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1.
Acta Neurochir Suppl ; 127: 105-119, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31407071

RESUMO

The protein kinase RNA-like endoplasmic reticulum kinase (PERK) pathway, which is a branch of the unfolded protein response, participates in a range of pathophysiological processes of neurological diseases. However, few studies have investigated the role of the PERK in intracerebral hemorrhage (ICH). The present study evaluated the role of the PERK pathway during the early phase of ICH-induced secondary brain injury (SBI) and its potential mechanisms. An autologous whole blood ICH model was established in rats, and cultured primary cortical neurons were treated with oxyhemoglobin to mimic ICH in vitro. We found that levels of phosphorylated alpha subunit of eukaryotic translation initiation factor 2 (p-eIF2α) and activating transcription factor 4 (ATF4) increased significantly and peaked at 12 h during the early phase of the ICH. To further elucidate the role of the PERK pathway, we assessed the effects of the PERK inhibitor, GSK2606414, and the eIF2α dephosphorylation antagonist, salubrinal, at 12 h after ICH both in vivo and in vitro. Inhibition of PERK with GSK2606414 suppressed the protein levels of p-eIF2α and ATF4, resulting in increase of transcriptional activator CCAAT/enhancer-binding protein homologous protein (CHOP) and caspase-12, which promoted apoptosis and reduced neuronal survival. Treatment with salubrinal yielded opposite results, which suggested that activation of the PERK pathway could promote neuronal survival and reduce apoptosis. In conclusion, the present study has demonstrated the neuroprotective effects of the PERK pathway during the early phase of ICH-induced SBI. These findings highlight the potential value of PERK pathway as a therapeutic target for ICH.


Assuntos
Lesões Encefálicas , Hemorragia Cerebral , RNA , eIF-2 Quinase , Animais , Lesões Encefálicas/metabolismo , Hemorragia Cerebral/metabolismo , Fator de Iniciação 2 em Eucariotos , Ratos , eIF-2 Quinase/metabolismo
2.
Anticancer Res ; 39(10): 5515-5524, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31570445

RESUMO

BACKGROUND/AIM: Administration of cisplatin in cancer patients is limited by the kidney-related adverse effects; however, a protective strategy is absent. We hypothesized that fucoidan protects the proximal tubule epithelial (TH-1) cells against the effects of cisplatin. MATERIALS AND METHODS: To assess the effect of fucoidan, its effect on reactive oxygen species (ROS) formation, endoplasmic reticulum (ER) stress response, DNA damage response (DDR), apoptosis, and cell-cycle arrest in TH-1 cells was investigated. RESULTS: Cisplatin increased the accumulation of ROS, leading to excessive ER stress. In presence of cisplatin, treatment of TH-1 cells with fucoidan significantly reduced the ER stress by maintaining the complex of GRP78 with PERK and IRE1α. In particular, fucoidan enhanced the antioxidative capacity through up-regulation of PrPC Furthermore, fucoidan suppressed cisplatin-induced apoptosis and cell-cycle arrest, whereas silencing of PRNP blocked these effects of fucoidan. CONCLUSION: Fucoidan may be a potential adjuvant therapy for cancer patients treated with cisplatin as it preserves renal functionality.


Assuntos
Cisplatino/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Túbulos Renais Proximais/efeitos dos fármacos , Polissacarídeos/farmacologia , Substâncias Protetoras/farmacologia , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Células Epiteliais/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Túbulos Renais Proximais/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Células Th1/efeitos dos fármacos , Células Th1/metabolismo , Regulação para Cima/efeitos dos fármacos , eIF-2 Quinase/metabolismo
3.
Life Sci ; 237: 116944, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31604108

RESUMO

AIMS: Endoplasmic reticulum stress (ERS) is an evolutionarily conserved cell stress response. Recently, it was found that ERS induces not only apoptosis but also endoplasmic reticulophagy (ER-phagy). A previous study demonstrated that inhibition of ER-phagy alleviates cell injury. The purpose of this study was to investigate the involvement of the protein kinase R-like ER kinase (PERK)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in ERS-induced ER-phagy in H9c2 cardiomyoblasts. To address this aim, cells were treated with ERS inhibitors and a Nrf2 inhibitor before establishment of thapsigargin (TG)- or tunicamycin (TM)-induced ERS models in H9c2 cardiomyoblasts. MAIN METHODS: Transmission electron microscopy and immunofluorescence staining were used to detect ER-phagy. Western blotting was employed to detect the levels of calreticulin (CRT), total and phosphorylated PERK, nuclear Nrf2, activated transcription factor 4 (ATF4), light chain 3B (LC3B)-II and Beclin 1. Immunofluorescence staining was used to assess subcellular location of Nrf2. KEY FINDING: TG or TM induced H9c2 cell injury and ER-phagy and upregulated CRT expression, PERK phosphorylation, Nrf2 nuclear translocation, and expression of ATF4, Beclin 1, and LC3B-II compared with control cells. Treatment with ERS inhibitors decreased TG- or TM-induced ER-phagy, downregulated CRT expression, PERK phosphorylation, Nrf2 nuclear translocation and the expression of ATF4, Beclin 1 and LC3B-II. Moreover, a Nrf2 inhibitor downregulated the expression of ATF4, Beclin 1 and LC3B-II and alleviated TG- or TM-induced ER-phagy and H9c2 cell injury. SIGNIFICANCE: These findings suggest that the PERK/Nrf2 pathway mediates upregulation of ER-phagy, thereby inducing cell injury in H9c2 cardiomyoblasts.


Assuntos
Apoptose , Estresse do Retículo Endoplasmático , Retículo Endoplasmático/patologia , Miócitos Cardíacos/patologia , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , eIF-2 Quinase/metabolismo , Fator 4 Ativador da Transcrição/metabolismo , Animais , Proteína Beclina-1/metabolismo , Células Cultivadas , Retículo Endoplasmático/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Miócitos Cardíacos/metabolismo , Fosforilação , Ratos , Transdução de Sinais
4.
Shanghai Kou Qiang Yi Xue ; 28(3): 259-263, 2019 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-31489412

RESUMO

PURPOSE: This study was aimed to figure out the way that cyclic-stretch influenced the apoptosis of myoblasts and evaluate the importance of PERK and its possible mechanism involved. METHODS: L6 rat myoblasts were cultured in vitro and mechanical stimulation model was constructed successfully. The myoblasts were imposed tension for 0, 2, 6, 12 and 24 hours respectively by multi-channel cell stress loading system. The force value was 15% cell deformation and the frequency was 10 cycles/min. Each cycle was consisted of stretch for 3 seconds and relaxation for 3 seconds, and the group without tension was used as the control group. The apoptotic myoblasts were dyed by DAPI and observed through fluorescence microscopy to detect the apoptosis rate; the mRNA levels of PERK and CHOP in different groups were detected by real-time PCR and protein levels of PERK and p-PERK in different groups were detected by Western blot. PERK inhibitor was used to clear the role of PERK in apoptosis induced by cyclic-stretch and clarify the relationship between the endoplasmic reticulum stress and apoptosis induced by cyclic-stretch. SPSS 17.0 software package was used to analyze the data statistically. RESULTS: DAPI nuclear stain showed that cyclical tensile stress can induce apoptosis in vitro cultured myoblast. Apoptosis rate showed a trend of rising gradually over time, peaked at 24 h. After dealt with the inhibitor of PERK, the apoptosis rate of the 24 h group under the cyclic stretch showed no difference compared with the control. The results of real- time PCR showed that the mRNA of CHOP was increased with the extension loading time, while the mRNA of PERK showed no difference compared with the control. Western blot results showed that the protein level of p-PERK was increased with the extension of loading time, while the expression of PERK showed no difference compared with the control group. When PERK inhibitor added, the mRNA level of CHOP along with the protein expression level of p-PERK showed no significant difference compared to the control. CONCLUSIONS: PERK signaling pathway is involved in the apoptosis of myoblasts induced by cyclic stretch, and the possible mechanism may be closely related to the phosphorylation of PERK.


Assuntos
Apoptose , Estresse do Retículo Endoplasmático , Mioblastos , eIF-2 Quinase , Animais , Estresse do Retículo Endoplasmático/fisiologia , Ratos , Transdução de Sinais , eIF-2 Quinase/metabolismo
5.
Cancer Sci ; 110(10): 3275-3287, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31368616

RESUMO

p97/VCP is an endoplasmic reticulum (ER)-associated protein that belongs to the AAA (ATPases associated with diverse cellular activities) ATPase family. It has a variety of cellular functions including ER-associated protein degradation, autophagy, and aggresome formation. Recent studies have shown emerging roles of p97/VCP and its potential as a therapeutic target in several cancer subtypes including multiple myeloma (MM). We conducted a cell-based compound screen to exploit novel small compounds that have cytotoxic activity in myeloma cells. Among approximately 2000 compounds, OSSL_325096 showed relatively strong antiproliferative activity in MM cell lines (IC50 , 100-500 nmol/L). OSSL_325096 induced apoptosis in myeloma cell lines, including a bortezomib-resistant cell line and primary myeloma cells purified from patients. Accumulation of poly-ubiquitinated proteins, PERK, CHOP, and IREα, was observed in MM cell lines treated with OSSL_325096, suggesting that it induces ER stress in MM cells. OSSL_325096 has a similar chemical structure to DBeQ, a known p97/VCP inhibitor. Knockdown of the gene encoding p97/VCP induced apoptosis in myeloma cells, accompanied by accumulation of poly-ubiquitinated protein. IC50 of OSSL_325096 to myeloma cell lines were found to be lower (0.1-0.8 µmol/L) than those of DBeQ (2-5 µmol/L). In silico protein-drug-binding simulation suggested possible binding of OSSL_325096 to the ATP binding site in the D2 domain of p97/VCP. In cell-free ATPase assays, OSSL_325096 showed dose-dependent inhibition of p97/VCP ATPase activity. Finally, OSSL_325096 inhibited the growth of subcutaneous myeloma cell tumors in vivo. The present data suggest that OSSL_325096 exerts anti-myeloma activity, at least in part through p97/VCP inhibition.


