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1.
Cell ; 184(4): 1064-1080.e20, 2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33606977

RESUMO

Understanding the functional consequences of single-nucleotide variants is critical to uncovering the genetic underpinnings of diseases, but technologies to characterize variants are limiting. Here, we leverage CRISPR-Cas9 cytosine base editors in pooled screens to scalably assay variants at endogenous loci in mammalian cells. We benchmark the performance of base editors in positive and negative selection screens, identifying known loss-of-function mutations in BRCA1 and BRCA2 with high precision. To demonstrate the utility of base editor screens to probe small molecule-protein interactions, we screen against BH3 mimetics and PARP inhibitors, identifying point mutations that confer drug sensitivity or resistance. We also create a library of single guide RNAs (sgRNAs) predicted to generate 52,034 ClinVar variants in 3,584 genes and conduct screens in the presence of cellular stressors, identifying loss-of-function variants in numerous DNA damage repair genes. We anticipate that this screening approach will be broadly useful to readily and scalably functionalize genetic variants.


Assuntos
Edição de Genes , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Alelos , Proteína BRCA1/genética , Proteína BRCA2/genética , Sequência de Bases , Domínio Catalítico , Linhagem Celular Tumoral , Humanos , Mutação com Perda de Função , Mutagênese/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Mutação Puntual/genética , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/genética , Reprodutibilidade dos Testes , Seleção Genética , Proteína bcl-X/genética
2.
Annu Rev Biochem ; 85: 431-54, 2016 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-26844395

RESUMO

Recent developments indicate that macrodomains, an ancient and diverse protein domain family, are key players in the recognition, interpretation, and turnover of ADP-ribose (ADPr) signaling. Crucial to this is the ability of macrodomains to recognize ADPr either directly, in the form of a metabolic derivative, or as a modification covalently bound to proteins. Thus, macrodomains regulate a wide variety of cellular and organismal processes, including DNA damage repair, signal transduction, and immune response. Their importance is further indicated by the fact that dysregulation or mutation of a macrodomain is associated with several diseases, including cancer, developmental defects, and neurodegeneration. In this review, we summarize the current insights into macrodomain evolution and how this evolution influenced their structural and functional diversification. We highlight some aspects of macrodomain roles in pathobiology as well as their emerging potential as therapeutic targets.


Assuntos
Reparo do DNA , Proteínas de Escherichia coli/química , Neoplasias/enzimologia , Poli(ADP-Ribose) Polimerases/química , Processamento de Proteína Pós-Traducional , Proteínas Repressoras/química , Viroses/enzimologia , Adenosina Difosfato Ribose/química , Adenosina Difosfato Ribose/metabolismo , Animais , Dano ao DNA , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Evolução Molecular , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Família Multigênica , Neoplasias/química , Neoplasias/genética , Neoplasias/patologia , Filogenia , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Domínios Proteicos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Homologia Estrutural de Proteína , Viroses/genética , Viroses/patologia , Viroses/virologia
3.
Nature ; 596(7873): 597-602, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34408320

RESUMO

ADP-ribosyltransferases use NAD+ to catalyse substrate ADP-ribosylation1, and thereby regulate cellular pathways or contribute to toxin-mediated pathogenicity of bacteria2-4. Reversible ADP-ribosylation has traditionally been considered a protein-specific modification5, but recent in vitro studies have suggested nucleic acids as targets6-9. Here we present evidence that specific, reversible ADP-ribosylation of DNA on thymidine bases occurs in cellulo through the DarT-DarG toxin-antitoxin system, which is found in a variety of bacteria (including global pathogens such as Mycobacterium tuberculosis, enteropathogenic Escherichia coli and Pseudomonas aeruginosa)10. We report the structure of DarT, which identifies this protein as a diverged member of the PARP family. We provide a set of high-resolution structures of this enzyme in ligand-free and pre- and post-reaction states, which reveals a specialized mechanism of catalysis that includes a key active-site arginine that extends the canonical ADP-ribosyltransferase toolkit. Comparison with PARP-HPF1, a well-established DNA repair protein ADP-ribosylation complex, offers insights into how the DarT class of ADP-ribosyltransferases evolved into specific DNA-modifying enzymes. Together, our structural and mechanistic data provide details of this PARP family member and contribute to a fundamental understanding of the ADP-ribosylation of nucleic acids. We also show that thymine-linked ADP-ribose DNA adducts reversed by DarG antitoxin (functioning as a noncanonical DNA repair factor) are used not only for targeted DNA damage to induce toxicity, but also as a signalling strategy for cellular processes. Using M. tuberculosis as an exemplar, we show that DarT-DarG regulates growth by ADP-ribosylation of DNA at the origin of chromosome replication.


Assuntos
ADP-Ribosilação , Proteínas de Bactérias/metabolismo , DNA/química , DNA/metabolismo , Timina/química , Timina/metabolismo , Adenosina Difosfato Ribose/metabolismo , Antitoxinas , Proteínas de Bactérias/química , Toxinas Bacterianas , Sequência de Bases , Biocatálise , DNA/genética , Adutos de DNA/química , Adutos de DNA/metabolismo , Dano ao DNA , Reparo do DNA , Elementos de DNA Transponíveis/genética , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Modelos Moleculares , Mycobacterium/enzimologia , Mycobacterium/genética , Nitrogênio/química , Nitrogênio/metabolismo , Poli(ADP-Ribose) Polimerases/química , Origem de Replicação/genética , Especificidade por Substrato , Thermus/enzimologia , Timidina/química , Timidina/metabolismo
4.
Trends Biochem Sci ; 47(5): 390-402, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-34366182

RESUMO

Poly-ADP-ribose-polymerases (PARPs) are a family of 17 enzymes that regulate a diverse range of cellular processes in mammalian cells. PARPs catalyze the transfer of ADP-ribose from NAD+ to target molecules, most prominently amino acids on protein substrates, in a process known as ADP-ribosylation. Identifying the direct protein substrates of individual PARP family members is an essential first step for elucidating the mechanism by which PARPs regulate a particular pathway in cells. Two distinct chemical genetic (CG) strategies have been developed for identifying the direct protein substrates of individual PARP family members. In this review, we discuss the design principles behind these two strategies and how target identification has provided novel insight into the cellular function of individual PARPs and PARP-mediated ADP-ribosylation.


Assuntos
ADP-Ribosilação , Inibidores de Poli(ADP-Ribose) Polimerases , Adenosina Difosfato Ribose/metabolismo , Animais , Mamíferos , Inibidores de Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas/metabolismo
5.
Nat Chem Biol ; 20(4): 463-472, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-37945894

RESUMO

Ubiquitination plays essential roles in eukaryotic cellular processes. The effector protein CteC from Chromobacterium violaceum blocks host ubiquitination by mono-ADP-ribosylation of ubiquitin (Ub) at residue T66. However, the structural basis for this modification is unknown. Here we report three crystal structures of CteC in complexes with Ub, NAD+ or ADP-ribosylated Ub, which represent different catalytic states of CteC in the modification. CteC adopts a special 'D-E' catalytic motif for catalysis and binds NAD+ in a half-ligand binding mode. The specific recognition of Ub by CteC is determined by a relatively separate Ub-targeting domain and a long loop L6, not the classic ADP-ribosylating turn-turn loop. Structural analyses with biochemical results reveal that CteC represents a large family of poly (ADP-ribose) polymerase (PARP)-like ADP-ribosyltransferases, which harbors chimeric features from the R-S-E and H-Y-E classes of ADP-ribosyltransferases. The family of CteC-like ADP-ribosyltransferases has a common 'D-E' catalytic consensus and exists extensively in bacteria and eukaryotic microorganisms.


Assuntos
Treonina , Ubiquitina , Ubiquitina/química , Treonina/metabolismo , NAD/metabolismo , ADP-Ribosilação , ADP Ribose Transferases/química , Poli(ADP-Ribose) Polimerases/química , Bactérias/metabolismo , Adenosina Difosfato Ribose
6.
Nature ; 585(7826): 609-613, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32939087

