Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.250
Filtrar
1.
Nat Commun ; 12(1): 4581, 2021 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-34321462

RESUMO

Poly(ADP-ribosyl)ation (PAR) is a versatile and complex posttranslational modification composed of repeating units of ADP-ribose arranged into linear or branched polymers. This scaffold is linked to the regulation of many of cellular processes including the DNA damage response, alteration of chromatin structure and Wnt signalling. Despite decades of research, the principles and mechanisms underlying all steps of PAR removal remain actively studied. In this work, we synthesise well-defined PAR branch point molecules and demonstrate that PARG, but not ARH3, can resolve this distinct PAR architecture. Structural analysis of ARH3 in complex with dimeric ADP-ribose as well as an ADP-ribosylated peptide reveal the molecular basis for the hydrolysis of linear and terminal ADP-ribose linkages. We find that ARH3-dependent hydrolysis requires both rearrangement of a catalytic glutamate and induction of an unusual, square-pyramidal magnesium coordination geometry.


Assuntos
Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Poli ADP Ribosilação/fisiologia , ADP-Ribosilação , Adenosina Difosfato Ribose/metabolismo , Animais , Catálise , Humanos , Hidrólise , Poli ADP Ribosilação/genética , Processamento de Proteína Pós-Traducional , Via de Sinalização Wnt
2.
Int J Mol Sci ; 22(14)2021 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-34299254

RESUMO

The transient receptor potential (TRP) melastatin-like subfamily member 2 (TRPM2) is a non-selective calcium-permeable cation channel. It is expressed by many mammalian tissues, including bone marrow, spleen, lungs, heart, liver, neutrophils, and endothelial cells. The best-known mechanism of TRPM2 activation is related to the binding of ADP-ribose to the nudix-box sequence motif (NUDT9-H) in the C-terminal domain of the channel. In cells, the production of ADP-ribose is a result of increased oxidative stress. In the context of endothelial function, TRPM2-dependent calcium influx seems to be particularly interesting as it participates in the regulation of barrier function, cell death, cell migration, and angiogenesis. Any impairments of these functions may result in endothelial dysfunction observed in such conditions as atherosclerosis or hypertension. Thus, TRPM2 seems to be an attractive therapeutic target for the conditions connected with the increased production of reactive oxygen species. However, before the application of TRPM2 inhibitors will be possible, some issues need to be resolved. The main issues are the lack of specificity, poor membrane permeabilization, and low stability in in vivo conditions. The article aims to summarize the latest findings on a role of TRPM2 in endothelial cells. We also show some future perspectives for the application of TRPM2 inhibitors in cardiovascular system diseases.


Assuntos
Células Endoteliais/metabolismo , Canais de Cátion TRPM/metabolismo , Adenosina Difosfato Ribose/metabolismo , Animais , Cálcio/metabolismo , Morte Celular , Movimento Celular , Células Endoteliais/fisiologia , Humanos , Ativação do Canal Iônico/fisiologia , Estresse Oxidativo/fisiologia , Pirofosfatases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/fisiologia
3.
Toxicol Sci ; 183(1): 154-169, 2021 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-34129049

