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1.
J Ethnopharmacol ; 301: 115806, 2023 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-36216198

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Qushi Huayu Decoction (QHD) is a traditional Chinese medicine formula consisting of five herbs, which has been used for non-alcoholic fatty liver disease (NAFLD) treatment in clinic for decades in China and validated in several NAFLD animal models. The hepatic de novo lipogenesis (DNL) is enhanced greatly to contribute to steatosis in NAFLD. The spliced form of X-box binding protein 1 (XBP1s) initiates DNL independently of sterol regulatory element-binding protein (SREBP) and carbohydrate-responsive element-binding protein (ChREBP). AIM OF THE STUDY: To disclose the mechanism of inhibition on hepatic DNL by QHD and the responsible compounds. METHODS: The effects of QHD on hepatic DNL were evaluated in mice induced by high-fructose diet (HFru). The effects of the serum-absorbed compounds of QHD on XBP1s were evaluated in HepG2 cells induced by tunicamycin. Hepatic histology, triglyceride (TG) and nonesterified fatty acids were observed. Hepatic apolipoprotein B100 and very low-density lipoprotein were measured to reflect lipid out-transport. The mRNA expression of XBP1s and its target genes were detected by real-time polymerase chain reaction. The protein expression of TG synthetases and DNL enzymes, and inositol requirement enzyme 1 alpha (IRE1α), phosphorylated IRE1α and XBP1s were detected in liver tissue and HepG2 cells by western-blot. The binding activity of SREBP1, protein expression of ChREBP and XBP1s were detected in the nuclear extracts of liver tissue. RESULTS: Dynamical observing suggested feeding with HFru for 2 weeks was sufficient to induce hepatic lipogenesis and XBP1s. QHD ameliorated liver steatosis without enhancing out-transport of lipids, accompanied with more inhibitory effects on DNL enzymes than TG synthetases. QHD inhibits the nuclear XBP1s without affecting ChREBP and SREBP1. In QHD, chlorogenic acid, geniposide and polydatin inhibit lipogenesis initiated by XPB1s. CONCLUSION: QHD probably decreases hepatic DNL by inhibiting XBP1s independent of SREBP1 and ChREBP. Chlorogenic acid, geniposide and polydatin are the potential responsible compounds.


Assuntos
Lipogênese , Hepatopatia Gordurosa não Alcoólica , Animais , Camundongos , Ácido Clorogênico/farmacologia , Endorribonucleases/metabolismo , Endorribonucleases/farmacologia , Endorribonucleases/uso terapêutico , Frutose , Ligases/metabolismo , Ligases/farmacologia , Ligases/uso terapêutico , Fígado , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/metabolismo , Proteínas Serina-Treonina Quinases , Triglicerídeos/metabolismo
2.
Cell Death Differ ; 29(1): 230-245, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34453119

RESUMO

Mounting evidence indicates that immunogenic therapies engaging the unfolded protein response (UPR) following endoplasmic reticulum (ER) stress favor proficient cancer cell-immune interactions, by stimulating the release of immunomodulatory/proinflammatory factors by stressed or dying cancer cells. UPR-driven transcription of proinflammatory cytokines/chemokines exert beneficial or detrimental effects on tumor growth and antitumor immunity, but the cell-autonomous machinery governing the cancer cell inflammatory output in response to immunogenic therapies remains poorly defined. Here, we profiled the transcriptome of cancer cells responding to immunogenic or weakly immunogenic treatments. Bioinformatics-driven pathway analysis indicated that immunogenic treatments instigated a NF-κB/AP-1-inflammatory stress response, which dissociated from both cell death and UPR. This stress-induced inflammation was specifically abolished by the IRE1α-kinase inhibitor KIRA6. Supernatants from immunogenic chemotherapy and KIRA6 co-treated cancer cells were deprived of proinflammatory/chemoattractant factors and failed to mobilize neutrophils and induce dendritic cell maturation. Furthermore, KIRA6 significantly reduced the in vivo vaccination potential of dying cancer cells responding to immunogenic chemotherapy. Mechanistically, we found that the anti-inflammatory effect of KIRA6 was still effective in IRE1α-deficient cells, indicating a hitherto unknown off-target effector of this IRE1α-kinase inhibitor. Generation of a KIRA6-clickable photoaffinity probe, mass spectrometry, and co-immunoprecipitation analysis identified cytosolic HSP60 as a KIRA6 off-target in the IKK-driven NF-κB pathway. In sum, our study unravels that HSP60 is a KIRA6-inhibitable upstream regulator of the NF-κB/AP-1-inflammatory stress responses evoked by immunogenic treatments. It also urges caution when interpreting the anti-inflammatory action of IRE1α chemical inhibitors.


