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1.
PLoS Biol ; 21(3): e3001977, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36862640

RESUMEN

Failures in mitophagy, a process by which damaged mitochondria are cleared, results in neurodegeneration, while enhancing mitophagy promotes the survival of dopaminergic neurons. Using an artificial intelligence platform, we employed a natural language processing approach to evaluate the semantic similarity of candidate molecules to a set of well-established mitophagy enhancers. Top candidates were screened in a cell-based mitochondrial clearance assay. Probucol, a lipid-lowering drug, was validated across several orthogonal mitophagy assays. In vivo, probucol improved survival, locomotor function, and dopaminergic neuron loss in zebrafish and fly models of mitochondrial damage. Probucol functioned independently of PINK1/Parkin, but its effects on mitophagy and in vivo depended on ABCA1, which negatively regulated mitophagy following mitochondrial damage. Autophagosome and lysosomal markers were elevated by probucol treatment in addition to increased contact between lipid droplets (LDs) and mitochondria. Conversely, LD expansion, which occurs following mitochondrial damage, was suppressed by probucol and probucol-mediated mitophagy enhancement required LDs. Probucol-mediated LD dynamics changes may prime the cell for a more efficient mitophagic response to mitochondrial damage.


Asunto(s)
Gotas Lipídicas , Probucol , Animales , Probucol/farmacología , Inteligencia Artificial , Mitofagia , Pez Cebra
2.
Biochem J ; 451(1): 45-53, 2013 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-23320500

RESUMEN

Reversible phosphorylation is a widespread molecular mechanism to regulate the function of cellular proteins, including transcription factors. Phosphorylation of the nuclear receptor PPARγ (peroxisome-proliferator-activated receptor γ) at two conserved serine residue (Ser(112) and Ser(273)) results in an altered transcriptional activity of this transcription factor. So far, only a very limited number of cellular enzymatic activities has been described which can dephosphorylate nuclear receptors. In the present study we used immunoprecipitation assays coupled to tandem MS analysis to identify novel PPARγ-regulating proteins. We identified the serine/threonine phosphatase PPM1B [PP (protein phosphatase), Mg(2+)/Mn(2+) dependent, 1B; also known as PP2Cß] as a novel PPARγ-interacting protein. Endogenous PPM1B protein is localized in the nucleus of mature 3T3-L1 adipocytes where it can bind to PPARγ. Furthermore we show that PPM1B can directly dephosphorylate PPARγ, both in intact cells and in vitro. In addition PPM1B increases PPARγ-mediated transcription via dephosphorylation of Ser(112). Finally, we show that knockdown of PPM1B in 3T3-L1 adipocytes blunts the expression of some PPARγ target genes while leaving others unaltered. These findings qualify the phosphatase PPM1B as a novel selective modulator of PPARγ activity.


Asunto(s)
Adipocitos/metabolismo , Núcleo Celular/metabolismo , PPAR gamma/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Transcripción Genética/fisiología , Células 3T3-L1 , Transporte Activo de Núcleo Celular/fisiología , Adipocitos/citología , Animales , Línea Celular Tumoral , Núcleo Celular/genética , Humanos , Magnesio/metabolismo , Manganeso/metabolismo , Ratones , PPAR gamma/genética , Fosfoproteínas Fosfatasas/genética , Fosforilación/fisiología , Proteína Fosfatasa 2C
3.
Cell Rep ; 42(8): 112895, 2023 08 29.
Artículo en Inglés | MEDLINE | ID: mdl-37498743

RESUMEN

Mitochondrial morphology is regulated by the post-translational modifications of the dynamin family GTPase proteins including mitofusin 1 (MFN1), MFN2, and dynamin-related protein 1 (DRP1). Mitochondrial phosphatase phosphoglycerate mutase 5 (PGAM5) is emerging as a regulator of these post-translational modifications; however, its precise role in the regulation of mitochondrial morphology is unknown. We show that PGAM5 interacts with MFN2 and DRP1 in a stress-sensitive manner. PGAM5 regulates MFN2 phosphorylation and consequently protects it from ubiquitination and degradation. Further, phosphorylation and dephosphorylation modification of MFN2 regulates its fusion ability. Phosphorylation enhances fission and degradation, whereas dephosphorylation enhances fusion. PGAM5 dephosphorylates MFN2 to promote mitochondrial network formation. Further, using a Drosophila genetic model, we demonstrate that the MFN2 homolog Marf and dPGAM5 are in the same biological pathway. Our results identify MFN2 dephosphorylation as a regulator of mitochondrial fusion and PGAM5 as an MFN2 phosphatase.


Asunto(s)
GTP Fosfohidrolasas , Monoéster Fosfórico Hidrolasas , GTP Fosfohidrolasas/metabolismo , Fosfoglicerato Mutasa , Dinámicas Mitocondriales/genética , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Dinaminas/metabolismo
4.
Bioorg Med Chem Lett ; 20(12): 3675-9, 2010 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-20471252

RESUMEN

In this study, a series of small molecule inhibitors of human FABP4 were identified through virtual screening. Compound 1 is the most potent hit against FABP4 with a selectivity of more than 144-fold preferences over human FABP3. In addition, MD simulation and mutation studies revealed key residues for inhibitory potency and selectivity, which provides a guideline for further drug design against obesity, diabetes and atherosclerosis.


Asunto(s)
Simulación por Computador , Proteínas de Unión a Ácidos Grasos/antagonistas & inhibidores , Aterosclerosis/tratamiento farmacológico , Sitios de Unión , Diabetes Mellitus/tratamiento farmacológico , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos/métodos , Proteína 3 de Unión a Ácidos Grasos , Proteínas de Unión a Ácidos Grasos/química , Proteínas de Unión a Ácidos Grasos/genética , Humanos , Mutación , Obesidad/tratamiento farmacológico , Compuestos Orgánicos/química , Compuestos Orgánicos/farmacología , Unión Proteica/genética , Relación Estructura-Actividad , Especificidad por Sustrato
5.
Methods Mol Biol ; 1388: 13-9, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27033067

RESUMEN

Discovered in 1997, PTEN remains one of the most studied tumor suppressors. In this issue of Methods in Molecular Biology, we assembled a series of papers describing various clinical and experimental approaches to studying PTEN function. Due to its broad expression, regulated subcellular localization, and intriguing phosphatase activity, methodologies aimed at PTEN study have often been developed in the context of mutations affecting various aspects of its regulation, found in patients burdened with PTEN loss-driven tumors. PTEN's extensive posttranslational modifications and dynamic localization pose unique challenges for studying PTEN features in isolation and necessitate considerable development of experimental systems to enable controlled characterization. Nevertheless, ongoing efforts towards the development of PTEN knockout and knock-in animals and cell lines, antibodies, and enzymatic assays have facilitated a huge body of work, which continues to unravel the fascinating biology of PTEN.


Asunto(s)
Fosfohidrolasa PTEN/metabolismo , Animales , Línea Celular , Técnicas de Sustitución del Gen , Técnicas de Inactivación de Genes , Humanos , Fosfohidrolasa PTEN/genética , Fosfatidilinositoles/metabolismo , Fosfoproteínas Fosfatasas/metabolismo
6.
Cell Death Dis ; 7(12): e2560, 2016 12 22.
Artículo en Inglés | MEDLINE | ID: mdl-28005077

RESUMEN

Signal transducer and activator of transcription 5 (STAT5) and nucleophosmin (NPM1) are critical regulators of multiple biological and pathological processes. Although a reciprocal regulatory relationship was established between STAT5A and a NPM-ALK fusion protein in T-cell lymphoma, no direct connection between STAT5 and wild-type NPM1 has been documented. Here we demonstrate a mutually regulatory relationship between STAT5 and NPM1. Induction of STAT5 phosphorylation at Y694 (P-STAT5) diminished NPM1 expression, whereas inhibition of STAT5 phosphorylation enhanced NPM1 expression. Conversely, NPM1 not only negatively regulated STAT5 phosphorylation but also preserved unphosphorylated STAT5 level. Mechanistically, we show that NPM1 downregulation by P-STAT5 is mediated by impairing the BRCA1-BARD1 ubiquitin ligase, which controls the stability of NPM1. In turn, decreased NPM1 levels led to suppression of p53 expression, resulting in enhanced cell survival. This study reveals a new STAT5 signaling pathway regulating p53 expression via NPM1 and uncovers new therapeutic targets for anticancer treatment in tumors driven by STAT5 signaling.


