RESUMEN
Biological membranes are partitioned into functional zones termed membrane microdomains, which contain specific lipids and proteins1-3. The composition and organization of membrane microdomains remain controversial because few techniques are available that allow the visualization of lipids in situ without disrupting their native behaviour3,4. The yeast eisosome, composed of the BAR-domain proteins Pil1 and Lsp1 (hereafter, Pil1/Lsp1), scaffolds a membrane compartment that senses and responds to mechanical stress by flattening and releasing sequestered factors5-9. Here we isolated near-native eisosomes as helical tubules made up of a lattice of Pil1/Lsp1 bound to plasma membrane lipids, and solved their structures by helical reconstruction. Our structures reveal a striking organization of membrane lipids, and, using in vitro reconstitutions and molecular dynamics simulations, we confirmed the positioning of individual PI(4,5)P2, phosphatidylserine and sterol molecules sequestered beneath the Pil1/Lsp1 coat. Three-dimensional variability analysis of the native-source eisosomes revealed a dynamic stretching of the Pil1/Lsp1 lattice that affects the sequestration of these lipids. Collectively, our results support a mechanism in which stretching of the Pil1/Lsp1 lattice liberates lipids that would otherwise be anchored by the Pil1/Lsp1 coat, and thus provide mechanistic insight into how eisosome BAR-domain proteins create a mechanosensitive membrane microdomain.
Asunto(s)
Microscopía por Crioelectrón , Microdominios de Membrana , Saccharomyces cerevisiae , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Microdominios de Membrana/química , Microdominios de Membrana/metabolismo , Microdominios de Membrana/ultraestructura , Modelos Moleculares , Simulación de Dinámica Molecular , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosfoproteínas/ultraestructura , Dominios Proteicos , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestructura , Esteroles/química , Esteroles/metabolismo , Estrés MecánicoRESUMEN
Target of rapamycin complex 1 (TORC1) is a protein kinase controlling cell homeostasis and growth in response to nutrients and stresses. In Saccharomyces cerevisiae, glucose depletion triggers a redistribution of TORC1 from a dispersed localization over the vacuole surface into a large, inactive condensate called TOROID (TORC1 organized in inhibited domains). However, the mechanisms governing this transition have been unclear. Here, we show that acute depletion and repletion of EGO complex (EGOC) activity is sufficient to control TOROID distribution, independently of other nutrient-signaling pathways. The 3.9-Å-resolution structure of TORC1 from TOROID cryo-EM data together with interrogation of key interactions in vivo provide structural insights into TORC1-TORC1' and TORC1-EGOC interaction interfaces. These data support a model in which glucose-dependent activation of EGOC triggers binding to TORC1 at an interface required for TOROID assembly, preventing TORC1 polymerization and promoting release of active TORC1.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Diana Mecanicista del Complejo 1 de la Rapamicina/química , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Polimerizacion , Factores de Transcripción/metabolismo , Saccharomyces cerevisiae/metabolismo , Glucosa/metabolismoRESUMEN
The SEA complex (SEAC) is a growth regulator that acts as a GTPase-activating protein (GAP) towards Gtr1, a Rag GTPase that relays nutrient status to the Target of Rapamycin Complex 1 (TORC1) in yeast1. Functionally, the SEAC has been divided into two subcomplexes: SEACIT, which has GAP activity and inhibits TORC1, and SEACAT, which regulates SEACIT2. This system is conserved in mammals: the GATOR complex, consisting of GATOR1 (SEACIT) and GATOR2 (SEACAT), transmits amino acid3 and glucose4 signals to mTORC1. Despite its importance, the structure of SEAC/GATOR, and thus molecular understanding of its function, is lacking. Here, we solve the cryo-EM structure of the native eight-subunit SEAC. The SEAC has a modular structure in which a COPII-like cage corresponding to SEACAT binds two flexible wings, which correspond to SEACIT. The wings are tethered to the core via Sea3, which forms part of both modules. The GAP mechanism of GATOR1 is conserved in SEACIT, and GAP activity is unaffected by SEACAT in vitro. In vivo, the wings are essential for recruitment of the SEAC to the vacuole, primarily via the EGO complex. Our results indicate that rather than being a direct inhibitor of SEACIT, SEACAT acts as a scaffold for the binding of TORC1 regulators.
