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1.
Cancer Cell ; 35(5): 738-751.e9, 2019 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-31085175

RESUMO

Ripretinib (DCC-2618) was designed to inhibit the full spectrum of mutant KIT and PDGFRA kinases found in cancers and myeloproliferative neoplasms, particularly in gastrointestinal stromal tumors (GISTs), in which the heterogeneity of drug-resistant KIT mutations is a major challenge. Ripretinib is a "switch-control" kinase inhibitor that forces the activation loop (or activation "switch") into an inactive conformation. Ripretinib inhibits all tested KIT and PDGFRA mutants, and notably is a type II kinase inhibitor demonstrated to broadly inhibit activation loop mutations in KIT and PDGFRA, previously thought only achievable with type I inhibitors. Ripretinib shows efficacy in preclinical cancer models, and preliminary clinical data provide proof-of-concept that ripretinib inhibits a wide range of KIT mutants in patients with drug-resistant GISTs.


Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-kit/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Animais , Células CHO , Linhagem Celular , Linhagem Celular Tumoral , Cricetulus , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Gastrointestinais/tratamento farmacológico , Neoplasias Gastrointestinais/genética , Células HCT116 , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Mutação/efeitos dos fármacos , Mutação/genética
2.
Methods Mol Biol ; 1805: 333-347, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29971726

RESUMO

Most eukaryotic DNA is tightly packaged into nucleosomes that render these sequences largely inaccessible for transcription or repair. Molecular motors called chromatin remodelers use an ATP-dependent mechanism to relieve the inhibition of these processes by sliding or disassembling the nucleosomes. This allows them to serve an essential role in the regulation of gene expression and genomic integrity. The sliding of nucleosomes along DNA can be studied directly by monitoring the associated changes in the fluorescence anisotropy of fluorophores attached to the ends of the DNA. Nucleosome repositioning can also be monitored indirectly through the ATP hydrolysis of the chromatin remodeler during the sliding reaction. Here we discuss how the kinetic data collected in these experiments can be analyzed by simultaneous global nonlinear least squares (NLLS) analysis using simple sequential "n-step" mechanisms to obtain estimates of the macroscopic rate of nucleosome repositioning and of the stoichiometry of coupling ATP binding and hydrolysis to this reaction.


Assuntos
Bioensaio/métodos , Nucleossomos/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Anisotropia , Sítios de Ligação , Montagem e Desmontagem da Cromatina , DNA/metabolismo , Hidrólise , Cinética , Especificidade por Substrato , Fatores de Tempo , Xenopus laevis
3.
Phys Rev E ; 97(3-1): 032422, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29776169

RESUMO

Chromatin remodelers are molecular motors that play essential roles in the regulation of nucleosome positioning and chromatin accessibility. These machines couple the energy obtained from the binding and hydrolysis of ATP to the mechanical work of manipulating chromatin structure through processes that are not completely understood. Here we present a quantitative analysis of nucleosome repositioning by the imitation switch (ISWI) chromatin remodeler and demonstrate that nucleosome stability significantly impacts the observed activity. We show how DNA damage induced changes in the affinity of DNA wrapping within the nucleosome can affect ISWI repositioning activity and demonstrate how assay-dependent limitations can bias studies of nucleosome repositioning. Together, these results also suggest that some of the diversity seen in chromatin remodeler activity can be attributed to the variations in the thermodynamics of interactions between the remodeler, the histones, and the DNA, rather than reflect inherent properties of the remodeler itself.


Assuntos
Biocatálise , Modelos Biológicos , Nucleossomos/metabolismo , DNA/genética , DNA/metabolismo , Dano ao DNA
4.
Mol Cancer Ther ; 16(11): 2486-2501, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28838996

RESUMO

Tumor-infiltrating myeloid cells promote tumor progression by mediating angiogenesis, tumor cell intravasation, and metastasis, which can offset the effects of chemotherapy, radiation, and antiangiogenic therapy. Here, we show that the kinase switch control inhibitor rebastinib inhibits Tie2, a tyrosine kinase receptor expressed on endothelial cells and protumoral Tie2-expressing macrophages in mouse models of metastatic cancer. Rebastinib reduces tumor growth and metastasis in an orthotopic mouse model of metastatic mammary carcinoma through reduction of Tie2+ myeloid cell infiltration, antiangiogenic effects, and blockade of tumor cell intravasation mediated by perivascular Tie2Hi/Vegf-AHi macrophages in the tumor microenvironment of metastasis (TMEM). The antitumor effects of rebastinib enhance the efficacy of microtubule inhibiting chemotherapeutic agents, either eribulin or paclitaxel, by reducing tumor volume, metastasis, and improving overall survival. Rebastinib inhibition of angiopoietin/Tie2 signaling impairs multiple pathways in tumor progression mediated by protumoral Tie2+ macrophages, including TMEM-dependent dissemination and angiopoietin/Tie2-dependent angiogenesis. Rebastinib is a promising therapy for achieving Tie2 inhibition in cancer patients. Mol Cancer Ther; 16(11); 2486-501. ©2017 AACR.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Neovascularização Patológica/tratamento farmacológico , Tumores Neuroendócrinos/tratamento farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Pirazóis/farmacologia , Piridinas/farmacologia , Quinolinas/farmacologia , Receptor TIE-2/antagonistas & inibidores , Angiopoietinas/antagonistas & inibidores , Angiopoietinas/genética , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Camundongos , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Tumores Neuroendócrinos/genética , Tumores Neuroendócrinos/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Pirazóis/uso terapêutico , Piridinas/uso terapêutico , Quinolinas/uso terapêutico , Receptor TIE-2/genética , Transdução de Sinais/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos
5.
Biochim Biophys Acta ; 1854(10 Pt A): 1487-93, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26116984

RESUMO

The motor protein ISWI (Imitation SWItch) is the conserved catalytic ATPase domain of the ISWI family of chromatin remodelers. Members of the ISWI family are involved in regulating the structure of cellular chromatin during times of transcription, translation, and repair. Current models for the nucleosome repositioning activity of ISWI and other chromatin remodelers require the translocation of the remodeling protein along double-stranded DNA through an ATP-dependent mechanism. Here we report results from spectrofluorometric stopped-flow experiments which demonstrate that ISWI displays very low processivity for free DNA translocation. By combining these results with those from experiments monitoring the DNA stimulated ATPase activity of ISWI we further demonstrate that the DNA translocation by ISWI is tightly coupled to ATP hydrolysis. The calculated coupling efficiency of 0.067±0.018 ATP/ISWI/bp is seemingly quite low in comparison to similar DNA translocases and we present potential models to account for this. Nevertheless, the tight coupling of ATP hydrolysis to DNA translocation suggests that DNA translocation is not energetically rate limiting for nucleosome repositioning by ISWI.


Assuntos
Adenosina Trifosfatases/química , Trifosfato de Adenosina/metabolismo , DNA/química , Nucleossomos/química , Fatores de Transcrição/química , Proteínas de Xenopus/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Montagem e Desmontagem da Cromatina , DNA/genética , DNA/metabolismo , Hidrólise , Cinética , Modelos Químicos , Nucleossomos/genética , Nucleossomos/metabolismo , Biossíntese de Proteínas , Transporte Proteico , Termodinâmica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismo
6.
Biochemistry ; 53(27): 4346-57, 2014 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-24898619