Assuntos
Adenosina Trifosfatases/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Inibidores Enzimáticos/administração & dosagem , Mieloma Múltiplo/tratamento farmacológico , Proteínas Nucleares/metabolismo , Bibliotecas de Moléculas Pequenas/administração & dosagem , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/química , Animais , Sítios de Ligação , Bortezomib/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático , Endorribonucleases/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Camundongos , Modelos Moleculares , Mieloma Múltiplo/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/química , Proteínas Serina-Treonina Quinases/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Fator de Transcrição CHOP/metabolismo , Ubiquitinação , Ensaios Antitumorais Modelo de Xenoenxerto , eIF-2 Quinase/metabolismo
6.
Nat Commun ; 10(1): 2421, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160600

RESUMO

Translation efficiency can be affected by mRNA stability and secondary structures, including G-quadruplex structures (G4s). The highly conserved DEAH-box helicase DHX36/RHAU resolves G4s on DNA and RNA in vitro, however a systems-wide analysis of DHX36 targets and function is lacking. We map globally DHX36 binding to RNA in human cell lines and find it preferentially interacting with G-rich and G4-forming sequences on more than 4500 mRNAs. While DHX36 knockout (KO) results in a significant increase in target mRNA abundance, ribosome occupancy and protein output from these targets decrease, suggesting that they were rendered translationally incompetent. Considering that DHX36 targets, harboring G4s, preferentially localize in stress granules, and that DHX36 KO results in increased SG formation and protein kinase R (PKR/EIF2AK2) phosphorylation, we speculate that DHX36 is involved in resolution of rG4 induced cellular stress.


Assuntos
RNA Helicases DEAD-box/metabolismo , Quadruplex G , RNA Mensageiro/metabolismo , Regiões não Traduzidas , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Fosforilação , Biossíntese de Proteínas , Ribossomos/metabolismo , Estresse Fisiológico , eIF-2 Quinase/metabolismo
7.
Nat Commun ; 10(1): 2871, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253805

RESUMO

Adenovirus Virus-Associated (VA) RNAs are the first discovered viral noncoding RNAs. By mimicking double-stranded RNAs (dsRNAs), the exceptionally abundant, multifunctional VA RNAs sabotage host machineries that sense, transport, process, or edit dsRNAs. How VA-I suppresses PKR activation despite its strong dsRNA character, and inhibits the crucial antiviral kinase to promote viral translation, remains largely unknown. Here, we report a 2.7 Å crystal structure of VA-I RNA. The acutely bent VA-I features an unusually structured apical loop, a wobble-enriched, coaxially stacked apical and tetra-stems necessary and sufficient for PKR inhibition, and a central domain pseudoknot that resembles codon-anticodon interactions and prevents PKR activation by VA-I. These global and local structural features collectively define VA-I as an archetypal PKR inhibitor made of RNA. The study provides molecular insights into how viruses circumnavigate cellular rules of self vs non-self RNAs to not only escape, but further compromise host innate immunity.


Assuntos
Conformação de Ácido Nucleico , RNA de Cadeia Dupla/química , RNA Viral/química , Adenovírus Humanos/genética , Sequência de Bases , Cristalização , Luz , RNA de Cadeia Dupla/genética , RNA Viral/genética , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
8.
J Dairy Sci ; 102(8): 7359-7370, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31155263

RESUMO

Disruption of endoplasmic reticulum (ER) homeostasis, often termed ER stress, is intrinsically linked with perturbation of lipid metabolism in humans and mice. Whether ER homeostasis is affected in cows experiencing fatty liver is unknown. The aim of this study was to investigate the potential role of ER stress in hepatic lipid accumulation in calf hepatocytes and ER stress status in dairy cows with severe fatty liver. In vitro experiments were conducted in which hepatocytes were isolated from calves and treated with different concentrations of fatty acids, tauroursodeoxycholic acid (TUDCA; a canonical inhibitor of ER stress), or both. The increase in phosphorylation level of protein kinase RNA-like ER kinase (PERK) and inositol requiring protein-1α (IRE1α) proteins, and the cleavage of activating transcription factor-6 (ATF6) protein in response to increasing doses of fatty acids (which were reversed by TUDCA treatment) in primary hepatocytes underscored a mechanistic link between fatty acids and ER stress. In addition, fatty acid treatment increased the abundance of sterol regulatory element-binding protein 1c, acetyl-CoA carboxylase-α, fatty acid synthase, and diacylglycerol acyltransferase 1, and lipid accumulation in calf primary hepatocytes, whereas inhibition of ER stress by incubating with TUDCA significantly weakened these effects. Overall, results in vitro indicate that inhibition of ER stress in calf hepatocytes alleviates fatty acid-induced lipid accumulation by downregulating the expression of lipogenic genes. In vivo experiments, liver and blood samples were collected from cows diagnosed as healthy (n = 15) or with severe fatty liver (n = 15). The phosphorylation level of PERK and IRE1α, the cleavage of ATF6 protein, and the abundance of several unfolded protein response genes (78 kDa glucose-regulated protein, AMP-dependent transcription factor 4, and spliced X-box binding protein 1) were greater in liver of cows with severe fatty liver. The present in vivo study confirms the occurrence of ER stress in dairy cows with severe fatty liver. Considering the causative role of fatty acid-induced ER stress in hepatic lipid accumulation in calf hepatocytes, the existence of ER stress in the liver of severe fatty liver cows may presage its participation in fatty liver progression in dairy cows. However, the mechanistic relationship between ER stress and fatty liver in dairy cows remain to be determined.


Assuntos
Doenças dos Bovinos/fisiopatologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Ácidos Graxos/administração & dosagem , Fígado Gorduroso/veterinária , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/metabolismo , Fator 6 Ativador da Transcrição/metabolismo , Animais , Bovinos , Células Cultivadas , Estresse do Retículo Endoplasmático/fisiologia , Endorribonucleases/metabolismo , Feminino , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Lipogênese/genética , Fígado/efeitos dos fármacos , Camundongos , Fosforilação , Ácido Tauroquenodesoxicólico/administração & dosagem , Resposta a Proteínas não Dobradas/genética , eIF-2 Quinase/metabolismo
9.
Appl Microbiol Biotechnol ; 103(15): 6153-6167, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31154490

RESUMO

MoHrip2, identified from Magnaporthe oryzae as an elicitor, can activate plant defense responses either in the form of recombinant protein in vitro or ectopic expressed protein in rice. However, its intrinsic function in the infective interaction of M. oryzae-rice is largely unknown. Here, we found that mohrip2 expression was significantly induced at stages of fungal penetration and colonization. Meanwhile, the induced MoHrip2 mainly accumulated in the rice apoplast by outlining the entire invasive hyphae during infection, and its secretion was via the conventional endoplasmic reticulum (ER)-to-Golgi pathway, demonstrating the nature of MoHrip2 as an apoplastic effector. What's more, the disease facilitating function of MoHrip2 was revealed by the significantly compromised virulence of Δmohrip2 mutants on rice seedlings and even on the wounded rice leaves. Inoculations of these mutant strains on rice leaf sheaths showed a reduction in penetration and subsequent expansion of fungal growth, which is probably due to activated host immunity including the expression of certain defense-related genes and the production of certain phytoalexins. Altogether, these results demonstrated the necessity of MoHrip2 in suppression of host immunity and the full virulence of M. oryzae.


Assuntos
Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Magnaporthe/patogenicidade , Oryza/microbiologia , Doenças das Plantas/microbiologia , Fatores de Virulência/metabolismo , eIF-2 Quinase/metabolismo , Deleção de Genes , Magnaporthe/enzimologia , Oryza/imunologia , Virulência , Fatores de Virulência/deficiência , eIF-2 Quinase/deficiência
10.
PLoS Negl Trop Dis ; 13(6): e0007500, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31216268

RESUMO

BACKGROUND: Leishmania parasites are transmitted to vertebrate hosts by phlebotomine sandflies and, in humans, may cause tegumentary or visceral leishmaniasis. The role of PKR (dsRNA activated kinase) and Toll-like receptor 3 (TLR3) activation in the control of Leishmania infection highlights the importance of the engagement of RNA sensors, which are usually involved in the antiviral cell response, in the fate of parasitism by Leishmania. We tested the hypothesis that Phlebovirus, a subgroup of the Bunyaviridae, transmitted by sandflies, would interfere with Leishmania infection. METHODOLOGY/PRINCIPAL FINDINGS: We tested two Phlebovirus isolates, Icoaraci and Pacui, from the rodents Nectomys sp. and Oryzomys sp., respectively, both natural sylvatic reservoir of Leishmania (Leishmania) amazonensis from the Amazon region. Phlebovirus coinfection with L. (L.) amazonensis in murine macrophages led to increased intracellular growth of L. (L.) amazonensis. Further studies with Icoaraci coinfection revealed the requirement of the PKR/IFN1 axis on the exacerbation of the parasite infection. L. (L.) amazonensis and Phlebovirus coinfection potentiated PKR activation and synergistically induced the expression of IFNß and IL-10. Importantly, in vivo coinfection of C57BL/6 mice corroborated the in vitro data. The exacerbation effect of RNA virus on parasite infection may be specific because coinfection with dengue virus (DENV2) exerted the opposite effect on parasite load. CONCLUSIONS: Altogether, our data suggest that coinfections with specific RNA viruses shared by vectors or reservoirs of Leishmania may enhance and sustain the activation of host cellular RNA sensors, resulting in aggravation of the parasite infection. The present work highlights new perspectives for the investigation of antiviral pathways as important modulators of protozoan infections.