Asunto(s)
Microscopía por Crioelectrón , Proteínas Activadoras de GTPasa , Complejos Multienzimáticos , Animales , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/metabolismo , GTP Fosfohidrolasas/ultraestructura , Proteínas Activadoras de GTPasa/química , Proteínas Activadoras de GTPasa/metabolismo , Proteínas Activadoras de GTPasa/ultraestructura , Mamíferos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Complejos Multienzimáticos/ultraestructura , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestructura , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Aminoácidos , Glucosa , Vesículas Cubiertas por Proteínas de Revestimiento/química , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismoRESUMEN
Centromeres are chromosomal regions that serve as sites for kinetochore formation and microtubule attachment, processes that are essential for chromosome segregation during mitosis. Centromeres are almost universally defined by the histone variant CENP-A. In the holocentric nematode C. elegans, CENP-A deposition depends on the loading factor KNL-2. Depletion of either CENP-A or KNL-2 results in defects in centromere maintenance, chromosome condensation and kinetochore formation, leading to chromosome segregation failure. Here, we show that KNL-2 is phosphorylated by CDK-1 in vitro, and that mutation of three C-terminal phosphorylation sites causes chromosome segregation defects and an increase in embryonic lethality. In strains expressing phosphodeficient KNL-2, CENP-A and kinetochore proteins are properly localised, indicating that the role of KNL-2 in centromere maintenance is not affected. Instead, the mutant embryos exhibit reduced mitotic levels of condensin II on chromosomes and significant chromosome condensation impairment. Our findings separate the functions of KNL-2 in CENP-A loading and chromosome condensation, and demonstrate that KNL-2 phosphorylation regulates the cooperation between centromeric regions and the condensation machinery in C. elegans. This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Centrómero/metabolismo , Proteína A Centromérica/genética , Proteína A Centromérica/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica , Humanos , Cinetocoros/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Mitosis , FosforilaciónRESUMEN
Centromere protein A (CENP-A) is a histone H3 variant that defines centromeric chromatin and is essential for centromere function. In most eukaryotes, CENP-A-containing chromatin is epigenetically maintained, and centromere identity is inherited from one cell cycle to the next. In the germ line of the holocentric nematode Caenorhabditis elegans, this inheritance cycle is disrupted. CENP-A is removed at the mitosis-to-meiosis transition and is reestablished on chromatin during diplotene of meiosis I. Here, we show that the N-terminal tail of CENP-A is required for the de novo establishment of centromeres, but then its presence becomes dispensable for centromere maintenance during development. Worms homozygous for a CENP-A tail deletion maintain functional centromeres during development but give rise to inviable offspring because they fail to reestablish centromeres in the maternal germ line. We identify the N-terminal tail of CENP-A as a critical domain for the interaction with the conserved kinetochore protein KNL-2 and argue that this interaction plays an important role in setting centromere identity in the germ line. We conclude that centromere establishment and maintenance are functionally distinct in C. elegans.
Asunto(s)
Caenorhabditis elegans/crecimiento & desarrollo , Proteína A Centromérica/genética , Centrómero , Impresión Genómica , Células Germinativas , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteína A Centromérica/química , Proteína A Centromérica/metabolismo , Cromatina/metabolismo , Cromosomas , Femenino , Homocigoto , Cinetocoros , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Unión Proteica , Dominios ProteicosRESUMEN
Replication-independent variant histones replace canonical histones in nucleosomes and act as important regulators of chromatin function. H3.3 is a major variant of histone H3 that is remarkably conserved across taxa and is distinguished from canonical H3 by just four key amino acids. Most genomes contain two or more genes expressing H3.3, and complete loss of the protein usually causes sterility or embryonic lethality. Here, we investigate the developmental expression patterns of the five Caenorhabditis elegans H3.3 homologs and identify two previously uncharacterized homologs to be restricted to the germ line. Despite these specific expression patterns, we find that neither loss of individual H3.3 homologs nor the knockout of all five H3.3-coding genes causes sterility or lethality. However, we demonstrate an essential role for the conserved histone chaperone HIRA in the nucleosomal loading of all H3.3 variants. This requirement can be bypassed by mutation of the H3.3-specific residues to those found in H3. While even removal of all H3.3 homologs does not result in lethality, it leads to reduced fertility and viability in response to high-temperature stress. Thus, our results show that H3.3 is nonessential in C. elegans but is critical for ensuring adequate response to stress.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Respuesta al Choque Térmico , Histonas/metabolismo , Animales , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Células Germinativas/citología , Células Germinativas/metabolismo , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Histonas/genética , Infertilidad/genética , Nucleosomas/metabolismoRESUMEN
The Target of Rapamycin Complex is a central controller of cell growth and differentiation in eukaryotes. Its global architecture has been described by cryoelectron microscopy, and regions of its central TOR protein have been described by X-ray crystallography. However, the N-terminal region of this protein, which consists of a series of HEAT repeats, remains uncharacterised at high resolution, most likely due to the absence of a suitable purification procedure. Here, we present a robust method for the preparation of the HEAT-repeat domain, utilizing the thermophilic fungus Chaetomium thermophilum as a source organism. We describe construct design and stable expression in insect cells. An efficient two-step purification procedure is presented, and the purified product is characterised by SEC and MALDI-TOF MS. The methods described pave the way for a complete high-resolution characterisation of this elusive region of the TOR protein.
