Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.338
Filtrar
1.
Nat Commun ; 11(1): 3532, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32669539

RESUMO

Asexual proliferation of the Plasmodium parasites that cause malaria follows a developmental program that alternates non-canonical intraerythrocytic replication with dissemination to new host cells. We carried out a functional analysis of the Plasmodium falciparum homolog of Protein Phosphatase 1 (PfPP1), a universally conserved cell cycle factor in eukaryotes, to investigate regulation of parasite proliferation. PfPP1 is indeed required for efficient replication, but is absolutely essential for egress of parasites from host red blood cells. By phosphoproteomic and chemical-genetic analysis, we isolate two functional targets of PfPP1 for egress: a HECT E3 protein-ubiquitin ligase; and GCα, a fusion protein composed of a guanylyl cyclase and a phospholipid transporter domain. We hypothesize that PfPP1 regulates lipid sensing by GCα and find that phosphatidylcholine stimulates PfPP1-dependent egress. PfPP1 acts as a key regulator that integrates multiple cell-intrinsic pathways with external signals to direct parasite egress from host cells.


Assuntos
Eritrócitos/parasitologia , Plasmodium falciparum/enzimologia , Proteína Fosfatase 1/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Proliferação de Células , GMP Cíclico/metabolismo , Regulação Enzimológica da Expressão Gênica , Humanos , Concentração Inibidora 50 , Camundongos , Camundongos Knockout , Fosfatidilcolinas/química , Domínios Proteicos , Proteoma , Ubiquitina-Proteína Ligases/metabolismo
2.
PLoS Genet ; 16(7): e1008484, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32673313

RESUMO

Yeast and fast-growing human tumor cells share metabolic similarities in that both cells use fermentation of glucose for energy and both are highly sensitive to the glucose analog 2-deoxyglucose. Spontaneous mutations in S. cerevisiae that conferred resistance to 2-deoxyglucose were identified by whole genome sequencing. Missense alleles of the HXK2, REG1, GLC7 and SNF1 genes were shown to confer significant resistance to 2-deoxyglucose and all had the potential to alter the activity and or target selection of the Snf1 kinase signaling pathway. All three missense alleles in HXK2 resulted in significantly reduced catalytic activity. Addition of 2DG promotes endocytosis of the glucose transporter Hxt3. All but one of the 2DG-resistant strains reduced the 2DG-mediated hexose transporter endocytosis by increasing plasma membrane occupancy of the Hxt3 protein. Increased expression of the DOG (deoxyglucose) phosphatases has been associated with resistance to 2-deoxyglucose. Expression of both the DOG1 and DOG2 mRNA was elevated after treatment with 2-deoxyglucose but induction of these genes is not associated with 2DG-resistance. RNAseq analysis of the transcriptional response to 2DG showed large scale, genome-wide changes in mRNA abundance that were greatly reduced in the 2DG resistant strains. These findings suggest the common adaptive response to 2DG is to limit the magnitude of the response. Genetic studies of 2DG resistance using the dominant SNF1-G53R allele in cells that are genetically compromised in both the endocytosis and DOG pathways suggest that at least one more mechanism for conferring resistance to this glucose analog remains to be discovered.


Assuntos
Metabolismo Energético/genética , Glucose/metabolismo , Hexoquinase/genética , Monoéster Fosfórico Hidrolases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas de Saccharomyces cerevisiae/genética , Desoxiglucose/efeitos adversos , Desoxiglucose/farmacologia , Endocitose/efeitos dos fármacos , Endocitose/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Proteínas Facilitadoras de Transporte de Glucose/genética , Humanos , Mutação/genética , Proteína Fosfatase 1/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Transdução de Sinais/efeitos dos fármacos , Sequenciamento Completo do Genoma
3.
PLoS Pathog ; 16(7): e1008669, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32702076