RESUMO

Breaks in DNA strands recruit the protein PARP1 and its paralogue PARP2 to modify histones and other substrates through the addition of mono- and poly(ADP-ribose) (PAR)1-5. In the DNA damage responses, this post-translational modification occurs predominantly on serine residues6-8 and requires HPF1, an accessory factor that switches the amino acid specificity of PARP1 and PARP2 from aspartate or glutamate to serine9,10. Poly(ADP) ribosylation (PARylation) is important for subsequent chromatin decompaction and provides an anchor for the recruitment of downstream signalling and repair factors to the sites of DNA breaks2,11. Here, to understand the molecular mechanism by which PARP enzymes recognize DNA breaks within chromatin, we determined the cryo-electron-microscopic structure of human PARP2-HPF1 bound to a nucleosome. This showed that PARP2-HPF1 bridges two nucleosomes, with the broken DNA aligned in a position suitable for ligation, revealing the initial step in the repair of double-strand DNA breaks. The bridging induces structural changes in PARP2 that signal the recognition of a DNA break to the catalytic domain, which licenses HPF1 binding and PARP2 activation. Our data suggest that active PARP2 cycles through different conformational states to exchange NAD+ and substrate, which may enable PARP enzymes to act processively while bound to chromatin. The processes of PARP activation and the PARP catalytic cycle we describe can explain mechanisms of resistance to PARP inhibitors and will aid the development of better inhibitors as cancer treatments12-16.


Assuntos
Proteínas de Transporte/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Biocatálise , Proteínas de Transporte/química , Proteínas de Transporte/ultraestrutura , Microscopia Crioeletrônica , DNA/metabolismo , Reparo do DNA , Ativação Enzimática , Humanos , Modelos Moleculares , NAD/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/ultraestrutura , Nucleossomos/química , Nucleossomos/ultraestrutura , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/ultraestrutura , Domínios Proteicos
7.
Nature ; 579(7800): 598-602, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32028527

RESUMO

The anti-cancer drug target poly(ADP-ribose) polymerase 1 (PARP1) and its close homologue, PARP2, are early responders to DNA damage in human cells1,2. After binding to genomic lesions, these enzymes use NAD+ to modify numerous proteins with mono- and poly(ADP-ribose) signals that are important for the subsequent decompaction of chromatin and the recruitment of repair factors3,4. These post-translational modifications are predominantly serine-linked and require the accessory factor HPF1, which is specific for the DNA damage response and switches the amino acid specificity of PARP1 and PARP2 from aspartate or glutamate to serine residues5-10. Here we report a co-structure of HPF1 bound to the catalytic domain of PARP2 that, in combination with NMR and biochemical data, reveals a composite active site formed by residues from HPF1 and PARP1 or PARP2 . The assembly of this catalytic centre is essential for the addition of ADP-ribose moieties after DNA damage in human cells. In response to DNA damage and occupancy of the NAD+-binding site, the interaction of HPF1 with PARP1 or PARP2 is enhanced by allosteric networks that operate within the PARP proteins, providing an additional level of regulation in the induction of the DNA damage response. As HPF1 forms a joint active site with PARP1 or PARP2, our data implicate HPF1 as an important determinant of the response to clinical PARP inhibitors.


Assuntos
ADP-Ribosilação , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Dano ao DNA , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerase-1/química , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/metabolismo , Regulação Alostérica , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Biocatálise , Proteínas de Transporte/genética , Domínio Catalítico , Células HEK293 , Humanos , Modelos Moleculares , Mutação , NAD/metabolismo , Ressonância Magnética Nuclear Biomolecular , Proteínas Nucleares/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Anêmonas-do-Mar
8.
Chem Rev ; 123(14): 9065-9093, 2023 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-37115110

RESUMO

Biomolecular condensates are reversible compartments that form through a process called phase separation. Post-translational modifications like ADP-ribosylation can nucleate the formation of these condensates by accelerating the self-association of proteins. Poly(ADP-ribose) (PAR) chains are remarkably transient modifications with turnover rates on the order of minutes, yet they can be required for the formation of granules in response to oxidative stress, DNA damage, and other stimuli. Moreover, accumulation of PAR is linked with adverse phase transitions in neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. In this review, we provide a primer on how PAR is synthesized and regulated, the diverse structures and chemistries of ADP-ribosylation modifications, and protein-PAR interactions. We review substantial progress in recent efforts to determine the molecular mechanism of PAR-mediated phase separation, and we further delineate how inhibitors of PAR polymerases may be effective treatments for neurodegenerative pathologies. Finally, we highlight the need for rigorous biochemical interrogation of ADP-ribosylation in vivo and in vitro to clarify the exact pathway from PARylation to condensate formation.