Asunto(s)
Chaetomium , Clonación Molecular , Proteínas Fúngicas , Expresión Génica , Chaetomium/enzimología , Chaetomium/genética , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/aislamiento & purificación , Dominios Proteicos , Proteínas Recombinantes , Secuencias Repetitivas de Aminoácido , Serina-Treonina Quinasas TOR/biosíntesis , Serina-Treonina Quinasas TOR/química , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/aislamiento & purificaciónRESUMEN
The Split Ends (SPEN) protein was originally discovered in Drosophila in the late 1990s. Since then, homologous proteins have been identified in eukaryotic species ranging from plants to humans. Every family member contains three predicted RNA recognition motifs (RRMs) in the N-terminal region of the protein. We have determined the crystal structure of the region of the human SPEN homolog that contains these RRMs-the SMRT/HDAC1 Associated Repressor Protein (SHARP), at 2.0 Å resolution. SHARP is a co-regulator of the nuclear receptors. We demonstrate that two of the three RRMs, namely RRM3 and RRM4, interact via a highly conserved interface. Furthermore, we show that the RRM3-RRM4 block is the main platform mediating the stable association with the H12-H13 substructure found in the steroid receptor RNA activator (SRA), a long, non-coding RNA previously shown to play a crucial role in nuclear receptor transcriptional regulation. We determine that SHARP association with SRA relies on both single- and double-stranded RNA sequences. The crystal structure of the SHARP-RRM fragment, together with the associated RNA-binding studies, extend the repertoire of nucleic acid binding properties of RRM domains suggesting a new hypothesis for a better understanding of SPEN protein functions.
Asunto(s)
Proteínas de Homeodominio/química , Proteínas Nucleares/química , Proteínas de Unión al ARN/química , ARN/química , Secuencias de Aminoácidos , Cristalografía por Rayos X , Proteínas de Unión al ADN , Proteínas de Homeodominio/metabolismo , Humanos , Modelos Moleculares , Proteínas Nucleares/metabolismo , Conformación de Ácido Nucleico , Unión Proteica , Proteínas de Unión al ARN/metabolismoRESUMEN
The addition of uridine nucleotide by the poly(U) polymerase (PUP) enzymes has a demonstrated impact on various classes of RNAs such as microRNAs (miRNAs), histone-encoding RNAs and messenger RNAs. Cid1 protein is a member of the PUP family. We solved the crystal structure of Cid1 in complex with non-hydrolyzable UMPNPP and a short dinucleotide compound ApU. These structures revealed new residues involved in substrate/product stabilization. In particular, one of the three catalytic aspartate residues explains the RNA dependence of its PUP activity. Moreover, other residues such as residue N165 or the ß-trapdoor are shown to be critical for Cid1 activity. We finally suggest that the length and sequence of Cid1 substrate RNA influence the balance between Cid1's processive and distributive activities. We propose that particular processes regulated by PUPs require the enzymes to switch between the two types of activity as shown for the miRNA biogenesis where PUPs can either promote DICER cleavage via short U-tail or trigger miRNA degradation by adding longer poly(U) tail. The enzymatic properties of these enzymes may be critical for determining their particular function in vivo.
