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1.
Int J Mol Sci ; 22(12)2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34208396

RESUMO

Non-small cell lung cancer (NSCLC) continues to be the leading cause of cancer death worldwide. Recently, targeting molecules whose functions are associated with tumorigenesis has become a game changing adjunct to standard anti-cancer therapy. As evidenced by the results of preclinical and clinical investigations, whole-body irradiations (WBI) with X-rays at less than 0.1-0.2 Gy per fraction can induce remissions of various neoplasms without inciting adverse side effects of conventional chemo- and radiotherapy. In the present study, a murine model of human NSCLC was employed to evaluate for the first time the anti-neoplastic efficacy of WBI combined with inactivation of CTLA-4, PD-1, and/or HSP90. The results indicate that WBI alone and in conjunction with the inhibition of the function of the cytotoxic T-lymphocyte antigen-4 (CTLA-4) and the programmed death-1 (PD-1) receptor immune checkpoints (ICs) and/or heat shock protein 90 (HSP90) markedly reduced tumorigenesis in mice implanted by three different routes with the syngeneic Lewis lung cancer cells and suppressed clonogenic potential of Lewis lung carcinoma (LLC1) cells in vitro. These results were associated with the relevant changes in the profile of pro- and anti-neoplastic immune cells recruited to the growing tumors and the circulating anti- and pro-inflammatory cytokines. In contrast, inhibition of the tested molecular targets used either separately or in combination with each other did not exert notable anti-neoplastic effects. Moreover, no significant synergistic effects were detected when the inhibitors were applied concurrently with WBI. The obtained results supplemented with further mechanistic explanations provided by future investigations will help design the effective strategies of treatment of lung and other cancers based on inactivation of the immune checkpoint and/or heat shock molecules combined with low-dose radiotherapy.


Assuntos
Proteínas de Choque Térmico/metabolismo , Proteínas de Checkpoint Imunológico/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/radioterapia , Transplante de Neoplasias , Dosagem Radioterapêutica , Irradiação Corporal Total , Animais , Células Clonais , Pulmão/patologia , Contagem de Linfócitos , Linfócitos do Interstício Tumoral , Macrófagos/patologia , Camundongos Endogâmicos C57BL , Ensaio Tumoral de Célula-Tronco
2.
Nat Commun ; 12(1): 3285, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34078899

RESUMO

In peripheral nerves, Schwann cells form myelin and provide trophic support to axons. We previously showed that the mitochondrial protein prohibitin 2 can localize to the axon-Schwann-cell interface and is required for developmental myelination. Whether the homologous protein prohibitin 1 has a similar role, and whether prohibitins also play important roles in Schwann cell mitochondria is unknown. Here, we show that deletion of prohibitin 1 in Schwann cells minimally perturbs development, but later triggers a severe demyelinating peripheral neuropathy. Moreover, mitochondria are heavily affected by ablation of prohibitin 1 and demyelination occurs preferentially in cells with apparent mitochondrial loss. Furthermore, in response to mitochondrial damage, Schwann cells trigger the integrated stress response, but, contrary to what was previously suggested, this response is not detrimental in this context. These results identify a role for prohibitin 1 in myelin integrity and advance our understanding about the Schwann cell response to mitochondrial damage.


Assuntos
Nervo Femoral/metabolismo , Mitocôndrias/metabolismo , Proteínas Repressoras/genética , Células de Schwann/metabolismo , Nervo Isquiático/metabolismo , Nervo Tibial/metabolismo , Animais , Aspartato-Amônia Ligase/genética , Aspartato-Amônia Ligase/metabolismo , Axônios/metabolismo , Axônios/ultraestrutura , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Feminino , Nervo Femoral/patologia , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/patologia , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/deficiência , Células de Schwann/patologia , Nervo Isquiático/patologia , Estresse Fisiológico , Nervo Tibial/patologia , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismo , gama-Glutamilciclotransferase/genética , gama-Glutamilciclotransferase/metabolismo
3.
Int J Mol Sci ; 22(11)2021 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-34074030

