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1.
ACS Chem Biol ; 8(11): 2538-49, 2013 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-24010878

RESUMEN

Histone deacetylase 3 (HDAC3) is a promising epigenetic drug target for multiple therapeutic applications. Direct interaction between the Deacetylase Activating Domain of the silencing mediator for retinoid or thyroid-hormone receptors (SMRT-DAD) is required for activation of enzymatic activity of HDAC3. The structure of this complex and the nature of interactions with HDAC inhibitors in solution are unknown. Using novel photoreactive HDAC probes, "nanorulers", we determined the distance between the catalytic site of the full-length HDAC3 and SMRT-DAD in solution at physiologically relevant conditions and found it to be substantially different from that predicted by the X-ray model with a Δ379-428 aa truncated HDAC3. Further experiments indicated that in solution this distance might change in response to chemical stimuli, while the enzymatic activity remained unaffected. These observations were further validated by Saturation Transfer Difference (STD) NMR experiments. We propose that the observed changes in the distance are an important part of the histone code that remains to be explored. Mapping direct interactions and distances between macromolecules with such "nanorulers" as a function of cellular events facilitates better understanding of basic biology and ways for its manipulation in a cell- and tissue-specific manner.


Asunto(s)
Histona Desacetilasas/química , Luz , Co-Represor 2 de Receptor Nuclear/química , Cristalografía por Rayos X , Humanos , Concentración 50 Inhibidora , Conformación Molecular , Nanotecnología
2.
Bioorg Med Chem Lett ; 22(21): 6621-7, 2012 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-23010266

RESUMEN

A novel series of HDAC8 inhibitors without a zinc-chelating hydroxamic acid moiety is reported. Photoaffinity labeling and molecular modeling studies suggest that these ligands are likely to bind in an 'upside-down' fashion in a secondary binding site proximal to the main catalytic site. The most potent ligand in the series exhibits an IC(50) of 28 µM for HDAC8 and is found to inhibit the deacetylation of H4 but not α-tubulin in SH-SY5Y cell line.


Asunto(s)
Quelantes , Histona Desacetilasas/metabolismo , Proteínas Represoras/metabolismo , Sitios de Unión , Western Blotting , Línea Celular , Línea Celular Tumoral , Quelantes/síntesis química , Quelantes/metabolismo , Quelantes/farmacología , Histona Desacetilasas/química , Humanos , Ácidos Hidroxámicos/química , Concentración 50 Inhibidora , Ligandos , Estructura Molecular , Unión Proteica/efectos de los fármacos , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/química , Zinc/química
3.
J Med Chem ; 54(13): 4350-64, 2011 Jul 14.
Artículo en Inglés | MEDLINE | ID: mdl-21548582

RESUMEN

The design, synthesis, docking, and biological evaluation of novel potent HDAC3 and HDAC8 isoxazole- and pyrazole-based diazide probes suitable for binding ensemble profiling with photoaffinity labeling (BEProFL) experiments in cells is described. Both the isoxazole- and pyrazole-based probes exhibit low nanomolar inhibitory activity against HDAC3 and HDAC8, respectively. The pyrazole-based probe 3f appears to be one of the most active HDAC8 inhibitors reported in the literature with an IC(50) of 17 nM. Our docking studies suggest that unlike the isoxazole-based ligands the pyrazole-based ligands are flexible enough to occupy the second binding site of HDAC8. Probes/inhibitors 2b, 3a, 3c, and 3f exerted the antiproliferative and neuroprotective activities at micromolar concentrations through inhibition of nuclear HDACs, indicating that they are cell permeable and the presence of an azide or a diazide group does not interfere with the neuroprotection properties, or enhance cellular cytotoxicity, or affect cell permeability.