Asunto(s)
Proteína BRCA1/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Factor de Transcripción STAT5/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Línea Celular , Supervivencia Celular , Regulación hacia Abajo , Técnicas de Silenciamiento del Gen , Humanos , Modelos Biológicos , Nucleofosmina , Fosforilación , Fosfotirosina/metabolismo , Unión Proteica , Proteolisis
7.
Eksp Onkol ; 26(1): 15-9, 2004 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-15112574

RESUMEN

AIM: To identify novel PTEN-binding partners. METHODS: The technique of yeast two-hybrid screening was used in this study. A panel of bait constructs was created, containing the C-terminal domain of PTEN, full length PTEN, activated and phosphatase-dead mutants. The expression of LexA-fused baits, their nuclear localization and autoactivation potential were tested according to the standard protocol of Duplex A system. CDNA libraries from Colon Cancer, HeLa and Mouse Embryo were screened with two selected bait constructs. Isolated positive clones were further analysed by mating assay and identified by automated DNA sequencing and database searching. RESULTS: Extensive screening of cDNA libraries with the full length and the C-terminal domain of PTEN led to the identification of 43 positive clones, which were confirmed in mating assay. Sequence analysis indicated that two clones encode AEBP1 (Adipocyte Enhancer Binding Protein 1). CONCLUSION: Our data indicate that the interaction between PTEN and AEBP1 is mediated by their C-terminal and N-terminal domains, respectively. The functional importance of PTEN-AEBP1 interaction is currently under investigation.


Asunto(s)
Proteínas Fúngicas/genética , Monoéster Fosfórico Hidrolasas/genética , Proteínas Recombinantes de Fusión/genética , Transcripción Genética , Proteínas Supresoras de Tumor/genética , Técnicas del Sistema de Dos Híbridos , ADN Complementario/genética , Proteínas Fúngicas/metabolismo , Genes Supresores de Tumor , Fosfohidrolasa PTEN , Monoéster Fosfórico Hidrolasas/metabolismo , Plásmidos/genética , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Levaduras/genética
8.
Eksp Onkol ; 26(1): 24-30, 2004 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-15112576

RESUMEN

AIM: To express recombinant S6K2 in baculovirus expression system; to purify large quantities of recombinant S6K2 for biochemical studies; to generate and characterise specific MABs against recombinant S6K2; to study the patterns S6K1 and S6K2 expression and subcellular localization in normal, benign and malignant breast tissues. METHODS: Recombinant baculovirus, expressing wild type S6K2 was generated using Bac-to-Bac system (Invitrogen); recombinant S6K was purified from infected Sf9 cells using affinity purification approach; monoclonal antibodies against recombinant S6K2 were generated; the specificity of generated MABs towards recombinant and endogenous S6K2 were examined by ELISA, Western blotting, immunoprecipitation and immuhohistochemical staining; immunohistochemical detection of S6K1 and S6K2 in human breast tissues was performed using specific monoclonal antibodies towards S6K1 and S6K2. RESULTS: Large amounts of enzymatically active S6K2 were purified using baculovirus expression system; highly purified preparations of S6K2 were used to generate and characterize anti-S6K2 MABs; elevated levels of S6K1 and S6K2 were found in breast tumors when compared to normal breast tissues; S6K2 is frequently localized in the nuclei of adenocarcinoma tissues, but rarely in fibroadenoma or "normal" breast tissues. CONCLUSION: Production of recombinant S6K2 in large amount and generation of specific monoclonal antibodies towards S6K2 has provided us with excellent tools to study the function and regulation of this important signalling molecule in normal and cancer cells. Immunnohistochemical analysis of S6K1 and S6K2 expression in normal and malignant breast clearly indicates that both kinases are overexpressed in breast tumors, when compared to "normal" tissues. The retention of S6K2 in the nuclei of malignant cells may be caused by disregulation of nucleocytoplasmic shuttling and could subsequently affect cell growth and proliferation.


Asunto(s)
Neoplasias de la Mama/enzimología , Proteínas Quinasas S6 Ribosómicas 90-kDa/biosíntesis , Anticuerpos Monoclonales , Western Blotting , Neoplasias de la Mama/patología , Humanos , Inmunohistoquímica , Pruebas de Precipitina , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Proteínas Quinasas S6 Ribosómicas 90-kDa/inmunología , Células Tumorales Cultivadas
9.
Hybridoma (Larchmt) ; 28(4): 287-93, 2009 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-19663702

RESUMEN

Fibroblast growth factor receptor 1 (FGFR1) is a member of the FGFR family of receptor tyrosine kinases, whose function has been implicated in diverse biological processes including cell proliferation, differentiation, survival, and tumorigenesis. This diversity is possibly mediated by the existence of multiple FGFR1 isoforms, generated by alternative splicing and post-translational modifications, mainly through glycosylation. In this study we report the generation and characterization of a panel of monoclonal antibodies directed towards FGFR1. To achieve this, we used as an antigen a fragment of FGFR1, corresponding to loop II-III of the extracellular domain, which shares low homology to other members of the FGFR family and possesses numerous antigentic determinants. Two rounds of ELISA screening and Western blot analysis allowed us to isolate a panel of monoclonal antibodies, which recognize specifically recombinant FGFR1 loop II-III. The ability of generated antibodies to recognize endogenous FGFR1 was examined in 3T3 L1 cells, which are known to express FGFR1, but not other members of FGFR family. Immunoblot analysis of 3T3 L1 cell lysates with hybridoma media of selected clones revealed a different, but overlapping pattern of immunoreactive bands, which might represent splicing and post-translationally modified forms of FGFR1. Furthermore, we also tested the cross-reactivity of generated antibodies towards recombinant full-length FGFR3 and their ability to recognize FGFR1 in 3T3 L1 cells by cyto- and immunocytochemistry. In summary, generated antibodies should be useful as tools for examining the expression pattern and biological functions of FGFR1 in normal and pathological tissues.


Asunto(s)
Anticuerpos Monoclonales/química , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/química , Células 3T3-L1 , Empalme Alternativo , Animales , Diferenciación Celular , Proliferación Celular , Supervivencia Celular , Femenino , Regulación de la Expresión Génica , Glicosilación , Humanos , Ratones , Ratones Endogámicos BALB C , Estructura Terciaria de Proteína
10.
Hybridoma (Larchmt) ; 28(4): 295-300, 2009 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-19663703

RESUMEN

Fibroblast growth factor receptor 3 (FGFR3) is a member of the FGFR family of receptor tyrosine kinases, whose function has been implicated in diverse biological processes, including cell proliferation, differentiation, survival, and tumorigenesis. Deregulation of FGFR3 signaling has been implicated with human pathologies, including cancer. Activating mutations in FGFR3 gene are frequently detected in bladder cancer, multiple myeloma, and noninvasive papillary urothelial cell carcinomas, while the overexpression of the receptor is observed in thyroid lymphoma and bladder cancer. The main aim of this study was to generate hybridoma clones producing antibody that could specifically recognize FGFR3/S249C mutant, but not the wild-type FGFR. To achieve this, we used for immunization bacterially expressed fragment of FGFR3 corresponding to loops II-III of the extracellular domain (GST-His/FGFR3/S249C-LII-III), which possesses oncogenic mutation at Ser249 detected in at least 50% of bladder cancers. Primary ELISA screening allowed us to isolate several hybridoma clones that showed specificity towards FGFR3/S249C, but not FGFR3wt protein. Unfortunately, these clones were not stable during single-cell cloning and expansion and lost the ability to recognize specifically FGFR3/S249C. However, this study allowed us to generate several monoclonal antibodies specific towards both FGFR3wt and FGFR3/S249C recombinant proteins. Produced hybridomas secreted MAbs that were specific in Western blotting towards bacterially expressed FGFR3wt and FGFR3/S249C, as well as the full-length receptors ectopically expressed in Sf21 and HEK293 cells. Moreover, transiently expressed wild-type and oncogenic forms of FGFR were efficiently immunoprecipitated with selected antibodies from the lysates of infected Sf21 and transiently transfected HEK293. In summary, generated antibodies should be useful as tools for examining the expression pattern and biological functions of FGFR3 in normal and pathological cells and tissues.