RESUMO

Hepatitis B virus (HBV) replicates its genomic DNA via viral DNA polymerase self-primed reverse transcription of a RNA pre-genome in the nucleocapsid assembled by 120 core protein (Cp) dimers. The arginine-rich carboxyl-terminal domain (CTD) of Cp plays an important role in the selective packaging of viral DNA polymerase-pregenomic (pg) RNA complex into nucleocapsid. Previous studies suggested that the CTD is initially phosphorylated at multiple sites to facilitate viral RNA packaging and subsequently dephosphorylated in association with viral DNA synthesis and secretion of DNA-containing virions. However, our recent studies suggested that Cp is hyper-phosphorylated as free dimers and its dephosphorylation is associated with pgRNA encapsidation. Herein, we provide further genetic and biochemical evidence supporting that extensive Cp dephosphorylation does take place during the assembly of pgRNA-containing nucleocapsids, but not empty capsids. Moreover, we found that cellular protein phosphatase 1 (PP1) is required for Cp dephosphorylation and pgRNA packaging. Interestingly, the PP1 catalytic subunits α and ß were packaged into pgRNA-containing nucleocapsids, but not empty capsids, and treatment of HBV replicating cells with core protein allosteric modulators (CpAMs) promoted empty capsid assembly and abrogated the encapsidation of PP1 α and ß. Our study thus identified PP1 as a host cellular factor that is co-packaged into HBV nucleocapsids, and plays an essential role in selective packaging of the viral DNA-polymerase-pgRNA complex through catalyzing Cp dephosphorylation.


Assuntos
Vírus da Hepatite B/fisiologia , Nucleocapsídeo/metabolismo , Proteína Fosfatase 1/metabolismo , RNA Viral/metabolismo , Montagem de Vírus/fisiologia , Linhagem Celular , Hepatite B/virologia , Humanos , Fragmentos de Peptídeos/metabolismo , Fosforilação , Proteínas do Core Viral/metabolismo
4.
Mol Cell ; 77(6): 1157-1158, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32200795

RESUMO

In this issue of Molecular Cell, Cossa et al. (2020) uncover the basis for a dependency of tumor cells with deregulated MYC on the kinase NUAK1, which acts through PP1 and PNUTS to ensure that splicing keeps up with MYC-driven transcription.


Assuntos
Spliceossomos , Proteína Fosfatase 1
5.
Mol Cell ; 77(6): 1322-1339.e11, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32006464

RESUMO

Deregulated expression of MYC induces a dependence on the NUAK1 kinase, but the molecular mechanisms underlying this dependence have not been fully clarified. Here, we show that NUAK1 is a predominantly nuclear protein that associates with a network of nuclear protein phosphatase 1 (PP1) interactors and that PNUTS, a nuclear regulatory subunit of PP1, is phosphorylated by NUAK1. Both NUAK1 and PNUTS associate with the splicing machinery. Inhibition of NUAK1 abolishes chromatin association of PNUTS, reduces spliceosome activity, and suppresses nascent RNA synthesis. Activation of MYC does not bypass the requirement for NUAK1 for spliceosome activity but significantly attenuates transcription inhibition. Consequently, NUAK1 inhibition in MYC-transformed cells induces global accumulation of RNAPII both at the pause site and at the first exon-intron boundary but does not increase mRNA synthesis. We suggest that NUAK1 inhibition in the presence of deregulated MYC traps non-productive RNAPII because of the absence of correctly assembled spliceosomes.


Assuntos
Núcleo Celular/metabolismo , Cromatina/metabolismo , Proteínas Quinases/metabolismo , Proteína Fosfatase 1/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Repressoras/metabolismo , Spliceossomos/metabolismo , Transcrição Genética , Animais , Núcleo Celular/genética , Cromatina/genética , Regulação da Expressão Gênica , Células HeLa , Humanos , Camundongos , Células NIH 3T3 , Fosforilação , Proteínas Quinases/genética , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Processamento de RNA , Proteínas Repressoras/genética , Spliceossomos/genética
6.
FASEB J ; 34(1): 1591-1601, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914597