Assuntos
Endorribonucleases , Retículo Endoplasmático/metabolismo , Endorribonucleases/metabolismo , Humanos , Imidazóis , Morte Celular Imunogênica , Inflamação/metabolismo , Naftalenos , Pirazinas
3.
Biochem Biophys Res Commun ; 636(Pt 1): 155-161, 2022 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-36334439

RESUMO

BACKGROUND: Peroxiredoxin 4 (Prdx4) in the endoplasmic reticulum (ER) is the only secretory member of the antioxidant Prdx family. Our previous studies demonstrated that Prdx4 in cumulus cells (CCs) ameliorated the maturation of oocytes in vitro and enhanced oocyte developmental competence by preventing CCs apoptosis caused by oxidative stress (OS) through gap junctions. In this study, we aimed to determine whether Prdx4 released by CCs can repair meiotic defects in mouse oocytes by co-culturing immature (germinal vesicle) oocytes with CCs from mature oocytes in the absence of gap junctions. RESULTS: The OS-induced meiotic defects in mouse oocytes were impeded by co-culture with CCs, as evidenced by the increased first polar body (PB1) extrusion rate and decreased ROS level. CCs increased Prdx4 expression and lowered IRE1α, Bip expression in H2O2-treated oocytes. After knockdown of Prdx4 expression in CCs, the rate of PB1 extrusion in the oocytes was significantly reduced to the level detected in H2O2 group, and ER stress was not alleviated. CO-IP and immunofluorescence co-localization experiments demonstrated that Prdx4 interacted with PDIA6 in the oocytes and the Pearson's R value was 0.69 calculated using ImageJ. CONCLUSIONS: Cumulus cells can promote the maturation of oocytes in vitro by secreting Prdx4 in a paracrine manner and serve as a promising therapeutic antioxidant for improving the quality of oocytes, especially aging oocytes, in clinical in vitro maturation (IVM).


Assuntos
Células do Cúmulo , Técnicas de Maturação in Vitro de Oócitos , Peroxirredoxinas , Animais , Feminino , Camundongos , Antioxidantes/metabolismo , Endorribonucleases/metabolismo , Peróxido de Hidrogênio/metabolismo , Oócitos/metabolismo , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Proteínas Serina-Treonina Quinases
4.
PLoS Pathog ; 18(11): e1010930, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36318584

RESUMO

The antiviral endoribonuclease, RNase L, is activated by the mammalian innate immune response to destroy host and viral RNA to ultimately reduce viral gene expression. Herein, we show that RNase L and RNase L-mediated mRNA decay are primarily localized to the cytoplasm. Consequently, RNA-binding proteins (RBPs) translocate from the cytoplasm to the nucleus upon RNase L activation due to the presence of intact nuclear RNA. The re-localization of RBPs to the nucleus coincides with global alterations to RNA processing in the nucleus. While affecting many host mRNAs, these alterations are pronounced in mRNAs encoding type I and type III interferons and correlate with their retention in the nucleus and reduction in interferon protein production. Similar RNA processing defects also occur during infection with either dengue virus or SARS-CoV-2 when RNase L is activated. These findings reveal that the distribution of RBPs between the nucleus and cytosol is dictated by the availability of RNA in each compartment. Thus, viral infections that trigger RNase L-mediated cytoplasmic RNA in the cytoplasm also alter RNA processing in the nucleus, resulting in an ingenious multi-step immune block to protein biogenesis.


Assuntos
COVID-19 , SARS-CoV-2 , Animais , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , COVID-19/genética , Endorribonucleases/genética , Endorribonucleases/metabolismo , Citoplasma/metabolismo , Mamíferos
5.
Molecules ; 27(21)2022 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-36364107

RESUMO

Hepatocellular carcinoma (HCC) is the most common primary liver malignant tumor, and the targeted therapy for HCC is very limited. Our previous study demonstrated that prodigiosin(PG), a secondary metabolite from Serratia marcescens found in the intestinal flora of cockroaches, inhibits the proliferation of HCC and increases the expression of CHOP, a marker protein for endoplasmic reticulum stress (ERS)-mediated apoptosis, in a dose-dependent manner. However, the mechanisms underlying the activity of PG in vivo and in vitro are unclear. This study explored the molecular mechanisms of PG-induced ERS against liver cancer in vitro and in vivo. The apoptosis of hepatocellular carcinoma cells induced by PG through endoplasmic reticulum stress was observed by flow cytometry, colony formation assay, cell viability assay, immunoblot analysis, and TUNEL assay. The localization of PG in cells was observed using laser confocal fluorescence microscopy. Flow cytometry was used to detect the intracellular Ca2+ concentration after PG treatment. We found that PG could promote apoptosis and inhibit the proliferation of HCC. It was localized in the endoplasmic reticulum of HepG2 cells, where it induces the release of Ca2+. PG also upregulated the expression of key unfolded response proteins, including PERK, IRE1α, Bip, and CHOP, and related apoptotic proteins, including caspase3, caspase9, and Bax, but down-regulated the expression of anti-apoptotic protein Bcl-2 in liver cancer. Alleviating ERS reversed the above phenomenon. PG had no obvious negative effects on the functioning of the liver, kidney, and other main organs in nude mice, but the growth of liver cancer cells was inhibited by inducing ERS in vivo. The findings of this study showed that PG promotes apoptosis of HCC by inducing ERS.


