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1.
J Am Chem Soc ; 143(39): 15998-16006, 2021 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-34559526

RESUMO

The extant complex proteins must have evolved from ancient short and simple ancestors. The double-ψ ß-barrel (DPBB) is one of the oldest protein folds and conserved in various fundamental enzymes, such as the core domain of RNA polymerase. Here, by reverse engineering a modern DPBB domain, we reconstructed its plausible evolutionary pathway started by "interlacing homodimerization" of a half-size peptide, followed by gene duplication and fusion. Furthermore, by simplifying the amino acid repertoire of the peptide, we successfully created the DPBB fold with only seven amino acid types (Ala, Asp, Glu, Gly, Lys, Arg, and Val), which can be coded by only GNN and ARR (R = A or G) codons in the modern translation system. Thus, the DPBB fold could have been materialized by the early translation system and genetic code.

2.
Sci Adv ; 7(30)2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34290093

RESUMO

The dedicator of cytokinesis (DOCK) family of guanine nucleotide exchange factors (GEFs) promotes cell motility, phagocytosis, and cancer metastasis through activation of Rho guanosine triphosphatases. Engulfment and cell motility (ELMO) proteins are binding partners of DOCK and regulate Rac activation. Here, we report the cryo-electron microscopy structure of the active ELMO1-DOCK5 complex bound to Rac1 at 3.8-Å resolution. The C-terminal region of ELMO1, including the pleckstrin homology (PH) domain, aids in the binding of the catalytic DOCK homology region 2 (DHR-2) domain of DOCK5 to Rac1 in its nucleotide-free state. A complex α-helical scaffold between ELMO1 and DOCK5 stabilizes the binding of Rac1. Mutagenesis studies revealed that the PH domain of ELMO1 enhances the GEF activity of DOCK5 through specific interactions with Rac1. The structure provides insights into how ELMO modulates the biochemical activity of DOCK and how Rac selectivity is achieved by ELMO.

3.
Plants (Basel) ; 10(3)2021 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-33802194

RESUMO

Here, we explored heat dependent thylakoid FtsH protease substrates and investigated proteotoxicity induced by thermal damage and processive protease dysfunction on the thylakoid membrane. Through our thylakoid enriched proteome analysis and biochemical experiments, carbonylated stromal proteins were suggested as possible FtsH targets. Furthermore, we observed in the thylakoid fractions in the absence of FtsH stromal reactive oxygen species-detoxifying enzymes, as well as heat shock proteins and chaperones, which are known to be upregulated at the transcriptional level when this protease is absent, which is called the damaged protein response, resembling unfolded protein response in eukaryotic cells. Interestingly, the thylakoid-enriched high-density fractions included stromal translation factors and RNA-binding proteins, along with aminoacyl-tRNA synthetase, reminiscent of the formation of stress granules. Unexpectedly, extraplastid proteins such as mitochondrial chaperones, peroxidase, tricarboxylic acid cycle and respiratory chain enzymes, as well as cytosolic ribosomes, translation factors, heat shock proteins, antioxidants and metabolic enzymes, were also found deposited in the high-density fractions depending on the loss of thylakoid FtsH, with more prominent effects of thermal stress on the cytosolic proteins. This may reflect intracellular adaptation to the proteotoxic influences from the organelle.

4.
Nat Commun ; 12(1): 146, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33420015

RESUMO

The microtubule-binding outer kinetochore is coupled to centromeric chromatin through CENP-CMif2, CENP-TCnn1, and CENP-UAme1 linker pathways originating from the constitutive centromere associated network (CCAN) of the inner kinetochore. Here, we demonstrate the recurrent loss of most CCAN components, including certain kinetochore linkers during the evolution of the fungal phylum of Basidiomycota. By kinetochore interactome analyses in a model basidiomycete and human pathogen Cryptococcus neoformans, a forkhead-associated domain containing protein "bridgin" was identified as a kinetochore component along with other predicted kinetochore proteins. In vivo and in vitro functional analyses of bridgin reveal its ability to connect the outer kinetochore with centromeric chromatin to ensure accurate chromosome segregation. Unlike established CCAN-based linkers, bridgin is recruited at the outer kinetochore establishing its role as a distinct family of kinetochore proteins. Presence of bridgin homologs in non-fungal lineages suggests an ancient divergent strategy exists to bridge the outer kinetochore with centromeric chromatin.


Assuntos
Cromatina/metabolismo , Segregação de Cromossomos , Cryptococcus neoformans/fisiologia , Proteínas Fúngicas/metabolismo , Cinetocoros/metabolismo , Imunoprecipitação da Cromatina , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Microscopia Intravital , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
5.
EMBO J ; 40(5): e105671, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33463726

RESUMO

The CENP-A nucleosome is a key structure for kinetochore assembly. Once the CENP-A nucleosome is established in the centromere, additional proteins recognize the CENP-A nucleosome to form a kinetochore. CENP-C and CENP-N are CENP-A binding proteins. We previously demonstrated that vertebrate CENP-C binding to the CENP-A nucleosome is regulated by CDK1-mediated CENP-C phosphorylation. However, it is still unknown how the phosphorylation of CENP-C regulates its binding to CENP-A. It is also not completely understood how and whether CENP-C and CENP-N act together on the CENP-A nucleosome. Here, using cryo-electron microscopy (cryo-EM) in combination with biochemical approaches, we reveal a stable CENP-A nucleosome-binding mode of CENP-C through unique regions. The chicken CENP-C structure bound to the CENP-A nucleosome is stabilized by an intramolecular link through the phosphorylated CENP-C residue. The stable CENP-A-CENP-C complex excludes CENP-N from the CENP-A nucleosome. These findings provide mechanistic insights into the dynamic kinetochore assembly regulated by CDK1-mediated CENP-C phosphorylation.


Assuntos
Proteína Centromérica A/metabolismo , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Microscopia Crioeletrônica/métodos , Cinetocoros/metabolismo , Nucleossomos/metabolismo , Animais , Centrômero/ultraestrutura , Proteína Centromérica A/ultraestrutura , Galinhas , Proteínas Cromossômicas não Histona/ultraestrutura , Cinetocoros/ultraestrutura , Modelos Moleculares , Nucleossomos/ultraestrutura , Fosforilação , Conformação Proteica
6.
Nat Commun ; 12(1): 628, 2021 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-33504795

RESUMO

Consolidated memory can be preserved or updated depending on the environmental change. Although such conflicting regulation may happen during memory updating, the flexibility of memory updating may have already been determined in the initial memory consolidation process. Here, we explored the gating mechanism for activity-dependent transcription in memory consolidation, which is unexpectedly linked to the later memory updating in Drosophila. Through proteomic analysis, we discovered that the compositional change in the transcriptional repressor, which contains the histone deacetylase Rpd3 and CoRest, acts as the gating mechanism that opens and closes the time window for activity-dependent transcription. Opening the gate through the compositional change in Rpd3/CoRest is required for memory consolidation, but closing the gate through Rpd3/CoRest is significant to limit future memory updating. Our data indicate that the flexibility of memory updating is determined through the initial activity-dependent transcription, providing a mechanism involved in defining memory state.


Assuntos
Proteínas Correpressoras/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Histona Desacetilase 1/metabolismo , Memória/fisiologia , Transcrição Genética , Acetilação , Animais , Comportamento Animal , Encéfalo/fisiologia , Loci Gênicos , Corpos Pedunculados/inervação , Ligação Proteica , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
7.
Elife ; 92020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32869745

