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1.
Blood ; 142(6): 574-588, 2023 08 10.
Artículo en Inglés | MEDLINE | ID: mdl-37192295

RESUMEN

Tyrosine kinase inhibitors (TKIs) are very effective in treating chronic myelogenous leukemia (CML), but primitive, quiescent leukemia stem cells persist as a barrier to the cure. We performed a comprehensive evaluation of metabolic adaptation to TKI treatment and its role in CML hematopoietic stem and progenitor cell persistence. Using a CML mouse model, we found that glycolysis, glutaminolysis, the tricarboxylic acid cycle, and oxidative phosphorylation (OXPHOS) were initially inhibited by TKI treatment in CML-committed progenitors but were restored with continued treatment, reflecting both selection and metabolic reprogramming of specific subpopulations. TKI treatment selectively enriched primitive CML stem cells with reduced metabolic gene expression. Persistent CML stem cells also showed metabolic adaptation to TKI treatment through altered substrate use and mitochondrial respiration maintenance. Evaluation of transcription factors underlying these changes helped detect increased HIF-1 protein levels and activity in TKI-treated stem cells. Treatment with an HIF-1 inhibitor in combination with TKI treatment depleted murine and human CML stem cells. HIF-1 inhibition increased mitochondrial activity and reactive oxygen species (ROS) levels, reduced quiescence, increased cycling, and reduced the self-renewal and regenerating potential of dormant CML stem cells. We, therefore, identified the HIF-1-mediated inhibition of OXPHOS and ROS and maintenance of CML stem cell dormancy and repopulating potential as a key mechanism of CML stem cell adaptation to TKI treatment. Our results identify a key metabolic dependency in CML stem cells persisting after TKI treatment that can be targeted to enhance their elimination.


Asunto(s)
Leucemia Mielógena Crónica BCR-ABL Positiva , Proteínas Tirosina Quinasas , Ratones , Humanos , Animales , Proteínas Tirosina Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Especies Reactivas de Oxígeno/metabolismo , Células Madre Neoplásicas/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Resistencia a Antineoplásicos
2.
Org Biomol Chem ; 18(10): 1957-1967, 2020 03 14.
Artículo en Inglés | MEDLINE | ID: mdl-32101244

RESUMEN

1,4- and 1,5-Disubstituted triazole amino acid monomers have gained increasing interest among peptidic foldamers, as they are easily prepared via Cu- and Ru-catalyzed click reactions, with the potential for side chain variation. While the latter is key to their applicability, the synthesis and structural properties of the chiral mono- or disubstituted triazole amino acids have only been partially addressed. We here present the synthesis of all eight possible chiral derivatives of a triazole monomer prepared via a ruthenium-catalyzed azide alkyne cycloaddition (RuAAC). To evaluate the conformational properties of the individual building units, a systematic quantum chemical study was performed on all monomers, indicating their capacity to form several low energy conformers. This feature may be used to effect structural diversity when the monomers are inserted into various peptide sequences. We envisage that these results will facilitate new applications for these artificial oligomeric compounds in diverse areas, ranging from pharmaceutics to biotechnology.


Asunto(s)
Peptidomiméticos/síntesis química , Triazoles/síntesis química , Alquinos/química , Azidas/química , Química Clic , Reacción de Cicloadición , Modelos Moleculares , Polimerizacion , Polímeros/síntesis química , Teoría Cuántica , Estereoisomerismo , Termodinámica
3.
Org Biomol Chem ; 15(28): 5993-6000, 2017 Jul 19.
Artículo en Inglés | MEDLINE | ID: mdl-28678298

RESUMEN

A catalytic meta selective C-H alkylation of arenes is described using a wide range of α-halo carbonyls as coupling partners. Previously unreported primary alkylations with high meta selectivity have been enabled by this methodology whereas using straight chain alkyl halides affords ortho substituted products. Mechanistic analysis reveals an activation pathway whereby cyclometalation with a ruthenium(ii) complex activates the substrate molecule and is responsible for the meta selectivity observed. A distinct second activation of the coupling partner allows site selective reaction between both components.

4.
Angew Chem Int Ed Engl ; 56(47): 15131-15135, 2017 11 20.
Artículo en Inglés | MEDLINE | ID: mdl-28968000

RESUMEN

The para-selective C-H alkylation of aniline derivatives furnished with a pyrimidine auxiliary is herein reported. This reaction is proposed to take place via an N-H-activated cyclometalate formed in situ. Experimental and DFT mechanistic studies elucidate a dual role of the ruthenium catalyst. Here the ruthenium catalyst can undergo cyclometalation by N-H metalation (as opposed to C-H metalation in meta-selective processes) and form a redox active ruthenium species, to enable site-selective radical addition at the para position.

5.
J Biol Chem ; 289(17): 12168-12176, 2014 Apr 25.
Artículo en Inglés | MEDLINE | ID: mdl-24616106

RESUMEN

The Drosophila Groucho protein and its mammalian orthologues the transducin-like enhancers of split (TLEs) are critical transcriptional corepressors that repress Wnt and other signaling pathways. Although it is known that Groucho/TLEs are recruited to target genes by pathway-specific transcription factors, molecular events after the corepressor recruitment are largely unclear. We report that association of TLEs with O-GlcNAc transferase, an enzyme that catalyzes posttranslational modification of proteins by O-linked N-acetylglucosamine, is essential for TLE-mediated transcriptional repression. Removal of O-GlcNAc from Wnt-responsive gene promoters is critical for gene activation from Wnt-responsive promoters. Thus, these studies identify a molecular mechanism by which Groucho/TLEs repress gene transcription and provide a model whereby O-GlcNAc may control distinct intracellular signaling pathways.


Asunto(s)
N-Acetilglucosaminiltransferasas/metabolismo , Transducción de Señal , Transducina/metabolismo , Vía de Señalización Wnt , Animales , Secuencia de Bases , Inmunoprecipitación de Cromatina , Cartilla de ADN , Drosophila , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa
6.
J Biol Chem ; 287(41): 34419-31, 2012 Oct 05.
Artículo en Inglés | MEDLINE | ID: mdl-22908225

RESUMEN

The posttranslational modification of nuclear and cytosolic proteins by O-linked ß-N-acetylglucosamine (O-GlcNAc) has been shown to play an important role in cellular response to stress. Although increases in O-GlcNAc levels have typically been thought to be substrate-driven, studies in several transformed cell lines reported that glucose deprivation increased O-GlcNAc levels by a number of different mechanisms. A major goal of this study therefore was to determine whether in primary cells, such as neonatal cardiomyocytes, glucose deprivation increases O-GlcNAc levels and if so by what mechanism. Glucose deprivation significantly increased cardiomyocyte O-GlcNAc levels in a time-dependent manner and was associated with decreased O-GlcNAcase (OGA) but not O-GlcNAc transferase (OGT) protein. This response was unaffected by either the addition of pyruvate as an alternative energy source or by the p38 MAPK inhibitor SB203580. However, the response to glucose deprivation was blocked completely by glucosamine, but not by inhibition of OGA with 2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate. Interestingly, the CaMKII inhibitor KN93 also significantly reduced the response to glucose deprivation. Lowering extracellular Ca(2+) with EGTA or blocking store operated Ca(2+) entry with SKF96365 also attenuated the glucose deprivation-induced increase in O-GlcNAc. In C2C12 and HEK293 cells both glucose deprivation and heat shock increased O-GlcNAc levels, and CaMKII inhibitor KN93 attenuated the response to both stresses. These results suggest that increased intracellular calcium and subsequent activation of CaMKII play a key role in regulating the stress-induced increase in cellular O-GlcNAc levels.


Asunto(s)
Calcio/metabolismo , Glucosa/metabolismo , Miocitos Cardíacos/metabolismo , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Inhibidores Enzimáticos , Glicosilación/efectos de los fármacos , Células HEK293 , Humanos , Proteínas Musculares/antagonistas & inhibidores , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miocitos Cardíacos/citología , N-Acetilglucosaminiltransferasas/antagonistas & inhibidores , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
7.
Leukemia ; 37(3): 560-570, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36550214

RESUMEN

Fms-like tyrosine kinase 3 (Flt3) tyrosine kinase inhibitors (Flt3-TKI) have improved outcomes for patients with Flt3-mutated acute myeloid leukemia (AML) but are limited by resistance and relapse, indicating persistence of leukemia stem cells (LSC). Here utilizing a Flt3-internal tandem duplication (Flt3-ITD) and Tet2-deleted AML genetic mouse model we determined that FLT3-ITD AML LSC were enriched within the primitive ST-HSC population. FLT3-ITD LSC showed increased expression of the CXCL12 receptor CXCR4. CXCL12-abundant reticular (CAR) cells were increased in Flt3-ITD AML marrow. CXCL12 deletion from the microenvironment enhanced targeting of AML cells by Flt3-TKI plus chemotherapy treatment, including enhanced LSC targeting. Both treatment and CXCL12 deletion partially reduced p38 mitogen-activated protein kinase (p38) signaling in AML cells and further reduction was seen after treatment in CXCL12 deleted mice. p38 inhibition reduced CXCL12-dependent and -independent maintenance of both murine and human Flt3-ITD AML LSC by MSC and enhanced their sensitivity to treatment. p38 inhibition in combination with chemotherapy plus TKI treatment leads to greater depletion of Flt3-ITD AML LSC compared with CXCL12 deletion. Our studies support roles for CXCL12 and p38 signaling in microenvironmental protection of AML LSC and provide a rationale for inhibiting p38 signaling to enhance Flt3-ITD AML targeting.


Asunto(s)
Leucemia Mieloide Aguda , Tirosina Quinasa 3 Similar a fms , Animales , Humanos , Ratones , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Tirosina Quinasa 3 Similar a fms/metabolismo , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Sistema de Señalización de MAP Quinasas , Mutación , Transducción de Señal , Células Madre/metabolismo , Microambiente Tumoral , Proteínas Quinasas p38 Activadas por Mitógenos
8.
J Am Heart Assoc ; 12(19): e029898, 2023 10 03.
Artículo en Inglés | MEDLINE | ID: mdl-37750556

RESUMEN

Background Lifestyle and metabolic diseases influence the severity and pathogenesis of cardiovascular disease through numerous mechanisms, including regulation via posttranslational modifications. A specific posttranslational modification, the addition of O-linked ß-N acetylglucosamine (O-GlcNAcylation), has been implicated in molecular mechanisms of both physiological and pathologic adaptations. The current study aimed to test the hypothesis that in cardiomyocytes, sustained protein O-GlcNAcylation contributes to cardiac adaptations, and its progression to pathophysiology. Methods and Results Using a naturally occurring dominant-negative O-GlcNAcase (dnOGA) inducible cardiomyocyte-specific overexpression transgenic mouse model, we induced dnOGA in 8- to 10-week-old mouse hearts. We examined the effects of 2-week and 24-week dnOGA overexpression, which progressed to a 1.8-fold increase in protein O-GlcNAcylation. Two-week increases in protein O-GlcNAc levels did not alter heart weight or function; however, 24-week increases in protein O-GlcNAcylation led to cardiac hypertrophy, mitochondrial dysfunction, fibrosis, and diastolic dysfunction. Interestingly, systolic function was maintained in 24-week dnOGA overexpression, despite several changes in gene expression associated with cardiovascular disease. Specifically, mRNA-sequencing analysis revealed several gene signatures, including reduction of mitochondrial oxidative phosphorylation, fatty acid, and glucose metabolism pathways, and antioxidant response pathways after 24-week dnOGA overexpression. Conclusions This study indicates that moderate increases in cardiomyocyte protein O-GlcNAcylation leads to a differential response with an initial reduction of metabolic pathways (2-week), which leads to cardiac remodeling (24-week). Moreover, the mouse model showed evidence of diastolic dysfunction consistent with a heart failure with preserved ejection fraction. These findings provide insight into the adaptive versus maladaptive responses to increased O-GlcNAcylation in heart.


Asunto(s)
Enfermedades Cardiovasculares , Miocitos Cardíacos , Ratones , Animales , Miocitos Cardíacos/metabolismo , Acetilglucosamina/metabolismo , Enfermedades Cardiovasculares/metabolismo , Glicosilación , Cardiomegalia/genética , Cardiomegalia/metabolismo , Procesamiento Proteico-Postraduccional , Mitocondrias/metabolismo , Modelos Animales de Enfermedad , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo
9.
Leukemia ; 36(11): 2621-2633, 2022 11.
Artículo en Inglés | MEDLINE | ID: mdl-36220999

RESUMEN

The FLT3-ITD mutation is associated with poor prognosis in acute myeloid leukemia (AML). FLT3 tyrosine kinase inhibitors (TKIs) demonstrate clinical efficacy but fail to target leukemia stem cells (LSC) and do not generate sustained responses. Autophagy is an important cellular stress response contributing to hematopoietic stem cells (HSC) maintenance and promoting leukemia development. Here we investigated the role of autophagy in regulating FLT3-ITD AML stem cell function and response to TKI treatment. We show that autophagy inhibition reduced quiescence and depleted repopulating potential of FLT3-ITD AML LSC, associated with mitochondrial accumulation and increased oxidative phosphorylation. However, TKI treatment reduced mitochondrial respiration and unexpectedly antagonized the effects of autophagy inhibition on LSC attrition. We further show that TKI-mediated targeting of AML LSC and committed progenitors was p53-dependent, and that autophagy inhibition enhanced p53 activity and increased TKI-mediated targeting of AML progenitors, but decreased p53 activity in LSC and reduced TKI-mediated LSC inhibition. These results provide new insights into the role of autophagy in differentially regulating AML stem and progenitor cells, reveal unexpected antagonistic effects of combined oncogenic tyrosine kinase inhibition and autophagy inhibition in AML LSC, and suggest an alternative approach to target AML LSC quiescence and regenerative potential.


Asunto(s)
Leucemia Mieloide Aguda , Proteínas Tirosina Quinasas , Humanos , Proteínas Tirosina Quinasas/genética , Proteína p53 Supresora de Tumor/genética , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Autofagia , Células Madre , Tirosina Quinasa 3 Similar a fms/genética , Tirosina Quinasa 3 Similar a fms/uso terapéutico , Mutación
10.
Am J Physiol Cell Physiol ; 300(3): C456-65, 2011 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-21178104

RESUMEN

The protein O-linked ß-N-acetylglucosamine (O-GlcNAc) modification plays an important role in skeletal muscle development and physiological function. In this study, bitransgenic mice were generated that overexpressed NCOAT(GK), an O-GlcNAcase-inactive spliced variant of the O-GlcNAcase gene, specifically in skeletal muscle using the muscle creatine kinase promoter. Expression of the chimeric enhanced green fluorescent protein-NCOAT(GK) transgene caused an increase of cellular O-GlcNAc levels, along with the accumulation and activation of proapoptotic factors in muscles of bitransgenic mice. The consequence of overexpressing the transgene for a 2-wk period was muscle atrophy and, in some cases, resulted in the death of male mice. Muscle atrophy is a common complication of many diseases, some of which correlate markedly with high cellular O-GlcNAc levels, such as diabetes. Our study provides direct evidence linking muscle atrophy and the disruption of O-GlcNAcase activity.


Asunto(s)
Empalme Alternativo/genética , Fibras Musculares Esqueléticas/patología , Músculo Esquelético/enzimología , Músculo Esquelético/patología , Atrofia Muscular/enzimología , Atrofia Muscular/patología , beta-N-Acetilhexosaminidasas/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Modelos Animales de Enfermedad , Regulación Enzimológica de la Expresión Génica/fisiología , Masculino , Ratones , Ratones Transgénicos , Fibras Musculares Esqueléticas/metabolismo , Regiones Promotoras Genéticas/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transgenes/genética , Regulación hacia Arriba/genética , beta-N-Acetilhexosaminidasas/química , beta-N-Acetilhexosaminidasas/genética
11.
Chem Commun (Camb) ; 57(53): 6518-6521, 2021 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-34105551

RESUMEN

A palladium-catalyzed domino arylation-cyclization of biocatalytically derived cyclic 1,3-dienes is demonstrated. The reaction introduces a high degree of structural complexity in a single step, giving access to tricyclic tetrahydrofluorenones with full regio- and stereoselectivity. The transformation proceeds through a novel acylation-terminated Heck-type sequence, and quantum chemical calculations indicate that C-H activation is involved in the terminating acylation step.

12.
Org Lett ; 22(6): 2464-2469, 2020 03 20.
Artículo en Inglés | MEDLINE | ID: mdl-32150420

RESUMEN

Microbial arene oxidation of benzoic acid with Ralstonia eutropha B9 provides a chiral highly functionalized cyclohexadiene, suitable for further structural diversification. Subjecting this scaffold to a Pd-catalyzed Heck reaction effects a regio- and stereoselective arylation of the cyclohexadiene ring, with 1,3-chirality transfer of stereogenic information installed in the microbial arene oxidation. Quantum chemical calculations explain the selectivity both by a kinetic preference for the observed arylation position and by reversible carbopalladation in competing positions. Further product transformation allowed the formation of a tricyclic ketone possessing four stereogenic centers. This demonstrates the capability of the method to introduce stereochemical complexity from planar nonchiral benzoic acid in just a few steps.


Asunto(s)
Cupriavidus necator/metabolismo , Ciclohexenos/síntesis química , Paladio/química , Benzoatos/química , Catálisis , Cupriavidus necator/química , Yodobencenos/química , Oxidación-Reducción , Estereoisomerismo
13.
J Am Heart Assoc ; 9(15): e013039, 2020 08 04.
Artículo en Inglés | MEDLINE | ID: mdl-32750298

RESUMEN

Background Perturbations in myocardial substrate utilization have been proposed to contribute to the pathogenesis of cardiac dysfunction in diabetic subjects. The failing heart in nondiabetics tends to decrease reliance on fatty acid and glucose oxidation, and increases reliance on ketone body oxidation. In contrast, little is known regarding the mechanisms mediating this shift among all 3 substrates in diabetes mellitus. Therefore, we tested the hypothesis that changes in myocardial glucose utilization directly influence ketone body catabolism. Methods and Results We examined ventricular-cardiac tissue from the following murine models: (1) streptozotocin-induced type 1 diabetes mellitus; (2) high-fat-diet-induced glucose intolerance; and transgenic inducible cardiac-restricted expression of (3) glucose transporter 4 (transgenic inducible cardiac restricted expression of glucose transporter 4); or (4) dominant negative O-GlcNAcase. Elevated blood glucose (type 1 diabetes mellitus and high-fat diet mice) was associated with reduced cardiac expression of ß-hydroxybutyrate-dehydrogenase and succinyl-CoA:3-oxoacid CoA transferase. Increased myocardial ß-hydroxybutyrate levels were also observed in type 1 diabetes mellitus mice, suggesting a mismatch between ketone body availability and utilization. Increased cellular glucose delivery in transgenic inducible cardiac restricted expression of glucose transporter 4 mice attenuated cardiac expression of both Bdh1 and Oxct1 and reduced rates of myocardial BDH1 activity and ß-hydroxybutyrate oxidation. Moreover, elevated cardiac protein O-GlcNAcylation (a glucose-derived posttranslational modification) by dominant negative O-GlcNAcase suppressed ß-hydroxybutyrate dehydrogenase expression. Consistent with the mouse models, transcriptomic analysis confirmed suppression of BDH1 and OXCT1 in patients with type 2 diabetes mellitus and heart failure compared with nondiabetic patients. Conclusions Our results provide evidence that increased glucose leads to suppression of cardiac ketolytic capacity through multiple mechanisms and identifies a potential crosstalk between glucose and ketone body metabolism in the diabetic myocardium.


Asunto(s)
Glucosa/metabolismo , Cuerpos Cetónicos/metabolismo , Miocardio/metabolismo , Animales , Western Blotting , Diabetes Mellitus Experimental/metabolismo , Intolerancia a la Glucosa/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Inmunoprecipitación , Masculino , Ratones , Miocitos Cardíacos/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ARN
14.
Mol Pharmacol ; 76(1): 47-57, 2009 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-19372209

RESUMEN

This study investigated the mechanism by which the transcription factor Sp1 is degraded in prostate cancer cells. We recently developed a thiazolidinedione derivative, (Z)-5-(4-hydroxy-3-trifluoromethylbenzylidene)-3-(1-methylcyclohexyl)-thiazolidine-2,4-dione (OSU-CG12), that induces Sp1 degradation in a manner paralleling that of glucose starvation. Based on our finding that thiazolidinediones suppress beta-catenin and cyclin D1 by up-regulating the E3 ligase SCF(beta-TrCP), we hypothesized that beta-transducin repeat-containing protein (beta-TrCP) targets Sp1 for proteasomal degradation in response to glucose starvation or OSU-CG12. Here we show that either treatment of LNCaP cells increased specific binding of Sp1 with beta-TrCP. This direct binding was confirmed by in vitro pull-down analysis with bacterially expressed beta-TrCP. Although ectopic expression of beta-TrCP enhanced the ability of OSU-CG12 to facilitate Sp1 degradation, suppression of endogenous beta-TrCP function by a dominant-negative mutant or small interfering RNA-mediated knockdown blocked OSU-CG12-facilitated Sp1 ubiquitination and/or degradation. Sp1 contains a C-terminal conventional DSG destruction box ((727)DSGAGS(732)) that mediates beta-TrCP recognition and encompasses a glycogen synthase kinase 3beta (GSK3beta) phosphorylation motif (SXXXS). Pharmacological and molecular genetic approaches and mutational analyses indicate that extracellular signal-regulated kinase-mediated phosphorylation of Thr739 and GSK3beta-mediated phosphorylation of Ser728 and Ser732 were critical for Sp1 degradation. The ability of OSU-CG12 to mimic glucose starvation to activate beta-TrCP-mediated Sp1 degradation has translational potential to foster novel strategies for cancer therapy.


Asunto(s)
Glucosa/deficiencia , Factor de Transcripción Sp1/metabolismo , Tiazolidinedionas/farmacología , Proteínas con Repetición de beta-Transducina/biosíntesis , Línea Celular Tumoral , Femenino , Glucógeno Sintasa Quinasa 3/fisiología , Glucógeno Sintasa Quinasa 3 beta , Humanos , MAP Quinasa Quinasa 1/fisiología , Masculino , Fosforilación , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/genética , Regulación hacia Arriba
15.
Biochem Biophys Res Commun ; 387(1): 70-6, 2009 Sep 11.
Artículo en Inglés | MEDLINE | ID: mdl-19577582

RESUMEN

Diminished proteolytic functionality in the lens may cause cataracts. We have reported that O-GlcNAc is an endogenous inhibitor of the proteasome. We hypothesize that in the lens there is a cause-and-effect relationship between proteasome inhibition by O-GlcNAc, and cataract formation. To demonstrate this, we established novel transgenic mouse models to over-express a dominant-negative form of O-GlcNAcase, GK-NCOAT, in the lens. Expression of GK-NCOAT suppresses removal of O-GlcNAc from proteins, resulting in increased levels of O-GlcNAc in the lenses of our transgenic mice, along with decreased proteasome function. We observed that transgenic mice developed markedly larger cataracts than controls and lens fiber cell denucleation was inhibited. Our study suggests that increased O-GlcNAc in the lens could lead to cataract formation and attenuation of lens fiber cell denucleation by inhibition of proteasome function. These findings may explain why cataract formation is a common complication of diabetes since O-GlcNAc is derived from glucose.


Asunto(s)
Acetilglucosamina/metabolismo , Catarata/patología , Diferenciación Celular , Cristalino/citología , Cristalino/patología , gamma-Cristalinas/metabolismo , Animales , Histona Acetiltransferasas/genética , Hialuronoglucosaminidasa/genética , Ratones , Ratones Transgénicos
16.
Mol Cell Biol ; 26(22): 8539-50, 2006 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-16966374

RESUMEN

Mechanisms controlling nuclear hormone receptors are a central question to mammalian developmental and disease processes. Herein, we show that a subtle increase in O-GlcNAc levels inhibits activation of nuclear hormone receptors. In vivo, increased levels of O-GlcNAc impair estrogen receptor activation and cause a decrease in mammary ductal side-branching morphogenesis associated with loss of progesterone receptors. Increased O-GlcNAc levels suppress transcriptional expression of coactivators and of the nuclear hormone receptors themselves. Surprisingly, increased O-GlcNAc levels are also associated with increased transcription of genes encoding corepressor proteins NCoR and SMRT. The association of the enzyme O-GlcNAc transferase with these corepressors contributes to specific regulation of nuclear hormone receptors by O-GlcNAc. Overall, transcriptional inhibition is related to the integrated effect of O-GlcNAc by direct modification of critical elements of the transcriptome and indirectly through O-GlcNAc modification of the proteasome.


Asunto(s)
Regulación de la Expresión Génica , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Transcripción Genética , Animales , Línea Celular Tumoral , Humanos , Glándulas Mamarias Humanas/embriología , Glándulas Mamarias Humanas/enzimología , Ratones , Ratones Transgénicos , Modelos Biológicos , N-Acetilglucosaminiltransferasas/fisiología , Proteínas Nucleares/metabolismo , Co-Represor 1 de Receptor Nuclear , Proteínas Represoras/metabolismo , Transfección
17.
Mol Cell Endocrinol ; 492: 110445, 2019 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-31100495

RESUMEN

Human growth hormone (GH) binds and activates GH receptor (GHR) and prolactin (PRL) receptor (PRLR). LNCaP human prostate cancer cells express only GHR. A soluble fragment of IGF-1 receptor (IGF-1R) extracellular domain (sol IGF-1R) interacts with GHR and blocks GH signaling. We now explore sol IGF-1R's specificity for inhibiting GH signaling via GHR vs. PRLR and test GHR and PRLR extracellular domain inhibition determinants. Although T47D human breast cancer cells express GHR and PRLR, GH signaling is largely PRLR-mediated. In T47D, sol IGF-1R inhibited neither GH- nor PRL-induced STAT5 activation. However, sol IGF-1R inhibited GH-induced STAT5 activation in T47D-shPRLR cells, which harbor reduced PRLR. In MIN6 mouse ß-cells, bovine GH (bGH) activates mouse GHR, not PRLR, while human GH activates mouse GHR and PRLR. In MIN6, sol IGF-1R inhibited bGH-induced STAT5 activation, but partially inhibited human GH-induced STAT5 activation. These findings suggest sol IGF-1R's inhibition is GHR-specific. Using a cellular reconstitution system, we compared effects of sol IGF-1R on signaling through GHR, PRLR, or chimeras in which extracellular subdomains 2 (S2) of the receptors were swapped. Sol IGF-1R inhibited GH-induced STAT5 activation in GHR-expressing, not PRLR-expressing cells, consistent with GHR specificity of sol IGF-1R. Interestingly, we found that GHR S2 (which harbors the GHR-GHR dimer interface) was required, but not sufficient for sol IGF-1R inhibition of GHR signaling. These results suggest sol IGF-1R specifically inhibits GH-induced GHR-mediated signaling, possibly through interaction with GHR S1 and S2 domains. Our findings have implications for GH antagonist development.


Asunto(s)
Neoplasias de la Mama/metabolismo , Proteínas Portadoras/metabolismo , Hormona de Crecimiento Humana/efectos de los fármacos , Neoplasias de la Próstata/metabolismo , Receptor IGF Tipo 1/metabolismo , Receptores de Prolactina/metabolismo , Animales , Sitios de Unión , Proteínas Portadoras/química , Bovinos , Línea Celular Tumoral , Femenino , Humanos , Masculino , Ratones , Dominios Proteicos , Receptor IGF Tipo 1/química , Factor de Transcripción STAT5/metabolismo , Transducción de Señal/efectos de los fármacos
18.
Cell Stem Cell ; 24(5): 769-784.e6, 2019 05 02.
Artículo en Inglés | MEDLINE | ID: mdl-30905620