RESUMO

The ability of organisms to quickly sense and transduce signals of environmental stresses is critical for their survival. Ca2+ is a versatile intracellular messenger involved in sensing a wide variety of stresses and regulating the subsequent cellular responses. So far, our understanding for calcium signaling was mostly obtained from ex vivo tissues and cultured cell lines, and the in vivo spatiotemporal dynamics of stress-triggered calcium signaling in a vertebrate remains to be characterized. Here, we describe the generation and characterization of a transgenic zebrafish line with ubiquitous expression of GCaMP6s, a genetically encoded calcium indicator (GECI). We developed a method to investigate the spatiotemporal patterns of Ca2+ events induced by heat stress. Exposure to heat stress elicited immediate and transient calcium signaling in developing zebrafish. Cells extensively distributed in the integument of the head and body trunk were the first batch of responders and different cell populations demonstrated distinct response patterns upon heat stress. Activity of the heat stress-induced calcium signaling peaked at 30 s and swiftly decreased to near the basal level at 120 s after the beginning of exposure. Inhibition of the heat-induced calcium signaling by LaCl3 and capsazepine and treatment with the inhibitors for CaMKII (Ca²2/calmodulin-dependent protein kinase II) and HSF1 (Heat shock factor 1) all significantly depressed the enhanced heat shock response (HSR). Together, we delineated the spatiotemporal dynamics of heat-induced calcium signaling and confirmed functions of the Ca2+-CaMKII-HSF1 pathway in regulating the HSR in zebrafish.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Calmodulina/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Resposta ao Choque Térmico/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/genética , Sinalização do Cálcio/fisiologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Calmodulina/genética , Capsaicina/análogos & derivados , Capsaicina/farmacologia , Proteínas de Fluorescência Verde/genética , Fatores de Transcrição de Choque Térmico/antagonistas & inibidores , Fatores de Transcrição de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico/fisiologia , Hibridização In Situ , Lantânio/farmacologia , Microscopia de Fluorescência , Análise Espaço-Temporal , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/metabolismo
4.
Int J Mol Sci ; 22(11)2021 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-34074034

RESUMO

In certain diseases of the pancreas, pancreatic stellate cells form an important part of fibrosis and are critical for the development of cancer cells. A hypoxic condition develops within the tumor, to which pancreatic stellate cells adapt and are able to proliferate. The consequence is the growth of the tumor. Melatonin, the product of the pineal gland, is gaining attention as an agent with therapeutic potential against pancreatic cancers. Its actions on tumor cells lead, in general, to a reduction in cell viability and proliferation. However, its effects on pancreatic stellate cells subjected to hypoxia are less known. In this study, we evaluated the actions of pharmacological concentrations of melatonin (1 mM-1 µM) on pancreatic stellate cells subjected to hypoxia. The results show that melatonin induced a decrease in cell viability at the highest concentrations tested. Similarly, the incorporation of BrdU into DNA was diminished by melatonin. The expression of cyclins A and D also was decreased in the presence of melatonin. Upon treatment of cells with melatonin, increases in the expression of major markers of ER stress, namely BIP, phospho-eIF2α and ATF-4, were detected. Modulation of apoptosis was noticed as an increase in caspase-3 activation. In addition, changes in the phosphorylated state of p44/42, p38 and JNK MAPKs were detected in cells treated with melatonin. A slight decrease in the content of α-smooth muscle actin was detected in cells treated with melatonin. Finally, treatment of cells with melatonin decreased the expression of matrix metalloproteinases 2, 3, 9 and 13. Our observations suggest that melatonin, at pharmacological concentrations, diminishes the proliferation of pancreatic stellate cells subjected to hypoxia through modulation of cell cycle, apoptosis and the activation of crucial MAPKs. Cellular responses might involve certain ER stress regulator proteins. In view of the results, melatonin could be taken into consideration as a potential therapeutic agent for pancreatic fibrosis.


Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ciclina A/metabolismo , Ciclina D/metabolismo , Melatonina/farmacologia , Células Estreladas do Pâncreas/efeitos dos fármacos , Células Estreladas do Pâncreas/metabolismo , Actinas/metabolismo , Fator 4 Ativador da Transcrição/metabolismo , Animais , Bromodesoxiuridina/metabolismo , Caspase 3/metabolismo , Hipóxia Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fator de Iniciação 2 em Eucariotos/metabolismo , Proteínas de Choque Térmico/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Metaloproteinases da Matriz/metabolismo , Ratos , Ratos Wistar , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
5.
J Biol Chem ; 296: 100759, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33965375

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID-19 global pandemic, utilizes the host receptor angiotensin-converting enzyme 2 (ACE2) for viral entry. However, other host factors might also play important roles in SARS-CoV-2 infection, providing new directions for antiviral treatments. GRP78 is a stress-inducible chaperone important for entry and infectivity for many viruses. Recent molecular docking analyses revealed putative interaction between GRP78 and the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein (SARS-2-S). Here we report that GRP78 can form a complex with SARS-2-S and ACE2 on the surface and at the perinuclear region typical of the endoplasmic reticulum in VeroE6-ACE2 cells and that the substrate-binding domain of GRP78 is critical for this interaction. In vitro binding studies further confirmed that GRP78 can directly bind to the RBD of SARS-2-S and ACE2. To investigate the role of GRP78 in this complex, we knocked down GRP78 in VeroE6-ACE2 cells. Loss of GRP78 markedly reduced cell surface ACE2 expression and led to activation of markers of the unfolded protein response. Treatment of lung epithelial cells with a humanized monoclonal antibody (hMAb159) selected for its safe clinical profile in preclinical models depleted cell surface GRP78 and reduced cell surface ACE2 expression, as well as SARS-2-S-driven viral entry and SARS-CoV-2 infection in vitro. Our data suggest that GRP78 is an important host auxiliary factor for SARS-CoV-2 entry and infection and a potential target to combat this novel pathogen and other viruses that utilize GRP78 in combination therapy.