RESUMO

The chromatin remodeler ISWI is capable of repositioning clusters of nucleosomes to create well-ordered arrays or moving single nucleosomes from the center of DNA fragments toward the ends without disrupting their integrity. Using standard electrophoresis assays, we have monitored the ISWI-catalyzed repositioning of different nucleosome samples each containing a different length of DNA symmetrically flanking the initially centrally positioned histone octamer. We find that ISWI moves the histone octamer between distinct and thermodynamically stable positions on the DNA according to a random walk mechanism. Through the application of a spectrophotometric assay for nucleosome repositioning, we further characterized the repositioning activity of ISWI using short nucleosome substrates and were able to determine the macroscopic rate of nucleosome repositioning by ISWI. Additionally, quantitative analysis of repositioning experiments performed at various ISWI concentrations revealed that a monomeric ISWI is sufficient to obtain the observed repositioning activity as the presence of a second ISWI bound had no effect on the rate of nucleosome repositioning. We also found that ATP hydrolysis is poorly coupled to nucleosome repositioning, suggesting that DNA translocation by ISWI is not energetically rate-limiting for the repositioning reaction. This is the first calculation of a microscopic ATPase coupling efficiency for nucleosome repositioning and also further supports our conclusion that a second bound ISWI does not contribute to the repositioning reaction.


Assuntos
Adenosina Trifosfatases/metabolismo , Nucleossomos/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Xenopus/metabolismo , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/metabolismo , Animais , Montagem e Desmontagem da Cromatina , DNA/metabolismo , Polarização de Fluorescência , Hidrólise , Pichia/metabolismo , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Tempo , Fatores de Transcrição/genética , Proteínas de Xenopus/genética , Xenopus laevis
7.
Biochemistry ; 53(27): 4334-45, 2014 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-24898734

RESUMO

The regulation of chromatin structure is controlled by a family of molecular motors called chromatin remodelers. The ability of these enzymes to remodel chromatin structure is dependent on their ability to couple ATP binding and hydrolysis into the mechanical work that drives nucleosome repositioning. The necessary first step in determining how these essential enzymes perform this function is to characterize both how they bind nucleosomes and how this interaction is regulated by ATP binding and hydrolysis. With this goal in mind, we monitored the interaction of the chromatin remodeler ISWI with fluorophore-labeled nucleosomes and DNA through associated changes in fluorescence anisotropy of the fluorophore upon binding of ISWI to these substrates. We determined that one ISWI molecule binds to a 20 bp double-stranded DNA substrate with an affinity of 18 ± 2 nM. In contrast, two ISWI molecules can bind to the core nucleosome with short linker DNA with stoichiometric macroscopic equilibrium constants: 1/ß1 = 1.3 ± 0.6 nM, and 1/ß2 = 13 ± 7 nM(2). Furthermore, to improve our understanding of the mechanism of DNA translocation by ISWI, and hence nucleosome repositioning, we determined the effect of nucleotide analogues on substrate binding by ISWI. While the affinity of ISWI for the nucleosome substrate with short lengths of flanking DNA was not affected by the presence of nucleotides, the affinity of ISWI for the DNA substrate is weakened in the presence of nonhydrolyzable ATP analogues but not by ADP.


Assuntos
Adenosina Trifosfatases/metabolismo , DNA/metabolismo , Nucleossomos/metabolismo , Nucleotídeos/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Xenopus/metabolismo , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Animais , Montagem e Desmontagem da Cromatina , Polarização de Fluorescência , Pichia/metabolismo , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/genética , Proteínas de Xenopus/genética , Xenopus laevis
8.
PLoS One ; 6(11): e27802, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22114698

RESUMO

Ether glycerolipids of Leishmania major are important membrane components as well as building blocks of various virulence factors. In L. major, the first enzyme of the ether glycerolipid biosynthetic pathway, LmDAT, is an unusual, glycosomal dihydroxyacetonephosphate acyltransferase important for parasite's growth and survival during the stationary phase, synthesis of ether lipids, and virulence. The present work extends our knowledge of this important biosynthetic enzyme in parasite biology. Site-directed mutagenesis of LmDAT demonstrated that an active enzyme was critical for normal growth and survival during the stationary phase. Deletion analyses showed that the large N-terminal extension of this initial acyltransferase may be important for its stability or activity. Further, abrogation of the C-terminal glycosomal targeting signal sequence of LmDAT led to extraglycosomal localization, did not impair its enzymatic activity but affected synthesis of the ether glycerolipid-based virulence factor lipophosphoglycan. In addition, expression of this recombinant form of LmDAT in a null mutant of LmDAT did not restore normal growth and survival during the stationary phase. These results emphasize the importance of this enzyme's compartmentalization in the glycosome for the generation of lipophosphoglycan and parasite's biology.


Assuntos
Aciltransferases/metabolismo , Glicoesfingolipídeos/metabolismo , Leishmania major/enzimologia , Microcorpos/metabolismo , Proteínas Recombinantes/metabolismo , Aciltransferases/genética , Imunofluorescência , Leishmania major/crescimento & desenvolvimento , Plasmídeos/genética , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética
9.
J Biol Chem ; 285(19): 14415-23, 2010 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-20228053

RESUMO

PIASy is a small ubiquitin-related modifier (SUMO) ligase that modifies chromosomal proteins in mitotic Xenopus egg extracts and plays an essential role in mitotic chromosome segregation. We have isolated a novel SUMO-2/3-modified mitotic chromosomal protein and identified it as poly(ADP-ribose) polymerase 1 (PARP1). PARP1 was robustly conjugated to SUMO-2/3 on mitotic chromosomes but not on interphase chromatin. PIASy promotes SUMOylation of PARP1 both in egg extracts and in vitro reconstituted SUMOylation assays. Through tandem mass spectrometry analysis of mitotically SUMOylated PARP1, we identified a residue within the BRCA1 C-terminal domain of PARP1 (lysine 482) as its primary SUMOylation site. Mutation of this residue significantly reduced PARP1 SUMOylation in egg extracts and enhanced the accumulation of species derived from modification of secondary lysine residues in assays using purified components. SUMOylation of PARP1 did not alter in vitro PARP1 enzyme activity, poly-ADP-ribosylation (PARylation), nor did inhibition of SUMOylation of PARP1 alter the accumulation of PARP1 on mitotic chromosomes, suggesting that SUMOylation regulates neither the intrinsic activity of PARP1 nor its localization. However, loss of SUMOylation increased PARP1-dependent PARylation on isolated chromosomes, indicating SUMOylation controls the capacity of PARP1 to modify other chromatin-associated proteins.


Assuntos
Cromossomos/fisiologia , Mitose , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animais , Cromatina/genética , Imunofluorescência , Oócitos/citologia , Oócitos/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes
10.
Mol Biochem Parasitol ; 168(2): 177-85, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19720088

RESUMO

Glycerolipid biosynthesis in Leishmania initiates with the acylation of glycerol-3-phosphate by a single glycerol-3-phosphate acyltransferase, LmGAT, or of dihydroxyacetonephosphate by a dihydroxyacetonephosphate acyltransferase, LmDAT. We previously reported that acylation of the precursor dihydroxyacetonephosphate rather than glycerol-3-phosphate is the physiologically relevant pathway for Leishmania parasites. We demonstrated that LmDAT is important for normal growth, survival during the stationary phase, and for virulence. Here, we assessed the role of LmDAT in glycerolipid metabolism and metacyclogenesis. LmDAT was found to be implicated in the biosynthesis of ether glycerolipids, including the ether lipid derived virulence factor lipophosphoglycan and glycosylphosphatidylinositol-anchored proteins. The null mutant produced longer lipophosphoglycan molecules that were not released in the medium, and augmented levels of glycosylphosphatidylinositol-anchored proteins. In addition, the integrity of detergent resistant membranes was not affected by the absence of the LmDAT gene. Further, our genetic analyses strongly suggest that LmDAT was synthetic lethal with the glycerol-3-phosphate acyltransferase encoding gene LmGAT, implying that Leishmania expresses only two acyltransferases that initiate the biosynthesis of its cellular glycerolipids. Last, despite the fact that LmDAT is important for virulence the null mutant still exhibited the typical characteristics of metacyclics.


Assuntos
Aciltransferases/fisiologia , Membrana Celular/efeitos dos fármacos , Detergentes/farmacologia , Éter/metabolismo , Leishmania/enzimologia , Lipídeos/biossíntese , Aciltransferases/genética , Animais , Deleção de Genes , Leishmania/efeitos dos fármacos
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