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1.
Nat Commun ; 15(1): 7984, 2024 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-39266569

RESUMO

Alterations in nuclear structure and function are hallmarks of cancer cells. Little is known about these changes in Cancer-Associated Fibroblasts (CAFs), crucial components of the tumor microenvironment. Loss of the androgen receptor (AR) in human dermal fibroblasts (HDFs), which triggers early steps of CAF activation, leads to nuclear membrane changes and micronuclei formation, independent of cellular senescence. Similar changes occur in established CAFs and are reversed by restoring AR activity. AR associates with nuclear lamin A/C, and its loss causes lamin A/C nucleoplasmic redistribution. AR serves as a bridge between lamin A/C and the protein phosphatase PPP1. Loss of AR decreases lamin-PPP1 association and increases lamin A/C phosphorylation at Ser 301, a characteristic of CAFs. Phosphorylated lamin A/C at Ser 301 binds to the regulatory region of CAF effector genes of the myofibroblast subtype. Expression of a lamin A/C Ser301 phosphomimetic mutant alone can transform normal fibroblasts into tumor-promoting CAFs.


Assuntos
Fibroblastos Associados a Câncer , Núcleo Celular , Lamina Tipo A , Receptores Androgênicos , Humanos , Receptores Androgênicos/metabolismo , Receptores Androgênicos/genética , Lamina Tipo A/metabolismo , Lamina Tipo A/genética , Fosforilação , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Núcleo Celular/metabolismo , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 1/genética , Fibroblastos/metabolismo , Membrana Nuclear/metabolismo , Masculino , Microambiente Tumoral
2.
Appl Microbiol Biotechnol ; 108(1): 459, 2024 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-39230729

RESUMO

The recombinant adeno-associated virus (rAAV) vector is among the most promising viral vectors in gene therapy. However, the limited manufacturing capacity in human embryonic kidney (HEK) cells is a barrier to rAAV commercialization. We investigated the impact of endoplasmic reticulum (ER) protein processing and apoptotic genes on transient rAAV production in HEK293 cells. We selected four candidate genes based on prior transcriptomic studies: XBP1, GADD34 / PPP1R15A, HSPA6, and BCL2. These genes were stably integrated into HEK293 host cells. Traditional triple-plasmid transient transfection was used to assess the vector production capability and the quality of both the overexpressed stable pools and the parental cells. We show that the overexpression of XBP1, HSPA6, and GADD34 increases rAAV productivity by up to 100% and increases specific rAAV productivity by up to 78% in HEK293T cells. Additionally, more prominent improvement associated with ER protein processing gene overexpression was observed when parental cell productivity was high, but no substantial variation was detected under low-producing conditions. We also confirmed genome titer improvement across different serotypes (AAV2 and AAV8) and different cell lines (HEK293T and HEK293); however, the extent of improvement may vary. This study unveiled the importance of ER protein processing pathways in viral particle synthesis, capsid assembly, and vector production. KEY POINTS: • Upregulation of endoplasmic reticulum (ER) protein processing (XBP1, HSPA6, and GADD34) leads to a maximum 100% increase in rAAV productivity and a maximum 78% boost in specific rAAV productivity in HEK293T cells • The enhancement in productivity can be validated across different HEK293 cell lines and can be used for the production of various AAV serotypes, although the extent of the enhancement might vary slightly • The more pronounced improvements linked to overexpressing ER protein processing genes were observed when parental cell productivity was high, with minimal variation noted under low-producing conditions.


Assuntos
Dependovirus , Retículo Endoplasmático , Vetores Genéticos , Proteína 1 de Ligação a X-Box , Humanos , Células HEK293 , Dependovirus/genética , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismo , Retículo Endoplasmático/metabolismo , Vetores Genéticos/genética , Expressão Gênica , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Capsídeo/metabolismo
3.
Proc Natl Acad Sci U S A ; 121(36): e2408787121, 2024 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-39207734

RESUMO

Protein phosphatase-1 catalytic subunit (PP1) joins diverse targeting subunits to form holophosphatases that regulate many cellular processes. Newly synthesized PP1 is known to be transiently sequestered in an inhibitory complex with Suppressor-of-Dis2-number-2 (SDS22) and Inhibitor-3 (I3), which is disassembled by the ATPases Associated with diverse cellular Activities plus (AAA+) protein p97. Here, we show that the SDS22-PP1-I3 complex also acts as a thermodynamic sink for mature PP1 and that cycles of SDS22-PP1-I3 formation and p97-driven disassembly regulate PP1 function and subunit exchange beyond PP1 biogenesis. Förster Resonance energy transfer (FRET) analysis of labeled proteins in vitro revealed that in the p97-mediated disassembly step, both SDS22 and I3 dissociate concomitantly, releasing PP1. In presence of a targeting subunit, for instance Growth Arrest and DNA Damage-inducible protein 34 (GADD34), liberated PP1 formed an active holophosphatase that dephosphorylated its substrate, eukaryotic translation initiation factor 2 alpha (eIF2α). Inhibition of p97 results in displacement of the GADD34 targeting subunit by rebinding of PP1 to SDS22 and I3 indicating that the SDS22-PP1-I3 complex is thermodynamically favored. Likewise, p97 inhibition in cells causes rapid sequestration of PP1 by free SDS22 and I3 at the expense of other subunits. This suggests that PP1 exists in a steady state maintained by spontaneous SDS22-PP1-I3 formation and adenosine triphosphate (ATP) hydrolysis, p97-driven disassembly that recycles active PP1 between different holophosphatase complexes to warrant a dynamic holophosphatase landscape.


Assuntos
Proteína Fosfatase 1 , Proteína Fosfatase 1/metabolismo , Humanos , Ligação Proteica , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Holoenzimas/metabolismo , Transferência Ressonante de Energia de Fluorescência , Fosforilação , Proteína Fosfatase 2C
4.
Commun Biol ; 7(1): 1073, 2024 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-39217250

RESUMO

The coordination between kinases and phosphatases is crucial for regulating the phosphorylation levels of essential signaling molecules. Methods enabling precise control of kinase activities are valuable for understanding the kinase functions and for developing targeted therapies. Here, we use the abscisic acid (ABA)-induced proximity system to reversibly control kinase signaling by recruiting phosphatases. Using this method, we found that the oncogenic tyrosine kinase BCR::ABL1 can be inhibited by recruiting various cytoplasmic phosphatases. We also discovered that the oncogenic serine/threonine kinase BRAF(V600E), which has been reported to bypass phosphorylation regulation, can be positively regulated by protein phosphatase 1 (PP1) and negatively regulated by PP5. Additionally, we observed that the dual-specificity kinase MEK1 can be inhibited by recruiting PP5. This suggests that bifunctional molecules capable of recruiting PP5 to MEK or RAF kinases could be promising anticancer drug candidates. Thus, the ABA-induced dephosphorylation method enables rapid screening of phosphatases to precisely control kinase signaling.


Assuntos
Ácido Abscísico , Transdução de Sinais , Fosforilação , Transdução de Sinais/efeitos dos fármacos , Humanos , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacologia , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , MAP Quinase Quinase 1/metabolismo , Proteínas de Fusão bcr-abl/metabolismo , Proteínas de Fusão bcr-abl/genética , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 1/genética
5.
Nat Commun ; 15(1): 6810, 2024 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-39122682

RESUMO

Multiple myeloma is a hematological malignancy arising from immunoglobulin-secreting plasma cells. It remains poorly understood how chromatin rewiring of regulatory elements contributes to tumorigenesis and therapy resistance in myeloma. Here we generate a high-resolution contact map of myeloma-associated super-enhancers by integrating H3K27ac ChIP-seq and HiChIP from myeloma cell lines, patient-derived myeloma cells and normal plasma cells. Our comprehensive transcriptomic and phenomic analyses prioritize candidate genes with biological and clinical implications in myeloma. We show that myeloma cells frequently acquire SE that transcriptionally activate an oncogene PPP1R15B, which encodes a regulatory subunit of the holophosphatase complex that dephosphorylates translation initiation factor eIF2α. Epigenetic silencing or knockdown of PPP1R15B activates pro-apoptotic eIF2α-ATF4-CHOP pathway, while inhibiting protein synthesis and immunoglobulin production. Pharmacological inhibition of PPP1R15B using Raphin1 potentiates the anti-myeloma effect of bortezomib. Our study reveals that myeloma cells are vulnerable to perturbation of PPP1R15B-dependent protein homeostasis, highlighting a promising therapeutic strategy.


Assuntos
Regulação Neoplásica da Expressão Gênica , Mieloma Múltiplo , Proteína Fosfatase 1 , Proteostase , Super Intensificadores , Fator de Transcrição CHOP , Animais , Humanos , Fator 4 Ativador da Transcrição/metabolismo , Fator 4 Ativador da Transcrição/genética , Bortezomib/farmacologia , Linhagem Celular Tumoral , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 2 em Eucariotos/genética , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 1/genética , Super Intensificadores/genética , Fator de Transcrição CHOP/metabolismo , Fator de Transcrição CHOP/genética
6.
Cells ; 13(13)2024 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-38994993

RESUMO

The reparative and regenerative capabilities of dental pulp stem cells (DPSCs) are crucial for responding to pulp injuries, with protein phosphatase 1 (PP1) playing a significant role in regulating cellular functions pertinent to tissue healing. Accordingly, this study aimed to explore the effects of a novel cell-penetrating peptide Modified Sperm Stop 1-MSS1, that disrupts PP1, on the proliferation and odontogenic differentiation of DPSCs. Employing MSS1 as a bioportide, DPSCs were cultured and characterized for metabolic activity, cell proliferation, and cell morphology alongside the odontogenic differentiation through gene expression and alkaline phosphatase (ALP) activity analysis. MSS1 exposure induced early DPSC proliferation, upregulated genes related to odontogenic differentiation, and increased ALP activity. Markers associated with early differentiation events were induced at early culture time points and those associated with matrix mineralization were upregulated at mid-culture stages. This investigation is the first to document the potential of a PP1-disrupting bioportide in modulating DPSC functionality, suggesting a promising avenue for enhancing dental tissue regeneration and repair.


Assuntos
Diferenciação Celular , Proliferação de Células , Polpa Dentária , Odontogênese , Proteína Fosfatase 1 , Células-Tronco , Polpa Dentária/citologia , Polpa Dentária/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/metabolismo , Humanos , Proteína Fosfatase 1/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Odontogênese/efeitos dos fármacos , Peptídeos/farmacologia , Peptídeos/metabolismo , Células Cultivadas , Fosfatase Alcalina/metabolismo
7.
CNS Neurosci Ther ; 30(7): e14830, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-39046182

RESUMO

N6-methyladenosine (m6A) methylation is a vital epigenetic mechanism associated with drug addiction. However, the relationship between m6A modification and oxycodone rewarding is less well explored. Based on an open field test, the present study evaluated oxycodone rewarding using chromatin immunoprecipitation PCR, immunofluorescence, and RNA sequencing. A marked increase in METTL14 protein and a decrease in PP1α protein due to oxycodone abundance in the striatal neurons were observed in a dose- and time-dependent manner. Oxycodone markedly increased LSD1 expression, and decreased H3K4me1 expression in the striatum. In the open field test, intra-striatal injection of METTL14 siRNA, HOTAIR siRNA, or LSD1 shRNA blocked oxycodone-induced increase in locomotor activity. The downregulation of PP1α was also inhibited after treatment with METTL14/HOTAIR siRNA and LSD1 shRNA. Enhanced binding of LSD1 with CoRest and of CoRest with the PP1α gene induced by oxycodone was also reversed by LSD1 shRNA. In addition, H3K4me1 demethylation was also blocked by the treatment. In summary, the investigation confirmed that METTL14-mediated upregulation of HOTAIR resulted in the repression of PP1α, which in turn facilitated the recruitment of LSD1, thus catalyzing H3K4me1 demethylation and promoting oxycodone addiction.


Assuntos
Metiltransferases , Oxicodona , RNA Longo não Codificante , Animais , Masculino , Camundongos , Corpo Estriado/metabolismo , Corpo Estriado/efeitos dos fármacos , Desmetilação , Histona Desmetilases/metabolismo , Histona Desmetilases/genética , Histonas/metabolismo , Lisina/análogos & derivados , Metiltransferases/metabolismo , Metiltransferases/genética , Camundongos Endogâmicos C57BL , Oxicodona/farmacologia , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 1/genética , RNA Longo não Codificante/metabolismo , RNA Longo não Codificante/genética , Regulação para Cima
8.
J Med Case Rep ; 18(1): 269, 2024 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-38835078

RESUMO

BACKGROUND: Acute myeloid leukemia (AML) is the second most common type of leukemia in children. Although prognostic and diagnostic tests of AML patients have improved, there is still a great demand for new reliable clinical biomarkers for AML. Read-through fusion transcripts (RTFTs) are complex transcripts of adjacent genes whose molecular mechanisms are poorly understood. This is the first report of the presence of the PPP1R1B::STARD3 fusion transcript in an AML patient. Here, we investigated the presence of PPP1R1B::STARD3 RTFT in a case of AML using paired-end RNA sequencing (RNA-seq). CASE PRESENTATION: A Persian 12-year-old male was admitted to Dr. Sheikh Hospital of Mashhad, Iran, in September 2019 with the following symptoms, including fever, convulsions, hemorrhage, and bone pain. The patient was diagnosed with AML (non-M3-FAB subtype) based on cell morphologies and immunophenotypical features. Chromosomal analysis using the G-banding technique revealed t (9;22) (q34;q13). CONCLUSIONS: Single-cell RNA sequencing (scRNA-seq) analysis suggested that the PPP1R1B promoter may be responsible for the PPP1R1B::STARD3 expression. Alterations in the level of lipid metabolites implicate cancer development, and this fusion can play a crucial role in the cholesterol movement in cancer cells. PPP1R1B::STARD3 may be considered a candidate for targeted therapies of the cholesterol metabolic and the PI3K/AKT signaling pathways involved in cancer development and progression.


Assuntos
Leucemia Mieloide Aguda , Humanos , Masculino , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/diagnóstico , Criança , Proteína Fosfatase 1/genética , Proteínas de Fusão Oncogênica/genética
9.
Int J Mol Sci ; 25(12)2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38928151

RESUMO

Valosin-containing protein (VCP), an ATPase-associated protein, is emerging as a crucial regulator in cardiac pathologies. However, the pivotal role of VCP in the heart under physiological conditions remains undetermined. In this study, we tested a hypothesis that sufficient VCP expression is required for cardiac development and physiological cardiac function. Thus, we generated a cardiac-specific VCP knockout (KO) mouse model and assessed the consequences of VCP suppression on the heart through physiological and molecular studies at baseline. Our results reveal that homozygous KO mice are embryonically lethal, whereas heterozygous KO mice with a reduction in VCP by ~40% in the heart are viable at birth but progressively develop heart failure and succumb to mortality at the age of 10 to 12 months. The suppression of VCP induced a selective activation of the mammalian target of rapamycin complex 1 (mTORC1) but not mTORC2 at the early age of 12 weeks. The prolonged suppression of VCP increased the expression (by ~2 folds) and nuclear translocation (by >4 folds) of protein phosphatase 1 (PP1), a key mediator of protein dephosphorylation, accompanied by a remarked reduction (~80%) in AKTSer473 phosphorylation in VCP KO mouse hearts at a later age but not the early stage. These temporal molecular alterations were highly associated with the progressive decline in cardiac function. Overall, our findings shed light on the essential role of VCP in the heart under physiological conditions, providing new insights into molecular mechanisms in the development of heart failure.


Assuntos
Insuficiência Cardíaca , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos Knockout , Proteína Fosfatase 1 , Proteína com Valosina , Animais , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/genética , Proteína com Valosina/metabolismo , Proteína com Valosina/genética , Camundongos , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 1/genética , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Miocárdio/metabolismo , Miocárdio/patologia , Masculino , Modelos Animais de Doenças
10.
PLoS Pathog ; 20(6): e1012355, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38935808

RESUMO

Stress granules (SGs), formed by untranslated messenger ribonucleoproteins (mRNPs) during cellular stress in eukaryotes, have been linked to flavivirus interference without clear understanding. This study reveals the role of Zika virus (ZIKV) NS2B as a scaffold protein mediating interaction between protein phosphatase 1α (PP1α) and eukaryotic initiation factor 2α (eIF2α). This interaction promotes eIF2α dephosphorylation by PP1α, inhibiting SG formation. The NS2B-PP1α complex exhibits remarkable stability, resisting ubiquitin-induced degradation and amplifying eIF2α dephosphorylation, thus promoting ZIKV replication. In contrast, the NS2BV35A mutant, interacting exclusively with eIF2α, fails to inhibit SG formation, resulting in reduced viral replication and diminished impact on brain organoid growth. These findings reveal PP1α's dual role in ZIKV infection, inducing interferon production as an antiviral factor and suppressing SG formation as a viral promoter. Moreover, we found that NS2B also serves as a versatile mechanism employed by flaviviruses to counter host antiviral defenses, primarily by broadly inhibiting SG formation. This research advances our comprehension of the complex interplay in flavivirus-host interactions, offering potential for innovative therapeutic strategies against flavivirus infections.


Assuntos
Fator de Iniciação 2 em Eucariotos , Proteína Fosfatase 1 , Grânulos de Estresse , Proteínas não Estruturais Virais , Replicação Viral , Infecção por Zika virus , Zika virus , Zika virus/fisiologia , Replicação Viral/fisiologia , Humanos , Infecção por Zika virus/virologia , Infecção por Zika virus/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas não Estruturais Virais/genética , Proteína Fosfatase 1/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Grânulos de Estresse/metabolismo , Animais
11.
Nat Commun ; 15(1): 5111, 2024 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-38877002

RESUMO

Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) is a determinant of cardiac myofilament function. Although cMyBP-C phosphorylation by various protein kinases has been extensively studied, the influence of protein phosphatases on cMyBP-C's multiple phosphorylation sites has remained largely obscure. Here we provide a detailed biochemical characterization of cMyBP-C dephosphorylation by protein phosphatases 1 and 2 A (PP1 and PP2A), and develop an integrated kinetic model for cMyBP-C phosphorylation using data for both PP1, PP2A and various protein kinases known to phosphorylate cMyBP-C. We find strong site-specificity and a hierarchical mechanism for both phosphatases, proceeding in the opposite direction of sequential phosphorylation by potein kinase A. The model is consistent with published data from human patients and predicts complex non-linear cMyBP-C phosphorylation patterns that are validated experimentally. Our results suggest non-redundant roles for PP1 and PP2A under both physiological and heart failure conditions, and emphasize the importance of phosphatases for cMyBP-C regulation.


Assuntos
Proteínas de Transporte , Miocárdio , Proteína Fosfatase 1 , Proteína Fosfatase 2 , Fosforilação , Humanos , Proteína Fosfatase 1/metabolismo , Proteínas de Transporte/metabolismo , Animais , Proteína Fosfatase 2/metabolismo , Miocárdio/metabolismo , Proteínas Quinases/metabolismo , Cinética
12.
Nat Commun ; 15(1): 5359, 2024 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-38918402

RESUMO

SDS22 forms an inactive complex with nascent protein phosphatase PP1 and Inhibitor-3. SDS22:PP1:Inhibitor-3 is a substrate for the ATPase p97/VCP, which liberates PP1 for binding to canonical regulatory subunits. The exact role of SDS22 in PP1-holoenzyme assembly remains elusive. Here, we show that SDS22 stabilizes nascent PP1. In the absence of SDS22, PP1 is gradually lost, resulting in substrate hyperphosphorylation and a proliferation arrest. Similarly, we identify a female individual with a severe neurodevelopmental disorder bearing an unstable SDS22 mutant, associated with decreased PP1 levels. We furthermore find that SDS22 directly binds to Inhibitor-3 and that this is essential for the stable assembly of SDS22:PP1: Inhibitor-3, the recruitment of p97/VCP, and the extraction of SDS22 during holoenzyme assembly. SDS22 with a disabled Inhibitor-3 binding site co-transfers with PP1 to canonical regulatory subunits, thereby forming non-functional holoenzymes. Our data show that SDS22, through simultaneous interaction with PP1 and Inhibitor-3, integrates the major steps of PP1 holoenzyme assembly.


Assuntos
Proteína Fosfatase 1 , Feminino , Humanos , Células HEK293 , Holoenzimas/metabolismo , Fosforilação , Ligação Proteica , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 1/genética , Proteína com Valosina/metabolismo , Proteína com Valosina/genética
13.
Ann Hematol ; 103(8): 2853-2863, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38842564

RESUMO

Acute Myeloid Leukemia (AML) is a life-threatening disease whose induction treatment consists of combination chemotherapy with Idarubicin and Cytarabine for fit patients. Treatment failures are frequent, urging the need for novel treatments for this disease. The DNA Damage Response Mechanism (DDR) comprises numerous molecules and pathways intended to arrest the cell cycle until DNA damage is repaired or else drive the cell to apoptosis. AML-derived cell lines after treatment with Idarubicin and Cytarabine were used for studying the expression profile of 84 DDR genes, through PCR arrays. Utilizing de novo AML patient and control samples we studied the expression of PPP1R15A, CDKN1A, GADD45A, GADD45G, and EXO1. Next, we performed PPP1R15A silencing in AML cell lines in two separate experiments using siRNA and CRISPR-cas9, respectively. Our findings highlight that DDR regulators demonstrate increased expression in patients with high cytogenetic risk possibly reflecting increased genotoxic stress. Especially, PPP1R15A is mainly involved in the recovery of the cells from stress and it was the only DDR gene upregulated in AML patients. The PPP1R15A silencing resulted in decreased viability of Idarubicin and Cytarabine-treated cell lines, in contrast to untreated cells. These findings shed light on new strategies to enhance chemotherapy efficacy and demonstrate that PPP1R15A is an important DDR regulator in AML and its downregulation might be a safe and effective way to increase sensitivity to chemotherapy in this disease.


Assuntos
Citarabina , Dano ao DNA , Inativação Gênica , Leucemia Mieloide Aguda , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Dano ao DNA/efeitos dos fármacos , Citarabina/farmacologia , Linhagem Celular Tumoral , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Idarubicina/farmacologia , Idarubicina/administração & dosagem , Masculino , Feminino , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Reparo do DNA/efeitos dos fármacos , Pessoa de Meia-Idade , Adulto , Idoso , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos
14.
Carcinogenesis ; 45(9): 673-684, 2024 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-38715543

RESUMO

Esophageal cancer is one of the most common malignant tumors, and the 5-year overall survival rate is only 20%. Esophageal squamous cell carcinoma (ESCC) is the primary histological type of esophageal carcinoma in China. Protein phosphatase 1 regulatory subunit 18 (PPP1r18) is one of the actin-regulatory proteins and is able to bind to protein phosphatase 1 catalytic subunit alpha (PPP1CA). Yet, little is known about the role of PPP1r18 in ESCC. This study aimed to elucidate the biological functions of PPP1r18 in the ESCC progression. Clinical samples first confirmed that PPP1r18 expression was upregulated in ESCC, and PPP1r18 was correlated with tumor invasion depth, lymph node metastasis, distant metastasis and reduced overall survival. We then observed that PPP1r18 overexpression enhanced cell proliferation in vitro and in vivo. Mechanistically, PPP1r18 regulated tumor progression of ESCC through activating the calcineurin-mediated ERK pathway, rather than binding to PPP1CA. Collectively, our results suggest that PPP1r18 promotes ESCC progression by regulating the calcineurin-mediated ERK pathway. PPP1r18 might be a potential target for the diagnosis and treatment of ESCC.


Assuntos
Calcineurina , Proliferação de Células , Progressão da Doença , Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Regulação Neoplásica da Expressão Gênica , Sistema de Sinalização das MAP Quinases , Humanos , Carcinoma de Células Escamosas do Esôfago/patologia , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/metabolismo , Calcineurina/metabolismo , Calcineurina/genética , Neoplasias Esofágicas/patologia , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/mortalidade , Proliferação de Células/genética , Animais , Camundongos , Masculino , Feminino , Sistema de Sinalização das MAP Quinases/genética , Pessoa de Meia-Idade , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Prognóstico , Linhagem Celular Tumoral , Camundongos Nus , Movimento Celular/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Metástase Linfática
15.
Open Biol ; 14(5): 230460, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38806145

RESUMO

The precise spatial and temporal control of histone phosphorylations is important for the ordered progression through the different phases of mitosis. The phosphorylation of H2B at S6 (H2B S6ph), which is crucial for chromosome segregation, reaches its maximum level during metaphase and is limited to the inner centromere. We discovered that the temporal and spatial regulation of this modification, as well as its intensity, are governed by the scaffold protein RepoMan and its associated catalytically active phosphatases, PP1α and PP1γ. Phosphatase activity is inhibited at the area of maximal H2B S6 phosphorylation at the inner centromere by site-specific Aurora B-mediated inactivation of the PP1/RepoMan complex. The motor protein Mklp2 contributes to the relocalization of Aurora B from chromatin to the mitotic spindle during anaphase, thus alleviating Aurora B-dependent repression of the PP1/RepoMan complex and enabling dephosphorylation of H2B S6. Accordingly, dysregulation of Mklp2 levels, as commonly observed in tumour cells, leads to the lack of H2B S6 dephosphorylation during early anaphase, which might contribute to chromosomal instability.


Assuntos
Aurora Quinase B , Proteínas de Ciclo Celular , Histonas , Mitose , Proteína Fosfatase 1 , Aurora Quinase B/metabolismo , Fosforilação , Humanos , Histonas/metabolismo , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 1/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Células HeLa , Fuso Acromático/metabolismo , Centrômero/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética
16.
Nucleic Acids Res ; 52(12): 6866-6885, 2024 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-38783162

RESUMO

The genomes of Leishmania and trypanosomes are organized into polycistronic transcription units flanked by a modified DNA base J involved in promoting RNA polymerase II (Pol II) termination. We recently characterized a Leishmania complex containing a J-binding protein, PP1 protein phosphatase 1, and PP1 regulatory protein (PNUTS) that controls transcription termination potentially via dephosphorylation of Pol II by PP1. While T. brucei contains eight PP1 isoforms, none purified with the PNUTS complex, complicating the analysis of PP1 function in termination. We now demonstrate that the PP1-binding motif of TbPNUTS is required for function in termination in vivo and that TbPP1-1 modulates Pol II termination in T. brucei and dephosphorylation of the large subunit of Pol II. PP1-1 knock-down results in increased cellular levels of phosphorylated RPB1 accompanied by readthrough transcription and aberrant transcription of the chromosome by Pol II, including Pol I transcribed loci that are typically silent, such as telomeric VSG expression sites involved in antigenic variation. These results provide important insights into the mechanism underlying Pol II transcription termination in primitive eukaryotes that rely on polycistronic transcription and maintain allelic exclusion of VSG genes.


Assuntos
Alelos , Proteína Fosfatase 1 , Proteínas de Protozoários , RNA Polimerase II , Terminação da Transcrição Genética , Trypanosoma brucei brucei , Glicoproteínas Variantes de Superfície de Trypanosoma , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/enzimologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Glicoproteínas Variantes de Superfície de Trypanosoma/genética , Glicoproteínas Variantes de Superfície de Trypanosoma/metabolismo , Fosforilação , Transcrição Gênica
17.
Antiviral Res ; 226: 105895, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38679165

RESUMO

Rift Valley fever virus (RVFV) is an arbovirus in the Phenuiviridae family identified initially by the large 'abortion storms' observed among ruminants; RVFV can also infect humans. In humans, there is a wide variation of clinical symptoms ranging from subclinical to mild febrile illness to hepatitis, retinitis, delayed-onset encephalitis, or even hemorrhagic fever. The RVFV is a tri-segmented negative-sense RNA virus consisting of S, M, and L segments. The L segment encodes the RNA-dependent RNA polymerase (RdRp), termed the L protein, which is responsible for both viral mRNA synthesis and genome replication. Phosphorylation of viral RdRps is known to regulate viral replication. This study shows that RVFV L protein is serine phosphorylated and identified Casein Kinase 1 alpha (CK1α) and protein phosphatase 1 alpha (PP1α) as L protein binding partners. Inhibition of CK1 and PP1 through small molecule inhibitor treatment, D4476 and 1E7-03, respectively, caused a change in the phosphorylated status of the L protein. Inhibition of PP1α resulted in increased L protein phosphorylation whereas inhibition of CK1α decreased L protein phosphorylation. It was also found that in RVFV infected cells, PP1α localized to the cytoplasmic compartment. Treatment of RVFV infected cells with CK1 inhibitors reduced virus production in both mammalian and mosquito cells. Lastly, inhibition of either CK1 or PP1 reduced viral genomic RNA levels. These data indicate that L protein is phosphorylated and that CK1 and PP1 play a crucial role in regulating the L protein phosphorylation cycle, which is critical to viral RNA production and viral replication.


Assuntos
Proteína Fosfatase 1 , Vírus da Febre do Vale do Rift , Replicação Viral , Vírus da Febre do Vale do Rift/fisiologia , Vírus da Febre do Vale do Rift/genética , Fosforilação , Humanos , Animais , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 1/genética , Genoma Viral , Proteínas Virais/metabolismo , Proteínas Virais/genética , Caseína Quinase Ialfa/metabolismo , Caseína Quinase Ialfa/genética , Chlorocebus aethiops , Linhagem Celular , RNA Polimerase Dependente de RNA/metabolismo , RNA Polimerase Dependente de RNA/genética , Células Vero , RNA Viral/genética , RNA Viral/metabolismo , Febre do Vale de Rift/virologia
18.
Life Sci ; 345: 122608, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38574885

RESUMO

BACKGROUND AND AIMS: The protein phosphatase 1 regulatory inhibitor subunit 1A (PPP1R1A) has been linked with insulin secretion and diabetes mellitus. Yet, its full significance in pancreatic ß-cell function remains unclear. This study aims to elucidate the role of the PPP1R1A gene in ß-cell biology using human pancreatic islets and rat INS-1 (832/13) cells. RESULTS: Disruption of Ppp1r1a in INS-1 cells was associated with reduced insulin secretion and impaired glucose uptake; however, cell viability, ROS, apoptosis or proliferation were intact. A significant downregulation of crucial ß-cell function genes such as Ins1, Ins2, Pcsk1, Cpe, Pdx1, Mafa, Isl1, Glut2, Snap25, Vamp2, Syt5, Cacna1a, Cacna1d and Cacnb3, was observed upon Ppp1r1a disruption. Furthermore, silencing Pdx1 in INS-1 cells altered PPP1R1A expression, indicating that PPP1R1A is a target gene for PDX1. Treatment with rosiglitazone increased Ppp1r1a expression, while metformin and insulin showed no effect. RNA-seq analysis of human islets revealed high PPP1R1A expression, with α-cells showing the highest levels compared to other endocrine cells. Muscle tissues exhibited greater PPP1R1A expression than pancreatic islets, liver, or adipose tissues. Co-expression analysis revealed significant correlations between PPP1R1A and genes associated with insulin biosynthesis, exocytosis machinery, and intracellular calcium transport. Overexpression of PPP1R1A in human islets augmented insulin secretion and upregulated protein expression of Insulin, MAFA, PDX1, and GLUT1, while silencing of PPP1R1A reduced Insulin, MAFA, and GLUT1 protein levels. CONCLUSION: This study provides valuable insights into the role of PPP1R1A in regulating ß-cell function and glucose homeostasis. PPP1R1A presents a promising opportunity for future therapeutic interventions.


Assuntos
Células Secretoras de Insulina , Ilhotas Pancreáticas , Proteína Fosfatase 1 , Animais , Humanos , Ratos , Canais de Cálcio/metabolismo , Linhagem Celular , Glucose/metabolismo , Insulina/metabolismo , Secreção de Insulina/genética , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo
19.
Trends Neurosci ; 47(5): 319-321, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38614892

RESUMO

In a recent study, Oliveira and colleagues revealed how growth arrest and DNA damage-inducible protein 34 (GADD34), an effector of the integrated stress response, initiates the translation of synaptic plasticity-related mRNAs following brain-derived neurotrophic factor (BDNF) stimulation. This work suggests that GADD34 may link transcriptional products with translation control upon neuronal activation, illuminating how protein synthesis is orchestrated in neuronal plasticity.


Assuntos
Plasticidade Neuronal , Neurônios , Biossíntese de Proteínas , Proteína Fosfatase 1 , Animais , Humanos , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Plasticidade Neuronal/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia , Biossíntese de Proteínas/fisiologia , Proteína Fosfatase 1/metabolismo , Estresse Fisiológico/fisiologia
20.
Cells ; 13(8)2024 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-38667309

RESUMO

Variants of mitochondrial DNA (mtDNA) have been identified as risk factors for the development of Parkinson's disease (PD). However, the underlying pathogenetic mechanisms remain unclear. Cybrid models carrying various genotypes of mtDNA variants were tested for resistance to PD-simulating MPP+ treatment. The most resistant line was selected for transcriptome profiling, revealing specific genes potentially influencing the resistant characteristic. We then conducted protein validation and molecular biological studies to validate the related pathways as the influential factor. Cybrids carrying the W3 mtDNA haplogroup demonstrated the most resistance to the MPP+ treatment. In the transcriptome study, PPP1R15A was identified, while further study noted elevated expressions of the coding protein GADD34 across all cybrids. In the study of GADD34-related mitochondrial unfolding protein response (mtUPR), we found that canonical mtUPR, launched by the phosphate eIF2a, is involved in the resistant characteristic of specific mtDNA to MPP+ treatment. Our study suggests that a lower expression of GADD34 in the late phase of mtUPR may prolong the mtUPR process, thereby benefitting protein homeostasis and facilitating cellular resistance to PD development. We herein demonstrate that GADD34 plays an important role in PD development and should be further investigated as a target for the development of therapies for PD.


Assuntos
DNA Mitocondrial , Haplótipos , Doença de Parkinson , Doença de Parkinson/genética , Humanos , DNA Mitocondrial/genética , Haplótipos/genética , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/genética , Resposta a Proteínas não Dobradas/genética
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