Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 16 de 16
Filtrar
Más filtros













Base de datos
Intervalo de año de publicación
1.
Curr Protoc ; 3(11): e920, 2023 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-37933593

RESUMEN

Human fertility is declining in Western countries, and it is becoming increasingly clear that male infertility plays a pivotal role in the overall fertility decline. To understand the process that drives successful male germ cell maturation, the study of spermatogenesis of model organisms, such as mice, is essential. Residual bodies (RBs) play an important role in the last stages of spermatogenesis. They are formed at the time when post-meiotic spermatids undergo sequential differentiation steps so that the acrosome and flagellum are developed, the nucleus is markedly condensed, and the cytoplasm is lost. The masses of lost cytoplasm become RBs. Our recent work has shown that RB dynamics are highly sensitive to even small fertility defects. It was also noted that the transcriptome and proteome of RBs changes in response to spermatogenic defects. Thus, RBs represent an excellent and highly sensitive entity for studying male fertility. Previously published protocols for RB purification had some major limitations: they produced an RB fraction that was heavily contaminated with spermatozoa and erythrocytes or required tens of grams of starting material. In addition, most of the available protocols were developed for purification of RBs from rat testes. Here, we present a protocol that allows the isolation of 2.5-3 × 106 RBs from mouse testes with a purity of 98% from only 1 g of starting material. The purified material can be used for various downstream applications to study male fertility, such as transcriptome and proteome analyses, super-resolution microscopy, and electron and cryo-electron microscopy, amongst many others. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: An improved method for purification of the residual bodies from the seminiferous tubules of mice.


Asunto(s)
Proteoma , Túbulos Seminíferos , Ratas , Ratones , Masculino , Animales , Humanos , Microscopía por Crioelectrón , Túbulos Seminíferos/fisiología , Espermatozoides , Espermátides
2.
EMBO Rep ; 22(8): e52462, 2021 08 04.
Artículo en Inglés | MEDLINE | ID: mdl-34350706

RESUMEN

Testis-specific regulators of chromatin function are commonly ectopically expressed in human cancers, but their roles are poorly understood. Examination of 81 primary Hodgkin lymphoma (HL) samples showed that the ectopic expression of the eutherian testis-specific histone variant H2A.B is an inherent feature of HL. In experiments using two HL cell lines derived from different subtypes of HL, H2A.B knockdown inhibited cell proliferation. H2A.B was enriched in both nucleoli of these HL cell lines and primary HL samples. We found that H2A.B enhanced ribosomal DNA (rDNA) transcription, was enriched at the rDNA promoter and transcribed regions, and interacted with RNA Pol I. Depletion of H2A.B caused the loss of RNA Pol I from rDNA chromatin. Remarkably, H2A.B was also required for high levels of ribosomal protein gene expression being located at the transcriptional start site and within the gene body. H2A.B knockdown reduced gene body chromatin accessibility of active RNA Pol II genes concurrent with a decrease in transcription. Taken together, our data show that in HL H2A.B has acquired a new function, the ability to increase ribosome biogenesis.


Asunto(s)
Histonas , Enfermedad de Hodgkin , Cromatina/genética , Histonas/genética , Enfermedad de Hodgkin/genética , Humanos , Masculino , Ribosomas/genética , Testículo
3.
Cells ; 9(4)2020 04 02.
Artículo en Inglés | MEDLINE | ID: mdl-32252453

RESUMEN

The dynamic packaging of DNA into chromatin regulates all aspects of genome function by altering the accessibility of DNA and by providing docking pads to proteins that copy, repair and express the genome. Different epigenetic-based mechanisms have been described that alter the way DNA is organised into chromatin, but one fundamental mechanism alters the biochemical composition of a nucleosome by substituting one or more of the core histones with their variant forms. Of the core histones, the largest number of histone variants belong to the H2A class. The most divergent class is the designated "short H2A variants" (H2A.B, H2A.L, H2A.P and H2A.Q), so termed because they lack a H2A C-terminal tail. These histone variants appeared late in evolution in eutherian mammals and are lineage-specific, being expressed in the testis (and, in the case of H2A.B, also in the brain). To date, most information about the function of these peculiar histone variants has come from studies on the H2A.B and H2A.L family in mice. In this review, we describe their unique protein characteristics, their impact on chromatin structure, and their known functions plus other possible, even non-chromatin, roles in an attempt to understand why these peculiar histone variants evolved in the first place.