Assuntos
Infecções por Bunyaviridae/complicações , Coinfecção/imunologia , Suscetibilidade a Doenças , Interferon beta/metabolismo , Interleucina-10/metabolismo , Leishmaniose/imunologia , eIF-2 Quinase/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Leishmania/imunologia , Camundongos Endogâmicos C57BL , Modelos Teóricos , Phlebovirus/imunologia
11.
RNA ; 25(9): 1192-1201, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31239298

RESUMO

Protein kinase RNA-activated (PKR) is an interferon-inducible kinase that is potently activated by long double-stranded RNA (dsRNA). In a previous study, we found that snoRNAs exhibit increased association with PKR in response to metabolic stress. While it was unclear if snoRNAs also activated PKR in cells, activation in vitro was observed. snoRNAs do not exhibit the double-stranded character typically required for activation of PKR, but some studies suggest such RNAs can activate PKR if triphosphorylated at the 5' terminus, or if they are able to form intermolecular dimers. To interrogate the mechanism of PKR activation by snoRNAs in vitro we focused on SNORD113. Using multiple methods for defining the 5'-phosphorylation state, we find that activation of PKR by SNORD113 does not require a 5'-triphosphate. Gel purification from a native gel followed by analysis using analytical ultracentrifugation showed that dimerization was also not responsible for activation. We isolated distinct conformers of SNORD113 from a native polyacrylamide gel and tracked the activating species to dsRNA formed from antisense RNA synthesized during in vitro transcription with T7 RNA polymerase. Similar studies with additional snoRNAs and small RNAs showed the generality of our results. Our studies suggest that a 5' triphosphate is not an activating ligand for PKR, and emphasize the insidious nature of antisense contamination.


Assuntos
Ativação Enzimática/genética , Polifosfatos/metabolismo , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Dimerização , Humanos , Ligantes , Fosforilação/genética , Ligação Proteica/genética , RNA de Cadeia Dupla/genética , RNA Nucleolar Pequeno/genética , Transcrição Genética/genética , Ultracentrifugação/métodos , Proteínas Virais/metabolismo
12.
Nat Commun ; 10(1): 2139, 2019 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-31086176

RESUMO

Trastuzumab is integral to HER2+ cancer treatment, but its therapeutic index is narrowed by the development of resistance. Phosphorylation of the translation initiation factor eIF2α (eIF2α-P) is the nodal point of the integrated stress response, which promotes survival or death in a context-dependent manner. Here, we show an anti-tumor function of the protein kinase PKR and its substrate eIF2α in a mouse HER2+ breast cancer model. The anti-tumor function depends on the transcription factor ATF4, which upregulates the CDK inhibitor P21CIP1 and activates JNK1/2. The PKR/eIF2α-P arm is induced by Trastuzumab in sensitive but not resistant HER2+ breast tumors. Also, eIF2α-P stimulation by the phosphatase inhibitor SAL003 substantially increases Trastuzumab potency in resistant HER2+ breast and gastric tumors. Increased eIF2α-P prognosticates a better response of HER2+ metastatic breast cancer patients to Trastuzumab therapy. Hence, the PKR/eIF2α-P arm antagonizes HER2 tumorigenesis whereas its pharmacological stimulation improves the efficacy of Trastuzumab therapy.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/patologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Neoplasias Gástricas/patologia , Trastuzumab/farmacologia , eIF-2 Quinase/metabolismo , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/metabolismo , Mama/patologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Fosforilação , Prognóstico , Receptor ErbB-2/metabolismo , Neoplasias Gástricas/tratamento farmacológico , Análise de Sobrevida , Análise Serial de Tecidos , Trastuzumab/uso terapêutico , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto , eIF-2 Quinase/antagonistas & inibidores
13.
Nat Commun ; 10(1): 2136, 2019 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-31086188

RESUMO

Protein synthesis in eukaryotes is controlled by signals and stresses via a common pathway, called the integrated stress response (ISR). Phosphorylation of the translation initiation factor eIF2 alpha at a conserved serine residue mediates translational control at the ISR core. To provide insight into the mechanism of translational control we have determined the structures of eIF2 both in phosphorylated and unphosphorylated forms bound with its nucleotide exchange factor eIF2B by electron cryomicroscopy. The structures reveal that eIF2 undergoes large rearrangements to promote binding of eIF2α to the regulatory core of eIF2B comprised of the eIF2B alpha, beta and delta subunits. Only minor differences are observed between eIF2 and eIF2αP binding to eIF2B, suggesting that the higher affinity of eIF2αP for eIF2B drives translational control. We present a model for controlled nucleotide exchange and initiator tRNA binding to the eIF2/eIF2B complex.