Assuntos
Enzima de Conversão de Angiotensina 2/genética , Proteínas de Choque Térmico/genética , Interações Hospedeiro-Patógeno/genética , SARS-CoV-2/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/genética , Internalização do Vírus/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Sítios de Ligação , Chlorocebus aethiops , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Regulação da Expressão Gênica , Proteínas de Choque Térmico/antagonistas & inibidores , Proteínas de Choque Térmico/metabolismo , Humanos , Mutação , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Transdução de Sinais , Glicoproteína da Espícula de Coronavírus/metabolismo , Resposta a Proteínas não Dobradas , Células Vero
6.
Biochem Biophys Res Commun ; 562: 89-93, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: covidwho-1240200

RESUMO

New SARS-CoV-2 variants emerged in the United Kingdom and South Africa in December 2020 in concomitant with the Brazillian variant in February 2021 (B.1.1.248 lineage) and currently sparking worldwide during the last few months. The new strain 501.V2 in South Africa bears three mutations in the spike receptor-binding domain (RBD); K417 N, E484K, and N501Y, while the Brazilian B.1.1.248 lineage has 12 mutations. In the current study, we simulate the complex ACE2-SARS-CoV-2 spike RBD system in which the RBD is in the wild-type and mutated isoforms. Additionally, the cell-surface Glucose Regulated Protein 78 (CS-GRP78) associated with the ACE2-SARS-CoV-2 spike RBD complex (ACE2-S RBD) is modeled at the presence of these mutant variants of the viral spike. The results showed that E484K and N501Y are critical in viral spike recognition through either ACE2 or CS-GRP78. The mutated variants (the UK, South African, and Brazilian) of the spike RBD tightly bind to GRP78 more than in the case of the wild-type RBD. These results point to the potent role of GRP78 with ACE2 in the attachment of the new variants, which could be a key for the design of inhibitors to block SARS-CoV-2 attachment and entry to the host cell.


Assuntos
Simulação por Computador , Proteínas de Choque Térmico/metabolismo , SARS-CoV-2/metabolismo , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , Brasil , Proteínas de Choque Térmico/química , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutação , Receptores Virais/química , Receptores Virais/metabolismo , SARS-CoV-2/química , SARS-CoV-2/genética , África do Sul , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Especificidade por Substrato , Reino Unido , Internalização do Vírus
7.
Cell Mol Life Sci ; 78(12): 5179-5195, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33974094

RESUMO

Despite new advances on the functions of ER chaperones at the cell surface, the translocation mechanisms whereby these chaperones can escape from the ER to the cell surface are just emerging. Previously we reported that in many cancer types, upon ER stress, IRE1α binds to and triggers SRC activation resulting in KDEL receptor dispersion from the Golgi and suppression of retrograde transport. In this study, using a combination of molecular, biochemical, and imaging approaches, we discovered that in colon and lung cancer, upon ER stress, ER chaperones, such as GRP78 bypass the Golgi and unconventionally traffic to the cell surface via endosomal transport mediated by Rab GTPases (Rab4, 11 and 15). Such unconventional transport is driven by membrane fusion between ER-derived vesicles and endosomes requiring the v-SNARE BET1 and t-SNARE Syntaxin 13. Furthermore, GRP78 loading into ER-derived vesicles requires the co-chaperone DNAJC3 that is regulated by ER-stress induced PERK-AKT-mTOR signaling.


Assuntos
Membrana Celular/metabolismo , Neoplasias do Colo/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Choque Térmico/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Proteínas de Choque Térmico/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mutagênese Sítio-Dirigida , Mutação , Transporte Proteico , Transdução de Sinais , Células Tumorais Cultivadas
8.
Nat Commun ; 12(1): 3007, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-34021140

RESUMO

Small heat shock proteins (sHsps) bind unfolding proteins, thereby playing a pivotal role in the maintenance of proteostasis in virtually all living organisms. Structural elucidation of sHsp-substrate complexes has been hampered by the transient and heterogeneous nature of their interactions, and the precise mechanisms underlying substrate recognition, promiscuity, and chaperone activity of sHsps remain unclear. Here we show the formation of a stable complex between Arabidopsis thaliana plastid sHsp, Hsp21, and its natural substrate 1-deoxy-D-xylulose 5-phosphate synthase (DXPS) under heat stress, and report cryo-electron microscopy structures of Hsp21, DXPS and Hsp21-DXPS complex at near-atomic resolution. Monomeric Hsp21 binds across the dimer interface of DXPS and engages in multivalent interactions by recognizing highly dynamic structural elements in DXPS. Hsp21 partly unfolds its central α-crystallin domain to facilitate binding of DXPS, which preserves a native-like structure. This mode of interaction suggests a mechanism of sHsps anti-aggregation activity towards a broad range of substrates.