Asunto(s)
Nucleotidiltransferasas/química , Proteínas de Schizosaccharomyces pombe/química , Cristalografía por Rayos X , Modelos Moleculares , Nucleótidos/química , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Polimerizacion , Unión Proteica , ARN/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
L1 retrotransposons have a prominent role in reshaping mammalian genomes. To replicate, the L1 ribonucleoprotein particle (RNP) first uses its endonuclease (EN) to nick the genomic DNA. The newly generated DNA end is subsequently used as a primer to initiate reverse transcription within the L1 RNA poly(A) tail, a process known as target-primed reverse transcription (TPRT). Prior studies demonstrated that most L1 insertions occur into sequences related to the L1 EN consensus sequence (degenerate 5'-TTTT/A-3' sites) and frequently preceded by imperfect T-tracts. However, it is currently unclear whether--and to which degree--the liberated 3'-hydroxyl extremity on the genomic DNA needs to be accessible and complementary to the poly(A) tail of the L1 RNA for efficient priming of reverse transcription. Here, we employed a direct assay for the initiation of L1 reverse transcription to define the molecular rules that guide this process. First, efficient priming is detected with as few as 4 matching nucleotides at the primer 3' end. Second, L1 RNP can tolerate terminal mismatches if they are compensated within the 10 last bases of the primer by an increased number of matching nucleotides. All terminal mismatches are not equally detrimental to DNA extension, a C being extended at higher levels than an A or a G. Third, efficient priming in the context of duplex DNA requires a 3' overhang. This suggests the possible existence of additional DNA processing steps, which generate a single-stranded 3' end to allow L1 reverse transcription. Based on these data we propose that the specificity of L1 reverse transcription initiation contributes, together with the specificity of the initial EN cleavage, to the distribution of new L1 insertions within the human genome.
Asunto(s)
Genoma Humano , Elementos de Nucleótido Esparcido Largo/genética , Retroelementos/genética , Transcripción Reversa/genética , Ribonucleoproteínas Nucleares Pequeñas/genética , Animales , Secuencia de Bases , Cartilla de ADN/genética , Endonucleasas/genética , Humanos , Docilidad , Poli T/genética , Sensibilidad y EspecificidadRESUMEN
The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells that involve most natural HIV-1 target cells. Vif counteracts the packaging of two cellular cytidine deaminases named APOBEC3G (A3G) and A3F by diverse mechanisms including the recruitment of an E3 ubiquitin ligase complex and the proteasomal degradation of A3G/A3F, the inhibition of A3G mRNA translation or by a direct competition mechanism. In addition, Vif appears to be an active partner of the late steps of viral replication by participating in virus assembly and Gag processing, thus regulating the final stage of virion formation notably genomic RNA dimerization and by inhibiting the initiation of reverse transcription. Vif is a small pleiotropic protein with multiple domains, and recent studies highlighted the importance of Vif conformation and flexibility in counteracting A3G and in binding RNA. In this review, we will focus on the oligomerization and RNA chaperone properties of Vif and show that the intrinsic disordered nature of some Vif domains could play an important role in virus assembly and replication. Experimental evidence demonstrating the RNA chaperone activity of Vif will be presented.
Asunto(s)
VIH-1/fisiología , Chaperonas Moleculares/metabolismo , Multimerización de Proteína , ARN de Transferencia de Lisina/metabolismo , ARN Viral/metabolismo , Ensamble de Virus , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
In eukaryotes, mRNA degradation begins with poly(A) tail removal, followed by decapping, and the mRNA body is degraded by exonucleases. In recent years, the major influence of 3'-end uridylation as a regulatory step within several RNA degradation pathways has generated significant attention toward the responsible enzymes, which are called poly(U) polymerases (PUPs). We determined the atomic structure of the Cid1 protein, the founding member of the PUP family, in its UTP-bound form, allowing unambiguous positioning of the UTP molecule. Our data also suggest that the RNA substrate accommodation and product translocation by the Cid1 protein rely on local and global movements of the enzyme. Supplemented by point mutations, the atomic model is used to propose a catalytic cycle. Our study underlines the Cid1 RNA binding properties, a feature with critical implications for miRNAs, histone mRNAs, and, more generally, cellular RNA degradation.
