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1.
J Biol Chem ; 299(12): 105455, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37949232

RESUMO

The Akt-Rheb-mTORC1 pathway plays a crucial role in regulating cell growth, but the mechanisms underlying the activation of Rheb-mTORC1 by Akt remain unclear. In our previous study, we found that CBAP was highly expressed in human T-ALL cells and primary tumors, and its deficiency led to reduced phosphorylation of TSC2/S6K1 signaling proteins as well as impaired cell proliferation and leukemogenicity. We also demonstrated that CBAP was required for Akt-mediated TSC2 phosphorylation in vitro. In response to insulin, CBAP was also necessary for the phosphorylation of TSC2/S6K1 and the dissociation of TSC2 from the lysosomal membrane. Here we report that CBAP interacts with AKT and TSC2, and knockout of CBAP or serum starvation leads to an increase in TSC1 in the Akt/TSC2 immunoprecipitation complexes. Lysosomal-anchored CBAP was found to override serum starvation and promote S6K1 and 4EBP1 phosphorylation and c-Myc expression in a TSC2-dependent manner. Additionally, recombinant CBAP inhibited the GAP activity of TSC2 complexes in vitro, leading to increased Rheb-GTP loading, likely due to the competition between TSC1 and CBAP for binding to the HBD domain of TSC2. Overexpression of the N26 region of CBAP, which is crucial for binding to TSC2, resulted in a decrease in mTORC1 signaling and an increase in TSC1 association with the TSC2/AKT complex, ultimately leading to increased GAP activity toward Rheb and impaired cell proliferation. Thus, we propose that CBAP can modulate the stability of TSC1-TSC2 as well as promote the translocation of TSC1/TSC2 complexes away from lysosomes to regulate Rheb-mTORC1 signaling.


Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina , Proteínas de Membrana , Proteínas Proto-Oncogênicas c-akt , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose Tuberosa , Humanos , Proliferação de Células , Guanosina Trifosfato/metabolismo , Imunoprecipitação , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Enriquecida em Homólogo de Ras do Encéfalo/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteína 1 do Complexo Esclerose Tuberosa/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo
2.
Proc Natl Acad Sci U S A ; 116(2): 566-574, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30587587

RESUMO

We report a patient who presented with congenital hypotonia, hypoventilation, and cerebellar histopathological alterations. Exome analysis revealed a homozygous mutation in the initiation codon of the NME3 gene, which encodes an NDP kinase. The initiation-codon mutation leads to deficiency in NME3 protein expression. NME3 is a mitochondrial outer-membrane protein capable of interacting with MFN1/2, and its depletion causes dysfunction in mitochondrial dynamics. Consistently, the patient's fibroblasts were characterized by a slow rate of mitochondrial dynamics, which was reversed by expression of wild-type or catalytic-dead NME3. Moreover, glucose starvation caused mitochondrial fragmentation and cell death in the patient's cells. The expression of wild-type and catalytic-dead but not oligomerization-attenuated NME3 restored mitochondrial elongation. However, only wild-type NME3 sustained ATP production and viability. Thus, the separate functions of NME3 in mitochondrial fusion and NDP kinase cooperate in metabolic adaptation for cell survival in response to glucose starvation. Given the critical role of mitochondrial dynamics and energy requirements in neuronal development, the homozygous mutation in NME3 is linked to a fatal mitochondrial neurodegenerative disorder.


Assuntos
Trifosfato de Adenosina , Metabolismo Energético/genética , Homozigoto , Dinâmica Mitocondrial/genética , Nucleosídeo NM23 Difosfato Quinases , Doenças Neurodegenerativas , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Linhagem Celular , Sobrevivência Celular , Feminino , Humanos , Masculino , Mitocôndrias/enzimologia , Mitocôndrias/genética , Mitocôndrias/patologia , Nucleosídeo NM23 Difosfato Quinases/genética , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Doenças Neurodegenerativas/enzimologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia
3.
Anal Chem ; 93(42): 14247-14255, 2021 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-34633808