Asunto(s)
Anticuerpos Monoclonales/química , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/química , Animales , Línea Celular Tumoral , Proliferación Celular , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Humanos , Hibridomas/inmunología , Insectos , Ratones , Ratones Endogámicos BALB C , Mutación , Estructura Terciaria de Proteína , Transducción de Señal
11.
Hybridoma (Larchmt) ; 25(2): 86-90, 2006 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-16704309

RESUMEN

Fatty acid binding protein 4 (FABP4) is a key mediator of intracellular transport and metabolism of fatty acids in adipose tissues. FABP4 binds fatty acids with high affinity and transports them to various compartments in the cell. When in complex with fatty acids, FABP4 interacts with and modulates the activity of two important regulators of metabolism: hormone-sensitive lipase and peroxisome proliferator-activated receptor gamma. Genetic studies in mice clearly indicated that deregulation of FABP4 function may lead to the development of severe diseases such as diabetes II type and atherosclerosis. In this study, we report the production and detailed characterization of monoclonal antibodies (MAbs) against FABP4. Recombinant glutathione S-transferase (GST)-FABP4 or His-FABP4 was expressed in bacteria, affinity purified, and used for immunization of mice, enzyme-linked immunosorbent assay (ELISA) screening, and characterization of selected clones. We have isolated two hybridoma clones that produced antibodies specific for recombinant and native FABP4, as shown by Western blotting and immunoprecipitation. The specificity of generated antibodies was further tested in a cell-based model of adipogenesis. In this analysis, the accumulation of FABP4 during NIH 3T3-L1 differentiation into adipocytes was detected by generated antibodies, which correlates well with previously published data. Taken together, we produced MAbs that will be useful for the scientific community working on fatty acid-binding proteins and lipid metabolism.


Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Proteínas de Unión a Ácidos Grasos/inmunología , Células 3T3-L1 , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Western Blotting , Proteínas de Unión a Ácidos Grasos/metabolismo , Humanos , Hibridomas/metabolismo , Inmunoprecipitación , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes/inmunología
12.
J Biol Chem ; 278(50): 50316-21, 2003 Dec 12.
Artículo en Inglés | MEDLINE | ID: mdl-14514684

RESUMEN

CoA synthase mediates the last two steps in the sequence of enzymatic reactions, leading to CoA biosynthesis. We have recently identified cDNA for CoA synthase and demonstrated that it encodes a bifunctional enzyme possessing 4'-phosphopantetheine adenylyltransferase and dephospho-CoA kinase activities. Molecular cloning of CoA synthase provided us with necessary tools to study subcellular localization and the regulation of this bifunctional enzyme. Transient expression studies and confocal microscopy allowed us to demonstrate that full-length CoA synthase is associated with the mitochondria, whereas the removal of the N-terminal region relocates the enzyme to the cytosol. In addition, we showed that the N-terminal sequence of CoA synthase (amino acids 1-29) exhibits a hydrophobic profile and targets green fluorescent protein exclusively to mitochondria. Further analysis, involving subcellular fractionation and limited proteolysis, indicated that CoA synthase is localized on the mitochondrial outer membrane. Moreover, we demonstrate for the first time that phosphatidylcholine and phosphatidylethanolamine, which are the main components of the mitochondrial outer membrane, are potent activators of both enzymatic activities of CoA synthase in vitro. Taken together, these data provide the evidence that the final stages of CoA biosynthesis take place on mitochondria and the activity of CoA synthase is regulated by phospholipids.


Asunto(s)
Coenzima A Ligasas/biosíntesis , Coenzima A Ligasas/genética , Regulación Enzimológica de la Expresión Génica , Secuencia de Aminoácidos , Animales , Línea Celular , Línea Celular Tumoral , Membrana Celular/metabolismo , Centrifugación por Gradiente de Densidad , Clonación Molecular , Coenzima A/metabolismo , ADN Complementario/metabolismo , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/metabolismo , Ratones , Microscopía Confocal , Microscopía Fluorescente , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Células 3T3 NIH , Nucleotidiltransferasas/química , Nucleotidiltransferasas/genética , Fosfolípidos/química , Plásmidos/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Fracciones Subcelulares/metabolismo
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