RESUMO

The Slack (KCNT1) gene encodes sodium-activated potassium channels that are abundantly expressed in the central nervous system. Human mutations alter the function of Slack channels, resulting in epilepsy and intellectual disability. Most of the disease-causing mutations are located in the extended cytoplasmic C-terminus of Slack channels and result in increased Slack current. Previous experiments have shown that the C-terminus of Slack channels binds a number of cytoplasmic signaling proteins. One of these is Phactr1, an actin-binding protein that recruits protein phosphatase 1 (PP1) to certain phosphoprotein substrates. Using co-immunoprecipitation, we found that Phactr1 is required to link the channels to actin. Using patch clamp recordings, we found that co-expression of Phactr1 with wild-type Slack channels reduces the current amplitude but has no effect on Slack channels in which a conserved PKC phosphorylation site (S407) that regulates the current amplitude has been mutated. Furthermore, a Phactr1 mutant that disrupts the binding of PP1 but not that of actin fails to alter Slack currents. Our data suggest that Phactr1 regulates the Slack by linking PP1 to the channel. Targeting Slack-Phactr1 interactions may therefore be helpful in developing the novel therapies for brain disorders associated with the malfunction of Slack channels.


Assuntos
Canais de Potássio Ativados por Sódio/metabolismo , Proteína Fosfatase 1/metabolismo , Actinas/metabolismo , Animais , Linhagem Celular , Células HEK293 , Humanos , Potenciais da Membrana/fisiologia , Camundongos , Mutação/genética , Neurônios/metabolismo , Técnicas de Patch-Clamp/métodos , Ratos , Transdução de Sinais/fisiologia
7.
Neuron ; 105(5): 855-866.e5, 2020 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-31924446

RESUMO

Recent interest in astrocyte activation states has raised the fundamental question of how these cells, normally essential for synapse and neuronal maintenance, become pathogenic. Here, we show that activation of the unfolded protein response (UPR), specifically phosphorylated protein kinase R-like endoplasmic reticulum (ER) kinase (PERK-P) signaling-a pathway that is widely dysregulated in neurodegenerative diseases-generates a distinct reactivity state in astrocytes that alters the astrocytic secretome, leading to loss of synaptogenic function in vitro. Further, we establish that the same PERK-P-dependent astrocyte reactivity state is harmful to neurons in vivo in mice with prion neurodegeneration. Critically, targeting this signaling exclusively in astrocytes during prion disease is alone sufficient to prevent neuronal loss and significantly prolongs survival. Thus, the astrocyte reactivity state resulting from UPR over-activation is a distinct pathogenic mechanism that can by itself be effectively targeted for neuroprotection.


Assuntos
Astrócitos/metabolismo , Fator de Iniciação 2B em Eucariotos/metabolismo , Doenças Neurodegenerativas/metabolismo , Doenças Priônicas/metabolismo , Sinapses/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , eIF-2 Quinase/metabolismo , Animais , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Técnicas In Vitro , Memória , Camundongos , Fosforilação , Biossíntese de Proteínas , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Transdução de Sinais , Tapsigargina/farmacologia , Transcriptoma , Tunicamicina/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos
9.
Cancer Invest ; 38(1): 1-12, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31797701

RESUMO

Purpose: The function of long noncoding RNAs (lncRNA) in breast cancer metastasis remains largely unknown. In this work, the role of HOXC-AS3 in breast cancer progression was investigated.Methods: By using Cancer Genome Atlas (TCGA) Database, we investigated the expression of HOXC-AS3 in breast cancer and explored the association between HOXC-AS3 expression and prognosis. Then, we studied the biological function of HOXC-AS3 in cell migration and invasion both in vitro and in vivo. Furthermore, the target miRNA of HOXC-AS3, and the target mRNA of miR-3922-5p were proved.Results: HOXC-AS3 is aberrantly overexpressed in breast cancers especially the HER2+ type. Moreover, high expression of HOXC-AS3 has a relationship with poor clinical outcomes of breast cancer. In addition, HOXC-AS3 regulates cell Invasion and migration both in vitro and in vivo. Our results demonstrated that miR-3922-5p was a direct target of HOXC-AS3, and PPP1R1A was a target of miR-3922-5p in breast cancer.Conclusions: The novel lncRNA HOXC-AS3 acts as a miR-3922-5p sponge to upregulate PPP1R1A protein expression, and thus results in promoting breast cancer metastasis. HOXC-AS3 could be a novel therapeutic target for breast cancer therapeutics.


Assuntos
Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Proteína Fosfatase 1/genética , RNA Longo não Codificante/metabolismo , Animais , Mama/patologia , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Conjuntos de Dados como Assunto , Feminino , Humanos , Camundongos , MicroRNAs/metabolismo , Prognóstico , Análise de Sobrevida , Fatores de Tempo , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto
10.
J Med Chem ; 63(6): 2807-2813, 2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-31874036

RESUMO

Heterobifunctional molecules have proven powerful tools to induce ligase-dependent ubiquitination of target proteins. We describe here a chemical strategy for controlling a different post-translational modification (PTM): phosphorylation. Heterobifunctional molecules were designed to promote the proximity of a protein phosphatase (PP1) to protein targets. The synthesized molecules induced the PP1-dependent dephosphorylation of AKT and EGFR. To our knowledge, this work represents the first examples of small molecules recruiting non-native partners to induce removal of a PTM.


Assuntos
Descoberta de Drogas , Fosforilação/efeitos dos fármacos , Fosfotransferases/metabolismo , Proteína Fosfatase 1/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Linhagem Celular , Receptores ErbB/metabolismo , Humanos , Ligantes , Estudo de Prova de Conceito , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Bibliotecas de Moléculas Pequenas/química
11.
Dis Markers ; 2019: 5946461, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31827636

RESUMO

Background: The hypoxic conditions at high altitudes are great threats to survival, causing pressure for adaptation. More and more high-altitude denizens are not adapted with the condition known as high-altitude polycythemia (HAPC) that featured excessive erythrocytosis. As a high-altitude sickness, the etiology of HAPC is still unclear. Methods: In this study, we reported the whole-genome sequencing-based study of 10 native Tibetans with HAPC and 10 control subjects followed by genotyping of selected 21 variants from discovered single nucleotide variants (SNVs) in an independent cohort (232 cases and 266 controls). Results: We discovered the egl nine homologue 3 (egln3/phd3) (14q13.1, rs1346902, P = 1.91 × 10-5) and PPP1R2P1 (Protein Phosphatase 1 Regulatory Inhibitor Subunit 2) gene (6p21.32, rs521539, P = 0.012). Our results indicated an unbiased framework to identify etiological mechanisms of HAPC and showed that egln3/phd3 and PPP1R2P1 may be associated with the susceptibility to HAPC. Egln3/phd3b is associated with hypoxia-inducible factor subunit α (HIFα). Protein Phosphatase 1 Regulatory Inhibitor is associated with reactive oxygen species (ROS) and oxidative stress. Conclusions: Our genome sequencing conducted in Tibetan HAPC patients identified egln3/phd3 and PPP1R2P1 associated with HAPC.


Assuntos
Doença da Altitude/diagnóstico , Biomarcadores/análise , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Policitemia/diagnóstico , Polimorfismo de Nucleotídeo Único , Proteína Fosfatase 1/genética , Sequenciamento Completo do Genoma/métodos , Adulto , Idoso , Doença da Altitude/epidemiologia , Doença da Altitude/genética , Estudos de Casos e Controles , Feminino , Seguimentos , Genoma Humano , Genótipo , Humanos , Hipóxia , Masculino , Pessoa de Meia-Idade , Policitemia/epidemiologia , Policitemia/genética , Prognóstico , Tibet/epidemiologia
12.
Oxid Med Cell Longev ; 2019: 2193019, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31885777

