RESUMO
Dose optimization of sirolimus may further improve outcomes in allogeneic hematopoietic cell transplant (HCT) patients receiving post-transplantation cyclophosphamide (PTCy) to prevent graft-versus-host disease (GVHD). Sirolimus exposure-response association studies in HCT patients (i.e., the association of trough concentration with clinical outcomes) have been conflicting. Sirolimus has important effects on T-cells, including conventional (Tcons) and regulatory T-cells (Tregs), both of which have been implicated in the mechanisms by which PTCy prevents GVHD, but there is an absence of validated biomarkers of sirolimus effects on these cell subsets. Considering the paucity of existing biomarkers and the complexities of the immune system, we conducted a literature review to inform a quantitative systems pharmacology (QSP) model of GVHD. The published literature presented multiple challenges. The sirolimus pharmacokinetic models insufficiently describe sirolimus distribution to relevant physiological compartments. Despite multiple publications describing sirolimus effects on Tcons and Tregs in preclinical and human ex vivo models, consistent parameters relating sirolimus concentrations to circulating Tcons and Tregs could not be found. Each aspect presents a challenge in building a QSP model of sirolimus and its temporal effects on T-cell subsets and GVHD prevention. To optimize GVHD prevention regimens, phase I studies and systematic studies of immunosuppressant concentration-effect association are needed for QSP modeling.
Assuntos
Ciclofosfamida , Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Imunossupressores , Sirolimo , Humanos , Sirolimo/administração & dosagem , Doença Enxerto-Hospedeiro/prevenção & controle , Doença Enxerto-Hospedeiro/imunologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Transplante de Células-Tronco Hematopoéticas/métodos , Ciclofosfamida/efeitos adversos , Imunossupressores/administração & dosagem , Imunossupressores/farmacocinética , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Modelos BiológicosRESUMO
Stimulator of interferon genes (STING) agonists have shown potent anti-tumor efficacy in various mouse tumor models and have the potential to overcome resistance to immune checkpoint inhibitors (ICI) by linking the innate and acquired immune systems. First-generation STING agonists are administered intratumorally; however, a systemic delivery route would greatly expand the clinical use of STING agonists. Biochemical and cell-based experiments, as well as syngeneic mouse efficacy models, were used to demonstrate the anti-tumoral activity of ALG-031048, a novel STING agonist. In vitro, ALG-031048 is highly stable in plasma and liver microsomes and is resistant to degradation via phosphodiesterases. The high stability in biological matrices translated to good cellular potency in a HEK 293 STING R232 reporter assay, efficient activation and maturation of primary human dendritic cells and monocytes, as well as long-lasting, antigen-specific anti-tumor activity in up to 90% of animals in the CT26 mouse colon carcinoma model. Significant reductions in tumor growth were observed in two syngeneic mouse tumor models following subcutaneous administration. Combinations of ALG-031048 and ICIs further enhanced the in vivo anti-tumor activity. This initial demonstration of anti-tumor activity after systemic administration of ALG-031048 warrants further investigation, while the combination of systemically administered ALG-031048 with ICIs offers an attractive approach to overcome key limitations of ICIs in the clinic.
Assuntos
Neoplasias do Colo , Neoplasias , Camundongos , Animais , Humanos , Células HEK293 , Neoplasias/patologia , Neoplasias do Colo/tratamento farmacológico , Modelos Animais de Doenças , Imunoterapia , Microambiente TumoralRESUMO
Busulfan is hepatically metabolized through glutathione (GSH) conjugation; in vitro, this process depletes hepatocyte GSH stores and generates the cytotoxic metabolite γ-glutamyldehydroalanylglycine, which is too unstable to be quantitated in vivo. We sought to evaluate if pre-graft (i.e., immediately before allograft infusion) concentrations of busulfan metabolites' and of endogenous metabolomic compounds (EMCs) representing the glutathione pathway were associated with clinical outcomes in hematopoietic cell transplant (HCT) recipients receiving busulfan. The clinical outcomes evaluated were relapse, acute graft versus host disease (GVHD), chronic GVHD, non-relapse mortality, and neutrophil nadir. In pre-graft samples obtained from patients immediately before allograft infusion, our objectives were to evaluate for: (1) the presence of busulfan and its metabolites tetrahydrothiophenium ion (THT+), tetrahydrothiophene 1-oxide, sulfolane, and 3-hydroxysulfolane (N = 124); (2) EMCs using a global metabolomics assay (N = 77); and (3) the association of the busulfan metabolites and the EMCs with clinical outcomes. In the pre-graft samples, busulfan and THT+ could not be detected. THT 1-oxide, sulfolane, and 3-hydroxysulfolane were quantitated in 9.6%, 26%, and 58% of pre-graft samples; their concentrations were not associated with clinical outcomes. Four pre-graft EMCs were statistically significantly associated with the neutrophil nadir. The pre-graft EMCs were not associated with the other clinical outcomes. In conclusion, busulfan's metabolites are present in patients' plasma immediately before allograft infusion; the neutrophil nadir is associated with pre-graft EMCs. Future research should investigate the association of clinical outcomes with the concentrations of busulfan's metabolites and EMCs in the pre-graft plasma from allogeneic HCT recipients.