Assuntos
Poli Adenosina Difosfato Ribose , Poli(ADP-Ribose) Polimerases , Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/metabolismo , Condensados Biomoleculares , Poli ADP Ribosilação , Processamento de Proteína Pós-Traducional
9.
Nat Rev Mol Cell Biol ; 14(7): 443-51, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23736681

RESUMO

ADP-ribosylation of proteins was first described in the early 1960's, and today the function and regulation of poly(ADP-ribosyl)ation (PARylation) is partially understood. By contrast, little is known about intracellular mono(ADP-ribosyl)ation (MARylation) by ADP-ribosyl transferase (ART) enzymes, such as ARTD10. Recent findings indicate that MARylation regulates signalling and transcription by modifying key components in these processes. Emerging evidence also suggests that specific macrodomain-containing proteins, including ARTD8, macroD1, macroD2 and C6orf130, which are distinct from those affecting PARylation, interact with MARylation on target proteins to 'read' and 'erase' this modification. Thus, studying macrodomain-containing proteins is key to understanding the function and regulation of MARylation.


Assuntos
Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Animais , Regulação da Expressão Gênica , Humanos , Neoplasias/metabolismo , Poli(ADP-Ribose) Polimerases/química , Estrutura Terciária de Proteína , Transdução de Sinais , Transcrição Gênica , Resposta a Proteínas não Dobradas
10.
Mol Cell ; 65(5): 777-778, 2017 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-28257697

RESUMO

In this issue of Molecular Cell, Bonfiglio et al. (2017) demonstrate that histone PARylation factor 1 (HPF1) is required for PARP1 to attach ADP-ribose groups onto the hydroxyl oxygen of the Ser residues of target substrates, including both PARP1 itself and histones. Here, mechanisms and implications of this unexpected, O-linked ADP-ribosylation are speculated on.


Assuntos
Histonas/química , Poli(ADP-Ribose) Polimerases/química , Adenosina Difosfato Ribose , Poli(ADP-Ribose) Polimerase-1
11.
Biochem J ; 481(17): 1097-1123, 2024 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-39178157

RESUMO

ADP-ribosylation is a prominent and versatile post-translational modification, which regulates a diverse set of cellular processes. Poly-ADP-ribose (PAR) is synthesised by the poly-ADP-ribosyltransferases PARP1, PARP2, tankyrase (TNKS), and tankyrase 2 (TNKS2), all of which are linked to human disease. PARP1/2 inhibitors have entered the clinic to target cancers with deficiencies in DNA damage repair. Conversely, tankyrase inhibitors have continued to face obstacles on their way to clinical use, largely owing to our limited knowledge of their molecular impacts on tankyrase and effector pathways, and linked concerns around their tolerability. Whilst detailed structure-function studies have revealed a comprehensive picture of PARP1/2 regulation, our mechanistic understanding of the tankyrases lags behind, and thereby our appreciation of the molecular consequences of tankyrase inhibition. Despite large differences in their architecture and cellular contexts, recent structure-function work has revealed striking parallels in the regulatory principles that govern these enzymes. This includes low basal activity, activation by intra- or inter-molecular assembly, negative feedback regulation by auto-PARylation, and allosteric communication. Here we compare these poly-ADP-ribosyltransferases and point towards emerging parallels and open questions, whose pursuit will inform future drug development efforts.


Assuntos
Poli(ADP-Ribose) Polimerase-1 , Tanquirases , Tanquirases/metabolismo , Tanquirases/antagonistas & inibidores , Tanquirases/genética , Tanquirases/química , Humanos , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/genética , Animais , Processamento de Proteína Pós-Traducional , Reparo do DNA , ADP-Ribosilação , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli ADP Ribosilação/genética
12.
Subcell Biochem ; 104: 531-548, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38963499