Asunto(s)
Variación Genética/genética , Histonas/genética , Animales , Humanos , Ratones , Análisis de Secuencia de Proteína
4.
Genome Biol ; 20(1): 23, 2019 01 31.
Artículo en Inglés | MEDLINE | ID: mdl-30704500

RESUMEN

BACKGROUND: Altering the biochemical makeup of chromatin by the incorporation of histone variants during development represents a key mechanism in regulating gene expression. The histone variant H2A.B, H2A.B.3 in mice, appeared late in evolution and is most highly expressed in the testis. In the mouse, it is encoded by three different genes. H2A.B expression is spatially and temporally regulated during spermatogenesis being most highly expressed in the haploid round spermatid stage. Active genes gain H2A.B where it directly interacts with polymerase II and RNA processing factors within splicing speckles. However, the importance of H2A.B for gene expression and fertility are unknown. RESULTS: Here, we report the first mouse knockout of this histone variant and its effects on fertility, nuclear organization, and gene expression. In view of the controversy related to the generation of off-target mutations by gene editing approaches, we test the specificity of TALENs by disrupting the H2A.B multi-copy gene family using only one pair of TALENs. We show that TALENs do display a high level of specificity since no off-target mutations are detected by bioinformatics analyses of exome sequences obtained from three consecutive generations of knockout mice and by Sanger DNA sequencing. Male H2A.B.3 knockout mice are subfertile and display an increase in the proportion of abnormal sperm and clogged seminiferous tubules. Significantly, a loss of proper RNA Pol II targeting to distinct transcription-splicing territories and changes to pre-mRNA splicing are observed. CONCLUSION: We have produced the first H2A.B knockout mouse using the TALEN approach.


Asunto(s)
Fertilidad/genética , Edición Génica/métodos , Histonas/genética , Infertilidad Masculina/etiología , Nucleasas de los Efectores Tipo Activadores de la Transcripción , Animales , Secuencia de Bases , Proteínas Cromosómicas no Histona/metabolismo , Femenino , Expresión Génica , Infertilidad Masculina/metabolismo , Infertilidad Masculina/patología , Masculino , Ratones Noqueados , Mutación , ARN Polimerasa II/metabolismo , Espermatozoides/metabolismo , Espermatozoides/patología
5.
Methods Mol Biol ; 1832: 169-184, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30073527

RESUMEN

Chromatin is a dynamic macromolecular structure comprised of histones and a wealth of non-histone proteins. Recently, it has become clear that RNA is also an integral component of chromatin playing an important role in regulating its structure and function. Central to the understanding of RNA function is the ability to identify and genomically map interactions between chromatin components and RNA.Here, we describe a new method, RChIP-seq (RNA-associated-Chromatin-Immuno Precipitation followed by next-generation sequencing) that allows the identification of RNA species that are directly bound to specific components of chromatin in the mouse testis.


Asunto(s)
Inmunoprecipitación de Cromatina/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ARN/métodos , Testículo/crecimiento & desarrollo , Testículo/metabolismo , Animales , Reactivos de Enlaces Cruzados/química , ADN Complementario/genética , Células Germinativas/metabolismo , Masculino , Ratones Endogámicos BALB C , Proteínas/metabolismo , ARN/metabolismo , Rayos Ultravioleta
6.
Cell Death Differ ; 24(6): 1029-1044, 2017 06.
Artículo en Inglés | MEDLINE | ID: mdl-28475176

RESUMEN

Sperm differentiation requires unique transcriptional regulation and chromatin remodeling after meiosis to ensure proper compaction and protection of the paternal genome. Abnormal sperm chromatin remodeling can induce sperm DNA damage, embryo lethality and male infertility, yet, little is known about the factors which regulate this process. Deficiency in Sly, a mouse Y chromosome-encoded gene expressed only in postmeiotic male germ cells, has been shown to result in the deregulation of hundreds of sex chromosome-encoded genes associated with multiple sperm differentiation defects and subsequent male infertility. The underlying mechanism remained, to date, unknown. Here, we show that SLY binds to the promoter of sex chromosome-encoded and autosomal genes highly expressed postmeiotically and involved in chromatin regulation. Specifically, we demonstrate that Sly knockdown directly induces the deregulation of sex chromosome-encoded H2A variants and of the H3K79 methyltransferase DOT1L. The modifications prompted by loss of Sly alter the postmeiotic chromatin structure and ultimately result in abnormal sperm chromatin remodeling with negative consequences on the sperm genome integrity. Altogether our results show that SLY is a regulator of sperm chromatin remodeling. Finally we identified that SMRT/N-CoR repressor complex is involved in gene regulation during sperm differentiation since members of this complex, in particular TBL1XR1, interact with SLY in postmeiotic male germ cells.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Ensamble y Desensamble de Cromatina , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Represoras/metabolismo , Cromosomas Sexuales/metabolismo , Espermatozoides/metabolismo , Proteínas Adaptadoras del Transporte Vesicular , Animales , Cromosomas de los Mamíferos/genética , Cromosomas de los Mamíferos/metabolismo , ADN/metabolismo , Regulación de la Expresión Génica , Masculino , Ratones , Cromosomas Sexuales/genética , Espermatogénesis , Espermatozoides/fisiología
7.
PLoS Genet ; 13(2): e1006633, 2017 02.
Artículo en Inglés | MEDLINE | ID: mdl-28234895

