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1.
Medicine (Baltimore) ; 99(40): e22544, 2020 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-33019464

RESUMO

BACKGROUND: Clinical studies have shown that celecoxib can significantly inhibit the development of tumors, and basic experiments and in vitro experiments also provide a certain basis, but it is not clear how celecoxib inhibits tumor development in detail. METHODS: A literature search of all major academic databases was conducted (PubMed, China National Knowledge Internet (CNKI), Wan-fang, China Science and Technology Journal Database (VIP), including the main research on the mechanisms of celecoxib on tumors. RESULTS: Celecoxib can intervene in tumor development and reduce the formation of drug resistance through multiple molecular mechanisms. CONCLUSION: Celecoxib mainly regulates the proliferation, migration, and invasion of tumor cells by inhibiting the cyclooxygenases-2/prostaglandin E2 signal axis and thereby inhibiting the phosphorylation of nuclear factor-κ-gene binding, Akt, signal transducer and activator of transcription and the expression of matrix metalloproteinase 2 and matrix metalloproteinase 9. Meanwhile, it was found that celecoxib could promote the apoptosis of tumor cells by enhancing mitochondrial oxidation, activating mitochondrial apoptosis process, promoting endoplasmic reticulum stress process, and autophagy. Celecoxib can also reduce the occurrence of drug resistance by increasing the sensitivity of cancer cells to chemotherapy drugs.


Assuntos
Celecoxib/farmacologia , Inibidores de Ciclo-Oxigenase 2/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Celecoxib/efeitos adversos , Celecoxib/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase 2/efeitos adversos , Inibidores de Ciclo-Oxigenase 2/uso terapêutico , Dinoprostona/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/efeitos dos fármacos , Metaloproteinase 9 da Matriz/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos
2.
Mol Cell ; 79(6): 874-875, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32946760

RESUMO

PARP enzymes are increasingly taking on important roles beyond DNA repair. Huang et al. (2020b) report how the NAD+-dependent ADP-ribosylation of histone H2B by PARP-1 in complex with a metabolic enzyme suppresses the phosphorylation of an adjacent residue, impacting adipogenesis.


Assuntos
Histonas , Inibidores de Poli(ADP-Ribose) Polimerases , ADP-Ribosilação , Adipogenia , Epigênese Genética , Humanos , Obesidade , Fosforilação , Poli(ADP-Ribose) Polimerases
3.
Anticancer Res ; 40(9): 4913-4919, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32878779

RESUMO

BACKGROUND/AIM: A new class of imidazo[2,1-b][1,3,4]thiadiazole compounds have recently been evaluated as inhibitors of phosphorylation of focal adhesion kinase (FAK) in pancreatic cancer. FAK is overexpressed in mesothelioma and has recently emerged as an interesting target for the treatment of this disease. MATERIALS AND METHODS: Ten imidazo[2,1-b][1,3,4]thiadiazole compounds characterized by indole bicycle and a thiophene ring, were evaluated for their cytotoxic activity in two primary cell cultures of peritoneal mesothelioma, MesoII and STO cells. RESULTS: Compounds 1a and 1b showed promising antitumor activity with IC50 values in the range of 0.59 to 2.81 µM in both cell lines growing as monolayers or as spheroids. Their antiproliferative and antimigratory activity was associated with inhibition of phospho-FAK, as detected by a specific ELISA assay in STO cells. Interestingly, these compounds potentiated the antiproliferative activity of gemcitabine, and these results might be explained by the increase in the mRNA expression of the key gemcitabine transporter human equilibrative nucleoside transporter-1 (hENT-1). CONCLUSION: These promising results support further studies on new imidazo[2,1-b][1,3,4]thiadiazole compounds as well as on the role of both FAK and hENT-1 modulation in order to develop new drug combinations for peritoneal mesothelioma.