Asunto(s)
Nucleotidiltransferasas/química , Proteínas de Schizosaccharomyces pombe/química , Schizosaccharomyces/enzimología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Dominio Catalítico , Secuencia Conservada , Cristalografía por Rayos X , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , ARN de Hongos/química , Especificidad por Sustrato , Propiedades de Superficie , Uridina Trifosfato/químicaRESUMEN
Shifts between epigenetic states of transcriptional activity are typically correlated with changes in epigenetic marks. However, exceptions to this rule suggest the existence of additional, as yet uncharacterized, layers of epigenetic regulation. MOM1, a protein of 2,001 amino acids that acts as a transcriptional silencer, represents such an exception. Here we define the 82 amino acid domain called CMM2 (Conserved MOM1 Motif 2) as a minimal MOM1 fragment capable of transcriptional regulation. As determined by X-ray crystallography, this motif folds into an unusual hendecad-based coiled-coil. Structure-based mutagenesis followed by transgenic complementation tests in plants demonstrate that CMM2 and its dimerization are effective for transcriptional suppression at chromosomal loci co-regulated by MOM1 and the siRNA pathway but not at loci controlled by MOM1 in an siRNA-independent fashion. These results reveal a surprising separation of epigenetic activities that enable the single, large MOM1 protein to coordinate cooperating mechanisms of epigenetic regulation.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Arabidopsis/química , Arabidopsis/genética , Epigénesis Genética/genética , Regulación de la Expresión Génica de las Plantas , Proteínas Nucleares/química , Proteínas Nucleares/genética , Factores de Transcripción/química , Factores de Transcripción/genética , Transcripción Genética , ATPasas Asociadas con Actividades Celulares Diversas , Secuencia de Aminoácidos , Cristalografía por Rayos X , Silenciador del Gen , Datos de Secuencia Molecular , Mutagénesis , Plantas Modificadas Genéticamente , Conformación Proteica , Pliegue de Proteína , Multimerización de Proteína , Estructura Terciaria de Proteína/genética , ARN Interferente Pequeño/genética , Relación Estructura-ActividadRESUMEN
Prion diseases are unique neurodegenerative illnesses associated with the conversion of the cellular prion protein (PrP(C)) into the aggregated misfolded scrapie isoform, named PrP(Sc). Recent studies on the physiological role of PrP(C) revealed that this protein has probably multiple functions, notably in cell-cell adhesion and signal transduction, and in assisting nucleic acid folding. In fact, in vitro findings indicated that the human PrP (huPrP) possesses nucleic acid binding and annealing activities, similarly to nucleic acid chaperone proteins that play essential roles in cellular DNA and RNA metabolism. Here, we show that a peptide, representing the N-terminal domain of huPrP, facilitates nucleic acid annealing by two parallel pathways nucleated through the stem termini. We also show that PrP of human or ovine origin facilitates DNA strand exchange, ribozyme-directed cleavage of an RNA template and RNA trans-splicing in a manner similar to the nucleocapsid protein of HIV-1. In an attempt to characterize inhibitors of PrP-chaperoning in vitro we discovered that the thioaptamer 5'-GACACAAGCCGA-3' was extensively inhibiting the PrP chaperoning activities. At the same time a recently characterized methylated oligoribonucleotide inhibiting the chaperoning activity of the HIV-1 nucleocapsid protein was poorly impairing the PrP chaperoning activities.
Asunto(s)
Aptámeros de Nucleótidos/farmacología , ADN/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas PrPC/metabolismo , ARN/metabolismo , Animales , ADN Viral/química , ADN Viral/metabolismo , Humanos , Cinética , Chaperonas Moleculares/antagonistas & inhibidores , Chaperonas Moleculares/química , Péptidos/metabolismo , Proteínas PrPC/antagonistas & inhibidores , Proteínas PrPC/química , ARN Catalítico/metabolismo , Ovinos , Trans-EmpalmeRESUMEN
Upon HIV-1 infection of a target cell, the viral reverse transcriptase (RT) copies the genomic RNA to synthesize the viral DNA. The genomic RNA is within the incoming HIV-1 core where it is coated by molecules of nucleocapsid (NC) protein that chaperones the reverse transcription process. Indeed, the RT chaperoning properties of NC extend from the initiation of cDNA synthesis to completion of the viral DNA. New and effective drugs against HIV-1 continue to be required, which prompted us to search for compounds aimed at inhibiting NC protein. Here, we report that the NC chaperoning activity is extensively inhibited in vitro by small methylated oligoribonucleotides (mODN). These mODNs were delivered intracellularly using a cell-penetrating-peptide and found to impede HIV-1 replication in primary human cells at nanomolar concentrations. Extensive analysis showed that viral cDNA synthesis was severely impaired by mODNs. Partially resistant viruses with mutations in NC and RT emerged after months of passaging in cell culture. A HIV-1 molecular clone (NL4.3) bearing these mutations was found to replicate at high concentrations of mODN, albeit with a reduced fitness. Small, methylated ODNs such as mODN-11 appear to be a new type of highly potent inhibitor of HIV-1.
Asunto(s)
Fármacos Anti-VIH/química , Transcriptasa Inversa del VIH/antagonistas & inhibidores , VIH-1/genética , Oligorribonucleótidos/química , Transcripción Reversa , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Células Cultivadas , ADN Complementario/biosíntesis , Transcriptasa Inversa del VIH/genética , VIH-1/fisiología , Humanos , Metilación , Mutación , Replicación Viral , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Of the known epigenetic control regulators found in plants, the Morpheus' molecule 1 (MOM1) protein is atypical in that the deletion of MOM1 does not affect the level of epigenetic marks controlling the transcriptional status of the genome. A short 197-amino-acid fragment of the MOM1 protein sequence can complement MOM1 deletion when coupled to a nuclear localization signal, suggesting that this region contains a functional domain that compensates for the loss of the full-length protein. Numerous constructs centred on the highly conserved MOM1 motif 2 (CMM2) present in these 197 residues have been generated and expressed in Escherichia coli. Following purification and crystallization screening, diamond-shaped single crystals were obtained that diffracted to approximately 3.2 A resolution. They belonged to the trigonal space group P3(1)21 (or P3(2)21), with unit-cell parameters a=85.64, c=292.74 A. Structure determination is ongoing.
Asunto(s)
Proteínas de Arabidopsis/química , Arabidopsis/química , Proteínas Nucleares/química , Factores de Transcripción/química , ATPasas Asociadas con Actividades Celulares Diversas , Secuencias de Aminoácidos , Proteínas de Arabidopsis/genética , Cristalización , Cristalografía por Rayos X , Expresión Génica , Proteínas Nucleares/genética , Factores de Transcripción/genéticaRESUMEN
The human immunodeficiency virus type 1 (HIV-1) is a primate lentivirus that causes the acquired immunodeficiency syndrome (AIDS). In addition to the virion structural proteins and enzyme precursors, that are Gag, Env and Pol, HIV-1 encodes several regulatory proteins, notably a small nuclear transcriptional activator named Tat. The Tat protein is absolutely required for virus replication since it controls proviral DNA transcription to generate the full-length viral mRNA. Tat can also regulate mRNA capping and splicing and was recently found to interfere with the cellular mi- and siRNA machinery. Because of its extensive interplay with nucleic acids, and its basic and disordered nature we speculated that Tat had nucleic acid-chaperoning properties. This prompted us to examine in vitro the nucleic acid-chaperoning activities of Tat and Tat peptides made by chemical synthesis. Here we report that Tat has potent nucleic acid-chaperoning activities according to the standard DNA annealing, DNA and RNA strand exchange, RNA ribozyme cleavage and trans-splicing assays. The active Tat(44-61) peptide identified here corresponds to the smallest known sequence with DNA/RNA chaperoning properties.
Asunto(s)
ADN Viral/metabolismo , VIH-1/genética , Chaperonas Moleculares/metabolismo , ARN/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Secuencia de Aminoácidos , Duplicado del Terminal Largo de VIH , Chaperonas Moleculares/química , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Péptidos/química , Péptidos/metabolismo , ARN Catalítico , Trans-Empalme , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/químicaRESUMEN
RNA chaperone proteins are essential partners of RNA in living organisms and viruses. They are thought to assist in the correct folding and structural rearrangements of RNA molecules by resolving misfolded RNA species in an ATP-independent manner. RNA chaperoning is probably an entropy-driven process, mediated by the coupled binding and folding of intrinsically disordered protein regions and the kinetically trapped RNA. Previously, we have shown that the core protein of hepatitis C virus (HCV) is a potent RNA chaperone that can drive profound structural modifications of HCV RNA in vitro. We now examined the RNA chaperone activity and the disordered nature of core proteins from different Flaviviridae genera, namely that of HCV, GBV-B (GB virus B), WNV (West Nile virus) and BVDV (bovine viral diarrhoea virus). Despite low-sequence similarities, all four proteins demonstrated general nucleic acid annealing and RNA chaperone activities. Furthermore, heat resistance of core proteins, as well as far-UV circular dichroism spectroscopy suggested that a well-defined 3D protein structure is not necessary for core-induced RNA structural rearrangements. These data provide evidence that RNA chaperoning-possibly mediated by intrinsically disordered protein segments-is conserved in Flaviviridae core proteins. Thus, besides nucleocapsid formation, core proteins may function in RNA structural rearrangements taking place during virus replication.
