RESUMEN
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.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Secuencias Repetitivas de Aminoácido , Secuencia de Aminoácidos , Animales , Humanos , Células Jurkat , Leucina , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Secundaria de ProteínaRESUMEN
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 2.10â Å resolution at 100â 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=108.3â 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 2.38â Å3â Da(-1) and a solvent content of 48.3%.
Asunto(s)
Isomerasas Aldosa-Cetosa/química , Bacteroides/enzimología , Cristalización , Cristalografía por Rayos X , Electroforesis en Gel de Poliacrilamida , Cetosas/química , Cetosas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
There has been considerable interest in virulence genes in the plasticity region of Helicobacter pylori, but little is known about many of these genes. JHP940, one of the virulence factors encoded by the plasticity region of H. pylori strain J99, is a proinflammatory protein that induces tumor necrosis factor-alpha and interleukin-8 secretion as well as enhanced translocation of NF-κB in cultured macrophages. Here we have characterized the structure and function of JHP940 to provide the framework for better understanding its role in inflammation by H. pylori. Our work demonstrates that JHP940 is the first example of a eukaryotic-type Ser/Thr kinase from H. pylori. We show that JHP940 is catalytically active as a protein kinase and translocates into cultured human cells. Furthermore, the kinase activity is indispensable for indirectly up-regulating phosphorylation of NF-κB p65 at Ser276. Our results, taken together, contribute significantly to understanding the molecular basis of the role of JHP940 in inflammation and subsequent pathogenesis caused by H. pylori. We propose to rename the jhp940 gene as ctkA (cell translocating kinase A).
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Helicobacter pylori/enzimología , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Factores de Virulencia/química , Factores de Virulencia/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/genética , Dominio Catalítico , Línea Celular Tumoral , Helicobacter pylori/genética , Helicobacter pylori/patogenicidad , Humanos , Inflamación/metabolismo , Inflamación/microbiología , Modelos Moleculares , FN-kappa B/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Factores de Virulencia/genéticaRESUMEN
BACKGROUND: Antibodies to aquaporin-4 (AQP4-Ab), known as NMO-IgG, are a sensitive and specific marker for neuromyelitis optica (NMO). METHODS: To develop an enzyme-linked immunosorbent assay (ELISA) for AQP4-Ab, we expressed M23 isoform of human AQP4 in a baculovirus system, and used it as an antigen. We measured AQP4-Ab in the sera of 300 individuals: 64 with definite NMO, 31 with high-risk NMO, 105 with multiple sclerosis (MS), 57 with other neurological diseases (ONDs), and 43 healthy controls. We also performed longitudinal measurements of AQP4-Ab in 787 samples collected from 51 patients with definite or high-risk NMO. RESULTS: AQP4-Abs were positive in 72% with definite NMO, 55% with high-risk NMO, and 4% with MS, but none of the OND patients and the healthy individuals. The longitudinal measurement showed AQP4-Ab levels correlating with disease activity. Out of 38 initially seropositive patients, 21 became seronegative under effective immunosuppressive therapy. During most relapses, the serum AQP4-Ab levels were either high or rising compared with the previous value, although rising AQP4-Ab levels did not always lead to acute exacerbation. Two of the 13 initially seronegative patients converted to seropositive following acute exacerbations. CONCLUSIONS: We established an AQP4-Ab ELISA, which could be a potential monitoring tool of disease activity.
Asunto(s)
Acuaporina 4/inmunología , Autoanticuerpos/sangre , Ensayo de Inmunoadsorción Enzimática , Neuromielitis Óptica/diagnóstico , Adolescente , Adulto , Biomarcadores/sangre , Estudios de Casos y Controles , Diagnóstico Diferencial , Progresión de la Enfermedad , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Inmunosupresores/uso terapéutico , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/sangre , Esclerosis Múltiple/diagnóstico , Esclerosis Múltiple/inmunología , Neuromielitis Óptica/sangre , Neuromielitis Óptica/tratamiento farmacológico , Neuromielitis Óptica/inmunología , Valor Predictivo de las Pruebas , Proteínas Recombinantes , Recurrencia , República de Corea , Sensibilidad y Especificidad , Factores de Tiempo , Resultado del Tratamiento , Adulto JovenRESUMEN
Pyrococcus furiosus PF2050, a putative uncharacterized protein, was overexpressed in Escherichia coli, purified and crystallized at 298â K using 2-methyl-2,4-pentanediol as the precipitant. X-ray diffraction data were collected to a resolution of 1.56â Å at 100â K using synchrotron X-rays. The crystal was found to belong to space group P2(1), with unit-cell parameters a = 41.76, b = 66.43, c = 46.38â Å, ß = 96.62°. The asymmetric unit contained one subunit of PF2050, with a crystal volume per protein weight (V(M)) of 2.21â Å(3)â Da(-1) and a solvent content of 44.41%.
Asunto(s)
Proteínas Arqueales/química , Pyrococcus furiosus/química , Proteínas Arqueales/genética , Cristalización , Cristalografía por Rayos X , Expresión GénicaRESUMEN
Transglutaminase 2 (TGase2) is a calcium-dependent, cross-linking enzyme that catalyzes iso-peptide bond formation between peptide-bound lysine and glutamine residues. TGase 2 can activate NF-kappaB through the polymerization-mediated depletion of I-kappaBalpha without IKK activation. This NF-kappaB activation mechanism is associated with drug resistance in cancer cells. However, the polymers cannot be detected in cells, while TGase 2 over-expression depletes free I-kappaBalpha, which raises the question of how the polymerized I-kappaBalpha can be metabolized in cells. Among proteasome, lysosome and calpain systems, calpain inhibition was found to effectively increase the accumulation of I-kappaBalpha polymers in MCF7 cells transfected with TGase 2, and induced high levels of I-kappaBalpha polymers as well in MDA-MB-231 breast cancer cells that naturally express a high level of TGase 2. Inhibition of calpain also boosted the level of I-kappaBalpha polymers in HEK-293 cells in case of TGase 2 transfection either with I-kappaBalpha or I-kappaBalpha mutant (S32A, S36A). Interestingly, the combined inhibition of calpain and the proteasome resulted in an increased accumulation of both I-kappaBalpha polymers and I-kappaBalpha, concurrent with an inhibition of NF-kappaB activity in MDA-MB-231 cells. This suggests that mu-calpain proteasome-dependent I-kappaBalpha polymer degradation may contribute to cancer progression through constitutive NF-kappaB activation.
Asunto(s)
Calpaína/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas I-kappa B/metabolismo , Neoplasias/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Transglutaminasas/metabolismo , Ubiquitina/metabolismo , Calpaína/genética , Línea Celular Tumoral , Cistamina/farmacología , Dipéptidos/farmacología , Proteínas de Unión al GTP/genética , Humanos , Proteínas I-kappa B/antagonistas & inhibidores , Inhibidor NF-kappaB alfa , Neoplasias/patología , Proteína Glutamina Gamma Glutamiltransferasa 2 , ARN Interferente Pequeño/genética , Transglutaminasas/genéticaRESUMEN
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.
Asunto(s)
Bioquímica/métodos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/aislamiento & purificación , Receptores de Ácido Retinoico/química , Receptores de Ácido Retinoico/aislamiento & purificación , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/aislamiento & purificación , Secuencia de Aminoácidos , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Datos de Secuencia Molecular , Fosfolipasas A2/metabolismo , Fosfolipasas A2 Calcio-Independiente , Estructura Terciaria de Proteína , Receptores de Ácido Retinoico/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0070358.].
RESUMEN
Spleen tyrosine kinase (SYK) is a cytosolic nonreceptor protein tyrosine kinase that mediates key signal transduction pathways following the activation of immune cell receptors. SYK regulates cellular events induced by the B-cell receptor and Fc receptors with high intrinsic activity. Furthermore, SYK has been regarded as an attractive target for the treatment of autoimmune diseases and cancers. Here, we report the crystal structures of SYK in complex with seven newly developed inhibitors (G206, G207, O178, O194, O259, O272, and O282) to provide structural insights into which substituents of the inhibitors and binding regions of SYK are essential for lead compound optimization. Our kinase inhibitors exhibited high inhibitory activities against SYK, with half-maximal inhibitory concentrations (IC50 ) of approximately 0.7-33 nm, but they showed dissimilar inhibitory activities against KDR, RET, JAK2, JAK3, and FLT3. Among the seven SYK inhibitors, O272 and O282 exhibited highly specific inhibitions against SYK, whereas O194 exhibited strong inhibition of both SYK and FLT3. Three inhibitors (G206, G207, and O178) more efficiently inhibited FLT3 while still substantially inhibiting SYK activity. The binding mode analysis suggested that a highly selective SYK inhibitor can be developed by optimizing the functional groups that facilitate direct interactions with Asn499. DATABASE: The atomic coordinates and structure factors for human SYK are in the Protein Data Bank under accession codes 4XG2 (inhibitor-free form), 4XG3 (G206), 4XG4 (G207), 5GHV (O178), 4XG6 (O194), 4XG7 (O259), 4XG8 (O272), and 4XG9 (O282).
Asunto(s)
Antineoplásicos/química , Inhibidores de Proteínas Quinasas/química , Quinasa Syk/antagonistas & inhibidores , Quinasa Syk/química , Antineoplásicos/farmacología , Cristalografía por Rayos X , Diseño de Fármacos , Indazoles/química , Modelos Moleculares , Oxazinas/química , Inhibidores de Proteínas Quinasas/farmacología , Pirazinas/química , Piridinas/químicaRESUMEN
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.
Asunto(s)
Inhibidores de Proteínas Quinasas/química , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-pim-1/química , Pirimidinas/química , Sitios de Unión , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Estructura Molecular , Unión Proteica , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-pim-1/metabolismo , Piridonas/química , Piridonas/metabolismo , Piridonas/farmacología , Pirimidinas/metabolismo , Pirimidinas/farmacología , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Proteínas Arqueales/química , Pyrococcus furiosus , Secuencia de Aminoácidos , Proteínas Arqueales/metabolismo , Cristalografía por Rayos X , ADN/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de ProteínaRESUMEN
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.
Asunto(s)
Adenosina Trifosfato/metabolismo , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/metabolismo , Transglutaminasas/química , Transglutaminasas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cristalografía por Rayos X , Bases de Datos de Proteínas , Guanosina Difosfato/metabolismo , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Proteína Glutamina Gamma Glutamiltransferasa 2RESUMEN
Helicobacter pylori infection is one of the highest risk factors for gastroduodenal diseases including gastric cancer. Tumor necrosis factor-alpha (TNF-alpha) is one of the essential cytokines for tumor promotion, and thus, an H. pylori protein that induces TNF-alpha is believed to play a significant role in gastric cancer development in humans. The HP0596 gene product of H. pylori strain 26695 was identified as the TNF-alpha-inducing protein (Tipalpha). Tipalpha is secreted from H. pylori as dimers and enters the gastric cells. It was shown to have a DNA-binding activity. Here, we have determined the crystal structure of Tipalpha from H. pylori. Its monomer consists of two structural domains ("mixed domain" and "helical domain"). Tipalpha exists as a dimer in the crystal, and the dimeric structure represents a novel scaffold for DNA binding. A positively charged surface patch formed across the two monomers of the Tipalpha dimer by the loop between helices alpha1 and alpha2 may be important in DNA binding.
Asunto(s)
Proteínas Bacterianas/química , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , ADN/metabolismo , Dimerización , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
It has been suggested that nucleophosmin has an anti-apoptotic function via Bax binding. We found that nucleophosmin is a substrate of transglutaminase 2 (TGase 2) in cancer cells. Increased expression of TGase 2 expression is highly associated with drug resistance, and polymerization of nucleophosmin by TGase 2 also can be correlated with the drug resistance of cancer cells. In the present study, an accumulation of nucleophosmin in cytosol was detected when doxorubicin was treated to cancer cells, and it was found, moreover, that an increase of cytosolic nucleophosmin can result in drug-induced apoptosis. Nucleophosmin was polymerized by TGase 2, and the polymerization was inhibited with the TGase 2 inhibitor, cystamine, in vitro. The nucleophosmin level in the cytosolic cell fraction was reduced when TGase 2 was expressed, and the reduced nucleophosmin level was rescued by cystamine treatment. Moreover, nucleophosmin cross-linked by TGase 2 was eradicated in MCF7 cells via the ubiquitin-proteasomal pathway. In parallel with this nucleophosmin-level restoration, the pro-apoptotic Bax protein level was increased. Therefore, depletion of cytosolic nucleophosmin by TGase 2 can decrease Bax protein stability and lead to anti-apoptosis. Drug-resistant cancer cells became sensitive to doxorubicin treatment when nucleophosmin was expressed in cytosol. Taking these results together, it can be concluded that TGase 2 inhibits accumulation of cytosolic nucleophosmin through polymerization, which results in drug resistance in cancer cells.