Assuntos
Antineoplásicos/farmacologia , Desoxicitidina/análogos & derivados , Transportador Equilibrativo 1 de Nucleosídeo/genética , Quinase 1 de Adesão Focal/metabolismo , Imidazóis/farmacologia , Tiadiazóis/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Desoxicitidina/farmacologia , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imidazóis/síntese química , Imidazóis/química , Mesotelioma/tratamento farmacológico , Mesotelioma/patologia , Estrutura Molecular , Neoplasias Peritoneais/tratamento farmacológico , Neoplasias Peritoneais/patologia , Fosforilação/efeitos dos fármacos , Tiadiazóis/síntese química , Tiadiazóis/química , Células Tumorais Cultivadas
4.
Nat Commun ; 11(1): 4607, 2020 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-32929081

RESUMO

Drug tolerance is the basis for acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) including osimertinib, through mechanisms that still remain unclear. Here, we show that while AXL-low expressing EGFR mutated lung cancer (EGFRmut-LC) cells are more sensitive to osimertinib than AXL-high expressing EGFRmut-LC cells, a small population emerge osimertinib tolerance. The tolerance is mediated by the increased expression and phosphorylation of insulin-like growth factor-1 receptor (IGF-1R), caused by the induction of its transcription factor FOXA1. IGF-1R maintains association with EGFR and adaptor proteins, including Gab1 and IRS1, in the presence of osimertinib and restores the survival signal. In AXL-low-expressing EGFRmut-LC cell-derived xenograft and patient-derived xenograft models, transient IGF-1R inhibition combined with continuous osimertinib treatment could eradicate tumors and prevent regrowth even after the cessation of osimertinib. These results indicate that optimal inhibition of tolerant signals combined with osimertinib may dramatically improve the outcome of EGFRmut-LC.


Assuntos
Acrilamidas/uso terapêutico , Compostos de Anilina/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Mutação/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptor IGF Tipo 1/antagonistas & inibidores , Acrilamidas/farmacologia , Idoso de 80 Anos ou mais , Compostos de Anilina/farmacologia , Animais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Receptores ErbB/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Humanos , Imidazóis/farmacologia , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Pirazinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Regulação para Cima/efeitos dos fármacos
5.
Nat Commun ; 11(1): 4586, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32934222

RESUMO

Frequent mutation of the tumour suppressor RNF43 is observed in many cancers, particularly colon malignancies. RNF43, an E3 ubiquitin ligase, negatively regulates Wnt signalling by inducing degradation of the Wnt receptor Frizzled. In this study, we discover that RNF43 activity requires phosphorylation at a triplet of conserved serines. This phospho-regulation of RNF43 is required for zebrafish development and growth of mouse intestinal organoids. Cancer-associated mutations that abrogate RNF43 phosphorylation cooperate with active Ras to promote tumorigenesis by abolishing the inhibitory function of RNF43 in Wnt signalling while maintaining its inhibitory function in p53 signalling. Our data suggest that RNF43 mutations cooperate with KRAS mutations to promote multi-step tumorigenesis via the Wnt-Ras-p53 axis in human colon cancers. Lastly, phosphomimetic substitutions of the serine trio restored the tumour suppressive activity of extracellular oncogenic mutants. Therefore, harnessing phospho-regulation of RNF43 might be a potential therapeutic strategy for tumours with RNF43 mutations.


Assuntos
Carcinogênese/metabolismo , Receptores Wnt/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Carcinogênese/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteína Oncogênica p21(ras)/genética , Proteína Oncogênica p21(ras)/metabolismo , Fosforilação , Proteólise , Receptores Wnt/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/genética , Via de Sinalização Wnt
6.
Nat Commun ; 11(1): 4486, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32900989

RESUMO

Centromeres are epigenetically determined nuclear domains strictly required for chromosome segregation and genome stability. However, the mechanisms regulating centromere and kinetochore chromatin modifications are not known. Here, we demonstrate that LSH is enriched at meiotic kinetochores and its targeted deletion induces centromere instability and abnormal chromosome segregation. Superresolution chromatin analysis resolves LSH at the inner centromere and kinetochores during oocyte meiosis. LSH knockout pachytene oocytes exhibit reduced HDAC2 and DNMT-1. Notably, mutant oocytes show a striking increase in histone H3 phosphorylation at threonine 3 (H3T3ph) and accumulation of major satellite transcripts in both prophase-I and metaphase-I chromosomes. Moreover, knockout oocytes exhibit centromere fusions, ectopic kinetochore formation and abnormal exchange of chromatin fibers between paired bivalents and asynapsed chromosomes. Our results indicate that loss of LSH affects the levels and chromosomal localization of H3T3ph and provide evidence that, by maintaining transcriptionally repressive heterochromatin, LSH may be essential to prevent deleterious meiotic recombination events at repetitive centromeric sequences.


Assuntos
DNA Helicases/metabolismo , Meiose/fisiologia , Oócitos/citologia , Oócitos/metabolismo , Animais , Centrômero/genética , Centrômero/metabolismo , DNA Helicases/deficiência , DNA Helicases/genética , Feminino , Histonas/metabolismo , Cinetocoros/metabolismo , Masculino , Meiose/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Transcrição Genética
7.
Nat Commun ; 11(1): 4634, 2020 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-32929078

RESUMO

The current opioid epidemic necessitates a better understanding of human addiction neurobiology to develop efficacious treatment approaches. Here, we perform genome-wide assessment of chromatin accessibility of the human striatum in heroin users and matched controls. Our study reveals distinct neuronal and non-neuronal epigenetic signatures, and identifies a locus in the proximity of the gene encoding tyrosine kinase FYN as the most affected region in neurons. FYN expression, kinase activity and the phosphorylation of its target Tau are increased by heroin use in the post-mortem human striatum, as well as in rats trained to self-administer heroin and primary striatal neurons treated with chronic morphine in vitro. Pharmacological or genetic manipulation of FYN activity significantly attenuates heroin self-administration and responding for drug-paired cues in rodents. Our findings suggest that striatal FYN is an important driver of heroin-related neurodegenerative-like pathology and drug-taking behavior, making FYN a promising therapeutic target for heroin use disorder.


Assuntos
Cromatina/metabolismo , Corpo Estriado/enzimologia , Dependência de Heroína/enzimologia , Terapia de Alvo Molecular , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Animais , Sequência de Bases , Comportamento Animal/efeitos dos fármacos , Sinais (Psicologia) , Genoma , Células HEK293 , Heroína/efeitos adversos , Humanos , Masculino , Neurônios/metabolismo , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-fyn/antagonistas & inibidores , Ratos Long-Evans , Autoadministração , Transcrição Genética/efeitos dos fármacos , Proteínas tau/metabolismo
8.
PLoS Pathog ; 16(8): e1008775, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866218

RESUMO

Small RNA viruses only have a very limited coding capacity, thus most viral proteins have evolved to fulfill multiple functions. The highly conserved matrix protein 1 (M1) of influenza A viruses is a prime example for such a multifunctional protein, as it acts as a master regulator of virus replication whose different functions have to be tightly regulated. The underlying mechanisms, however, are still incompletely understood. Increasing evidence points towards an involvement of posttranslational modifications in the spatio-temporal regulation of M1 functions. Here, we analyzed the role of M1 tyrosine phosphorylation in genuine infection by using recombinant viruses expressing M1 phosphomutants. Presence of M1 Y132A led to significantly decreased viral replication compared to wildtype and M1 Y10F. Characterization of phosphorylation dynamics by mass spectrometry revealed the presence of Y132 phosphorylation in M1 incorporated into virions that is most likely mediated by membrane-associated Janus kinases late upon infection. Molecular dynamics simulations unraveled a potential phosphorylation-induced exposure of the positively charged linker domain between helices 4 and 5, supposably acting as interaction platform during viral assembly. Consistently, M1 Y132A showed a defect in lipid raft localization due to reduced interaction with viral HA protein resulting in a diminished structural stability of viral progeny and the formation of filamentous particles. Importantly, reduced M1-RNA binding affinity resulted in an inefficient viral genome incorporation and the production of non-infectious virions that interferes with virus pathogenicity in mice. This study advances our understanding of the importance of dynamic phosphorylation as a so far underestimated level of regulation of multifunctional viral proteins and emphasizes the potential feasibility of targeting posttranslational modifications of M1 as a novel antiviral intervention.


Assuntos
Vírus da Influenza A/metabolismo , Mutação de Sentido Incorreto , Proteínas da Matriz Viral/metabolismo , Células A549 , Substituição de Aminoácidos , Animais , Cães , Feminino , Células HEK293 , Humanos , Vírus da Influenza A/genética , Células Madin Darby de Rim Canino , Masculino , Camundongos , Camundongos Transgênicos , Fosforilação , Proteínas da Matriz Viral/genética
9.
Wei Sheng Yan Jiu ; 49(4): 534-539, 2020 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-32928344

RESUMO

OBJECTIVE: To explore the role of calcium/calmodulin-dependent protein kinase Ⅱ(CaMK-Ⅱ) and protein phosphatase-2 A(PP2 A) on hyperphosphorylation of tau induced by AlCl_3 in SH-SY5 Y cell. METHODS: SH-SY5 Y cells were assigned to the control group, AlCl_3 low, middle, high exposed groups(200, 400800 µmol/L Al~(3+)), KN93 intervention group(800 µmol/L Al~(3+)+0. 5 µmol/L KN93) and sphingosine intervention group(800 µmol/L Al~(3+)+5 nmol/L sphingosine). After 48 h of exposure and intervention, the viabilities of cells were measured by CCK-8 assay, the contents of CaMK-Ⅱ and PP2 A were determined by ELISA, and the expression of tau5 and phosphorylation of Thr-181, Thr-231, Ser-262 and Ser-396 were detected by Western-Blot. RESULTS: The viabilities of cells in AlCl_3 middle and high exposed groups were significantly lower than that of the control group(P<0. 05). Compared with the AlCl_3 high exposed group, the viabilities of cells in KN93 intervention group and sphingosine intervention group were significantly increased(P<0. 05), as well as the difference of the cell viability between sphingosine intervention group and the control group was not statistically significant. Compared with the control group, the contents of CaMK-Ⅱin AlCl_3 low, middle, high exposed groups were significantly increased(P<0. 05), while the contents of PP2 A in those groups were significantly decreased(P<0. 05). Compared with AlCl_3 high exposed group, the contents of CaMK-Ⅱ in KN93 intervention group and sphingosine intervention group were significantly decreased(P<0. 05), and PP2 A in those groups were significantly increased(P<0. 05). The expression of tau5 and phosphorylation of Thr-181, Thr-231, Ser-396 in AlCl_3 low, middle, high exposed group showed significantly higher than those of the control group(P<0. 05), while the phosphorylation of Ser-262 in AlCl_3 high exposed group showed significantly higher than that of the control group(P<0. 05). After intervention with KN93 and sphingosine, the expression of tau5, and phosphorylation of Thr-181, Thr-231, Ser-262, Ser-396 in KN93 intervention group and sphingosine intervention group were significantly lower than those of AlCl_3 high exposed group( P<0. 05), as well as compared with the control group, the phosphorylation of Ser-262 in KN93 intervention group and Thr-181, Thr-231 and Ser-396 in sphingosine intervention group were not statistical difference. CONCLUSION: AlCl_3 could increase the phosphorylation of Thr-181, Thr-231, Ser-262 and Ser396 in SH-SY5 Y cells, which mechanism may relate to the changes of CaMK-Ⅱand PP2 A. CaMK-Ⅱ mainly regulates the phosphorylation of Ser-262 induced by AlCl_3, while PP2 A mainly regulates the phosphorylation of Thr-181, Thr-231 and Ser-396 induced by AlCl_3. It is suggested that hyperphosphorylated tau protein induced by AlCl_3 is closely related to PP2 A.


Assuntos
Proteínas tau , Linhagem Celular Tumoral , Sobrevivência Celular , Fosforilação
10.
Nat Commun ; 11(1): 4859, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32978401

RESUMO

Cell death is intrinsically linked with immunity. Disruption of an immune-activated MAPK cascade, consisting of MEKK1, MKK1/2, and MPK4, triggers cell death and autoimmunity through the nucleotide-binding leucine-rich repeat (NLR) protein SUMM2 and the MAPK kinase kinase MEKK2. In this study, we identify a Catharanthus roseus receptor-like kinase 1-like (CrRLK1L), named LETUM2/MEDOS1 (LET2/MDS1), and the glycosylphosphatidylinositol (GPI)-anchored protein LLG1 as regulators of mekk1-mkk1/2-mpk4 cell death. LET2/MDS1 functions additively with LET1, another CrRLK1L, and acts genetically downstream of MEKK2 in regulating SUMM2 activation. LET2/MDS1 complexes with LET1 and promotes LET1 phosphorylation, revealing an intertwined regulation between different CrRLK1Ls. LLG1 interacts with the ectodomain of LET1/2 and mediates LET1/2 transport to the plasma membrane, corroborating its function as a co-receptor of LET1/2 in the mekk1-mkk1/2-mpk4 cell death pathway. Thus, our data suggest that a trimeric complex consisting of two CrRLK1Ls LET1, LET2/MDS1, and a GPI-anchored protein LLG1 that regulates the activation of NLR SUMM2 for initiating cell death and autoimmunity.


Assuntos
Autoimunidade/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas Ligadas por GPI/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Autoimunidade/fisiologia , Proteínas de Transporte/imunologia , Catharanthus/genética , Catharanthus/metabolismo , Morte Celular/genética , Proteínas Ligadas por GPI/genética , Regulação da Expressão Gênica de Plantas , Glicosilfosfatidilinositóis , MAP Quinase Quinase Quinases/genética , Sistema de Sinalização das MAP Quinases , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fosforilação , Proteínas de Plantas/imunologia , Plantas Geneticamente Modificadas , Interferência de RNA , Transcriptoma
11.
Life Sci ; 259: 118383, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32896555

RESUMO

AIMS: Previous studies have shown that the widespread use of estrogen preparations can cause adverse outcomes such as thrombosis and cardiovascular disease. Autophagy is a biochemical process necessary to maintain cell homeostasis. The present study investigated whether E-2 mediates autophagy-induced endothelial cell dysfunction. The role of aspirin in this process was then studied. MAIN METHODS: Western blot, fluorescence microscopy, electron transmission microscopy, plasma construction and transfection, vasoreactivity study in wire myograph are all used in this study. KEY FINDINGS: We found that E-2 activated the PI3K/mTOR signaling pathway and inhibited the formation of the Atg14L-Beclin1-Vps34-Vps15 complex, thereby inhibiting autophagy. Aspirin promoted Beclin1 phosphorylation in autophagy initiation complexes and enhanced autophagy. Furthermore, E-2 treatment of HAECs resulted in endothelial dysfunction by inhibiting autophagy and leading to accumulation of α-smooth muscle actin (α-SMA). E-2 inhibited the activation of eNOS and reduced the expression of eNOS protein. In the mouse aortic vascular function test, E-2 disrupted endothelium-dependent vasodilation. An α-SMA-shRNA lentivirus eliminated the disruption to endothelium-dependent vasodilation by E-2. Aspirin inhibited α-SMA accumulation by enhancing autophagy, reversed endothelial functional impairment caused by E-2, and promoted endothelium-dependent vasodilation. SIGNIFICANCE: This study provides new evidence that E-2 inhibits autophagy and induces abnormal accumulation of α-SMA, resulting in endothelial cell dysfunction and affecting vasodilation. Aspirin can effectively restore the endothelial cell function disrupted E-2.


Assuntos
Actinas/metabolismo , Aspirina/farmacologia , Autofagia/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Estradiol/metabolismo , Proteína VPS15 de Distribuição Vacuolar/metabolismo , Animais , Western Blotting , Células Cultivadas , Endotélio Vascular/ultraestrutura , Feminino , Humanos , Imunoprecipitação , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Fosforilação/efeitos dos fármacos
12.
Mol Genet Genomics ; 295(6): 1489-1500, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32948893

RESUMO

Glucose, fructose and mannose are the preferred carbon/energy sources for the yeast Saccharomyces cerevisiae. Absence of preferred energy sources activates glucose derepression, which is regulated by the kinase Snf1. Snf1 phosphorylates the transcriptional repressor Mig1, which results in its exit from the nucleus and subsequent derepression of genes. In contrast, Snf1 is inactive when preferred carbon sources are available, which leads to dephosphorylation of Mig1 and its translocation to the nucleus where Mig1 acts as a transcription repressor. Here we revisit the role of the three hexose kinases, Hxk1, Hxk2 and Glk1, in glucose de/repression. We demonstrate that all three sugar kinases initially affect Mig1 nuclear localization upon addition of glucose, fructose and mannose. This initial import of Mig1 into the nucleus was temporary; for continuous nucleocytoplasmic shuttling of Mig1, Hxk2 is required in the presence of glucose and mannose and in the presence of fructose Hxk2 or Hxk1 is required. Our data suggest that Mig1 import following exposure to preferred energy sources is controlled via two different pathways, where (1) the initial import is regulated by signals derived from metabolism and (2) continuous shuttling is regulated by the Hxk2 and Hxk1 proteins. Mig1 nucleocytoplasmic shuttling appears to be important for the maintenance of the repressed state in which Hxk1/2 seems to play an essential role.


Assuntos
Núcleo Celular/metabolismo , Frutose/metabolismo , Glucose/metabolismo , Hexoquinase/metabolismo , Manose/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Regulação Fúngica da Expressão Gênica , Hexoquinase/genética , Fosforilação , Transporte Proteico , Proteínas Repressoras/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética
13.
Anticancer Res ; 40(10): 5545-5556, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32988878

RESUMO

BACKGROUND/AIM: The p38 family of mitogen-activated protein kinases (MAPK) includes four isoforms: p38α, -ß, -γ and -δ. The aim of this study was to elucidate possible functions of p38α and p38ß in human pancreatic cancer. MATERIALS AND METHODS: Isoform expression was determined in seven human pancreatic cancer cell lines. After shRNA based selective knockdown of p38α and p38ß, in vitro growth and migration as well as in vivo tumorigenicity were assessed. RESULTS: All pancreatic cancer cells expressed p38 isoforms. Knockdown of p38α and p38ß inhibited in vitro growth. Migration was markedly reduced in p38α shRNA expressing clones, but not altered by p38ß knockdown. While in vivo inhibition of p38ß decreased tumor formation and growth, the knockdown of p38α significantly enhanced tumorigenicity. CONCLUSION: p38 MAPKs may exert isoform specific functions in pancreatic cancer. Selective targeting may contribute to individualized treatment of pancreatic cancer in the future.