Assuntos
Arabidopsis/metabolismo , Proteínas de Choque Térmico Pequenas/química , Proteínas de Choque Térmico Pequenas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Microscopia Crioeletrônica , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico Pequenas/genética , Resposta ao Choque Térmico , Modelos Moleculares , Dobramento de Proteína , Transferases/química , Transferases/metabolismo
9.
Biochem Biophys Res Commun ; 562: 89-93, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34049205

RESUMO

New SARS-CoV-2 variants emerged in the United Kingdom and South Africa in December 2020 in concomitant with the Brazillian variant in February 2021 (B.1.1.248 lineage) and currently sparking worldwide during the last few months. The new strain 501.V2 in South Africa bears three mutations in the spike receptor-binding domain (RBD); K417 N, E484K, and N501Y, while the Brazilian B.1.1.248 lineage has 12 mutations. In the current study, we simulate the complex ACE2-SARS-CoV-2 spike RBD system in which the RBD is in the wild-type and mutated isoforms. Additionally, the cell-surface Glucose Regulated Protein 78 (CS-GRP78) associated with the ACE2-SARS-CoV-2 spike RBD complex (ACE2-S RBD) is modeled at the presence of these mutant variants of the viral spike. The results showed that E484K and N501Y are critical in viral spike recognition through either ACE2 or CS-GRP78. The mutated variants (the UK, South African, and Brazilian) of the spike RBD tightly bind to GRP78 more than in the case of the wild-type RBD. These results point to the potent role of GRP78 with ACE2 in the attachment of the new variants, which could be a key for the design of inhibitors to block SARS-CoV-2 attachment and entry to the host cell.


Assuntos
Simulação por Computador , Proteínas de Choque Térmico/metabolismo , SARS-CoV-2/metabolismo , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , Brasil , Proteínas de Choque Térmico/química , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutação , Receptores Virais/química , Receptores Virais/metabolismo , SARS-CoV-2/química , SARS-CoV-2/genética , África do Sul , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Especificidade por Substrato , Reino Unido , Internalização do Vírus
10.
Adv Clin Exp Med ; 30(5): 545-554, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-34004082

RESUMO

BACKGROUND: Cervical cancer is the 2nd most frequently diagnosed gynecological cancer. Therefore, it is clinically significant to discover an effective anti-cancer approach. OBJECTIVES: This study aimed to investigate the effects of low-intensity ultrasound irradiation (USI) on cervical cancer cells and associated mechanisms of cell death. MATERIAL AND METHODS: Normal human cervical HaCaT cells and cervical cancer cell lines C33A, Hela and Siha were cultured and γ-rays applied at a dosage of 2.0 Gy/min. The MTT assay was then used to assess viability (proliferation) of HaCaT, C33A, Hela, and Siha cells. Small interfering RNA (siRNA) sequences that silence the glucose-related protein (GRP78) gene were synthesized. Structural changes to cells exposed to USI were observed with scanning electron microscopy. Immunocytochemistry and western blotting were utilized to examine GRP78, C/EBP-homologous protein (CHOP), phosphorylated JNK (p-JNK), and caspase-12 expression in cervical cancer cells. RESULTS: Ultrasound irradiation reduced the viability of cervical cancer cells and increased apoptosis, compared to untreated tumor cells (p < 0.05). This effect was not apparent on HaCaT cells. Ultrasound irradiation also induced formation of apoptotic bodies compared to untreated tumor cells (p < 0.05), and activated endoplasmic reticulum (ER) stress-associated apoptosis compared to untreated tumor cells (p < 0.05), by triggering GRP78, CHOP and caspase-12 expression. Moreover, USI triggered ER stress by upregulating GRP78 expression. Remarkably, USI triggered phosphorylation of JNK compared to untreated tumor cells (p < 0.05). Ultrasound irradiation initiated phosphorylation of JNK by increasing GRP78 expression. Silencing of GRP78 further enhanced the effect of USI on tumor cells. CONCLUSIONS: Ultrasound irradiation significantly inhibited proliferation and induced apoptosis of cervical cancer cells by initiating ER stress associated with apoptosis signaling pathways and triggering phosphorylation of JNK.


Assuntos
Estresse do Retículo Endoplasmático , Neoplasias do Colo do Útero , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Proteínas de Choque Térmico/metabolismo , Humanos , Fosforilação , Neoplasias do Colo do Útero/radioterapia
11.
Arch Insect Biochem Physiol ; 107(3): e21794, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33948968

RESUMO

Bombyx mori as a representative in Lepidoptera is an important economic insect in agriculture production. Bacillus thuringiensis (Bt) is a bacterial pathogen in silkworm production. Understanding how silkworm respond to Bt-toxin can provide guidance to cultivate resistant silkworm strains. Cry1Ac is one type of Bt-toxin. In current research, Dazao, a susceptible B. mori strain to Bt-toxin, was treated by Cry1Ac toxin and compared its transcriptome with untreated samples. This analysis detected 1234 differentially expressed genes (DEGs). Gene Ontology, KEGG, and UniProt keyword enrichment analysis showed that DEGs include ATP-binding cassette (ABC) transporter, stress response, cuticle, and protein synthesis, and folding process. Five ABC genes were upregulated after Cry1Ac treatment including ABCA2, ABCA3, and ABCC4. They are also known as the transporters of Bt-toxin in lepidopteran insect. Expression of cuticle proteins was significantly increased at 6 h after Cry1Ac treatment. Sex-specific storage-proteins and heat shock protein were also upregulated in Cry1Ac treated samples. Our data provide an expression profile about the response of Cry1Ac toxin in susceptible B. mori strain.


Assuntos
Toxinas de Bacillus thuringiensis/farmacologia , Bombyx/efeitos dos fármacos , Endotoxinas/farmacologia , Proteínas Hemolisinas/farmacologia , Transcriptoma/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Bombyx/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Insetos/metabolismo
12.
Oxid Med Cell Longev ; 2021: 6378568, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33815655

RESUMO

Objective: To investigate the impact of different-intensity exercise on lipid metabolism, oxidative stress, hepatocyte injury, and apoptosis and the related protein expression of endoplasmic reticulum stress on nonalcoholic fatty liver disease rats. Method: 50 male Sprague-Dawley rats, 2 months old, were randomly divided into the normal control (CON) group, high-fat diet (HFD) group, low-intensity exercise (LIE) group, moderate-intensity exercise (MIE) group, and incremental-intensity exercise (IIE) group. Blood lipids were tested by the automatic biochemical analyzer. The changes in liver tissues were observed by hematoxylin-eosin staining (HE). The protein expression of Bax and Bcl-2 was detected by the immunohistochemical method. The apoptosis of hepatocytes was detected by the TUNEL method. The protein expression of GRP78, Caspase-3, IRE1, p-IRE1, JNK1, CHOP, PERK, eIF2α, and ATF4 was detected by Western blotting. Results: Our study showed that compared with the HFD group, TG, TC, FFA, and LDL-c were reduced in all exercise groups. The different exercise intensities could reduce the protein expression of ATF4, Bax, and hepatocyte apoptosis. Meanwhile, the antioxidant function and Bcl-2 were increased. However, the moderate-intensity exercise demonstrated more effect on improving the antioxidant capacity and inhibiting hepatocyte apoptosis. Compared with the HFD group, Caspase-3 and JNK were significantly decreased in all exercise groups (P < 0.01) and CHOP was decreased in the LIE and MIE groups (P < 0.05). IRE1, eIF2α, the ratio of p-IRE1/IRE1 (P < 0.01), and ATF4 were decreased (P < 0.05) in the MIE group. Compared with the IIE group, p-IRE1 was decreased (P < 0.05) in the MIE group. GRP78 had no significant difference among the exercise groups. Conclusion: Exercise at different intensities improved blood lipid and hepatic injury in NAFLD rats. However, the body weight of the rats in each exercise group was not significantly different. Moderate-intensity exercise demonstrated more effect on improving the antioxidant ability and inhibiting hepatocyte apoptosis. The possible mechanism depends on the regulation of endoplasmic reticulum stress signaling pathways IRE1/JNK and eIF2α/CHOP.


Assuntos
Apoptose , Estresse do Retículo Endoplasmático , Fator de Iniciação 2 em Eucariotos/metabolismo , Hepatócitos/patologia , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Condicionamento Físico Animal , Proteínas Serina-Treonina Quinases/metabolismo , Fator de Transcrição CHOP/metabolismo , Animais , Peso Corporal , Caspase 3/metabolismo , Dieta Hiperlipídica , Comportamento Alimentar , Proteínas de Choque Térmico/metabolismo , Metabolismo dos Lipídeos , Fígado/patologia , Masculino , Tamanho do Órgão , Estresse Oxidativo , Ratos Sprague-Dawley , Proteína X Associada a bcl-2/metabolismo , eIF-2 Quinase/metabolismo
13.
Int J Mol Sci ; 22(9)2021 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-33925631

RESUMO

In Drosophila, endoplasmic reticulum (ER) stress activates the protein kinase R-like endoplasmic reticulum kinase (dPerk). dPerk can also be activated by defective mitochondria in fly models of Parkinson's disease caused by mutations in pink1 or parkin. The Perk branch of the unfolded protein response (UPR) has emerged as a major toxic process in neurodegenerative disorders causing a chronic reduction in vital proteins and neuronal death. In this study, we combined microarray analysis and quantitative proteomics analysis in adult flies overexpressing dPerk to investigate the relationship between the transcriptional and translational response to dPerk activation. We identified tribbles and Heat shock protein 22 as two novel Drosophila activating transcription factor 4 (dAtf4) regulated transcripts. Using a combined bioinformatics tool kit, we demonstrated that the activation of dPerk leads to translational repression of mitochondrial proteins associated with glutathione and nucleotide metabolism, calcium signalling and iron-sulphur cluster biosynthesis. Further efforts to enhance these translationally repressed dPerk targets might offer protection against Perk toxicity.


Assuntos
eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo , Fator 4 Ativador da Transcrição/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Biologia Computacional/métodos , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Retículo Endoplasmático/patologia , Estresse do Retículo Endoplasmático , Fator de Iniciação 2 em Eucariotos/metabolismo , Proteínas de Choque Térmico/metabolismo , Mitocôndrias/metabolismo , Doenças Neurodegenerativas/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Proteômica/métodos , Transdução de Sinais , Fatores de Transcrição/metabolismo , Transcriptoma , Ubiquitina-Proteína Ligases/metabolismo , Resposta a Proteínas não Dobradas/genética , Resposta a Proteínas não Dobradas/fisiologia
14.
Arch Insect Biochem Physiol ; 107(2): e21791, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33860954

RESUMO

In the last decade, unexpected high temperatures have been frequent in spring and early summer. Numerous studies have shown that such thermal stress has substantial effects on life-history traits that influence fitness of insects, but few have examined expression dynamics of heat shock proteins (Hsps) across developmental stages, especially as regards potential carry-over effects at the transcriptional level across metamorphosis. We exposed pupae of the oriental fruit moth ("OFM," Grapholita molesta Busck) to mild heat stress (38°C, 6 h) and then quantified expression patterns of six Hsps (Hsp90, 70, 60, 40, 21, and 11) from pupal through adult stages. Almost all Hsps showed a higher expression immediately after pupae were heat-stressed, but later dropped to normal levels after metamorphosis. Although upregulation of Hsps is transient and the effects carry over longer to early adult stage, upregulation will nonetheless have positive effects on adult fitness. The fitness of some insects may benefit from higher expression of chaperon genes after mild stress, in the form of higher fecundity and longer lifespan, as a carry-over effect. These results suggest that mild thermal stress can change genetic expression that later boosts adult fitness through a cascade effect.


Assuntos
Proteínas de Choque Térmico/metabolismo , Metamorfose Biológica/fisiologia , Mariposas/metabolismo , Animais , Fertilidade/fisiologia , Regulação da Expressão Gênica/fisiologia , Resposta ao Choque Térmico/fisiologia , Proteínas de Insetos/metabolismo , Longevidade/fisiologia , Pupa/metabolismo
15.
Int J Mol Sci ; 22(8)2021 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-33916919

RESUMO

Reactive intermediate deaminase (Rid) proteins are enzymes conserved in all domains of life. UK114, a mammalian member of RidA subfamily, has been firstly identified as a component of liver perchloric acid-soluble proteins (L-PSP). Although still poorly defined, several functions have been attributed to the mammalian protein UK114/RIDA, including the reactive intermediate deamination activity. The expression of UK114/RIDA has been observed in some tumors, arousing interest in this protein as an evaluable tumor marker. However, other studies reported a negative correlation between UK114/RIDA expression, tumor differentiation degree and cell proliferation. This work addressed the question of UK114/RIDA expression in human non-tumor HEK293 cell lines and in some human tumor cell lines. Here we reported that human RIDA (hRIDA) was expressed in all the analyzed cell line and subjected to lysine (K-)succinylation. In HEK293, hRIDA K-succinylation was negatively correlated to the cell proliferation rate and was under the control of SIRT5. Moreover, K-succinylation clearly altered hRIDA quantification by immunoblotting, explaining, at least in part, some discrepancies about RIDA expression reported in previous studies. We found that hRIDA was able to deaminate reactive enamine-imine intermediates and that K-succinylation drastically reduced deaminase activity. As predicted by in silico analysis, the observed reduction of deaminase activity has been related to the drastic alterations of hRIDA structure inferred by K-succinylation. The role of hRIDA and the importance of its K-succinylation in cell metabolism, especially in cancer biology, have been discussed.