RESUMEN

The replacement of histone H2A with its variant forms is critical for regulating all aspects of genome organisation and function. The histone variant H2A.B appeared late in evolution and is most highly expressed in the testis followed by the brain in mammals. This raises the question of what new function(s) H2A.B might impart to chromatin in these important tissues. We have immunoprecipitated the mouse orthologue of H2A.B, H2A.B.3 (H2A.Lap1), from testis chromatin and found this variant to be associated with RNA processing factors and RNA Polymerase (Pol) II. Most interestingly, many of these interactions with H2A.B.3 (Sf3b155, Spt6, DDX39A and RNA Pol II) were inhibited by the presence of endogenous RNA. This histone variant can bind to RNA directly in vitro and in vivo, and associates with mRNA at intron-exon boundaries. This suggests that the ability of H2A.B to bind to RNA negatively regulates its capacity to bind to these factors (Sf3b155, Spt6, DDX39A and RNA Pol II). Unexpectedly, H2A.B.3 forms highly decompacted nuclear subdomains of active chromatin that co-localizes with splicing speckles in male germ cells. H2A.B.3 ChIP-Seq experiments revealed a unique chromatin organization at active genes being not only enriched at the transcription start site (TSS), but also at the beginning of the gene body (but being excluded from the +1 nucleosome) compared to the end of the gene. We also uncover a general histone variant replacement process whereby H2A.B.3 replaces H2A.Z at intron-exon boundaries in the testis and the brain, which positively correlates with expression and exon inclusion. Taken together, we propose that a special mechanism of splicing may occur in the testis and brain whereby H2A.B.3 recruits RNA processing factors from splicing speckles to active genes following its replacement of H2A.Z.


Asunto(s)
Histonas/genética , Precursores del ARN/genética , Empalme del ARN , Sitio de Iniciación de la Transcripción , Transcripción Genética/genética , Animales , Western Blotting , Encéfalo/metabolismo , Cromatina/genética , Cromatina/metabolismo , Exones/genética , Técnica del Anticuerpo Fluorescente , Variación Genética , Histonas/metabolismo , Intrones/genética , Masculino , Espectrometría de Masas , Ratones Endogámicos BALB C , Unión Proteica , ARN/genética , ARN/metabolismo , ARN Polimerasa II/metabolismo , Precursores del ARN/metabolismo , Sitios de Empalme de ARN/genética , Proteínas de Unión al ARN/metabolismo , Testículo/citología , Testículo/metabolismo
8.
Trends Genet ; 30(5): 199-209, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24768041

RESUMEN

The function of a eukaryotic cell crucially depends on accurate gene transcription to ensure the right genes are expressed whereas unrequired genes are repressed. Therefore, arguably, one of the most important regions in the genome is the transcription start-site (TSS) of protein-coding and non-coding genes. Until recently, understanding the mechanisms that define the location of the TSS and how it is created has largely focused on the role of DNA sequence-specific transcription factors. However, within the nucleus of a eukaryotic cell, transcription occurs in a highly compacted nucleosomal environment, and it is becoming clear that accessibility of the TSS is a key controlling step in transcriptional regulation. It has traditionally been thought that transcription can only proceed once the nucleosomes at the TSS have been evicted. New work suggests otherwise, however, and the focus of this review is to challenge this belief.


