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1.
Nat Commun ; 11(1): 2289, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32385263

RESUMO

The osteoblast differentiation capacity of skeletal stem cells (SSCs) must be tightly regulated, as inadequate bone formation results in low bone mass and skeletal fragility, and over-exuberant osteogenesis results in heterotopic ossification (HO) of soft tissues. RUNX2 is essential for tuning this balance, but the mechanisms of posttranslational control of RUNX2 remain to be fully elucidated. Here, we identify that a CK2/HAUSP pathway is a key regulator of RUNX2 stability, as Casein kinase 2 (CK2) phosphorylates RUNX2, recruiting the deubiquitinase herpesvirus-associated ubiquitin-specific protease (HAUSP), which stabilizes RUNX2 by diverting it away from ubiquitin-dependent proteasomal degradation. This pathway is important for both the commitment of SSCs to osteoprogenitors and their subsequent maturation. This CK2/HAUSP/RUNX2 pathway is also necessary for HO, as its inhibition blocked HO in multiple models. Collectively, active deubiquitination of RUNX2 is required for bone formation and this CK2/HAUSP deubiquitination pathway offers therapeutic opportunities for disorders of inappropriate mineralization.


Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Ossificação Heterotópica/metabolismo , Osteogênese , Adulto , Idoso , Animais , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Diferenciação Celular , Displasia Cleidocraniana/genética , Displasia Cleidocraniana/patologia , Feminino , Deleção de Genes , Haploinsuficiência/genética , Membro Posterior/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Ossificação Heterotópica/genética , Ossificação Heterotópica/patologia , Osteoblastos/metabolismo , Fosforilação , Estabilidade Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Peptidase 7 Específica de Ubiquitina/metabolismo
2.
PLoS One ; 15(1): e0227340, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31910234

RESUMO

The PI3K/Akt pathway is interconnected to protein kinase CK2, which directly phosphorylates Akt1 at S129. We have previously found that, in HK-2 renal cells, downregulation of the CK2 regulatory subunit ß (shCK2ß cells) reduces S129 Akt phosphorylation. Here, we investigated in more details how the different CK2 isoforms impact on Akt and other signaling pathways. We found that all CK2 isoforms phosphorylate S129 in vitro, independently of CK2ß. However, in HK-2 cells the dependence on CK2ß was confirmed by rescue experiments (CK2ß re-expression in shCK2ß HK-2 cells), suggesting the presence of additional components that drive Akt recognition by CK2 in cells. We also found that CK2ß downregulation altered the phosphorylation ratio between the two canonical Akt activation sites (pT308 strongly reduced, pS473 slightly increased) in HK-2 cells. Similar results were found in other cell lines where CK2ß was stably knocked out by CRISPR-Cas9 technology. The phosphorylation of rpS6 S235/S236, a downstream effector of Akt, was strongly reduced in shCK2ß HK-2 cells, while the phosphorylation of two Akt direct targets, PRAS40 T246 and GSK3ß S9, was increased. Differently to what observed in response to CK2ß down-regulation, the chemical inhibition of CK2 activity by cell treatment with the specific inhibitor CX-4945 reduced both the Akt canonical sites, pT308 and pS473. In CX-4945-treated cells, the changes in rpS6 pS235/S236 and GSK3ß pS9 mirrored those induced by CK2ß knock-down (reduction and slight increase, respectively); on the contrary, the effect on PRAS40 pT246 phosphorylation was sharply different, being strongly reduced by CK2 inhibition; this suggests that this Akt target might be dependent on Akt pS473 status in HK-2 cells. Since PI3K/Akt and ERK1/2/p90rsk pathways are known to be interconnected and both modulated by CK2, with GSK3ß pS9 representing a convergent point, we investigated if ERK1/2/p90rsk signaling was affected by CK2ß knock-down and CX-4945 treatment in HK-2 cells. We found that p90rsk was insensitive to any kind of CK2 targeting; therefore, the observation that, similarly, GSK3ß pS9 was not reduced by CK2 blockade suggests that GSK3ß phosphorylation is mainly under the control of p90rsk in these cells. However, we found that the PI3K inhibitor LY294002 reduced GSK3ß pS9, and concomitantly decreased Snail1 levels (a GSK3ß target and Epithelial-to-Mesenchymal transition marker). The effects of LY294002 were observed also in CK2ß-downregulated cells, suggesting that reducing GSK3ß pS9 could be a strategy to control Snail1 levels in any situation where CK2ß is defective, as possibly occurring in cancer cells.


Assuntos
Caseína Quinase II/genética , Glicogênio Sintase Quinase 3 beta/genética , Proteína Oncogênica v-akt/genética , Fatores de Transcrição da Família Snail/genética , Sistemas CRISPR-Cas/genética , Linhagem Celular , Cromonas/farmacologia , Transição Epitelial-Mesenquimal/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Morfolinas/farmacologia , Naftiridinas/farmacologia , Fosfatidilinositol 3-Quinases/genética , Fosforilação/efeitos dos fármacos , Isoformas de Proteínas , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Transdução de Sinais/efeitos dos fármacos
3.
Biochem J ; 477(2): 431-444, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-31904830

RESUMO

Protein Ser/Thr phosphatase-6 (PP6) regulates pathways for activation of NF-kB, YAP1 and Aurora A kinase (AURKA). PP6 is a heterotrimer comprised of a catalytic subunit, one of three different SAPS subunits and one of three different ankyrin-repeat ANKRD subunits. Here, we show FLAG-PP6C expressed in cells preferentially binds endogenous SAPS3, and the complex is active with the chemical substrate DiFMUP. SAPS3 has multiple acidic sequence motifs recognized by protein kinase CK2 (CK2) and SAPS3 is phosphorylated by purified CK2, without affecting its associated PP6 phosphatase activity. However, HA3-SAPS3-PP6 phosphatase activity using pT288 AURKA as substrate is significantly increased by phosphorylation with CK2. The substitution of Ala in nine putative phosphorylation sites in SAPS3 was required to prevent CK2 activation of the phosphatase. Different CK2 chemical inhibitors equally increased phosphorylation of endogenous AURKA in living cells, consistent with reduction in PP6 activity. CRISPR/Cas9 deletion or siRNA knockdown of SAPS3 resulted in highly activated endogenous AURKA, and a high proportion of cells with abnormal nuclei. Activation of PP6 by CK2 can form a feedback loop with bistable changes in substrates.


Assuntos
Aurora Quinase A/genética , Caseína Quinase II/química , Fosfoproteínas Fosfatases/genética , Alanina/genética , Substituição de Aminoácidos/genética , Aurora Quinase A/química , Sistemas CRISPR-Cas/genética , Caseína Quinase II/genética , Domínio Catalítico/genética , Inibidores Enzimáticos/farmacologia , Células HeLa , Humanos , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/química , Fosforilação/genética , Ligação Proteica/efeitos dos fármacos , RNA Interferente Pequeno/genética , Especificidade por Substrato/efeitos dos fármacos
4.
Int J Med Sci ; 17(1): 13-20, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31929734

RESUMO

Diabetes mellitus (DM) is a chronic disease found worldwide. Notably, BKS.Cg- Dock7m +/+ Leprdb/JNarl mice are useful animal models for studying type 2 diabetes mellitus (T2DM). In this study, we investigated casein kinase 2 alpha 1 (CSNK2A1) gene and protein expression in the liver tissues of mice at different ages (4, 16, and 32 weeks) using real-time quantitative polymerase chain reactions, western blotting, immunohistochemistry, and enzyme-linked immunosorbent assay. Our data paved the way for exploring BKS.Cg- Dock7m +/+ Leprdb/JNarl in the mouse model by demonstrating a significant increase in gene and protein expression in T2DM (+Leprdb/+Leprdb) mouse liver when compared to control (+Dock7m/+Dock7m) mouse liver. We also observed that CSNK2A1 protein level in the serum of T2DM patient group was higher than that of the control group, although the data was not statistically significant. Based on our findings, we can now understand the role of CSNK2A1 gene upregulation when encountering T2DM pathologies.


Assuntos
Diabetes Mellitus Tipo 2/genética , Predisposição Genética para Doença , Obesidade/genética , Receptores para Leptina/genética , Idoso , Animais , Glicemia , Caseína Quinase II/genética , Diabetes Mellitus Tipo 2/patologia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/genética , Humanos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Obesidade/patologia
5.
EBioMedicine ; 51: 102603, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31901862

RESUMO

BACKGROUND: Tumor necrosis factor α-induced protein 1 (TNFAIP1) is frequently downregulated in cancer cell lines and promotes cancer cell apoptosis. However, its role, clinical significance and molecular mechanisms in hepatocellular carcinoma (HCC) are unknown. METHODS: The expression of TNFAIP1 in HCC tumor tissues and cell lines was measured by Western blot and immunohistochemistry. The effects of TNFAIP1 on HCC proliferation, apoptosis, metastasis, angiogenesis and tumor formation were evaluated by Cell Counting Kit-8 (CCK8), Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL), transwell, tube formation assay in vitro and nude mice experiments in vivo. The interaction between TNFAIP1 and CSNK2B was validated by liquid chromatography-tandem mass spectrometry (LC-MS/MS), Co-immunoprecipitation and Western blot. The mechanism of how TNFAIP1 regulated nuclear factor-kappaB (NF-κB) pathway was analyzed by dual-luciferase reporter, immunofluorescence, quantitative Real-time polymerase chain reaction (RT-qPCR) and Western blot. FINDINGS: The TNFAIP1 expression is significantly decreased in HCC tissues and cell lines, and negatively correlated with the increased HCC histological grade. Overexpression of TNFAIP1 inhibits HCC cell proliferation, metastasis, angiogenesis and promotes cancer cell apoptosis both in vitro and in vivo, whereas the knockdown of TNFAIP1 in HCC cell displays opposite effects. Mechanistically, TNFAIP1 interacts with CSNK2B and promotes its ubiquitin-mediated degradation with Cul3, causing attenuation of CSNK2B-dependent NF-κB trans-activation in HCC cell. Moreover, the enforced expression of CSNK2B counteracts the inhibitory effects of TNFAIP1 on HCC cell proliferation, migration, and angiogenesis in vitro and in vivo. INTERPRETATION: Our results support that TNFAIP1 can act as a tumor suppressor of HCC by modulating TNFAIP1/CSNK2B/NF-κB pathway, implying that TNFAIP1 may represent a potential marker and a promising therapeutic target for HCC.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Carcinoma Hepatocelular/metabolismo , Caseína Quinase II/genética , Regulação para Baixo , Neoplasias Hepáticas/metabolismo , NF-kappa B/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Apoptose , Carcinogênese/genética , Carcinogênese/patologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Camundongos Nus , Pessoa de Meia-Idade , Invasividade Neoplásica , Metástase Neoplásica , Neovascularização Patológica/genética , Proteólise , Ubiquitina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
6.
Int J Mol Sci ; 20(23)2019 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-31779225

RESUMO

Protein kinase CK2 (CK2) is a highly conserved and ubiquitous kinase is involved in crucial biological processes, including proliferation, migration, and differentiation. CK2 holoenzyme is a tetramer composed by two catalytically active (α/α') and two regulatory (ß) subunits and exerts its function on a broad range of targets. In the brain, it regulates different steps of neurodevelopment, such as neural differentiation, neuritogenesis, and synaptic plasticity. Interestingly, CK2 mutations have been recently linked to neurodevelopmental disorders; however, the functional requirements of the individual CK2 subunits in neurodevelopment have not been yet investigated. Here, we disclose the role of CK2 on the migration and adhesion properties of GN11 cells, an established model of mouse immortalized neurons, by different in vitro experimental approaches. Specifically, the cellular requirement of this kinase has been assessed pharmacologically and genetically by exploiting CK2 inhibitors and by generating subunit-specific CK2 knockout GN11 cells (with a CRISPR/Cas9-based approach). We show that CK2α' subunit has a primary role in increasing cell adhesion and reducing migration properties of GN11 cells by activating the Akt-GSK3ß axis, whereas CK2α subunit is dispensable. Further, the knockout of the CK2ß regulatory subunits counteracts cell migration, inducing dramatic alterations in the cytoskeleton not observed in CK2α' knockout cells. Collectively taken, our data support the view that the individual subunits of CK2 play different roles in cell migration and adhesion properties of GN11 cells, supporting independent roles of the different subunits in these processes.


Assuntos
Caseína Quinase II/genética , Neurônios/citologia , Animais , Caseína Quinase II/metabolismo , Adesão Celular , Linhagem Celular , Movimento Celular , Técnicas de Silenciamento de Genes , Glicogênio Sintase Quinase 3 beta/metabolismo , Camundongos , Mutação , Neurônios/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais
7.
Mol Cells ; 42(11): 773-782, 2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31617338

RESUMO

Cellular senescence is an irreversible form of cell cycle arrest. Senescent cells have a unique gene expression profile that is frequently accompanied by senescence-associated heterochromatic foci (SAHFs). Protein kinase CK2 (CK2) downregulation can induce trimethylation of histone H3 Lys 9 (H3K9me3) and SAHFs formation by activating SUV39h1. Here, we present evidence that the PI3K-AKTmTOR-reactive oxygen species-p53 pathway is necessary for CK2 downregulation-mediated H3K9me3 and SAHFs formation. CK2 downregulation promotes SUV39h1 stability by inhibiting its proteasomal degradation in a p53dependent manner. Moreover, the dephosphorylation status of Ser 392 on p53, a possible CK2 target site, enhances the nuclear import and subsequent stabilization of SUV39h1 by inhibiting the interactions between p53, MDM2, and SUV39h1. Furthermore, p21Cip1/WAF1 is required for CK2 downregulation-mediated H3K9me3, and dephosphorylation of Ser 392 on p53 is important for efficient transcription of p21Cip1/WAF1. Taken together, these results suggest that CK2 downregulation induces dephosphorylation of Ser 392 on p53, which subsequently increases the stability of SUV39h1 and the expression of p21Cip1/WAF1, leading to H3K9me3 and SAHFs formation.


Assuntos
Caseína Quinase II/metabolismo , Senescência Celular , Histonas/metabolismo , Metiltransferases/metabolismo , Proteínas Repressoras/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Caseína Quinase II/genética , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação para Baixo , Células HCT116 , Heterocromatina/genética , Humanos , Lisina/metabolismo , Células MCF-7 , Metilação , Fosforilação , Estabilidade Proteica , Interferência de RNA , Serina/metabolismo , Transdução de Sinais/genética , Proteína Supressora de Tumor p53/genética
8.
Fish Shellfish Immunol ; 94: 643-653, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31563555

RESUMO

Protein kinase CK2 (CK2) is a ubiquitous serine/threonine kinase with multiple cellular functions in vertebrates including apoptosis, differentiation, proliferation, survival, tumorigenesis, signal transduction, immune regulation and inflammation. In the current study, the catalytic and regulatory subunit homologs of Litopenaeus vannamei protein kinase CK2 (LvCK2α and LvCK2ß) were cloned and characterized. LvCK2α has a full-length cDNA sequence of 1764 bp with a 1053 bp open reading frame (ORF) encoding a putative protein of 351 amino acids, which contains a typical serine/threonine kinase domain. On the other hand, LvCK2ß has a 1394 bp full-length cDNA with an ORF of 663 bp encoding a protein with 221 amino acids, which contains a Casein kinase II regulatory subunit domain. Sequence and phylogenetic analysis revealed that LvCK2 was evolutionary related with the CK2 of invertebrates. Quantitative reverse transcription PCR (RT-qPCR) analysis showed that LvCK2α and LvCK2ß transcripts were widely expressed in all shrimp tissues tested, and were both induced in hemocytes and hepatopancreas upon challenge with Vibrio parahaemolyticus, Streptoccocus iniae, lipopolysaccharide (LPS), and white spot syndrome virus (WSSV), suggesting their involvement in shrimp immune response. Moreover, RNA interference (RNAi) of LvCK2α resulted in increased hemocytes apoptosis, shown by high caspase 3/7 activity, increased number of apoptotic cells, coupled with an elevation in transcript levels of pro-apoptotic LvCaspase3 and LvCytochrome C, and a reduction in mRNA levels of pro-survival LvBcl2, LvIAP1, and LvIAP2. In addition, LvCK2α knockdown followed by V. parahaemolyticus challenge resulted in higher cumulative mortality of shrimp. Taken together, our current findings suggest that LvCK2 modulates shrimp hemocytes apoptosis as part of the innate immune response to pathogens.


Assuntos
Caseína Quinase II/genética , Caseína Quinase II/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Penaeidae/genética , Penaeidae/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Caseína Quinase II/química , Perfilação da Expressão Gênica , Lipopolissacarídeos/fisiologia , Filogenia , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/imunologia , Alinhamento de Sequência , Streptococcus iniae/fisiologia , Vibrio parahaemolyticus/fisiologia , Vírus da Síndrome da Mancha Branca 1/fisiologia
9.
Plant Cell Physiol ; 60(12): 2785-2796, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31424513

RESUMO

Phosphorus is one of the most important macronutrients required for plant growth and development. The importance of phosphorylation modification in regulating phosphate (Pi) homeostasis in plants is emerging. We performed phosphoproteomic profiling to characterize proteins whose degree of phosphorylation is altered in response to Pi starvation in rice root. A subset of 554 proteins, including 546 down-phosphorylated and eight up-phosphorylated proteins, exhibited differential phosphorylation in response to Pi starvation. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis with the differentially phosphorylated proteins indicated that RNA processing, transport, splicing and translation and carbon metabolism played critical roles in response to Pi starvation in rice. Levels of phosphorylation of four mitogen-activated protein kinases (MAPKs), including OsMAPK6, five calcium-dependent protein kinases (CDPKs) and OsCK2ß3 decreased in response to Pi starvation. The decreased phosphorylation level of OsMAPK6 was confirmed by Western blotting. Mutation of OsMAPK6 led to Pi accumulation under Pi-sufficient conditions. Motif analysis indicated that the putative MAPK, casein kinase 2 (CK2) and CDPK substrates represented about 54.4%, 21.5% and 4.7%, respectively, of the proteins exhibiting differential phosphorylation. Based on the motif analysis, 191, 151 and 46 candidate substrates for MAPK, CK2 and CDPK were identified. These results indicate that modification of phosphorylation profiles provides complementary information on Pi-starvation-induced processes, with CK2, MAPK and CDPK protein kinase families playing key roles in these processes in rice.


Assuntos
Caseína Quinase II/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oryza/metabolismo , Fosfatos/metabolismo , Proteínas de Plantas/metabolismo , Caseína Quinase II/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Proteínas Quinases Ativadas por Mitógeno/genética , Oryza/fisiologia , Fosfatos/deficiência , Proteínas de Plantas/genética
10.
J Biol Chem ; 294(40): 14546-14561, 2019 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-31371453

RESUMO

Many viral factors manipulate the host post-translational modification (PTM) machinery for efficient viral replication. In particular, phosphorylation and SUMOylation can distinctly regulate the activity of the human cytomegalovirus (HCMV) transactivator immediate early 2 (IE2). However, the molecular mechanism of this process is unknown. Using various structural, biochemical, and cell-based approaches, here we uncovered that IE2 exploits a cross-talk between phosphorylation and SUMOylation. A scan for small ubiquitin-like modifier (SUMO)-interacting motifs (SIMs) revealed two SIMs in IE2, and a real-time SUMOylation assay indicated that the N-terminal SIM (IE2-SIM1) enhances IE2 SUMOylation up to 4-fold. Kinetic analysis and structural studies disclosed that IE2 is a SUMO cis-E3 ligase. We also found that two putative casein kinase 2 (CK2) sites adjacent to IE2-SIM1 are phosphorylated in vitro and in cells. The phosphorylation drastically increased IE2-SUMO affinity, IE2 SUMOylation, and cis-E3 activity of IE2. Additional salt bridges between the phosphoserines and SUMO accounted for the increased IE2-SUMO affinity. Phosphorylation also enhanced the SUMO-dependent transactivation activity and auto-repression activity of IE2. Together, our findings highlight a novel mechanism whereby SUMOylation and phosphorylation of the viral cis-E3 ligase and transactivator protein IE2 work in tandem to enable transcriptional regulation of viral gene.


Assuntos
Caseína Quinase II/genética , Proteínas Imediatamente Precoces/genética , Fosforilação/genética , Proteína SUMO-1/genética , Sumoilação/genética , Transativadores/genética , Sítios de Ligação , Caseína Quinase II/química , Citomegalovirus/enzimologia , Citomegalovirus/genética , Regulação Viral da Expressão Gênica/genética , Humanos , Proteínas Imediatamente Precoces/química , Proteínas Imediatamente Precoces/metabolismo , Cinética , Domínios e Motivos de Interação entre Proteínas/genética , Processamento de Proteína Pós-Traducional , Proteína SUMO-1/química , Proteína SUMO-1/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Transativadores/química , Transativadores/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Replicação Viral/genética
11.
Prep Biochem Biotechnol ; 49(7): 659-670, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31066619

RESUMO

Small interfering RNA (siRNA)-based gene silencing strategy has high potential on suppressing specific molecular targets, involved in cancer progression. However, the lack of an effective nanocarrier system that safely delivers siRNA to its target still limits the clinical applications of siRNA. This study aimed to develop albumin-sericin nanoparticles (Alb-Ser NPs) as a novel siRNA delivery system for laryngeal cancer treatment. Nanoparticle formulations composed of albumin and sericin at different ratios (1:1, 2:1, 1:2 w/w) were synthesized by desolvation method. The nanoparticles were modified with poly-L-lysine (PLL) for siRNA binding and decorated with hyaluronic acid (HA) to target laryngeal cancer cell line, Hep-2. HA/PLL/Alb-Ser NPs were individually loaded with siRNAs for casein kinase 2 (CK2), Absent, Small, or Homeotic-Like (ASH2L), and Cyclin D1 genes, which are overexpressed in Hep-2 cells. Downregulation of genes was confirmed by real-time PCR (RT-PCR). Size, morphological, and thermogravimetric characterizations revealed that Alb-Ser NPs having 2:1 (w/w) ratio are the most optimized formulation. Between 36.8 and 61.3% of siRNA entrapment efficiencies were achieved. HA/PLL-siRNA/Alb-Ser (2:1) NPs-mediated gene silencing resulted in a significant inhibition of cell growth and induction of apoptosis in cells. Our findings showed that HA/PLL/Alb-Ser (2:1) NPs were promising as a siRNA carrier.


Assuntos
Técnicas de Transferência de Genes , Neoplasias Laríngeas/terapia , Nanopartículas/química , RNA Interferente Pequeno/administração & dosagem , Terapêutica com RNAi , Sericinas/química , Albumina Sérica Humana/química , Caseína Quinase II/genética , Linhagem Celular Tumoral , Ciclina D1/genética , Proteínas de Ligação a DNA/genética , Portadores de Fármacos/química , Humanos , Neoplasias Laríngeas/genética , Nanopartículas/ultraestrutura , Proteínas Nucleares/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/uso terapêutico , Terapêutica com RNAi/métodos , Fatores de Transcrição/genética
12.
Zhonghua Er Ke Za Zhi ; 57(5): 368-372, 2019 May 02.
Artigo em Chinês | MEDLINE | ID: mdl-31060130

RESUMO

Objective: To summarize the clinical features and gene variation characteristics of a child with Okur-Chung syndrome caused by CSNK2A1 gene variation. Methods: The medical records of one patient who was diagnosed with Okur-Chung syndrome in Department of Pediatrics, Xiangya Hospital of Central South University in July 2018 were analyzed. Using "CSNK2A1" gene as the keyword, relevant information about CSNK2A1 gene was searched at CNKI, Wangfang Data, OMIM, PubMed, ClinVar, Decipher (until August 2018). The characteristics of CSNK2A1 gene variation and the clinical phenotype of children with Okur-Chung syndrome were summarized. Results: The boy, 1 year and 8 months old, was sent to hospital at the age of 1 year and 6 months because of delayed growth for more than 1 year. He was susceptible to cough while eating or drinking. He was also suffering from constipation and poor sleep. Physical examination showed that his body weight was 10.2 kg, microcephalus, broad nasal bridge, micrognathia and hypotonia were observed. Whole exome-sequencing test identified a de novo heterozygous variation c.524A>G (p.D175G) in CSNK2A1 gene. This was the first case report of CSNK2A1 gene variation in the mainland of China. So far, a total of 52 cases were reported worldwide (52 single nucleotide variants), including 35 cases in 7 articles, 9 cases in Decipher database and 14 cases in ClinVar database, 6 of which were also reported in PubMed. In previously reported 52 cases, there were 48 missense variants, whereas, splice and frameshift variations were found in 3 cases and 1 case, respectively. Among the variation sites, p.K198R was the most common sites (12 cases), followed by p.R47 (6 cases), p.R80H (4 cases) and p.S51 (4 cases). Among these 52 cases, only 27 cases have been elaborately described in other studies, so the clinical characteristics were summarized in 28 cases eventually (including 27 cases in the articles and this patient), 27 of whom presented severe intellectual disability or global development delay, 1 case with mild language development delay, and 19 had hypotonia; 8 had autism spectrum disorders, 5 had attention deficit hyperactivity disorder, and 9 had sleep problems. 20 had dysmorphic facial features, 10 of them had microcephalus. 16 had failure to thrive or short stature, 12 had gastrointestinal or oromotor problem, 5 had immunological problem, and 4 had skin abnormalities. Conclusions: The main clinical feature of patients with CSNK2A1 gene variations is intellectual disability with multiple systems involved, such as microcephalus, abnormal facial shape and hypotonia. The variation of CSNK2A1 gene is the cause of Okur-Chung syndrome. Missense variation is the main cause, and P. K198R is the hotspot variation.


Assuntos
Deficiência Intelectual , Hipotonia Muscular , Mutação/genética , Caseína Quinase II/genética , China , Humanos , Lactente , Masculino , Mutação de Sentido Incorreto , Síndrome , Sequenciamento Completo do Genoma
13.
PLoS Pathog ; 15(5): e1007769, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31116803

RESUMO

The Human Papillomavirus E7 oncoprotein plays an essential role in the development and maintenance of malignancy, which it achieves through targeting a number of critical cell control pathways. An important element in the ability of E7 to contribute towards cell transformation is the presence of a Casein Kinase II phospho-acceptor site within the CR2 domain of the protein. Phosphorylation is believed to enhance E7 interaction with a number of different cellular target proteins, and thereby increase the ability of E7 to enhance cell proliferation and induce malignancy. However, there is little information on how important this site in E7 is, once the tumour cells have become fully transformed. In this study, we have performed genome editing of the HPV-18 E7 CKII recognition site in C4-1 cervical tumour-derived cells. We first show that mutation of HPV18 E7 S32/S34 to A32/A34 abolishes CKII phosphorylation of E7, and subsequently we have isolated C4-1 clones containing these mutations in E7. The cells continue to proliferate, but are somewhat more slow-growing than wild type cells, reach lower saturation densities, and are also more susceptible to low nutrient conditions. These cells are severely defective in matrigel invasion assays, partly due to downregulation of matrix metalloproteases (MMPs). Mechanistically, we find that phosphorylation of E7 plays a direct role in the ability of E7 to activate AKT signaling, which in turn is required for optimal levels of MMP secretion. These results demonstrate that the E7 CKII phospho-acceptor site thus continues to play an important role for E7's activity in cells derived from cervical cancers, and suggests that blocking this activity of E7 could be expected to have therapeutic potential.


Assuntos
Caseína Quinase II/metabolismo , Proliferação de Células , Transformação Celular Viral , Proteínas de Ligação a DNA/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Neoplasias do Colo do Útero/patologia , Caseína Quinase II/genética , Proteínas de Ligação a DNA/genética , Feminino , Humanos , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 13 da Matriz/metabolismo , Proteínas Oncogênicas Virais/genética , Fenótipo , Fosforilação , Células Tumorais Cultivadas , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo
14.
PLoS Pathog ; 15(5): e1007788, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31091289

RESUMO

Inhibition of human papillomavirus (HPV) replication is a promising therapeutic approach for intervening with HPV-related pathologies. Primary targets for interference are two viral proteins, E1 and E2, which are required for HPV replication. Both E1 and E2 are phosphoproteins; thus, the protein kinases that phosphorylate them might represent secondary targets to achieve inhibition of HPV replication. In the present study, we show that CX4945, an ATP-competitive small molecule inhibitor of casein kinase 2 (CK2) catalytic activity, suppresses replication of different HPV types, including novel HPV5NLuc, HPV11NLuc and HPV18NLuc marker genomes, but enhances the replication of HPV16 and HPV31. We further corroborate our findings using short interfering RNA (siRNA)-mediated knockdown of CK2 α and α' subunits in U2OS and CIN612 cells; we show that while both subunits are expressed in these cell lines, CK2α is required for HPV replication, but CK2α' is not. Furthermore, we demonstrate that CK2α acts in a kinase activity-dependent manner and regulates the stability and nuclear retention of endogenous E1 proteins of HPV11 and HPV18. This unique feature of CK2α makes it an attractive target for developing antiviral agents.


Assuntos
Papillomaviridae/fisiologia , Infecções por Papillomavirus/virologia , Fosfoproteínas/metabolismo , Proteínas Virais/metabolismo , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Neoplasias Ósseas/virologia , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Humanos , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Osteossarcoma/virologia , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/metabolismo , Fosfoproteínas/genética , Fosforilação , Células Tumorais Cultivadas , Proteínas Virais/genética
15.
FEBS J ; 286(8): 1561-1575, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30834696

RESUMO

The acronym CK2 (derived from the misnomer 'casein kinase-2') denotes a pleiotropic acidophilic protein kinase implicated in a plethora of cellular functions, whose abnormally high expression correlates with malignancy. CK2 holoenzyme is composed of two catalytic (α and/or α') and two noncatalytic ß-subunits. The ß-subunits are not responsible for either activation or inactivation of the catalytic ones. Hence, to gain additional information about the roles of the individual CK2 subunits, we have generated C2C12 myoblasts entirely devoid either of both catalytic subunits, or of the ß-subunit. Here, we show that while CK2α/α'(-/-) cells grow similarly to wild-type cells, the growth of CK2ß(-/-) cells is severely impaired, consistent with the hypothesis that not all cellular functions of the ß-subunit are mediated by CK2 holoenzyme. To get a deeper insight into the functional implications of the ß-subunit, a quantitative proteomics study of CK2ß(-/-) cells was performed, leading to the identification and quantification of more than 1200 proteins. Of these, 187 showed a significantly altered expression (fold change ≥ 1.5 or ≤ -1.5) as compared to wild-type cells. A functional analysis of these proteins discloses the implication of CK2ß in many processes, for example, cell cycle, proliferation, transport, metabolic processes, etc., and in some of which the catalytic subunits of CK2 do not seem to play a relevant role. On the other hand, the pool of ecto-CK2 is not apparently affected by the lack of the ß-subunit. Collectively, our data corroborate the concept that the cellular functions of the ß-subunit of CK2 are partially independent of CK2 holoenzyme.


Assuntos
Caseína Quinase II/metabolismo , Mioblastos/metabolismo , Proteômica/métodos , Animais , Caseína Quinase II/genética , Domínio Catalítico , Linhagem Celular , Proliferação de Células , Técnicas de Inativação de Genes , Camundongos , Subunidades Proteicas , Espectrometria de Massas em Tandem
16.
Sci Adv ; 5(3): eaav5078, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30906869

RESUMO

Defective nuclear lamina protein lamin A is associated with premature aging. Casein kinase 2 (CK2) binds the nuclear lamina, and inhibiting CK2 activity induces cellular senescence in cancer cells. Thus, it is feasible that lamin A and CK2 may cooperate in the aging process. Nuclear CK2 localization relies on lamin A and the lamin A carboxyl terminus physically interacts with the CK2α catalytic core and inhibits its kinase activity. Loss of lamin A in Lmna-knockout mouse embryonic fibroblasts (MEFs) confers increased CK2 activity. Conversely, prelamin A that accumulates in Zmpste24-deficent MEFs exhibits a high CK2α binding affinity and concomitantly reduces CK2 kinase activity. Permidine treatment activates CK2 by releasing the interaction between lamin A and CK2, promoting DNA damage repair and ameliorating progeroid features. These data reveal a previously unidentified function for nuclear lamin A and highlight an essential role for CK2 in regulating senescence and aging.


Assuntos
Envelhecimento/genética , Caseína Quinase II/genética , Lamina Tipo A/genética , Proteínas de Membrana/genética , Metaloendopeptidases/genética , Progéria/genética , Envelhecimento/efeitos dos fármacos , Envelhecimento/patologia , Animais , Caseína Quinase II/química , Núcleo Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Senescência Celular/genética , Dano ao DNA/genética , Reparo do DNA/efeitos dos fármacos , Modelos Animais de Doenças , Fibroblastos/efeitos dos fármacos , Humanos , Lamina Tipo A/química , Camundongos , Camundongos Knockout , Progéria/tratamento farmacológico , Progéria/patologia , Ligação Proteica/genética , Espermidina/farmacologia
17.
Apoptosis ; 24(5-6): 499-510, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30850922

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a deadly and progressive fibrotic lung disease, but the precise etiology remains elusive. IPF is characterized by the presence of apoptosis-resistant (myo)fibroblasts that relentlessly produce a collagen-rich extracellular matrix (ECM). Recent studies showed that an anti-cancer chemotherapy drug cisplatin is implicated in the development of pulmonary fibrosis, suggesting that the treatment of cancer patients with cisplatin may alter fibroblast viability. To address this possibility, we investigated the cisplatin-induced cell death mechanism in lung fibroblasts derived from IPF and non-IPF patients in response to a collagen matrix. IPF fibroblasts showed enhanced resistance to cisplatin-induced cell death compared to non-IPF fibroblasts in a time- and dose-dependent manner. Molecular study showed that the expression of γH2AX, PUMA and caspase-3/7 activity was abnormally reduced in IPF fibroblasts, suggesting that DNA damage-induced apoptosis caused by cisplatin was suppressed in IPF fibroblasts. Our study further revealed that DNA repair protein XRCC1 activity was aberrantly increased as a result of CK2 hyper-activation in cisplatin-treated IPF fibroblasts, and this alteration protected IPF fibroblasts from cisplatin-induced cell death. Our results showed that IPF fibroblasts residing in a collagen rich matrix are resistance to cisplatin-induced cell death due to the aberrantly high CK2/XRCC1-dependent DNA repair activity. This finding suggests that pulmonary fibrosis may develop and worsen due to the presence of apoptosis-resistant lung fibroblasts in cisplatin-treated cancer patients.


Assuntos
Antineoplásicos/farmacologia , Caseína Quinase II/metabolismo , Morte Celular/efeitos dos fármacos , Cisplatino/farmacologia , Fibroblastos/efeitos dos fármacos , Fibrose Pulmonar Idiopática/patologia , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/metabolismo , Apoptose/efeitos dos fármacos , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/genética , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Dano ao DNA , Fibroblastos/metabolismo , Fibroblastos/patologia , Expressão Gênica , Humanos , RNA Interferente Pequeno , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genética
18.
J Pathol ; 248(3): 363-376, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30883733

RESUMO

Ten-eleven translocation methylcytosine dioxygenase-1, TET1, takes part in active DNA demethylation. However, our understanding of DNA demethylation in cancer biology and its clinical significance remain limited. This study showed that TET1 expression correlated with poor survival in advanced-stage epithelial ovarian carcinoma (EOC), and with cell migration, anchorage-independent growth, cancer stemness, and tumorigenicity. In particular, TET1 was highly expressed in serous tubal intraepithelial carcinoma (STIC), a currently accepted type II EOC precursor, and inversely correlated with TP53 mutations. Moreover, TET1 could demethylate the epigenome and activate multiple oncogenic pathways, including an immunomodulation network having casein kinase II subunit alpha (CK2α) as a hub. Patients with TET1high CK2αhigh EOCs had the worst outcomes, and TET1-expressing EOCs were more sensitive to a CK2 inhibitor, both in vitro and in vivo. Our findings uncover the oncogenic and poor prognostic roles of TET1 in EOC and suggest an unexplored role of epigenetic reprogramming in early ovarian carcinogenesis. Moreover, the immunomodulator CK2α represents a promising new therapeutic target, warranting clinical trials of the tolerable CK2 inhibitor, CX4945, for precision medicine against EOC. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Assuntos
Caseína Quinase II/genética , Cistadenocarcinoma Seroso/patologia , Regulação Neoplásica da Expressão Gênica/genética , Oxigenases de Função Mista/genética , Proteínas Proto-Oncogênicas/genética , Animais , Carcinoma Epitelial do Ovário/genética , Carcinoma Epitelial do Ovário/patologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Cistadenocarcinoma Seroso/genética , Transição Epitelial-Mesenquimal/genética , Neoplasias das Tubas Uterinas/genética , Neoplasias das Tubas Uterinas/patologia , Feminino , Humanos , Camundongos Nus , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Prognóstico
19.
J Exp Clin Cancer Res ; 38(1): 131, 2019 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-30885251

RESUMO

BACKGROUND: Dysfunction of p53 is a key cause of cancer development, while CCDC106 can reduce p53 stability and is associated with lung cancer. However, the roles of CCDC106 in other cancer types and its upstream regulators have not been investigated. METHODS: The phosphorylation status was investigated by in vitro kinase assay and Western blotting using phosphorylation-specific antibodies. Co-immunoprecipitation assay and GST-pulldown were used to detect protein interaction. Cell viability, apoptosis, colony formation, wound-healing and invasion assays were measured for in vitro functional analyses. The in vivo effect of CCDC106 on tumor growth was investigated using a subcutaneous xenograft tumor mouse model. RESULTS: We demonstrated that CCDC106 knockdown enhanced apoptosis by stabilizing p53 and suppressed cell viability, colony formation, migration and invasion in cervical cancer HeLa and breast cancer MCF7 cells with wild-type p53 (wtp53), whereas CCDC106 overexpression exerted the opposite effects in normal breast epithelial HBL100 and cervical cancer SiHa cells with wtp53. However, CCDC106 had no similar effects on p53-mutant cervical and breast cancer cells (C33A and MDA-MB-231). Further study showed that CK2 interacts with CCDC106 through its regulatory ß subunit and then phosphorylates CCDC106 at Ser-130 and Ser-147. The phosphorylation of CCDC106 at Ser-130 and Ser-147 is required for its interaction with p53 and nuclear localization, respectively. Inhibiting CCDC106 phosphorylation by substituting both Ser-130 and Ser-147 with alanine or treating cells with the CK2 inhibitor CX-4945 abrogated CCDC106-induced p53 degradation and its oncogenic function in cells with wtp53. Wildtype CCDC106, but not Ser-130/- 147 mutant CCDC106, enhanced tumor growth and p53 degradation in a xenograft mouse model. Moreover, suppression of CCDC106 increased CX-4945 sensitivity of cancer cells with wtp53. CONCLUSION: This study revealed a CK2/CCDC106/p53 signaling axis in the progression of breast and cervical cancers, which may provide a new therapeutic target for cancer treatment.


Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Transporte/metabolismo , Caseína Quinase II/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Neoplasias do Colo do Útero/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Caseína Quinase II/genética , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Progressão da Doença , Feminino , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Xenoenxertos , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosforilação , Transfecção , Proteína Supressora de Tumor p53/antagonistas & inibidores , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia
20.
ChemMedChem ; 14(8): 833-841, 2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30786177

RESUMO

The ubiquitously expressed Ser/Thr kinase CK2 is a key regulator in a variety of key processes in normal and malignant cells. Due to its distinctive anti-apoptotic and tumor-driving properties, elevated levels of CK2 have frequently been found in tumors of different origin. In recent years, development of CK2 inhibitors has largely been focused on ATP-competitive compounds; however, targeting the CK2α/CK2ß interface has emerged as a further concept that might avoid selectivity issues. To address the CK2 subunit interaction site, we have synthesized halogenated CK2ß-mimicking cyclic peptides modified with the cell-penetrating peptide sC18 to mediate cellular uptake. We investigated the binding of the resulting chimeric peptides to recombinant human CK2α using a recently developed fluorescence anisotropy assay. The iodinated peptide sC18-I-Pc was identified as a potent CK2α ligand (Ki =0.622 µm). It was internalized in cells to a high extent and exhibited significant cytotoxicity toward cancerous HeLa cells (IC50 =37 µm) in contrast to non-cancerous HEK-293 cells. The attractive features and functionalities of sC18-I-Pc offer the opportunity for further improvement.


Assuntos
Caseína Quinase II/metabolismo , Desenho de Fármacos , Peptídeos/metabolismo , Inibidores de Proteínas Quinases/química , Sequência de Aminoácidos , Caseína Quinase II/química , Caseína Quinase II/genética , Sobrevivência Celular/efeitos dos fármacos , Polarização de Fluorescência , Células HEK293 , Células HeLa , Humanos , Peptídeos/química , Peptídeos/farmacologia , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação
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