RESUMO

Ca2+/calmodulin-dependent protein kinase II (CaMKII), regulated by inhibitor 1 of protein phosphatase 1 (I1PP1), is vital for maintaining cardiovascular homeostasis. However, the role and mechanism of I1PP1 against hypoxia-reoxygenation (H/R) injury in cardiomyocytes remain a question. In our study, after I1PP1 overexpression by adenovirus infection in the neonatal cardiomyocytes followed by hypoxia for 4 h and reoxygenation for 12 h, the CaMKIIδ alternative splicing subtype, ATP content, and lactate dehydrogenase (LDH) release were determined. CaMKII activity was evaluated by phosphoprotein phosphorylation at Thr17 (p-PLB Thr17), CaMKII phosphorylation (p-CaMKII), and CaMKII oxidation (ox-CaMKII). Reactive oxygen species (ROS), mitochondrial membrane potential, dynamin-related protein 1 (DRP1), and optic atrophy 1 (OPA1) expressions were assessed. Our study verified that I1PP1 overexpression attenuated the CaMKIIδ alternative splicing disorder; suppressed PLB phosphorylation at Thr17, p-CaMKII, and ox-CaMKII; decreased cell LDH release; increased ATP content; attenuated ROS production; increased mitochondrial membrane potential; and decreased DRP1 expression but increased OPA1 expression in the cardiomyocytes after H/R. Contrarily, CaMKIIδ alternative splicing disorder, LDH release, ATP reduction, and ROS accumulation were aggravated after H/R injury with the I1PP1 knockdown. Collectively, I1PP1 overexpression corrected disorders of CaMKIIδ alternative splicing, inhibited CaMKII phosphorylation, repressed CaMKII oxidation, suppressed ROS production, and attenuated cardiomyocyte H/R injury.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Hipóxia Celular/fisiologia , Traumatismo por Reperfusão Miocárdica/enzimologia , Miócitos Cardíacos/enzimologia , Estresse Oxidativo/fisiologia , Proteína Fosfatase 1/metabolismo , Processamento Alternativo , Animais , Benzilaminas/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/biossíntese , Células Cultivadas , Dinaminas/biossíntese , GTP Fosfo-Hidrolases/biossíntese , Potencial da Membrana Mitocondrial , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Oxirredução , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteína Fosfatase 1/biossíntese , Proteína Fosfatase 1/genética , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Sulfonamidas/farmacologia
13.
Cesk Patol ; 55(4): 239-243, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31842556

RESUMO

Inflammatory myofibroblastic tumor (IMT) of the uterus is rare but probably underdiagnosed tumor. It is usually benign but small fraction of cases may locally recur or rarely metastasize. Herein, we present a case report of 66-year-old patient with uterine IMT originally diagnosed as leiomyosarcoma of the uterus. The patient died within few months due to local tumor progression with skeletal metastases. Macroscopically, this was a voluminous locally aggressive yellowish-grey tumor of soft consistency limited to myometrium. Microscopically, the tumor was characterized by polymorphic spindle cell proliferation with marked nuclear atypia and numerous mitoses. Small geographic necroses was noticed. Typical histologic features of IMT were represented by lymphocytic infiltrate which was only very small and focal. Myxoid stroma was absent. Immunohistochemically, there was strong and diffuse cytoplasmic positivity of ALK (anaplastic lymphoma kinase). The presence of PPP1CB-ALK fusion transcript was confirmed by molecular-genetic methods. Proper diagnosis of uterine IMT is of importance as there is an option of targeted ALK inhibitor therapy in cases of aggressive tumor behaviour. Currently it is thought that histomorphology of uterine IMT may overlap with that of leiomyosarcoma and STUMP (smooth muscle tumor of uncertain malignant potential). The presence of ALK rearrangement is probably the only reliable diagnostic marker. Thus, ALK immunohistochemistry followed by molecular-genetic testing seems to represent suitable screening tool for the detection of uterine IMT.