Asunto(s)
Histonas/metabolismo , Sitio de Iniciación de la Transcripción , Secuencia de Aminoácidos , Animales , Histonas/química , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Nucleosomas/metabolismo , Regiones Promotoras Genéticas , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo
9.
Nucleus ; 4(6): 431-8, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24213378

RESUMEN

Considerable attention has been given to the understanding of how nucleosomes are altered or removed from the transcription start site of RNA polymerase II genes to enable transcription to proceed. This has led to the view that for transcriptional activation to occur, the transcription start site (TSS) must become depleted of nucleosomes. However, we have shown that this is not the case with different unstable histone H2A variant-containing nucleosomes occupying the TSS under different physiological settings. For example, during mouse spermatogenesis we found that the mouse homolog of human H2A.Bbd, H2A.Lap1, is targeted to the TSS of active genes expressed during specific stages of spermatogenesis. On the other hand, we observed in trophoblast stem cells, a H2A.Z-containing nucleosome occupying the TSS of genes active in the G 1 phase of the cell cycle. Notably, this H2A.Z-containing nucleosome was different compared with other promoter specific H2A.Z nucleosomes by being heterotypic rather than being homotypic. In other words, it did not contain the expected two copies of H2A.Z per nucleosome but only one (i.e., H2A.Z/H2A rather than H2A.Z/H2A.Z). Given these observations, we wondered whether the histone variant composition of a nucleosome at an active TSS could in fact vary in the same cell type. To investigate this possibility, we performed H2A.Z ChIP-H2A reChIP assays in the mouse testis and compared this data with our testis H2A.Lap1 ChIP-seq data. Indeed, we find that different promoters involved in the expression of genes involved in distinct biological processes can contain either H2A.Z/H2A or H2A.Lap1. This argues that specific mechanisms exist, which can determine whether H2A.Z or H2A.Lap1 is targeted to the TSS of an active gene.


Asunto(s)
Ciclo Celular/fisiología , Regulación de la Expresión Génica , Histonas/genética , Histonas/metabolismo , Modelos Biológicos , Nucleosomas/metabolismo , Regiones Promotoras Genéticas/genética , Sitio de Iniciación de la Transcripción/fisiología , Trofoblastos/fisiología , Animales , Masculino
10.
Nat Struct Mol Biol ; 19(11): 1076-83, 2012 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-23085713

RESUMEN

Although it has been clearly established that well-positioned histone H2A.Z-containing nucleosomes flank the nucleosome-depleted region (NDR) at the transcriptional start site (TSS) of active mammalian genes, how this chromatin-based information is transmitted through the cell cycle is unknown. We show here that in mouse trophoblast stem cells, the amount of histone H2A.Z at promoters decreased during S phase, coinciding with homotypic (H2A.Z-H2A.Z) nucleosomes flanking the TSS becoming heterotypic (H2A.Z-H2A). To our surprise these nucleosomes remained heterotypic at M phase. At the TSS, we identified an unstable heterotypic histone H2A.Z-containing nucleosome in G1 phase that was lost after DNA replication. These dynamic changes at the TSS mirror a global expansion of the NDR at S and M phases, which, unexpectedly, is unrelated to transcriptional activity. Coincident with the loss of histone H2A.Z at promoters, histone H2A.Z is targeted to the centromere when mitosis begins.


Asunto(s)
Ciclo Celular/fisiología , Histonas/metabolismo , Modelos Biológicos , Nucleosomas/metabolismo , Regiones Promotoras Genéticas/genética , Sitio de Iniciación de la Transcripción/fisiología , Trofoblastos/fisiología , Animales , Western Blotting , Células Cultivadas , Centrómero/genética , Inmunoprecipitación de Cromatina , Cartilla de ADN/genética , Citometría de Flujo , Ratones , Nucleosomas/genética , Análisis de Secuencia de ADN , Trofoblastos/metabolismo
11.
Nat Struct Mol Biol ; 19(1): 25-30, 2011 Dec 04.
Artículo en Inglés | MEDLINE | ID: mdl-22139013

RESUMEN

Transcriptional activation is controlled by chromatin, which needs to be unfolded and remodeled to ensure access to the transcription start site (TSS). However, the mechanisms that yield such an 'open' chromatin structure, and how these processes are coordinately regulated during differentiation, are poorly understood. We identify the mouse (Mus musculus) H2A histone variant H2A.Lap1 as a previously undescribed component of the TSS of active genes expressed during specific stages of spermatogenesis. This unique chromatin landscape also includes a second histone variant, H2A.Z. In the later stages of round spermatid development, H2A.Lap1 dynamically loads onto the inactive X chromosome, enabling the transcriptional activation of previously repressed genes. Mechanistically, we show that H2A.Lap1 imparts unique unfolding properties to chromatin. We therefore propose that H2A.Lap1 coordinately regulates gene expression by directly opening the chromatin structure of the TSS at genes regulated during spermatogenesis.