Assuntos
Proliferação de Células/fisiologia , Proteínas de Choque Térmico/metabolismo , Ribonucleases/metabolismo , Linhagem Celular , Ativação Enzimática , Expressão Gênica , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Humanos , Lisina/metabolismo , Modelos Moleculares , Conformação Proteica , Processamento de Proteína Pós-Traducional , Ribonucleases/química , Ribonucleases/genética , Sirtuínas/metabolismo , Relação Estrutura-Atividade
16.
Nat Commun ; 12(1): 2426, 2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33893288

RESUMO

To adapt to fluctuating protein folding loads in the endoplasmic reticulum (ER), the Hsp70 chaperone BiP is reversibly modified with adenosine monophosphate (AMP) by the ER-resident Fic-enzyme FICD/HYPE. The structural basis for BiP binding and AMPylation by FICD has remained elusive due to the transient nature of the enzyme-substrate-complex. Here, we use thiol-reactive derivatives of the cosubstrate adenosine triphosphate (ATP) to covalently stabilize the transient FICD:BiP complex and determine its crystal structure. The complex reveals that the TPR-motifs of FICD bind specifically to the conserved hydrophobic linker of BiP and thus mediate specificity for the domain-docked conformation of BiP. Furthermore, we show that both AMPylation and deAMPylation of BiP are not directly regulated by the presence of unfolded proteins. Together, combining chemical biology, crystallography and biochemistry, our study provides structural insights into a key regulatory mechanism that safeguards ER homeostasis.


Assuntos
Proteínas de Choque Térmico/metabolismo , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/metabolismo , Processamento de Proteína Pós-Traducional , Repetições de Tetratricopeptídeos , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Retículo Endoplasmático/metabolismo , Células HEK293 , Proteínas de Choque Térmico/química , Homeostase , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Simulação de Dinâmica Molecular , Nucleotidiltransferases/química , Nucleotidiltransferases/genética , Ligação Proteica , Conformação Proteica , Especificidade por Substrato
17.
BMC Plant Biol ; 21(1): 185, 2021 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-33865315

RESUMO

BACKGROUND: Heat shock factors (Hsfs) and Heat shock proteins (Hsps) belong to an essential group of molecular regulators involved in controlling cellular processes under normal and stress conditions. The role of Hsfs and Hsps is well known in model plant species under diverse stress conditions. While plants Hsfs are vital components of the signal transduction response to maintain cellular homeostasis, Hsps function as chaperones helping to maintain folding of damaged and newly formed proteins during stress conditions. In lettuce (Lactuca sativa), a highly consumed vegetable crop grown in the field and in hydroponic systems, the role of these gene families in response to artificial light is not well characterized. RESULTS: Using a genome-wide analysis approach, we identified 32 Hsfs and 22 small heat shock proteins (LsHsps) in lettuce, some of which do not have orthologs in Arabidopsis, poplar, and rice. LsHsp60s, LsHsp90s, and LsHsp100s are highly conserved among dicot and monocot species. Surprisingly, LsHsp70s have three times more members than Arabidopsis and two times more than rice. Interestingly, the lettuce genome triplication did not contribute to the increased number of LsHsp70s genes. The large number of LsHsp70s was the result of genome tandem duplication. Chromosomal distribution analysis shows larger tandem repeats of LsHsp70s genes in Chr1, Chr7, Chr8, and Chr9. At the transcriptional level, some genes of the LsHsfs, LsHsps, LsHsp60s, and LsHsp70s families were highly responsive to UV and high intensity light stress, in contrast to LsHsp90s and LsHsp100s which did not respond to a light stimulus. CONCLUSIONS: Our genome-wide analysis provides a detailed identification of Hsfs and Hsps in lettuce. Chromosomal location and syntenic region analysis together with our transcriptional analysis under different light conditions provide candidate genes for breeding programs aiming to produce lettuce varieties able to grow healthy under hydroponic systems that use artificial light.


Assuntos
Estudo de Associação Genômica Ampla , Fatores de Transcrição de Choque Térmico/genética , Proteínas de Choque Térmico/genética , Alface/genética , Proteínas de Plantas/genética , Raios Ultravioleta , Fatores de Transcrição de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Alface/metabolismo , Alface/efeitos da radiação , Proteínas de Plantas/metabolismo
18.
Int J Mol Sci ; 22(6)2021 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-33803511

RESUMO

Rhizoctonia solani is the causer of black scurf disease on potatoes and is responsible for high economical losses in global agriculture. In order to increase the limited knowledge of the plants' molecular response to this pathogen, we inoculated potatoes with R. solani AG3-PT isolate Ben3 and carried out RNA sequencing with total RNA extracted from potato sprouts at three and eight days post inoculation (dpi). In this dual RNA-sequencing experiment, the necrotrophic lifestyle of R. solani AG3-PT during early phases of interaction with its host has already been characterised. Here the potato plants' comprehensive transcriptional response to inoculation with R. solani AG3 was evaluated for the first time based on significantly different expressed plant genes extracted with DESeq analysis. Overall, 1640 genes were differentially expressed, comparing control (-Rs) and with R. solani AG3-PT isolate Ben3 inoculated plants (+Rs). Genes involved in the production of anti-fungal proteins and secondary metabolites with antifungal properties were significantly up regulated upon inoculation with R. solani. Gene ontology (GO) terms involved in the regulation of hormone levels (i.e., ethylene (ET) and jasmonic acid (JA) at 3 dpi and salicylic acid (SA) and JA response pathways at 8 dpi) were significantly enriched. Contrastingly, the GO term "response to abiotic stimulus" was down regulated at both time points analysed. These results may support future breeding efforts toward the development of cultivars with higher resistance level to black scurf disease or the development of new control strategies.