Assuntos
Proteína Quinase 11 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/genética , Neoplasias Pancreáticas/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Carcinogênese/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Neoplasias Pancreáticas/patologia , Fosforilação , Isoformas de Proteínas/genética , RNA Interferente Pequeno/genética
14.
Nat Commun ; 11(1): 4845, 2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32973148

RESUMO

Herpesviruses encode conserved protein kinases (CHPKs) to stimulate phosphorylation-sensitive processes during infection. How CHPKs bind to cellular factors and how this impacts their regulatory functions is poorly understood. Here, we use quantitative proteomics to determine cellular interaction partners of human herpesvirus (HHV) CHPKs. We find that CHPKs can target key regulators of transcription and replication. The interaction with Cyclin A and associated factors is identified as a signature of ß-herpesvirus kinases. Cyclin A is recruited via RXL motifs that overlap with nuclear localization signals (NLS) in the non-catalytic N termini. This architecture is conserved in HHV6, HHV7 and rodent cytomegaloviruses. Cyclin A binding competes with NLS function, enabling dynamic changes in CHPK localization and substrate phosphorylation. The cytomegalovirus kinase M97 sequesters Cyclin A in the cytosol, which is essential for viral inhibition of cellular replication. Our data highlight a fine-tuned and physiologically important interplay between a cellular cyclin and viral kinases.


Assuntos
Replicação do DNA/fisiologia , Infecções por Herpesviridae/metabolismo , Herpesviridae/metabolismo , Proteínas Quinases/metabolismo , Animais , Ciclina A/genética , Ciclina A/metabolismo , Citomegalovirus/genética , DNA/metabolismo , Células HEK293 , Herpesviridae/enzimologia , Herpesviridae/genética , Infecções por Herpesviridae/virologia , Humanos , Camundongos , Células NIH 3T3 , Sinais de Localização Nuclear/metabolismo , Fosforilação , Mapas de Interação de Proteínas , Proteínas Virais/genética , Proteínas Virais/metabolismo
15.
Chem Biol Interact ; 330: 109245, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32866465

RESUMO

The calcineurin inhibitor, cyclosporin A (CsA) is one of the most common immunosuppressive agents used in organ transplantation. However, its clinical use is often limited by several unwanted effects including nephrotoxicity and hepatotoxicity. By using immunohistochemical and ELISA techniques, it was found that CsA administration causes a rapid activation of a disintegrin and metalloproteases-17 (ADAM-17), epidermal growth factor receptor (EGFR) and subsequent ERK1/2 phosphorylation in the liver and kidney of albino mice. Furthermore, this study presents mechanistic relevance of this signaling cascade involving reactive oxygen species (ROS)-mediated ADAM-17/EGFR/ERK1/2 activation as indicated by a clear reduction in ADAM-17 and EGFR activities as well as ERK1/2 phosphorylation when the animals pretreated with Polyethylene glycol-superoxide dismutase (PEG-SOD) before CsA administration. Collectively, our findings demonstrate that CsA has the ability to activate ADAM-17-mediated EGFR/ERK1/2 phosphorylation in the liver and kidney of albino mice in ROS-dependent manner. Finally, these data may support the concept of using antioxidant therapy as a valuable approach for the prevention of CsA-induced nephrotoxicity and hepatotoxicity.


Assuntos
Ciclosporina/toxicidade , Rim/metabolismo , Fígado/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Polietilenoglicóis/farmacologia , Superóxido Dismutase/farmacologia , Proteína ADAM17/metabolismo , Animais , Ciclosporina/farmacologia , Interações Medicamentosas , Receptores ErbB/metabolismo , Rim/efeitos dos fármacos , Fígado/efeitos dos fármacos , Camundongos , Fosforilação/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
16.
Nan Fang Yi Ke Da Xue Xue Bao ; 40(7): 1001-1007, 2020 Jul 30.
Artigo em Chinês | MEDLINE | ID: mdl-32895154

RESUMO

OBJECTIVE: To explore the effects of taurolithocholic acid (tLCA) and chenodeoxycholic acid (CDCA) on the expression of aorexigenic neuropeptide in mouse hypothalamus GT1-7 cells. METHODS: Mouse hypothalamic GT1-7 cells were treated with culture medium containing 10% FBS (control group, n=3) or with 10 nmol/L, 100 nmol/L, 1 µmol/L and 10 µmol/L tLCA (tLCA group, n=3) or CDCA (CDCA group, n=3) for 12, 24 or 48 h. Real-time PCR was performed to determine the expression levels of proopiomelanocortin (POMC) mRNA in the cells, and the production levels of α-melanocyte-stimulating hormone (α-MSH) were assessed using an ELISA kit. Signal transduction and activator of transcription 3 phosphorylation (p-STAT3), threonine kinase phosphorylation (p-AKT), suppressor of cytokine signaling 3 (SOCS3), G protein-coupled bile acid receptor-1 (TGR5) and farnesoid X receptor (FXR) protein were detected by Western blotting. RESULTS: Western blotting results showed that mouse hypothalamic GT1-7 cells expressed two bile acid receptors, TGR5 and FXR, whose expressions were regulated by bile acids. Real-time PCR showed that the expression of POMC mRNA was significantly increased in the cells after treatment with 10 µmol/L tLCA or CDCA for 24 h. POMC-derived anorexigenic peptide α-MSH increased significantly in GT1-7 cells after treatment with 10 µmol/L tLCA or CDCA for 24 h. Treatment of the cells with tLCA or CDCA significantly increased the expressions of intracellular signaling proteins including p-STAT3, p-AKT and SOCS3. CONCLUSIONS: Mouse hypothalamic GT1-7 cells express bile acid receptors TGR5 and FXR. Bile acids tLCA or CDCA can promote the expression of POMC mRNA and increase the production of the anorexigenic peptide α-MSH. The intracellular signaling proteins p-AKT, p-STAT3 and SOCS3 are likely involved in bile acid-induced anorexigenic peptide production.


Assuntos
Transdução de Sinais , Animais , Ácidos e Sais Biliares , Ácido Quenodesoxicólico , Hipotálamo , Camundongos , Neuropeptídeos , Fosforilação , Fator de Transcrição STAT3 , Proteína 3 Supressora da Sinalização de Citocinas
17.
Mol Cell ; 80(1): 164-174.e4, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32877642

RESUMO

SARS-CoV-2 infections are rapidly spreading around the globe. The rapid development of therapies is of major importance. However, our lack of understanding of the molecular processes and host cell signaling events underlying SARS-CoV-2 infection hinders therapy development. We use a SARS-CoV-2 infection system in permissible human cells to study signaling changes by phosphoproteomics. We identify viral protein phosphorylation and define phosphorylation-driven host cell signaling changes upon infection. Growth factor receptor (GFR) signaling and downstream pathways are activated. Drug-protein network analyses revealed GFR signaling as key pathways targetable by approved drugs. The inhibition of GFR downstream signaling by five compounds prevents SARS-CoV-2 replication in cells, assessed by cytopathic effect, viral dsRNA production, and viral RNA release into the supernatant. This study describes host cell signaling events upon SARS-CoV-2 infection and reveals GFR signaling as a central pathway essential for SARS-CoV-2 replication. It provides novel strategies for COVID-19 treatment.


Assuntos
Antivirais/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/genética , Fosfatidilinositol 3-Quinase/genética , Receptores de Fatores de Crescimento/genética , Proteínas Virais/genética , Corticosteroides/uso terapêutico , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Betacoronavirus/imunologia , Betacoronavirus/patogenicidade , Células CACO-2 , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Humanos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Receptores de Fatores de Crescimento/antagonistas & inibidores , Receptores de Fatores de Crescimento/metabolismo , Transdução de Sinais , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacos
18.
PLoS Pathog ; 16(9): e1008841, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32881973

RESUMO

The influenza virus polymerase transcribes and replicates the viral genome. The proper timing and balance of polymerase activity is important for successful replication. Genome replication is controlled in part by phosphorylation of NP that regulates assembly of the replication machinery. However, it remains unclear whether phosphorylation directly regulated polymerase activity. Here we identified polymerase phosphosites that control its function. Mutating phosphosites in the catalytic subunit PB1 altered polymerase activity and virus replication. Biochemical analyses revealed phosphorylation events that disrupted global polymerase function by blocking the NTP entry channel or preventing RNA binding. We also identified a regulatory site that split polymerase function by specifically suppressing transcription. These experiments show that host kinases phospho-regulate viral RNA synthesis directly by modulating polymerase activity and indirectly by controlling assembly of replication machinery. Further, they suggest polymerase phosphorylation may bias replication versus transcription at discrete times or locations during the infectious cycle.


Assuntos
Vírus da Influenza A/fisiologia , RNA Viral/biossíntese , Transcrição Genética , Proteínas Virais/metabolismo , Replicação Viral , Células A549 , Animais , Cães , Células HEK293 , Humanos , Células Madin Darby de Rim Canino , Fosforilação , RNA Viral/genética , Proteínas Virais/genética
19.
Nat Commun ; 11(1): 4766, 2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32958778

RESUMO

Germline telomere maintenance defects are associated with an increased incidence of inflammatory diseases in humans, yet whether and how telomere dysfunction causes inflammation are not known. Here, we show that telomere dysfunction drives pATM/c-ABL-mediated activation of the YAP1 transcription factor, up-regulating the major pro-inflammatory factor, pro-IL-18. The colonic microbiome stimulates cytosolic receptors activating caspase-1 which cleaves pro-IL-18 into mature IL-18, leading to recruitment of interferon (IFN)-γ-secreting T cells and intestinal inflammation. Correspondingly, patients with germline telomere maintenance defects exhibit DNA damage (γH2AX) signaling together with elevated YAP1 and IL-18 expression. In mice with telomere dysfunction, telomerase reactivation in the intestinal epithelium or pharmacological inhibition of ATM, YAP1, or caspase-1 as well as antibiotic treatment, dramatically reduces IL-18 and intestinal inflammation. Thus, telomere dysfunction-induced activation of the ATM-YAP1-pro-IL-18 pathway in epithelium is a key instigator of tissue inflammation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Inflamação/patologia , Telômero/patologia , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Antibacterianos/uso terapêutico , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Caspase 1/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Criança , Colo/metabolismo , Colo/microbiologia , Colo/patologia , Gastroenteropatias/patologia , Microbioma Gastrointestinal/efeitos dos fármacos , Microbioma Gastrointestinal/fisiologia , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/microbiologia , Interleucina-18/genética , Interleucina-18/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Camundongos , Camundongos Mutantes , Fosforilação , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Transdução de Sinais , Telomerase/genética , Telomerase/metabolismo
20.
Nat Commun ; 11(1): 4765, 2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32958780

RESUMO

Fatty acids (FAs) are essential nutrients, but how they are transported into cells remains unclear. Here, we show that FAs trigger caveolae-dependent CD36 internalization, which in turn delivers FAs into adipocytes. During the process, binding of FAs to CD36 activates its downstream kinase LYN, which phosphorylates DHHC5, the palmitoyl acyltransferase of CD36, at Tyr91 and inactivates it. CD36 then gets depalmitoylated by APT1 and recruits another tyrosine kinase SYK to phosphorylate JNK and VAVs to initiate endocytic uptake of FAs. Blocking CD36 internalization by inhibiting APT1, LYN or SYK abolishes CD36-dependent FA uptake. Restricting CD36 at either palmitoylated or depalmitoylated state eliminates its FA uptake activity, indicating an essential role of dynamic palmitoylation of CD36. Furthermore, blocking endocytosis by targeting LYN or SYK inhibits CD36-dependent lipid droplet growth in adipocytes and high-fat-diet induced weight gain in mice. Our study has uncovered a dynamic palmitoylation-regulated endocytic pathway to take up FAs.


Assuntos
Antígenos CD36/metabolismo , Endocitose/fisiologia , Ácidos Graxos/metabolismo , Lipoilação , Células 3T3-L1 , Aciltransferases/metabolismo , Adipócitos/metabolismo , Animais , Antígenos CD36/deficiência , Antígenos CD36/genética , Cavéolas/metabolismo , Células Cultivadas , Dieta Hiperlipídica/efeitos adversos , Humanos , Gotículas Lipídicas/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Obesidade/tratamento farmacológico , Fosforilação , Transdução de Sinais , Quinase Syk/antagonistas & inibidores , Quinase Syk/metabolismo , Ganho de Peso/efeitos dos fármacos , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismo
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