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1.
Sci Adv ; 5(11): eaax9778, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31723605

RESUMO

A homozygous mutation of human tyrosyl-DNA phosphodiesterase 1 (TDP1) causes the neurodegenerative syndrome, spinocerebellar ataxia with axonal neuropathy (SCAN1). TDP1 hydrolyzes the phosphodiester bond between DNA 3'-end and a tyrosyl moiety within trapped topoisomerase I (Top1)-DNA covalent complexes (Top1cc). TDP1 is critical for mitochondrial DNA (mtDNA) repair; however, the role of mitochondria remains largely unknown for the etiology of SCAN1. We demonstrate that mitochondria in cells expressing SCAN1-TDP1 (TDP1H493R) are selectively trapped on mtDNA in the regulatory non-coding region and promoter sequences. Trapped TDP1H493R-mtDNA complexes were markedly increased in the presence of the Top1 poison (mito-SN38) when targeted selectively into mitochondria in nanoparticles. TDP1H493R-trapping accumulates mtDNA damage and triggers Drp1-mediated mitochondrial fission, which blocks mitobiogenesis. TDP1H493R prompts PTEN-induced kinase 1-dependent mitophagy to eliminate dysfunctional mitochondria. SCAN1-TDP1 in mitochondria creates a pathological state that allows neurons to turn on mitophagy to rescue fit mitochondria as a mechanism of survival.

2.
Nucleic Acids Res ; 46(11): 5601-5617, 2018 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-29718323

RESUMO

Human tyrosyl-DNA phosphodiesterases (TDP) hydrolyze the phosphodiester bond between DNA and the catalytic tyrosine of Top1 to excise topoisomerase I cleavage complexes (Top1cc) that are trapped by camptothecin (CPT) and by genotoxic DNA alterations. Here we show that the protein arginine methyltransferase PRMT5 enhances the repair of Top1cc by direct binding to TDP1 and arginine dimethylation of TDP1 at residues R361 and R586. Top1-induced replication-mediated DNA damage induces TDP1 arginine methylation, enhancing its 3'- phosphodiesterase activity. TDP1 arginine methylation also increases XRCC1 association with TDP1 in response to CPT, and the recruitment of XRCC1 to Top1cc DNA damage foci. PRMT5 knockdown cells exhibit defective TDP1 activity with marked elevation in replication-coupled CPT-induced DNA damage and lethality. Finally, methylation of R361 and R586 stimulate TDP1 repair function and promote cell survival in response to CPT. Together, our findings provide evidence for the importance of PRMT5 for the post-translational regulation of TDP1 and repair of Top1cc.


Assuntos
Reparo do DNA , DNA Topoisomerases Tipo I/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Animais , Arginina/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Dano ao DNA , Replicação do DNA , Células HEK293 , Humanos , Metilação , Camundongos , Diester Fosfórico Hidrolases/química , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/metabolismo
3.
J Med Chem ; 61(3): 804-817, 2018 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-29290109

RESUMO

Camptothecin (CPT) selectively traps topoisomerase 1-DNA cleavable complexes (Top1cc) to promote anticancer activity. Here, we report the design and synthesis of a new class of neutral porphyrin derivative 5,10-bis(4-carboxyphenyl)-15, 20-bis(4-dimethylaminophenyl)porphyrin (compound 8) as a potent catalytic inhibitor of human Top1. In contrast to CPT, compound 8 reversibly binds with the free enzyme and inhibits the formation of Top1cc and promotes reversal of the preformed Top1cc with CPT. Compound 8 induced inhibition of Top1cc formation in live cells was substantiated by fluorescence recovery after photobleaching (FRAP) assays. We established that MCF7 cells treated with compound 8 trigger proteasome-mediated Top1 degradation, accumulate higher levels of reactive oxygen species (ROS), PARP1 cleavage, oxidative DNA fragmentation, and stimulate apoptotic cell death without stabilizing apoptotic Top1-DNA cleavage complexes. Finally, compound 8 shows anticancer activity by targeting cellular Top1 and preventing the enzyme from directly participating in the apoptotic process.


Assuntos
Apoptose/efeitos dos fármacos , Clivagem do DNA/efeitos dos fármacos , DNA Topoisomerases Tipo I/metabolismo , Porfirinas/química , Porfirinas/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Biocatálise/efeitos dos fármacos , Quebras de DNA/efeitos dos fármacos , Humanos , Células MCF-7 , Espécies Reativas de Oxigênio/metabolismo , Inibidores da Topoisomerase I/química , Inibidores da Topoisomerase I/farmacologia
4.
Anal Biochem ; 528: 53-56, 2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28416394

RESUMO

Rapid diagnostic tests can be developed using ELISA for detection of diseases in emergency conditions. Conventional ELISA takes 1-2 days, making it unsuitable for rapid diagnostics. Here, we report the effect of reagents mixing via shaking or vortexing on the assay timing of ELISA. A 48-min protocol of ELISA involving 12-min incubations with reagent mixing at 750 rpm for every step was optimized. Contrary to this, time-optimized control ELISA performed without mixing produced similar results in 8 h, leaving a time gain of 7 h using the developed protocol. Collectively, the findings suggest the development of ELISA-based rapid diagnostics.


Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Imunoglobulina G/análise , Concanavalina A/análise , Humanos , Imunoglobulina E/análise , Indicadores e Reagentes
5.
Small ; 13(15)2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28134490

RESUMO

Aggregation-induced emission (AIE) is commonly observed in irregular bulk form. Herein, unique aggregation properties of an AIE-active complex into branched supramolecular wires are reported for the first time. Mono-cyclometalated Ir(III) complex shows in-plane J-aggregation at the air-water interface owing to the restriction of intramolecular vibration of bidentate phenylpyridinato and intramolecular rotations of monodentate triphenylphosphine ligands at air-water interface. As a consequence, a large enhancement of luminescence comparable to the solid state is obtained from the monolayers of supramolecular wires. This unique feature is utilized for the fabrication of light-emitting diodes with low threshold voltage using supramolecular wires as active layer. This study opens up the need of ordered assembly of AIE complexes to achieve optimal luminescence characteristics.

6.
Nucleic Acids Res ; 44(17): 8363-75, 2016 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-27466387

RESUMO

Topoisomerase 1 (Top1) is essential for removing the DNA supercoiling generated during replication and transcription. Anticancer drugs like camptothecin (CPT) and its clinical derivatives exert their cytotoxicity by reversibly trapping Top1 in covalent complexes on the DNA (Top1cc). Poly(ADP-ribose) polymerase (PARP) catalyses the addition of ADP-ribose polymers (PAR) onto itself and Top1. PARP inhibitors enhance the cytotoxicity of CPT in the clinical trials. However, the molecular mechanism by which PARylation regulates Top1 nuclear dynamics is not fully understood. Using live-cell imaging of enhanced green fluorescence tagged-human Top1, we show that PARP inhibitors (Veliparib, ABT-888) delocalize Top1 from the nucleolus to the nucleoplasm, which is independent of Top1-PARP1 interaction. Using fluorescence recovery after photobleaching and subsequent fitting of the data employing kinetic modelling we demonstrate that ABT-888 markedly increase CPT-induced bound/immobile fraction of Top1 (Top1cc) across the nuclear genome, suggesting Top1-PARylation counteracts CPT-induced stabilization of Top1cc. We further show Trp205 and Asn722 of Top1 are critical for subnuclear dynamics. Top1 mutant (N722S) was restricted to the nucleolus in the presence of CPT due to its deficiency in the accumulation of CPT-induced Top1-PARylation and Top1cc formation. This work identifies ADP-ribose polymers as key determinant for regulating Top1 subnuclear dynamics.


Assuntos
Camptotecina/farmacologia , Núcleo Celular/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Benzimidazóis/farmacologia , Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/metabolismo , Núcleo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , DNA/metabolismo , Difusão , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Recuperação de Fluorescência Após Fotodegradação , Proteínas de Fluorescência Verde/metabolismo , Células HCT116 , Humanos , Cinética , Proteínas Mutantes/metabolismo , Plasmídeos/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia
7.
Nucleic Acids Res ; 42(7): 4435-49, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24493735

RESUMO

Poly(ADP-ribose) polymerases (PARP) attach poly(ADP-ribose) (PAR) chains to various proteins including themselves and chromatin. Topoisomerase I (Top1) regulates DNA supercoiling and is the target of camptothecin and indenoisoquinoline anticancer drugs, as it forms Top1 cleavage complexes (Top1cc) that are trapped by the drugs. Endogenous and carcinogenic DNA lesions can also trap Top1cc. Tyrosyl-DNA phosphodiesterase 1 (TDP1), a key repair enzyme for trapped Top1cc, hydrolyzes the phosphodiester bond between the DNA 3'-end and the Top1 tyrosyl moiety. Alternative repair pathways for Top1cc involve endonuclease cleavage. However, it is unknown what determines the choice between TDP1 and the endonuclease repair pathways. Here we show that PARP1 plays a critical role in this process. By generating TDP1 and PARP1 double-knockout lymphoma chicken DT40 cells, we demonstrate that TDP1 and PARP1 are epistatic for the repair of Top1cc. The N-terminal domain of TDP1 directly binds the C-terminal domain of PARP1, and TDP1 is PARylated by PARP1. PARylation stabilizes TDP1 together with SUMOylation of TDP1. TDP1 PARylation enhances its recruitment to DNA damage sites without interfering with TDP1 catalytic activity. TDP1-PARP1 complexes, in turn recruit X-ray repair cross-complementing protein 1 (XRCC1). This work identifies PARP1 as a key component driving the repair of trapped Top1cc by TDP1.


Assuntos
Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Linhagem Celular Tumoral , DNA Topoisomerases Tipo I/metabolismo , Epistasia Genética , Humanos , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/genética , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/genética , Domínios e Motivos de Interação entre Proteínas , Sumoilação , Proteína 1 Complementadora Cruzada de Reparo de Raio-X
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