RESUMO

Vault ribonucleoprotein particles are naturally designed nanocages, widely found in the eukaryotic kingdom. Vaults consist of 78 copies of the major vault protein (MVP) that are organized in 2 symmetrical cup-shaped halves, of an approximate size of 70x40x40 nm, leaving a huge internal cavity which accommodates the vault poly(ADP-ribose) polymerase (vPARP), the telomerase-associated protein-1 (TEP1) and some small untranslated RNAs. Diverse hypotheses have been developed on possible functions of vaults, based on their unique capsular structure, their rapid movements and the distinct subcellular localization of the particles, implicating transport of cargo, but they are all pending confirmation. Vault particles also possess many attributes that can be exploited in nanobiotechnology, particularly in the creation of vehicles for the delivery of multiple molecular cargoes. Here we review what is known about the structure and dynamics of the vault complex and discuss a possible mechanism for the vault opening process. The recent findings in the characterization of the vaults in cells and in its natural microenvironment will be also discussed.


Assuntos
Partículas de Ribonucleoproteínas em Forma de Abóbada , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo , Partículas de Ribonucleoproteínas em Forma de Abóbada/química , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética , Humanos , Animais , Poli(ADP-Ribose) Polimerases/metabolismo , Poli(ADP-Ribose) Polimerases/química
13.
Proc Natl Acad Sci U S A ; 119(11): e2121979119, 2022 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-35259019

RESUMO

SignificancePARP is an important target in the treatment of cancers, particularly in patients with breast, ovarian, or prostate cancer that have compromised homologous recombination repair (i.e., BRCA-/-). This review about inhibitors of PARP (PARPi) is for readers interested in the development of next-generation drugs for the treatment of cancer, providing insights into structure-activity relationships, in vitro vs. in vivo potency, PARP trapping, and synthetic lethality.


Assuntos
Inibidores de Poli(ADP-Ribose) Polimerases/química , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proteína BRCA1/genética , Proteína BRCA2/genética , Reparo do DNA , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Humanos , Modelos Moleculares , Estrutura Molecular , Mutação , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-Atividade , Mutações Sintéticas Letais
14.
Biochem Biophys Res Commun ; 720: 150101, 2024 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-38749191

RESUMO

Poly(ADP-ribose) polymerases (PARPs) are critical to regulating cellular activities, such as the response to DNA damage and cell death. PARPs catalyze a reversible post-translational modification (PTM) in the form of mono- or poly(ADP-ribosyl)ation. This type of modification is known to form a ubiquitin-ADP-ribose (Ub-ADPR) conjugate that depends on the actions of Deltex family of E3 ubiquitin ligases (DTXs). In particular, DTXs add ubiquitin to the 3'-OH of adenosine ribose' in ADP-ribose, which effectively sequesters ubiquitin and impedes ubiquitin-dependent signaling. Previous work demonstrates DTX function for ubiquitination of protein-free ADPR, mono-ADP-ribosylated peptides, and ADP-ribosylated nucleic acids. However, the dynamics of DTX-mediated ubiquitination of poly(ADP-ribosyl)ation remains to be defined. Here we show that the ADPR ubiquitination function is not found in other PAR-binding E3 ligases and is conserved across DTX family members. Importantly, DTXs specifically target poly(ADP-ribose) chains for ubiquitination that can be cleaved by PARG, the primary eraser of poly(ADP-ribose), leaving the adenosine-terminal ADPR unit conjugated to ubiquitin. Our collective results demonstrate the DTXs' specific ubiquitination of the adenosine terminus of poly(ADP-ribosyl)ation and suggest the unique Ub-ADPR conjugation process as a basis for PARP-DTX control of cellular activities.


Assuntos
Adenosina Difosfato Ribose , Ubiquitina-Proteína Ligases , Ubiquitinação , Ubiquitina-Proteína Ligases/metabolismo , Humanos , Adenosina Difosfato Ribose/metabolismo , Poli ADP Ribosilação , Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/genética , Ubiquitina/metabolismo , ADP-Ribosilação , Células HEK293
15.
Arch Biochem Biophys ; 756: 110010, 2024 06.
Artigo em Inglês | MEDLINE | ID: mdl-38642632