Asunto(s)
Azidas/síntesis química , Inhibidores de Histona Desacetilasas/síntesis química , Histona Desacetilasas/química , Ácidos Hidroxámicos/síntesis química , Isoxazoles/síntesis química , Modelos Moleculares , Etiquetas de Fotoafinidad/síntesis química , Pirazoles/síntesis química , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Azidas/química , Azidas/farmacología , Línea Celular Tumoral , Permeabilidad de la Membrana Celular , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores de Histona Desacetilasas/química , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Humanos , Ácidos Hidroxámicos/química , Ácidos Hidroxámicos/farmacología , Isoxazoles/química , Isoxazoles/farmacología , Inhibidores de la Metaloproteinasa de la Matriz , Metaloproteinasas de la Matriz/química , Fármacos Neuroprotectores/síntesis química , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/farmacología , Etiquetas de Fotoafinidad/química , Etiquetas de Fotoafinidad/farmacología , Unión Proteica , Pirazoles/química , Pirazoles/farmacología , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Relación Estructura-Actividad
4.
J Biol Chem ; 286(11): 8729-39, 2011 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-21239500

RESUMEN

Mineralized matrix formation is a well orchestrated event requiring several players. Glucose-regulated protein-78 (GRP-78) is an endoplasmic reticulum chaperone protein that has been implicated in functional roles ranging from involvement in cancer biology to serving as a receptor for viruses. In the present study we explored the role of GRP-78 in mineralized matrix formation. Differential expression of GRP-78 mRNA and protein was observed upon in vitro differentiation of primary mouse calvarial cells. An interesting observation was that GRP-78 was identified in the secretome of these cells and in the bone matrix, suggesting an extracellular function during matrix formation. In vitro nucleation experiments under physiological concentrations of calcium and phosphate ions indicated that GRP-78 can induce the formation of calcium phosphate polymorphs by itself, when bound to immobilized type I collagen and on demineralized collagen wafers. We provide evidence that GRP-78 can bind to DMP1 and type I collagen independent of each other in a simulated extracellular environment. Furthermore, we demonstrate the cell surface localization of GRP-78 and provide evidence that it functions as a receptor for DMP1 endocytosis in pre-osteoblasts and primary calvarial cells. Overall, this study represents a paradigm shift in the biological function of GRP-78.


Asunto(s)
Calcificación Fisiológica/fisiología , Diferenciación Celular/fisiología , Matriz Extracelular/metabolismo , Proteínas de Choque Térmico/metabolismo , Osteoblastos/metabolismo , Cráneo/metabolismo , Animales , Fosfatos de Calcio/metabolismo , Línea Celular , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Endocitosis/fisiología , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Chaperón BiP del Retículo Endoplásmico , Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de Choque Térmico/genética , Ratones , Osteoblastos/citología , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Unión Proteica , Ratas , Cráneo/citología
5.
Arch Pharm (Weinheim) ; 343(3): 167-72, 2010 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-20217840

RESUMEN

The histone deacetylase (HDAC) enzyme plays an important role in gene transcription. Inhibitors of histone deacetylases induce cell differentiation and suppress cell proliferation in tumor cells. Hydroxamates with rigid linker have displayed better inhibition profiles than those with linear and flexible aliphatic linkers. We have designed and synthesized several potential histone deacetylase inhibitors with a piperazine moiety in the linker region to test the effect of reduced linker flexibility. Inhibitors were evaluated for their inhibitory action on human HDAC3/NCoR2 and HDAC8. N-Hydroxycarboxamide derivatives (compounds 4a-d) were found to be better than N-hydroxyacetamide derivatives (compounds 6a-d) against HDAC8. Amongst the synthesized compounds, 4a (HDAC8, IC50: 3.15 microM) with no substitution in the aryl cap was the most active and promising lead for further investigations.


Asunto(s)
Pruebas de Enzimas/métodos , Inhibidores de Histona Desacetilasas/síntesis química , Inhibidores de Histona Desacetilasas/farmacología , Piperazinas/síntesis química , Piperazinas/farmacología , Química Farmacéutica/métodos , Evaluación Preclínica de Medicamentos , Inhibidores de Histona Desacetilasas/química , Humanos , Ácidos Hidroxámicos/química , Técnicas In Vitro , Modelos Moleculares , Estructura Molecular , Piperazinas/química , Unión Proteica , Relación Estructura-Actividad
6.
J Med Chem ; 52(22): 7003-13, 2009 Nov 26.
Artículo en Inglés | MEDLINE | ID: mdl-19886628

RESUMEN

A binding ensemble profiling with (f)photoaffinity labeling (BEProFL) approach that utilizes photolabeling of HDAC8 with a probe containing a UV-activated aromatic azide, mapping of the covalent modifications by liquid chromatography-tandem mass spectrometry, and a computational method to characterize the multiple binding poses of the probe is described. By use of the BEProFL approach, two distinct binding poses of the HDAC8 probe were identified. The data also suggest that an "upside-down" pose with the surface binding group of the probe bound in an alternative pocket near the catalytic site may contribute to the binding.


Asunto(s)
Inhibidores de Histona Desacetilasas/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Etiquetas de Fotoafinidad/metabolismo , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/metabolismo , Secuencia de Aminoácidos , Azidas/química , Azidas/metabolismo , Azidas/farmacología , Cromatografía Liquida , Inhibidores de Histona Desacetilasas/química , Histona Desacetilasas/química , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión Proteica , Conformación Proteica , Proteínas Represoras/química , Espectrometría de Masas en Tándem
7.
J Mol Model ; 15(12): 1463-79, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19440740

RESUMEN

Molecular modeling and docking studies along with three-dimensional quantitative structure relationships (3D-QSAR) studies have been used to determine the correct binding mode of glycogen synthase kinase 3beta (GSK-3beta) inhibitors. The approaches of comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) are used for the 3D-QSAR of 51 substituted benzofuran-3-yl-(indol-3-yl)maleimides as GSK-3beta inhibitors. Two binding modes of the inhibitors to the binding site of GSK-3beta are investigated. The binding mode 1 yielded better 3D-QSAR correlations using both CoMFA and CoMSIA methodologies. The three-component CoMFA model from the steric and electrostatic fields for the experimentally determined pIC(50) values has the following statistics: R(2)(cv) = 0.386 nd SE(cv) = 0.854 for the cross-validation, and R(2) = 0.811 and SE = 0.474 for the fitted correlation. F (3,47) = 67.034, and probability of R(2) = 0 (3,47) = 0.000. The binding mode suggested by the results of this study is consistent with the preliminary results of X-ray crystal structures of inhibitor-bound GSK-3beta. The 3D-QSAR models were used for the estimation of the inhibitory potency of two additional compounds.


Asunto(s)
Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Maleimidas/química , Maleimidas/farmacología , Modelos Moleculares , Relación Estructura-Actividad Cuantitativa , Glucógeno Sintasa Quinasa 3 beta , Lisinopril/química , Lisinopril/farmacología
8.
J Med Chem ; 52(7): 1853-63, 2009 Apr 09.
Artículo en Inglés | MEDLINE | ID: mdl-19338355

RESUMEN

Recent studies have demonstrated that glycogen synthase kinase 3beta (GSK-3beta) is overexpressed in human colon and pancreatic carcinomas, contributing to cancer cell proliferation and survival. Here, we report the design, synthesis, and biological evaluation of benzofuran-3-yl-(indol-3-yl)maleimides, potent GSK-3beta inhibitors. Some of these compounds show picomolar inhibitory activity toward GSK-3beta and an enhanced selectivity against cyclin-dependent kinase 2 (CDK-2). Selected GSK-3beta inhibitors were tested in the pancreatic cancer cell lines MiaPaCa-2, BXPC-3, and HupT3. We determined that some of these compounds, namely compounds 5, 6, 11, 20, and 26, demonstrate antiproliferative activity against some or all of the pancreatic cancer cells at low micromolar to nanomolar concentrations. We found that the treatment of pancreatic cancer cells with GSK-3beta inhibitors 5 and 26 resulted in suppression of GSK-3beta activity and a distinct decrease of the X-linked inhibitor of apoptosis (XIAP) expression, leading to significant apoptosis. The present data suggest a possible role for GSK-3beta inhibitors in cancer therapy, in addition to their more prominent applications in CNS disorders.