RESUMO

The Polycomb repressive complex 2 (PRC2) is a multicomponent histone H3K27 methyltransferase complex, best known for silencing the Hox genes during embryonic development. The Polycomb-like proteins PHF1, MTF2, and PHF19 are critical components of PRC2 by stimulating its catalytic activity in embryonic stem cells. The Tudor domains of PHF1/19 have been previously shown to be readers of H3K36me3 in vitro. However, some other studies suggest that PHF1 and PHF19 co-localize with the H3K27me3 mark but not H3K36me3 in cells. Here, we provide further evidence that PHF1 co-localizes with H3t in testis and its Tudor domain preferentially binds to H3tK27me3 over canonical H3K27me3 in vitro. Our complex structures of the Tudor domains of PHF1 and PHF19 with H3tK27me3 shed light on the molecular basis for preferential recognition of H3tK27me3 by PHF1 and PHF19 over canonical H3K27me3, implicating that H3tK27me3 might be a physiological ligand of PHF1/19.


Assuntos
Proteínas de Ligação a DNA/química , Histonas/química , Proteínas do Grupo Polycomb/química , Fatores de Transcrição/química , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Histonas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Complexo Repressor Polycomb 2/química , Complexo Repressor Polycomb 2/metabolismo , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Ligação Proteica , Testículo/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Domínio Tudor
8.
Org Lett ; 22(14): 5434-5438, 2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32615768

RESUMO

The irradiation of red light-emitting-diode light (λ = 660 nm) to 3-acyl-2-methoxyindolizines in the presence of a catalytic amount of methylene blue triggered the photooxidation of the indolizine ring, resulting in a nearly quantitative release of alcohols or carboxylic acids within a few minutes. The method was applicable for photouncaging various functional molecules such as a carboxylic anticancer drug and a phenolic fluorescent dye from the corresponding indolizine conjugates, including an insulin-indolizine-dye conjugate.

9.
Front Microbiol ; 10: 2189, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31611853

RESUMO

Human norovirus (HuNoV) GII.P17-GII.17 (Kawasaki2014 variant) reportedly emerged in 2014 and caused gastroenteritis outbreaks worldwide. To clarify the evolution of both VP1 and RNA-dependent RNA polymerase (RdRp) regions of GII.P17-GII.17, we analyzed both global and novel Japanese strains detected during 2013-2017. Time-scaled phylogenetic trees revealed that the ancestral GII.17 VP1 region diverged around 1949, while the ancestral GII.P17 RdRp region diverged around 2010. The evolutionary rates of the VP1 and RdRp regions were estimated at ~2.7 × 10-3 and ~2.3 × 10-3 substitutions/site/year, respectively. The phylogenetic distances of the VP1 region exhibited no overlaps between intra-cluster and inter-cluster peaks in the GII.17 strains, whereas those of the RdRp region exhibited a unimodal distribution in the GII.P17 strains. Conformational epitope positions in the VP1 protein of the GII.P17-GII.17 strains were similar, although some substitutions, insertions and deletions had occurred. Strains belonging to the same cluster also harbored substitutions around the binding sites for the histo-blood group antigens of the VP1 protein. Moreover, some amino acid substitutions were estimated to be near the interface between monomers and the active site of the RdRp protein. These results suggest that the GII.P17-GII.17 virus has produced variants with the potential to alter viral antigenicity, host-binding capability, and replication property over the past 10 years.

10.
EMBO Rep ; 20(10): e48111, 2019 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-31468675

RESUMO

The methylation of histone H3 at lysine 9 (H3K9me), performed by the methyltransferase Clr4/SUV39H, is a key event in heterochromatin assembly. In fission yeast, Clr4, together with the ubiquitin E3 ligase Cul4, forms the Clr4 methyltransferase complex (CLRC), whose physiological targets and biological role are currently unclear. Here, we show that CLRC-dependent H3 ubiquitylation regulates Clr4's methyltransferase activity. Affinity-purified CLRC ubiquitylates histone H3, and mass spectrometric and mutation analyses reveal that H3 lysine 14 (H3K14) is the preferred target of the complex. Chromatin immunoprecipitation analysis shows that H3K14 ubiquitylation (H3K14ub) is closely associated with H3K9me-enriched chromatin. Notably, the CLRC-mediated H3 ubiquitylation promotes H3K9me by Clr4, suggesting that H3 ubiquitylation is intimately linked to the establishment and/or maintenance of H3K9me. These findings demonstrate a cross-talk mechanism between histone ubiquitylation and methylation that is involved in heterochromatin assembly.


Assuntos
Heterocromatina/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Schizosaccharomyces/metabolismo , Ubiquitinação , Sequência de Aminoácidos , Histonas/química , Metilação , Metiltransferases/metabolismo , Mutação/genética , Proteínas de Schizosaccharomyces pombe/metabolismo
11.
PLoS Pathog ; 15(5): e1007749, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31121004

RESUMO

The regulation of paramyxovirus RNA synthesis by host proteins is poorly understood. Here, we identified a novel regulation mechanism of paramyxovirus RNA synthesis by the Hsp90 co-chaperone R2TP complex. We showed that the R2TP complex interacted with the paramyxovirus polymerase L protein and that silencing of the R2TP complex led to uncontrolled upregulation of mumps virus (MuV) gene transcription but not genome replication. Regulation by the R2TP complex was critical for MuV replication and evasion of host innate immune responses. The R2TP complex also regulated measles virus (MeV) RNA synthesis, but its function was inhibitory and not beneficial to MeV, as MeV evaded host innate immune responses in the absence of the R2TP complex. The identification of the R2TP complex as a critical host factor sheds new light on the regulation of paramyxovirus RNA synthesis.


Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Interações Hospedeiro-Patógeno , Vírus da Caxumba/genética , Caxumba/genética , RNA Viral/biossíntese , Proteínas Virais/metabolismo , Replicação Viral , Células A549 , Proteínas de Choque Térmico HSP90/genética , Humanos , Caxumba/virologia , Proteínas Virais/genética
12.
Vaccine ; 37(14): 1964-1971, 2019 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-30827736

RESUMO

In Japan, the oral poliovirus vaccine (OPV) was changed to 2 types of inactivated poliovirus vaccine (IPV), the standalone conventional IPV (cIPV) and the Sabin-derived IPV combined with diphtheria-tetanus-acellular pertussis vaccine (DTaP-sIPV), for routine immunization in 2012. We evaluated polio vaccination coverage and the seroprevalence of poliovirus antibodies using data from the National Epidemiological Surveillance of Vaccine-Preventable Diseases (NESVPD) from 2011 to 2015. Several years before the introduction of IPV in 2012, OPV administration for children was refused by some parents because of concerns about the risk of vaccine-associated paralytic poliomyelitis. Consequently, in children aged <1 years who were surveyed in 2011-2012, polio vaccination coverage (45.0-48.8%) and seropositivity rates for poliovirus (type 1: 51.7-65.9%, type 2: 48.3-53.7%, and type 3: 15.0-29.3%) were decreased compared to those surveyed in 2009. However, after IPV introduction, the vaccination coverage (95.5-100%) and seropositivity rates (type 1: 93.2-96.6%, type 2: 93.1-100%, and type 3: 88.6-93.9%) increased among children aged <1 years in 2013-2015. In particular, seropositivity rates and geometric mean titers (GMTs) for poliovirus type 3 in <5-year-old children who received 4 doses of IPV (98.5% and 247.4, respectively) were significantly higher than in those who received 2 doses of OPV (72.5% and 22.9, respectively). Furthermore, in <5-year-old children who received 4 doses of either DTaP-sIPV or cIPV, the seropositivity rates and the GMTs for all 3 types of poliovirus were similarly high (96.5-100% and 170.3-368.8, respectively). Our findings from the NESVPD demonstrate that both the vaccination coverage and seropositivity rates for polio remained high in children after IPV introduction.


Assuntos
Poliomielite/epidemiologia , Poliomielite/prevenção & controle , Vacina Antipólio de Vírus Inativado/imunologia , Poliovirus/imunologia , Vacinação , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Humanos , Japão/epidemiologia , Estudos Soroepidemiológicos , Cobertura Vacinal
13.
J Biochem ; 165(5): 433-446, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30590679

RESUMO

Heterochromatin protein 1 (HP1) is an evolutionarily conserved chromosomal protein that plays a crucial role in heterochromatin-mediated gene silencing. We previously showed that mammalian HP1α is constitutively phosphorylated at its N-terminal serine residues by casein kinase II (CK2), and that this phosphorylation enhances HP1α's binding specificity for nucleosomes containing lysine 9-methylated histone H3 (H3K9me). Although the presence of additional HP1α phosphorylation during mitosis was reported more than a decade ago, its biological significance remains largely elusive. Here we found that mitosis-specific HP1α phosphorylation affected HP1α's ability to bind chromatin. Using biochemical and mutational analyses, we showed that HP1α's mitotic phosphorylation was located in its hinge region and was reversibly regulated by Aurora B kinase and serine/threonine phosphatases. In addition, chromatin fractionation and electrophoretic mobility shift assays revealed that hinge region-phosphorylated HP1α was preferentially dissociated from mitotic chromatin and exhibited a reduced DNA-binding activity. Although HP1's mitotic behaviour was previously linked to H3 serine 10 phosphorylation, which blocks the binding of HP1's chromodomain to H3K9me3, our findings suggest that mitotic phosphorylation in HP1α's hinge region also contributes to changes in HP1α's association with mitotic chromatin.


Assuntos
Ciclo Celular , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Mitose , Aurora Quinase B/metabolismo , DNA/metabolismo , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Fosforilação , Ligação Proteica , Proteína Fosfatase 2/metabolismo , Proteína Fosfatase 2C/metabolismo
14.
Nat Ecol Evol ; 2(11): 1761-1771, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30297745

RESUMO

Modern cartilaginous fishes are divided into elasmobranchs (sharks, rays and skates) and chimaeras, and the lack of established whole-genome sequences for the former has prevented our understanding of early vertebrate evolution and the unique phenotypes of elasmobranchs. Here we present de novo whole-genome assemblies of brownbanded bamboo shark and cloudy catshark and an improved assembly of the whale shark genome. These relatively large genomes (3.8-6.7 Gbp) contain sparse distributions of coding genes and regulatory elements and exhibit reduced molecular evolutionary rates. Our thorough genome annotation revealed Hox C genes previously hypothesized to have been lost, as well as distinct gene repertories of opsins and olfactory receptors that would be associated with adaptation to unique underwater niches. We also show the early establishment of the genetic machinery governing mammalian homoeostasis and reproduction at the jawed vertebrate ancestor. This study, supported by genomic, transcriptomic and epigenomic resources, provides a foundation for the comprehensive, molecular exploration of phenotypes unique to sharks and insights into the evolutionary origins of vertebrates.


Assuntos
Evolução Biológica , Genoma , Tubarões/genética , Animais , Elasmobrânquios/genética , Vertebrados/genética
15.
Front Microbiol ; 8: 705, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28487679

RESUMO

The RNA-dependent RNA polymerase (RdRp) and capsid (VP1) genes of 51 GII.2 human norovirus (HuNoV) strains collected during the period of 2004-2015 in Japan were analyzed. Full-length analyses of the genes were performed using next-generation sequencing. Based on the gene sequences, we constructed the time-scale evolutionary trees by Bayesian Markov chain Monte Carlo methods. Time-scale phylogenies showed that the RdRp and VP1 genes evolved uniquely and independently. Four genotypes of GII.2 (major types: GII.P2-GII.2 and GII.P16-GII.2) were detected. A common ancestor of the GII.2 VP1 gene existed until about 1956. The evolutionary rates of the genes were high (over 10-3 substitutions/site/year). Moreover, the VP1 gene evolution may depend on the RdRp gene. Based on these results, we hypothesized that transfer of the RdRp gene accelerated the VP1 gene evolution of HuNoV genotype GII.2. Consequently, recombination between ORF1 (polymerase) and ORF2 (capsid) might promote changes of GII.2 antigenicity.

16.
Sci Rep ; 6: 29400, 2016 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-27384324

RESUMO

Capsid protein of norovirus genogroup II (GII) plays crucial roles in host infection. Although studies on capsid gene evolution have been conducted for a few genotypes of norovirus, the molecular evolution of norovirus GII is not well understood. Here we report the molecular evolution of all GII genotypes, using various bioinformatics techniques. The time-scaled phylogenetic tree showed that the present GII strains diverged from GIV around 1630CE at a high evolutionary rate (around 10(-3) substitutions/site/year), resulting in three lineages. The GII capsid gene had large pairwise distances (maximum > 0.39). The effective population sizes of the present GII strains were large (>10(2)) for about 400 years. Positive (20) and negative (over 450) selection sites were estimated. Moreover, some linear and conformational B-cell epitopes were found in the deduced GII capsid protein. These results suggested that norovirus GII strains rapidly evolved with high divergence and adaptation to humans.


Assuntos
Proteínas do Capsídeo/genética , Evolução Molecular , Genótipo , Norovirus/genética , Proteínas do Capsídeo/classificação , Genes Virais , Filogenia , Probabilidade , Conformação Proteica
17.
Infect Genet Evol ; 41: 160-173, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27071530

RESUMO

Rotaviruses C (RVCs) circulate worldwide as an enteric pathogen in both humans and animals. Most studies of their genetic diversity focus on the VP7 and VP4 genes, but the complete genomes of 18 human RVCs have been described in independent studies. The genetic background of the Far East Asian RVCs is different than other human RVCs that were found in India and Bangladesh. Recently, a RVC detected in 2010 in South Korea had genetic background similar to the Indian-Bangladeshi RVCs. This study was undertaken to determine the whole genome of eight Japanese RVCs detected in 2005-2012, and to compare them with other human and animal global RVCs to better understand the genetic background of contemporary Far East Asian RVC. By phylogenetic analysis, the human RVCs appeared to be distinct from animal RVCs. Among human RVCs, three lineage constellations had prolonged circulation. The genetic background of the Far East Asian RVC was distinguished from Indian-Bangladeshi RVC as reported earlier. However, we found one Japanese RVC in 2012 that carried the genetic background of Indian-Bangladeshi RVC, whereas the remaining seven Japanese RVCs carried the typical genetic background of Far East Asian RVC. This is the first report of the Indian-Bangladeshi RVC in Japan. With that observation and the reassortment event of human RVCs in Hungary, our study indicates that the RVCs are spreading from one region to another.


Assuntos
Genoma Viral , Filogenia , RNA Viral/genética , Infecções por Rotavirus/epidemiologia , Rotavirus/genética , Doenças dos Suínos/epidemiologia , Animais , Proteínas do Capsídeo/genética , Bovinos , Cães , Europa Oriental/epidemiologia , Extremo Oriente/epidemiologia , Biblioteca Gênica , Variação Genética , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Rotavirus/classificação , Rotavirus/isolamento & purificação , Infecções por Rotavirus/transmissão , Infecções por Rotavirus/virologia , Suínos/virologia , Doenças dos Suínos/transmissão , Doenças dos Suínos/virologia , Proteínas não Estruturais Virais/genética
18.
Nucleic Acids Res ; 42(20): 12498-511, 2014 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-25332400

RESUMO

Heterochromatin protein 1 (HP1) is an evolutionarily conserved chromosomal protein that binds to lysine 9-methylated histone H3 (H3K9me), a hallmark of heterochromatin. Although HP1 phosphorylation has been described in several organisms, the biological implications of this modification remain largely elusive. Here we show that HP1's phosphorylation has a critical effect on its nucleosome binding properties. By in vitro phosphorylation assays and conventional chromatography, we demonstrated that casein kinase II (CK2) is the kinase primarily responsible for phosphorylating the N-terminus of human HP1α. Pull-down assays using in vitro-reconstituted nucleosomes showed that unmodified HP1α bound H3K9-methylated and H3K9-unmethylated nucleosomes with comparable affinity, whereas CK2-phosphorylated HP1α showed a high specificity for H3K9me3-modified nucleosomes. Electrophoretic mobility shift assays showed that CK2-mediated phosphorylation diminished HP1α's intrinsic DNA binding, which contributed to its H3K9me-independent nucleosome binding. CK2-mediated phosphorylation had a similar effect on the nucleosome-binding specificity of fly HP1a and S. pombe Swi6. These results suggested that HP1 phosphorylation has an evolutionarily conserved role in HP1's recognition of H3K9me-marked nucleosomes.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Nucleossomos/metabolismo , Caseína Quinase II/metabolismo , Linhagem Celular , Proteínas Cromossômicas não Histona/química , DNA/metabolismo , Histonas/metabolismo , Humanos , Fosforilação , Ligação Proteica , Serina/metabolismo
19.
J Biochem ; 155(3): 159-71, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24307402

RESUMO

Eukaryotic chromosomes are organized into heterochromatin and euchromatin domains. Heterochromatin domains are transcriptionally repressed and prevented from spreading into neighbouring genes by chromatin boundaries. Previously, we identified 55 boundary-related genes in Saccharomyces cerevisiae. In this study, we describe the characterization of one of these boundary genes, named SGF29, which was previously reported as a component of the SAGA, SLIK, ADA and HAT-A2 complex. A domain analysis of Sgf29 identified two minimal regions that can function as individual boundaries. The N-terminal minimal region comprising amino acids 1-12, which has not been defined as a functional domain, showed stronger boundary formation ability than the C-terminal minimal region comprising amino acids 110-255, which contains Tudor domains. Together with Ada2, Ada3 and Sgf29, which are all components of SAGA, Gcn5 acetylates multiple lysine residues on nucleosomal histone H3, which is associated with an open chromatin structure. However, the results presented in this study suggest that the boundary formation ability of the Sgf29 minimal regions is independent of Gcn5. An in vivo analysis also revealed that Sgf29 and Gcn5 perform distinct functions at native telomere boundary regions on the chromosome.


Assuntos
Heterocromatina/metabolismo , Histona Acetiltransferases/química , Histona Acetiltransferases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Genes Fúngicos , Ligação Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Relação Estrutura-Atividade , Telômero/metabolismo
20.
J Biol Chem ; 288(40): 28630-40, 2013 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-23928302

RESUMO

Up-frameshift (Upf) factors eliminate aberrant mRNAs containing a specific premature termination codon (PTC). Here, we show that Upf complex facilitates the ubiquitin-dependent degradation of products derived from mRNA containing specific PTCs in Saccharomyces cerevisiae. The efficiency of recruitment of the Upf complex to a PTC product was correlated with the decay of the PTC product. Upf factors promoted the degradation of the human von Hippel-Lindau (VHL) protein, which is an unfolded protein in yeast cells, in a manner that depends on the presence of a faux 3'-UTR. Mass spectrometric analysis and Western blot analysis revealed that Hsp70 was associated with the PTC product. These findings suggest that the Upf complex may be recruited to ribosomes in a faux 3'-UTR-dependent manner and then associates with aberrant products to facilitate their degradation by the proteasome.


Assuntos
Códon sem Sentido/metabolismo , Complexos Multiproteicos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Regiões 3' não Traduzidas/genética , Células HEK293 , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Modelos Biológicos , Fosfoglicerato Quinase/metabolismo , Ligação Proteica , Desdobramento de Proteína , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ubiquitinação , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo
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