RESUMO

PARP1 plays a pivotal role in DNA repair within the base excision pathway, making it a promising therapeutic target for cancers involving BRCA mutations. Current study is focused on the discovery of PARP inhibitors with enhanced selectivity for PARP1. Concurrent inhibition of PARP1 with PARP2 and PARP3 affects cellular functions, potentially causing DNA damage accumulation and disrupting immune responses. In step 1, a virtual library of 593 million compounds has been screened using a shape-based screening approach to narrow down the promising scaffolds. In step 2, hierarchical docking approach embedded in Schrödinger suite was employed to select compounds with good dock score, drug-likeness and MMGBSA score. Analysis supplemented with decomposition energy, molecular dynamics (MD) simulations and hydrogen bond frequency analysis, pinpointed that active site residues; H862, G863, R878, M890, Y896 and F897 are crucial for specific binding of ZINC001258189808 and ZINC000092332196 with PARP1 as compared to PARP2 and PARP3. The binding of ZINC000656130962, ZINC000762230673, ZINC001332491123, and ZINC000579446675 also revealed interaction involving two additional active site residues of PARP1, namely N767 and E988. Weaker or no interaction was observed for these residues with PARP2 and PARP3. This approach advances our understanding of PARP-1 specific inhibitors and their mechanisms of action, facilitating the development of targeted therapeutics.


Assuntos
Antineoplásicos , Desenho de Fármacos , Simulação de Dinâmica Molecular , Poli(ADP-Ribose) Polimerase-1 , Inibidores de Poli(ADP-Ribose) Polimerases , Humanos , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/química , Inibidores de Poli(ADP-Ribose) Polimerases/química , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Simulação de Acoplamento Molecular , Domínio Catalítico , Poli(ADP-Ribose) Polimerases/metabolismo , Poli(ADP-Ribose) Polimerases/química , Ligação de Hidrogênio
16.
Protein Expr Purif ; 224: 106580, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-39154924

RESUMO

Poly-ADP-ribose polymerase-14 (PARP14) can modify proteins and nucleic acids by the reversible addition of a single ADP-ribose molecule. Aberrant PARP14 functions have been related to cancer and inflammation, and its domains are involved in processes related to viral infection. Previous research indicates that PARP14 functions might be mediated via a multitude of target proteins. In vitro studies of this large multidomain enzyme have been complicated by difficulties to obtain biochemical quantities of pure protein. Here we present a strategy that allows bacterial expression and purification of a functional multidomain construct of PARP14. We substituted an internal KH domain and its neighboring unstructured region with a SUMO domain to obtain a protein construct that encompasses three macrodomains, a WWE domain, and a PARP catalytic domain. We show that the resulting construct retains both ADP-ribosyltransferase and de-MARylase activities. This construct will be useful in structural and functional studies of PARP14.


Assuntos
Escherichia coli , Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Domínios Proteicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/biossíntese , Expressão Gênica , Clonagem Molecular
17.
Bioessays ; 44(7): e2200015, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35532219

RESUMO

The lysine demethylase KDM5A collaborates with PARP1 and the histone variant macroH2A1.2 to modulate chromatin to promote DNA repair. Indeed, KDM5A engages poly(ADP-ribose) (PAR) chains at damage sites through a previously uncharacterized coiled-coil domain, a novel binding mode for PAR interactions. While KDM5A is a well-known transcriptional regulator, its function in DNA repair is only now emerging. Here we review the molecular mechanisms that regulate this PARP1-macroH2A1.2-KDM5A axis in DNA damage and consider the potential involvement of this pathway in transcription regulation and cancer. Using KDM5A as an example, we discuss how multifunctional chromatin proteins transition between several DNA-based processes, which must be coordinated to protect the integrity of the genome and epigenome. The dysregulation of chromatin and loss of genome integrity that is prevalent in human diseases including cancer may be related and could provide opportunities to target multitasking proteins with these pathways as therapeutic strategies.


Assuntos
Inibidores de Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerases , Cromatina/genética , Dano ao DNA/genética , Reparo do DNA/genética , Humanos , Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Proteína 2 de Ligação ao Retinoblastoma/genética , Proteína 2 de Ligação ao Retinoblastoma/metabolismo
18.
Genes Cells ; 27(4): 305-312, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35124853

RESUMO

Mono(ADP-ribosyl)ation and poly(ADP-ribosyl)ation are posttranslational modifications evolutionarily conserved in prokaryotes and eukaryotes. They entail transfer of one or more ADP-ribose moieties from NAD+ to acceptor proteins with the simultaneous release of nicotinamide. The resultant ADP-ribosylated acceptor proteins regulate diverse cellular functions. For instance, ADP-ribosyltransferase 1 (ART1) catalyzes mono(ADP-ribosyl)ation of arginine residues in Trim72, a protein specifically expressed in muscle cells and involved in cell membrane repair, which is enhanced upon its ADP-ribosylation. By contrast, the contribution made by ADP-ribosylation to membrane repair in epithelial cells remains unclear. In this study, we investigated the involvement of ADP-ribosylation in cell membrane repair in HEK293T and HeLa cells. We found that upon induction of membrane damage using streptolysin-O, poly(ADP-ribose) polymerase 1 (PARP1) catalyzed poly(ADP-ribosyl)ation. In scratch assays, inhibition of PARP1 activity using the nonspecific PARP inhibitor PJ34 or shRNA targeting PARP1 delayed wound healing, suggesting that PARP1-catalyzed poly(ADP-ribosyl)ation plays a key role in membrane repair in epithelial cells.


Assuntos
Poli(ADP-Ribose) Polimerase-1 , Poli ADP Ribosilação , Poli Adenosina Difosfato Ribose , Células HEK293 , Células HeLa , Humanos , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo
19.
Mol Cell ; 60(5): 742-754, 2015 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-26626479

RESUMO

Poly(ADP-ribose)polymerase 1 (PARP-1) is a key eukaryotic stress sensor that responds in seconds to DNA single-strand breaks (SSBs), the most frequent genomic damage. A burst of poly(ADP-ribose) synthesis initiates DNA damage response, whereas PARP-1 inhibition kills BRCA-deficient tumor cells selectively, providing the first anti-cancer therapy based on synthetic lethality. However, the mechanism underlying PARP-1's function remained obscure; inherent dynamics of SSBs and PARP-1's multi-domain architecture hindered structural studies. Here we reveal the structural basis of SSB detection and how multi-domain folding underlies the allosteric switch that determines PARP-1's signaling response. Two flexibly linked N-terminal zinc fingers recognize the extreme deformability of SSBs and drive co-operative, stepwise self-assembly of remaining PARP-1 domains to control the activity of the C-terminal catalytic domain. Automodification in cis explains the subsequent release of monomeric PARP-1 from DNA, allowing repair and replication to proceed. Our results provide a molecular framework for understanding PARP inhibitor action and, more generally, allosteric control of dynamic, multi-domain proteins.


Assuntos
Quebras de DNA de Cadeia Simples , DNA/metabolismo , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/metabolismo , Domínio Catalítico , Cristalografia por Raios X , DNA/química , Reparo do DNA , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação de Ácido Nucleico , Poli(ADP-Ribose) Polimerase-1 , Dobramento de Proteína , Dedos de Zinco
20.
Mol Cell ; 60(5): 755-768, 2015 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-26626480

RESUMO

Poly(ADP-ribose) polymerase-1 (PARP-1) creates the posttranslational modification PAR from substrate NAD(+) to regulate multiple cellular processes. DNA breaks sharply elevate PARP-1 catalytic activity to mount a cell survival repair response, whereas persistent PARP-1 hyperactivation during severe genotoxic stress is associated with cell death. The mechanism for tight control of the robust catalytic potential of PARP-1 remains unclear. By monitoring PARP-1 dynamics using hydrogen/deuterium exchange-mass spectrometry (HXMS), we unexpectedly find that a specific portion of the helical subdomain (HD) of the catalytic domain rapidly unfolds when PARP-1 encounters a DNA break. Together with biochemical and crystallographic analysis of HD deletion mutants, we show that the HD is an autoinhibitory domain that blocks productive NAD(+) binding. Our molecular model explains how PARP-1 DNA damage detection leads to local unfolding of the HD that relieves autoinhibition, and has important implications for the design of PARP inhibitors.


Assuntos
DNA/metabolismo , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/metabolismo , Desdobramento de Proteína , Domínio Catalítico , Cristalografia por Raios X , Quebras de DNA , Reparo do DNA , Medição da Troca de Deutério , Humanos , Modelos Moleculares , Mutação , NAD/metabolismo , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/genética , Estrutura Secundária de Proteína
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