RESUMO

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly-adenosine diphosphate (ADP)-ribose polymerase (TIPARP/PARP7), an aryl hydrocarbon receptor (AHR) target gene and mono-ADP-ribosyltransferase, acts as part of a negative feedback loop to repress AHR signaling. This process is prevented by a single H532A mutation in TIPARP that destroys its catalytic activity. We hypothesized that the loss of TIPARP catalytic activity would increase sensitivity to TCDD-induced toxicity in vivo. To test this, we created a catalytically deficient mouse line (TiparpH532A) by introducing a single H532A mutation in TIPARP. Treatment of mouse embryonic fibroblasts or hepatocytes isolated from TiparpH532A mice confirmed the increased TCDD-induced expression of the AHR target genes Cyp1a1, Cyp1b1, and Tiparp. TiparpH532A mice given a single injection of 10 µg/kg TCDD, a nonlethal dose in Tiparp+/+ mice, did not survive beyond day 10. All Tiparp+/+ mice survived the 30-day treatment. TCDD-treated TiparpH532A mice displayed increased expression of AHR target genes, increased steatohepatitis and hepatotoxicity. Hepatic RNA-sequencing revealed 7-fold more differentially expressed genes in TiparpH532A mice than in Tiparp+/+ mice (4542 vs 647 genes) 6 days after TCDD treatment. Differentially expressed genes included genes involved in xenobiotic metabolism, lipid homeostasis and inflammation. Taken together, these data further support TIPARP as a critical negative regulator of AHR activity and show that loss of its catalytic activity is sufficient to increase sensitivity to TCDD-induced steatohepatitis and lethality. Since TIPARP inhibition has recently emerged as a potential anticancer therapy, the impact on AHR signaling, TCDD and polycyclic aromatic hydrocarbon toxicity will need to be carefully considered under conditions of therapeutic TIPARP inhibition.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Dibenzodioxinas Policloradas , Adenosina Difosfato Ribose , Animais , Doença Hepática Induzida por Substâncias e Drogas/genética , Fibroblastos , Camundongos , Dibenzodioxinas Policloradas/toxicidade , Receptores de Hidrocarboneto Arílico/genética
4.
Methods Mol Biol ; 2276: 165-171, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34060040

RESUMO

ADP-ribosylation is a posttranslational protein modification, involved in various cellular processes, ranging from DNA-damage repair to apoptosis. While its function has been studied amply with respect to genotoxic stress-associated nuclear ADP-ribosylation, the functional relevance of mitochondrial ADP-ribosylation remains so far poorly studied. This is mainly attributed to the absence of powerful techniques able to detect the modification. However, the usage of recently developed anti-ADP-ribose-specific antibodies allows now to investigate mitochondrial ADP-ribosylation under physiological and pathophysiological conditions. In the below method, we describe in detail how to efficiently detect and quantify mitochondrial ADP-ribosylation via immunofluorescence.


Assuntos
Adenosina Difosfato Ribose/metabolismo , Imunofluorescência/métodos , Mitocôndrias/metabolismo , ADP-Ribosilação , Animais , Células Cultivadas , Humanos
5.
PLoS One ; 16(6): e0254022, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34191856

RESUMO

ADP-ribosylation is a key post-translational modification that regulates a wide variety of cellular stress responses. The ADP-ribosylation cycle is maintained by writers and erasers. For example, poly(ADP-ribosyl)ation cycles consist of two predominant enzymes, poly(ADP-ribose) polymerases (PARPs) and poly(ADP-ribose) glycohydrolase (PARG). However, historically, mechanisms of erasers of ADP-ribosylations have been understudied, primarily due to the lack of quantitative tools to selectively monitor specific activities of different ADP-ribosylation reversal enzymes. Here, we developed a new NUDT5-coupled AMP-Glo (NCAG) assay to specifically monitor the protein-free ADP-ribose released by ADP-ribosylation reversal enzymes. We found that NUDT5 selectively cleaves protein-free ADP-ribose, but not protein-bound poly- and mono-ADP-ribosylations, protein-free poly(ADP-ribose) chains, or NAD+. As a proof-of-concept, we successfully measured the kinetic parameters for the exo-glycohydrolase activity of PARG, which releases monomeric ADP-ribose, and monitored activities of site-specific mono-ADP-ribosyl-acceptor hydrolases, such as ARH3 and TARG1. This NCAG assay can be used as a general platform to study the mechanisms of diverse ADP-ribosylation reversal enzymes that release protein-free ADP-ribose as a product. Furthermore, this assay provides a useful tool to identify small-molecule probes targeting ADP-ribosylation metabolism and to quantify ADP-ribose concentrations in cells.


