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1.
Food Chem Toxicol ; 172: 113592, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36587836

RESUMO

Ochratoxin A (OTA), a secondary fungal metabolite with nephrotoxicity, is widespread in numerous kinds of feeds and foodstuffs. Ursolic acid (UA), a water-insoluble pentacyclic triterpene acid, exists in a wide range of food materials and medicinal plants. Our earlier researches provided preliminary evidence that mitochondria- and mitochondria-associated endoplasmic reticulum membranes (MAMs)-located stress-responsive Lon protease 1 (Lonp1) had a protective function in OTA-induced nephrotoxicity, and the renoprotective function of UA against OTA partially due to Lonp1. However, whether other MAMs-located protiens, such as endoplasmic reticulum stress (ERS)-responsive Sigma 1-type opioid receptor (Sig-1R), contribute to the protection of UA against OTA-induced nephrotoxicity together with Lonp1 needs further investigation. In this study, the cell viability, reactive oxygen species, and protein expressions of human proximal tubule epithelial-originated kidney-2 (HK-2) cells varied with OTA and/or UA/CDDO-me/AVex-73/Sig-1R siRNA treatments were determined. Results indicated that a 24 h-treatment of 5 µM OTA could significantly induce mitochondrial-mediated apoptosis via repressing Lonp1 and Sig-1R, thereby enhancing the protein expressions of GRP78, p-PERK, p-eIF2α, CHOP, IRE1α, and Bax, and inhibiting the protein expression of Bcl-2 in HK-2 cells, which could be remarkably relieved by a 2 h-pre-treatment of 4 µM UA (P < 0.05). In conclusion, through mutual promotion between Lonp1 and Sig-1R, UA could effectively relieve OTA-induced apoptosis in vitro and break the vicious cycle between oxidative stress and ERS, which activated the mitochondrial apoptosis pathway.


Assuntos
Protease La , Humanos , Endorribonucleases , Proteínas Serina-Treonina Quinases , Mitocôndrias , Apoptose , Estresse do Retículo Endoplasmático , Proteínas Mitocondriais , Proteases Dependentes de ATP
2.
Int J Mol Sci ; 24(2)2023 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-36674822

RESUMO

A spinal cord injury (SCI) is the devastating trauma associated with functional deterioration due to apoptosis. Most laboratory SCI models are generated by a direct impact on an animal's spinal cord; however, our model does not involve the direct impact on the spinal cord. Instead, we use a clamp compression to create an ischemia in the descending aortas of mice. Following the success of inducing an ischemic SCI (ISCI), we hypothesized that this model may show apoptosis via an endoplasmic reticulum (ER) stress pathway. This apoptosis by the ER stress pathway is enhanced by the inducible nitric oxide synthase (iNOS). The ER is used for the protein folding in the cell. When the protein folding capacity is overloaded, the condition is termed the ER stress and is characterized by the accumulation of misfolded proteins inside the ER lumen. The unfolded protein response (UPR) signaling pathways that deal with the ER stress response then become activated. This UPR activates the three signal pathways that are regulated by the inositol-requiring enzyme 1α (IRE1α), the activating transcription factor 6 (ATF6), and the protein kinase RNA-like ER kinase (PERK). IRE1α and PERK are associated with the expression of the apoptotic proteins. Apoptosis caused by an ISCI is assessed using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) test. An ISCI also reduces synaptophysin and the neuronal nuclear protein (NeuN) in the spinal cord. In conclusion, an ISCI increases the ER stress proteins, resulting in apoptosis in neuronal cells in the spinal cord.


Assuntos
Proteínas Serina-Treonina Quinases , Traumatismos da Medula Espinal , Camundongos , Animais , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Endorribonucleases/metabolismo , Chaperona BiP do Retículo Endoplasmático , Apoptose , Estresse do Retículo Endoplasmático/fisiologia , Resposta a Proteínas não Dobradas , Modelos Animais de Doenças , Isquemia , Traumatismos da Medula Espinal/metabolismo
3.
J Agric Food Chem ; 71(3): 1499-1509, 2023 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-36630614

RESUMO

Osteoarthritis (OA) is a common joint disease, and studies have reported that the endoplasmic reticulum stress (ERS) in chondrocytes caused by the cartilage tissue damage could mediate the activation of Nod-like receptor protein 3 (NLRP3) inflammasomes through inositol-requiring enzyme 1 alpha (IRE1α) and thioredoxin interacting protein (TXNIP). Ginsenoside compound K (CK) has an inhibitory effect on IRE1α activation. However, the role of IRE1α-TXNIP and its interaction with CK are still unclear. In this study, we examined the role and mechanism of action of CK in OA. We found that CK ameliorated OA and ERS in IL-1ß-treated chondrocytes and a monoiodoacetate-induced rat OA model. The effect of CK on inflammation, pyroptosis, and ERS was blocked by the ERS inducer tunicamycin. In conclusion, CK hindered OA progression by inhibiting the ERS-IRE1α-TXNIP-NLRP3 axis. Overall, our data indicate that CK could be useful in the treatment of OA and other chronic inflammatory diseases.


Assuntos
Osteoartrite , Proteínas Serina-Treonina Quinases , Ratos , Animais , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Piroptose , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas NLR/metabolismo , Endorribonucleases/genética , Endorribonucleases/metabolismo , Endorribonucleases/farmacologia , Condrócitos , Apoptose , Estresse do Retículo Endoplasmático , Inflamassomos , Osteoartrite/tratamento farmacológico , Osteoartrite/genética , Osteoartrite/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/farmacologia
4.
PLoS Biol ; 21(1): e3001942, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36603027

RESUMO

RNA processing and degradation shape the transcriptome by generating stable molecules that are necessary for translation (rRNA and tRNA) and by facilitating the turnover of mRNA, which is necessary for the posttranscriptional control of gene expression. In bacteria and the plant chloroplast, RNA degradosomes are multienzyme complexes that process and degrade RNA. In many bacterial species, the endoribonuclease RNase E is the central component of the RNA degradosome. RNase E-based RNA degradosomes are inner membrane proteins in a large family of gram-negative bacteria (ß- and γ-Proteobacteria). Until now, the reason for membrane localization was not understood. Here, we show that a mutant strain of Escherichia coli, in which the RNA degradosome is localized to the interior of the cell, has high levels of 20S and 40S particles that are defective intermediates in ribosome assembly. These particles have aberrant protein composition and contain rRNA precursors that have been cleaved by RNase E. After RNase E cleavage, rRNA fragments are degraded to nucleotides by exoribonucleases. In vitro, rRNA in intact ribosomes is resistant to RNase E cleavage, whereas protein-free rRNA is readily degraded. We conclude that RNA degradosomes in the nucleoid of the mutant strain interfere with cotranscriptional ribosome assembly. We propose that membrane-attached RNA degradosomes in wild-type cells control the quality of ribosome assembly after intermediates are released from the nucleoid. That is, the compact structure of mature ribosomes protects rRNA against cleavage by RNase E. Turnover of a proportion of intermediates in ribosome assembly explains slow growth of the mutant strain. Competition between mRNA and rRNA degradation could be the cause of slower mRNA degradation in the mutant strain. We conclude that attachment of the RNA degradosome to the bacterial inner cytoplasmic membrane prevents wasteful degradation of rRNA precursors, thus explaining the reason for conservation of membrane-attached RNA degradosomes throughout the ß- and γ-Proteobacteria.


Assuntos
Proteínas de Escherichia coli , RNA Ribossômico , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Endorribonucleases/genética , Endorribonucleases/metabolismo , Ribossomos/metabolismo , Complexos Multienzimáticos/metabolismo , RNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Membrana Celular/metabolismo , Bactérias/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Bacteriano/genética
5.
Food Chem Toxicol ; 172: 113602, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36610474

RESUMO

The endoplasmic reticulum (ER) controls many biological functions besides maintaining the function of liver cells. Various studies reported the role of the ER stress and UPR signaling pathway in various liver diseases via triggering hepatocytes apoptosis. This study aims to investigate the suppressive effect of ß-sitosterol (ßS) on apoptosis associated with liver injury and ER stress. METHODS: Liver damage in rats was induced by TAA (150 mg/kg I.P twice a week/3 weeks) and γ-irradiation (single dose 3.5 Gy) and treated with ßS (20 mg/kg daily for 30 days). Serum aminotransferase activity, lipid profile and lipid metabolic factors were measured beside liver oxidative stress and inflammatory markers. Moreover, the hepatic expression of ER stress markers (inositol-requiring enzyme 1 alpha (IRE1α), X-box-binding protein 1 (XBP1) and CCAAT/enhancer binding protein homologous protein (CHOP) and apoptotic markers were detected together with histopathological examination. RESULTS: ßS diminished the aminotransferase activity, the oxidative stress markers as well as the inflammatory mediators. Furthermore, ßS lowered the circulating TG and TC and the hepatic lipotoxicity via the suppression of lipogenesis (Srebp-1c) and improved the ß-oxidation (Pparα and Cpt1a) together with the mitochondrial biogenesis (Pgc-1 α). Moreover, the upregulated levels of ER stress markers were reduced upon treatment with ßS, which consequently attenuated hepatic apoptosis. CONCLUSION: ßS relieves hepatic injury, ameliorates mitochondrial biogenesis, and reduces lipotoxicity and apoptosis via inhibition of CHOP and ER stress response.


Assuntos
Endorribonucleases , Tioacetamida , Ratos , Animais , Endorribonucleases/metabolismo , Tioacetamida/metabolismo , Tioacetamida/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Fígado , Hepatócitos , Apoptose , Sitosteroides/farmacologia , Estresse do Retículo Endoplasmático , Transaminases/metabolismo
6.
FASEB J ; 37(2): e22788, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36692424

RESUMO

Ischemic stroke is known to cause the accumulation of misfolded proteins and loss of calcium homeostasis, leading to impairment of endoplasmic reticulum (ER) function and activating the unfolded protein response (UPR). PARP16 is an active (ADP-ribosyl)transferase known tail-anchored ER transmembrane protein with a cytosolic catalytic domain. Here, we find PARP16 is highly expressed in ischemic cerebral hemisphere and oxygen-glucose deprivation/reoxygenation (OGD/R)-treated immortalized hippocampal neuronal cell HT22. Using an adeno-associated virus-mediated PARP16 knockdown approach in mice, we find PARP16 knockdown decreases infarct demarcations and has a better neurological outcome after ischemic stroke. Our data indicate PARP16 knockdown decreases ER stress and neuronal death caused by OGD/R, whereas PARP16 overexpression promotes ER stress-mediated cell damage in primary cortical neurons. Furthermore, PARP16 functions mechanistically as ADP-ribosyltransferase to modulate the level of ADP-ribosylation of the corresponding PERK and IRE1α arm of the UPR, and such modifications mediate activation of PERK and IRE1α. Indeed, pharmacological stimulation of the UPR using Brefeldin A partly counteracts PARP16 knockdown-mediated neuronal protection upon OGD/R treatment. In conclusion, PARP16 plays a crucial role in post-ischemic UPR and PARP16 knockdown alleviates brain injury after ischemic stroke. This study demonstrates the potential of the PARP16-PERK/IRE1α axis as a target for neuronal survival in ischemic stroke.


Assuntos
Isquemia Encefálica , AVC Isquêmico , Poli(ADP-Ribose) Polimerases , Traumatismo por Reperfusão , Animais , Camundongos , Apoptose , Isquemia Encefálica/metabolismo , Infarto Cerebral/metabolismo , Estresse do Retículo Endoplasmático , Endorribonucleases/metabolismo , AVC Isquêmico/metabolismo , Neurônios/metabolismo , Oxigênio/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Traumatismo por Reperfusão/metabolismo , Resposta a Proteínas não Dobradas , Poli(ADP-Ribose) Polimerases/metabolismo
7.
Nucleic Acids Res ; 51(1): 365-379, 2023 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-36594161

RESUMO

RNase E is an endoribonuclease found in many bacteria, including important human pathogens. Within Escherichia coli, it has been shown to have a major role in both the maturation of all classes of RNA involved in translation and the initiation of mRNA degradation. Thus, knowledge of the major determinants of RNase E cleavage is central to our understanding and manipulation of bacterial gene expression. We show here that the binding of RNase E to structured RNA elements is crucial for the processing of tRNA, can activate catalysis and may be important in mRNA degradation. The recognition of structured elements by RNase E is mediated by a recently discovered groove that is distant from the domains associated with catalysis. The functioning of this groove is shown here to be essential for E. coli cell viability and may represent a key point of evolutionary divergence from the paralogous RNase G family, which we show lack amino acid residues conserved within the RNA-binding groove of members of the RNase E family. Overall, this work provides new insights into the recognition and cleavage of RNA by RNase E and provides further understanding of the basis of RNase E essentiality in E. coli.


Assuntos
Proteínas de Escherichia coli , Escherichia coli , Humanos , Escherichia coli/genética , Escherichia coli/metabolismo , Endorribonucleases/metabolismo , RNA/genética , RNA/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Catálise , RNA Bacteriano/metabolismo
8.
Nat Commun ; 14(1): 120, 2023 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-36624093

RESUMO

IRE1α-XBP1 signaling is emerging as a central orchestrator of malignant progression and immunosuppression in various cancer types. Employing a computational XBP1s detection method applied to TCGA datasets, we demonstrate that expression of the XBP1s mRNA isoform predicts poor survival in non-small cell lung cancer (NSCLC) patients. Ablation of IRE1α in malignant cells delays tumor progression and extends survival in mouse models of NSCLC. This protective effect is accompanied by alterations in intratumoral immune cell subsets eliciting durable adaptive anti-cancer immunity. Mechanistically, cancer cell-intrinsic IRE1α activation sustains mPGES-1 expression, enabling production of the immunosuppressive lipid mediator prostaglandin E2. Accordingly, restoring mPGES-1 expression in IRE1αKO cancer cells rescues normal tumor progression. We have developed an IRE1α gene signature that predicts immune cell infiltration and overall survival in human NSCLC. Our study unveils an immunoregulatory role for cancer cell-intrinsic IRE1α activation and suggests that targeting this pathway may help enhance anti-tumor immunity in NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Endorribonucleases , Neoplasias Pulmonares , Proteínas Serina-Treonina Quinases , Animais , Humanos , Camundongos , Carcinoma Pulmonar de Células não Pequenas/genética , Endorribonucleases/genética , Endorribonucleases/metabolismo , Neoplasias Pulmonares/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismo
9.
Chem Biol Interact ; 370: 110332, 2023 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-36581200

RESUMO

Transforming growth factor-ß1 (TGF-ß1) associated epithelial-mesenchymal transition (EMT) contributes to multiple respiration diseases via Smad or MAPKs pathway. Our previous study has demonstrated that the typical macrolide antibiotic, azithromycin (AZM) played a notable anti-EMT role in ovalbumin (OVA)-challenged mice. However, the precise mechanism of AZM on TGF-ß1 mediated EMT in bronchial epithelial cells is still unclear. The purpose of this study was to elucidate whether azithromycin targeting RACK1 inhibits TGF-ß1 mediated EMT in vitro. The results showed that AZM significantly inhibited the expression of RACK1 and the activation of the downstream JNK, ERK, and Smad3 signaling pathways, thereby suppressing the migration of bronchial epithelial cells and reversing the TGF-ß1-induced EMT. The effect of AZM on TGF-ß1 mediated EMT in vitro is dependent on the dose of AZM. Although RACK1 has been shown to regulate IRE1α expression with siRACK1 transfection, there was no direct interaction between IRE1α and AZM. On the contrary, weak interaction between AZM and RACK1 was predicted with molecular docking. In summary, AZM targets RACK1 to trigger downstream JNK, ERK, and Smad3 signaling pathways and is an effective anti-EMT drug for bronchial epithelial cells in a dose-dependent manner.


Assuntos
Azitromicina , Fator de Crescimento Transformador beta1 , Camundongos , Animais , Fator de Crescimento Transformador beta1/metabolismo , Azitromicina/farmacologia , Azitromicina/metabolismo , Endorribonucleases/metabolismo , Endorribonucleases/farmacologia , Transição Epitelial-Mesenquimal , Simulação de Acoplamento Molecular , Proteínas Serina-Treonina Quinases/metabolismo , Células Epiteliais/metabolismo , Receptores de Quinase C Ativada/metabolismo , Receptores de Quinase C Ativada/farmacologia
10.
Food Chem Toxicol ; 172: 113577, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36563925

RESUMO

Microplastics (MPs) have raised health concerns in public for its potential reproductive toxicity. In this study, we subjected the Kunming mice to 0.01, 0.1 and 1.0 mg/day polystyrene MPs (10 µm, PS-MPs) for 35 days, aiming to investigate the relevant male reproductive toxicity and latent molecular mechanism. The results showed the decreased sperm counts and motility, while the elevated sperm abnormality in PS-MPs-exposed mice. Testicular H&E staining displayed the vacuolization, atrophy, and even shedding of germ cells in seminiferous tubule. And the testosterone content in serum also decreased with PS-MPs treatment. Moreover, molecular analysis indicated that PS-MPs upregulated the expression trait factors for ERS (e.g., immunoglobulin-binding protein [BIP], inositol-requiring protein 1α [IRE1α], X-box-binding protein 1 splicing [XBP1s], Jun kinase [JNK], and the transcription of CCAAT/enhancer-binding protein (C/EBP) homologous protein [CHOP]) and downstream apoptotic modulator (e.g., Caspase-12, -9, and -3) in the testis. The steroidogenic acute regulatory protein (StAR), the testosterone synthetic initiator, was also downregulated. With the supplementation of ERS inhibitor, the MPs-induced testicular damage and decreased testosterone were improved to almost normal level. Overall, this study suggested that PS-MPs generate reproductive toxicity possibly via activating ERS and apoptosis signaling pathway.


Assuntos
Microplásticos , Poliestirenos , Camundongos , Masculino , Animais , Poliestirenos/toxicidade , Plásticos , Endorribonucleases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sêmen/metabolismo , Transdução de Sinais , Estresse do Retículo Endoplasmático , Apoptose , Testosterona
11.
EMBO J ; 42(2): e112574, 2023 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-36504162

RESUMO

Biogenesis of the essential precursor of the bacterial cell envelope, glucosamine-6-phosphate (GlcN6P), is controlled by intricate post-transcriptional networks mediated by GlmZ, a small regulatory RNA (sRNA). GlmZ stimulates translation of the mRNA encoding GlcN6P synthtase in Escherichia coli, but when bound by RapZ protein, the sRNA becomes inactivated through cleavage by the endoribonuclease RNase E. Here, we report the cryoEM structure of the RapZ:GlmZ complex, revealing a complementary match of the RapZ tetrameric quaternary structure to structural repeats in the sRNA. The nucleic acid is contacted by RapZ mostly through a highly conserved domain that shares an evolutionary relationship with phosphofructokinase and suggests links between metabolism and riboregulation. We also present the structure of a precleavage intermediate formed between the binary RapZ:GlmZ complex and RNase E that reveals how GlmZ is presented and recognised by the enzyme. The structures provide a framework for understanding how other encounter complexes might guide recognition and action of endoribonucleases on target transcripts, and how structured substrates in polycistronic precursors may be recognised for processing by RNase E.


Assuntos
Proteínas de Escherichia coli , Pequeno RNA não Traduzido , Endorribonucleases/genética , Endorribonucleases/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Ribonucleoproteínas/genética , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/genética
12.
Front Endocrinol (Lausanne) ; 13: 1001349, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36465658

RESUMO

Objective: Thyroid-associated ophthalmopathy (TAO) is a disfiguring autoimmune disease, which destroys the structure of orbital tissues and even threatens vision. Metabolic reprograming is critical in autoimmune diseases; however, the metabolic basis of TAO remains to be clarified. Our study aimed to reveal the metabolic profile of TAO. Methods: Orbital adipose/connective tissues from eleven TAO patients and twelve control subjects were collected during surgeries and analyzed with liquid chromatograph-mass spectrometer. Orthogonal partial least-squares discrimination analysis (OPLS-DA), variable importance in projection (VIP), heat map, and volcano plot were used to reveal metabolic profile in TAO. Pathway analysis and metabolites-gene analysis were utilized to explore potential metabolic metabolism in TAO. Results: 3038 metabolites were detected in samples from the TAO patients and the controls. OPLS-DA analysis of the metabolomics results showed two distinguished groups, demonstrating that TAO has a unique metabolome. Univariate tests identified 593 dysregulated metabolites (P < 0.05), including 367 increased metabolites and 226 decreased metabolites. Pathway analysis showed that changed metabolites were enriched in cholesterol metabolism, choline metabolism in cancer, fat digestion and absorption, regulation of lipolysis in adipocytes, and insulin resistance. In addition, metabolites-gene analysis illustrated that cholesterol metabolism was involved in the pathogenesis of TAO. Endoplasmic reticulum stress-related genes (ATF6, PERK, and IRE1α) expressions were higher in TAO orbital tissues than in control orbital tissues verified by western blot. Additionally, the expression level of diacylglycerol acyltransferase 1 (DGAT1), a key metabolic protein for triacylglycerol synthesis, was increased in orbital tissues of TAO detected by qRT-PCR, indicating disrupted cholesterol metabolism in TAO. Conclusion: The present study demonstrated different metabolite profiles and potential metabolic mechanisms in TAO.


Assuntos
Doenças Autoimunes , Oftalmopatia de Graves , Humanos , Oftalmopatia de Graves/genética , Endorribonucleases , Proteínas Serina-Treonina Quinases , Tecido Adiposo , Colesterol
13.
Zhen Ci Yan Jiu ; 47(12): 1068-72, 2022 Dec 25.
Artigo em Chinês | MEDLINE | ID: mdl-36571221

RESUMO

OBJECTIVE: To observe the effect of electroacupuncture (EA) on the cognitive impairment and expressions of inositol-dependent kinase 1α (IRE1α)/c-Jun N-terminal kinase (JNK) pathway-related proteins in diabetic mice, so as to explore its underlying mechanism. METHODS: Thirty db/db mice were randomly divided into model group (n=15) and EA group (n=15), and 15 db/m mice were chosen as the control group. EA was applied to "Baihui" (GV20) and "Shenting" (GV24), bilateral "Pishu" (BL20) and "Shenshu" (BL23), "Zusanli" (ST36) and "Sanyinjiao" (SP6) for 20 min, and bilateral "Feishu" (BL13), "Hegu" (LI4) and "Taichong" (LR3) were stimulated with filiform needles, with the needles retained for 20 min, once daily, 6 days a week for 4 weeks. The daily food intake, water intake, and weekly body weight and blood glucose of the mice in each group were recorded. The learning and memory abilities were detected by Morris water maze, the morphology of hippocampal cells was observed by HE staining, and IRE1α-JNK pathway-related proteins IRE1α, JNK, anti-apoptotic protein (Bcl-2) were detected by Western blot. RESULTS: Compared with the control group, the food intake, water intake, body weight, blood glucose in the mo-del group were significantly increased (P<0.01), the escape latency was significantly prolonged (P<0.05), the times of cros-sing platform were significantly reduced (P<0.01), and the expression levels of IRE1α and JNK proteins were significantly increased (P<0.01), while the expression of Bcl-2 protein was significantly decreased (P<0.01). Compared with the model group, the food and water intake in the EA group were significantly decreased (P<0.01), the body weight and blood glucose were significantly decreased (P<0.05, P<0.01), the escape latency was significantly shortened (P<0.05), the times of crossing platform significantly increased (P<0.05), and the expression levels of IRE1α and JNK proteins were significantly decreased (P<0.05), while the Bcl-2 expression was significantly increased (P<0.01). The cells in hippocampal CA1 area of mice in the model group are in disorder, with unclear nuclei and obvious vacuoles; while the morphology of nerve cells in EA group was significantly improved. CONCLUSION: EA can improve the cognitive impairment of db/db mice, as well as regulate body weight, blood glucose, and improve the cell morphology in the hippocampus, which may be related to its function in regulating the IRE1α-JNK pathway and related proteins.


Assuntos
Disfunção Cognitiva , Diabetes Mellitus Experimental , Eletroacupuntura , Ratos , Camundongos , Animais , Ratos Sprague-Dawley , Sistema de Sinalização das MAP Quinases , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/terapia , Endorribonucleases , Glicemia , Proteínas Serina-Treonina Quinases/genética , Hipocampo , Proteínas Proto-Oncogênicas c-bcl-2 , Disfunção Cognitiva/genética , Disfunção Cognitiva/terapia
14.
Cells ; 11(23)2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36497127

RESUMO

Hepatocellular carcinoma (HCC) commonly possesses chronical elevation of IRE1α-ASK1 signaling. Orphan nuclear receptor Nur77, a promising therapeutic target in various cancer types, is frequently silenced in HCC. In this study, we show that cryptomeridiol (Bkh126), a naturally occurring sesquiterpenoid derivative isolated from traditional Chinese medicine Magnolia officinalis, has therapeutic efficacy in HCC by aggravating the pre-activated UPR and activating the silenced Nur77. Mechanistically, Nur77 is induced to sense IRE1α-ASK1-JNK signaling and translocate to the mitochondria, which leads to the loss of mitochondrial membrane potential (Δψm). The Bkh126-induced aggravation of ER stress and mitochondrial dysfunction result in increased cytotoxic product of reactive oxygen species (ROS). The in vivo anti-HCC activity of Bkh126 is superior to that of sorafenib, currently used to treat advanced HCC. Our study shows that Bkh126 induces Nur77 to connect ER stress to mitochondria-mediated cell killing. The identification of Nur77 as a molecular target of Bhk126 provides a basis for improving the leads for the further development of anti-HCC drugs.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Receptores Nucleares Órfãos , Humanos , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Estresse do Retículo Endoplasmático , Endorribonucleases , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Receptores Nucleares Órfãos/metabolismo , Proteínas Serina-Treonina Quinases
15.
Elife ; 112022 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-36542058

RESUMO

Class switch recombination generates distinct antibody isotypes critical to a robust adaptive immune system, and defects are associated with autoimmune disorders and lymphomagenesis. Transcription is required during class switch recombination to recruit the cytidine deaminase AID-an essential step for the formation of DNA double-strand breaks-and strongly induces the formation of R loops within the immunoglobulin heavy-chain locus. However, the impact of R loops on double-strand break formation and repair during class switch recombination remains unclear. Here, we report that cells lacking two enzymes involved in R loop removal-senataxin and RNase H2-exhibit increased R loop formation and genome instability at the immunoglobulin heavy-chain locus without impacting its transcriptional activity, AID recruitment, or class switch recombination efficiency. Senataxin and RNase H2-deficient cells also exhibit increased insertion mutations at switch junctions, a hallmark of alternative end joining. Importantly, these phenotypes were not observed in cells lacking senataxin or RNase H2B alone. We propose that senataxin acts redundantly with RNase H2 to mediate timely R loop removal, promoting efficient repair while suppressing AID-dependent genome instability and insertional mutagenesis.


The immune system is a complex network of cells and molecules, which helps to protect the body from invaders. The adaptive immune system can recognise millions of assailants, kill them, and 'learn' from this experience to mount an even quicker defence the next time the body is infected. To achieve this level of protection, specific immune cells, called B cells, divide when they come into contact with a molecule from a foreign particle, the antigen. The cloned B cells then produce millions of protective proteins, the antibodies, which patrol the blood stream and tag harmful particles for destruction. An antibody resembles a Y-shaped structure that contains a 'variable' region, which gives it the specificity to interact with an antigen, and a 'constant' region, which interacts with components of the immune system and determines the mechanisms used to destroy a pathogen. Based on the constant region, antibodies can be divided into five main classes. B cells are able to switch their production from one antibody class to another in an event known as class switch recombination, by making changes to the constant region. They do this by cutting out a portion of the genes for the constant region from their DNA and fusing the remaining DNA. The resulting antibodies still recognise the same target, but interact with different components of the immune system, ensuring that all the body's forces are mobilised. R-loops are temporary structures that form when a cell 'reads' the instructions in its DNA to make proteins. R-loops provide physical support by anchoring the transcription template to the DNA. They help control the activity of genes, but if they stay on the DNA for too long they could interfere with any form of. DNA repair ­ including the cutting and fusing mechanisms during class switch recombination. To find out more about this process, Zhao et al. used B-cells from mice lacking two specific proteins that usually help to remove R-loops. Without these proteins, the B cells generated more R-loops than normal. Nevertheless, the B-cells were able to undergo class switch recombination, even though their chromosomes showed large areas of DNA damage, and DNA sections that had been repaired contained several mistakes. Errors that occur during class switch recombination have been linked to immune disorders and B cell cancers. The study of Zhao et al. shows that even if R-loops do not affect some processes in B cells, they could still impact the overall health of their DNA. A next step would be to test if an inability to remove R-loops could indeed play a role in immune disorders and B-cell cancers.


Assuntos
Recombinação Genética , Ribonucleases , Humanos , Ribonucleases/genética , Switching de Imunoglobulina/genética , Endorribonucleases/genética , Isotipos de Imunoglobulinas/genética , Instabilidade Genômica , Citidina Desaminase/genética
16.
Elife ; 112022 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-36479971

RESUMO

CRISPR-Cas systems are a family of adaptive immune systems that use small CRISPR RNAs (crRNAs) and CRISPR-associated (Cas) nucleases to protect prokaryotes from invading plasmids and viruses (i.e., phages). Type III systems launch a multilayered immune response that relies upon both Cas and non-Cas cellular nucleases, and although the functions of Cas components have been well described, the identities and roles of non-Cas participants remain poorly understood. Previously, we showed that the type III-A CRISPR-Cas system in Staphylococcus epidermidis employs two degradosome-associated nucleases, PNPase and RNase J2, to promote crRNA maturation and eliminate invading nucleic acids (Chou-Zheng and Hatoum-Aslan, 2019). Here, we identify RNase R as a third 'housekeeping' nuclease critical for immunity. We show that RNase R works in concert with PNPase to complete crRNA maturation and identify specific interactions with Csm5, a member of the type III effector complex, which facilitate nuclease recruitment/stimulation. Furthermore, we demonstrate that RNase R and PNPase are required to maintain robust anti-plasmid immunity, particularly when targeted transcripts are sparse. Altogether, our findings expand the known repertoire of accessory nucleases required for type III immunity and highlight the remarkable capacity of these systems to interface with diverse cellular pathways to ensure successful defense.


Assuntos
Sistemas CRISPR-Cas , Endorribonucleases , Endonucleases/metabolismo , Endorribonucleases/metabolismo , Ribonucleases/metabolismo , RNA/genética , Staphylococcus epidermidis
17.
Int J Mol Sci ; 23(24)2022 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-36555805

RESUMO

Endoplasmic reticulum stress activates inositol-requiring enzyme 1α (IRE1α) and protein kinase, R-like endoplasmic reticulum kinase (PERK), the two principal regulators of the unfolded protein response (UPR). In multiple myeloma, adaptive IRE1α signaling is predominantly activated and regulates cell fate along with PERK. Recently, we demonstrated that GNF-2, an allosteric c-Abl inhibitor, rheostatically enhanced IRE1α activity and induced apoptosis through c-Abl conformational changes in pancreatic ß cells. Herein, we analyzed whether the pharmacological modulation of c-Abl conformation resulted in anti-myeloma effects. First, we investigated the effects of GNF-2 on IRE1α activity and cell fate, followed by an investigation of the anti-myeloma effects of asciminib, a new allosteric c-Abl inhibitor. Finally, we performed RNA sequencing to characterize the signaling profiles of asciminib. We observed that both GNF-2 and asciminib decreased cell viability and induced XBP1 mRNA splicing in primary human myeloma cells and myeloma cell lines. RNA sequencing identified the induction of UPR- and apoptosis-related genes by asciminib. Asciminib re-localized c-Abl to the endoplasmic reticulum, and its combination with a specific IRE1α inhibitor, KIRA8, enhanced cell death with the reciprocal induction of CHOP mRNA expression. Together, the allosteric inhibition of c-Abl-activated UPR with anti-myeloma effects; this could be a novel therapeutic target for multiple myeloma.


Assuntos
Mieloma Múltiplo , Humanos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Endorribonucleases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Resposta a Proteínas não Dobradas , Estresse do Retículo Endoplasmático , Morte Celular , RNA Mensageiro/genética , Proteína 1 de Ligação a X-Box/metabolismo
18.
BMC Cancer ; 22(1): 1369, 2022 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-36585626

RESUMO

BACKGROUND: Attenuated Oxaliplatin efficacy is a challenge in treating colorectal cancer (CRC) patients, contributory to the failure in chemotherapy and the risks in relapse and metastasis. However, the mechanism of Oxaliplatin de-efficacy during CRC treatment has not been completely elucidated. METHODS: Microarray screening, western blot and qPCR on clinic CRC samples were conducted to select the target gene ABCC10 transporter. The Cancer Genome Atlas data was analyzed to figure out the correlation between the clinical manifestation and ABCC10 expression. ABCC10 knock-down in CRC cells was conducted to identify its role in the Oxaliplatin resistance. Cell counting kit-8 assay was conducted to identify the CRC cell viability and Oxaliplatin IC50. Flow cytometry was conducted to detect the cell apoptosis exposed to Oxaliplatin. The intracellular Oxaliplatin accumulation was measured by ultra-high performance liquid chromatography coupled to tandem mass spectrometry. RESULTS: CRC patients with higher ABCC10 were prone to relapse and metastasis. Differential ABCC10 expression in multiple CRC cell lines revealed a strong positive correlation between ABCC10 expression level and decreased Oxaliplatin response. In ABCC10 knock-down CRC cells the Oxaliplatin sensitivity was evidently elevated due to an increase of intracellular Oxaliplatin accumulation resulted from the diminished drug efflux. To explore a strategy to block ABCC10 in CRC cells, we paid a special interest in the endoplasmic reticulum stress (ERS) / unfolded protein response (UPR) that plays a dual role in tumor development. We found that neither the inhibition of ERS nor the induction of mild ERS had anti-CRC effect. However, the CRC cell viability was profoundly decreased and the pro-apoptotic factor CHOP and apoptosis were increased by the induction of intense ERS. Significantly, the Oxaliplatin sensitivity of CRC cells was enhanced in response to the intense ERS, which was blocked by inhibiting IRE1α branch of UPR. Finally, we figured out that the intense ERS down-regulated ABCC10 expression via regulated IRE1-dependent decay activity. CONCLUSION: Oxaliplatin was a substrate of ABCC10 efflux transporter. The intense ERS/IRE1α enhanced Oxaliplatin efficacy through down-regulating ABCC10 in addition to inducing CHOP. We suggested that introduction of intense ERS/UPR could be a promising strategy to restore chemo-sensitivity when used in combination with Oxaliplatin or other chemotherapeutic drugs pumped out by ABCC10.


Assuntos
Neoplasias Colorretais , Proteínas Serina-Treonina Quinases , Humanos , Oxaliplatina/farmacologia , Oxaliplatina/uso terapêutico , Proteínas Serina-Treonina Quinases/metabolismo , Endorribonucleases/genética , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Recidiva Local de Neoplasia , Apoptose , Estresse do Retículo Endoplasmático , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética
19.
PLoS One ; 17(12): e0279016, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36520816

RESUMO

BACKGROUND: Increased intrahepatic bile acids cause endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) is activated to maintain homeostasis. UPR dysregulation, including the inositol-requiring enzyme 1α/X-box protein 1 (IRE1α/XBP1) pathway, is associated with adult liver diseases but has not been characterized in pediatric liver diseases. We evaluated hepatic UPR expression in pediatric cholestatic liver disease (CLD) explants and hypothesize that an inability to appropriately activate the hepatic IRE1α/XBP1 pathway is associated with the pathogenesis of CLD. METHODS: We evaluated 34 human liver explants, including: pediatric CLD (Alagille, ALGS, and progressive familial intrahepatic cholestasis, PFIC), pediatric non-cholestatic liver disease controls (autoimmune hepatitis, AIH), adult CLD, and normal controls. We performed RNA-seq, quantitative PCR, and western blotting to measure expression differences of the hepatic UPR and other signaling pathways. RESULTS: Pathway analysis demonstrated that the KEGG 'protein processing in ER' pathway was downregulated in pediatric CLD compared to normal controls. Pediatric CLD had decreased hepatic IRE1α/XBP1 pathway gene expression and decreased protein expression of phosphorylated IRE1α compared to normal controls. IRE1α/XBP1 pathway gene expression was also decreased in pediatric CLD compared to AIH disease controls. CONCLUSIONS: Pediatric CLD explants have decreased expression of the protective IRE1α/XBP1 pathway and down-regulated KEGG protein processing in the ER pathways. IRE1α/XBP1 pathway expression differences occur when compared to both normal and non-cholestatic disease controls. Attenuated expression of the IRE1α/XBP1 pathway is associated with cholestatic diseases and may be a target for future therapeutics.


Assuntos
Endorribonucleases , Proteínas Serina-Treonina Quinases , Adulto , Humanos , Criança , Endorribonucleases/genética , Endorribonucleases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismo , Resposta a Proteínas não Dobradas , Estresse do Retículo Endoplasmático/genética , Inositol
20.
Nucleic Acids Res ; 50(22): 12913-12923, 2022 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-36484100

RESUMO

The type III-E CRISPR-Cas systems are newly identified adaptive immune systems in prokaryotes that use a single Cas7-11 protein to specifically cleave target RNA. Cas7-11 could associate with Csx29, a putative caspase-like protein encoded by the gene frequently found in the type III-E loci, suggesting a functional linkage between the RNase and protease activities in type III-E systems. Here, we demonstrated that target RNA recognition would stimulate the proteolytic activity of Csx29, and protein Csx30 is the endogenous substrate. More interestingly, while the cognate target RNA recognition would activate Csx29, non-cognate target RNA with the complementary 3' anti-tag sequence inhibits the enzymatic activity. Csx30 could bind to the sigma factor RpoE, which may initiate the stress response after proteolytic cleavage. Combined with biochemical and structural studies, we have elucidated the mechanisms underlying the target RNA-guided proteolytic activity of Csx29. Our work will guide further developments leveraging this simple RNA targeting system for RNA and protein-related applications.


Assuntos
Proteínas Associadas a CRISPR , RNA , RNA/genética , Sistemas CRISPR-Cas , Endorribonucleases/metabolismo , Ribonucleases/metabolismo , Peptídeo Hidrolases/genética , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo
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