Assuntos
Interações Hospedeiro-Patógeno/genética , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Rhizoctonia/fisiologia , Solanum tuberosum/genética , Solanum tuberosum/microbiologia , Transcrição Genética , Regulação para Baixo/genética , Regulação da Expressão Gênica de Plantas , Ontologia Genética , Genes de Plantas , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Doenças das Plantas/genética , Análise de Componente Principal , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Solanum tuberosum/imunologia , Transcriptoma/genética , Regulação para Cima/genética
19.
Int J Mol Sci ; 22(6)2021 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-33799579

RESUMO

Diabetic kidney disease (DKD) is the leading cause of kidney failure. RhoA/Rho-associated protein kinase (ROCK) signaling is a recognized mediator of its pathogenesis, largely through mediating the profibrotic response. While RhoA activation is not feasible due to the central role it plays in normal physiology, ROCK inhibition has been found to be effective in attenuating DKD in preclinical models. However, this has not been evaluated in clinical studies as of yet. Alternate means of inhibiting RhoA/ROCK signaling involve the identification of disease-specific activators. This report presents evidence showing the activation of RhoA/ROCK signaling both in vitro in glomerular mesangial cells and in vivo in diabetic kidneys by two recently described novel pathogenic mediators of fibrosis in DKD, activins and cell-surface GRP78. Neither are present in normal kidneys. Activin inhibition with follistatin and neutralization of cell-surface GRP78 using a specific antibody blocked RhoA activation in mesangial cells and in diabetic kidneys. These data identify two novel RhoA/ROCK activators in diabetic kidneys that can be evaluated for their efficacy in inhibiting the progression of DKD.


Assuntos
Ativinas/genética , Diabetes Mellitus Experimental/genética , Proteínas de Choque Térmico/genética , Células Mesangiais/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Ativinas/antagonistas & inibidores , Ativinas/metabolismo , Animais , Anticorpos Neutralizantes/farmacologia , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Modelos Animais de Doenças , Folistatina/farmacologia , Regulação da Expressão Gênica , Proteínas de Choque Térmico/antagonistas & inibidores , Proteínas de Choque Térmico/metabolismo , Humanos , Masculino , Células Mesangiais/efeitos dos fármacos , Células Mesangiais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Nefrectomia/métodos , Cultura Primária de Células , Transdução de Sinais , Estreptozocina/administração & dosagem , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
20.
Int J Mol Sci ; 22(4)2021 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-33672387

RESUMO

Parasitic organisms especially those of the Apicomplexan phylum, harbour a cytosol localised canonical Hsp70 chaperone. One of the defining features of this protein is the presence of GGMP repeat residues sandwiched between α-helical lid and C-terminal EEVD motif. The role of the GGMP repeats of Hsp70s remains unknown. In the current study, we introduced GGMP mutations in the cytosol localised Hsp70-1 of Plasmodium falciparum (PfHsp70-1) and a chimeric protein (KPf), constituted by the ATPase domain of E. coli DnaK fused to the C-terminal substrate binding domain of PfHsp70-1. A complementation assay conducted using E. coli dnaK756 cells demonstrated that the GGMP motif was essential for chaperone function of the chimeric protein, KPf. Interestingly, insertion of GGMP motif of PfHsp70-1 into DnaK led to a lethal phenotype in E. coli dnaK756 cells exposed to elevated growth temperature. Using biochemical and biophysical assays, we established that the GGMP motif accounts for the elevated basal ATPase activity of PfHsp70-1. Furthermore, we demonstrated that this motif is important for interaction of the chaperone with peptide substrate and a co-chaperone, PfHop. Our findings suggest that the GGMP may account for both the specialised chaperone function and reportedly high catalytic efficiency of PfHsp70-1.


Assuntos
Proteínas de Choque Térmico HSP72/genética , Proteínas de Choque Térmico HSP72/metabolismo , Mutação , Plasmodium falciparum , Proteínas de Protozoários/genética , Dicroísmo Circular , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Teste de Complementação Genética , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP72/química , Proteínas de Choque Térmico/metabolismo , Modelos Moleculares , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Plasmodium falciparum/metabolismo , Estabilidade Proteica , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sequências Repetitivas de Aminoácidos , Espectrometria de Fluorescência
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