Assuntos
Fator de Iniciação 2B em Eucariotos/ultraestrutura , Fator de Iniciação 2 em Eucariotos/ultraestrutura , RNA de Transferência de Metionina/ultraestrutura , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Simulação por Computador , Microscopia Crioeletrônica , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 2B em Eucariotos/metabolismo , Modelos Moleculares , Nucleotídeos/metabolismo , Fosforilação/fisiologia , Ligação Proteica/fisiologia , Biossíntese de Proteínas/fisiologia , RNA de Transferência de Metionina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , eIF-2 Quinase/metabolismo
14.
Int J Mol Sci ; 20(7)2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30939763

RESUMO

The demyelinating canine distemper virus (CDV)-leukoencephalitis represents a translational animal model for multiple sclerosis. The present study investigated the expression of type I interferon (IFN-I) pathway members in CDV-induced cerebellar lesions to gain an insight into their role in lesion development. Gene expression of 110 manually selected genes in acute, subacute and chronic lesions was analyzed using pre-existing microarray data. Interferon regulatory factor (IRF) 3, IRF7, signal transducer and activator of transcription (STAT) 1, STAT2, MX protein, protein kinase R (PKR), 2'-5'-oligoadenylate synthetase (OAS) 1 and interferon-stimulated gene (ISG) 15 expression were also evaluated using immunohistochemistry. Cellular origin of STAT1, STAT2, MX and PKR were determined using immunofluorescence. CDV infection caused an increased expression of the antiviral effector proteins MX, PKR, OAS1 and ISG15, which probably contributed to a restricted viral replication, particularly in neurons and oligodendrocytes. This increase might be partly mediated by IRF-dependent pathways due to the lack of changes in IFN-I levels and absence of STAT2 in astrocytes. Nevertheless, activated microglia/macrophages showed a strong expression of STAT1, STAT2 and MX proteins in later stages of the disease, indicating a strong activation of the IFN-I signaling cascade, which might be involved in the aggravation of bystander demyelination.


Assuntos
Cinomose/genética , Imunidade Inata/genética , Interferons/farmacologia , 2',5'-Oligoadenilato Sintetase/genética , 2',5'-Oligoadenilato Sintetase/metabolismo , Animais , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Cinomose/imunologia , Cães , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/metabolismo , Proteínas de Resistência a Myxovirus/genética , Proteínas de Resistência a Myxovirus/metabolismo , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismo , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
15.
MBio ; 10(2)2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015330

RESUMO

Protein kinase R (PKR) plays a major role in activating host immunity during infection by sensing double-stranded RNA (dsRNA) produced by viruses. Once activated by dsRNA, PKR phosphorylates the translation factor eukaryotic initiation factor 2α (eIF2α), halting cellular translation. Many viruses have methods of inhibiting PKR activation or its downstream effects, circumventing protein synthesis shutdown. These include sequestering dsRNA or producing proteins that bind to and inhibit PKR activation. Here we describe our finding that in multiple cell types, PKR was depleted during mouse adenovirus type 1 (MAV-1) infection. MAV-1 did not appear to be targeting PKR at the transcriptional or translational level, because total PKR mRNA levels and levels of PKR mRNA bound to polysomes were unchanged or increased during MAV-1 infection. However, inhibiting the proteasome reduced the PKR depletion seen in MAV-1-infected cells, whereas inhibiting the lysosome had no effect. This suggests that proteasomal degradation alone is responsible for PKR degradation during MAV-1 infection. Time course experiments indicated that the degradation occurs early after infection. Infecting cells with UV-inactivated virus prevented PKR degradation, whereas inhibiting viral DNA replication did not. Together, these results suggest that an early viral gene is responsible. Degradation of PKR is a rare mechanism to oppose PKR activity, and it has been described in only six RNA viruses. To our knowledge, this is the first example of a DNA virus counteracting PKR by degrading it.IMPORTANCE The first line of defense in cells during viral infection is the innate immune system, which is activated by different viral products. PKR is a part of this innate immune system and is induced by interferon and activated by dsRNA produced by DNA and RNA viruses. PKR is such an important part of the antiviral response that many viral families have gene products to counteract its activation or the resulting effects of its activity. Although a few RNA viruses degrade PKR, this method of counteracting PKR has not been reported for any DNA viruses. MAV-1 does not encode virus-associated RNAs, a human adenoviral defense against PKR activation. Instead, MAV-1 degrades PKR, and it is the first DNA virus reported to do so. The innate immune evasion by PKR degradation is a previously unidentified way for a DNA virus to circumvent the host antiviral response.


Assuntos
Interações Hospedeiro-Patógeno , Mastadenovirus/crescimento & desenvolvimento , Proteólise , Replicação Viral , eIF-2 Quinase/metabolismo , Animais , Linhagem Celular , Evasão da Resposta Imune , Imunidade Inata , Camundongos , Complexo de Endopeptidases do Proteassoma/metabolismo
16.
Mar Drugs ; 17(4)2019 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-31022939

RESUMO

Vasculogenic mimicry (VM) formed by tumor cells plays a vital role in the progress of tumor, because it provides nutrition for tumor cells and takes away the metabolites. Therefore, the inhibition of VM is crucial to the clinical treatment of tumors. In this study, we investigated the anti-tumor effect of a novel peptide, KVEPQDPSEW (AATP), isolated from abalone (Haliotis discus hannai) on HT1080 cells by migration, invasion analysis and the mode of action. The results showed that AATP effectively inhibited MMPs by blocking MAPKs and NF-κB pathways, leading to the downregulation of metastasis of tumor cells. Moreover, AATP significantly inhibited VM and pro-angiogenic factors, including VEGF and MMPs by suppression of AKT/mTOR signaling. In addition, molecular docking was used to study the interaction of AATP and HIF-1α, and the results showed that AATP was combined with an active site of HIF-1α by a hydrogen bond. The effect of AATP on anti-metastatic and anti-vascular in HT1080 cells revealed that AATP may be a potential lead compound for treatment of tumors in the future.


Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos/farmacologia , Gastrópodes/química , Neoplasias/tratamento farmacológico , Peptídeos/farmacologia , Adulto , Inibidores da Angiogênese/química , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Metaloproteinases da Matriz/metabolismo , NF-kappa B/metabolismo , Metástase Neoplásica , Neoplasias/irrigação sanguínea , Neoplasias/metabolismo , Neoplasias/patologia , Proteína Oncogênica v-akt/metabolismo , Peptídeos/química , Peptídeos/isolamento & purificação , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Esferoides Celulares/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , eIF-2 Quinase/metabolismo
17.
Toxicol Lett ; 308: 24-33, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30910607

RESUMO

Dibutyl phthalate (DBP)-induced germ cell apoptosis contributes to male reproductive toxicity, however, the primary target organelle of DBP or the molecular events triggered by DBP to initiate germ cell apoptosis remain unclear. Our previous studies demonstrated DBP could stimulate the production of intracellular reactive oxygen species (ROS), which served as an upstream mediator of activation of endoplasmic reticulum (ER) stress in mouse spermatocyte-derived GC-2 cells. In the present study, the impacts of DBP-induced ROS generation on the mitochondria-related damage and the associations between ER stress and mitochondrial-related damage were investigated in GC-2 cells. We observed significant decreases of mitochondrial mass, mtDNA copy number, COX IV protein level, and ATP level in DBP-treated GC-2 cells in a dose-dependent manner. And DBP activated mitochondrial-related apoptosis, indicated by the elevation of cytoplasmic cytochrome C (Cyt C) and the activation of caspase-9/3 cascade. Pretreatment with antioxidant melatonin obviously attenuated DBP-induced mitochondrial damage and mitochondrial-dependent apoptosis in GC-2 cells, indicating the role of ROS in DBP-caused testicular toxicity. In response to oxidative stress, the Nrf2/ARE axis was activated in DBP-treated GC-2 cells to counteract ROS overproduction and subsequent mitochondrial damage. Further experiments showed DBP treatment increased the phosphorylated expression of ER stress-related protein PERK. GSK2606414, a specific inhibitor of PERK, partly attenuated the expression of Nrf2. And both DBP-induced mitochondrial damage in GC-2 cells and mitochondrial-dependent apoptosis of the germ cells in rat testes were further aggravated by PERK inhibition. Taken together, our data suggest that PERK regulates the Nrf2/ARE antioxidant pathway functioning as a self-defense mechanism against ROS-related mitochondrial damage induced by DBP in male germ cells.


Assuntos
Elementos de Resposta Antioxidante/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Dibutilftalato/toxicidade , Mitocôndrias/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Espermatócitos/efeitos dos fármacos , eIF-2 Quinase/metabolismo , Animais , Linhagem Celular , Masculino , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Espermatócitos/metabolismo , Espermatócitos/patologia , Testículo/efeitos dos fármacos , Testículo/metabolismo , Testículo/patologia
18.
Arch Virol ; 164(5): 1323-1334, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30877450

RESUMO

Porcine circovirus type 2 (PCV2) is the essential infectious agent causing porcine circovirus-associated disease (PCVD) in pigs and one of the important viruses that severely jeopardize the swine husbandry industry. PCV2 elicits the unfolded protein response (UPR) via activation of the PERK pathway, and its capsid protein (Cap) has also been found to induce UPR with subsequent activation of apoptosis. The open reading frame 5 (ORF5) protein is a recently discovered non-structural protein, and its function in PCV2 pathogenesis remains unknown. The aim of this study was to determine whether the PCV2 ORF5 protein could induce endoplasmic reticulum stress (ERS) and UPR in porcine alveolar macrophages (PAMs). pEGFP-tagged ORF5 protein was transiently overexpressed in PAMs. Transmission electron microscopy (TEM) was employed to examine changes in ER morphology, and quantitative real-time PCR and western blotting analysis were used to measure UPR-related cell signaling alterations. We found that the ORF5 protein triggers swelling and degranulation of the ER and upregulates the expression of ERS markers. Further experiments demonstrated that the PCV2 ORF5 protein induces ERS and UPR via the PERK (RNA-activated protein kinase-like endoplasmic reticulum kinase), ATF6 (activating transcription factor 6) and IRE1 (inositol requiring enzyme 1) signaling pathways. Together with previous studies, we provide new information on the ERS-UPR induced by the PCV2 ORF5 protein.


