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1.
Nat Commun ; 12(1): 833, 2021 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-33547280

RESUMO

The structure of proline prevents it from adopting an optimal position for rapid protein synthesis. Poly-proline-tract (PPT) associated ribosomal stalling is resolved by highly conserved eIF5A, the only protein to contain the amino acid hypusine. We show that de novo heterozygous EIF5A variants cause a disorder characterized by variable combinations of developmental delay, microcephaly, micrognathia and dysmorphism. Yeast growth assays, polysome profiling, total/hypusinated eIF5A levels and PPT-reporters studies reveal that the variants impair eIF5A function, reduce eIF5A-ribosome interactions and impair the synthesis of PPT-containing proteins. Supplementation with 1 mM spermidine partially corrects the yeast growth defects, improves the polysome profiles and restores expression of PPT reporters. In zebrafish, knockdown eif5a partly recapitulates the human phenotype that can be rescued with 1 µM spermidine supplementation. In summary, we uncover the role of eIF5A in human development and disease, demonstrate the mechanistic complexity of EIF5A-related disorder and raise possibilities for its treatment.


Assuntos
Deficiências do Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento , Microcefalia/genética , Micrognatismo/genética , Fatores de Iniciação de Peptídeos/genética , Proteínas de Ligação a RNA/genética , Adolescente , Sequência de Aminoácidos , Animais , Criança , Deficiências do Desenvolvimento/metabolismo , Deficiências do Desenvolvimento/patologia , Embrião não Mamífero , Feminino , Humanos , Lisina/análogos & derivados , Lisina/genética , Lisina/metabolismo , Masculino , Microcefalia/metabolismo , Microcefalia/patologia , Micrognatismo/metabolismo , Micrognatismo/patologia , Fatores de Iniciação de Peptídeos/deficiência , Peptídeos/genética , Peptídeos/metabolismo , Biossíntese de Proteínas , Conformação Proteica , Isoformas de Proteínas/deficiência , Isoformas de Proteínas/genética , Ribossomos/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Espermidina/farmacologia , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
2.
Nat Commun ; 12(1): 499, 2021 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-33479238

RESUMO

The human genome is partitioned into a collection of genomic features, inclusive of genes, transposable elements, lamina interacting regions, early replicating control elements and cis-regulatory elements, such as promoters, enhancers, and anchors of chromatin interactions. Uneven distribution of these features within chromosomes gives rise to clusters, such as topologically associating domains (TADs), lamina-associated domains, clusters of cis-regulatory elements or large organized chromatin lysine (K) domains (LOCKs). Here we show that LOCKs from diverse histone modifications discriminate primitive from differentiated cell types. Active LOCKs (H3K4me1, H3K4me3 and H3K27ac) cover a higher fraction of the genome in primitive compared to differentiated cell types while repressive LOCKs (H3K9me3, H3K27me3 and H3K36me3) do not. Active LOCKs in differentiated cells lie proximal to highly expressed genes while active LOCKs in primitive cells tend to be bivalent. Genes proximal to bivalent LOCKs are minimally expressed in primitive cells. Furthermore, bivalent LOCKs populate TAD boundaries and are preferentially bound by regulators of chromatin interactions, including CTCF, RAD21 and ZNF143. Together, our results argue that LOCKs discriminate primitive from differentiated cell populations.


Assuntos
Diferenciação Celular/genética , Cromatina/genética , Código das Histonas/genética , Histonas/genética , Lisina/genética , Animais , Sítios de Ligação/genética , Elementos de DNA Transponíveis/genética , Elementos Facilitadores Genéticos/genética , Genoma Humano/genética , Humanos , Regiões Promotoras Genéticas/genética
3.
Adv Exp Med Biol ; 1283: 85-96, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33155140

RESUMO

Histone modification stands for a vital genetic information form, which shows tight correlation with the modulation of normal physiological activities by genes. Abnormal regulation of histone methylation due to histone modification enzyme changes and histone mutations plays an important role in the development of cancer. Histone mutations, especially H3K27M and H3K36M, have been identified in various cancers such as pediatric DIPG (diffuse intrinsic pontine glioma) and chondroblastoma respectively. "K to M" mutation results overall downregulation of methylation on these lysine residues. Also, "K to M" mutant histones can inhibit the enzymatic activity of the responsible HMT (histone methyltransferase); for instance, SETD2 indicates H3K36 methylation, and Ezh2 represents H3K27 methylation. In-depth analysis of the mechanism of tumor formation triggered by the K to M mutation results in possible targeted therapies. This chapter is going to briefly introduce the mechanism of histone lysine-to-methionine mutation and review the recently identified targeted therapeutic strategies.


Assuntos
Antineoplásicos , Histonas , Condroblastoma/genética , Glioma/genética , Histonas/genética , Humanos , Lisina/genética , Metionina/genética , Mutação
4.
PLoS One ; 15(12): e0241576, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33362225

RESUMO

Mitophagy, the process that degrades mitochondria selectively through autophagy, is involved in the quality control of mitochondria in cells grown under respiratory conditions. In yeast, the presence of the Atg32 protein on the outer mitochondrial membrane allows for the recognition and targeting of superfluous or damaged mitochondria for degradation. Post-translational modifications such as phosphorylation are crucial for the execution of mitophagy. In our study we monitor the stability of Atg32 protein in the yeast S. cerevisiae and show that Atg32 is degraded under normal growth conditions, upon starvation or rapamycin treatment. The Atg32 turnover can be prevented by inhibition of the proteasome activity, suggesting that Atg32 is also ubiquitinated. Mass spectrometry analysis of purified Atg32 protein revealed that at least lysine residue in position 282 is ubiquitinated. Interestingly, the replacement of lysine 282 with alanine impaired Atg32 degradation only partially in the course of cell growth, suggesting that additional lysine residues on Atg32 might also be ubiquitinated. Our results provide the foundation to further elucidate the physiological significance of Atg32 turnover and the interplay between mitophagy and the proteasome.


Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Mitocôndrias/metabolismo , Mitofagia , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Alanina/genética , Alanina/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/isolamento & purificação , Lisina/genética , Lisina/metabolismo , Membranas Mitocondriais/metabolismo , Mutagênese Sítio-Dirigida , Estabilidade Proteica , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Ubiquitinação/fisiologia
5.
PLoS One ; 15(10): e0236612, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33104714

RESUMO

A precisely balanced activity of canonical Wnt signaling is essential for a number of biological processes and its perturbation leads to developmental defects or diseases. Here, we demonstrate that alternative isoforms of the KDM2A and KDM2B lysine demethylases have the ability to negatively regulate canonical Wnt signaling. These KDM2A and KDM2B isoforms (KDM2A-SF and KDM2B-SF) lack the N-terminal demethylase domain, but they still have the ability to bind to CpG islands in promoters and to interact with their protein partners via their other functional domains. We have observed that KDM2A-SF and KDM2B-SF bind to the promoters of axin 2 and cyclin D1, two canonical Wnt signaling target genes, and repress their activity. Moreover, KDM2A-SF and KDM2B-SF are both able to strongly repress a Wnt-responsive luciferase reporter. The transcriptional repression mediated by KDM2A-SF and KDM2B-SF, but also by KDM2A-LF, is dependent on their DNA binding domain, while the N-terminal demethylase domain is dispensable for this process. Surprisingly, KDM2B-LF is unable to repress both the endogenous promoters and the luciferase reporter. Finally, we show that both KDM2A-SF and KDM2B-SF are able to interact with TCF7L1, one of the transcriptional mediators of canonical Wnt signaling. KDM2A-SF and KDM2B-SF are thus likely to negatively affect the transcription of canonical Wnt signaling target genes by binding to their promoters and by interacting with TCF7L1 and other co-repressors.


Assuntos
Ciclina D1/metabolismo , Proteínas F-Box/metabolismo , Regulação da Expressão Gênica , Histona Desmetilases com o Domínio Jumonji/metabolismo , Regiões Promotoras Genéticas , Proteína 1 Semelhante ao Fator 7 de Transcrição/metabolismo , Via de Sinalização Wnt , Ilhas de CpG , Ciclina D1/genética , Proteínas F-Box/genética , Células HEK293 , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Lisina/genética , Lisina/metabolismo , Isoformas de Proteínas , Proteína 1 Semelhante ao Fator 7 de Transcrição/genética
6.
Science ; 370(6514): 346-350, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33060360

RESUMO

Mutualisms, or reciprocally beneficial interspecific interactions, constitute the foundation of many ecological communities and agricultural systems. Mutualisms come in different forms, from pairwise interactions to extremely diverse communities, and they are continually challenged with exploitation by nonmutualistic community members (exploiters). Thus, understanding how mutualisms persist remains an essential question in ecology. Theory suggests that high species richness and functional redundancy could promote mutualism persistence in complex mutualistic communities. Using a yeast system (Saccharomyces cerevisiae), we experimentally show that communities with the greatest mutualist richness and functional redundancy are nearly two times more likely to survive exploitation than are simple communities. Persistence increased because diverse communities were better able to mitigate the negative effects of competition with exploiters. Thus, large mutualistic networks may be inherently buffered from exploitation.


Assuntos
Saccharomyces cerevisiae/fisiologia , Simbiose/fisiologia , Adenina/metabolismo , Biota , Lisina/genética , Lisina/metabolismo , Saccharomyces cerevisiae/genética , Simbiose/genética
7.
J Transl Med ; 18(1): 329, 2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32867854

RESUMO

BACKGROUND: The new Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), which was first detected in Wuhan (China) in December of 2019 is responsible for the current global pandemic. Phylogenetic analysis revealed that it is similar to other betacoronaviruses, such as SARS-CoV and Middle-Eastern Respiratory Syndrome, MERS-CoV. Its genome is ∼ 30 kb in length and contains two large overlapping polyproteins, ORF1a and ORF1ab that encode for several structural and non-structural proteins. The non-structural protein 1 (nsp1) is arguably the most important pathogenic determinant, and previous studies on SARS-CoV indicate that it is both involved in viral replication and hampering the innate immune system response. Detailed experiments of site-specific mutagenesis and in vitro reconstitution studies determined that the mechanisms of action are mediated by (a) the presence of specific amino acid residues of nsp1 and (b) the interaction between the protein and the host's small ribosomal unit. In fact, substitution of certain amino acids resulted in reduction of its negative effects. METHODS: A total of 17,928 genome sequences were obtained from the GISAID database (December 2019 to July 2020) from patients infected by SARS-CoV-2 from different areas around the world. Genomes alignment was performed using MAFFT (REFF) and the nsp1 genomic regions were identified using BioEdit and verified using BLAST. Nsp1 protein of SARS-CoV-2 with and without deletion have been subsequently modelled using I-TASSER. RESULTS: We identified SARS-CoV-2 genome sequences, from several Countries, carrying a previously unknown deletion of 9 nucleotides in position 686-694, corresponding to the AA position 241-243 (KSF). This deletion was found in different geographical areas. Structural prediction modelling suggests an effect on the C-terminal tail structure. CONCLUSIONS: Modelling analysis of a newly identified deletion of 3 amino acids (KSF) of SARS-CoV-2 nsp1 suggests that this deletion could affect the structure of the C-terminal region of the protein, important for regulation of viral replication and negative effect on host's gene expression. In addition, substitution of the two amino acids (KS) from nsp1 of SARS-CoV was previously reported to revert loss of interferon-alpha expression. The deletion that we describe indicates that SARS-CoV-2 is undergoing profound genomic changes. It is important to: (i) confirm the spreading of this particular viral strain, and potentially of strains with other deletions in the nsp1 protein, both in the population of asymptomatic and pauci-symptomatic subjects, and (ii) correlate these changes in nsp1 with potential decreased viral pathogenicity.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Deleção de Sequência , Proteínas não Estruturais Virais/genética , Sequência de Aminoácidos , Sequência de Bases , Betacoronavirus/patogenicidade , Doenças Transmissíveis Emergentes/virologia , Infecções por Coronavirus/epidemiologia , Frequência do Gene , Genoma Viral , Geografia , Humanos , Lisina/genética , Modelos Moleculares , Pandemias/estatística & dados numéricos , Fenilalanina/genética , Pneumonia Viral/epidemiologia , Domínios Proteicos/genética , Serina/genética , Proteínas não Estruturais Virais/química , Virulência/genética , Replicação Viral/genética
8.
Proc Natl Acad Sci U S A ; 117(41): 25517-25522, 2020 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-32973095

RESUMO

Escherichia coli NhaA is a prototypical sodium-proton antiporter responsible for maintaining cellular ion and volume homeostasis by exchanging two protons for one sodium ion; despite two decades of research, the transport mechanism of NhaA remains poorly understood. Recent crystal structure and computational studies suggested Lys300 as a second proton-binding site; however, functional measurements of several K300 mutants demonstrated electrogenic transport, thereby casting doubt on the role of Lys300. To address the controversy, we carried out state-of-the-art continuous constant pH molecular dynamics simulations of NhaA mutants K300A, K300R, K300Q/D163N, and K300Q/D163N/D133A. Simulations suggested that K300 mutants maintain the electrogenic transport by utilizing an alternative proton-binding residue Asp133. Surprisingly, while Asp133 is solely responsible for binding the second proton in K300R, Asp133 and Asp163 jointly bind the second proton in K300A, and Asp133 and Asp164 jointly bind two protons in K300Q/D163N. Intriguingly, the coupling between Asp133 and Asp163 or Asp164 is enabled through the proton-coupled hydrogen-bonding network at the flexible intersection of two disrupted helices. These data resolve the controversy and highlight the intricacy of the compensatory transport mechanism of NhaA mutants. Alternative proton-binding site and proton sharing between distant aspartates may represent important general mechanisms of proton-coupled transport in secondary active transporters.