Assuntos
ADP-Ribosilação , Adenosina Difosfato Ribose/metabolismo , Enzimas/metabolismo , Aminoácidos/metabolismo , Glicosídeo Hidrolases/metabolismo , Humanos , Hidrolases/metabolismo , Cinética , Pirofosfatases/metabolismo
6.
Mol Cell ; 81(12): 2640-2655.e8, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34019811

RESUMO

ARH3/ADPRHL2 and PARG are the primary enzymes reversing ADP-ribosylation in vertebrates, yet their functions in vivo remain unclear. ARH3 is the only hydrolase able to remove serine-linked mono(ADP-ribose) (MAR) but is much less efficient than PARG against poly(ADP-ribose) (PAR) chains in vitro. Here, by using ARH3-deficient cells, we demonstrate that endogenous MARylation persists on chromatin throughout the cell cycle, including mitosis, and is surprisingly well tolerated. Conversely, persistent PARylation is highly toxic and has distinct physiological effects, in particular on active transcription histone marks such as H3K9ac and H3K27ac. Furthermore, we reveal a synthetic lethal interaction between ARH3 and PARG and identify loss of ARH3 as a mechanism of PARP inhibitor resistance, both of which can be exploited in cancer therapy. Finally, we extend our findings to neurodegeneration, suggesting that patients with inherited ARH3 deficiency suffer from stress-induced pathogenic increase in PARylation that can be mitigated by PARP inhibition.


Assuntos
Glicosídeo Hidrolases/metabolismo , Poli ADP Ribosilação/fisiologia , ADP-Ribosilação , Adenosina Difosfato Ribose/metabolismo , Linhagem Celular Tumoral , Cromatina , DNA , Dano ao DNA , Fibroblastos/metabolismo , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/fisiologia , Células HEK293 , Células HeLa , Humanos , Poli Adenosina Difosfato Ribose/metabolismo , Cultura Primária de Células
7.
J Proteome Res ; 20(6): 3090-3101, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34032442

RESUMO

Blood and plasma proteins are heavily investigated as biomarkers for different diseases. However, the post-translational modification states of these proteins are rarely analyzed since blood contains many enzymes that rapidly remove these modifications after sampling. In contrast to the well-described role of protein ADP-ribosylation in cells and organs, its role in blood remains mostly uncharacterized. Here, we discovered that plasma phosphodiesterases and/or ADP-ribosylhydrolases rapidly demodify in vitro ADP-ribosylated proteins. Thus, to identify the in vivo whole blood and plasma ADP-ribosylomes, we established a mass-spectrometry-based workflow that was applied to blood samples collected from LPS-treated pigs (Sus scrofa domesticus), which serves as a model for human systemic inflammatory response syndrome. These analyses identified 60 ADP-ribosylated proteins, 17 of which were ADP-ribosylated plasma proteins. This new protocol provides an important step forward for the rapidly developing field of ADP-ribosylation and defines the blood and plasma ADP-ribosylomes under both healthy and disease conditions.


Assuntos
ADP-Ribosilação , Processamento de Proteína Pós-Traducional , Difosfato de Adenosina , Adenosina Difosfato Ribose/metabolismo , Animais , Espectrometria de Massas , Proteínas/metabolismo , Suínos
8.
Cells ; 10(3)2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33808662

RESUMO

Adenosine diphosphate (ADP)-ribosylation is a nicotinamide adenine dinucleotide (NAD+)-dependent post-translational modification that is found on proteins as well as on nucleic acids. While ARTD1/PARP1-mediated poly-ADP-ribosylation has extensively been studied in the past 60 years, comparably little is known about the physiological function of mono-ADP-ribosylation and the enzymes involved in its turnover. Promising technological advances have enabled the development of innovative tools to detect NAD+ and NAD+/NADH (H for hydrogen) ratios as well as ADP-ribosylation. These tools have significantly enhanced our current understanding of how intracellular NAD dynamics contribute to the regulation of ADP-ribosylation as well as to how mono-ADP-ribosylation integrates into various cellular processes. Here, we discuss the recent technological advances, as well as associated new biological findings and concepts.


Assuntos
Adenosina Difosfato Ribose/metabolismo , NAD/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , ADP-Ribosilação/fisiologia , Humanos , Poli(ADP-Ribose) Polimerase-1/metabolismo
10.
Commun Biol ; 4(1): 123, 2021 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-33504944

RESUMO

The macro domain is an ADP-ribose (ADPR) binding module, which is considered to act as a sensor to recognize nicotinamide adenine dinucleotide (NAD) metabolites, including poly ADPR (PAR) and other small molecules. The recognition of macro domains with various ligands is important for a variety of biological functions involved in NAD metabolism, including DNA repair, chromatin remodeling, maintenance of genomic stability, and response to viral infection. Nevertheless, how the macro domain binds to moieties with such structural obstacles using a simple cleft remains a puzzle. We systematically investigated the Middle East respiratory syndrome-coronavirus (MERS-CoV) macro domain for its ligand selectivity and binding properties by structural and biophysical approaches. Of interest, NAD, which is considered not to interact with macro domains, was co-crystallized with the MERS-CoV macro domain. Further studies at physiological temperature revealed that NAD has similar binding ability with ADPR because of the accommodation of the thermal-tunable binding pocket. This study provides the biochemical and structural bases of the detailed ligand-binding mode of the MERS-CoV macro domain. In addition, our observation of enhanced binding affinity of the MERS-CoV macro domain to NAD at physiological temperature highlights the need for further study to reveal the biological functions.


Assuntos
Coronavírus da Síndrome Respiratória do Oriente Médio/química , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , NAD/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Adenosina Difosfato Ribose/metabolismo , Sítios de Ligação , Fenômenos Biofísicos , Cristalização , Cristalografia por Raios X , Humanos , Ligantes , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Poli Adenosina Difosfato Ribose/metabolismo , Ligação Proteica , Domínios Proteicos , Estabilidade Proteica , Termodinâmica
11.
Proc Natl Acad Sci U S A ; 118(2)2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33397718

RESUMO

Macrodomains are proteins that recognize and hydrolyze ADP ribose (ADPR) modifications of intracellular proteins. Macrodomains are implicated in viral genome replication and interference with host cell immune responses. They are important to the infectious cycle of Coronaviridae and Togaviridae viruses. We describe crystal structures of the conserved macrodomain from the bat coronavirus (CoV) HKU4 in complex with ligands. The structures reveal a binding cavity that accommodates ADPR and analogs via local structural changes within the pocket. Using a radioactive assay, we present evidence of mono-ADPR (MAR) hydrolase activity. In silico analysis presents further evidence on recognition of the ADPR modification for hydrolysis. Mutational analysis of residues within the binding pocket resulted in diminished enzymatic activity and binding affinity. We conclude that the common structural features observed in the macrodomain in a bat CoV contribute to a conserved function that can be extended to other known macrodomains.


Assuntos
Adenosina Difosfato Ribose/química , Coronavirus/enzimologia , Pirofosfatases/química , Proteínas não Estruturais Virais/química , Animais , Sítios de Ligação , Quirópteros , Coronavirus/genética , Cristalografia por Raios X , Hidrólise , Pirofosfatases/genética , Proteínas não Estruturais Virais/genética
12.
Toxins (Basel) ; 13(1)2021 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-33450958

RESUMO

Scabin is a mono-ADP-ribosyltransferase toxin/enzyme and possible virulence factor produced by the agriculture pathogen, Streptomyces scabies. Recently, molecular dynamic approaches and MD simulations revealed its interaction with both NAD+ and DNA substrates. An Essential Dynamics Analysis identified a crab-claw-like mechanism, including coupled changes in the exposed motifs, and the Rß1-RLa-NLc-STTß2-WPN-WARTT-(QxE)ARTT sequence motif was proposed as a catalytic signature of the Pierisin family of DNA-acting toxins. A new fluorescence assay was devised to measure the kinetics for both RNA and DNA substrates. Several protein variants were prepared to probe the Scabin-NAD-DNA molecular model and to reveal the reaction mechanism for the transfer of ADP-ribose to the guanine base in the DNA substrate. The results revealed that there are several lysine and arginine residues in Scabin that are important for binding the DNA substrate; also, key residues such as Asn110 in the mechanism of ADP-ribose transfer to the guanine base were identified. The DNA-binding residues are shared with ScARP from Streptomyces coelicolor but are not conserved with Pierisin-1, suggesting that the modification of guanine bases by ADP-ribosyltransferases is divergent even in the Pierisin family.