RESUMO

Measurement of four dNTP pools is important for investigating metabolism, genome stability, and drug action. In this report, we developed a two-step method for quantitating dNTPs by the combination of rolling circle amplification (RCA) and quantitative polymerase chain reaction (qPCR). We used CircLigase to generate a single-strand DNA in circular monomeric configuration, which was then used for the first step of RCA reaction that contained three dNTPs in excess for quantification of one dNTP at limiting levels. The second step is the amplification of RCA products by qPCR, in which one primer was designed to be completely annealed with the polymeric ssDNA product but not the monomeric template DNA. Using 1 amol of the template in the assay, each dNTP from 0.02 to 2.5 pmol gave a linearity with r2 > 0.99, and the quantification was not affected by the presence of rNTPs. We further found that the preparation of biological samples for the RCA reaction required methanol and chloroform extraction. The method was so sensitive that 1 × 104 cells were sufficient for dNTP quantification with the results similar to those determined by a radio-isotope method using 2 × 105 cells. Thus, the RCA/qPCR method is convenient, cost-effective, and highly sensitive for dNTP quantification.


Assuntos
DNA , Polifosfatos , Bioensaio , DNA/genética , Técnicas de Amplificação de Ácido Nucleico , Reação em Cadeia da Polimerase
4.
J Biol Chem ; 294(27): 10686-10697, 2019 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-31152062

RESUMO

The yeast Candida albicans is the most prevalent opportunistic fungal pathogen in humans. Drug resistance among C. albicans isolates poses a common challenge, and overcoming this resistance represents an unmet need in managing this common pathogen. Here, we investigated CDC8, encoding thymidylate kinase (TMPK), as a potential drug target for the management of C. albicans infections. We found that the region spanning amino acids 106-123, namely the Ca-loop of C. albicans TMPK (CaTMPK), contributes to the hyperactivity of this enzyme compared with the human enzyme (hTMPK) and to the utilization of deoxyuridine monophosphate (dUMP)/deoxy-5-fluorouridine monophosphate (5-FdUMP) as a substrate. Notably, expression of CaTMPK, but not of hTMPK, produced dUTP/5-FdUTP-mediated DNA toxicity in budding yeast (Saccharomyces cerevisiae). CRISPR-mediated deletion of this Ca-loop in C. albicans revealed that the Ca-loop is critical for fungal growth and susceptibility to 5-fluorouridine (5-FUrd). Of note, pathogenic and drug-resistant C. albicans clones were similarly sensitive to 5-FUrd, and we also found that CaTMPK is essential for the growth of C. albicans In conclusion, these findings not only identified a target site for the development of CaTMPK-selective drugs, but also revealed that 5-FUrd may have potential utility as drug for managing C. albicans infections.


Assuntos
Candida albicans/enzimologia , Proteínas Fúngicas/química , Núcleosídeo-Fosfato Quinase/química , Pirimidinas/farmacologia , Sequência de Aminoácidos , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Candida albicans/crescimento & desenvolvimento , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Edição de Genes , Humanos , Cinética , Testes de Sensibilidade Microbiana , Núcleosídeo-Fosfato Quinase/genética , Núcleosídeo-Fosfato Quinase/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Especificidade por Substrato , Uridina/análogos & derivados , Uridina/farmacologia , Uridina Monofosfato/química , Uridina Monofosfato/metabolismo
6.
FASEB J ; 33(2): 2017-2025, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30199284