Assuntos
Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Humanos , Bussulfano , Transplantados , Doença Enxerto-Hospedeiro/etiologia , Glutationa/metabolismo , AloenxertosRESUMO
Brown adipose tissue (BAT) is a major site of non-shivering thermogenesis in mammals and plays an important role in energy homeostasis. Nuclear factor-erythroid 2-related factor 1 (NFE2L1, also known as Nrf1), a master regulator of cellular metabolic homeostasis and numerous stress responses, has been found to function as a critical driver in BAT thermogenic adaption to cold or obesity by providing proteometabolic quality control. Our recent studies using adipocyte-specific Nfe2l1 knockout [Nfe2l1(f)-KO] mice demonstrated that NFE2L1-dependent transcription of lipolytic genes is crucial for white adipose tissue (WAT) homeostasis and plasticity. In the present study, we found that Nfe2l1(f)-KO mice develop an age-dependent whitening and shrinking of BAT, with signatures of down-regulation of proteasome, impaired mitochondrial function, reduced thermogenesis, pro-inflammation, and elevated regulatory cell death (RCD). Mechanistic studies revealed that deficiency of Nfe2l1 in brown adipocytes (BAC) primarily results in down-regulation of lipolytic genes, which decelerates lipolysis, making BAC unable to fuel thermogenesis. These changes lead to BAC hypertrophy, inflammation-associated RCD, and consequently cold intolerance. Single-nucleus RNA-sequencing of BAT reveals that deficiency of Nfe2l1 induces significant transcriptomic changes leading to aberrant expression of a variety of genes involved in lipid metabolism, proteasome, mitochondrial stress, inflammatory responses, and inflammation-related RCD in distinct subpopulations of BAC. Taken together, our study demonstrated that NFE2L1 serves as a vital transcriptional regulator that controls the lipid metabolic homeostasis in BAC, which in turn determines the metabolic dynamics, cellular heterogeneity and subsequently cell fates in BAT.
Assuntos
Tecido Adiposo Marrom , Complexo de Endopeptidases do Proteassoma , Animais , Camundongos , Adipócitos Marrons/metabolismo , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Inflamação/metabolismo , Mamíferos/genética , Camundongos Knockout , Complexo de Endopeptidases do Proteassoma/metabolismo , RNA , Termogênese/genéticaRESUMO
A functional cure of chronic hepatitis B requires eliminating the hepatitis B virus (HBV)-encoded surface antigen (HBsAg), which can suppress immune responses. STOPS are phosphorothioated single-stranded oligonucleotides containing novel chemistries that significantly reduce HBsAgs produced by HBV-infected liver cells. The STOPS molecule ALG-10000 functions inside cells to reduce the levels of multiple HBV-encoded molecules. However, it does not bind HBV molecules. An affinity resin coupled with ALG-10000 was found to bind several proteins from liver cells harboring replicating HBV. Silencing RNAs targeting host factors SRSF1, HNRNPA2B1, GRP78 (HspA5), RPLP1, and RPLP2 reduced HBsAg levels and other HBV molecules that are concomitantly reduced by STOPS. Host proteins RPLP1/RPLP2 and GRP78 function in the translation of membrane proteins, protein folding, and degradation. ALG-10000 and the knockdowns of RPLP1/2 and GRP78 decreased the levels of HBsAg and increased their ubiquitination and proteasome degradation. GRP78, RPLP1, and RPLP2 affected HBsAg production only when HBsAg was expressed with HBV regulatory sequences, suggesting that HBV has evolved to engage with these STOPS-interacting molecules. The STOPS inhibition of HBsAg levels in HBV-infected cells occurs by sequestering cellular proteins needed for proper expression and folding of HBsAg.
RESUMO
Fine-tuning of osteoclast differentiation (OD) and bone remodeling is crucial for bone homeostasis. Dissecting the mechanisms regulating osteoclastogenesis is fundamental to understanding the pathogenesis of various bone disorders including osteoporosis and arthritis. Nuclear factor erythroid 2-related factor 1 (NFE2L1, also known as NRF1), which belongs to the CNC-bZIP family of transcription factors, orchestrates a variety of physiological processes and stress responses. While Nfe2l1 gene may be transcribed into multiple alternatively spliced isoforms, the biological function of the different isoforms of NFE2L1 in bone metabolism, osteoclastogenesis in particular, has not been reported. Here we demonstrate that knockout of all isoforms of Nfe2l1 transcripts specifically in the myeloid lineage in mice [Nfe2l1(M)-KO] results in increased activity of osteoclasts, decreased bone mass and worsening of osteoporosis induced by ovariectomy and aging. In comparison, LysM-Cre-mediated Nfe2l1 deletion has no significant effect on the osteoblast and osteocytes. Mechanistic investigations using bone marrow cells and RAW 264.7 cells revealed that deficiency of Nfe2l1 leads to accelerated and elevated OD, which is attributed, at least in part, to enhanced accumulation of ROS in the early stage of OD and expression of nuclear factor of activated T cells, cytoplasmic, calcineurin dependent 1α (Nfatc1/α). In addition, NFE2L1 regulates the transcription of multiple antioxidant genes and Nfatc1/α and OD in an isoform-specific manner. While long isoforms of NFE2L1 function as accelerators of induction of Nfatc1/α and antioxidant genes and OD, the short isoform NFE2L1-453 serves as a brake that keeps the long isoforms' accelerator effects in check. These findings provide a novel insight into the regulatory roles of NFE2L1 in osteoclastogenesis and highlight that NFE2L1 is essential in regulating bone remodeling and thus may be a valuable therapeutic target for bone disorders.
RESUMO
As well as their ion transportation function, the voltage-dependent potassium channels could act as the cell signal inducer in a variety of pathogenic processes. However, their roles in neurogenesis after stroke insults have not been clearly illustrated. In our preliminary study, the expressions of voltage-dependent potassium channels Kv4.2 was significantly decreased after stroke in cortex, striatum and hippocampus by real-time quantitative PCR assay. To underlie the neuroprotection of Kv4.2 in stroke rehabilitation, recombinant plasmids encoding the cDNAs of mouse Kv4.2 was constructed. Behavioral tests showed that the increased Kv4.2 could be beneficial to the recovery of the sensory, the motor functions and the cognitive deficits after stroke. Temozolomide (TMZ), an inhibitor of neurogenesis, could partially abolish the mentioned protections of Kv4.2. The immunocytochemical staining showed that Kv4.2 could promote the proliferations of neural stem cells and induce the neural stem cells to differentiate into neurons in vitro and in vivo. And Kv4.2 could up-regulate the expressions of ERK1/2, p-ERK1/2, p-STAT3, NGF, p-TrkA, and BDNF, CAMKII and the concentration of intracellular Ca2+. Namely, we concluded that Kv4.2 promoted neurogenesis through ERK1/2/STAT3, NGF/TrkA, Ca2+/CAMKII signal pathways and rescued the ischemic impairments. Kv4.2 might be a potential drug target for ischemic stroke intervention.