Asunto(s)
Antineoplásicos/síntesis química , Benzofuranos/síntesis química , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Indoles/síntesis química , Maleimidas/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Apoptosis , Benzofuranos/química , Benzofuranos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Ensayos de Selección de Medicamentos Antitumorales , Glucógeno Sintasa Quinasa 3/química , Glucógeno Sintasa Quinasa 3 beta , Humanos , Indoles/química , Indoles/farmacología , Maleimidas/química , Maleimidas/farmacología , Modelos Moleculares , Neoplasias Pancreáticas , Relación Estructura-Actividad , Proteína Inhibidora de la Apoptosis Ligada a X/antagonistas & inhibidores , Proteína Inhibidora de la Apoptosis Ligada a X/biosíntesis
9.
J Biol Chem ; 283(47): 32880-8, 2008 Nov 21.
Artículo en Inglés | MEDLINE | ID: mdl-18796433

RESUMEN

Human glutathione transferase pi (GST pi) has been crystallized as a homodimer, with a subunit molecular mass of approximately 23 kDa; however, in solution the average molecular mass depends on protein concentration, approaching that of monomer at <0.03 mg/ml, concentrations typically used to measure catalytic activity of the enzyme. Electrostatic interaction at the subunit interface greatly influences the dimer-monomer equilibrium of the enzyme and is an important force for holding subunits together. Arg-70, Arg-74, Asp-90, Asp-94, and Thr-67 were selected as target sites for mutagenesis, because they are at the subunit interface. R70Q, R74Q, D90N, D94N, and T67A mutant enzymes were constructed, expressed in Escherichia coli, and purified. The construct of N-terminal His tag enzyme facilitates the purification of GST pi, resulting in a high yield of enzyme, but does not alter the kinetic parameters or secondary structure of the enzyme. Our results indicate that these mutant enzymes show no appreciable changes in K(m) for 1-chloro-2,4-dinitrobenzene and have similar CD spectra to that of wild-type enzyme. However, elimination of the charges of either Arg-70, Arg-74, Asp-90, or Asp-94 shifts the dimer-monomer equilibrium toward monomer. In addition, replacement of Asp-94 or Arg-70 causes a large increase in the K(m)(GSH), whereas substitution for Asp-90 or Arg-74 primarily results in a marked decrease in V(max). The GST pi retains substantial catalytic activity as a monomer probably because the glutathione and electrophilic substrate sites (such as for 1-chloro-2,4-dinitrobenzene) are predominantly located within each subunit.


Asunto(s)
Gutatión-S-Transferasa pi/química , Secuencia de Aminoácidos , Catálisis , Dicroismo Circular , Dimerización , Gutatión-S-Transferasa pi/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Cinética , Conformación Molecular , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Electricidad Estática
10.
J Biol Chem ; 283(44): 29658-70, 2008 Oct 31.
Artículo en Inglés | MEDLINE | ID: mdl-18757373

RESUMEN

Dentin matrix protein 1 (DMP1), a phosphorylated protein present in the mineral phase of both vertebrates and invertebrates, is a key regulatory protein during biogenic formation of mineral deposits. Previously we showed that DMP1 is localized in the nuclear compartment of preosteoblasts and preodontoblasts. In the nucleus DMP1 might play an important role in the regulation of genes that control osteoblast or odontoblast differentiation. Here, we show that cellular uptake of DMP1 occurs through endocytosis. Interestingly, this process is initiated by DMP1 binding to the glucose-regulated protein-78 (GRP-78) localized on the plasma membrane of preodontoblast cells. Binding of DMP1 to GRP-78 receptor was determined to be specific and saturable with a binding dissociation constant K(D)=85 nm. We further depict a road map for the endocytosed DMP1 and demonstrate that the internalization is mediated primarily by caveolae and that the vesicles containing DMP1 are routed to the nucleus along microtubules. Immunohistochemical analysis and binding studies performed with biotin-labeled DMP1 confirm spatial co-localization of DMP1 and GRP-78 in the preodontoblasts of a developing mouse molar. Co-localization of DMP1 with GRP-78 was also observed in T4-4 preodontoblast cells, dental pulp stem cells, and primary preodontoblasts. By small interfering RNA techniques, we demonstrate that the receptor for DMP1 is GRP-78. Therefore, binding of DMP1 with GRP-78 receptor might be an important mechanism by which DMP1 is internalized and transported to the nucleus during bone and tooth development.