Assuntos
Proteínas de Escherichia coli , Prótons , Trocadores de Sódio-Hidrogênio , Ácido Aspártico/química , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Sítios de Ligação , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ligação de Hidrogênio , Lisina/química , Lisina/genética , Lisina/metabolismo , Simulação de Dinâmica Molecular , Mutação , Trocadores de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Eletricidade Estática
9.
Proc Natl Acad Sci U S A ; 117(31): 18439-18447, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32675241

RESUMO

In mammals, repressive histone modifications such as trimethylation of histone H3 Lys9 (H3K9me3), frequently coexist with DNA methylation, producing a more stable and silenced chromatin state. However, it remains elusive how these epigenetic modifications crosstalk. Here, through structural and biochemical characterizations, we identified the replication foci targeting sequence (RFTS) domain of maintenance DNA methyltransferase DNMT1, a module known to bind the ubiquitylated H3 (H3Ub), as a specific reader for H3K9me3/H3Ub, with the recognition mode distinct from the typical trimethyl-lysine reader. Disruption of the interaction between RFTS and the H3K9me3Ub affects the localization of DNMT1 in stem cells and profoundly impairs the global DNA methylation and genomic stability. Together, this study reveals a previously unappreciated pathway through which H3K9me3 directly reinforces DNMT1-mediated maintenance DNA methylation.


Assuntos
DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA , Heterocromatina/metabolismo , Histonas/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , Heterocromatina/genética , Histonas/química , Histonas/genética , Humanos , Lisina/genética , Lisina/metabolismo , Metilação , Processamento de Proteína Pós-Traducional
10.
Am J Physiol Cell Physiol ; 319(2): C359-C370, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32520610

RESUMO

SLC4A11 is the only member of the SLC4 family that transports protons rather than bicarbonate. SLC4A11 is expressed in corneal endothelial cells, and its mutation causes corneal endothelial dystrophy, although the mechanism of pathogenesis is unknown. We previously demonstrated that the magnitude of the H+ conductance (Gm) mediated by SLC4A11 is increased by rises in intracellular as well as extracellular pH (pHi and pHe). To better understand this feature and whether it is altered in disease, we studied the pH dependence of wild-type and mutant mouse Slc4a11 expressed in Xenopus oocytes. Using voltage-clamp circuitry in conjunction with a H+-selective microelectrode and a microinjector loaded with NaHCO3, we caused incremental rises in oocyte pHi and measured the effect on Gm. We find that the rise of Gm has a steeper pHi dependence at pHe =8.50 than at pHe =7.50. Data gathered at pHe =8.50 can be fit to the Hill equation enabling the calculation of a pK value that reports pHi dependence. We find that mutation of lysine residues that are close to the first transmembrane span (TM1) causes an alkaline shift in pK. Furthermore, two corneal-dystrophy-causing mutations close to the extracellular end of TM1, E399K and T401K (E368K and T370K in mouse), cause an acidic shift in pK, while a third mutation in the fourth intracellular loop, R804H (R774H in mouse), causes an alkaline shift in pK. This is the first description of determinants of SLC4A11 pH dependence and the first indication that a shift in pH dependence could modify disease expressivity in some cases of corneal dystrophy.


Assuntos
Proteínas de Transporte de Ânions/genética , Transporte Biológico/genética , Distrofias Hereditárias da Córnea/genética , Lisina/genética , Simportadores/genética , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Bicarbonatos/metabolismo , Distrofias Hereditárias da Córnea/metabolismo , Distrofias Hereditárias da Córnea/patologia , Modelos Animais de Doenças , Epitélio Posterior/metabolismo , Epitélio Posterior/patologia , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Transporte de Íons/genética , Lisina/metabolismo , Camundongos , Mutação/genética , Oócitos/metabolismo , Oócitos/patologia , Sódio , Xenopus/genética
11.
Mol Pharmacol ; 98(2): 88-95, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32487734

RESUMO

Arylamine N-acetyltransferase 1 (NAT1) is a phase II xenobiotic-metabolizing enzyme that also has a role in cancer cell growth and metabolism. Recently, it was reported that NAT1 undergoes lysine acetylation, an important post-translational modification that can regulate protein function. In the current study, we use site-directed mutagenesis to identify K100 and K188 as major sites of lysine acetylation in the NAT1 protein. Acetylation of ectopically expressed NAT1 in HeLa cells was decreased by C646, an inhibitor of the protein acetyltransferases p300/CREB-binding protein (CBP). Recombinant p300 directly acetylated NAT1 in vitro. Acetylation of NAT1 was enhanced by the sirtuin (SIRT) inhibitor nicotinamide but not by the histone deacetylase inhibitor trichostatin A. Cotransfection of cells with NAT1 and either SIRT 1 or 2, but not SIRT3, significantly decreased NAT1 acetylation. NAT1 activity was evaluated in cells after nicotinamide treatment to enhance acetylation or cotransfection with SIRT1 to inhibit acetylation. The results indicated that NAT1 acetylation impaired its enzyme kinetics, suggesting decreased acetyl coenzyme A binding. In addition, acetylation attenuated the allosteric effects of ATP on NAT1. Taken together, this study shows that NAT1 is acetylated by p300/CBP in situ and is deacetylated by the sirtuins SIRT1 and 2. It is hypothesized that post-translational modification of NAT1 by acetylation at K100 and K188 may modulate NAT1 effects in cells. SIGNIFICANCE STATEMENT: There is growing evidence that arylamine N-acetyltransferase 1 has an important cellular role in addition to xenobiotic metabolism. Here, we show that NAT1 is acetylated at K100 and K188 and that changes in protein acetylation equilibrium can modulate its activity in cells.


Assuntos
Arilamina N-Acetiltransferase/química , Arilamina N-Acetiltransferase/metabolismo , Proteína de Ligação a CREB/genética , Proteína p300 Associada a E1A/genética , Isoenzimas/química , Isoenzimas/metabolismo , Sirtuína 1/genética , Sirtuína 2/genética , Acetilcoenzima A/metabolismo , Acetilação/efeitos dos fármacos , Arilamina N-Acetiltransferase/genética , Benzoatos/farmacologia , Proteína de Ligação a CREB/metabolismo , Cristalografia por Raios X , Proteína p300 Associada a E1A/metabolismo , Células HeLa , Humanos , Ácidos Hidroxâmicos/farmacologia , Isoenzimas/genética , Lisina/química , Lisina/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Niacinamida/farmacologia , Conformação Proteica , Pirazóis/farmacologia , Sirtuína 1/metabolismo , Sirtuína 2/metabolismo , Transfecção
12.
Mol Cell ; 78(3): 506-521.e6, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32386543

RESUMO

Higher-order chromatin structure and DNA methylation are implicated in multiple developmental processes, but their relationship to cell state is unknown. Here, we find that large (>7.3 kb) DNA methylation nadirs (termed "grand canyons") can form long loops connecting anchor loci that may be dozens of megabases (Mb) apart, as well as inter-chromosomal links. The interacting loci cover a total of ∼3.5 Mb of the human genome. The strongest interactions are associated with repressive marks made by the Polycomb complex and are diminished upon EZH2 inhibitor treatment. The data are suggestive of the formation of these loops by interactions between repressive elements in the loci, forming a genomic subcompartment, rather than by cohesion/CTCF-mediated extrusion. Interestingly, unlike previously characterized subcompartments, these interactions are present only in particular cell types, such as stem and progenitor cells. Our work reveals that H3K27me3-marked large DNA methylation grand canyons represent a set of very-long-range loops associated with cellular identity.


Assuntos
Cromatina/química , Cromatina/genética , Metilação de DNA , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Diferenciação Celular , Cromatina/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Epigênese Genética , Regulação da Expressão Gênica , Histonas/genética , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Hibridização in Situ Fluorescente , Lisina/genética , Lisina/metabolismo , Proteínas Nucleares/genética , Fatores de Transcrição SOXB1/genética , Proteína de Homoeobox de Baixa Estatura/genética , Fatores de Transcrição/genética
13.
Tunis Med ; 98(2): 161-163, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32395807

RESUMO

We report the case of a 23-year-old woman with a not yet described (to the best of our knowledge) association of left ventricle non-compaction with both atrial and ventricular defects. Family genetic survey concluded to, a probably sporadic, E101K gene mutation.


Assuntos
Cardiopatias Congênitas/diagnóstico , Comunicação Interatrial/diagnóstico , Ventrículos do Coração/anormalidades , Substituição de Aminoácidos/genética , Proteínas Associadas à Distrofina/genética , Feminino , Ácido Glutâmico/genética , Cardiopatias Congênitas/genética , Comunicação Interatrial/complicações , Comunicação Interatrial/genética , Humanos , Lisina/genética , Mutação de Sentido Incorreto , Neuropeptídeos/genética , Adulto Jovem
14.
Nat Commun ; 11(1): 2193, 2020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32366851

RESUMO

Innate immunity to nucleic acids forms the backbone for anti-viral immunity and several inflammatory diseases. Upon sensing cytosolic viral RNA, retinoic acid-inducible gene-I-like receptors (RLRs) interact with the mitochondrial antiviral signaling protein (MAVS) and activate TANK-binding kinase 1 (TBK1) to induce type I interferon (IFN-I). TRAF3-interacting protein 3 (TRAF3IP3, T3JAM) is essential for T and B cell development. It is also well-expressed by myeloid cells, where its role is unknown. Here we report that TRAF3IP3 suppresses cytosolic poly(I:C), 5'ppp-dsRNA, and vesicular stomatitis virus (VSV) triggers IFN-I expression in overexpression systems and Traf3ip3-/- primary myeloid cells. The mechanism of action is through the interaction of TRAF3IP3 with endogenous TRAF3 and TBK1. This leads to the degradative K48 ubiquitination of TBK1 via its K372 residue in a DTX4-dependent fashion. Mice with myeloid-specific gene deletion of Traf3ip3 have increased RNA virus-triggered IFN-I production and reduced susceptibility to virus. These results identify a function of TRAF3IP3 in the regulation of the host response to cytosolic viral RNA in myeloid cells.


Assuntos
Proteínas de Transporte/genética , Regulação da Expressão Gênica , Interferon Tipo I/genética , Proteínas de Membrana/genética , Células Mieloides/metabolismo , Proteínas Serina-Treonina Quinases/genética , RNA Viral/genética , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular , Células Cultivadas , Chlorocebus aethiops , Citosol/metabolismo , Citosol/virologia , Células HEK293 , Células HeLa , Humanos , Interferon Tipo I/metabolismo , Células Jurkat , Lisina/genética , Lisina/metabolismo , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células Mieloides/virologia , Proteínas Serina-Treonina Quinases/metabolismo , RNA Viral/metabolismo , Células THP-1 , Ubiquitinação , Células Vero , Vírus da Estomatite Vesicular Indiana/genética , Vírus da Estomatite Vesicular Indiana/fisiologia
15.
Nat Cell Biol ; 22(6): 621-629, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32393884

RESUMO

Epigenetic marks are reprogrammed in the gametes to reset genomic potential in the next generation. In mammals, paternal chromatin is extensively reprogrammed through the global erasure of DNA methylation and the exchange of histones with protamines1,2. Precisely how the paternal epigenome is reprogrammed in flowering plants has remained unclear since DNA is not demethylated and histones are retained in sperm3,4. Here, we describe a multi-layered mechanism by which H3K27me3 is globally lost from histone-based sperm chromatin in Arabidopsis. This mechanism involves the silencing of H3K27me3 writers, activity of H3K27me3 erasers and deposition of a sperm-specific histone, H3.10 (ref. 5), which we show is immune to lysine 27 methylation. The loss of H3K27me3 facilitates the transcription of genes essential for spermatogenesis and pre-configures sperm with a chromatin state that forecasts gene expression in the next generation. Thus, plants have evolved a specific mechanism to simultaneously differentiate male gametes and reprogram the paternal epigenome.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Reprogramação Celular , Cromatina/genética , Metilação de DNA , Epigênese Genética , Histonas/genética , Sequência de Aminoácidos , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Cromatina/metabolismo , Histonas/metabolismo , Lisina/genética , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Homologia de Sequência
16.
Sci Rep ; 10(1): 8521, 2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32444613

RESUMO

LSD1/KDM1A is a widely conserved lysine-specific demethylase that removes methyl groups from methylated proteins, mainly histone H3. We previously isolated the zebrafish LSD1 gene and demonstrated that it is required for primitive hematopoiesis. Recently, a neuron-specific splicing variant of LSD1 was found in mammals and its specific functions and substrate specificities were reported. To our surprise, zebrafish LSD1 cDNA, which we previously analyzed, was corresponded to the neuron-specific variant in mammals. In this study, we investigated the structures and expression of LSD1 splicing variants in zebrafish and found all 4 types of LSD1 isoforms: LSD1, LSD1+2al, LSD1+8al and LSD1+2al8al. Interestingly, LSD1+8al/LSD1+2al8al, which correspond to mammalian neuron-specific variants, expressed ubiquitously in zebrafish. We also performed phenotypic rescue experiments of a zebrafish LSD1 mutant (kdm1ait627) using human and zebrafish LSD1 variants to identify which variant is involved in primitive hematopoiesis. Unexpectedly, the overexpression of all types of human and zebrafish variants was able to rescue the hematopoietic phenotypes in LSD1 mutants. Furthermore, enzymatic-deficient LSD1K661A (human) and K638A (zebrafish) were also able to rescue the mutant phenotypes. These results suggest that the LSD1 functions in zebrafish primitive hematopoiesis are free from any splicing-dependent regulation or demethylation reaction.


Assuntos
Embrião não Mamífero/fisiologia , Hematopoese , Histona Desmetilases/metabolismo , Lisina/genética , Processamento de RNA , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Embrião não Mamífero/citologia , Histona Desmetilases/genética , Humanos , Lisina/metabolismo , Metilação , Mutação , Isoformas de Proteínas , Homologia de Sequência , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
17.
Proc Natl Acad Sci U S A ; 117(21): 11459-11470, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32385148

RESUMO

Genomic regions preferentially associate with regions of similar transcriptional activity, partitioning genomes into active and inactive compartments within the nucleus. Here we explore mechanisms controlling genome compartment organization in Caenorhabditis elegans and investigate roles for compartments in regulating gene expression. Distal arms of C. elegans chromosomes, which are enriched for heterochromatic histone modifications H3K9me1/me2/me3, interact with each other both in cis and in trans, while interacting less frequently with central regions, leading to genome compartmentalization. Arms are anchored to the nuclear periphery via the nuclear envelope protein CEC-4, which binds to H3K9me. By performing genome-wide chromosome conformation capture experiments (Hi-C), we showed that eliminating H3K9me1/me2/me3 through mutations in the methyltransferase genes met-2 and set-25 significantly impaired formation of inactive Arm and active Center compartments. cec-4 mutations also impaired compartmentalization, but to a lesser extent. We found that H3K9me promotes compartmentalization through two distinct mechanisms: Perinuclear anchoring of chromosome arms via CEC-4 to promote their cis association, and an anchoring-independent mechanism that compacts individual chromosome arms. In both met-2 set-25 and cec-4 mutants, no dramatic changes in gene expression were found for genes that switched compartments or for genes that remained in their original compartment, suggesting that compartment strength does not dictate gene-expression levels. Furthermore, H3K9me, but not perinuclear anchoring, also contributes to formation of another prominent feature of chromosome organization, megabase-scale topologically associating domains on X established by the dosage compensation condensin complex. Our results demonstrate that H3K9me plays crucial roles in regulating genome organization at multiple levels.


Assuntos
Caenorhabditis elegans/genética , Cromossomos/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos/genética , Regulação da Expressão Gênica , Genoma , Heterocromatina/genética , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Lisina/genética , Metilação , Mutação , Cromossomo X/genética , Cromossomo X/metabolismo
18.
Technol Cancer Res Treat ; 19: 1533033820914295, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32347192

RESUMO

OBJECTIVE: This study aimed to explore whether eukaryotic translation elongation factor 1 alpha 2 affected cell proliferation, migration, and apoptosis via regulating the dimethylation of eukaryotic translation elongation factor 1 alpha at lysine 55 in acute myeloid leukemia. METHODS: The expressions of eukaryotic translation elongation factor 1 alpha 2 and dimethylation of eukaryotic translation elongation factor 1 alpha at lysine 55 in acute myeloid leukemia cell lines and human normal bone marrow mononuclear cells (as control) were assessed. Control CRISPR-Cas9 lentivirus, eukaryotic translation elongation factor 1 alpha 2 knockout CRISPR-Cas9 lentivirus, vector plasmid, eukaryotic translation elongation factor 1 alpha 2 wild type overexpression plasmid, and eukaryotic translation elongation factor 1 alpha 2 with a K55R substitution overexpression plasmid were transfected into AML-193 and Kasumi-1 cells combined or alone, and were accordingly divided into 4 groups (Sgcontrol + vector group, SgeEF1A2 + vector group, SgeEF1A2 + eEF1A2WT group, and SgeEFIA2 + eEF1A2K55R group). RESULTS: Eukaryotic translation elongation factor 1 alpha 2 and dimethylation of eukaryotic translation elongation factor 1 alpha at lysine 55 expressions were higher in AML-193, Kasumi-1, and KG-1 cell lines compared to the control. In AML-193 and Kasumi-1 cells, the knockout and compensated experiments revealed that eukaryotic translation elongation factor 1 alpha 2 promoted cell proliferation and migration but repressed apoptosis. Additionally, the knockout of eukaryotic translation elongation factor 1 alpha 2 decreased dimethylation of eukaryotic translation elongation factor 1 alpha at lysine 55 expression, meanwhile, eukaryotic translation elongation factor 1 alpha 2 wild type overexpression enhanced while eukaryotic translation elongation factor 1 alpha 2 with a K55R substitution overexpression did not influence the dimethylation of eukaryotic translation elongation factor 1 alpha at lysine 55 expression. Furthermore, eukaryotic translation elongation factor 1 alpha 2 wild type overexpression promoted cell proliferation, enhanced migration, and decreased apoptosis, but eukaryotic translation elongation factor 1 alpha 2 with a K55R substitution overexpression did not influence these cellular functions in AML-193 and Kasumi-1 cells, suggesting the implication of dimethylation of eukaryotic translation elongation factor 1 alpha at lysine 55 in eukaryotic translation elongation factor 1 alpha 2 mediated oncogenesis of acute myeloid leukemia. CONCLUSION: Eukaryotic translation elongation factor 1 alpha 2 and its dimethylated product may serve as therapeutic targets, and these findings may provide support for exploring novel strategies in acute myeloid leukemia treatment.


Assuntos
Leucemia Mieloide Aguda/metabolismo , Lisina/metabolismo , Fator 1 de Elongação de Peptídeos/genética , Fator 1 de Elongação de Peptídeos/metabolismo , Apoptose/fisiologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Lisina/genética , Metilação , Processamento de Proteína Pós-Traducional
19.
PLoS One ; 15(4): e0230930, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32267870

RESUMO

Human epidemiological studies have shown that paternal aging as one of the risk factors for neurodevelopmental disorders, such as autism, in offspring. A recent study has suggested that factors other than de novo mutations due to aging can influence the biology of offspring. Here, we focused on epigenetic alterations in sperm that can influence developmental programs in offspring. In this study, we qualitatively and semiquantitatively evaluated histone modification patterns in male germline cells throughout spermatogenesis based on immunostaining of testes taken from young (3 months old) and aged (12 months old) mice. Although localization patterns were not obviously changed between young and aged testes, some histone modification showed differences in their intensity. Among histone modifications that repress gene expression, histone H3 lysine 9 trimethylation (H3K9me3) was decreased in the male germline cells of the aged testis, while H3K27me2/3 was increased. The intensity of H3K27 acetylation (ac), an active mark, was lower/higher depending on the stages in the aged testis. Interestingly, H3K27ac was detected on the putative sex chromosomes of round spermatids, while other chromosomes were occupied by a repressive mark, H3K27me3. Among other histone modifications that activate gene expression, H3K4me2 was drastically decreased in the male germline cells of the aged testis. In contrast, H3K79me3 was increased in M-phase spermatocytes, where it accumulates on the sex chromosomes. Therefore, aging induced alterations in the amount of histone modifications and in the differences of patterns for each modification. Moreover, histone modifications on the sex chromosomes and on other chromosomes seems to be differentially regulated by aging. These findings will help elucidate the epigenetic mechanisms underlying the influence of paternal aging on offspring development.


Assuntos
Histonas/genética , Meiose/genética , Espermatócitos/fisiologia , Espermatogênese/genética , Testículo/fisiologia , Acetilação , Animais , Epigênese Genética/genética , Epigenômica/métodos , Expressão Gênica/genética , Código das Histonas/genética , Humanos , Lisina/genética , Masculino , Metilação , Camundongos , Processamento de Proteína Pós-Traducional/genética , Cromossomos Sexuais/genética , Espermátides/fisiologia
20.
Biochim Biophys Acta Mol Cell Res ; 1867(7): 118694, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32151656

RESUMO

Dot1 enzymes are histone methyltransferases that mono-, di- and trimethylate lysine 79 of histone H3 to affect several nuclear processes. The functions of these different methylation states are still largely unknown. Trypanosomes, which are flagellated protozoa that cause several parasitic diseases, have two Dot1 homologues. Dot1A catalyzes the mono- and dimethylation of lysine 76 during late G2 and mitosis, and Dot1B catalyzes trimethylation, which is a modification found in all stages of the cell cycle. Here, we generated Trypanosoma cruzi lines lacking Dot1B. Deletion of one allele resulted in parasites with increased levels of mono- and dimethylation and a reduction in H3K76me3. In the full knockout (DKO), no trimethylation was observed. Both the DKO and the single knockout (SKO) showed aberrant morphology and decreased growth due to cell cycle arrest after G2. This phenotype could be rescued by caffeine in the DKO, as caffeine is a checkpoint inhibitor of the cell cycle. The knockouts also phosphorylated γH2A without producing extensive DNA breaks, and Dot1B-depleted cells were more susceptible to general checkpoint kinase inhibitors, suggesting that a lack of H3K76 trimethylation prevents the initiation and/or completion of cytokinesis.


Assuntos
Doença de Chagas/genética , Histona-Lisina N-Metiltransferase/genética , Mitose/genética , Trypanosoma cruzi/genética , Ciclo Celular/genética , Doença de Chagas/parasitologia , Histonas/genética , Lisina/genética , Metilação/efeitos dos fármacos , Proteínas Nucleares/genética , Fosforilação/genética , Trypanosoma cruzi/patogenicidade
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