Assuntos
Carcinoma Hepatocelular , Baratas , Neoplasias Hepáticas , Camundongos , Animais , Carcinoma Hepatocelular/metabolismo , Estresse do Retículo Endoplasmático , Neoplasias Hepáticas/patologia , Prodigiosina/farmacologia , Endorribonucleases/metabolismo , Serratia marcescens/metabolismo , Camundongos Nus , Baratas/metabolismo , Proteínas Serina-Treonina Quinases , Apoptose , Proliferação de Células
6.
Viruses ; 14(11)2022 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-36366584

RESUMO

Many viruses are known to trigger endoplasmic reticulum (ER) stress in host cells, which in turn can develop a protective unfolded protein response (UPR). Depending on the conditions, the UPR may lead to either cell survival or programmed cell death. One of three UPR branches involves the upregulation of Xbp1 transcription factor caused by the unconventional cytoplasmic splicing of its mRNA. This process is accomplished by the phosphorylated form of the endoribonuclease/protein kinase Ire1/ERN1. Here, we show that the phosphorylation of Ire1 is up-regulated in HeLa cells early in enterovirus infection but down-regulated at later stages. We also find that Ire1 is cleaved in poliovirus- and coxsackievirus-infected HeLa cells 4-6 h after infection. We further show that the Ire1-mediated Xbp1 mRNA splicing is repressed in infected cells in a time-dependent manner. Thus, our results demonstrate the ability of enteroviruses to actively modulate the Ire1-Xbp1 host defensive pathway by inducing phosphorylation and proteolytic cleavage of the ER stress sensor Ire1, as well as down-regulating its splicing activity. Inactivation of Ire1 could be a novel mode of the UPR manipulation employed by viruses to modify the ER stress response in the infected cells.


Assuntos
Infecções por Enterovirus , Enterovirus , Humanos , Estresse do Retículo Endoplasmático , Endorribonucleases/genética , Endorribonucleases/metabolismo , Enterovirus/genética , Células HeLa , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , Transdução de Sinais , Resposta a Proteínas não Dobradas , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismo
7.
Proc Natl Acad Sci U S A ; 119(48): e2208022119, 2022 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-36409892

RESUMO

The C-terminal domain (CTD) of the major endoribonuclease RNase E not only serves as a scaffold for the central RNA decay machinery in gram-negative bacteria but also mediates coupled degradation of small regulatory RNAs (sRNAs) and their cognate target transcripts following RNA chaperone Hfq-facilitated sRNA-mRNA base pairing. Despite the crucial role of RNase E CTD in sRNA-dependent gene regulation, the contribution of particular residues within this domain in recruiting sRNAs and mRNAs upon base pairing remains unknown. We have previously shown that in Escherichia coli, the highly conserved 3'-5'-exoribonuclease polynucleotide phosphorylase (PNPase) paradoxically stabilizes sRNAs by limiting access of RNase E to Hfq-bound sRNAs and by degrading target mRNA fragments that would otherwise promote sRNA decay. Here, we report that in the absence of PNPase, the RNA-binding region AR2 in the CTD is required for RNase E to initiate degradation of the Hfq-dependent sRNAs CyaR and RyhB. Additionally, we show that introducing mutations in either hfq that disrupts target mRNA binding to Hfq or the AR2 coding region of rne impairs RNase E binding to sRNAs. Altogether, our data support a model where sRNAs are recruited via bound mRNA targets to RNase E by its AR2 domain after Hfq catalyzes sRNA-mRNA pairing. These results also support our conclusion that in a PNPase-deficient strain, more rapid decay of sRNAs occurs due to accelerated pairing with mRNA targets as a consequence of their accumulation. Our findings provide insights into the mechanisms by which sRNAs and mRNAs are regulated by RNase E.


Assuntos
Endorribonucleases , Escherichia coli , Endorribonucleases/genética , Endorribonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Motivos de Ligação ao RNA , RNA Mensageiro/metabolismo , RNA/metabolismo
8.
Biomed Pharmacother ; 155: 113741, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36271543

RESUMO

Melanoma is the most lethal skin cancer with rising incidence worldwide. Despite significant advances in target therapy and immunotherapy, low response rates and the development of drug resistance remain key clinical barriers affecting patient prognosis. The complex interplay between multiple signaling molecules and pathways has brought little understanding of melanoma pathogenesis and resistance. The genetic mutation and hypermetabolic environment of melanoma cells lead to increasing demands for protein synthesis and perturb proteostasis resulting in endoplasmic reticulum (ER) stress. Subsequently, three unfolded protein response (UPR) signaling branches, represented by IRE1α, PERK and ATF6, are activated to direct cell fate towards pro-survival or pro-apoptosis depending on the intensity and duration of ER stress. In this review, we summarize ER stress and UPR in melanoma cells and tumor-infiltrating immune cells along with the crosstalk among these pathways. We provide the latest advances in understanding melanoma pathogenesis and resistance and discuss the potential of targeting the ER stress or UPR process for melanoma therapy.


Assuntos
Estresse do Retículo Endoplasmático , Melanoma , Humanos , Endorribonucleases/metabolismo , Proteínas Serina-Treonina Quinases , Apoptose/fisiologia , Melanoma/patologia
9.
Int J Mol Sci ; 23(19)2022 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-36232496

RESUMO

Natively monomeric RNase A can oligomerize upon lyophilization from 40% acetic acid solutions or when it is heated at high concentrations in various solvents. In this way, it produces many dimeric or oligomeric conformers through the three-dimensional domain swapping (3D-DS) mechanism involving both RNase A N- or/and C-termini. Here, we found many of these oligomers evolving toward not negligible amounts of large derivatives after being stored for up to 15 months at 4 °C in phosphate buffer. We call these species super-aggregates (SAs). Notably, SAs do not originate from native RNase A monomer or from oligomers characterized by the exclusive presence of the C-terminus swapping of the enzyme subunits as well. Instead, the swapping of at least two subunits' N-termini is mandatory to produce them. Through immunoblotting, SAs are confirmed to derive from RNase A even if they retain only low ribonucleolytic activity. Then, their interaction registered with Thioflavin-T (ThT), in addition to TEM analyses, indicate SAs are large and circular but not "amyloid-like" derivatives. This confirms that RNase A acts as an "auto-chaperone", although it displays many amyloid-prone short segments, including the 16-22 loop included in its N-terminus. Therefore, we hypothesize the opening of RNase A N-terminus, and hence its oligomerization through 3D-DS, may represent a preliminary step favoring massive RNase A aggregation. Interestingly, this process is slow and requires low temperatures to limit the concomitant oligomers' dissociation to the native monomer. These data and the hypothesis proposed are discussed in the light of protein aggregation in general, and of possible future applications to contrast amyloidosis.


Assuntos
Amiloidose , Ribonuclease Pancreático , Acetatos , Amiloide , Endorribonucleases/metabolismo , Humanos , Fosfatos , Agregados Proteicos , Estrutura Terciária de Proteína , Ribonuclease Pancreático/metabolismo , Ribonucleases/metabolismo , Solventes
10.
Int J Mol Sci ; 23(19)2022 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-36233127

RESUMO

The endoplasmic reticulum (ER) is a dynamic organelle that responds to demand in secretory proteins by undergoing expansion. The mechanisms that control the homeostasis of ER size and function involve the activation of the unfolded protein response (UPR). The UPR plays a role in various effector functions of immune cells. Mast cells (MCs) are highly granular tissue-resident cells and key drivers of allergic inflammation. Their diverse secretory functions in response to activation through the high-affinity receptor for IgE (FcεRI) suggest a role for the UPR in their function. Using human cord blood-derived MCs, we found that FcεRI triggering elevated the expression level and induced activation of the UPR transducers IRE1α and PERK, accompanied by expansion of the ER. In mouse bone marrow-derived MCs and peritoneal MCs, the ER underwent a more moderate expansion, and the UPR was not induced following MC activation. The deletion of IRE1α in mouse MCs did not affect proliferation, survival, degranulation, or cytokine stimulation following FcεRI triggering, but it did diminish the surface expression of TLR4 and the consequent response to LPS. A similar phenotype was observed in human MCs using an IRE1α inhibitor. Our data indicate that the ER of MCs, primarily of humans, undergoes a rapid remodeling in response to activation that promotes responses to TLR4. We suggest that IRE1α inhibition can be a strategy for inhibiting the hyperactivation of MCs by LPS over the course of allergic responses.