Asunto(s)
Regulación de la Expresión Génica , Histonas/genética , Histonas/metabolismo , Sitio de Iniciación de la Transcripción , Secuencia de Aminoácidos , Animales , Western Blotting , Cromatina/genética , Cromatina/metabolismo , Cristalografía por Rayos X , Perfilación de la Expresión Génica , Variación Genética , Histonas/química , Masculino , Meiosis/genética , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Nucleosomas/genética , Nucleosomas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Unión Proteica , Estructura Secundaria de Proteína , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Espermátides/metabolismo , Espermatogénesis/genética , Cromosoma X/genética , Cromosoma Y/genética
12.
Cytokine ; 42(2): 234-242, 2008 May.
Artículo en Inglés | MEDLINE | ID: mdl-18374598

RESUMEN

Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3 and IL-5 are related cytokines that play key roles in regulating the differentiation, proliferation, survival and activation of myeloid blood cells. The cell surface receptors for these cytokines are composed of cytokine-specific alpha-subunits and a common beta-receptor (betac), a shared subunit that is essential for receptor signaling in response to GM-CSF, IL-3 and IL-5. Previous studies have reached conflicting conclusions as to whether N-glycosylation of the betac-subunit is necessary for functional GM-CSF, IL-3 and IL-5 receptors. We sought to clarify whether betac N-glycosylation plays a role in receptor function, since all structural studies of human betac to date have utilized recombinant protein lacking N-glycosylation at Asn(328). Here, by eliminating individual N-glycans in human betac and the related murine homolog, beta(IL-3), we demonstrate unequivocally that ligand-binding and receptor activation are not critically dependent on individual N-glycosylation sites within the beta-subunit although the data do not preclude the possibility that N-glycans may exert some sort of fine control. These studies support the biological relevance of the X-ray crystal structures of the human betac domain 4 and the complete ectodomain, both of which lack N-glycosylation at Asn(328).


Asunto(s)
Subunidad beta Común de los Receptores de Citocinas/fisiología , Polisacáridos/fisiología , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , Receptores de Interleucina-3/fisiología , Receptores de Interleucina-5/fisiología , Animales , Células COS , Chlorocebus aethiops , Subunidad beta Común de los Receptores de Citocinas/química , Subunidad beta Común de los Receptores de Citocinas/genética , Humanos , Interleucina-3/metabolismo , Interleucina-5/metabolismo , Ratones , Mutagénesis Sitio-Dirigida , Polisacáridos/química , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/química , Receptores de Interleucina-3/química , Receptores de Interleucina-5/química
13.
Methods Enzymol ; 398: 540-54, 2005.
Artículo en Inglés | MEDLINE | ID: mdl-16275357

RESUMEN

Ubiquitin is synthesized in eukaryotes as a linear fusion with a normal peptide bond either to itself or to one of two ribosomal proteins and, in the latter case, enhances the yield of these ribosomal proteins and/or their incorporation into the ribosome. Such fusions are cleaved rapidly by a variety of deubiquitylating enzymes. Expression of heterologous proteins as linear ubiquitin fusions has been found to significantly increase the yield of unstable or poorly expressed proteins in either bacterial or eukaryotic hosts. If expressed in bacterial cells, the fusion is not cleaved due to the absence of deubiquitylating activity and can be purified intact. We have developed an efficient expression system, utilizing the ubiquitin fusion technique and a robust deubiquitylating enzyme, which allows convenient high yield and easy purification of authentic proteins. An affinity purification tag on both the ubiquitin fusion and the deubiquitylating enzyme allows their easy purification and the easy removal of unwanted components after cleavage, leaving the desired protein as the only soluble product. Ubiquitin is also conjugated to epsilon amino groups in lysine side chains of target proteins to form a so-called isopeptide linkage. Either a single ubiquitin can be conjugated or other lysines within ubiquitin can be acceptors for further conjugation, leading to formation of a branched, isopeptide-linked ubiquitin chain. Removal of these ubiquitin moieties or chains in vitro would be a valuable tool in the ubiquitinologists tool kit to simplify downstream studies on ubiquitylated targets. The robust deubiquitylating enzyme described earlier is also very useful for this task.