RESUMO

Cellular supply of deoxythymidine triphosphate (dTTP) is crucial for DNA replication and repair. Thymidylate kinase (TMPK) catalyzes the conversion of thymidine monophosphate to thymidine diphosphate, which is an essential step for dTTP synthesis. Despite their major cellular localization in cytosol, TMPK and ribonucleotide reductase (RNR) are detected at DNA damage sites for local dNDP formation. Because deoxyuridine diphosphate is synthesized by RNR, the simultaneous recruitment of TMPK and RNR to DNA damage sites is critical for preventing deoxyuridine triphosphate-mediated toxic repair. This study investigates the mechanism responsible for the recruitment of TMPK to DNA damage sites. Our data demonstrate the requirement of ataxia telangiectasia mutated (ATM) kinase activity for TMPK recruitment to DNA lesion sites. Moreover, we find that TMPK is able to form the complex with histone acetyltransferase Tip60 and RNR. Inhibition of ATM kinase reduces the complex formation and TMPK phosphorylation. Our analysis further shows the presence of TMPK phosphorylation at serine 88, which is an ATM kinase consensus site. A phosphorylation-defective mutation at this site suppresses TMPK recruitment to DNA damage sites and the complex formation with Tip60. Finally, we provide evidence that this site is critical for the function of TMPK in DNA repair but not for catalytic activity. Together, these findings suggest that Tip60-ATM signaling has a functional contribution to the recruitment of TMPK to DNA damage sites, thereby increasing local dTTP synthesis for DNA repair.-Hu, C.-M., Tsao, N., Wang, Y.-T., Chen, Y.-J., Chang, Z.-F. Thymidylate kinase is critical for DNA repair via ATM-dependent Tip60 complex formation.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Dano ao DNA , Reparo do DNA , Lisina Acetiltransferase 5/metabolismo , Complexos Multienzimáticos/metabolismo , Núcleosídeo-Fosfato Quinase/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Células HEK293 , Células HeLa , Humanos , Lisina Acetiltransferase 5/genética , Complexos Multienzimáticos/genética , Núcleosídeo-Fosfato Quinase/genética , Fosforilação/genética , Ribonucleotídeo Redutases/genética , Ribonucleotídeo Redutases/metabolismo
7.
Int J Mol Sci ; 21(14)2020 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-32708927

RESUMO

NME3 is a member of the nucleoside diphosphate kinase (NDPK) family that binds to the mitochondrial outer membrane to stimulate mitochondrial fusion. In this study, we showed that NME3 knockdown delayed DNA repair without reducing the cellular levels of nucleotide triphosphates. Further analyses revealed that NME3 knockdown increased fragmentation of mitochondria, which in turn led to mitochondrial oxidative stress-mediated DNA single-strand breaks (SSBs) in nuclear DNA. Re-expression of wild-type NME3 or inhibition of mitochondrial fission markedly reduced SSBs and facilitated DNA repair in NME3 knockdown cells, while expression of N-terminal deleted mutant defective in mitochondrial binding had no rescue effect. We further showed that disruption of mitochondrial fusion by knockdown of NME4 or MFN1 also caused mitochondrial oxidative stress-mediated genome instability. In conclusion, the contribution of NME3 to redox-regulated genome stability lies in its function in mitochondrial fusion.


Assuntos
Dano ao DNA , Mitocôndrias/metabolismo , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Técnicas de Silenciamento de Genes , Instabilidade Genômica , Células HEK293 , Células HeLa , Humanos , Mitocôndrias/genética , Nucleosídeo NM23 Difosfato Quinases/genética
8.
Int J Mol Sci ; 21(21)2020 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-33126676

RESUMO

Peroxisomes proliferate by sequential processes comprising elongation, constriction, and scission of peroxisomal membrane. It is known that the constriction step is mediated by a GTPase named dynamin-like protein 1 (DLP1) upon efficient loading of GTP. However, mechanism of fuelling GTP to DLP1 remains unknown in mammals. We earlier show that nucleoside diphosphate (NDP) kinase-like protein, termed dynamin-based ring motive-force organizer 1 (DYNAMO1), generates GTP for DLP1 in a red alga, Cyanidioschyzon merolae. In the present study, we identified that nucleoside diphosphate kinase 3 (NME3), a mammalian homologue of DYNAMO1, localizes to peroxisomes. Elongated peroxisomes were observed in cells with suppressed expression of NME3 and fibroblasts from a patient lacking NME3 due to the homozygous mutation at the initiation codon of NME3. Peroxisomes proliferated by elevation of NME3 upon silencing the expression of ATPase family AAA domain containing 1, ATAD1. In the wild-type cells expressing catalytically-inactive NME3, peroxisomes were elongated. These results suggest that NME3 plays an important role in peroxisome division in a manner dependent on its NDP kinase activity. Moreover, the impairment of peroxisome division reduces the level of ether-linked glycerophospholipids, ethanolamine plasmalogens, implying the physiological importance of regulation of peroxisome morphology.


