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4.
PLoS One ; 11(3): e0152611, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27018598

RESUMO

Myotubularin-related protein 1 (MTMR1) is a phosphatase that belongs to the tyrosine/dual-specificity phosphatase superfamily. MTMR1 has been shown to use phosphatidylinositol 3-monophosphate (PI(3)P) and/or phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) as substrates. Here, we determined the crystal structure of human MTMR1. The refined model consists of the Pleckstrin homology (PH)-GRAM and phosphatase (PTP) domains. The overall structure was highly similar to the previously reported MTMR2 structure. Interestingly, two phosphate molecules were coordinated by strictly conserved residues located in the C(X)5R motif of the active site. Additionally, our biochemical studies confirmed the substrate specificity of MTMR1 for PI(3)P and PI(3,5)P2 over other phosphatidylinositol phosphates. Our structural and enzymatic analyses provide insight into the catalytic mechanism and biochemical properties of MTMR1.


Assuntos
Proteínas Tirosina Fosfatases não Receptoras/química , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Humanos , Dados de Sequência Molecular , Fosfatos de Fosfatidilinositol/metabolismo , Estrutura Terciária de Proteína , Proteínas Tirosina Fosfatases não Receptoras/genética , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Especificidade por Substrato
5.
Artigo em Inglês | MEDLINE | ID: mdl-24100564

RESUMO

Bacteroides thetaiotaomicron BT0793, a putative xylose isomerase, was overexpressed in Escherichia coli, purified and crystallized using polyethylene glycol monomethyl ether 550 as the precipitant. X-ray diffraction data were collected to 2.10 Šresolution at 100 K using synchrotron X-rays. The crystal was found to belong to space group P1, with unit-cell parameters a=96.3, b=101.7, c=108.3 Å, α=82.8, ß=68.2, γ=83.0°. The asymmetric unit contained eight subunits of xylose isomerase with a crystal volume per protein weight (VM) of 2.38 Å3 Da(-1) and a solvent content of 48.3%.


Assuntos
Aldose-Cetose Isomerases/química , Bacteroides/enzimologia , Cristalização , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Cetoses/química , Cetoses/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
6.
PLoS One ; 8(7): e70358, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23936194

RESUMO

Human Pim1 kinase is a serine/threonine protein kinase that plays important biological roles in cell survival, apoptosis, proliferation, and differentiation. Moreover, Pim1 is up-regulated in various hematopoietic malignancies and solid tumors. Thus, Pim1 is an attractive target for cancer therapeutics, and there has been growing interest in developing small molecule inhibitors for Pim1. Here, we describe the crystal structure of Pim1 in complex with a newly developed pyrido[4,3-d]pyrimidine-derivative inhibitor (SKI-O-068). Our inhibitor exhibits a half maximum inhibitory concentration (IC50) of 123 (±14) nM and has an unusual binding mode in complex with Pim1 kinase. The interactions between SKI-O-068 and the Pim1 active site pocket residue are different from those of other scaffold inhibitor-bound structures. The binding mode analysis suggests that the SKI-O-068 inhibitor can be improved by introducing functional groups that facilitate direct interaction with Lys67, which aid in the design of an optimized inhibitor.


Assuntos
Inibidores de Proteínas Quinases/química , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-pim-1/química , Pirimidinas/química , Sítios de Ligação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Piridonas/química , Piridonas/metabolismo , Piridonas/farmacologia , Pirimidinas/metabolismo , Pirimidinas/farmacologia , Especificidade por Substrato
7.
FEBS Lett ; 586(9): 1384-8, 2012 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-22616997

RESUMO

Pyrococcus furiosus PF2050 is an uncharacterized putative protein that contains two DUF2666 domains. Functional and structural studies of PF2050 have not previously been performed. In this study, we determined the crystal structure of PF2050. The structure of PF2050 showed that the two DUF2666 domains interact tightly, forming a globular structure. Each DUF2666 domain comprises an antiparallel ß-sheet and an α-helical bundle. One side of the PF2050 structure has a positively charged basic cleft, which may have a DNA-binding function. Furthermore, we confirmed that PF2050 interacts with circular and linear dsDNA.


Assuntos
Proteínas Arqueais/química , Pyrococcus furiosus , Sequência de Aminoácidos , Proteínas Arqueais/metabolismo , Cristalografia por Raios X , DNA/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína
8.
J Biol Chem ; 287(14): 10727-37, 2012 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-22334682

RESUMO

Apoptosis inhibitor 5 (API5) is an anti-apoptotic protein that is up-regulated in various cancer cells. Here, we present the crystal structure of human API5. API5 exhibits an elongated all α-helical structure. The N-terminal half of API5 is similar to the HEAT repeat and the C-terminal half is similar to the ARM (Armadillo-like) repeat. HEAT and ARM repeats have been implicated in protein-protein interactions, suggesting that the cellular roles of API5 may be to mediate protein-protein interactions. Various components of multiprotein complexes have been identified as API5-interacting protein partners, suggesting that API5 may act as a scaffold for multiprotein complexes. API5 exists as a monomer, and the functionally important heptad leucine repeat does not exhibit the predicted a dimeric leucine zipper. Additionally, Lys-251, which can be acetylated in cells, plays important roles in the inhibition of apoptosis under serum deprivation conditions. The acetylation of this lysine also affects the stability of API5 in cells.


Assuntos
Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Sequências Repetitivas de Aminoácidos , Sequência de Aminoácidos , Animais , Humanos , Células Jurkat , Leucina , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína
9.
Int J Biol Macromol ; 47(2): 190-5, 2010 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-20450932

RESUMO

Transglutaminase 2 (TG2) is a calcium-dependent multifunctional protein associated with various human diseases. We determined the crystal structure of human TG2 in complex with adenosine triphosphate (ATP). The ATP molecule binds to the previously identified guanosine diphosphate (GDP) binding pocket but has different hydrogen bonds and ion interaction with protein. The four residues Arg476, Arg478, Val479 and Tyr583, all of which are involved in both ATP and GDP binding by hydrogen bonds, might play important roles in the stabilization of TG2 by ATP or GDP. However, Ser482 and Arg580, which are involved in GDP binding, do not form hydrogen bond with ATP. Additionally, we newly discovered an intramolecular disulfide bond between Cys230 and Cys370, which formation might regulate the enzymatic activity of TG2.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Transglutaminases/química , Transglutaminases/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Bases de Dados de Proteínas , Guanosina Difosfato/metabolismo , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Proteína 2 Glutamina gama-Glutamiltransferase
10.
Protein Expr Purif ; 71(1): 103-7, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20100577

RESUMO

Tarzarotene-induced gene 3 (TIG3) and HRAS-like suppressor (HRASLS3) are members of the HREV107 family of class II tumor suppressors, which are down-regulated in various cancer cells. TIG3 and HRASLS3 also exhibit phospholipase activities. Both proteins share a common domain architecture with hydrophilic N-terminal and hydrophobic C-terminal regions. The hydrophobic C-terminal region is important for tumor suppression. However, the function of the hydrophilic N-terminal region remains elusive. To facilitate biochemical characterizations of TIG3 and HRASLS3, we expressed and purified the N-terminal regions of TIG3 and HRASLS3, designated TIG3 (1-134) and HRASLS3 (1-133), in a bacterial system. We found that the N-terminal regions of TIG3 and HRASLS3 have calcium-independent phospholipase A(2) activities. Limited proteolysis revealed that TIG3 (1-132) is a structural domain in the N-terminal region of TIG3. Our data suggest that the hydrophobic C-terminal regions might be crucial for cellular localization, while the hydrophilic N-terminal regions are sufficient for the enzymatic activity of both TIG3 and HRASLS3.


Assuntos
Bioquímica/métodos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/isolamento & purificação , Receptores do Ácido Retinoico/química , Receptores do Ácido Retinoico/isolamento & purificação , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/isolamento & purificação , Sequência de Aminoácidos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Dados de Sequência Molecular , Fosfolipases A2/metabolismo , Fosfolipases A2 Independentes de Cálcio , Estrutura Terciária de Proteína , Receptores do Ácido Retinoico/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Supressoras de Tumor/metabolismo
11.
Toxicology ; 264(3): 145-54, 2009 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-19720108

RESUMO

Potential applications of embryonic stem (ES) cells are not limited to regenerative medicine but can also include in vitro screening of various toxicants. In this study, we established mouse ES cell lines from isolated blastomeres of two-cell stage embryos and examined their potential use as an in vitro system for the study of developmental toxicity. Two ES cell lines were established from 69 blastomere-derived blastocysts (2.9%). The blastomere-derived ES (bm-ES) cells were treated with mono-(2-ethylhexyl) phthalate (MEHP) in an undifferentiated state or after directed differentiation into early neural cell types. We observed significantly decreased cell viability when undifferentiated bm-ES cells were exposed to a high dose of MEHP (1000 microM). The cytotoxic effects of MEHP were accompanied by increased DNA fragmentation, nuclear condensation, and activation of Caspase-3, which are biochemical and morphological features of apoptosis. Compared to undifferentiated bm-ES cells, considerably lower doses of MEHP (50 and 100 microM) were sufficient to induce cell death in early neurons differentiated from bm-ES cells. At the lower doses, the number of neural cells positive for the active form of Caspase-3 was greater than that for undifferentiated bm-ES cells. Thus, our data indicate that differentiating neurons are more sensitive to MEHP than undifferentiated ES cells, and that undifferentiated ES cells may have more efficient defense systems against cytotoxic stresses. These findings might contribute to the development of a new predictive screening method for assessment of hazards for developmental toxicity.


Assuntos
Blastômeros/efeitos dos fármacos , Diferenciação Celular , Dietilexilftalato/análogos & derivados , Células-Tronco Embrionárias/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Plastificantes/toxicidade , Testes de Toxicidade , Animais , Apoptose/efeitos dos fármacos , Blastômeros/metabolismo , Blastômeros/patologia , Caspase 3/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Fragmentação do DNA , Dietilexilftalato/toxicidade , Relação Dose-Resposta a Droga , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/patologia , Ativação Enzimática , Camundongos , Neurônios/metabolismo , Neurônios/patologia , Medição de Risco , Fatores de Tempo
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