Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 6 de 6
Filtrar
1.
Hum Mutat ; 34(2): 317-29, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23169578

RESUMEN

Schnyder corneal dystrophy (SCD) is an autosomal dominant disease characterized by germline variants in UBIAD1 introducing missense alterations leading to deposition of cholesterol in the cornea, progressive opacification, and loss of visual acuity. UBIAD1 was recently shown to synthesize menaquinone-4 (MK-4, vitamin K(2) ), but causal mechanisms of SCD are unknown. We report a novel c.864G>A UBIAD1 mutation altering glycine 177 to glutamic acid (p.G177E) in six SCD families, including four families from Finland who share a likely founder mutation. We observed reduced MK-4 synthesis by UBIAD1 altered by SCD mutations p.N102S, p.G177R/E, and p.D112N, and molecular models showed p.G177-mutant UBIAD1 disrupted transmembrane helices and active site residues. We show UBIAD1 interacts with HMGCR and SOAT1, enzymes catalyzing cholesterol synthesis and storage, respectively, using yeast two-hybrid screening and immunoprecipitation. Docking simulations indicate cholesterol binds to UBIAD1 in the substrate-binding cleft and substrate-binding overlaps with GGPP binding, an MK-4 substrate, suggesting potential competition between these metabolites. Impaired MK-4 synthesis is a biochemical defect identified in SCD suggesting UBIAD1 links vitamin K and cholesterol metabolism through physical contact between enzymes and metabolites. Our data suggest a role for endogenous MK-4 in maintaining cornea health and visual acuity.


Asunto(s)
Colesterol/metabolismo , Distrofias Hereditarias de la Córnea/genética , Dimetilaliltranstransferasa/genética , Vitamina K 2/análogos & derivados , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Córnea/enzimología , Dimetilaliltranstransferasa/metabolismo , Femenino , Finlandia , Variación Genética , Ácido Glutámico/metabolismo , Glicina/metabolismo , Humanos , Hidroximetilglutaril-CoA Reductasas/genética , Hidroximetilglutaril-CoA Reductasas/metabolismo , Inmunoprecipitación , Japón , Metabolismo de los Lípidos , Masculino , Persona de Mediana Edad , Modelos Moleculares , Datos de Secuencia Molecular , Mutación Missense , Linaje , Conformación Proteica , Análisis de Secuencia de ADN , Esterol O-Aciltransferasa/genética , Esterol O-Aciltransferasa/metabolismo , Turquía , Vitamina K 2/metabolismo
2.
J Proteome Res ; 9(12): 6696-704, 2010 Dec 03.
Artículo en Inglés | MEDLINE | ID: mdl-20968308

RESUMEN

Affinity purification of protein complexes followed by identification using liquid chromatography/mass spectrometry (LC-MS/MS) is a robust method to study the fundamental process of protein interaction. Although affinity isolation reduces the complexity of the sample, fractionation prior to LC-MS/MS analysis is still necessary to maximize protein coverage. In this study, we compared the protein coverage obtained via LC-MS/MS analysis of protein complexes prefractionated using two commonly employed methods, SDS-PAGE and strong cation exchange chromatography (SCX). The two complexes analyzed focused on the nuclear proteins Bmi-1 and GATA3 that were expressed within the cells at low and high levels, respectively. Prefractionation of the complexes at the peptide level using SCX consistently resulted in the identification of approximately 3-fold more proteins compared to separation at the protein level using SDS-PAGE. The increase in the number of identified proteins was especially pronounced for the Bmi-1 complex, where the target protein was expressed at a low level. The data show that prefractionation of affinity isolated protein complexes using SCX prior to LC-MS/MS analysis significantly increases the number of identified proteins and individual protein coverage, particularly for target proteins expressed at low levels.


Asunto(s)
Fraccionamiento Químico/métodos , Cromatografía por Intercambio Iónico/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Complejos Multiproteicos/análisis , Cationes , Línea Celular Tumoral , Cromatografía Liquida , Factor de Transcripción GATA3/análisis , Factor de Transcripción GATA3/genética , Células HEK293 , Humanos , Espectrometría de Masas , Proteínas Nucleares/análisis , Proteínas Nucleares/genética , Complejo Represivo Polycomb 1 , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/análisis , Proteínas Represoras/genética , Reproducibilidad de los Resultados , Transfección
3.
Biomol Eng ; 22(1-3): 57-61, 2005 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-15857784

RESUMEN

The creation of protein libraries by random mutagenesis and cassette mutagenesis has proven to be a successful method of protein engineering. Appropriate statistical analysis is important for the proper construction of these libraries and even more important for the interpretation of data from these libraries. We present simple mathematical expressions useful in the creation and evaluation of such libraries. These equations are useful in estimating the distribution of mutations, the degeneracy of the library and the frequency of a particular clone in the library. In addition, general equations addressing the probability that a particular clone is in a library, the probability that a library is complete, and as the consequences of retransformation of the library on these probabilities are presented.