Assuntos
Dinaminas/metabolismo , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Peroxissomos/metabolismo , Frações Subcelulares/metabolismo , Sequência de Aminoácidos , Animais , Células HeLa , Homozigoto , Humanos , Rodófitas , Homologia de Sequência
9.
J Cell Sci ; 128(20): 3757-68, 2015 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-26359301

RESUMO

Dexamethasone, a synthetic glucocorticoid, is often used to induce osteoblast commitment of mesenchymal stem cells (MSCs), and this process requires RhoA-dependent cellular tension. The underlying mechanism is unclear. In this study, we show that dexamethasone stimulates expression of fibronectin and integrin α5 (ITGA5), accompanied by an increase in the interaction of GEF-H1 (also known as ARHGEF2) with Sec5 (also known as EXOC2), a microtubule (MT)-regulated RhoA activator and a component of the exocyst, respectively. Disruption of this interaction abolishes dexamethasone-induced cellular tension and GEF-H1 targeting to focal adhesion sites at the cell periphery without affecting dexamethasone-induced levels of ITGA5 and fibronectin, and the extracellular deposition of fibronectin at adhesion sites is specifically inhibited. We demonstrate that dexamethasone stimulates the expression of serum-glucocorticoid-induced protein kinase 1 (SGK1), which is necessary and sufficient for the induction of the Sec5-GEF-H1 interaction. Given the function of SGK1 in suppressing MT growth, our data suggest that the induction of SGK1 through treatment with dexamethasone alters MT dynamics to increase Sec5-GEF-H1 interactions, which promote GEF-H1 targeting to adhesion sites. This mechanism is essential for the formation of fibronectin fibrils and their attachment to integrins at adhesion sites in order to generate cellular tension.


Assuntos
Dexametasona/farmacologia , Indução Enzimática/efeitos dos fármacos , Proteínas Imediatamente Precoces/metabolismo , Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Adesão Celular/efeitos dos fármacos , Humanos , Proteínas Imediatamente Precoces/genética , Microtúbulos/genética , Proteínas Serina-Treonina Quinases/genética , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Proteínas de Transporte Vesicular/genética
10.
Biochem J ; 473(9): 1237-45, 2016 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-26945015

RESUMO

Cellular supply of dNTPs via RNR (ribonucleotide reductase) is crucial for DNA replication and repair. It has been shown that DNA-damage-site-specific recruitment of RNR is critical for DNA repair efficiency in quiescent cells. The catalytic function of RNR produces dNDPs. The subsequent step of dNTP formation requires the function of NDP kinase. There are ten isoforms of NDP kinase in human cells. In the present study, we identified NME3 as one specific NDP kinase that interacts directly with Tip60, a histone acetyltransferase, to form a complex with RNR. Our data reveal that NME3 recruitment to DNA damage sites depends on this interaction. Disruption of interaction of NME3 with Tip60 suppressed DNA repair in serum-deprived cells. Thus Tip60 interacts with RNR and NME3 to provide site-specific synthesis of dNTP for facilitating DNA repair in serum-deprived cells which contain low levels of dNTPs.


Assuntos
Reparo do DNA/fisiologia , Histona Acetiltransferases/metabolismo , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Ribonucleotídeo Redutases/metabolismo , Células HeLa , Histona Acetiltransferases/genética , Humanos , Lisina Acetiltransferase 5 , Células MCF-7 , Nucleosídeo NM23 Difosfato Quinases/genética , Ribonucleotídeo Redutases/genética
11.
J Cell Sci ; 127(Pt 19): 4186-200, 2014 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-25107365