Asunto(s)
Retículo Endoplásmico/metabolismo , Proteínas de la Matriz Extracelular/fisiología , Proteínas de Choque Térmico/fisiología , Chaperonas Moleculares/fisiología , Fosfoproteínas/fisiología , Secuencia de Aminoácidos , Animales , Desarrollo Óseo , Endocitosis , Proteínas de la Matriz Extracelular/química , Proteínas de Choque Térmico/metabolismo , Humanos , Microscopía Confocal , Modelos Biológicos , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Fosfoproteínas/química , Ratas , Proteínas Recombinantes/química , Diente/embriología
11.
J Am Chem Soc ; 129(26): 8328-32, 2007 Jul 04.
Artículo en Inglés | MEDLINE | ID: mdl-17552518

RESUMEN

More than two million American adults, or approximately one percent of the population 18 years or older, suffer from bipolar disorder. Current treatments include the so-called "mood stabilizers," lithium and valproic acid. Both are relatively dated drugs that are only partially effective and produce various undesirable side effects including weight gain. Based upon continued efforts to understand the molecular target for lithium, it now appears that specific inhibitors of the enzyme glycogen synthase kinase-3beta (GSK-3beta) may mimic the therapeutic action of mood stabilizers and might therefore allow for the design of improved drugs for treating patients with bipolar disorder as well as certain neurodegenerative disorders. Furthermore, the pro-apoptotic properties of the GSK-3 enzyme suggest the possible use of such inhibitors as neuroprotective agents. In fact, neuroprotection may contribute to the treatment of mood disorders. The present chemistry, modeling, and biology efforts have identified 3-benzofuranyl-4-indolylmaleimides as potent and relatively selective GSK-3beta inhibitors. The best ligand in this series (having a Ki value of 4.6 nM against GSK-3beta) was studied in a novel mouse model of mania that has recently been validated with several clinically effective mood stabilizers. This study presents the first demonstration of the efficacy of a GSK-3beta inhibitor in this mouse model of mania. Selective brain penetrable GSK-3 ligands like those described herein become valuable research tools in better defining the role of this multifaceted kinase in both physiological and pathophysiological events.


Asunto(s)
Antimaníacos/química , Antimaníacos/farmacología , Trastorno Bipolar/tratamiento farmacológico , Diseño de Fármacos , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Litio , Anfetamina , Animales , Trastorno Bipolar/inducido químicamente , Clordiazepóxido , Glucógeno Sintasa Quinasa 3 beta , Humanos , Ratones , Estructura Molecular , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad
12.
ChemMedChem ; 1(2): 256-66, 2006 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-16892358

RESUMEN

Research by Klein and co-workers suggests that the inhibition of GSK-3beta by small molecules may offer an important strategy in the treatment of a number of central nervous system (CNS) disorders including Alzheimer's disease, Parkinson's disease, and bipolar disorders. Based on results from kinase-screening assays that identified a staurosporine analogue as a modest inhibitor of GSK-3beta, a series of 3-indolyl-4-indazolylmaleimides was prepared for study in both enzymatic and cell-based assays. Most strikingly, whereas we identified ligands having poor to high potency for GSK-3beta inhibition, only ligands with a Ki value of less than 8 nM, namely maleimides 18 and 22, were found to inhibit Tau phosphorylation at a GSK-3beta-specific site (Ser 396/404). Accordingly, maleimides 18 and 22 may protect neuronal cells against cell death by decreasing the level of alpha-Syn protein expression. We conclude that the GSK-3beta inhibitors described herein offer promise in defending cells against MPP+-induced neurotoxicity and that such compounds will be valuable to explore in animal models of Parkinson's disease as well as in other Tau-related neurodegenerative disease states.


Asunto(s)
Inhibidores Enzimáticos/farmacología , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Enfermedad de Parkinson/metabolismo , alfa-Sinucleína/metabolismo , Proteínas tau/antagonistas & inhibidores , Línea Celular Tumoral , Inhibidores Enzimáticos/química , Glucógeno Sintasa Quinasa 3 beta , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Fosforilación , Espectrometría de Masa Bombardeada por Átomos Veloces , Relación Estructura-Actividad , Proteínas tau/metabolismo
13.
J Biol Chem ; 281(13): 8796-805, 2006 Mar 31.
Artículo en Inglés | MEDLINE | ID: mdl-16418174

RESUMEN

GRP78 is a major protein regulated by the mammalian endoplasmic reticulum stress response, and up-regulation has been shown to be important in protecting cells from challenge with cytotoxic agents. GRP78 has ATPase activity, acts as a chaperone, and interacts specifically with other proteins, such as caspases, as part of a mechanism regulating apoptosis. GRP78 is also reported to have a possible role as a Ca2+ storage protein. In order to understand the potential biological effects of Ca2+ and ATP/ADP binding on the biology of GRP78, we have determined its ligand binding properties. We show here for the first time that GRP78 can bind Ca2+, ATP, and ADP, each with a 1:1 stoichiometry, and that the binding of cation and nucleotide is cooperative. These observations do not support the hypothesis that GRP78 is a dynamic Ca2+ storage protein. Furthermore, we demonstrate that whereas Mg2+ enhances GRP78 binding to ADP and ATP to the same extent, Ca2+ shows a differential enhancement. In the presence of Ca2+, the KD for ATP is lowered approximately 11-fold, and the KD for ADP is lowered around 930-fold. The KD for Ca2+ is lowered approximately 40-fold in the presence of ATP and around 880-fold with ADP. These findings may explain the biological requirement for a nucleotide exchange factor to remove ADP from GRP78. Taken together, our data suggest that the Ca2+-binding property of GRP78 may be part of a signal transduction pathway that modulates complex interactions between GRP78, ATP/ADP, secretory proteins, and caspases, and this ultimately has important consequences for cell viability.


Asunto(s)
Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Calcio/metabolismo , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Adenosina Difosfato/farmacología , Adenosina Trifosfato/farmacología , Animales , Sitios de Unión , Calorimetría , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Relación Dosis-Respuesta a Droga , Chaperón BiP del Retículo Endoplásmico , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/aislamiento & purificación , Análisis de los Mínimos Cuadrados , Magnesio/metabolismo , Ratones , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/aislamiento & purificación , Mutación , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Análisis de Regresión , Espectrofotometría Ultravioleta , Termodinámica
14.
J Mol Biol ; 356(2): 469-82, 2006 Feb 17.
Artículo en Inglés | MEDLINE | ID: mdl-16376939

RESUMEN

Carriers of the D18G transthyretin (TTR) mutation display an unusual central nervous system (CNS) phenotype with late onset of disease. D18G TTR is monomeric and highly prone to misfold and aggregate even at physiological conditions. Extremely low levels of mutant protein circulate both in human serum and cerebrospinal fluid, indicating impaired secretion of D18G TTR. Recent data show efficient selective ER-associated degradation (ERAD) of D18G TTR. One essential component of the ER-assisted folding machinery is the molecular chaperone BiP. Co-expression of BiP and D18G TTR, or BiP and wild-type (wt) TTR, or mutants A25T TTR and L55P TTR in Escherichia coli showed that only D18G TTR was significantly captured by BiP. Negligible capture of wt TTR and L55P TTR was seen and a sixfold smaller amount of A25T TTR bound to BiP compared to D18G TTR. These data correlate very well with thermodynamic and kinetic stability of the TTR variants, indicating that folding efficiency is inversely correlated to BiP capture. The complexes between BiP and D18G TTR were stable and could be isolated through affinity chromatography. Analytical ultracentrifugation and size-exclusion chromatography revealed that D18G TTR and BiP formed a mixture of 1:1 complexes and large soluble oligomers. The stoichiometry of captured D18G TTR versus BiP increased with increasing size of the oligomers. This indicates that BiP either worked as a molecular shepherd collecting the aggregation-prone mutant into stable oligomers or that BiP could bind to oligomers formed from misfolded mutant protein. Sequence analysis of bound TTR peptides to BiP revealed a bound sequence corresponding to residues 88-103 of TTR, comprising beta-strand F in the folded TTR monomer constituting part of the hydrogen bonding tetramer interface in native TTR. The F-strand has also been suggested as a possible elongation region of amyloid fibrils, implicating how substoichiomeric amounts of BiP could sequester prefibrillar amyloidogenic oligomers through binding to this part of TTR. BiP binding to D18G TTR was abolished by addition of ATP. The released D18G TTR completely misfolded into amyloid aggregates as shown by ThT fluorescence and Congo red binding.


Asunto(s)
Amiloide/metabolismo , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Prealbúmina/química , Prealbúmina/genética , Pliegue de Proteína , Secuencia de Aminoácidos , Amiloide/ultraestructura , Chaperón BiP del Retículo Endoplásmico , Proteínas de Choque Térmico/genética , Humanos , Modelos Moleculares , Chaperonas Moleculares/genética , Datos de Secuencia Molecular , Complejos Multiproteicos , Prealbúmina/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína
15.
Biochem Biophys Res Commun ; 338(3): 1467-77, 2005 Dec 23.
Artículo en Inglés | MEDLINE | ID: mdl-16271702

RESUMEN

MTJ1/ERdj1 and its human homologue HTJ1 are membrane proteins that interact with the molecular chaperone BiP through their J-domain. HTJ1 also contains a C-terminal cytosolic region of unknown function that consists of two SANT domains separated by a spacer region. We recently showed that the second SANT domain of HTJ1 (SANT2) binds to alpha1-antichymotrypsin and alters its serpin activity [B. Kroczynska, C.M. Evangelista, S.S. Samant, E.C. Elguindi, S.Y. Blond, The SANT2 domain of the murine tumor cell DnaJ-like protein 1 human homologue interacts with alpha1-antichymotrypsin and kinetically interferes with its serpin inhibitory activity, J. Biol. Chem. 279 (2004) 11432-11443]. Here, we identified a new variant of human inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) that also interacts with the SANT2 domain of HTJ1. Biochemical, mutagenesis, and fluorescence studies demonstrate that SANT2 binds to a carboxyl-terminal fragment that corresponds to the last third of the new ITIH4 isoform sequence (residues 588-930). ITIH4 and MTJ1 co-immunoprecipitate from total liver protein extracts and SANT2 protects the ITIH4(588-930) recombinant fragment from being processed by kallikrein in vitro. This work reveals that the SANT2 domain of HTJ1 is a genuine protein-protein interaction module.


Asunto(s)
Proteínas Sanguíneas/metabolismo , Glicoproteínas/metabolismo , Chaperonas Moleculares/metabolismo , Inhibidores de Tripsina/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Sanguíneas/química , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/aislamiento & purificación , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/aislamiento & purificación , Proteínas del Choque Térmico HSP40 , Histidina/genética , Histidina/metabolismo , Humanos , Calicreínas/metabolismo , Proteínas de la Membrana , Ratones , Chaperonas Moleculares/genética , Datos de Secuencia Molecular , Oligopéptidos/genética , Oligopéptidos/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Unión Proteica , Pliegue de Proteína , Proteínas Inhibidoras de Proteinasas Secretoras , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Inhibidores de Tripsina/química , Inhibidores de Tripsina/genética , Inhibidores de Tripsina/aislamiento & purificación , Técnicas del Sistema de Dos Híbridos
16.
J Biol Chem ; 279(12): 11432-43, 2004 Mar 19.
Artículo en Inglés | MEDLINE | ID: mdl-14668352

RESUMEN

The murine tumor cell DnaJ-like protein 1 or MTJ1/ERdj1 is a membrane J-domain protein enriched in microsomal and nuclear fractions. We previously showed that its lumenal J-domain stimulates the ATPase activity of the molecular chaperone BiP/GRP78 (Chevalier, M., Rhee, H., Elguindi, E. C., and Blond, S. Y. (2000) J. Biol. Chem. 275, 19620-19627). MTJ1/ERdj1 also contains a large carboxyl-terminal cytosolic extension composed of two tryptophan-mediated repeats or SANT domains for which the function(s) is unknown. Here we describe the cloning of the human homologue HTJ1 and its interaction with alpha(1)-antichymotrypsin (ACT), a member of the serine proteinase inhibitor (serpin) family. The interaction was initially identified in a two-hybrid screening and further confirmed in vitro by dot blots, native electrophoresis, and fluorescence studies. The second SANT domain of HTJ1 (SANT2) was found to be sufficient for binding to ACT, both in yeast and in vitro. Single tryptophan-alanine substitutions at two strictly conserved residues significantly (Trp-497) or totally (Trp-520) abolished the interaction with ACT. SANT2 binds to human ACT with an intrinsic affinity equal to 0.5 nm. Preincubation of ACT with nearly stoichiometric concentrations of SANT2 wild-type but not SANT2: W520A results in an apparent loss of ACT inhibitory activity toward chymotrypsin. Kinetic analysis indicates that the formation of the covalent inhibitory complex ACT-chymotrypsin is significantly delayed in the presence of SANT2 with no change on the catalytic efficiency of the enzyme. This work demonstrates for the first time that the SANT2 domain of MTJ1/HTJ1/ERdj1 mediates stable and high affinity protein-protein interactions.


Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de la Membrana/metabolismo , Serpinas/metabolismo , alfa 1-Antiquimotripsina/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Proteínas Portadoras/química , Proteínas Portadoras/genética , Línea Celular Tumoral , Clonación Molecular , Cartilla de ADN , ADN Complementario , Chaperón BiP del Retículo Endoplásmico , Proteínas del Choque Térmico HSP40 , Proteínas de Choque Térmico/química , Humanos , Cinética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido
17.
Chem Res Toxicol ; 15(7): 935-42, 2002 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-12119004

RESUMEN

Glutathione S-transferases (GSTs) are a family of detoxification isozymes that protect cells by conjugating GSH to a variety of toxic compounds, and they may also play a role in the regulation of both cellular proliferation and apoptosis. We have previously shown that human GST P1-1, which is the most widely distributed extrahepatic isozyme, could be inactivated by the catechol estrogen metabolite 4-hydroxyequilenin (4-OHEN) in vitro [Chang, M., Shin, Y. G., van Breemen, R. B., Blond, S. Y., and Bolton, J. L. (2001) Biochemistry 40, 4811-4820]. In the present study, we found that 4-OHEN and another catechol estrogen, 4,17beta-hydroxyequilenin (4,17beta-OHEN), significantly decreased GSH levels and the activity of GST within minutes in both estrogen receptor (ER) negative (MDA-MB-231) and ER positive (S30) human breast cancer cells. In addition, 4-OHEN caused significant decreases in GST activity in nontransformed human breast epithelial cells (MCF-10A) but not in the human hepatoma HepG2 cells, which lack GST P1-1. We also showed that GSH partially protected the inactivation of GST P1-1 by 4-OHEN in vitro, and depletion of cellular GSH enhanced the 4-OHEN-induced inhibition of GST activity. In addition, 4-OHEN GSH conjugates contributed about 27% of the inactivation of GST P1-1 by 4-OEHN in vitro. Our in vitro kinetic inhibition experiments with 4-OHEN showed that GST P1-1 had a lower K(i) value (20.8 microM) compared to glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 52.4 microM), P450 reductase (PR, 77.4 microM), pyruvate kinase (PK, 159 microM), glutathione reductase (GR, 230 microM), superoxide dismutase (SOD, 448 microM), catalase (562 microM), GST M1-1 (620 microM), thioredoxin reductase (TR, 694 microM), and glutathione peroxidase (GPX, 1410 microM). In contrast to the significant inhibition of total GST activity in these human breast cancer cells, 4-OHEN only slightly inhibited the cellular GAPDH activity, and other cellular enzymes including PR, PK, GR, SOD, catalase, TR, and GPX were resistant to 4-OHEN-induced inhibition. These data suggest that GST P1-1 may be a preferred protein target for equine catechol estrogens in vivo.


Asunto(s)
Neoplasias de la Mama/enzimología , Inhibidores Enzimáticos/farmacología , Equilenina/análogos & derivados , Equilenina/farmacología , Estrógenos de Catecol/farmacología , Glutatión Transferasa/antagonistas & inhibidores , Isoenzimas/antagonistas & inhibidores , Animales , Neoplasias de la Mama/patología , Gutatión-S-Transferasa pi , Caballos , Humanos , Cinética , Especificidad por Sustrato , Células Tumorales Cultivadas
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