Asunto(s)
Algoritmos , Biblioteca de Genes , Clonación Molecular/métodos , Ingeniería de Proteínas/métodos
4.
Antioxid Redox Signal ; 21(4): 551-64, 2014 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-24252090

RESUMEN

AIMS: Adenosine triphosphate (ATP) synthase uses chemiosmotic energy across the inner mitochondrial membrane to convert adenosine diphosphate and orthophosphate into ATP, whereas genetic deletion of Sirt3 decreases mitochondrial ATP levels. Here, we investigate the mechanistic connection between SIRT3 and energy homeostasis. RESULTS: By using both in vitro and in vivo experiments, we demonstrate that ATP synthase F1 proteins alpha, beta, gamma, and Oligomycin sensitivity-conferring protein (OSCP) contain SIRT3-specific reversible acetyl-lysines that are evolutionarily conserved and bind to SIRT3. OSCP was further investigated and lysine 139 is a nutrient-sensitive SIRT3-dependent deacetylation target. Site directed mutants demonstrate that OSCP(K139) directs, at least in part, mitochondrial ATP production and mice lacking Sirt3 exhibit decreased ATP muscle levels, increased ATP synthase protein acetylation, and an exercise-induced stress-deficient phenotype. INNOVATION: This work connects the aging and nutrient response, via SIRT3 direction of the mitochondrial acetylome, to the regulation of mitochondrial energy homeostasis under nutrient-stress conditions by deacetylating ATP synthase proteins. CONCLUSION: Our data suggest that acetylome signaling contributes to mitochondrial energy homeostasis by SIRT3-mediated deacetylation of ATP synthase proteins.


Asunto(s)
Complejos de ATP Sintetasa/metabolismo , Sirtuina 3/metabolismo , Estrés Fisiológico , Acetilación , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Proteínas Portadoras/metabolismo , Línea Celular , Activación Enzimática , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , ATPasas de Translocación de Protón Mitocondriales , Músculo Esquelético/metabolismo , Condicionamiento Físico Animal , Unión Proteica , Sirtuina 3/genética , Estrés Fisiológico/genética
5.
Cancer Res ; 74(10): 2785-95, 2014 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-24648346

RESUMEN

Genome-wide association studies (GWAS) of 10 different cancers have identified pleiotropic cancer predisposition loci across a region of chromosome 5p15.33 that includes the TERT and CLPTM1L genes. Of these, susceptibility alleles for pancreatic cancer have mapped to the CLPTM1L gene, thus prompting an investigation of the function of CLPTM1L in the pancreas. Immunofluorescence analysis indicated that CLPTM1L localized to the endoplasmic reticulum where it is likely embedded in the membrane, in accord with multiple predicted transmembrane domains. Overexpression of CLPTM1L enhanced growth of pancreatic cancer cells in vitro (1.3-1.5-fold; PDAY7 < 0.003) and in vivo (3.46-fold; PDAY68 = 0.039), suggesting a role in tumor growth; this effect was abrogated by deletion of two hydrophilic domains. Affinity purification followed by mass spectrometry identified an interaction between CLPTM1L and non-muscle myosin II (NMM-II), a protein involved in maintaining cell shape, migration, and cytokinesis. The two proteins colocalized in the cytoplasm and, after treatment with a DNA-damaging agent, at the centrosomes. Overexpression of CLPTM1L and depletion of NMM-II induced aneuploidy, indicating that CLPTM1L may interfere with normal NMM-II function in regulating cytokinesis. Immunohistochemical analysis revealed enhanced staining of CLPTM1L in human pancreatic ductal adenocarcinoma (n = 378) as compared with normal pancreatic tissue samples (n = 17; P = 1.7 × 10(-4)). Our results suggest that CLPTM1L functions as a growth-promoting gene in the pancreas and that overexpression may lead to an abrogation of normal cytokinesis, indicating that it should be considered as a plausible candidate gene that could explain the effect of pancreatic cancer susceptibility alleles on chr5p15.33.


Asunto(s)
Carcinoma Ductal Pancreático/patología , Proteínas de la Membrana/biosíntesis , Proteínas de Neoplasias/biosíntesis , Neoplasias Pancreáticas/patología , Aneuploidia , Animales , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Femenino , Células HEK293 , Xenoinjertos , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Desnudos , Miosina Tipo II/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Fracciones Subcelulares/metabolismo
6.
PLoS One ; 8(11): e80746, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24260471

RESUMEN

Recent studies suggest that BET inhibitors are effective anti-cancer therapeutics. Here we show that BET inhibitors are effective against murine primary mammary tumors, but not pulmonary metastases. BRD4, a target of BET inhibitors, encodes two isoforms with opposite effects on tumor progression. To gain insights into why BET inhibition was ineffective against metastases the pro-metastatic short isoform of BRD4 was characterized using mass spectrometry and cellular fractionation. Our data show that the pro-metastatic short isoform interacts with the LINC complex and the metastasis-associated proteins RRP1B and SIPA1 at the inner face of the nuclear membrane. Furthermore, histone binding arrays revealed that the short isoform has a broader acetylated histone binding pattern relative to the long isoform. These differential biochemical and nuclear localization properties revealed in our study provide novel insights into the opposing roles of BRD4 isoforms in metastatic breast cancer progression.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Animales , Proteínas de Ciclo Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Modelos Animales de Enfermedad , Femenino , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Histonas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Acetiltransferasa E N-Terminal/metabolismo , Acetiltransferasas N-Terminal , Metástasis de la Neoplasia , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Nucleares/genética , Unión Proteica , Isoformas de Proteínas , Transporte de Proteínas , Factores de Transcripción/genética , Carga Tumoral/efectos de los fármacos
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA