substrate proteolysis in tumor biology and progression, the focus and emphasis placed on therapeutic design and development is coming into fruition. Underpinning this approach is the invariable progression from the direction of fully characterizing cathepsin protease members and their substrate targets, towards targeting such an interaction with tangible therapeutics. The two groups of such substrates that have gained much attention over the years are the pro- and anti- apoptotic protein intermediates from the extrinsic and intrinsic signaling arms of the apoptosis pathway. As proteins that are central to determining cellular fate, some of them present themselves as very favorable candidates for therapeutic targeting. However, considering that both anti- and pro- apoptotic signaling intermediates have been reported to be downstream substrates for certain activated cathepsin proteases, therapeutic targeting approaches based on greater selectivity do need to be given greater consideration. Herein, we review the relationships shared by the cathepsin proteases and the Bcl-2 homology domain proteins, in the context of how the topical approach of adopting 'BH3-mimetics' can be explored further in modulating the relationship between the anti- and pro- apoptotic signaling intermediates from the intrinsic apoptosis pathway and their upstream cathepsin protease regulators. Based on this, we highlight important future considerations for improved therapeutic design.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Catepsinas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Materiais Biomiméticos/farmacologia , Humanos , Mitocôndrias/metabolismo , Terapia de Alvo Molecular , Fragmentos de Peptídeos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/metabolismo
18.
Protein Sci ; 30(6): 1131-1143, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33786919

RESUMO

SARS-CoV-2 is the coronavirus responsible for the COVID-19 pandemic. Proteases are central to the infection process of SARS-CoV-2. Cleavage of the spike protein on the virus's capsid causes the conformational change that leads to membrane fusion and viral entry into the target cell. Since inhibition of one protease, even the dominant protease like TMPRSS2, may not be sufficient to block SARS-CoV-2 entry into cells, other proteases that may play an activating role and hydrolyze the spike protein must be identified. We identified amino acid sequences in all regions of spike protein, including the S1/S2 region critical for activation and viral entry, that are susceptible to cleavage by furin and cathepsins B, K, L, S, and V using PACMANS, a computational platform that identifies and ranks preferred sites of proteolytic cleavage on substrates, and verified with molecular docking analysis and immunoblotting to determine if binding of these proteases can occur on the spike protein that were identified as possible cleavage sites. Together, this study highlights cathepsins B, K, L, S, and V for consideration in SARS-CoV-2 infection and presents methodologies by which other proteases can be screened to determine a role in viral entry. This highlights additional proteases to be considered in COVID-19 studies, particularly regarding exacerbated damage in inflammatory preconditions where these proteases are generally upregulated.


Assuntos
COVID-19/metabolismo , Catepsinas/metabolismo , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Sítios de Ligação , COVID-19/virologia , Interações Hospedeiro-Patógeno , Humanos , Simulação de Acoplamento Molecular , Proteólise , Proteínas Recombinantes/metabolismo , SARS-CoV-2/química , Glicoproteína da Espícula de Coronavírus/química , Internalização do Vírus
19.
Theranostics ; 11(10): 4672-4687, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33754020

RESUMO

Rationale: Oxaliplatin-induced peripheral neuropathy (OIPN) is a common adverse effect that causes delayed treatment and poor prognosis among colorectal cancer (CRC) patients. However, its mechanism remains elusive, and no effective treatment is available. Methods: We employed a prospective cohort study of adult patients with pathologically confirmed stage III CRC receiving adjuvant chemotherapy with an oxaliplatin-based regimen for investigating OIPN. To further validate the clinical manifestations and identify a potential therapeutic strategy, animal models, and in vitro studies on the mechanism of OIPN were applied. Results: Our work found that (1) consistent with clinical findings, OIPN was observed in animal models. Targeting the enzymatic activity of cathepsin S (CTSS) by pharmacological blockade and gene deficiency strategy alleviates the manifestations of OIPN. (2) Oxaliplatin treatment increases CTSS expression by enhancing cytosol translocation of interferon response factor 1 (IRF1), which then facilitates STIM-dependent store-operated Ca2+ entry homeostasis. (3) The cytokine array demonstrated an increase in anti-inflammatory cytokines and suppression of proinflammatory cytokines in mice treated with RJW-58. (4) Mechanistically, inhibiting CTSS facilitated olfactory receptors transcription factor 1 release from P300/CBP binding, which enhanced binding to the interleukin-10 (IL-10) promoter region, driving IL-10 downstream signaling pathway. (5) Serum CTSS expression is increased in CRC patients with oxaliplatin-induced neurotoxicity. Conclusions: We highlighted the critical role of CTSS in OIPN, which provides a therapeutic strategy for the common adverse side effects of oxaliplatin.


Assuntos
Catepsinas/genética , Neurônios/metabolismo , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Catepsinas/antagonistas & inibidores , Catepsinas/efeitos dos fármacos , Quimioterapia Adjuvante , Estudos de Coortes , Neoplasias Colorretais/tratamento farmacológico , Citocinas/metabolismo , Modelos Animais de Doenças , Inibidores Enzimáticos , Feminino , Fluoruracila/uso terapêutico , Gânglios Espinais , Humanos , Técnicas In Vitro , Leucovorina/uso terapêutico , Masculino , Camundongos , Camundongos Knockout , Microglia/efeitos dos fármacos , Microglia/metabolismo , Terapia de Alvo Molecular , Condução Nervosa , Neurônios/efeitos dos fármacos , Compostos Organoplatínicos/uso terapêutico , Oxaliplatina/efeitos adversos , Oxaliplatina/farmacologia , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Estudos Prospectivos
20.
Int J Mol Sci ; 22(4)2021 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-33670411

RESUMO

Pycnodysostosis, a rare autosomal recessive skeletal dysplasia, is caused by a deficiency of cathepsin K. Patients have impaired bone resorption in the presence of normal or increased numbers of multinucleated, but dysfunctional, osteoclasts. Cathepsin K degrades collagen type I and generates N-telopeptide (NTX) and the C-telopeptide (CTX) that can be quantified. Levels of these telopeptides are increased in lactating women and are associated with increased bone resorption. Nothing is known about the consequences of cathepsin K deficiency in lactating women. Here we present for the first time normalized blood and CTX measurements in a patient with pycnodysostosis, exclusively related to the lactation period. In vitro studies using osteoclasts derived from blood monocytes during lactation and after weaning further show consistent bone resorption before and after lactation. Increased expression of cathepsins L and S in osteoclasts derived from the lactating patient suggests that other proteinases could compensate for the lack of cathepsin K during the lactation period of pycnodysostosis patients.


Assuntos
Reabsorção Óssea/enzimologia , Catepsina K/deficiência , Catepsina L/metabolismo , Catepsinas/metabolismo , Lactação/metabolismo , Osteoclastos/enzimologia , Picnodisostose/enzimologia , Adulto , Reabsorção Óssea/genética , Reabsorção Óssea/patologia , Catepsina K/metabolismo , Catepsina L/genética , Catepsinas/genética , Feminino , Humanos , Osteoclastos/patologia , Picnodisostose/genética , Picnodisostose/patologia
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