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1.
Nat Commun ; 15(1): 1606, 2024 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-38383525

RESUMO

African Swine Fever Virus is a Nucleo-Cytoplasmic Large DNA Virus that causes an incurable haemorrhagic fever in pigs with a high impact on global food security. ASFV replicates in the cytoplasm of the infected cell and encodes its own transcription machinery that is independent of cellular factors, however, not much is known about how this system works at a molecular level. Here, we present methods to produce recombinant ASFV RNA polymerase, functional assays to screen for inhibitors, and high-resolution cryo-electron microscopy structures of the ASFV RNAP in different conformational states. The ASFV RNAP bears a striking resemblance to RNAPII with bona fide homologues of nine of its twelve subunits. Key differences include the fusion of the ASFV assembly platform subunits RPB3 and RPB11, and an unusual C-terminal domain of the stalk subunit vRPB7 that is related to the eukaryotic mRNA cap 2´-O-methyltransferase 1. Despite the high degree of structural conservation with cellular RNA polymerases, the ASFV RNAP is resistant to the inhibitors rifampicin and alpha-amanitin. The cryo-EM structures and fully recombinant RNAP system together provide an important tool for the design, development, and screening of antiviral drugs in a low biosafety containment environment.


Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , RNA , Suínos , Animais , Vírus da Febre Suína Africana/genética , Febre Suína Africana/genética , Febre Suína Africana/prevenção & controle , Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA/genética , Sus scrofa
2.
Microorganisms ; 10(9)2022 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-36144426

RESUMO

Multisubunit RNA polymerases (RNAPs) carry out transcription in all domains of life; during virus infection, RNAPs are targeted by transcription factors encoded by either the cell or the virus, resulting in the global repression of transcription with distinct outcomes for different host-virus combinations. These repressors serve as versatile molecular probes to study RNAP mechanisms, as well as aid the exploration of druggable sites for the development of new antibiotics. Here, we review the mechanisms and structural basis of RNAP inhibition by the viral repressor RIP and the crenarchaeal negative regulator TFS4, which follow distinct strategies. RIP operates by occluding the DNA-binding channel and mimicking the initiation factor TFB/TFIIB. RIP binds tightly to the clamp and locks it into one fixed position, thereby preventing conformational oscillations that are critical for RNAP function as it progresses through the transcription cycle. TFS4 engages with RNAP in a similar manner to transcript cleavage factors such as TFS/TFIIS through the NTP-entry channel; TFS4 interferes with the trigger loop and bridge helix within the active site by occlusion and allosteric mechanisms, respectively. The conformational changes in RNAP described above are universally conserved and are also seen in inactive dimers of eukaryotic RNAPI and several inhibited RNAP complexes of both bacterial and eukaryotic RNA polymerases, including inactive states that precede transcription termination. A comparison of target sites and inhibitory mechanisms reveals that proteinaceous repressors and RNAP-specific antibiotics use surprisingly common ways to inhibit RNAP function.

3.
Nat Commun ; 12(1): 5523, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34535646

RESUMO

RNA polymerase inhibition plays an important role in the regulation of transcription in response to environmental changes and in the virus-host relationship. Here we present the high-resolution structures of two such RNAP-inhibitor complexes that provide the structural bases underlying RNAP inhibition in archaea. The Acidianus two-tailed virus encodes the RIP factor that binds inside the DNA-binding channel of RNAP, inhibiting transcription by occlusion of binding sites for nucleic acid and the transcription initiation factor TFB. Infection with the Sulfolobus Turreted Icosahedral Virus induces the expression of the host factor TFS4, which binds in the RNAP funnel similarly to eukaryotic transcript cleavage factors. However, TFS4 allosterically induces a widening of the DNA-binding channel which disrupts trigger loop and bridge helix motifs. Importantly, the conformational changes induced by TFS4 are closely related to inactivated states of RNAP in other domains of life indicating a deep evolutionary conservation of allosteric RNAP inhibition.


Assuntos
RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/química , Vírus/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Proteínas Arqueais/metabolismo , Microscopia Crioeletrônica , DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Modelos Moleculares , Ligação Proteica , Estrutura Secundária de Proteína , Fatores de Tempo , Proteínas Virais/metabolismo , Viroides/metabolismo
4.
J Chem Inf Model ; 59(9): 3927-3937, 2019 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-31408337

RESUMO

NPAC is a cytokine-like nuclear factor involved in chromatin modification and regulation of gene expression. In humans, the C-terminal domain of NPAC has the conserved structure of the ß-hydroxyacid dehydrogenases (ß-HAD) protein superfamily, which forms a stable tetrameric core scaffold for demethylase enzymes and organizes multiple sites for chromatin interactions. In spite of the close structural resemblance to other ß-HAD family members, the human NPAC dehydrogenase domain lacks a highly conserved catalytic lysine, substituted by a methionine. The reintroduction of the catalytic lysine by M437 K mutation results in a significant decrease of stability of the tetramer. Here, we have computationally investigated the molecular determinants of the functional differences between methionine and lysine-containing NPAC proteins. We find that the single mutation can determine strong consequences in terms of dynamics, stability, and ultimately ability to assemble in supramolecular complexes: the higher stability and lower flexibility of the methionine variant structurally preorganizes the monomer for tetramerization, whereas lysine increases flexibility and favors conformations that, while catalytically active, are not optimal for tetrameric assembly. We combine structure-dynamics analysis to an evolutionary study of NPAC sequences, showing that the methionine mutation occurs in a specifically flexible region of the lysine-containing protein, flanked by two domains that concentrate most of the stabilizing interactions. In our model, such separation of stability nuclei and flexible regions appears to favor the functional innovability of the protein.


Assuntos
Simulação de Dinâmica Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Sequência de Aminoácidos , Animais , Evolução Molecular , Humanos , Proteínas Mutantes/genética , Proteínas Nucleares/genética , Oxirredutases/genética , Conformação Proteica , Termodinâmica
5.
Cell Rep ; 27(2): 387-399.e7, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30970244

RESUMO

LSD1 and LSD2 are homologous histone demethylases with opposite biological outcomes related to chromatin silencing and transcription elongation, respectively. Unlike LSD1, LSD2 nucleosome-demethylase activity relies on a specific linker peptide from the multidomain protein NPAC. We used single-particle cryoelectron microscopy (cryo-EM), in combination with kinetic and mutational analysis, to analyze the mechanisms underlying the function of the human LSD2/NPAC-linker/nucleosome complex. Weak interactions between LSD2 and DNA enable multiple binding modes for the association of the demethylase to the nucleosome. The demethylase thereby captures mono- and dimethyl Lys4 of the H3 tail to afford histone demethylation. Our studies also establish that the dehydrogenase domain of NPAC serves as a catalytically inert oligomerization module. While LSD1/CoREST forms a nucleosome docking platform at silenced gene promoters, LSD2/NPAC is a multifunctional enzyme complex with flexible linkers, tailored for rapid chromatin modification, in conjunction with the advance of the RNA polymerase on actively transcribed genes.


Assuntos
Histona Desmetilases/metabolismo , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Oxirredutases/metabolismo , Sequência de Aminoácidos , Desmetilação , Histona Desmetilases/química , Histona Desmetilases/genética , Histonas/metabolismo , Humanos , Modelos Moleculares , Enzimas Multifuncionais/química , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Nucleossomos/enzimologia , Nucleossomos/genética , Oxirredutases/química , Oxirredutases/genética , Domínios Proteicos
6.
Emerg Top Life Sci ; 2(4): 517-533, 2018 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-33525828

RESUMO

The archaeal RNA polymerase (RNAP) is a double-psi ß-barrel enzyme closely related to eukaryotic RNAPII in terms of subunit composition and architecture, promoter elements and basal transcription factors required for the initiation and elongation phase of transcription. Understanding archaeal transcription is, therefore, key to delineate the universally conserved fundamental mechanisms of transcription as well as the evolution of the archaeo-eukaryotic transcription machineries. The dynamic interplay between RNAP subunits, transcription factors and nucleic acids dictates the activity of RNAP and ultimately gene expression. This review focusses on recent progress in our understanding of (i) the structure, function and molecular mechanisms of known and less characterized factors including Elf1 (Elongation factor 1), NusA (N-utilization substance A), TFS4, RIP and Eta, and (ii) their evolution and phylogenetic distribution across the expanding tree of Archaea.

7.
Sci Adv ; 2(9): e1601017, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27626075

RESUMO

Because of its involvement in the progression of several malignant tumors, the histone lysine-specific demethylase 1 (LSD1) has become a prominent drug target in modern medicinal chemistry research. We report on the discovery of two classes of noncovalent inhibitors displaying unique structural features. The antibiotics polymyxins bind at the entrance of the substrate cleft, where their highly charged cyclic moiety interacts with a cluster of positively charged amino acids. The same site is occupied by quinazoline-based compounds, which were found to inhibit the enzyme through a most peculiar mode because they form a pile of five to seven molecules that obstruct access to the active center. These data significantly indicate unpredictable strategies for the development of epigenetic inhibitors.


Assuntos
Histona Desmetilases/química , Neoplasias/tratamento farmacológico , Polimixinas/farmacologia , Quinazolinas/farmacologia , Animais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Epigenômica , Histona Desmetilases/antagonistas & inibidores , Humanos , Neoplasias/genética , Neoplasias/patologia , Polimixinas/química , Ligação Proteica , Quinazolinas/química
8.
Hum Mol Genet ; 25(12): 2578-2587, 2016 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27094131

RESUMO

Genetic diseases often lead to rare and severe syndromes and the identification of the genetic and protein alterations responsible for the pathogenesis is essential to understand both the physiological and pathological role of the gene product. Recently, de novo variants have been mapped on the gene encoding for the lysine-specific histone demethylase 1 (LSD1)/lysine(K)-specific histone demethylase 1A in three patients characterized by a new genetic disorder. We have analyzed the effects of these pathological mutations on the structure, stability and activity of LSD1 using both in vitro and cellular approaches. The three mutations (Glu403Lys, Asp580Gly and Tyr785His) affect active-site residues and lead to a partial impairment of catalytic activity. They also differentially perturb the ability of LSD1 to engage transcription factors that orchestrate key developmental programs. Moreover, cellular data indicate a decrease in the protein cellular half-life. Taken together, these results demonstrate the relevance of LSD1 in gene regulation and how even moderate alterations in its stability, catalytic activity and binding properties can strongly affect organism development. This depicts a perturbed interplay of catalytic and non-catalytic processes at the origin of the pathology.


Assuntos
Histona Desmetilases/química , Histona Desmetilases/genética , Deficiência Intelectual/genética , Transcrição Gênica , Catálise , Domínio Catalítico/genética , Regulação da Expressão Gênica , Histona Desmetilases/metabolismo , Humanos , Deficiência Intelectual/patologia , Mutação , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Fatores de Transcrição/química , Fatores de Transcrição/genética
9.
EMBO J ; 35(4): 376-88, 2016 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-26787641

RESUMO

Chromatin-associated enzymes are responsible for the installation, removal and reading of precise post-translation modifications on DNA and histone proteins. They are specifically recruited to the target gene by associated factors, and as a result of their activity, they contribute in modulating cell identity and differentiation. Structural and biophysical approaches are broadening our knowledge on these processes, demonstrating that DNA, histone tails and histone surfaces can each function as distinct yet functionally interconnected anchoring points promoting nucleosome binding and modification. The mechanisms underlying nucleosome recognition have been described for many histone modifiers and related readers. Here, we review the recent literature on the structural organization of these nucleosome-associated proteins, the binding properties that drive nucleosome modification and the methodological advances in their analysis. The overarching conclusion is that besides acting on the same substrate (the nucleosome), each system functions through characteristic modes of action, which bring about specific biological functions in gene expression regulation.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Animais , Humanos , Modelos Moleculares , Biologia Molecular/métodos
10.
Proc Natl Acad Sci U S A ; 112(9): 2752-7, 2015 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-25730864

RESUMO

With its noncatalytic domains, DNA-binding regions, and a catalytic core targeting the histone tails, LSD1-CoREST (lysine-specific demethylase 1; REST corepressor) is an ideal model system to study the interplay between DNA binding and histone modification in nucleosome recognition. To this end, we covalently associated LSD1-CoREST to semisynthetic nucleosomal particles. This enabled biochemical and biophysical characterizations of nucleosome binding and structural elucidation by small-angle X-ray scattering, which was extensively validated through binding assays and site-directed mutagenesis of functional interfaces. Our results suggest that LSD1-CoREST functions as an ergonomic clamp that induces the detachment of the H3 histone tail from the nucleosomal DNA to make it available for capture by the enzyme active site. The key notion emerging from these studies is the inherently competitive nature of the binding interactions because nucleosome tails, chromatin modifiers, transcription factors, and DNA represent sites for multiple and often mutually exclusive interactions.


Assuntos
Proteínas Correpressoras/química , DNA/química , Histona Desmetilases/química , Histonas/química , Modelos Moleculares , Proteínas do Tecido Nervoso/química , Nucleossomos/química , Domínio Catalítico , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , DNA/genética , DNA/metabolismo , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Metilação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , Espalhamento a Baixo Ângulo , Difração de Raios X
11.
J Neurochem ; 128(5): 603-16, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24111946

RESUMO

Epigenetic mechanisms play important roles in brain development, orchestrating proliferation, differentiation, and morphogenesis. Lysine-Specific Demethylase 1 (LSD1 also known as KDM1A and AOF2) is a histone modifier involved in transcriptional repression, forming a stable core complex with the corepressors corepressor of REST (CoREST) and histone deacetylases (HDAC1/2). Importantly, in the mammalian CNS, neuronal LSD1-8a, an alternative splicing isoform of LSD1 including the mini-exon E8a, sets alongside LSD1 and is capable of enhancing neurite growth and morphogenesis. Here, we describe that the morphogenic properties of neuronal LSD1-8a require switching off repressive activity and this negative modulation is mediated in vivo by phosphorylation of the Thr369b residue coded by exon E8a. Three-dimensional crystal structure analysis using a phospho-mimetic mutant (Thr369bAsp), indicate that phosphorylation affects the residues surrounding the exon E8a-coded amino acids, causing a local conformational change. We suggest that phosphorylation, without affecting demethylase activity, causes in neurons CoREST and HDAC1/2 corepressors detachment from LSD1-8a and impairs neuronal LSD1-8a repressive activity. In neurons, Thr369b phosphorylation is required for morphogenic activity, converting neuronal LSD1-8a in a dominant-negative isoform, challenging LSD1-mediated transcriptional repression on target genes.


Assuntos
Proteínas Correpressoras/biossíntese , Proteínas Correpressoras/genética , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Histona Desmetilases/biossíntese , Histona Desmetilases/genética , Transcrição Gênica/genética , Animais , Química Encefálica/fisiologia , Células Cultivadas , Cromatina/metabolismo , Repressão Enzimática , Éxons/genética , Regulação Enzimológica da Expressão Gênica/genética , Genes Reporter , Imunoprecipitação , Isoenzimas/metabolismo , Espectrometria de Massas , Mutagênese Sítio-Dirigida , Neuritos/metabolismo , Fosforilação , Conformação Proteica , Ratos
12.
ACS Chem Biol ; 8(8): 1677-82, 2013 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-23721412

RESUMO

The combinatorial assembly of protein complexes is at the heart of chromatin biology. Lysine demethylase LSD1(KDM1A)/CoREST beautifully exemplifies this concept. The active site of the enzyme tightly associates to the N-terminal domain of transcription factors of the SNAIL1 family, which therefore can competitively inhibit the binding of the N-terminal tail of the histone substrate. Our enzymatic, crystallographic, spectroscopic, and computational studies reveal that LSD1/CoREST can bind to a hexapeptide derived from the SNAIL sequence through recognition of a positively charged α-helical turn that forms upon binding to the enzyme. Variations in sequence and length of this six amino acid ligand modulate affinities enabling the same binding site to differentially interact with proteins that exert distinct biological functions. The discovered short peptide inhibitors exhibit antiproliferative activities and lay the foundation for the development of peptidomimetic small molecule inhibitors of LSD1.


Assuntos
Inibidores Enzimáticos/farmacologia , Histona Desmetilases/antagonistas & inibidores , Proteínas do Tecido Nervoso/antagonistas & inibidores , Peptídeos/farmacologia , Proteínas Repressoras/antagonistas & inibidores , Sequência de Aminoácidos , Sítios de Ligação , Proliferação de Células/efeitos dos fármacos , Proteínas Correpressoras , Inibidores Enzimáticos/química , Histona Desmetilases/química , Humanos , Modelos Moleculares , Proteínas do Tecido Nervoso/metabolismo , Peptídeos/química , Ligação Proteica/efeitos dos fármacos , Proteínas Repressoras/metabolismo , Relação Estrutura-Atividade
13.
J Am Chem Soc ; 132(19): 6827-33, 2010 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-20415477

RESUMO

LSD1 and LSD2 histone demethylases are implicated in a number of physiological and pathological processes, ranging from tumorigenesis to herpes virus infection. A comprehensive structural, biochemical, and cellular study is presented here to probe the potential of these enzymes for epigenetic therapies. This approach employs tranylcypromine as a chemical scaffold for the design of novel demethylase inhibitors. This drug is a clinically validated antidepressant known to target monoamine oxidases A and B. These two flavoenzymes are structurally related to LSD1 and LSD2. Mechanistic and crystallographic studies of tranylcypromine inhibition reveal a lack of selectivity and differing covalent modifications of the FAD cofactor depending on the enantiomeric form. These findings are pharmacologically relevant, since tranylcypromine is currently administered as a racemic mixture. A large set of tranylcypromine analogues were synthesized and screened for inhibitory activities. We found that the common evolutionary origin of LSD and MAO enzymes, despite their unrelated functions and substrate specificities, is reflected in related ligand-binding properties. A few compounds with partial enzyme selectivity were identified. The biological activity of one of these new inhibitors was evaluated with a cellular model of acute promyelocytic leukemia chosen since its pathogenesis includes aberrant activities of several chromatin modifiers. Marked effects on cell differentiation and an unprecedented synergistic activity with antileukemia drugs were observed. These data demonstrate that these LSD1/2 inhibitors are of potential relevance for the treatment of promyelocytic leukemia and, more generally, as tools to alter chromatin state with promise of a block of tumor progression.


Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Histona Desmetilases/antagonistas & inibidores , Tranilcipromina/química , Tranilcipromina/farmacologia , Animais , Antineoplásicos/farmacologia , Linhagem Celular , Sinergismo Farmacológico , Histona Desmetilases/química , Humanos , Camundongos , Modelos Moleculares , Conformação Molecular , Estereoisomerismo , Especificidade por Substrato
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