Assuntos
ADP Ribose Transferases/química , Proteínas de Bactérias/química , Toxinas Bacterianas/química , Streptomyces/enzimologia , Adenosina Difosfato Ribose/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Proteínas de Ligação a DNA/química , Guanina/metabolismo , Cinética , Simulação de Dinâmica Molecular , NAD/química , NAD/metabolismo , Ligação Proteica , Conformação Proteica , RNA/química , RNA/metabolismo , Relação Estrutura-Atividade
13.
J Am Soc Mass Spectrom ; 32(1): 157-168, 2021 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-33140951

RESUMO

ADP-ribosylation is a reversible post-translational modification of proteins that has been linked to many biological processes. The identification of ADP-ribosylated proteins and particularly of their acceptor amino acids remains a major challenge. The attachment sites of the modification are difficult to localize by mass spectrometry (MS) because of the labile nature of the linkage and the complex fragmentation pattern of the ADP-ribose in MS/MS experiments. In this study we performed a comprehensive analysis of higher-energy collisional dissociation (HCD) spectra acquired from ADP-ribosylated peptides which were modified on arginine, serine, glutamic acid, aspartic acid, tyrosine, or lysine residues. In addition to the fragmentation of the peptide backbone, various cleavages of the ADP-ribosylated amino acid side chains were investigated. We focused on gas-phase fragmentations that were specific either to ADP-ribosylated arginine or to ADP-ribosylated serine and other O-linked ADP-ribosylations. The O-glycosidic linkage between ADP-ribose and serine, glutamic acid, or aspartic acid was the major cleavage site, making localization of these modification sites difficult. In contrast, the bond between ADP-ribose and arginine was relatively stable. The main cleavage site was the inner bond of the guanidine group, which resulted in the formation of ADP-ribosylated carbodiimide and of ornithine in place of modified arginine. Taking peptide fragment ions resulting from this specific cleavage into account, a considerably larger number of peptides containing ADP-ribosylated arginine were identified in database searches. Furthermore, the presence of diagnostic ions and of losses of fragments from peptide ions allowed us, in most cases, to distinguish between ADP-ribosylated arginine and serine residues.


Assuntos
Arginina/química , Espectrometria de Massas/métodos , Peptídeos/química , ADP-Ribosilação , Adenosina Difosfato Ribose/química , Adenosina Difosfato Ribose/metabolismo , Arginina/metabolismo , Bases de Dados de Proteínas , Gases , Guanidina/química , Processamento de Proteína Pós-Traducional , Serina/química , Serina/metabolismo
14.
J Virol ; 95(3)2021 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-33158944

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-related CoVs encode 3 tandem macrodomains within nonstructural protein 3 (nsp3). The first macrodomain, Mac1, is conserved throughout CoVs and binds to and hydrolyzes mono-ADP-ribose (MAR) from target proteins. Mac1 likely counters host-mediated antiviral ADP-ribosylation, a posttranslational modification that is part of the host response to viral infections. Mac1 is essential for pathogenesis in multiple animal models of CoV infection, implicating it as a virulence factor and potential therapeutic target. Here, we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose. SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV) Mac1 domains exhibit similar structural folds, and all 3 proteins bound to ADP-ribose with affinities in the low micromolar range. Importantly, using ADP-ribose-detecting binding reagents in both a gel-based assay and novel enzyme-linked immunosorbent assays (ELISAs), we demonstrated de-MARylating activity for all 3 CoV Mac1 proteins, with the SARS-CoV-2 Mac1 protein leading to a more rapid loss of substrate than the others. In addition, none of these enzymes could hydrolyze poly-ADP-ribose. We conclude that the SARS-CoV-2 and other CoV Mac1 proteins are MAR-hydrolases with similar functions, indicating that compounds targeting CoV Mac1 proteins may have broad anti-CoV activity.IMPORTANCE SARS-CoV-2 has recently emerged into the human population and has led to a worldwide pandemic of COVID-19 that has caused more than 1.2 million deaths worldwide. With no currently approved treatments, novel therapeutic strategies are desperately needed. All coronaviruses encode a highly conserved macrodomain (Mac1) that binds to and removes ADP-ribose adducts from proteins in a dynamic posttranslational process that is increasingly being recognized as an important factor that regulates viral infection. The macrodomain is essential for CoV pathogenesis and may be a novel therapeutic target. Thus, understanding its biochemistry and enzyme activity are critical first steps for these efforts. Here, we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose and describe its ADP-ribose binding and hydrolysis activities in direct comparison to those of SARS-CoV and MERS-CoV Mac1 proteins. These results are an important first step for the design and testing of potential therapies targeting this unique protein domain.