RESUMO

Focal adhesions (FAs) undergo maturation that culminates in size and composition changes that modulate adhesion, cytoskeleton remodeling and differentiation. Although it is well recognized that stimuli for osteogenesis of mesenchymal stem cells (MSCs) drive FA maturation, actin organization and stress fiber polarization, the extent to which FA-mediated signals regulated by the FA protein composition specifies MSC commitment remains largely unknown. Here, we demonstrate that, upon dexamethasone (osteogenic induction) treatment, guanine nucleotide exchange factor H1 (GEF-H1, also known as Rho guanine nucleotide exchange factor 2, encoded by ARHGEF2) is significantly enriched in FAs. Perturbation of GEF-H1 inhibits FA formation, anisotropic stress fiber orientation and MSC osteogenesis in an actomyosin-contractility-independent manner. To determine the role of GEF-H1 in MSC osteogenesis, we explore the GEF-H1-modulated FA proteome that reveals non-muscle myosin-II heavy chain-B (NMIIB, also known as myosin-10, encoded by MYH10) as a target of GEF-H1 in FAs. Inhibition of targeting NMIIB into FAs suppresses FA formation, stress fiber polarization, cell stiffness and osteogenic commitments in MSCs. Our data demonstrate a role for FA signaling in specifying MSC commitment.


Assuntos
Adesões Focais/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Diferenciação Celular/fisiologia , Linhagem da Célula , Humanos , Osteogênese , Transdução de Sinais
12.
Nucleic Acids Res ; 42(8): 4972-84, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24561807

RESUMO

In quiescent fibroblasts, the expression levels of cytosolic enzymes for thymidine triphosphate (dTTP) synthesis are down-regulated, causing a marked reduction in the dTTP pool. In this study, we provide evidence that mitochondrial thymidylate synthesis via thymidine kinase 2 (TK2) is a limiting factor for the repair of ultraviolet (UV) damage in the nuclear compartment in quiescent fibroblasts. We found that TK2 deficiency causes secondary DNA double-strand breaks formation in the nuclear genome of quiescent cells at the late stage of recovery from UV damage. Despite slower repair of quiescent fibroblast deficient in TK2, DNA damage signals eventually disappeared, and these cells were capable of re-entering the S phase after serum stimulation. However, these cells displayed severe genome stress as revealed by the dramatic increase in 53BP1 nuclear body in the G1 phase of the successive cell cycle. Here, we conclude that mitochondrial thymidylate synthesis via TK2 plays a role in facilitating the quality repair of UV damage for the maintenance of genome integrity in the cells that are temporarily arrested in the quiescent state.


Assuntos
Núcleo Celular/genética , Reparo do DNA , Mitocôndrias/enzimologia , Estresse Fisiológico/genética , Timidina Quinase/fisiologia , Nucleotídeos de Timina/biossíntese , Ciclo Celular , Linhagem Celular , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Desoxirribonucleotídeos/metabolismo , Genoma , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/análise , Timidina Quinase/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Raios Ultravioleta
13.
J Cell Sci ; 126(Pt 2): 657-66, 2013 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-23178938

RESUMO

Podosomes are actin-enriched membrane protrusions that play important roles in extracellular matrix degradation and invasive cell motility. Podosomes undergo self-assembly into large rosette-like structures in Src-transformed fibroblasts, osteoclasts and certain highly invasive cancer cells. Several protein tyrosine kinases have been shown to be important for the formation of podosome rosettes, but little is known regarding the role of protein tyrosine phosphatases in this process. We found that knockdown of the Src homolog domain-containing phosphatase 2 (SHP2) significantly increased podosome rosette formation in Src-transformed fibroblasts. By contrast, SHP2 overexpression suppressed podosome rosette formation in these cells. The phosphatase activity of SHP2 was essential for the suppression of podosome rosette formation. SHP2 selectively suppressed the tyrosine phosphorylation of Tks5, a scaffolding protein required for podosome formation. The inhibitory effect of SHP2 on podosome rosette formation was associated with the increased activation of Rho-associated kinase (ROCK) and the enhanced polymerization of vimentin filaments. A higher content of polymerized vimentin filaments was correlated with a lower content of podosome rosettes. Taken together, our findings indicate that SHP2 serves as a negative regulator of podosome rosette formation through the dephosphorylation of Tks5 and the activation of ROCK-mediated polymerization of vimentin in Src-transformed fibroblasts.