Asunto(s)
Endopeptidasas/metabolismo , Ubiquitina/metabolismo , Secuencia de Aminoácidos , Animales , Escherichia coli/genética , Vectores Genéticos , Humanos , Concentración de Iones de Hidrógeno , Ratones , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Cloruro de Sodio , Ubiquitina/genética , Proteasas Ubiquitina-Específicas
14.
J Biol Chem ; 280(1): 745-52, 2005 Jan 07.
Artículo en Inglés | MEDLINE | ID: mdl-15494318

RESUMEN

The oncogenic deubiquitylating enzyme (DUB) Unp/Usp4, which binds to the retinoblastoma family of tumor suppressor proteins, was originally described as a nuclear protein. However, more recent studies have shown it to be cytoplasmic. In addition, analysis of its subcellular localization has been complicated by the existence of the paralog Usp15. In this study, we resolved this controversy by investigating the localization of exogenously expressed Usp4 (using red fluorescent protein-Usp4) and of endogenous Usp4 (using highly specific antibodies that can distinguish Usp4 from Usp15). We found that by inhibiting nuclear export with leptomycin B, both exogenous and endogenous Usp4 accumulate in the nucleus. Further, using a Rev-green fluorescent protein-based export assay, we confirmed the existence of a nuclear export signal ((133)VEVYLLELKL(142)) in Usp4. In addition, a functional nuclear import signal ((766)QPQKKKK(772)) was also identified, which was specifically recognized by importin alpha/beta. Finally, we show that the equilibrium of Usp4 subcellular localization varies between different cell types. Usp4 is thus the first DUB reported to have nucleocytoplasmic shuttling properties. The implications of this shuttling for its function as a DUB are discussed.


Asunto(s)
Proteínas Oncogénicas/metabolismo , Animales , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes , Ratones , Especificidad de Órganos , Transporte de Proteínas , Proteínas Proto-Oncogénicas , Ubiquitina Tiolesterasa , Proteasas Ubiquitina-Específicas , Proteína Fluorescente Roja
15.
Curr Protein Pept Sci ; 5(3): 191-200, 2004 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-15180524

RESUMEN

Conjugation of one or more molecules of ubiquitin to target proteins can signify one of several fates, including degradation by the 26S proteasome, or trafficking via the secretory or endocytic pathways. Whereas much attention in recent years has focussed on the mechanisms of forming these different ubiquitin conjugates, far less is known about the removal of ubiquitin, which is performed by deubiquitinating enzymes (DUBs). While it has been appreciated for some 10 years that DUBs constitute large gene families in eukaryotes, and known for much longer that ubiquitination is a reversible process, information on the exact role of DUBs has been slow in coming. This review will attempt to summarise results from the last few years that shows that DUBs are an essential regulatory step of both protein degradation by the proteasome, and of other ubiquitin-dependent processes, by virtue of their ability to regulate protein ubiquitination in a target-specific manner.


Asunto(s)
Cisteína Endopeptidasas/metabolismo , Complejos Multienzimáticos/metabolismo , Péptido Hidrolasas/metabolismo , Ubiquitina/metabolismo , Animales , Activadores de Enzimas/metabolismo , Humanos , Complejo de la Endopetidasa Proteasomal , Desnaturalización Proteica , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Transporte de Proteínas , Especificidad por Sustrato
16.
Protein Sci ; 13(5): 1331-9, 2004 May.
Artículo en Inglés | MEDLINE | ID: mdl-15096636

RESUMEN

Expression of recombinant proteins as fusions to the eukaryotic protein ubiquitin has been found to significantly increase the yield of unstable or poorly expressed proteins. The benefit of this technique is further enhanced by the availability of naturally occurring deubiquitylating enzymes, which remove ubiquitin from the fusion product. However, the versatility of the system has been constrained due to the lack of a robust, easily purified deubiquitylating enzyme. Here we report the development of an efficient expression system, utilizing the ubiquitin fusion technique, which allows convenient high yield and easy purification of authentic protein. An Escherichia coli vector (pHUE) was constructed for the expression of proteins as histidine-tagged ubiquitin fusions, and a histidine-tagged deubiquitylating enzyme to cleave these fusions was expressed and purified. The expression system was tested using several proteins varying in size and complexity. These results indicate that this procedure will be suitable for the expression and rapid purification of a broad range of proteins and peptides, and should be amenable to high-throughput applications.


Asunto(s)
Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Ubiquitina/genética , Secuencia de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos , Glutatión Transferasa/química , Glutatión Transferasa/genética , Glutatión Transferasa/aislamiento & purificación , Carioferinas/química , Carioferinas/aislamiento & purificación , Carioferinas/metabolismo , Datos de Secuencia Molecular , Péptidos/genética , Péptidos/aislamiento & purificación , Plásmidos/genética , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de Proteína , Ubiquitina/química
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA