Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 67
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Artigo em Inglês | MEDLINE | ID: mdl-34807800

RESUMO

Orai1 is a plasma membrane Ca2+ channel that mediates store operated Ca2+ entry (SOCE) and regulates inflammation. Short palate lung and nasal epithelial clone 1 (SPLUNC1) is an asthma gene modifier which inhibits Orai1/SOCE via its C-terminal α6 region. SPLUNC1 levels are diminished in asthma patient airways. Thus, we hypothesized that inhaled α6 peptidomimetics could inhibit Orai1 and reduce airway inflammation in a murine asthma model. To evaluate α6-Orai1 interactions, we used fluorescent assays to measure Ca2+ signalling, Förster resonance energy transfer (FRET), fluorescent recovery after photobleaching, immunostaining, total internal reflection (TIRF) microscopy and Western blotting. To test whether α6 peptidomimetics inhibited SOCE and decreased inflammation in vivo, wild-type and SPLUNC1-/- mice were exposed to house dust mite (HDM) extract ± α6 peptide. We also performed nebulization, jet milling and scanning electron microscopy to evaluate α6 for inhalation. SPLUNC1-/- mice had an exaggerated response to HDM. In bronchoalveolar lavage (BAL)-derived immune cells, Orai1 levels increased after HDM exposure in SPLUNC1-/- but not wild-type mice. Inhaled α6 reduced Orai1 levels in mice regardless of genotype. In HDM-exposed mice, α6 dose-dependently reduced eosinophilia and neutrophilia. In vitro, α6 inhibited SOCE in multiple immune cell types and α6 could be nebulized or jet milled without loss of function. These data suggest that α6 peptidomimetics may be a novel, effective anti-inflammatory therapy for asthma patients.

2.
J Biol Chem ; 297(4): 101145, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34473995

RESUMO

Bromodomains (BD) are conserved reader modules that bind acetylated lysine residues on histones. Although much has been learned regarding the in vitro properties of these domains, less is known about their function within chromatin complexes. SWI/SNF chromatin-remodeling complexes modulate transcription and contribute to DNA damage repair. Mutations in SWI/SNF subunits have been implicated in many cancers. Here we demonstrate that the BD of Caenorhabditis elegans SMARCA4/BRG1, a core SWI/SNF subunit, recognizes acetylated lysine 14 of histone H3 (H3K14ac), similar to its Homo sapiens ortholog. We identify the interactions of SMARCA4 with the acetylated histone peptide from a 1.29 Å-resolution crystal structure of the CeSMARCA4 BD-H3K14ac complex. Significantly, most of the SMARCA4 BD residues in contact with the histone peptide are conserved with other proteins containing family VIII bromodomains. Based on the premise that binding specificity is conserved among bromodomain orthologs, we propose that loop residues outside of the binding pocket position contact residues to recognize the H3K14ac sequence. CRISPR-Cas9-mediated mutations in the SMARCA4 BD that abolish H3K14ac binding in vitro had little or no effect on C. elegans viability or physiological function in vivo. However, combining SMARCA4 BD mutations with knockdown of the SWI/SNF accessory subunit PBRM-1 resulted in severe developmental defects in animals. In conclusion, we demonstrated an essential function for the SWI/SNF bromodomain in vivo and detected potential redundancy in epigenetic readers in regulating chromatin remodeling. These findings have implications for the development of small-molecule BD inhibitors to treat cancers and other diseases.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Histonas/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Histonas/genética , Humanos , Ligação Proteica , Fatores de Transcrição/genética
3.
Genome Med ; 13(1): 101, 2021 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-34127050

RESUMO

BACKGROUND: Early in the pandemic, we designed a SARS-CoV-2 peptide vaccine containing epitope regions optimized for concurrent B cell, CD4+ T cell, and CD8+ T cell stimulation. The rationale for this design was to drive both humoral and cellular immunity with high specificity while avoiding undesired effects such as antibody-dependent enhancement (ADE). METHODS: We explored the set of computationally predicted SARS-CoV-2 HLA-I and HLA-II ligands, examining protein source, concurrent human/murine coverage, and population coverage. Beyond MHC affinity, T cell vaccine candidates were further refined by predicted immunogenicity, sequence conservation, source protein abundance, and coverage of high frequency HLA alleles. B cell epitope regions were chosen from linear epitope mapping studies of convalescent patient serum, followed by filtering for surface accessibility, sequence conservation, spatial localization near functional domains of the spike glycoprotein, and avoidance of glycosylation sites. RESULTS: From 58 initial candidates, three B cell epitope regions were identified. From 3730 (MHC-I) and 5045 (MHC-II) candidate ligands, 292 CD8+ and 284 CD4+ T cell epitopes were identified. By combining these B cell and T cell analyses, as well as a manufacturability heuristic, we proposed a set of 22 SARS-CoV-2 vaccine peptides for use in subsequent murine studies. We curated a dataset of ~ 1000 observed T cell epitopes from convalescent COVID-19 patients across eight studies, showing 8/15 recurrent epitope regions to overlap with at least one of our candidate peptides. Of the 22 candidate vaccine peptides, 16 (n = 10 T cell epitope optimized; n = 6 B cell epitope optimized) were manually selected to decrease their degree of sequence overlap and then synthesized. The immunogenicity of the synthesized vaccine peptides was validated using ELISpot and ELISA following murine vaccination. Strong T cell responses were observed in 7/10 T cell epitope optimized peptides following vaccination. Humoral responses were deficient, likely due to the unrestricted conformational space inhabited by linear vaccine peptides. CONCLUSIONS: Overall, we find our selection process and vaccine formulation to be appropriate for identifying T cell epitopes and eliciting T cell responses against those epitopes. Further studies are needed to optimize prediction and induction of B cell responses, as well as study the protective capacity of predicted T and B cell epitopes.


Assuntos
Vacinas contra COVID-19/administração & dosagem , COVID-19/prevenção & controle , Biologia Computacional/métodos , Epitopos de Linfócito B/química , Epitopos de Linfócito T/química , Sequência de Aminoácidos , Animais , COVID-19/virologia , Vacinas contra COVID-19/química , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Feminino , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe II/química , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos/química , Peptídeos/imunologia , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia
4.
ACS Chem Biol ; 16(1): 27-34, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33373188

RESUMO

Chemical proteomics provides a powerful strategy for the high-throughput assignment of enzyme function or inhibitor selectivity. However, identifying optimized probes for an enzyme family member of interest and differentiating signal from the background remain persistent challenges in the field. To address this obstacle, here we report a physiochemical discernment strategy for optimizing chemical proteomics based on the coenzyme A (CoA) cofactor. First, we synthesize a pair of CoA-based sepharose pulldown resins differentiated by a single negatively charged residue and find this change alters their capture properties in gel-based profiling experiments. Next, we integrate these probes with quantitative proteomics and benchmark analysis of "probe selectivity" versus traditional "competitive chemical proteomics." This reveals that the former is well-suited for the identification of optimized pulldown probes for specific enzyme family members, while the latter may have advantages in discovery applications. Finally, we apply our anionic CoA pulldown probe to evaluate the selectivity of a recently reported small molecule N-terminal acetyltransferase inhibitor. These studies further validate the use of physical discriminant strategies in chemoproteomic hit identification and demonstrate how CoA-based chemoproteomic probes can be used to evaluate the selectivity of small molecule protein acetyltransferase inhibitors, an emerging class of preclinical therapeutic agents.


Assuntos
Acetiltransferases/química , Sondas Moleculares/química , Proteômica/métodos , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Reprodutibilidade dos Testes , Especificidade por Substrato , Espectrometria de Massas em Tandem/métodos
5.
Polymers (Basel) ; 14(1)2021 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-35012177

RESUMO

Due to the content of lignocellulosic particles, wood plastic composites (WPC) composites can be attacked by both domestic and mold fungi. Household fungi reduce the mechanical properties of composites, while mold fungi reduce the aesthetics of products by changing their color and surface decomposition of the wood substance. As part of this study, the impact of lignocellulosic fillers in the form of sawdust and bark in poly (lactic acid) (PLA)-based biocomposites on their susceptibility to mold growth was determined. The evaluation of the samples fouled with mold fungi was performed by computer analysis of the image. For comparison, tests were carried out on analogous high-density polyethylene (HDPE) composites. Three levels of composites' filling were used with two degrees of comminution of lignocellulosic fillers and the addition of bonding aids to selected variants. The composites were produced in two stages employing extrusion and flat pressing. The research revealed that PLA composites were characterized by a higher fouling rate by Aspergillus niger Tiegh fungi compared to HDPE composites. In the case of HDPE composites. The type of filler (bark, sawdust) affected this process much more in the case of HDPE composites than for PLA composites. In addition, the use of filler with smaller particles enhanced the fouling process.

6.
Epigenetics Chromatin ; 13(1): 44, 2020 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-33097091

RESUMO

The chromatin-binding E3 ubiquitin ligase ubiquitin-like with PHD and RING finger domains 1 (UHRF1) contributes to the maintenance of aberrant DNA methylation patterning in cancer cells through multivalent histone and DNA recognition. The tandem Tudor domain (TTD) of UHRF1 is well-characterized as a reader of lysine 9 di- and tri-methylation on histone H3 (H3K9me2/me3) and, more recently, lysine 126 di- and tri-methylation on DNA ligase 1 (LIG1K126me2/me3). However, the functional significance and selectivity of these interactions remain unclear. In this study, we used protein domain microarrays to search for additional readers of LIG1K126me2, the preferred methyl state bound by the UHRF1 TTD. We show that the UHRF1 TTD binds LIG1K126me2 with high affinity and selectivity compared to other known methyllysine readers. Notably, and unlike H3K9me2/me3, the UHRF1 plant homeodomain (PHD) and its N-terminal linker (L2) do not contribute to multivalent LIG1K126me2 recognition along with the TTD. To test the functional significance of this interaction, we designed a LIG1K126me2 cell-penetrating peptide (CPP). Consistent with LIG1 knockdown, uptake of the CPP had no significant effect on the propagation of DNA methylation patterning across the genomes of bulk populations from high-resolution analysis of several cancer cell lines. Further, we did not detect significant changes in DNA methylation patterning from bulk cell populations after chemical or genetic disruption of lysine methyltransferase activity associated with LIG1K126me2 and H3K9me2. Collectively, these studies identify UHRF1 as a selective reader of LIG1K126me2 in vitro and further implicate the histone and non-histone methyllysine reader activity of the UHRF1 TTD as a dispensable domain function for cancer cell DNA methylation maintenance.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Código das Histonas , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/química , Epigênese Genética , Células HCT116 , Células HeLa , Histonas/química , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilação , Processamento de Proteína Pós-Traducional , Domínio Tudor , Ubiquitina-Proteína Ligases/química
7.
Int J Mol Sci ; 21(15)2020 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-32722282

RESUMO

FLICE-associated huge protein (FLASH), Yin Yang 1-Associated Protein-Related Protein (YARP) and Nuclear Protein, Ataxia-Telangiectasia Locus (NPAT) localize to discrete nuclear structures called histone locus bodies (HLBs) where they control various steps in histone gene expression. Near the C-terminus, FLASH and YARP contain a highly homologous domain that interacts with the C-terminal region of NPAT. Structural aspects of the FLASH-NPAT and YARP-NPAT complexes and their role in histone gene expression remain largely unknown. In this study, we used multidimensional NMR spectroscopy and in silico modeling to analyze the C-terminal domain in FLASH and YARP in an unbound form and in a complex with the last 31 amino acids of NPAT. Our results demonstrate that FLASH and YARP domains share the same fold of a triple α -helical bundle that resembles the DNA binding domain of Myb transcriptional factors and the SANT domain found in chromatin-modifying and remodeling complexes. The NPAT peptide contains a single α -helix that makes multiple contacts with α -helices I and III of the FLASH and YARP domains. Surprisingly, in spite of sharing a significant amino acid similarity, each domain likely binds NPAT using a unique network of interactions, yielding two distinct complexes. In silico modeling suggests that both complexes are structurally compatible with DNA binding, raising the possibility that they may function in identifying specific sequences within histone gene clusters, hence initiating the assembly of HLBs and regulating histone gene expression during cell cycle progression.


Assuntos
Proteínas Reguladoras de Apoptose/química , Proteínas de Ligação ao Cálcio/química , Proteínas de Ciclo Celular/química , Proteínas Correpressoras/química , Simulação por Computador , Proteínas de Ligação a DNA/química , Espectroscopia de Ressonância Magnética , Complexos Multiproteicos/química , Humanos , Conformação Proteica em alfa-Hélice , Domínios Proteicos
8.
Animals (Basel) ; 10(4)2020 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-32244432

RESUMO

The aim was to investigate the impact of an automatic in ovo injection of the raffinose family oligosaccharides (RFO) extracted from the seeds of Lupinus luteus L, on the chicken performance and resistance in a production environment. At day 12 of incubation, a total of 57,900 eggs (Ross 308) were divided into two groups: 1/ Control, injected with 0.9% NaCl and 2/ RFO group, injected with 1.9 mg/egg of the lupin seed extract, dissolved in 0.2 mL NaCl. The performance parameters, biochemical indices (lipid profile, hepatic parameters), gut histomorphology and duodenum structure, oxidative stability of the meat and microbiological counts of the major commensal microbiota species were analyzed. Mortality, body weight, and feed conversion ratio (FCR) were not affected. By day 42, several health indices were improved with RFO and were reflected in a beneficial lipid blood profile, increased villi surface and better combating opportunistic pathogens through reduction of Clostridia and decreased coccidia counts. The RFO increased meat oxidation, but only at the beginning of the storage. The RFO sourced from local legumes can be considered a promising prebiotic for broiler chickens. In ovo delivery of prebiotics and/or synbiotics should be further optimized as an important strategy for the earliest possible modulation of chicken resistance.

9.
Epigenetics Chromatin ; 13(1): 3, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980037

RESUMO

BACKGROUND: Plant homeodomain (PHD) fingers are central "readers" of histone post-translational modifications (PTMs) with > 100 PHD finger-containing proteins encoded by the human genome. Many of the PHDs studied to date bind to unmodified or methylated states of histone H3 lysine 4 (H3K4). Additionally, many of these domains, and the proteins they are contained in, have crucial roles in the regulation of gene expression and cancer development. Despite this, the majority of PHD fingers have gone uncharacterized; thus, our understanding of how these domains contribute to chromatin biology remains incomplete. RESULTS: We expressed and screened 123 of the annotated human PHD fingers for their histone binding preferences using reader domain microarrays. A subset (31) of these domains showed strong preference for the H3 N-terminal tail either unmodified or methylated at H3K4. These H3 readers were further characterized by histone peptide microarrays and/or AlphaScreen to comprehensively define their H3 preferences and PTM cross-talk. CONCLUSIONS: The high-throughput approaches utilized in this study establish a compendium of binding information for the PHD reader family with regard to how they engage histone PTMs and uncover several novel reader domain-histone PTM interactions (i.e., PHRF1 and TRIM66). This study highlights the usefulness of high-throughput analyses of histone reader proteins as a means of understanding how chromatin engagement occurs biochemically.


Assuntos
Histonas/metabolismo , Proteínas de Homeodomínio/metabolismo , Sítios de Ligação , Histonas/química , Proteínas de Homeodomínio/química , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Metilação , Ligação Proteica , Processamento de Proteína Pós-Traducional
11.
iScience ; 21: 773-788, 2019 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-31727574

RESUMO

Acetylation of the histone variant H2A.Z (H2A.Zac) occurs at active regulatory regions associated with gene expression. Although the Tip60 complex is proposed to acetylate H2A.Z, functional studies suggest additional enzymes are involved. Here, we show that p300 acetylates H2A.Z at multiple lysines. In contrast, we found that although Tip60 does not efficiently acetylate H2A.Z in vitro, genetic inhibition of Tip60 reduces H2A.Zac in cells. Importantly, we found that interaction between the p300-bromodomain and H4 acetylation (H4ac) enhances p300-driven H2A.Zac. Indeed, H2A.Zac and H4ac show high genomic overlap, especially at active promoters. We also reveal unique chromatin features and transcriptional states at enhancers correlating with co-occurrence or exclusivity of H4ac and H2A.Zac. We propose that differential H4 and H2A.Z acetylation signatures can also define the enhancer state. In conclusion, we show both Tip60 and p300 contribute to H2A.Zac and reveal molecular mechanisms of writer/reader crosstalk between H2A.Z and H4 acetylation through p300.

12.
Sci Rep ; 9(1): 14226, 2019 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-31578417

RESUMO

Covalent post-translational modification (PTM) of proteins with acyl groups of various carbon chain-lengths regulates diverse biological processes ranging from chromatin dynamics to subcellular localization. While the YEATS domain has been found to be a prominent reader of acetylation and other short acyl modifications, whether additional acyl-lysine reader domains exist, particularly for longer carbon chains, is unclear. Here, we employed a quantitative proteomic approach using various modified peptide baits to identify reader proteins of various acyl modifications. We discovered that proteins harboring HEAT and ARM repeats bind to lysine myristoylated peptides. Recombinant HEAT and ARM repeats bind to myristoylated peptides independent of the peptide sequence or the position of the myristoyl group. Indeed, HEAT and ARM repeats bind directly to medium- and long-chain free fatty acids (MCFA and LCFA). Lipidomic experiments suggest that MCFAs and LCFAs interact with HEAT and ARM repeat proteins in mammalian cells. Finally, treatment of cells with exogenous MCFAs and inhibitors of MCFA-CoA synthases increase the transactivation activity of the ARM repeat protein ß-catenin. Taken together, our results suggest an unappreciated role for fatty acids in the regulation of proteins harboring HEAT or ARM repeats.


Assuntos
Ácidos Graxos/metabolismo , Sequências Repetitivas de Aminoácidos , Acilação , Linhagem Celular , Cromatografia Gasosa , Cromatografia Líquida de Alta Pressão , Ontologia Genética , Humanos , Lipidômica/métodos , Lisina/química , Ácido Mirístico/química , Peptídeos/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Espectrometria de Massas em Tandem , beta Catenina/química , beta Catenina/metabolismo
13.
Nat Commun ; 10(1): 4724, 2019 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-31624313

RESUMO

Acetylation of histone H3K23 has emerged as an essential posttranslational modification associated with cancer and learning and memory impairment, yet our understanding of this epigenetic mark remains insufficient. Here, we identify the native MORF complex as a histone H3K23-specific acetyltransferase and elucidate its mechanism of action. The acetyltransferase function of the catalytic MORF subunit is positively regulated by the DPF domain of MORF (MORFDPF). The crystal structure of MORFDPF in complex with crotonylated H3K14 peptide provides mechanistic insight into selectivity of this epigenetic reader and its ability to recognize both histone and DNA. ChIP data reveal the role of MORFDPF in MORF-dependent H3K23 acetylation of target genes. Mass spectrometry, biochemical and genomic analyses show co-existence of the H3K23ac and H3K14ac modifications in vitro and co-occupancy of the MORF complex, H3K23ac, and H3K14ac at specific loci in vivo. Our findings suggest a model in which interaction of MORFDPF with acylated H3K14 promotes acetylation of H3K23 by the native MORF complex to activate transcription.


Assuntos
Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Acetilação , Acilação , Sítios de Ligação/genética , Linhagem Celular Tumoral , Cristalografia por Raios X , Células HEK293 , Histona Acetiltransferases/química , Histona Acetiltransferases/genética , Histonas/química , Humanos , Células K562 , Simulação de Dinâmica Molecular , Ligação Proteica , Domínios Proteicos
14.
Anal Chem ; 91(18): 11606-11613, 2019 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-31418558

RESUMO

Protein phosphorylation is a critical post-translational modification (PTM). Despite recent technological advances in reversed-phase liquid chromatography (RPLC)-mass spectrometry (MS)-based proteomics, comprehensive phosphoproteomic coverage in complex biological systems remains challenging, especially for hydrophilic phosphopeptides with enriched regions of serines, threonines, and tyrosines that often orchestrate critical biological functions. To address this issue, we developed a simple, easily implemented method to introduce a commonly used tandem mass tag (TMT) to increase peptide hydrophobicity, effectively enhancing RPLC-MS analysis of hydrophilic peptides. Different from conventional TMT labeling, this method capitalizes on using a nonprimary amine buffer and TMT labeling occurring before C18-based solid phase extraction. Through phosphoproteomic analyses of MCF7 cells, we have demonstrated that this method can greatly increase the number of identified hydrophilic phosphopeptides and improve MS detection signals. We applied this method to study the peptide QPSSSR, a very hydrophilic tryptic peptide located on the C-terminus of the G protein-coupled receptor (GPCR) CXCR3. Identification of QPSSSR has never been reported, and we were unable to detect it by traditional methods. We validated our TMT labeling strategy by comparative RPLC-MS analyses of both a hydrophilic QPSSSR peptide library as well as common phosphopeptides. We further confirmed the utility of this method by quantifying QPSSSR phosphorylation abundances in HEK 293 cells under different treatment conditions predicted to alter QPSSSR phosphorylation. We anticipate that this simple TMT labeling method can be broadly used not only for decoding GPCR phosphoproteome but also for effective RPLC-MS analysis of other highly hydrophilic analytes.


Assuntos
Sondas Moleculares/química , Fosfopeptídeos/análise , Sequência de Aminoácidos , Cromatografia de Fase Reversa , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Células MCF-7 , Biblioteca de Peptídeos , Fosfopeptídeos/química , Fosforilação , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Succinimidas/química , Espectrometria de Massas em Tandem/métodos
15.
Nat Commun ; 10(1): 2314, 2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-31127101

RESUMO

Histone methyltransferase MLL4 is centrally involved in transcriptional regulation and is often mutated in human diseases, including cancer and developmental disorders. MLL4 contains a catalytic SET domain that mono-methylates histone H3K4 and seven PHD fingers of unclear function. Here, we identify the PHD6 finger of MLL4 (MLL4-PHD6) as a selective reader of the epigenetic modification H4K16ac. The solution NMR structure of MLL4-PHD6 in complex with a H4K16ac peptide along with binding and mutational analyses reveal unique mechanistic features underlying recognition of H4K16ac. Genomic studies show that one third of MLL4 chromatin binding sites overlap with H4K16ac-enriched regions in vivo and that MLL4 occupancy in a set of genomic targets depends on the acetyltransferase activity of MOF, a H4K16ac-specific acetyltransferase. The recognition of H4K16ac is conserved in the PHD7 finger of paralogous MLL3. Together, our findings reveal a previously uncharacterized acetyllysine reader and suggest that selective targeting of H4K16ac by MLL4 provides a direct functional link between MLL4, MOF and H4K16 acetylation.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Histona Acetiltransferases/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Dedos de Zinco PHD/fisiologia , Acetilação , Animais , Sítios de Ligação , Cromatina/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Técnicas de Inativação de Genes , Células HEK293 , Histona Acetiltransferases/genética , Histona-Lisina N-Metiltransferase/química , Histonas/química , Humanos , Camundongos Transgênicos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
16.
Sci Adv ; 4(11): eaav2623, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30498785

RESUMO

Lysine methylation is a key regulator of histone protein function. Beyond histones, few connections have been made to the enzymes responsible for the deposition of these posttranslational modifications. Here, we debut a high-throughput functional proteomics platform that maps the sequence determinants of lysine methyltransferase (KMT) substrate selectivity without a priori knowledge of a substrate or target proteome. We demonstrate the predictive power of this approach for identifying KMT substrates, generating scaffolds for inhibitor design, and predicting the impact of missense mutations on lysine methylation signaling. By comparing KMT selectivity profiles to available lysine methylome datasets, we reveal a disconnect between preferred KMT substrates and the ability to detect these motifs using standard mass spectrometry pipelines. Collectively, our studies validate the use of this platform for guiding the study of lysine methylation signaling and suggest that substantial gaps exist in proteome-wide curation of lysine methylomes.


Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Proteoma/análise , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Humanos , Metilação , Mutação de Sentido Incorreto , Especificidade por Substrato
17.
Epigenetics ; 13(7): 683-692, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30045670

RESUMO

Histone posttranslational modifications control the organization and function of chromatin. In particular, methylation of lysine 36 in histone H3 (H3K36me) has been shown to mediate gene transcription, DNA repair, cell cycle regulation, and pre-mRNA splicing. Notably, mutations at or near this residue have been causally linked to the development of several human cancers. These observations have helped to illuminate the role of histones themselves in disease and to clarify the mechanisms by which they acquire oncogenic properties. This perspective focuses on recent advances in discovery and characterization of histone H3 mutations that impact H3K36 methylation. We also highlight findings that the common cancer-related substitution of H3K36 to methionine (H3K36M) disturbs functions of not only H3K36me-writing enzymes but also H3K36me-specific readers. The latter case suggests that the oncogenic effects could also be linked to the inability of readers to engage H3K36M.


Assuntos
Epigenômica , Histonas/genética , Mutação , Neoplasias/genética , Neoplasias/patologia , Humanos , Metilação , Transcrição Genética
19.
J Vestib Res ; 28(5-6): 409-415, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30714985

RESUMO

BACKGROUND: Recently, two types of movement sensors have been introduced into Virtual Reality (VR) therapy: motion trackers and force-plate platforms. Combining these two methods could produce better rehabilitation outcomes. Such devices, encompassing motion trackers and force platforms, are referred to as "hybrid" VR units. OBJECTIVE: To assess the effectiveness of a low-cost hybrid VR based vestibular rehabilitation programMETHODS: A prospective, non-randomized, controlled group study comparing training using a hybrid VR unit (Group 1 n = 25) vs. static posturography with visual feedback (Group 2 n = 25) in patients with peripheral vestibular dysfunction was conducted. The subjects underwent 10 training sessions over 10 days (30 minute sessions). All were examined on a posturography platform at the start and 1 month after rehabilitation and completed the Vertigo Symptom Scale - Short Form (VSS-SF) questionnaire. RESULTS: Both groups demonstrated improvement in posturographic parameters, which were statistically significant, but when comparing results between both groups there were no differences. The patients reported improvement in their subjective perception of symptoms on the VSS-SF scale, which were statistically significant in both groups, but greater in the VR group. CONCLUSIONS: Both methods reduce postural sway, however subjective reduction of symptoms was greater in the VR group.


Assuntos
Terapia por Exercício/métodos , Doenças Vestibulares/terapia , Realidade Virtual , Adulto , Idoso , Tontura/fisiopatologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Equilíbrio Postural , Estudos Prospectivos , Inquéritos e Questionários , Resultado do Tratamento , Vertigem/fisiopatologia , Doenças Vestibulares/fisiopatologia , Vestíbulo do Labirinto/fisiopatologia
20.
Epigenetics Chromatin ; 10: 12, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28293301

RESUMO

BACKGROUND: Histone posttranslational modifications (PTMs) function to regulate chromatin structure and function in part through the recruitment of effector proteins that harbor specialized "reader" domains. Despite efforts to elucidate reader domain-PTM interactions, the influence of neighboring PTMs and the target specificity of many reader domains is still unclear. The aim of this study was to use a high-throughput histone peptide microarray platform to interrogate 83 known and putative histone reader domains from the chromo and Tudor domain families to identify their interactions and characterize the influence of neighboring PTMs on these interactions. RESULTS: Nearly a quarter of the chromo and Tudor domains screened showed interactions with histone PTMs by peptide microarray, revealing known and several novel methyllysine interactions. Specifically, we found that the CBX/HP1 chromodomains that recognize H3K9me also recognize H3K23me2/3-a poorly understood histone PTM. We also observed that, in addition to their interaction with H3K4me3, Tudor domains of the Spindlin family also recognized H4K20me3-a previously uncharacterized interaction. Several Tudor domains also showed novel interactions with H3K4me as well. CONCLUSIONS: These results provide an important resource for the epigenetics and chromatin community on the interactions of many human chromo and Tudor domains. They also provide the basis for additional studies into the functional significance of the novel interactions that were discovered.


Assuntos
Histonas/metabolismo , Lisina/metabolismo , Análise Serial de Proteínas , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Células HEK293 , Histonas/química , Humanos , Metilação , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Domínios e Motivos de Interação entre Proteínas , Domínio Tudor
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...