Assuntos
Fibroblastos/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Quinases da Família src/metabolismo , Animais , Fibroblastos/citologia , Fibroblastos/enzimologia , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Células NIH 3T3 , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética
14.
BMC Cancer ; 15: 943, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26626121

RESUMO

BACKGROUND: Two isoforms of Rho-associated coiled-coil kinase (ROCK), ROCKI and ROCKII, play an important role in many cellular processes. Despite the accumulating evidence showing that ROCK could be a potential cancer therapeutic target, the relevant tumor types to ROCK activation are not well clarified. The aim of this study was to evaluate the ROCK activation status in different tumor types of breast cancer. RESULTS: We evaluated the immunoreactivities of phosphorylation-specific antibodies of ROCKI and ROCKII to inform their kinase activation in 275 of breast carcinoma tissues, including 56 of carcinoma in situ, 116 of invasive carcinoma, and 103 of invasive carcinoma with metastasis. ROCKII activation signal detected in nucleus was significantly correlated with tumor metastasis, while ROCKI and cytosolic ROCKII activation signals made no significant difference in that metastasis. Furthermore, nuclear ROCKII activation signal was associated with poor clinical outcome and correlated with late tumor stage, low expression of estrogen receptor (ER) and progesterone receptor (PR), overexpression of human epidermal growth factor receptor 2 (HER2) and high Ki67 labeling index. CONCLUSIONS: Nuclear ROCKII activation signal might contribute to the tumor metastasis in breast cancer. Differences in ROCK activation that underlie the phenotypes of breast cancer could enhance our understanding for the use of ROCK inhibitors in cancer therapy.


Assuntos
Neoplasias da Mama/enzimologia , Núcleo Celular/enzimologia , Quinases Associadas a rho/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/patologia , Ativação Enzimática , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Metástase Neoplásica , Prognóstico
15.
Calcif Tissue Int ; 97(5): 466-75, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26126938

RESUMO

Uremic patients are predisposed to atrophy of the alveolar bone and narrowing of the dental pulp chamber. Such pulp chamber changes have only been diagnosed radiologically; however, this has not been supported by any pathological evidence. We used a uremic rat model with secondary hyperparathyroidism induced by 5/6 nephrectomy surgery and high-phosphate diet to examine the dental pulp and adjacent alveolar bone pathology. In addition, we collected pulp tissues for real-time PCR. We found an opposite histopathological presentation of the ossified dental pulp and the osteomalacic adjacent alveolar bone. Furthermore, pulp cells with positive staining for Thy-1, a surrogate stem cell marker, were significantly reduced in the pulp of uremic rats compared to the controls, indicating a paucity of stem cells. This was further evidenced by the reduced pulp expression of dickkopf-1 (Dkk-1), a Wnt/ß-catenin signaling inhibitor produced by mesenchymal stem cells. In contrast, expressions of receptor activator of nuclear factor κB ligand (RANKL) and RANK in uremic pulp were up-regulated, probably to counteract the ossifying process of uremic pulp. In conclusion, uremic pulp ossifications were associated with a paucity of stem cells and dysregulated Dkk-1 and RANKL signaling systems, further shifting the imbalance toward osteogenesis. Strategies to counteract such an imbalance may offer a potential therapeutic target to improve dental health in uremic patients, which warrants further interventional studies.


Assuntos
Perda do Osso Alveolar/etiologia , Processo Alveolar/patologia , Polpa Dentária/patologia , Ossificação Heterotópica/etiologia , Uremia/complicações , Animais , Densidade Óssea , Modelos Animais de Doenças , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Osteogênese/fisiologia , Ligante RANK/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Células-Tronco/patologia , Microtomografia por Raio-X
16.
Nephrol Dial Transplant ; 30(8): 1356-63, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25817223