Assuntos
Retículo Endoplasmático , Endorribonucleases , Proteínas Serina-Treonina Quinases , Receptor 4 Toll-Like , Animais , Citocinas/metabolismo , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Endorribonucleases/metabolismo , Homeostase , Humanos , Imunoglobulina E/metabolismo , Lipopolissacarídeos , Mastócitos/metabolismo , Camundongos , Proteínas Serina-Treonina Quinases/genética , Receptores de IgE/metabolismo , Receptor 4 Toll-Like/metabolismo , Resposta a Proteínas não Dobradas
11.
Int J Mol Sci ; 23(20)2022 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-36293008

RESUMO

Endoplasmic reticulum (ER) function is dedicated to multiple essential processes in eukaryotes, including the processing of secretory proteins and the biogenesis of most membrane lipids. These roles implicate a heavy burden to the organelle, and it is thus prone to fluctuations in the homeostasis of molecules which govern these processes. The unfolded protein response (UPR) is a general ER stress response tasked with maintaining the ER for optimal function, mediated by the master activator Ire1. Ire1 is an ER transmembrane protein that initiates the UPR, forming characteristic oligomers in response to irregularities in luminal protein folding and in the membrane lipid environment. The role of lipids in regulating the UPR remains relatively obscure; however, recent research has revealed a potent role for sphingolipids in its activity. Here, we identify a major role for the oxysterol-binding protein Kes1, whose activity is of consequence to the sphingolipid profile in cells resulting in an inhibition of UPR activity. Using an mCherry-tagged derivative of Ire1, we observe that this occurs due to inhibition of Ire1 to form oligomers. Furthermore, we identify that a sphingolipid presence is required for Ire1 activity, and that specific sphingolipid profiles are of major consequence to Ire1 function. In addition, we highlight cases where Ire1 oligomerization is absent despite an active UPR, revealing a potential mechanism for UPR induction where Ire1 oligomerization is not necessary. This work provides a basis for the role of sphingolipids in controlling the UPR, where their metabolism harbors a crucial role in regulating its onset.


Assuntos
Oxisteróis , Proteínas Serina-Treonina Quinases , Proteínas Serina-Treonina Quinases/genética , Esfingolipídeos , Resposta a Proteínas não Dobradas , Estresse do Retículo Endoplasmático , Análise por Conglomerados , Endorribonucleases/metabolismo
12.
Endocr Regul ; 56(4): 254-264, 2022 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-36270342

RESUMO

Objective. The aim of the present study was to investigate the expression of pyruvate dehydrogenase genes such as PDHA1, PDHB, DLAT, DLD, and PDHX in U87 glioma cells in response to glutamine and glucose deprivations in control glioma cells and endoplasmic reticulum to nucleus signaling 1 (ERN1) knockdown cells, the major endoplasmic reticulum (ER) stress signaling pathway, to find out whether there exists a possible dependence of these important regulatory genes expression on both glutamine and glucose supply as well as ERN1 signaling. Methods. The expression level of PDHA1, PDHB, DLAT, DLD, and PDHX genes was studied by real-time quantitative polymerase chain reaction in control U87 glioma cells (transfected by empty vector) and cells with inhibition of ERN1(transfected by dnERN1) after cells exposure to glucose and glutamine deprivations. Results. The data showed that the expression level of PDHA1, PDHB, DLAT, and DLD genes was down-regulated (more profound in PDHB gene) in control glioma cells treated with glutamine deprivation. At the same time, ERN1 knockdown modified the impact of glutamine deprivation on the expression level of all these genes in glioma cells: suppressed the sensitivity of PDHB and DLD genes expression and removed the impact of glutamine deprivation on the expression of PDHA1 and DLAT genes. Glucose deprivation did not significantly change the expression level of all studied genes in control glioma cells, but ERN1 knockdown is suppressed the impact of glucose deprivation on PDHX and DLD genes expression and significantly enhanced the expression of PDHA1 and PDHB genes. No significant changes were observed in the sensitivity of PDHX gene expression to glutamine deprivation neither in control nor ERN1 knock-down glioma cells. The knock-down of ERN1 removed the sensitivity of DLAT gene expression to glucose deprivation. Conclusion. The results of this investigation demonstrate that the exposure of control U87 glioma cells under glutamine deprivation significantly affected the expression of PDHA1, PDHB, DLAT, and DLD genes in a gene specific manner and that impact of glutamine deprivation was modified by inhibition of the ER stress signaling mediated by ERN1. At the same time, glucose deprivation affected the expression of PDHA1, PDHB, PDHX, and DLD genes in ERN1 knockdown glioma cells only. Thus, the expression of pyruvate dehydrogenase genes under glutamine and glucose deprivation conditions appears to be controlled by the ER stress signaling through ERN1.


Assuntos
Glioma , Glutamina , Humanos , Glutamina/farmacologia , Endorribonucleases/genética , Endorribonucleases/metabolismo , Glucose/farmacologia , Proteínas Serina-Treonina Quinases/genética , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Glioma/genética , Glioma/metabolismo , Transdução de Sinais , Oxirredutases/metabolismo , Piruvatos
13.
Food Funct ; 13(22): 11489-11502, 2022 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-36190121

RESUMO

Endoplasmic reticulum stress (ERS) plays a vital role in the pathogenesis of the alcoholic liver disease (ALD). Betulinic acid (BA) has been reported to be effective in the attenuation of ALD; however, its role in ERS and associated stress-signaling pathways remains elusive. Here, we found that the BA pretreatment significantly reduced the alcohol-induced liver injury by decreasing the activities of serum alanine aminotransferase and aspartate aminotransferase, alleviating fat deposition and rupturing the ER in hepatocytes. Moreover, the protective effect of BA on ALD was associated with the inhibition of reactive oxygen species accumulation and ERS, accompanied by the downregulation of glucose-regulated protein 78 (Grp78), Grp94, phosphorylation-inositol-requiring enzyme 1α (p-IRE1α), and phosphorylation-protein kinase R-like endoplasmic reticulum kinase (p-PERK), activating the transcription factor 6 (ATF6) and C/EBP homologous protein (CHOP). Moreover, the alcohol-induced hepatocyte apoptosis was reduced, along with the downregulation of the mitogen-activated protein kinase pathway, caspase-12, caspase-3, and caspase-7, following BA administration. Additionally, the BA-mediated mitigation of alcohol-induced liver injury and deactivation of the ER pathways were the same with 4-PBA, an inhibitor of ERS, indicating that the protective effect of BA on ALD may be regulated by ERS-associated pathways. Collectively, BA is a potentially desirable agent for the ALD, which may reduce hepatocyte apoptosis by suppressing excessive ERS in the liver.


Assuntos
Anti-Inflamatórios não Esteroides , Apoptose , Estresse do Retículo Endoplasmático , Hepatócitos , Hepatopatias Alcoólicas , Triterpenos Pentacíclicos , Animais , Camundongos , Apoptose/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Endorribonucleases/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Hepatopatias Alcoólicas/tratamento farmacológico , Hepatopatias Alcoólicas/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Triterpenos Pentacíclicos/farmacologia , Triterpenos Pentacíclicos/uso terapêutico , Anti-Inflamatórios não Esteroides/farmacologia , Anti-Inflamatórios não Esteroides/uso terapêutico
14.
Proc Natl Acad Sci U S A ; 119(43): e2123187119, 2022 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-36252035

RESUMO

Disruption of alveolar type 2 cell (AEC2) protein quality control has been implicated in chronic lung diseases, including pulmonary fibrosis (PF). We previously reported the in vivo modeling of a clinical surfactant protein C (SP-C) mutation that led to AEC2 endoplasmic reticulum (ER) stress and spontaneous lung fibrosis, providing proof of concept for disruption to proteostasis as a proximal driver of PF. Using two clinical SP-C mutation models, we have now discovered that AEC2s experiencing significant ER stress lose quintessential AEC2 features and develop a reprogrammed cell state that heretofore has been seen only as a response to lung injury. Using single-cell RNA sequencing in vivo and organoid-based modeling, we show that this state arises de novo from intrinsic AEC2 dysfunction. The cell-autonomous AEC2 reprogramming can be attenuated through inhibition of inositol-requiring enzyme 1 (IRE1α) signaling as the use of an IRE1α inhibitor reduced the development of the reprogrammed cell state and also diminished AEC2-driven recruitment of granulocytes, alveolitis, and lung injury. These findings identify AEC2 proteostasis, and specifically IRE1α signaling through its major product XBP-1, as a driver of a key AEC2 phenotypic change that has been identified in lung fibrosis.


Assuntos
Células Epiteliais Alveolares , Reprogramação Celular , Lesão Pulmonar , Proteínas de Membrana , Proteínas Serina-Treonina Quinases , Fibrose Pulmonar , Células Epiteliais Alveolares/metabolismo , Estresse do Retículo Endoplasmático , Endorribonucleases/genética , Endorribonucleases/metabolismo , Inositol/metabolismo , Lesão Pulmonar/patologia , Proteínas Serina-Treonina Quinases/genética , Proteostase , Fibrose Pulmonar/genética , Proteínas de Membrana/genética , Proteína C Associada a Surfactante Pulmonar/metabolismo
15.
Exp Cell Res ; 421(1): 113376, 2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36209899

RESUMO

Mounting evidence indicates that activation of unfolded protein response (UPR) and metabolic reprogramming contribute to cancer cell migration and invasion, but the molecular mechanism of pro-EMT program through a coordinated action of UPR with metabolism has not been defined. In this study, we utilized ER stress-inducing reagent, thapsigargin (TG), to induced pharmacologic ER stress in lung cancer cells. Here. We report that the branch of UPR, IRE1α-XBP1 pathway plays a pivotal role in reprogramming lung cancer cell metabolism. At the molecular level, the expression of pyruvate dehydrogenase kinase-1 (PDK-1) is directly induced by XBP1 as a consequence of UPR activation, thus facilitating aerobic glycolysis and lactate production. We also demonstrated that PDK1 serves as a downstream element of UPR activation in induction of Snail and EMT program. In addition, PDK1-induced Snail was dependent on the lactate production derived from metabolic reprogramming. Our findings reveal a critical role of lactate in pro-invasion events and establishes a direct connection between ER-stress and metabolic reprogramming in facilitating cancer cell progression.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Endorribonucleases , Transição Epitelial-Mesenquimal , Piruvato Desidrogenase Quinase de Transferência de Acetil , Proteína 1 de Ligação a X-Box , Humanos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Estresse do Retículo Endoplasmático , Endorribonucleases/genética , Endorribonucleases/metabolismo , Transição Epitelial-Mesenquimal/genética , Lactatos , Neoplasias Pulmonares/genética , Proteínas Serina-Treonina Quinases/genética , Piruvato Desidrogenase Quinase de Transferência de Acetil/genética , Piruvato Desidrogenase Quinase de Transferência de Acetil/metabolismo , Tapsigargina , Resposta a Proteínas não Dobradas , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismo
16.
Poult Sci ; 101(12): 102190, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36252503

RESUMO

This study investigated the effect of nano-selenium (nano-Se) in protecting laying hens from mercury (Hg)-induced prehierarchical follicular atresia. Furthermore, the endoplasmic reticulum stress (ERS) was explored to reveal the molecular mechanism. In vivo, 720 Hyline-Brown laying hens were treated with Hg and nano-Se alone or in combination. In vitro, the prehierarchical follicles were treated with Hg, nano-Se and 4-phenyl butyric acid (4-PBA) alone or in combination (Control, 25 µM Hg group, 10 µM nano-Se group, 20 µM nano-Se group, 25 µM Hg + 10 µM nano-Se group, 25 µM Hg + 20 µM nano-Se group, 25 µM Hg + 4-PBA group, and 25 µM Hg + 20 µM nano-Se + 4-PBA group). The GCs were treated with Hg and nano-Se alone or in combination (Control, 15 µM Hg group, 6 µM nano-Se group, 12 µM nano-Se group, 15 µM Hg + 6 µM nano-Se group, 15 µM Hg + 12 µM nano-Se group). The results revealed that dietary Hg significantly reduced laying performance (P < 0.05) and egg quality (P < 0.05), whereas nano-Se addition prevented these reductions (P < 0.05). Hg exposure significantly induced the accumulation of Hg in PHFs (P < 0.05), prehierarchical follicular atresia (P < 0.05) and apoptosis in PHFs, whereas nano-Se addition significantly prevented these effects (P < 0.05). The levels of sex hormones (P < 0.05) were significantly decreased after Hg exposure in vivo and in vitro, while nano-Se addition prevented the reductions. Furthermore, the RNA-Seq results showed that the key factors of the ERS presented differential expression, including C/EBP homologous protein, protein kinase RNA-like endoplasmic reticulum kinase (PERK) and activating transcription factor 6 (ATF6) in GCs. Hg exposure significantly increased the key gene expression of endoplasmic reticulum stress in GCs, whereas nano-Se addition prevented the induction of expression of these genes. In addition, the protein levels of PERK, inositol requiring protein 1α (IRE1α) and ATF6 were significantly increased, whereas nano-Se addition prevented the enhancements of protein expression in GCs. In conclusion, this study shows that Hg exposure can reduce induce prehierarchical follicular atresia, whereas nano-Se can prevent these effects. Our results also elucidate a key role of ERS in these protective effects of nano-Se in laying hens.


Assuntos
Mercúrio , Selênio , Feminino , Animais , Selênio/farmacologia , Selênio/metabolismo , Galinhas/fisiologia , Endorribonucleases/metabolismo , Atresia Folicular , Mercúrio/metabolismo , Proteínas Serina-Treonina Quinases
17.
Biochem Biophys Res Commun ; 632: 158-164, 2022 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-36209584

RESUMO

Endoplasmic reticulum (ER) stress is enhanced in non-alcoholic steatohepatitis (NASH). Among three signalling pathways, the IRE1α/XBP1 signalling pathway is strongly implicated in the pathogenesis of NASH but its significance is still largely uncharacterised. In this report, we constructed a hepatocyte-specific XBP1-Luciferase knock-in mouse model that allows in vivo monitoring of the IRE1α/XBP1 activity in hepatocytes. Using this mouse model, we found that IRE1α/XBP1 was activated within hepatocytes during the pathogenesis of NASH. Significantly, a specific IRE1α kinase-inhibiting RNase attenuator, KIRA8, attenuated NASH in mice. In conclusion, our hepatocyte-specific XBP1 splicing reporter mouse represents a valid model for research and drug development of NASH, which showed that the IRE1α-induced XBP splicing is potentiated in hepatocytes during pathogenesis of NASH. Furthermore, we carried out the proof-of-concept study to demonstrate that the allosteric IRE1α RNase inhibitor serves as a promising therapeutic agent for the treatment of NASH.


Assuntos
Endorribonucleases , Hepatopatia Gordurosa não Alcoólica , Animais , Camundongos , Endorribonucleases/antagonistas & inibidores , Endorribonucleases/efeitos dos fármacos , Endorribonucleases/metabolismo , Luciferases/metabolismo , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/metabolismo , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Proteína 1 de Ligação a X-Box/efeitos dos fármacos , Proteína 1 de Ligação a X-Box/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo
18.
Methods Enzymol ; 675: 323-350, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36220275

RESUMO

RNA 5' ends are remarkably heterogeneous. In addition to the eukaryotic 5' methyl-7-Guanosine (m7G) cap, a number of primarily metabolite-based cap structures have been identified both in prokaryotic and eukaryotic systems. These metabolite caps include Nicotinamide Adenine Dinucleotide (NAD+/NADH), dephosphoCoenzyme A (dpCoA), Flavin Adenine Dinucleotide (FAD), dinucleotide polyphosphates and Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) (Chen et al., 2009; Kowtoniuk et al., 2009; Wang et al., 2019). The most highly studied of these new cap structures, 5' NAD, has significant effects on RNA stability (Bird et al., 2016; Jiao et al., 2017). Both prokaryotes and eukaryotes have decapping enzymes specific to these metabolite caps and decapping is an integral step in the control of RNA stability (Cahová et al., 2015; Jiao et al., 2017; Sharma et al., 2020; Zhang et al., 2020). To better study how these 5' metabolite RNAs are decapped, we present a method to (1) generate radiolabeled dinucleotide and "full length" 5' capped RNA substrates for use in decapping assays, (2) a simple decapping assay to test the activity of various enzymes on different 5' capped transcripts and (3) a gel electrophoresis-based method for the visualization and differentiation of 5' capped transcripts.


Assuntos
NAD , Capuzes de RNA , Eletroforese , Endorribonucleases/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Guanosina , NAD/metabolismo , Polifosfatos , Capuzes de RNA/química , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , Estabilidade de RNA , Uridina Difosfato N-Acetilglicosamina
19.
Pharmacol Res ; 185: 106513, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36252772

RESUMO

cytohesin-2 is a guanine nucleotide exchange factor to activate ARF1 and ARF6, which are involved in various biological processes, including signal transduction, cell differentiation, cell structure organization, and survival. Nevertheless, there is a lack of evidence revealing the role of cytohesin-2 in osteoclast differentiation and in the development of osteoporosis. In this study, we find cytohesin-2 and ARF1 positively regulate osteoclast differentiation and function. Blocking the cytohesin-2 /ARF1 axis with SecinH3 or by genetic silencing of cytohesin-2 inhibits osteoclast formation and function in vitro. In vivo treatment with SecinH3 ameliorates ovariectomy-induced osteoporosis. Mechanistically, RNA-sequencing combined with molecular biological methodologies reveal that the regulatory function of cythohesin-2/ARF1 axis in osteoclast differentiation is mainly dependent on activating the JNK pathway. Further, in addition to the common viewpoint that JNK is activated by IRE1 via its kinase activity, we found that JNK can act upstream and regulate the endoribonuclease activity of IRE1 to promote XBP1 splicing. Both SecinH3 and silencing of cytohesin-2 inhibit JNK activation and IRE1 endoribonuclease activity, leading to the suppression of osteoclast differentiation. Taken together, our findings add new insights into the regulation between JNK and IRE1, and reveal that inhibiting the cytohesin-2/ARF1/JNK/IRE1 axis might represent a potential new strategy for the treatment of post-menopause osteoporosis.


Assuntos
Fatores de Ribosilação do ADP , Osteoporose , Humanos , Fatores de Ribosilação do ADP/fisiologia , Osteoclastos/metabolismo , Fator 6 de Ribosilação do ADP , Osteoporose/tratamento farmacológico , Endorribonucleases/metabolismo , Proteínas Serina-Treonina Quinases
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