Assuntos
Leiomiossarcoma , Neoplasias Uterinas , Idoso , Biomarcadores Tumorais , Feminino , Humanos , Imuno-Histoquímica , Recidiva Local de Neoplasia , Proteína Fosfatase 1
14.
PLoS Genet ; 15(11): e1008475, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31710605

RESUMO

Circadian rhythms are generated by endogenous pacemakers that rely on transcriptional-translational feedback mechanisms conserved among species. In Drosophila, the stability of a key pacemaker protein PERIOD (PER) is tightly controlled by changes in phosphorylation status. A number of molecular players have been implicated in PER destabilization by promoting PER progressive phosphorylation. On the other hand, there have been few reports describing mechanisms that stabilize PER by delaying PER hyperphosphorylation. Here we report that the protein Suppressor of Ras (SUR-8) regulates circadian locomotor rhythms by stabilizing PER. Depletion of SUR-8 from circadian neurons lengthened the circadian period by about 2 hours and decreased PER abundance, whereas its overexpression led to arrhythmia and an increase in PER. Specifically SUR-8 promotes the stability of PER through phosphorylation regulation. Interestingly, downregulation of the protein phosphatase 1 catalytic subunit PP1-87B recapitulated the phenotypes of SUR-8 depletion. We found that SUR-8 facilitates interactions between PP1-87B and PER. Depletion of SUR-8 decreased the interaction of PER and PP1-87B, which supports the role of SUR-8 as a scaffold protein. Interestingly, the interaction between SUR-8 and PER is temporally regulated: SUR-8 has more binding to PER at night than morning. Thus, our results indicate that SUR-8 interacts with PP1-87B to control PER stability to regulate circadian rhythms.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Ritmo Circadiano/genética , Proteínas de Drosophila/genética , Proteínas Circadianas Period/genética , Fosfoproteínas Fosfatases/genética , Proteína Fosfatase 1/genética , Animais , Domínio Catalítico/genética , Drosophila melanogaster/genética , Neurônios/metabolismo , Fosforilação
15.
Mol Cell ; 76(6): 896-908.e4, 2019 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-31677974

RESUMO

Control of transcription speed, which influences many co-transcriptional processes, is poorly understood. We report that PNUTS-PP1 phosphatase is a negative regulator of RNA polymerase II (Pol II) elongation rate. The PNUTS W401A mutation, which disrupts PP1 binding, causes genome-wide acceleration of transcription associated with hyper-phosphorylation of the Spt5 elongation factor. Immediately downstream of poly(A) sites, Pol II decelerates from >2 kb/min to <1 kb/min, which correlates with Spt5 dephosphorylation. Pol II deceleration and Spt5 dephosphorylation require poly(A) site recognition and the PNUTS-PP1 complex, which is in turn necessary for transcription termination. These results lead to a model for termination, the "sitting duck torpedo" mechanism, where poly(A) site-dependent deceleration caused by PNUTS-PP1 and Spt5 dephosphorylation is required to convert Pol II into a viable target for the Xrn2 terminator exonuclease. Spt5 and its bacterial homolog NusG therefore have related functions controlling kinetic competition between RNA polymerases and the termination factors that pursue them.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Exorribonucleases/metabolismo , Proteína Fosfatase 1/metabolismo , Processamento de Proteína Pós-Traducional , RNA Polimerase II/metabolismo , RNA Mensageiro/biossíntese , Proteínas de Ligação a RNA/metabolismo , Terminação da Transcrição Genética , Sítios de Ligação , Proteínas de Ligação a DNA/genética , Exorribonucleases/genética , Células HEK293 , Humanos , Cinética , Proteínas Nucleares/genética , Fosforilação , Poli A/metabolismo , Ligação Proteica , Proteína Fosfatase 1/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Transdução de Sinais , Fatores de Elongação da Transcrição/genética
16.
PLoS Pathog ; 15(10): e1008068, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31648236

RESUMO

Ebola virus (EBOV) infections are characterized by a pronounced lymphopenia that is highly correlative with fatalities. However, the mechanisms leading to T-cell depletion remain largely unknown. Here, we demonstrate that both viral mRNAs and antigens are detectable in CD4+ T cells despite the absence of productive infection. A protein phosphatase 1 inhibitor, 1E7-03, and siRNA-mediated suppression of viral antigens were used to demonstrate de novo synthesis of viral RNAs and antigens in CD4+ T cells, respectively. Cell-to-cell fusion of permissive Huh7 cells with non-permissive Jurkat T cells impaired productive EBOV infection suggesting the presence of a cellular restriction factor. We determined that viral transcription is partially impaired in the fusion T cells. Lastly, we demonstrate that exposure of T cells to EBOV resulted in autophagy through activation of ER-stress related pathways. These data indicate that exposure of T cells to EBOV results in an abortive infection, which likely contributes to the lymphopenia observed during EBOV infections.


Assuntos
Linfócitos T CD4-Positivos/virologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Linfopenia/imunologia , Replicação Viral/fisiologia , Animais , Antígenos Virais/biossíntese , Antígenos Virais/genética , Autofagia/fisiologia , Linfócitos T CD4-Positivos/imunologia , Linhagem Celular , Chlorocebus aethiops , Estresse do Retículo Endoplasmático/fisiologia , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Indóis/farmacologia , Células Jurkat , Proteína Fosfatase 1/antagonistas & inibidores , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Viral/biossíntese , RNA Viral/genética , Fatores de Transcrição/metabolismo , Ureia/análogos & derivados , Ureia/farmacologia , Células Vero , Proteínas Virais/metabolismo
17.
Oncol Rep ; 42(6): 2644-2654, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31638256

RESUMO

Activin A, a multifunctional cytokine, is a member of transforming growth factor­ß (TGF­ß) superfamily. It is associated with a variety of pathophysiological processes, including inflammation, fibrosis, and tumorigenesis. Chronic or prolonged endoplasmic reticulum (ER) stress can lead to cells apoptosis. However, whether ER stress­related proteins, such as CHOP, GADD34 are involved in activin A­induced myeloma cell apoptosis remains unknown. In the present study, it was revealed that activin A inhibited the proliferation of myeloma cell line NS­1 cells and induced NS­1 cell apoptosis. Activin A upregulated the expression of CHOP, GADD34, caspase­3, and caspase­12. Moreover, both Smad3 and p­Smad3 levels were increased with treatment of activin A. Further studies revealed that the overexpression of activin signaling protein Smad3 in NS­1 cells increased the levels of CHOP, caspase­3, and p­Smad3. These data indicated that the CHOP protein of the ER stress pathway may be involved in activin A­induced NS­1 cell apoptosis, and also indicated the potential therapy of activin A­induced apoptosis via CHOP signaling for multiple myeloma.


Assuntos
Ativinas/metabolismo , Estresse do Retículo Endoplasmático/genética , Regulação Neoplásica da Expressão Gênica , Mieloma Múltiplo/patologia , Fator de Transcrição CHOP/metabolismo , Ativinas/administração & dosagem , Animais , Caspase 12/metabolismo , Linhagem Celular Tumoral/transplante , Proliferação de Células/genética , Sobrevivência Celular/genética , Modelos Animais de Doenças , Humanos , Injeções Intralesionais , Masculino , Camundongos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Proteína Fosfatase 1/metabolismo , Regulação para Cima
18.
Nat Commun ; 10(1): 4513, 2019 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-31586073

RESUMO

The midbody is an organelle assembled at the intercellular bridge between the two daughter cells at the end of mitosis. It controls the final separation of the daughter cells and has been involved in cell fate, polarity, tissue organization, and cilium and lumen formation. Here, we report the characterization of the intricate midbody protein-protein interaction network (interactome), which identifies many previously unknown interactions and provides an extremely valuable resource for dissecting the multiple roles of the midbody. Initial analysis of this interactome revealed that PP1ß-MYPT1 phosphatase regulates microtubule dynamics in late cytokinesis and de-phosphorylates the kinesin component MKLP1/KIF23 of the centralspindlin complex. This de-phosphorylation antagonizes Aurora B kinase to modify the functions and interactions of centralspindlin in late cytokinesis. Our findings expand the repertoire of PP1 functions during mitosis and indicate that spatiotemporal changes in the distribution of kinases and counteracting phosphatases finely tune the activity of cytokinesis proteins.


Assuntos
Citocinese/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Mapas de Interação de Proteínas/fisiologia , Proteína Fosfatase 1/metabolismo , Aurora Quinase B/metabolismo , Sítios de Ligação/genética , Células HeLa , Humanos , Microscopia Intravital , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/metabolismo , Mitose/fisiologia , Mutagênese Sítio-Dirigida , Fosforilação/fisiologia , Proteína Fosfatase 1/genética , RNA Interferente Pequeno/metabolismo , Fuso Acromático/metabolismo , Imagem com Lapso de Tempo
19.
EBioMedicine ; 48: 236-247, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31521612

RESUMO

BACKGROUND: USP11 is an ubiquitin-specific protease that plays an important role in tumor progression via different mechanisms. However, the expression and prognostic significance of USP11 in colorectal cancer (CRC) remain unknown. METHODS: Bioinformatics analyses, qRT-PCR, western blotting, and immunohistochemistry were applied for investigating USP11 expression in CRC tissues. Kaplan-Meier analysis with log-rank test was used for survival analyses. LC-MS/MS was performed for identifying potential protein interactions with USP11. In vitro and in vivo assays were used for exploring the function of USP11 during the progression of CRC. FINDINGS: USP11 was overexpressed in CRC tissues and functioned as an oncogene. Overexpression or knockdown of USP11 promoted or inhibited, respectively, the growth and metastasis of CRC cells in vitro and in vivo. Mechanically, USP11 stabilized PPP1CA by deubiquitinating and protecting it from proteasome-mediated degradation. Moreover, the USP11/PPP1CA complex promoted CRC progression by activating the ERK/MAPK signaling pathway. INTERPRETATION: USP11 promoted tumor growth and metastasis in CRC via the ERK/MAPK pathway by stabilizing PPP1CA, suggesting USP11 is a potential prognostic marker. FUND: This work was supported by National Natural Science Foundation of China (NSFC81530044, NSFC81220108021, NSFC81802343), Technology Major Project of China Grants 2017ZX10203206, Shanghai Sailing Program (19YF1409600) and The project of Shanghai Jiaotong University (YG2017QN30).


Assuntos
Neoplasias Colorretais/etiologia , Neoplasias Colorretais/metabolismo , Sistema de Sinalização das MAP Quinases , Proteína Fosfatase 1/metabolismo , Tioléster Hidrolases/genética , Idoso , Idoso de 80 Anos ou mais , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Camundongos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Ligação Proteica , Proteína Fosfatase 1/genética , Proteólise , Tioléster Hidrolases/metabolismo , Transcriptoma
20.
Proc Natl Acad Sci U S A ; 116(41): 20472-20481, 2019 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-31548429

RESUMO

The metalloenzyme protein phosphatase 1 (PP1), which is responsible for ≥50% of all dephosphorylation reactions, is regulated by scores of regulatory proteins, including the highly conserved SDS22 protein. SDS22 has numerous diverse functions, surprisingly acting as both a PP1 inhibitor and as an activator. Here, we integrate cellular, biophysical, and crystallographic studies to address this conundrum. We discovered that SDS22 selectively binds a unique conformation of PP1 that contains a single metal (M2) at its active site, i.e., SDS22 traps metal-deficient inactive PP1. Furthermore, we showed that SDS22 dissociation is accompanied by a second metal (M1) being loaded into PP1, as free metal cannot dissociate the complex and M1-deficient mutants remain constitutively trapped by SDS22. Together, our findings reveal that M1 metal loading and loss are essential for PP1 regulation in cells, which has broad implications for PP1 maturation, activity, and holoenzyme subunit exchange.


Assuntos
Metais/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteína Fosfatase 1/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Metais/química , Modelos Moleculares , Proteínas Nucleares/química , Fosfoproteínas Fosfatases/química , Fosforilação , Conformação Proteica , Proteína Fosfatase 1/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA