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1.
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
2.
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
3.
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
4.
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
5.
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
6.
J Hum Genet ; 64(4): 313-322, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30655572

RESUMO

Casein kinase 2 (CK2) is a serine threonine kinase ubiquitously expressed in eukaryotic cells and involved in various cellular processes. In recent studies, de novo variants in CSNK2A1 and CSNK2B, which encode the subunits of CK2, have been identified in individuals with intellectual disability syndrome. In this study, we describe four patients with neurodevelopmental disorders possessing de novo variants in CSNK2A1 or CSNK2B. Using whole-exome sequencing, we detected two de novo variants in CSNK2A1 in two unrelated Japanese patients, a novel variant c.571C>T, p.(Arg191*) and a recurrent variant c.593A>G, p.(Lys198Arg), and two novel de novo variants in CSNK2B in Japanese and Malaysian patients, c.494A>G, p.(His165Arg) and c.533_534insGT, p.(Pro179Tyrfs*49), respectively. All four patients showed mild to profound intellectual disabilities, developmental delays, and various types of seizures. This and previous studies have found a total of 20 CSNK2A1 variants in 28 individuals with syndromic intellectual disability. The hotspot variant c.593A>G, p.(Lys198Arg) was found in eight of 28 patients. Meanwhile, only five CSNK2B variants were identified in five individuals with neurodevelopmental disorders. We reviewed the previous literature to verify the phenotypic spectrum of CSNK2A1- and CSNK2B-related syndromes.


Assuntos
Caseína Quinase II/genética , Deficiências do Desenvolvimento/genética , Convulsões/genética , Adolescente , Criança , Pré-Escolar , Deficiências do Desenvolvimento/complicações , Deficiências do Desenvolvimento/fisiopatologia , Feminino , Humanos , Lactente , Deficiência Intelectual/genética , Deficiência Intelectual/fisiopatologia , Masculino , Mutação , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/fisiopatologia , Linhagem , Convulsões/complicações , Convulsões/fisiopatologia , Sequenciamento Completo do Exoma
7.
J Biol Chem ; 294(7): 2365-2374, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30617183

RESUMO

The mammalian lipin 1 phosphatidate phosphatase is a key regulatory enzyme in lipid metabolism. By catalyzing phosphatidate dephosphorylation, which produces diacylglycerol, the enzyme plays a major role in the synthesis of triacylglycerol and membrane phospholipids. The importance of lipin 1 to lipid metabolism is exemplified by cellular defects and lipid-based diseases associated with its loss or overexpression. Phosphorylation of lipin 1 governs whether it is associated with the cytoplasm apart from its substrate or with the endoplasmic reticulum membrane where its enzyme reaction occurs. Lipin 1ß is phosphorylated on multiple sites, but less than 10% of them are ascribed to a specific protein kinase. Here, we demonstrate that lipin 1ß is a bona fide substrate for casein kinase II (CKII), a protein kinase that is essential to viability and cell cycle progression. Phosphoamino acid analysis and phosphopeptide mapping revealed that lipin 1ß is phosphorylated by CKII on multiple serine and threonine residues, with the former being major sites. Mutational analysis of lipin 1ß and its peptides indicated that Ser-285 and Ser-287 are both phosphorylated by CKII. Substitutions of Ser-285 and Ser-287 with nonphosphorylatable alanine attenuated the interaction of lipin 1ß with 14-3-3ß protein, a regulatory hub that facilitates the cytoplasmic localization of phosphorylated lipin 1. These findings advance our understanding of how phosphorylation of lipin 1ß phosphatidate phosphatase regulates its interaction with 14-3-3ß protein and intracellular localization and uncover a mechanism by which CKII regulates cellular physiology.


Assuntos
Caseína Quinase II/química , Fosfatidato Fosfatase/química , Fosfoproteínas/química , Proteínas 14-3-3 , Substituição de Aminoácidos , Animais , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Retículo Endoplasmático/química , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Camundongos , Mutação de Sentido Incorreto , Fosfatidato Fosfatase/genética , Fosfatidato Fosfatase/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação/genética , Serina/química , Serina/genética , Serina/metabolismo
8.
Int J Mol Med ; 43(2): 1033-1040, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30535443

RESUMO

Protein kinase casein kinase 2 (CK2) is important in the regulation of cell proliferation and death, even under pathological conditions. Previously, we reported that CK2 regulates the expression of heme oxygenase­1 (HO­1) in stress­induced chondrocytes. In the present study, it was shown that CK2 is involved in the dedifferentiation and cellular senescence of chondrocytes. Treatment of primary articular chondrocytes with CK2 inhibitors, 4,5,6,7­terabromo­2­azabenzimidazole (TBB) or 5,6­dichlorobenzimidazole 1­ß­D­ribofuranoside (DRB), induced an increase in senescence­associated ß­galactosidase (SA­ß­gal) staining. In addition, TBB reduced the expression of type II collagen and stimulated the accumulation of ß­catenin, phenotypic markers of chondrocyte differentiation and dedifferentiation, respectively. It was also observed that the abrogation of CK2 activity by CK2 small interfering RNA induced phenotypes of chondrocyte senescence. The association between HO­1 and cellular senescence was also examined in CK2 inhibitor­treated chondrocytes. Pretreatment with 3­morpholinosydnonimine hydrochloride, an inducer of the HO­1 expression, or overexpression of the HO­1 gene significantly delayed chondrocyte senescence. These results show that CK2 is associated with chondrocyte differentiation and cellular senescence and that this is due to regulation of the expression of HO­1. Furthermore, the findings suggest that CK2 is crucial as an anti­aging factor during chondrocyte senescence.


Assuntos
Cartilagem Articular/citologia , Cartilagem Articular/metabolismo , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/metabolismo , Condrócitos/metabolismo , Regulação da Expressão Gênica , Heme Oxigenase-1/genética , Animais , Caseína Quinase II/genética , Senescência Celular/genética , Heme Oxigenase-1/metabolismo , Masculino , Ratos , Triazóis/farmacologia
9.
Proc Natl Acad Sci U S A ; 115(30): E7081-E7090, 2018 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-29987005

RESUMO

The huntingtin N17 domain is a modulator of mutant huntingtin toxicity and is hypophosphorylated in Huntington's disease (HD). We conducted high-content analysis to find compounds that could restore N17 phosphorylation. One lead compound from this screen was N6-furfuryladenine (N6FFA). N6FFA was protective in HD model neurons, and N6FFA treatment of an HD mouse model corrects HD phenotypes and eliminates cortical mutant huntingtin inclusions. We show that N6FFA restores N17 phosphorylation levels by being salvaged to a triphosphate form by adenine phosphoribosyltransferase (APRT) and used as a phosphate donor by casein kinase 2 (CK2). N6FFA is a naturally occurring product of oxidative DNA damage. Phosphorylated huntingtin functionally redistributes and colocalizes with CK2, APRT, and N6FFA DNA adducts at sites of induced DNA damage. We present a model in which this natural product compound is salvaged to provide a triphosphate substrate to signal huntingtin phosphorylation via CK2 during low-ATP stress under conditions of DNA damage, with protective effects in HD model systems.


Assuntos
Adenina , Adutos de DNA/metabolismo , Dano ao DNA , Doença de Huntington/tratamento farmacológico , Neurônios/metabolismo , Transdução de Sinais/efeitos dos fármacos , Adenina/análogos & derivados , Adenina/farmacocinética , Adenina/farmacologia , Adenina Fosforribosiltransferase/genética , Adenina Fosforribosiltransferase/metabolismo , Animais , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Linhagem Celular Transformada , Adutos de DNA/genética , Modelos Animais de Doenças , Humanos , Doença de Huntington/genética , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Camundongos , Camundongos Transgênicos , Neurônios/patologia , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Transdução de Sinais/genética
10.
Planta ; 248(3): 571-578, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29799081

RESUMO

MAIN CONCLUSION: Our transient gene expression analyses in Arabidopsis protoplasts support the view that CK2αs and CK2ßs positively and negatively modulate ABRE-dependent gene expression, respectively. The phytohormone abscisic acid (ABA) regulates the expression of thousands of genes via ABA-responsive elements (ABREs), and has a crucial role in abiotic stress response. Casein kinase II (CK2), a conserved Ser/Thr protein kinase in eukaryotes, is essential for plant viability. Although the CK2 has been known as a tetrameric holoenzyme comprised of two catalytic α and two regulatory ß subunits, each of the two types of subunits has been proposed to have independent functions. The Arabidopsis genome encodes four α subunits (CK2α1, CK2α2, CK2α3, CK2α4) and four ß subunits (CK2ß1, CK2ß2, CK2ß3, CK2ß4). There is a growing body of evidence linking CK2 to ABA signaling and abiotic stress responses. However, the roles of each CK2 subunit in ABA signaling remain largely elusive. Using the transient expression system with the core ABA signaling components in Arabidopsis leaf mesophyll protoplasts, we show here that CK2α1 and CK2α2 (CK2α1/2) positively modulate ABRE-dependent gene expression as ABA signal output in ABA signaling, whereas all four CK2ßs negatively modulate the ABRE-dependent gene expression mediated by subclass III SnRK2-AREB/ABF pathway and by CK2α1/2. These data indicate that CK2α1/2 and CK2ßs positively and negatively modulate ABA signal output, respectively, suggesting that the quantitative balance of CK2 subunits determines the ABA signal output in plants. Given that CK2s act as pleiotropic enzymes involved in multiple developmental and stress-responsive processes, our findings suggest that CK2 subunits may be involved in integration and coordination of ABA-dependent and -independent signaling.


Assuntos
Ácido Abscísico/metabolismo , Arabidopsis/metabolismo , Caseína Quinase II/metabolismo , Reguladores de Crescimento de Planta/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Caseína Quinase II/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Filogenia , Folhas de Planta/metabolismo , Protoplastos/metabolismo , Transdução de Sinais
11.
Mol Med Rep ; 17(6): 8397-8402, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29658601

RESUMO

Casein kinase 2 (CK2) is a serine/threonine protein kinase that has been considered to represent an important factor in mammary tumorigenesis. Increased expression of matrix metalloproteinase­9 (MMP­9) via nuclear factor­κB (NF­κB) activation has been demonstrated to promote breast cancer cell invasion. In the present study, the involvement of CK2 in protein kinase C (PKC) induced cell invasion in MCF­7 breast cancer cells was investigated as well as the underlying molecular mechanisms. The mRNA and protein levels of MMP­9 in MCF­7 cells were investigated using reverse transcription­quantitative polymerase chain reaction, western blot analyses and a zymography assay. Cell invasiveness was investigated using a Matrigel invasion assay, and it was revealed that small interfering RNA specific for CK2 suppressed PKC induced cell invasion by regulating MMP­9 expression via activation of the p38 kinase/c­Jun N­terminal kinase/NF­κB pathway. In addition, it was demonstrated that CK2 inhibitors [apigenin (20 µM), emodin (20 µM) or 2­dimethylamino­4,5,6,7­tetrabromo­1H­benzimidazole (2 µM)] suppressed PKC induced cell invasion and MMP­9 expression. The results of the present study suggested that CK2 is an important factor involved in the induction of MCF­7 breast cancer cell invasion by PKC. Therefore, CK2 may represent novel candidates for therapy intended to inhibit invasion in breast cancer.


Assuntos
Caseína Quinase II/genética , Inativação Gênica , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Proteína Quinase C/metabolismo , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Sobrevivência Celular/genética , Expressão Gênica , Humanos , Células MCF-7 , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Interferência de RNA
12.
Cell Physiol Biochem ; 45(5): 1818-1826, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29510389

RESUMO

BACKGROUND/AIMS: Cerebral ischemia-reperfusion (I/R) injury involves multiple independently fatal terminal pathways. CK2α/NADPH oxidase is an important signaling pathway associated with ischemia-reperfusion injury, and miR-125b can regulate oxidative stress-related injury. In this study, we investigated whether the effect of miR-125b in rat brain I/R injury occurs through its modulation of the CK2α/NADPH oxidase pathway. METHODS: Rats were subjected to 2 h of cerebral ischemia followed by 24 h of reperfusion to establish an I/R injury model. Neurological deficit was evaluated using a five-point score. Infarct volume was evaluated with 2, 3, 5-triphenyltetrazolium chloride (TTC) staining, and RT-PCR was used to detect expressions of miR125b and CK2α. We then examined the association between miR-125b expression and the CK2α/NADPH oxidative signaling pathway in a PC-12 cell oxygen-glucose deprivation and reoxygenation (OGD/R) injury model. Transfection with miR-125b mimics, an miR-125b inhibitor, and luciferase reporter gene plasmid was accomplished using commercial kits. In these cells, Western blots were used to detect the levels of expression of CK2α, cleaved caspase-3, NOX2, and NOX4. RT-PCR was used to detect the expressions of CK2α, miR125b, NOX2, and NOX4. We evaluated Lactate Dehydrogenase (LDH) level, NADPH oxidase activity, and caspase-3 activity using commercial kits. Mitochondrial reactive oxygen species (ROS) were measured by fluorescence microscopy. For both PC-12 cells and rat brains, histological analyses were conducted to observe morphological changes, and apoptosis was measured using a commercial kit. RESULTS: I/R rats exhibited an increase in neurological deficit score, infarct volume, and cellular apoptosis, along with miR-125b elevation and CK2α downregulation. OGD/R treatment increased PC-12 cells' injuries, cellular apoptosis, and ROS levels. These changes were associated with miR-125b elevation, CK2α downregulation and activations of NOX2 and NOX4, mimicking our in vivo findings. All of these effects were reversed by the inhibition of miR-125b, confirming a strong correlation between miR-125b activity and the CK2α/NADPH oxidase signaling pathway. CONCLUSIONS: Based on these observations, we conclude that inhibition of miR-125b protects the rat brain from I/R injury by regulating the CK2α/NADPH oxidative signaling pathway.


Assuntos
Caseína Quinase II/metabolismo , MicroRNAs/metabolismo , NADPH Oxidases/metabolismo , Animais , Antagomirs/metabolismo , Apoptose , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/genética , Caspase 3/metabolismo , Hipóxia Celular , Modelos Animais de Doenças , Regulação para Baixo , L-Lactato Desidrogenase/metabolismo , Masculino , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/genética , Células PC12 , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão , Transdução de Sinais
13.
Nat Commun ; 9(1): 838, 2018 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-29483533

RESUMO

Recent genome-wide association studies (GWAS) have identified multiple risk loci that show strong associations with schizophrenia. However, pinpointing the potential causal genes at the reported loci remains a major challenge. Here we identify candidate causal genes for schizophrenia using an integrative genomic approach. Sherlock integrative analysis shows that ALMS1, GLT8D1, and CSNK2B are schizophrenia risk genes, which are validated using independent brain expression quantitative trait loci (eQTL) data and integrative analysis method (SMR). Consistently, gene expression analysis in schizophrenia cases and controls further supports the potential role of these three genes in the pathogenesis of schizophrenia. Finally, we show that GLT8D1 and CSNK2B knockdown promote the proliferation and inhibit the differentiation abilities of neural stem cells, and alter morphology and synaptic transmission of neurons. These convergent lines of evidence suggest that the ALMS1, CSNK2B, and GLT8D1 genes may be involved in pathophysiology of schizophrenia.


Assuntos
Caseína Quinase II/genética , Predisposição Genética para Doença , Glicosiltransferases/genética , Esquizofrenia/genética , Animais , Encéfalo/metabolismo , Caseína Quinase II/metabolismo , Diferenciação Celular , Proliferação de Células , Técnicas de Silenciamento de Genes , Glicosiltransferases/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Proteínas/genética , Proteínas/metabolismo , Locos de Características Quantitativas , Esquizofrenia/metabolismo , Esquizofrenia/fisiopatologia
14.
Photosynth Res ; 137(1): 69-83, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29330702

RESUMO

In higher plant chloroplasts, the plastid-encoded RNA polymerase (PEP) consists of four catalytic subunits and numerous nuclear-encoded accessory proteins, including pTAC10, an S1-domain-containing protein. In this study, pTAC10 knockout lines were characterized. Two ptac10 mutants had an albino phenotype and severely impaired chloroplast development. The pTAC10 genomic sequence fused to a four-tandem MYC tag driven by its own promoter functionally complemented the ptac10-1 mutant phenotype. pTAC10 was present in both the chloroplast stroma and thylakoids. Two-dimensional blue native polyacrylamide gel electrophoresis (BN-PAGE), and immunoblotting assays showed that pTAC10:MYC co-migrates with one of the PEP core subunits, RpoB. A comprehensive investigation of the plastid gene expression profiles by quantitative RT-PCR revealed that, compared with wild-type plants, the abundance of PEP-dependent plastid transcripts is severely decreased in the ptac10-1 mutant, while the amount of plastid transcripts exclusively transcribed by NEP either barely changes or even increases. RNA blot analysis confirmed that PEP-dependent chloroplast transcripts, including psaB, psbA and rbcL, substantially decrease in the ptac10-1 mutant. Immunoblotting showed reduced accumulation of most chloroplast proteins in the ptac10 mutants. These data indicate the essential role of pTAC10 in plastid gene expression and plastid development. pTAC10 interacts with chloroplast-targeted casein kinase 2 (cpCK2) in vitro and in vivo and can be phosphorylated by Arabidopsis cpCK2 in vitro at sites Ser95, Ser396 and Ser434. RNA-EMSA assays showed that pTAC10 is able to bind to the psbA, atpE and accD transcripts, suggesting a non-specific RNA-binding activity of pTAC10. The RNA affinity of pTAC10 was enhanced by phosphorylation and decreased by the amino acid substitution Ser434-Ala of pTAC10. These data show that pTAC10 is essential for plastid gene expression in Arabidopsis and that it can be phosphorylated by cpCK2.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Caseína Quinase II/metabolismo , Proteínas de Cloroplastos/metabolismo , Plastídeos/genética , Substituição de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Caseína Quinase II/genética , Proteínas de Cloroplastos/genética , Cloroplastos/genética , Cloroplastos/metabolismo , Regulação da Expressão Gênica de Plantas , Mutação , Fosforilação , Complexo de Proteína do Fotossistema II/genética , Complexo de Proteína do Fotossistema II/metabolismo , Plantas Geneticamente Modificadas , Domínios Proteicos , RNA de Plantas/metabolismo , Tilacoides/metabolismo
15.
Cell Mol Life Sci ; 75(11): 2011-2026, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29119230

RESUMO

CK2 denotes a ubiquitous and pleiotropic protein kinase whose holoenzyme is composed of two catalytic (α and/or α') and two regulatory ß subunits. The CK2 consensus sequence, S/T-x-x-D/E/pS/pT is present in numerous phosphosites, but it is not clear how many of these are really generated by CK2. To gain information about this issue, advantage has been taken of C2C12 cells entirely deprived of both CK2 catalytic subunits by the CRISPR/Cas9 methodology. A comparative SILAC phosphoproteomics analysis reveals that, although about 30% of the quantified phosphosites do conform to the CK2 consensus, only one-third of these are substantially reduced in the CK2α/α'(-/-) cells, consistent with their generation by CK2. A parallel study with C2C12 cells deprived of the regulatory ß subunit discloses a role of this subunit in determining CK2 targeting. We also find that phosphosites notoriously generated by CK2 are not fully abrogated in CK2α/α'(-/-) cells, while some phosphosites unrelated to CK2 are significantly altered. Collectively taken our data allow to conclude that the phosphoproteome generated by CK2 is not as ample and rigidly pre-determined as it was believed before. They also show that the lack of CK2 promotes phosphoproteomics perturbations attributable to kinases other than CK2.


Assuntos
Caseína Quinase II/metabolismo , Fosfopeptídeos/metabolismo , Animais , Caseína Quinase II/genética , Linhagem Celular , Deleção de Genes , Técnicas de Inativação de Genes , Camundongos , Fosfopeptídeos/análise , Fosforilação , Proteômica/métodos
16.
PLoS One ; 12(12): e0188854, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29206231

RESUMO

A multitude of proteins are aberrantly expressed in cancer cells, including the oncogenic serine-threonine kinase CK2. In a previous report, we found increases in CK2 transcript expression that could explain the increased CK2 protein levels found in tumors from lung and bronchus, prostate, breast, colon and rectum, ovarian and pancreatic cancers. We also found that, contrary to the current notions about CK2, some CK2 transcripts were downregulated in several cancers. Here, we investigate all other cancers using Oncomine to determine whether they also display significant CK2 transcript dysregulation. As anticipated from our previous analysis, we found cancers with all CK2 transcripts upregulated (e.g. cervical), and cancers where there was a combination of upregulation and/or downregulation of the CK2 transcripts (e.g. sarcoma). Unexpectedly, we found some cancers with significant downregulation of all CK2 transcripts (e.g. testicular cancer). We also found that, in some cases, CK2 transcript levels were already dysregulated in benign lesions (e.g. Barrett's esophagus). We also found that CK2 transcript upregulation correlated with lower patient survival in most cases where data was significant. However, there were two cancer types, glioblastoma and renal cell carcinoma, where CK2 transcript upregulation correlated with higher survival. Overall, these data show that the expression levels of CK2 genes is highly variable in cancers and can lead to different patient outcomes.


Assuntos
Caseína Quinase II/genética , Neoplasias/genética , RNA Mensageiro/genética , Regulação para Baixo , Humanos , Neoplasias/classificação , Regulação para Cima
17.
Exp Mol Med ; 49(9): e375, 2017 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-28883547

RESUMO

Th17 cells promote inflammatory reactions, whereas regulatory T (Treg) cells inhibit them. Thus, the Th17/Treg cell balance is critically important in inflammatory diseases. However, the molecular mechanisms underlying this balance are unclear. Here, we demonstrate that casein kinase 2 (CK2) is a critical determinant of the Th17/Treg cell balance. Both the inhibition of CK2 with a specific pharmacological inhibitor, CX-4945, and its small hairpin RNA (shRNA)-mediated knockdown suppressed Th17 cell differentiation but reciprocally induced Treg cell differentiation in vitro. Moreover, CX-4945 ameliorated the symptoms of experimental autoimmune encephalomyelitis and reduced Th17 cell infiltration into the central nervous system. Mechanistically, CX-4945 inhibited the IL-6/STAT3 and Akt/mTOR signaling pathways. Thus, CK2 has a crucial role in regulating the Th17/Treg balance.


Assuntos
Caseína Quinase II/metabolismo , Diferenciação Celular , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/metabolismo , Células Th17/citologia , Células Th17/metabolismo , Animais , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/genética , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Encefalomielite Autoimune Experimental , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Contagem de Linfócitos , Camundongos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Receptores de Interleucina-6/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/imunologia , Serina-Treonina Quinases TOR , Células Th17/imunologia
18.
J Biol Chem ; 292(45): 18592-18607, 2017 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-28939766

RESUMO

Transcriptional regulation is modulated in part by chromatin-remodeling enzymes that control gene accessibility by altering chromatin compaction or nucleosome positioning. Brahma-related gene 1 (Brg1), a catalytic subunit of the mammalian SWI/SNF chromatin-remodeling enzymes, is required for both myoblast proliferation and differentiation, and the control of Brg1 phosphorylation by calcineurin, PKCß1, and p38 regulates the transition to differentiation. However, we hypothesized that Brg1 activity might be regulated by additional kinases. Here, we report that Brg1 is also a target of casein kinase 2 (CK2), a serine/threonine kinase, in proliferating myoblasts. We found that CK2 interacts with Brg1, and mutation of putative phosphorylation sites to non-phosphorylatable (Ser to Ala, SA) or phosphomimetic residues (Ser to Glu, SE) reduced Brg1 phosphorylation by CK2. Although BRG1-deleted myoblasts that ectopically express the SA-Brg1 mutant proliferated similarly to the parental cells or cells ectopically expressing wild-type (WT) Brg1, ectopic expression of the SE-Brg1 mutant reduced proliferation and increased cell death, similar to observations from cells lacking Brg1. Moreover, pharmacological inhibition of CK2 increased myoblast proliferation. Furthermore, the Pax7 promoter, which controls expression of a key transcription factor required for myoblast proliferation, was in an inaccessible chromatin state in the SE-Brg1 mutant, suggesting that hyperphosphorylated Brg1 cannot remodel chromatin. WT-, SA-, and SE-Brg1 exhibited distinct differences in interacting with and affecting expression of the SWI/SNF subunits Baf155 and Baf170 and displayed differential sub-nuclear localization. Our results indicate that CK2-mediated phosphorylation of Brg1 regulates myoblast proliferation and provides insight into one mechanism by which composition of the mammalian SWI/SNF enzyme complex is regulated.


Assuntos
Caseína Quinase II/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA Helicases/metabolismo , Regulação da Expressão Gênica , Mioblastos Esqueléticos/metabolismo , Proteínas Nucleares/metabolismo , Processamento de Proteína Pós-Traducional , Fatores de Transcrição/metabolismo , Substituição de Aminoácidos , Animais , Caseína Quinase II/efeitos dos fármacos , Caseína Quinase II/genética , Células Cultivadas , Proteínas Cromossômicas não Histona/química , DNA Helicases/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Mioblastos Esqueléticos/citologia , Mioblastos Esqueléticos/efeitos dos fármacos , Proteínas Nucleares/genética , Fator de Transcrição PAX7/agonistas , Fator de Transcrição PAX7/genética , Fator de Transcrição PAX7/metabolismo , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Multimerização Proteica/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/efeitos dos fármacos , Células Satélites de Músculo Esquelético/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética
19.
Vascul Pharmacol ; 99: 34-44, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28927755

RESUMO

Neointimal hyperplasia is a product of VSMC replication and consequent accumulation within the blood vessel wall. In this study, we determined whether inhibition of protein kinase CK2 and the resultant stabilisation of proline-rich homeodomain (PRH) could suppress VSMC proliferation. Both silencing and pharmacological inhibition of CK2 with K66 antagonised replication of isolated VSMCs. SiRNA-induced knockdown as well as ectopic overexpression of proline-rich homeodomain indicated that PRH disrupts cell cycle progression. Mutation of CK2 phosphorylation sites Ser163 and Ser177 within the PRH homeodomain enabled prolonged cell cycle arrest by PRH. Concomitant knockdown of PRH and inhibition of CK2 with K66 indicated that the anti-proliferative action of K66 required the presence of PRH. Both K66 and adenovirus-mediated gene transfer of S163C:S177C PRH impaired neointima formation in human saphenous vein organ cultures. Importantly, neither intervention had notable effects on cell cycle progression, cell survival or migration in cultured endothelial cells.


Assuntos
Proliferação de Células/efeitos dos fármacos , Proteínas de Homeodomínio/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Neointima , Inibidores de Proteínas Quinases/farmacologia , Fatores de Transcrição/metabolismo , Animais , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Proteínas de Homeodomínio/genética , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Hiperplasia , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/patologia , Mutação , Miócitos de Músculo Liso/enzimologia , Miócitos de Músculo Liso/patologia , Fosforilação , Domínios Proteicos Ricos em Prolina , Interferência de RNA , Ratos , Veia Safena/efeitos dos fármacos , Veia Safena/enzimologia , Veia Safena/patologia , Transdução de Sinais/efeitos dos fármacos , Técnicas de Cultura de Tecidos , Fatores de Transcrição/genética , Transfecção
20.
MBio ; 8(4)2017 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-28851847

RESUMO

The rotavirus nonstructural protein NSP1 repurposes cullin-RING E3 ubiquitin ligases (CRLs) to antagonize innate immune responses. By functioning as substrate adaptors of hijacked CRLs, NSP1 causes ubiquitination and proteasomal degradation of host proteins that are essential for expression of interferon (IFN) and IFN-stimulated gene products. The target of most human and porcine rotaviruses is the ß-transducin repeat-containing protein (ß-TrCP), a regulator of NF-κB activation. ß-TrCP recognizes a phosphorylated degron (DSGΦXS) present in the inhibitor of NF-κB (IκB); phosphorylation of the IκB degron is mediated by IκB kinase (IKK). Because NSP1 contains a C-terminal IκB-like degron (ILD; DSGXS) that recruits ß-TrCP, we investigated whether the NSP1 ILD is similarly activated by phosphorylation and whether this modification is required to trigger the incorporation of NSP1 into CRLs. Based on mutagenesis and phosphatase treatment studies, we found that both serine residues of the NSP1 ILD are phosphorylated, a pattern mimicking phosphorylation of IκB. A three-pronged approach using small-molecule inhibitors, small interfering RNAs, and mutagenesis demonstrated that NSP1 phosphorylation is mediated by the constitutively active casein kinase II (CKII), rather than IKK. In coimmunoprecipitation assays, we found that this modification was essential for NSP1 recruitment of ß-TrCP and induced changes involving the NSP1 N-terminal RING motif that allowed formation of Cul3-NSP1 complexes. Taken together, our results indicate a highly regulated stepwise process in the formation of NSP1-Cul3 CRLs that is initiated by CKII phosphorylation of NSP1, followed by NSP1 recruitment of ß-TrCP and ending with incorporation of the NSP1-ß-TrCP complex into the CRL via interactions dependent on the highly conserved NSP1 RING motif.IMPORTANCE Rotavirus is a segmented double-stranded RNA virus that causes severe diarrhea in young children. A primary mechanism used by the virus to inhibit host innate immune responses is to hijack cellular cullin-RING E3 ubiquitin ligases (CRLs) and redirect their targeting activity to the degradation of cellular proteins crucial for interferon expression. This task is accomplished through the rotavirus nonstructural protein NSP1, which incorporates itself into a CRL and serves as a substrate recognition subunit. The substrate recognized by the NSP1 of many human and porcine rotaviruses is ß-TrCP, a protein that regulates the transcription factor NF-κB. In this study, we show that formation of NSP1 CRLs is a highly regulated stepwise process initiated by CKII phosphorylation of the ß-TrCP recognition motif in NSP1. This modification triggers recruitment of the ß-TrCP substrate and induces subsequent changes in a highly conserved NSP1 RING domain that allow anchoring of the NSP1-ß-TrCP complex to a cullin scaffold.


Assuntos
Caseína Quinase II/metabolismo , Proteínas Culina/metabolismo , Rotavirus/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Caseína Quinase II/genética , Proteínas Culina/genética , Interações Hospedeiro-Patógeno , Humanos , Proteínas I-kappa B/metabolismo , Imunidade Inata , Mutagênese , NF-kappa B/metabolismo , Fosforilação , Proteólise , RNA Interferente Pequeno , Rotavirus/genética , Rotavirus/imunologia , Infecções por Rotavirus/imunologia , Infecções por Rotavirus/virologia , Transdução de Sinais , Suínos , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitinação , Proteínas não Estruturais Virais/genética , Proteínas Contendo Repetições de beta-Transducina/genética , Proteínas Contendo Repetições de beta-Transducina/metabolismo
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