Assuntos
Isquemia Encefálica/metabolismo , Isquemia Encefálica/prevenção & controle , AVC Isquêmico/metabolismo , AVC Isquêmico/prevenção & controle , Neurogênese/fisiologia , Canais de Potássio Shal/biossíntese , Animais , Isquemia Encefálica/genética , Linhagem Celular Transformada , AVC Isquêmico/genética , Masculino , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Canais de Potássio Shal/análise , Canais de Potássio Shal/genéticaRESUMO
G-quadruplexes (G4s) formed by guanine-rich nucleic acids play a role in essential biological processes such as transcription and replication. Besides the >1.5 million putative G-4-forming sequences (PQSs), the human genome features >640 million single-nucleotide variations (SNVs), the most common type of genetic variation among people or populations. An SNV may alter a G4 structure when it falls within a PQS motif. To date, genome-wide PQS-SNV interactions and their impact have not been investigated. Herein, we present a study on the PQS-SNV interactions and the impact they can bring to G4 structures and, subsequently, gene expressions. Based on build 154 of the Single Nucleotide Polymorphism Database (dbSNP), we identified 5 million gains/losses or structural conversions of G4s that can be caused by the SNVs. Of these G4 variations (G4Vs), 3.4 million are within genes, resulting in an average load of >120 G4Vs per gene, preferentially enriched near the transcription start site. Moreover, >80% of the G4Vs overlap with transcription factor-binding sites and >14% with enhancers, giving an average load of 3 and 7.5 for the two regulatory elements, respectively. Our experiments show that such G4Vs can significantly influence the expression of their host genes. These results reveal genome-wide G4Vs and their impact on gene activity, emphasizing an understanding of genetic variation, from a structural perspective, of their physiological function and pathological implications. The G4Vs may also provide a unique category of drug targets for individualized therapeutics, health risk assessment, and drug development.
Assuntos
Proteínas de Ligação a DNA/ultraestrutura , Quadruplex G , Genoma Humano/genética , Conformação de Ácido Nucleico , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica/genética , Humanos , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , Sequências Reguladoras de Ácido Nucleico/genética , Sítio de Iniciação de Transcrição , Ativação Transcricional/genéticaRESUMO
Adipocytes play pivotal roles in maintaining energy homeostasis by storing lipids in adipose tissue (AT), regulating the flux of lipids between AT and the circulation in response to the body's energy requirements and secreting a variety of hormones, cytokines and other factors. Proper AT development and function ensure overall metabolic health. Nuclear factor erythroid 2-related factor 1 (NFE2L1, also known as NRF1) belongs to the CNC-bZIP family and plays critical roles in regulating a wide range of essential cellular functions and varies stress responses in many cells and tissues. Human and rodent Nfe2l1 genes can be transcribed into multiple splice variants resulting in various protein isoforms, which may be further modified by a variety of post-translational mechanisms. While the long isoforms of NFE2L1 have been established as master regulators of cellular adaptive antioxidant response and proteasome homeostasis, the exact tissue distribution and physiological function of NFE2L1 isoforms, the short isoforms in particular, are still under intense investigation. With regard to key roles of NFE2L1 in adipocytes, emerging data indicates that deficiency of Nfe2l1 results in aberrant adipogenesis and impaired AT functioning. Intriguingly, a single nucleotide polymorphism (SNP) of the human NFE2L1 gene is associated with obesity. In this review, we summarize the most significant findings regarding the specific roles of the multiple isoforms of NFE2L1 in AT formation and function. We highlight that NFE2L1 plays a fundamental regulatory role in the expression of multiple genes that are crucial to AT metabolism and function and thus could be an important target to improve disease states involving aberrant adipose plasticity and lipid homeostasis.
Assuntos
Adipócitos , Fator 1 Relacionado a NF-E2 , Adipogenia , Animais , Homeostase , Camundongos , Isoformas de ProteínasRESUMO
There is an urgent need for antivirals targeting the SARS-CoV-2 virus to fight the current COVID-19 pandemic. The SARS-CoV-2 main protease (3CLpro) represents a promising target for antiviral therapy. The lack of selectivity for some of the reported 3CLpro inhibitors, specifically versus cathepsin L, raises potential safety and efficacy concerns. ALG-097111 potently inhibited SARS-CoV-2 3CLpro (IC50 = 7 nM) without affecting the activity of human cathepsin L (IC50 > 10 µM). When ALG-097111 was dosed in hamsters challenged with SARS-CoV-2, a robust and significant 3.5 log10 (RNA copies/mg) reduction of the viral RNA copies and 3.7 log10 (TCID50/mg) reduction in the infectious virus titers in the lungs was observed. These results provide the first in vivo validation for the SARS-CoV-2 3CLpro as a promising therapeutic target for selective small molecule inhibitors.
Assuntos
Amidas/farmacologia , Tratamento Farmacológico da COVID-19 , Proteases 3C de Coronavírus/antagonistas & inibidores , Inibidores de Cisteína Proteinase/farmacologia , Modelos Animais de Doenças , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , Amidas/farmacocinética , Animais , COVID-19/virologia , Catepsina L/antagonistas & inibidores , Linhagem Celular , Cricetinae , Inibidores de Cisteína Proteinase/farmacocinética , Feminino , Humanos , Concentração Inibidora 50 , Masculino , Mesocricetus/virologia , Reprodutibilidade dos Testes , SARS-CoV-2/crescimento & desenvolvimento , Serina Endopeptidases , Especificidade por Substrato , Replicação Viral/efeitos dos fármacosRESUMO
Nuclear factor-erythroid 2-related factor 1 (NFE2L1) is a key transcription factor that regulates cellular adaptive responses to various stresses. Our previous studies revealed that adult adipocyte-specific Nfe2l1-knockout [Nfe2l1(f)-KO] mice show adipocyte hypertrophy and severe adipose inflammation, which can be worsened by rosiglitazone, a peroxisome proliferator-activated receptor γ agonist. To further assess the crucial roles of NFE2L1 in adipocytes, we investigated the effect of CL316243, a ß3 adrenergic agonist that promotes lipolysis via a post-translational mechanism, on adipose inflammation in juvenile Nfe2l1(f)-KO mice. In contrast to adult mice, 4-week-old juvenile Nfe2l1(f)-KO mice displayed a normal fat distribution but reduced fasting plasma glycerol levels and elevated adipocyte hypertrophy and macrophage infiltration in inguinal and gonadal WAT. In addition, Nfe2l1(f)-KO mice had decreased expression of multiple lipolytic genes and reduced lipolytic activity in WAT. While 7 days of CL316243 treatment showed no significant effect on adipose inflammation in Nfe2l1-Floxed control mice, the same treatment dramatically alleviated macrophage infiltration and mRNA expression of inflammation and pyroptosis-related genes in WAT of Nfe2l1(f)-KO mice. Together with previous findings in adult mice, the current study highlights that NFE2L1 plays a fundamental regulatory role in lipolytic gene expression and thus might be an important target to improve adipose plasticity and lipid homeostasis.
Assuntos
Adipócitos , Tecido Adiposo Branco , Animais , Dioxóis , Inflamação/tratamento farmacológico , Inflamação/genética , Camundongos , Camundongos Knockout , Fator 1 Relacionado a NF-E2RESUMO
Prolonged treatment with rifampicin (RFP), a first-line antibacterial agent used in the treatment of drug-sensitive tuberculosis, may cause various side effects, including metabolic disorders. The nuclear factor (erythroid-derived 2)-like 2 (NFE2L2, also known as NRF2) plays an essential regulatory role in cellular adaptive responses to stresses via the antioxidant response element (ARE). Our previous studies discovered that NRF2 regulates the expression of CCAAT-enhancer-binding protein ß (Cebpb) and peroxisome proliferator-activated receptor gamma (Pparg) in the process of adipogenesis. Here, we found that prolonged RFP treatment in adult male mice fed a high-fat diet developed insulin resistance, but reduced fat accumulation and decreased expression of multiple adipogenic genes in white adipose tissues. In 3 T3-L1 preadipocytes, RFP reduced the induction of Cebpb, Pparg and Cebpa at mRNA and protein levels in the early and/or later stage of hormonal cocktail-induced adipogenesis. Mechanistic investigations demonstrated that RFP inhibits NRF2-ARE luciferase reporter activity and expression of NRF2 downstream genes under normal culture condition and in the early stage of adipogenesis in 3 T3-L1 preadipocytes, suggesting that RFP can disturb adipogenic differentiation via NRF2-ARE interference. Taken together, we demonstrate a potential mechanism that RFP impairs adipose function by which RFP likely inhibits NRF2-ARE pathway and thereby interrupts its downstream adipogenic transcription network.
Assuntos
Adipócitos Brancos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Tecido Adiposo Branco/efeitos dos fármacos , Antibióticos Antituberculose/toxicidade , Elementos de Resposta Antioxidante , Fator 2 Relacionado a NF-E2/metabolismo , Obesidade/metabolismo , Rifampina/toxicidade , Células 3T3-L1 , Adipócitos Brancos/metabolismo , Adipócitos Brancos/patologia , Adipogenia/genética , Tecido Adiposo Branco/metabolismo , Tecido Adiposo Branco/patologia , Tecido Adiposo Branco/fisiopatologia , Adiposidade/efeitos dos fármacos , Animais , Dieta Hiperlipídica , Modelos Animais de Doenças , Resistência à Insulina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2/genética , Obesidade/genética , Obesidade/patologia , Obesidade/fisiopatologia , Transdução de Sinais , Transcrição GênicaRESUMO
Nuclear factor-erythroid 2-related factor 1 (NFE2L1, also known as NRF1) belongs to the CNC-bZIP family and is a master regulator of cellular adaptive responses to various stresses in many cells and tissues. Rosiglitazone (RGZ), a thiazolidinedione agonist of PPARγ, is widely used in the treatment of type 2 diabetes mellitus by stimulating genes which favor storage of triglycerides. Our previous studies demonstrated that loss of Nfe2l1 in adipocytes [Nfe2l1(f)-KO] resulted in diminished subcutaneous white adipose tissue (WAT) mass with adipocyte hypertrophy and severe adipose inflammation, which might be attributed, at least in part, to impaired lipolysis. However, the exact mechanism underlying this phenotype remains unclear. To further clarify the regulatory role of NFE2L1 in adipocyte lipid metabolism, we used protracted RGZ treatment to facilitate lipid accumulation in mice. In Nfe2l1flox/flox control mice, three weeks of RGZ treatment significantly downregulated mRNA levels of a group of inflammation-related genes in WAT. In contrast, the phenotype of Nfe2l1(f)-KO mice was aggravated showing increased transcript expression related to inflammation and pyroptosis in their shrunk WAT. These findings provide deeper insight into the mechanisms by which NFE2L1 regulates the expression of a set of lipolysis-related genes and controls WAT plasticity and global energy homeostasis.
Assuntos
Adipócitos/metabolismo , Tecido Adiposo Branco/efeitos dos fármacos , Inflamação/induzido quimicamente , Fator 1 Relacionado a NF-E2/metabolismo , Rosiglitazona/toxicidade , Adipogenia , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Homeostase/efeitos dos fármacos , Hipoglicemiantes/toxicidade , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Knockout , Fator 1 Relacionado a NF-E2/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic that began in 2019. The coronavirus 3-chymotrypsin-like cysteine protease (3CLpro) controls replication and is therefore considered a major target for antiviral discovery. This study describes the evaluation of SARS-CoV-2 3CLpro inhibitors in a novel self-assembled monolayer desorption ionization mass spectrometry (SAMDI-MS) enzymatic assay. Compared with a traditional FRET readout, the label-free SAMDI-MS assay offers greater sensitivity and eliminates false positive inhibition from compound interference with the optical signal. The SAMDI-MS assay was optimized and validated with known inhibitors of coronavirus 3CLpro such as GC376 (IC50 = 0.060 µM), calpain inhibitors II and XII (IC50 ~20-25 µM). The FDA-approved drugs shikonin, disulfiram, and ebselen did not inhibit SARS-CoV-2 3CLpro activity in the SAMDI-MS assay under physiologically relevant reducing conditions. The three drugs did not directly inhibit human ß-coronavirus OC-43 or SARS-CoV-2 in vitro, but instead induced cell death. In conclusion, the SAMDI-MS 3CLpro assay, combined with antiviral and cytotoxic assessment, provides a robust platform to evaluate antiviral agents directed against SARS-CoV-2.
Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/enzimologia , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/farmacologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Proteínas não Estruturais Virais/antagonistas & inibidores , COVID-19 , Proteases 3C de Coronavírus , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Glicoproteínas/farmacologia , Células HeLa , Humanos , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , SARS-CoV-2 , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Tratamento Farmacológico da COVID-19RESUMO
Type III interferon (IFN-λ) is currently considered to be largely nonredundant to type I interferon (IFN-α) in antivirus infection, especially in epithelial cells. Previous studies reported that, compared with IFN-α, IFN-λ exhibited stronger induction of interferon-stimulated genes (ISGs) at the transcriptional level in intestinal epithelial cells and stronger inhibition of porcine epidemic diarrhea virus (PEDV). In this study, the different mechanisms of ISG upregulation induced by IFN-α and IFN-λ1 were compared at the mRNA and protein levels in the porcine intestinal epithelial cell model (IPEC-J2). It was proved that IFN-λ1 consistently exhibited stronger stimulation effects at both levels. At the mRNA level, 132 genes were significantly upregulated upon IFN-λ1 stimulation, while 42 genes upon IFN-α stimulation. At the protein level, 47 proteins were significantly upregulated upon IFN-λ1 stimulation, but only 8 proteins were upregulated upon IFN-α stimulation. The shared upregulated genes/proteins by IFN-λ1 in both transcriptional and translational omics, especially the regulation factors of ISG15, were involved in the JAK-STAT signaling pathway. Compared to IFN-α, IFN-λ1 could induce more consistent upregulation of the key ISGs (ISG15, USP18, OASL, and RSAD2) at 3-24 h postinduction as measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) validation. It was further confirmed through functional analysis that ISG15 and RSAD2 could inhibit PEDV infection in dose-dependent manners. This study provided solid evidence that IFN-λ1 could induce a more unique and higher ISG expression level, which exhibited anti-PEDV effects on porcine intestinal epithelial cells.
Assuntos
Infecções por Coronavirus , Vírus da Diarreia Epidêmica Suína , Animais , Infecções por Coronavirus/genética , Infecções por Coronavirus/veterinária , Células Epiteliais , Vírus da Diarreia Epidêmica Suína/genética , Proteômica , Suínos , TranscriptomaRESUMO
White adipocytes play a key role in maintaining whole body energy homeostasis by forming white adipose tissue (WAT). The impairment of WAT formation or WAT dysfunction is clearly associated with severe metabolic disorders. Mature adipocytes are derived from differentiated preadipocytes and are pivotal in energy storage and metabolism. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a member of a family of CNC-bZIP proteins which exert their transcriptional control on genes harboring antioxidant response elements (ARE) in partnership with small musculoaponeurotic fibrosarcoma proteins. The activation of Nrf2-ARE coordinated by specific repressor Kelch-like ECH-associated protein 1 (Keap1) regulates networks of genes controlling diverse homeostatic processes involving adaptive antioxidant response and detoxification among many other adaptive responses. Interestingly, accumulating evidence indicates that Nrf2 may act as a transcription factor in regulating the formation and function of adipose tissues, including adipogenesis, lipid metabolism and insulin sensitivity. In this mini-review, an overview on the distinct roles of Nrf2 in adipocytes is provided. While highlighting the regulatory role of Nrf2 in adipogenesis, recent key findings on Nrf2 in insulin signal transduction and energy metabolism of adipocytes are also summarized.
Assuntos
Adipócitos/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Animais , Metabolismo Energético , Humanos , Insulina/metabolismo , Transdução de SinaisRESUMO
Nuclear factor erythroid 2-related factor 1 (NRF1), a ubiquitously expressed CNC-bZIP transcription factor, plays a critical role in white adipocyte (WAC) biology, whereas the underlying mechanisms remain unknown. The mouse Nrf1 gene is transcribed in a number of alternatively spliced forms, resulting in two long protein isoforms (L-NRF1) containing 741 and 742 amino acids (aa) and multiple short isoforms (S-NRF1). Our previous study found that adipocyte-specific knockout of Nrf1 [Nrf1(f)-KO] in mice disturbs the expression of lipolytic genes in adipocytes, leading to adipocyte hypertrophy followed by inflammation, pyroptosis and insulin resistance. In the present study, we found that the stromal vascular fraction (SVF) cells isolated from white adipose tissues (WAT) of Nrf1(f)-KO mice display augmented adipogenesis showing elevated mRNA and protein expression of adipogenic markers and lipid accumulation. In 3T3-L1 cells, stable knockdown (KD) of all or long isoforms of Nrf1 (termed as A-Nrf1-KD and L-Nrf1-KD, respectively) using lentiviral shRNAs resulted in enhanced and accelerated adipogenic differentiation. Conversely, overexpression of L-NRF1-741, but not any of the S-NRF1, substantially attenuated adipogenesis in 3T3-L1 cells. These findings indicate that L-NRF1 might serve as a critical negative regulator of adipogenesis. Mechanistic investigation revealed that L-NRF1 may negatively regulates the transcription of peroxisome proliferator-activated receptor γ (PPARγ), in particular the master regulator of adipogenesis PPARγ2. Taken all together, the findings in the present study provide further evidence for a novel role of NRF1 beyond its participation in cellular antioxidant response and suggest that L-NRF1 is a negative regulator of PPARγ2 expression and thereby can suppress adipogenesis.
Assuntos
Adipogenia , Fator 1 Relacionado a NF-E2/genética , Fator 1 Relacionado a NF-E2/metabolismo , PPAR gama/genética , PPAR gama/metabolismo , Células 3T3-L1 , Animais , Diferenciação Celular , Células Cultivadas , Feminino , Técnicas de Inativação de Genes , Masculino , Camundongos , Regiões Promotoras Genéticas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive lung disorder and lacks effective treatments because of unclear mechanisms. Aberrant function of alveolar macrophages is directly linked to pulmonary fibrosis. Here, we show TIM-3 (T-cell immunoglobulin domain and mucin domain-3), a key regulator of macrophage function, aggravates pulmonary fibrosis. TIM-3 mRNA of patients with IPF was analyzed based on the Gene Expression Omnibus and Array Express databases. Lung pathology and profibrotic molecules were assessed in a bleomycin (BLM)-induced pulmonary fibrosis model using wild-type (WT) and TIM-3 transgenic (TIM-3-TG) mice. Macrophage cells, RAW264.7, were then applied to investigate the effect of macrophage TIM-3 under BLM exposure in vitro. Macrophage depletion and adoptive-transfer experiments were finally performed to examine lung morphology and profibrotic molecules. TIM-3 expression was increased both in patients with IPF and in our BLM-induced mouse model. TIM-3-TG mice developed more serious pathological changes in lung tissue and higher expressions of TGF-ß1 (transforming growth factor-ß1) and IL-10 than WT mice. After BLM treatment, TGF-ß1 and IL-10 expression was significantly decreased in RAW264.7 cells after TIM-3 knock-out, whereas it was increased in TIM-3-TG peritoneal macrophages. The scores of pulmonary fibrosis in WT and TIM-3-TG mice were significantly reduced, and there was no difference between them after macrophage depletion. Furthermore, WT mice receiving adoptive macrophages from TIM-3-TG mice also had more serious lung fibrosis and increased expression of TGF-ß1 and IL-10 than those receiving macrophages from WT mice. Our findings revealed that overexpressed TIM-3 in alveolar macrophages aggravated pulmonary fibrosis.
Assuntos
Receptor Celular 2 do Vírus da Hepatite A/sangue , Receptor Celular 2 do Vírus da Hepatite A/fisiologia , Fibrose Pulmonar Idiopática/patologia , Macrófagos Alveolares/metabolismo , Transferência Adotiva , Animais , Bleomicina/toxicidade , Sistemas CRISPR-Cas , Modelos Animais de Doenças , Receptor Celular 2 do Vírus da Hepatite A/deficiência , Receptor Celular 2 do Vírus da Hepatite A/genética , Humanos , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/genética , Interleucina-10/biossíntese , Pulmão/patologia , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/transplante , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células RAW 264.7 , RNA Mensageiro/biossíntese , Fator de Crescimento Transformador beta1/biossínteseRESUMO
Myeloid cell leukemia 1 (Mcl-1) overexpression is found in various human tumors and has emerged as a promising new target for pancreatic cancer treatment. Recent research has found that most pancreatic cancers develop resistance to the current first-line chemotherapeutic drug, gemcitabine (Gem), and high expression of Mcl-1 can reduce the sensitivity of pancreatic cancer cells to Gem chemotherapy. Therefore, novel strategies, such as combination therapy, to overcome resistance of Gem chemotherapy are needed urgently. Here, we employed a lipid-based delivery system (LPs) to codeliver Mcl-1 siRNA and Gem for pancreatic cancer treatment, named LP-Gem-siMcl-1. LP-Gem-siMcl-1 exhibited an increased cellular uptake, enhanced Mcl-1 down-regulation efficacy, and significant cytotoxicity in the human pancreatic carcinoma cell lines PANC-1 and BxPC-3. Furthermore, tumor inhibition in vivo proved that LP-Gem-siMcl-1 has higher anti-tumor efficiency than LP-siMcl-1 plus LP-Gem, indicating the synergistic anti-tumor effects of Gem and siMcl-1. Meanwhile, histological analysis demonstrated that LPs could efficiently co-deliver Gem and Mcl-1 siRNA to cancerous cells and overcome the resistance of Gem. Taken together, our results offer proof that LP-Gem-siMcl-1 is an effective co-delivery system to treat pancreatic cancers and may serve as a valuable tool for developing new strategies for cancer therapy.