Asunto(s)
Flaviviridae , Chaperonas Moleculares/química , ARN/química , Proteínas del Núcleo Viral/química , Dicroismo Circular , ADN/química , Chaperonas Moleculares/metabolismo , Desnaturalización Proteica , Estructura Secundaria de Proteína , ARN Catalítico/metabolismo , Proteínas de Unión al ARN/química , Proteínas del Núcleo Viral/metabolismoRESUMEN
Mobile LTR-retroelements comprising retroviruses and LTR-retrotransposons form a large part of eukaryotic genomes. Their mode of replication and abundance favour the notion that they are major actors in eukaryote evolution. The Gypsy retroelement can spread in the germ line of the fruit fly Drosophila melanogaster via both env-independent and env-dependent processes. Thus, Gypsy is both an active retrotransposon and an infectious retrovirus resembling the gammaretrovirus MuLV. However, unlike gammaretroviruses, the Gypsy Gag structural precursor is not processed into Matrix, Capsid and Nucleocapsid (NC) proteins. In contrast, it has features in common with Gag of the ancient yeast TY1 retroelement. These characteristics of Gypsy make it a very interesting model to study replication of a retroelement at the frontier between ancient retrotransposons and retroviruses. We investigated Gypsy replication using an in vitro model system and transfection of insect cells. Results show that an unstructured domain of Gypsy Gag has all the properties of a retroviral NC. This NC-like peptide forms ribonucleoparticle-like complexes upon binding Gypsy RNA and directs the annealing of primer tRNA(Lys,2) to two distinct primer binding sites (PBS) at the genome 5' and 3' ends. Only the 5' PBS is indispensable for cDNA synthesis in vitro and in Drosophila cells.
Asunto(s)
Retrovirus Endógenos/genética , Productos del Gen gag/química , Proteínas de la Nucleocápside/química , ARN de Transferencia de Lisina/química , ARN Viral/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , ADN Complementario/biosíntesis , Drosophila/citología , Datos de Secuencia Molecular , Proteínas de la Nucleocápside/metabolismo , Sistemas de Lectura Abierta , Péptidos/química , ARN/química , ARN de Transferencia de Lisina/metabolismo , ARN Viral/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismoRESUMEN
The core protein of hepatitis C virus (HCV) has been shown previously to act as a potent nucleic acid chaperone in vitro, promoting the dimerization of the 3'-untranslated region (3'-UTR) of the HCV genomic RNA, a process probably mediated by a small, highly conserved palindromic RNA motif, named DLS (dimer linkage sequence) [G. Cristofari, R. Ivanyi-Nagy, C. Gabus, S. Boulant, J. P. Lavergne, F. Penin and J. L. Darlix (2004) Nucleic Acids Res., 32, 2623-2631]. To investigate in depth HCV RNA dimerization, we generated a series of point mutations in the DLS region. We find that both the plus-strand 3'-UTR and the complementary minus-strand RNA can dimerize in the presence of core protein, while mutations in the DLS (among them a single point mutation that abolished RNA replication in a HCV subgenomic replicon system) completely abrogate dimerization. Structural probing of plus- and minus-strand RNAs, in their monomeric and dimeric forms, indicate that the DLS is the major if not the sole determinant of UTR RNA dimerization. Furthermore, the N-terminal basic amino acid clusters of core protein were found to be sufficient to induce dimerization, suggesting that they retain full RNA chaperone activity. These findings may have important consequences for understanding the HCV replicative cycle and the genetic variability of the virus.