Assuntos
Circovirus/genética , Estresse do Retículo Endoplasmático/genética , Retículo Endoplasmático/ultraestrutura , Macrófagos Alveolares/patologia , Resposta a Proteínas não Dobradas/genética , Proteínas do Envelope Viral/genética , Proteínas não Estruturais Virais/genética , Fator 6 Ativador da Transcrição/metabolismo , Animais , Linhagem Celular , Infecções por Circoviridae/patologia , Infecções por Circoviridae/veterinária , Retículo Endoplasmático/virologia , Endorribonucleases/metabolismo , Macrófagos Alveolares/virologia , Microscopia Eletrônica de Transmissão , Suínos , Doenças dos Suínos , Proteínas do Envelope Viral/metabolismo , eIF-2 Quinase/metabolismo
19.
Neurosci Lett ; 703: 125-131, 2019 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-30905823

RESUMO

The accumulation of ß-amyloid protein (Aß) in the brain is one of major pathological hallmarks of Alzheimer's disease (AD). Overactivation of the unfolded protein response (UPR) signaling has been reported to lead to ß-amyloidogenesis. The current study aimed to investigate the effects of treadmill exercise on UPR signaling and the Aß production and to demonstrate whether exercise-induced Aß reduction was associated with changes in UPR signaling. Three-month old male APP/PS1 transgenic and wild-type mice were subjected to treadmill exercise for 3 months. At the end of exercise (6 months old), the levels of Aß plaques and soluble forms of Aß, and proteins involve in the unfolded protein response (UPR) were analyzed in the hippocampus. Three months of treadmill exercise resulted in a robust reduction in Aß plaques and soluble forms of Aß in the hippocampus of APP/PS1 mice. This was accompanied by a significant decrease in ß-site amyloid precursor protein-cleaving enzyme 1 (BACE1) and presenilin-1 (PS1) expression. Meanwhile, we found that treadmill exercise down-regulated the expression of GRP78 and inhibited activation of PERK, eIF2α, and ATF4, reflecting the involvement of the UPR signaling. Overall, our findings suggest that treadmill exercise may suppresse the overactivation of the UPR signaling as well as inhibit the amyloidogenic pathway in APP/PS1 mice, thus may serve as an useful approach for the prevention and treatment of AD.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Condicionamento Físico Animal , Presenilina-1/genética , Resposta a Proteínas não Dobradas , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Ácido Aspártico Endopeptidases/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Teste de Esforço , Proteínas de Choque Térmico/metabolismo , Hipocampo/metabolismo , Hipocampo/patologia , Camundongos Transgênicos , Fragmentos de Peptídeos/metabolismo , Placa Amiloide/patologia , Presenilina-1/metabolismo , Transdução de Sinais , eIF-2 Quinase/metabolismo
20.
Int J Mol Med ; 43(5): 2015-2023, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30864682

RESUMO

Barbaloin (Bar) has a myocardial protective effect, but its mechanism of action is uncertain. The endoplasmic reticulum stress (ERS)­mediated apoptosis pathway serves an important role in the pathogenesis of myocardial ischemia­reperfusion injury (MIRI). Inhibiting ERS may significantly improve the progression of MIRI and serve a role in its prevention. Therefore, based on current knowledge of ERS­mediated cardiomyocyte apoptosis and the cardioprotective effect of Bar, the purpose of the present study was to further evaluate the myocardial protective effect and potential mechanisms of Bar pretreatment in MIRI. The present study established a MIR rat model and randomly divided these rats into four groups. Prior to myocardial ischemia, Bar (20 mg/kg) was administered to rats once daily for 1 week. Myocardial blood serum lactate dehydrogenase and creatine kinase were subsequently measured. A terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assay was used to evaluate the myocardial protective effect of Bar pretreatment on MIRI. To assess whether the ERS signaling pathway was involved in the myocardial protection mechanism of Bar pretreatment, the expression levels of ERS­associated proteins, protein canopy homolog 2 (CNPY2), glucose regulatory protein 78, transcriptional activator 4, C/EBP­homologous protein (CHOP), PKR endoplasmic reticulum kinase (PERK), caspase­12 and caspase­3 were detected by western blot analysis, immunohistochemistry or reverse transcription­quantitative polymerase chain reaction. The results confirmed that Bar pretreatment significantly reduced the damage and the level of apoptosis caused by MIR. Bar pretreatment significantly inhibited the expression of ERS­associated proteins in cardiomyocytes. In addition, the immunohistochemistry results demonstrated that Bar pretreatment significantly inhibited the CNPY2­positive cell apoptosis ratio of cardiomyocytes. Therefore, the results of the current study suggested that CNPY2 is present in cardiomyocytes and participates in the development of MIRI by initiating the PERK­CHOP signaling pathway. Bar pretreatment may attenuate MIRI by inhibiting the CNPY2­PERK apoptotic pathway.


Assuntos
Antracenos/uso terapêutico , Cardiotônicos/uso terapêutico , Proteínas de Membrana/metabolismo , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/metabolismo , eIF-2 Quinase/metabolismo , Animais , Antracenos/farmacologia , Apoptose/efeitos dos fármacos , Cardiotônicos/farmacologia , Creatina Quinase/sangue , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático/efeitos dos fármacos , L-Lactato Desidrogenase/sangue , Masculino , Traumatismo por Reperfusão Miocárdica/sangue , Miócitos Cardíacos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley
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