RESUMEN

Chronic myeloid leukemia (CML) originates in a hematopoietic stem cell (HSC) transformed by the breakpoint cluster region (BCR)-abelson (ABL) oncogene and is effectively treated with tyrosine kinase inhibitors (TKIs). TKIs do not eliminate disease-propagating leukemic stem cells (LSCs), suggesting a deeper understanding of niche-dependent regulation of CML LSCs is required to eradicate disease. Cxcl12 is expressed in bone marrow niches and controls HSC maintenance, and here, we show that targeted deletion of Cxcl12 from mesenchymal stromal cells (MSCs) reduces normal HSC numbers but promotes LSC expansion by increasing self-renewing cell divisions, possibly through enhanced Ezh2 activity. In contrast, endothelial cell-specific Cxcl12 deletion decreases LSC proliferation, suggesting niche-specific effects. During CML development, abnormal clusters of colocalized MSCs and LSCs form but disappear upon Cxcl12 deletion. Moreover, MSC-specific deletion of Cxcl12 increases LSC elimination by TKI treatment. These findings highlight a critical role of niche-specific effects of Cxcl12 expression in maintaining quiescence of TKI-resistant LSC populations.


Asunto(s)
Células Madre Hematopoyéticas/fisiología , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Células Madre Mesenquimatosas/fisiología , Células Madre Neoplásicas/fisiología , Nicho de Células Madre/fisiología , Animales , Apoptosis , Línea Celular Tumoral , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Resistencia a Antineoplásicos , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Especificidad de Órganos , Inhibidores de Proteínas Quinasas/uso terapéutico
19.
J Vasc Surg Cases Innov Tech ; 5(4): 529-531, 2019 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-31799480

RESUMEN

A 67-year-old woman presented to our vein clinic for chronic left lower extremity edema, pain, and varicosities. After failed conservative management, a computed tomography scan revealed central venous stenosis secondary to compression of the left common iliac vein by a large osteophyte along the anterolateral aspect of the L5-S1 disk space. An anterior osteophytectomy was performed, followed by iliac venous stenting at a 1-month interval. The patient had resolution of symptoms and remains symptom free at 15 months of follow-up. This report describes a spinal exostosis causing symptomatic venous compression successfully relieved by surgical decompression.

20.
Biochim Biophys Acta ; 1760(5): 829-39, 2006 May.
Artículo en Inglés | MEDLINE | ID: mdl-16517082

RESUMEN

NCOAT is a bifunctional nucleo-cytoplasmic protein with both O-GlcNAcase and histone acetyltransferase domains. The O-GlcNAcase domain catalyzes the removal of O-linked GlcNAc modifications from proteins and we have found that it resides in the N-terminal third of NCOAT. The recognition of the substrate GlcNAc suggests that the O-GlcNAcase is related in structure and catalytic mechanism to chitinases, hexosaminidases and hyaluronidases. These families of glycosidases all possess a catalytic doublet of carboxylate-containing residues, with one providing an acid-base function, and the second acting to orient and use the N-acetyl group of GlcNAc during catalysis. Indeed, we show that the O-GlcNAcase also possesses the catalytic doublet motif shared among these enzymes and that these two essential residues are aspartic acids at positions 175 and 177, respectively, in mouse NCOAT. In addition, a conserved cysteine at 166 and a conserved aspartic acid at 174 were also found to be necessary for fully efficient enzymatic activity. Given this information, we propose that the O-GlcNAcase active site resembles those of the above glycosidases which carry out the hydrolysis of GlcNAc linkages in a substrate-assisted acid-base manner.


Asunto(s)
Acetilglucosaminidasa/química , Acetilglucosaminidasa/genética , Histona Acetiltransferasas/química , Histona Acetiltransferasas/genética , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión , Concentración de Iones de Hidrógeno , Ratones , Datos de Secuencia Molecular , Mutagénesis , Mutación , beta-N-Acetilhexosaminidasas
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