Assuntos
N-Glicosil Hidrolases/metabolismo , SARS-CoV-2/enzimologia , Proteínas não Estruturais Virais/metabolismo , Adenosina Difosfato Ribose/química , Adenosina Difosfato Ribose/metabolismo , Sequência de Aminoácidos , Coronavirus/química , Coronavirus/enzimologia , Coronavirus/metabolismo , Cristalografia por Raios X , Humanos , Hidrólise , Cinética , N-Glicosil Hidrolases/química , Ligação Proteica , Domínios Proteicos , SARS-CoV-2/química , SARS-CoV-2/metabolismo , Proteínas não Estruturais Virais/química
15.
Nucleic Acids Res ; 49(D1): D261-D265, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33137182

RESUMO

ADP-ribosylation is a protein modification responsible for biological processes such as DNA repair, RNA regulation, cell cycle and biomolecular condensate formation. Dysregulation of ADP-ribosylation is implicated in cancer, neurodegeneration and viral infection. We developed ADPriboDB (adpribodb.leunglab.org) to facilitate studies in uncovering insights into the mechanisms and biological significance of ADP-ribosylation. ADPriboDB 2.0 serves as a one-stop repository comprising 48 346 entries and 9097 ADP-ribosylated proteins, of which 6708 were newly identified since the original database release. In this updated version, we provide information regarding the sites of ADP-ribosylation in 32 946 entries. The wealth of information allows us to interrogate existing databases or newly available data. For example, we found that ADP-ribosylated substrates are significantly associated with the recently identified human protein interaction networks associated with SARS-CoV-2, which encodes a conserved protein domain called macrodomain that binds and removes ADP-ribosylation. In addition, we create a new interactive tool to visualize the local context of ADP-ribosylation, such as structural and functional features as well as other post-translational modifications (e.g. phosphorylation, methylation and ubiquitination). This information provides opportunities to explore the biology of ADP-ribosylation and generate new hypotheses for experimental testing.


Assuntos
Adenosina Difosfato Ribose/metabolismo , Biologia Computacional/estatística & dados numéricos , Bases de Dados de Proteínas/estatística & dados numéricos , Proteínas/metabolismo , ADP-Ribosilação , Sítios de Ligação , COVID-19/epidemiologia , COVID-19/prevenção & controle , COVID-19/virologia , Biologia Computacional/métodos , Humanos , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Proteínas/química , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiologia , Proteínas Virais/química , Proteínas Virais/metabolismo
16.
IEEE/ACM Trans Comput Biol Bioinform ; 18(4): 1262-1270, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33306471

RESUMO

SARS-CoV-2 encodes the Mac1 domain within the large nonstructural protein 3 (Nsp3), which has an ADP-ribosylhydrolase activity conserved in other coronaviruses. The enzymatic activity of Mac1 makes it an essential virulence factor for the pathogenicity of coronavirus (CoV). They have a regulatory role in counteracting host-mediated antiviral ADP-ribosylation, which is unique part of host response towards viral infections. Mac1 shows highly conserved residues in the binding pocket for the mono and poly ADP-ribose. Therefore, SARS-CoV-2 Mac1 enzyme is considered as an ideal drug target and inhibitors developed against them can possess a broad antiviral activity against CoV. ADP-ribose-1 phosphate bound closed form of Mac1 domain is considered for screening with large database of ZINC. XP docking and QPLD provides strong potential lead compounds, that perfectly fits inside the binding pocket. Quantum mechanical studies expose that, substrate and leads have similar electron donor ability in the head regions, that allocates tight binding inside the substrate-binding pocket. Molecular dynamics study confirms the substrate and new lead molecules presence of electron donor and acceptor makes the interactions tight inside the binding pocket. Overall binding phenomenon shows both substrate and lead molecules are well-adopt to bind with similar binding mode inside the closed form of Mac1.


Assuntos
COVID-19/tratamento farmacológico , COVID-19/virologia , Proteases Semelhantes à Papaína de Coronavírus/antagonistas & inibidores , Proteases Semelhantes à Papaína de Coronavírus/química , SARS-CoV-2/efeitos dos fármacos , Adenosina Difosfato Ribose/metabolismo , Sequência de Aminoácidos , Antivirais/farmacologia , Biologia Computacional , Proteases Semelhantes à Papaína de Coronavírus/genética , Ensaios de Triagem em Larga Escala/métodos , Ensaios de Triagem em Larga Escala/estatística & dados numéricos , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Domínios Proteicos , Teoria Quântica , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Interface Usuário-Computador
17.
Nucleic Acids Res ; 48(22): 12746-12750, 2020 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-33264406

RESUMO

As nucleotidyl transferases, formation of a covalent enzyme-adenylate intermediate is a common first step of all DNA ligases. While it has been shown that eukaryotic DNA ligases utilize ATP as the adenylation donor, it was recently reported that human DNA ligase IV can also utilize NAD+ and, to a lesser extent ADP-ribose, as the source of the adenylate group and that NAD+, unlike ATP, enhances ligation by supporting multiple catalytic cycles. Since this unexpected finding has significant implications for our understanding of the mechanisms and regulation of DNA double strand break repair, we attempted to confirm that NAD+ and ADP-ribose can be used as co-factors by human DNA ligase IV. Here, we provide evidence that NAD+ does not enhance ligation by pre-adenylated DNA ligase IV, indicating that this co-factor is not utilized for re-adenylation and subsequent cycles of ligation. Moreover, we find that ligation by de-adenylated DNA ligase IV is dependent upon ATP not NAD+ or ADP-ribose. Thus, we conclude that human DNA ligase IV cannot use either NAD+ or ADP-ribose as adenylation donor for ligation.


Assuntos
Adenosina Difosfato Ribose/genética , DNA Ligase Dependente de ATP/genética , DNA/genética , NAD/genética , Monofosfato de Adenosina/genética , Trifosfato de Adenosina/genética , Sequência de Aminoácidos/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Humanos
18.
Nat Commun ; 11(1): 5600, 2020 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-33154359

RESUMO

The α-type ADP-ribosylated peptides represent a class of important molecular tools in the field of protein ADP-ribosylation, however, they are difficult to access because of their inherent complicated structures and the lack of effective synthetic tools. In this paper, we present a biomimetic α-selective ribosylation reaction to synthesize a key intermediate, α-ADP-ribosyl azide, directly from native ß-nicotinamide adenine dinucleotide in a clean ionic liquid system. This reaction in tandem with click chemistry then offers a two-step modular synthesis of α-ADP-ribosylated peptides. These syntheses can be performed open air in eppendorf tubes, without the need for specialized instruments or training. Importantly, we demonstrate that the synthesized α-ADP-ribosylated peptides show high binding affinity and desirable stability for enriching protein partners, and reactivity in post-stage poly ADP-ribosylations. Owing to their simple chemistry and multidimensional bio-applications, the presented methods may provide a powerful platform to produce general molecular tools for the study of protein ADP-ribosylation.


Assuntos
Adenosina Difosfato Ribose/química , Materiais Biomiméticos/síntese química , Peptídeos/síntese química , ADP-Ribosilação , Materiais Biomiméticos/química , Materiais Biomiméticos/metabolismo , Catálise , Química Click , Histonas/metabolismo , Líquidos Iônicos/química , NAD/química , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica
19.
J Transl Med ; 18(1): 439, 2020 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-33213473

RESUMO

BACKGROUND: Poly(ADP-ribose) polymerase inhibitors (PARPis) specifically target homologous recombination deficiency (HRD) cells and display good therapeutic effect in women with advanced-stage BRCA1/2-mutated breast and epithelial ovarian cancer (EOC). However, about 50% of high grade serous ovarian cancers (HGSOC) present with HRD due to epigenetic BRCA1 inactivation, as well as genetic/epigenetic inactivation(s) of other HR genes, a feature known as "BRCAness". Therefore, there is a potential for extending the use of PARPis to these patients if HR status can be identified. METHODS: We have developed a 3D (spheroid) functional assay to assess the sensitivity of two PARPis (niraparib and olaparib) in ascites-derived primary cell cultures (AsPCs) from HGSOC patients. A method for AsPCs preparation was established based on a matrix (agarose), allowing for easy isolation and successive propagation of monolayer and 3D AsPCs. Based on this method, we performed cytotoxicity assays on 42 AsPCs grown both as monolayers and spheroids. RESULTS: The response to PARPis treatment in monolayer AsPCs, was significantly higher, compared to 3D AsPCs, as 88% and 52% of the monolayer AsPCs displayed sensitivity to niraparib and olaparib respectively, while 66% of the 3D AsPCs were sensitive to niraparib and 38% to olaparib, the latter being more consistent with previous estimates of HRD (40%-60%) in EOC. Moreover, niraparib displayed a significantly stronger cytotoxic effect in both in 3D and monolayer AsPCs, which was confirmed by consecutive analyses of the HR pathway activity (γH2AX foci formation) in PARPis-sensitive and resistant AsPCs. Global gene expression comparison of 6 PARPi-resistant and 6 PARPi-sensitive 3D AsPCs was indicative for the predominant downregulation of numerous genes and networks with previously demonstrated roles in EOC chemoresistance, suggesting that the PARPis-sensitive AsPCs could display enhanced sensitivity to other chemotherapeutic drugs, commonly applied in cancer management. Microarray data validation identified 24 potential gene biomarkers associated with PARPis sensitivity. The differential expression of 7 selected biomarkers was consecutively confirmed by immunohistochemistry in matched EOC tumor samples. CONCLUSION: The application of this assay and the potential biomarkers with possible predictive significance to PARPis therapy of EOC patients now need testing in the setting of a clinical trial.


Assuntos
Neoplasias Ovarianas , Inibidores de Poli(ADP-Ribose) Polimerases , Adenosina Difosfato Ribose/uso terapêutico , Biomarcadores , Carcinoma Epitelial do Ovário/tratamento farmacológico , Carcinoma Epitelial do Ovário/genética , Feminino , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico
20.
Am J Case Rep ; 21: e927008, 2020 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-33191394

RESUMO

BACKGROUND Thrombocytopenia is a potentially treatment-limiting adverse event of particular interest with the PARP inhibitor niraparib. This adverse event may necessitate niraparib dose reduction or treatment discontinuation, resulting in suboptimal treatment outcomes. Here, we report on niraparib dose optimization in 2 patients with breast cancer and 4 patients with ovarian cancer through concurrent administration of the thrombopoietin receptor stimulating agent avatrombopag to mitigate thrombocytopenia, enabling niraparib reescalation and improved clinical response. CASE REPORT Three of 6 patients received niraparib 300 mg daily, the highest recommended dose, for a sustained period. Avatrombopag therapy enabled niraparib dose escalation that led to reductions in biomarkers associated with disease progression. Before initiation of avatrombopag, increases in CA-125 levels, a marker for ovarian cancer, were observed in association with niraparib dose interruption, and in 2 patients with ovarian cancer CA-125 levels fell in response to niraparib dose escalation enabled by concurrent avatrombopag therapy. Further, in 2 patients with metastatic breast cancer, intracranial response was observed in association with avatrombopag-enabled niraparib therapy. In 1 patient with metastatic breast cancer, niraparib induced an intracranial response, while previous use of talazoparib had not, confirming preclinical findings of superior blood-brain-barrier penetrance with niraparib. CONCLUSIONS Avatrombopag is currently approved for use in chronic immune thrombocytopenia and thrombocytopenia associated with chronic liver disease in patients undergoing a surgical procedure. A clinical trial of avatrombopag for chemotherapy-induced thrombocytopenia is ongoing. Preliminary results in these 6 patient cases demonstrate the need for a confirmatory trial of avatrombopag for optimizing the dose of niraparib.


Assuntos
Neoplasias da Mama , Neoplasias Ovarianas , Trombocitopenia , Adenosina Difosfato Ribose/uso terapêutico , Neoplasias da Mama/complicações , Neoplasias da Mama/tratamento farmacológico , Feminino , Humanos , Indazóis/uso terapêutico , Neoplasias Ovarianas/complicações , Neoplasias Ovarianas/tratamento farmacológico , Piperidinas , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Poli(ADP-Ribose) Polimerases/uso terapêutico , Tiazóis , Tiofenos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...