RESUMO

BACKGROUND: The process of vascular calcification has been associated with the canonical Wnt/ß-catenin signalling pathway in cell cultures and animal studies. The relationship between circulating Wnt/ß-catenin inhibitors and vascular calcification in dialysis patients is unknown. The aim of this study was to investigate the associations between serum dickkopf-1 (Dkk-1) and sclerostin, two circulating inhibitors of the Wnt/ß-catenin signalling pathway, and the severity of aortic calcification (AoC) and cardiovascular outcomes in dialysis patients. METHODS: This was a prospective observational cohort study. One hundred and twenty-five patients on maintenance haemodialysis participated in the study. Serum levels of Dkk-1 and sclerostin were measured. AoC scores were calculated from plain films of both posterior-anterior and lateral views. The patients were followed up for 2 years or until death or withdrawal. RESULTS: The circulating sclerostin level was inversely associated with the severity of AoC (P = 0.035) and indicators of the bone turnover rate including serum alkaline phosphatase (ALP) (r = -0.235, P = 0.008) and intact parathyroid hormone (r = -0.523, P < 0.001). Furthermore, Cox regression analysis indicated that the patients with high circulating sclerostin levels were less likely to experience future cardiovascular events [1 pmol/L sclerostin increase, hazard ratio 0.982 (95% CI, 0.967-0.996), P = 0.015] after adjusting for a propensity score. In contrast, serum Dkk-1 was not associated with AoC and clinical outcomes. CONCLUSIONS: In long-term haemodialysis patients, circulating sclerostin but not Dkk-1 is inversely associated with AoCs and future cardiovascular events. Our findings suggest that sclerostin, as a bone-related protein, might act as a communicator between uraemic bone and vasculature.


Assuntos
Biomarcadores/sangue , Proteínas Morfogenéticas Ósseas/sangue , Doenças Cardiovasculares/sangue , Peptídeos e Proteínas de Sinalização Intercelular/sangue , Diálise Renal/efeitos adversos , Uremia/sangue , Calcificação Vascular/sangue , Proteínas Adaptadoras de Transdução de Sinal , Doenças Cardiovasculares/etiologia , Estudos Transversais , Feminino , Marcadores Genéticos , Humanos , Masculino , Pessoa de Meia-Idade , Hormônio Paratireóideo/sangue , Estudos Prospectivos , Uremia/etiologia , Calcificação Vascular/etiologia , Via de Sinalização Wnt
17.
Commun Biol ; 7(1): 843, 2024 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-38987326

RESUMO

Bcr-Abl transformation leads to chronic myeloid leukemia (CML). The acquirement of T315I mutation causes tyrosine kinase inhibitors (TKI) resistance. This study develops a compound, JMF4073, inhibiting thymidylate (TMP) and cytidylate (CMP) kinases, aiming for a new therapy against TKI-resistant CML. In vitro and in vivo treatment of JMF4073 eliminates WT-Bcr-Abl-32D CML cells. However, T315I-Bcr-Abl-32D cells are less vulnerable to JMF4073. Evidence is presented that ATF4-mediated upregulation of GSH causes T315I-Bcr-Abl-32D cells to be less sensitive to JMF4073. Reducing GSH biosynthesis generates replication stress in T315I-Bcr-Abl-32D cells that require dTTP/dCTP synthesis for survival, thus enabling JMF4073 susceptibility. It further shows that the levels of ATF4 and GSH in several human CML blast-crisis cell lines are inversely correlated with JMF4073 sensitivity, and the combinatory treatment of JMF4073 with GSH reducing agent leads to synthetic lethality in these CML blast-crisis lines. Altogether, the investigation indicates an alternative option in CML therapy.


Assuntos
Glutationa , Leucemia Mielogênica Crônica BCR-ABL Positiva , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Glutationa/metabolismo , Humanos , Animais , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Linhagem Celular Tumoral , Proteínas de Fusão bcr-abl/metabolismo , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/antagonistas & inibidores
18.
Nat Commun ; 15(1): 2264, 2024 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-38480688

RESUMO

NME3 is a member of the nucleoside diphosphate kinase (NDPK) family localized on the mitochondrial outer membrane (MOM). Here, we report a role of NME3 in hypoxia-induced mitophagy dependent on its active site phosphohistidine but not the NDPK function. Mice carrying a knock-in mutation in the Nme3 gene disrupting NME3 active site histidine phosphorylation are vulnerable to ischemia/reperfusion-induced infarction and develop abnormalities in cerebellar function. Our mechanistic analysis reveals that hypoxia-induced phosphatidic acid (PA) on mitochondria is essential for mitophagy and the interaction of DRP1 with NME3. The PA binding function of MOM-localized NME3 is required for hypoxia-induced mitophagy. Further investigation demonstrates that the interaction with active NME3 prevents DRP1 susceptibility to MUL1-mediated ubiquitination, thereby allowing a sufficient amount of active DRP1 to mediate mitophagy. Furthermore, MUL1 overexpression suppresses hypoxia-induced mitophagy, which is reversed by co-expression of ubiquitin-resistant DRP1 mutant or histidine phosphorylatable NME3. Thus, the site-specific interaction with active NME3 provides DRP1 a microenvironment for stabilization to proceed the segregation process in mitophagy.


Assuntos
Dinaminas , Mitofagia , Animais , Camundongos , Dinaminas/genética , Dinaminas/metabolismo , Histidina/metabolismo , Hipóxia , Mitofagia/genética , Ubiquitinação
19.
J Biomed Sci ; 20: 83, 2013 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-24168723

RESUMO

BACKGROUND: Two isoforms of Rho-associated protein kinase (ROCK), ROCKI and ROCKII, play a pivotal role in regulation of cytoskeleton and are involved in multiple cellular processes in mammalian cells. Knockout mice experiments have indicated that the functions of ROCKI and II are probably non-redundant in physiology. However, it is difficult to differentiate the activation status of ROCKI and ROCKII in biological samples. Previously, we have identified phosphorylation site of ROCKII at Ser1366 residue sensitive to ROCK inhibition. We further investigated the activity-dependent phosphorylation site in ROCKI to establish the reagents that can be used to detect their individual activation. RESULTS: The phosphorylation site of ROCKI sensitive to its inhibition was identified to be the Ser1333 residue. The ROCKI pSer1333-specific antibody does not cross-react with phosphorylated ROCKII. The extent of S1333 phosphorylation of ROCKI correlates with myosin II light chain phosphorylation in cells in response to RhoA stimulation. CONCLUSIONS: Active ROCKI is phosphorylated at Ser1333 site. Antibodies that recognize phospho-Ser1333 of ROCKI and phospho-S1366 residues of ROCKII offer a means to discriminate their individual active status in cells and tissues.


Assuntos
Ativação Enzimática , Serina/metabolismo , Quinases Associadas a rho/genética , Animais , Anticorpos/isolamento & purificação , Células HEK293 , Humanos , Imunoprecipitação , Camundongos , Fosforilação , Ligação Proteica , Transfecção , Quinases Associadas a rho/metabolismo
20.
Biochem J ; 443(1): 145-51, 2012 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-22273145

RESUMO

ROCK (Rho-associated protein kinase), a downstream effector of RhoA, plays an important role in many cellular processes. Accumulating evidence has shown the involvement of ROCK activation in the pathogenesis of many diseases. However, a reagent capable of detecting ROCK activation directly is lacking. In the present study, we show autophosphorylation of ROCKII in an in vitro kinase reaction. The phosphorylation sites were identified by MS, and the major phosphorylation site was found to be at the highly conserved residue Ser1366. A phospho-specific antibody was generated that can specifically recognize ROCKII Ser1366 phosphorylation. We found that the extent of Ser1366 phosphorylation of endogenous ROCKII is correlated with that of myosin light chain phosphorylation in cells in response to RhoA stimulation, showing that Ser1366 phosphorylation reflects its kinase activity. In addition, ROCKII Ser1366 phosphorylation could be detected in human breast tumours by immunohistochemical staining. The present study provides a new approach for revealing the ROCKII activation status by probing ROCKII Ser1366 phosphorylation directly in cells or tissues.


Assuntos
Ativação Enzimática , Serina/metabolismo , Quinases Associadas a rho/metabolismo , Amidas/farmacologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Anticorpos/isolamento & purificação , Western Blotting , Células HEK293 , Humanos , Imunoprecipitação , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Ligação Proteica , Piridinas/farmacologia , Coelhos , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/genética , Quinases Associadas a rho/imunologia , Proteína rhoA de Ligação ao GTP/metabolismo
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