Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 158
Filtrar
Mais filtros










Filtros aplicados
Base de dados
Intervalo de ano de publicação
1.
Sci Rep ; 14(1): 3875, 2024 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-38365924

RESUMO

ADP-ribosyltransferases PARP1 and PARP2 play a major role in DNA repair mechanism by detecting the DNA damage and inducing poly-ADP-ribosylation dependent chromatin relaxation and recruitment of repair proteins. Catalytic PARP inhibitors are used as anticancer drugs especially in the case of tumors arising from sensitizing mutations. Recently, a study showed that Histone PARylation Factor (HPF1) forms a joint active site with PARP1/2. The interaction of HPF1 with PARP1/2 alters the modification site from Aspartate/Glutamate to Serine, which has been shown to be a key ADP-ribosylation event in the context of DNA damage. Therefore, disruption of PARP1/2-HPF1 interaction could be an alternative strategy for drug development to block the PARP1/2 activity. In this study, we describe a FRET based high-throughput screening assay to screen inhibitor libraries against PARP-HPF1 interaction. We optimized the conditions for FRET signal and verified the interaction by competing the FRET pair in multiple ways. The assay is robust and easy to automate. Validatory screening showed the robust performance of the assay, and we discovered two compounds Dimethylacrylshikonin and Alkannin, with µM inhibition potency against PARP1/2-HPF1 interaction. The assay will facilitate the discovery of inhibitors against HPF1-PARP1/2 complex and to develop potentially new effective anticancer agents.


Assuntos
Antineoplásicos , Histonas , Inibidores de Poli(ADP-Ribose) Polimerases , Antineoplásicos/química , Antineoplásicos/farmacologia , Dano ao DNA , Reparo do DNA , Ensaios de Triagem em Larga Escala , Histonas/efeitos dos fármacos , Histonas/metabolismo , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli ADP Ribosilação , Inibidores de Poli(ADP-Ribose) Polimerases/química , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia
2.
Eur J Pharmacol ; 967: 176377, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38346469

RESUMO

Poly (ADP-ribose) polymerase-1 (PARP-1) activity significantly increases during cerebral ischemia/reperfusion. PARP-1 is an NAD+-consumption enzyme. PARP-1 hyperactivity causes intracellular NAD+ deficiency and bioenergetic collapse, contributing to neuronal death. Besides, the powerful trigger of PARP-1 causes the catalyzation of poly (ADP-ribosyl)ation (PARylation), a posttranslational modification of proteins. Here, we found that PARP-1 was activated in the ischemic brain tissue during middle-cerebral-artery occlusion and reperfusion (MCAO/R) for 24 h, and PAR accumulated in the neurons in mice. Using immunoprecipitation, Western blotting, liquid chromatography-mass spectrometry, and 3D-modeling analysis, we revealed that the activation of PARP-1 caused PARylation of hexokinase-1 and lactate dehydrogenase-B, which, therefore, caused the inhibition of these enzyme activities and the resulting cell energy metabolism collapse. PARP-1 inhibition significantly reversed the activity of hexokinase and lactate dehydrogenase, decreased infarct volume, and improved neuronal deficiency. PARP-1 inhibitor combined with pyruvate further alleviated MCAO/R-induced ischemic brain injury in mice. As such, we conclude that PARP-1 inhibitor alleviates neuronal death partly by inhibiting the PARylation of metabolic-related enzymes and reversing metabolism reprogramming during cerebral ischemia/reperfusion injury in mice. PARP-1 inhibitor combined with pyruvate might be a promising therapeutic approach against brain ischemia/reperfusion injury.


Assuntos
Isquemia Encefálica , Traumatismo por Reperfusão , Camundongos , Animais , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Poli(ADP-Ribose) Polimerases/metabolismo , Poli ADP Ribosilação , Hexoquinase/metabolismo , NAD/metabolismo , Traumatismo por Reperfusão/tratamento farmacológico , Isquemia Encefálica/tratamento farmacológico , Piruvatos , Lactato Desidrogenases/metabolismo
3.
Nat Commun ; 15(1): 1000, 2024 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-38307862

RESUMO

The chromatin remodeler ALC1 is activated by DNA damage-induced poly(ADP-ribose) deposited by PARP1/PARP2 and their co-factor HPF1. ALC1 has emerged as a cancer drug target, but how it is recruited to ADP-ribosylated nucleosomes to affect their positioning near DNA breaks is unknown. Here we find that PARP1/HPF1 preferentially initiates ADP-ribosylation on the histone H2B tail closest to the DNA break. To dissect the consequences of such asymmetry, we generate nucleosomes with a defined ADP-ribosylated H2B tail on one side only. The cryo-electron microscopy structure of ALC1 bound to such an asymmetric nucleosome indicates preferential engagement on one side. Using single-molecule FRET, we demonstrate that this asymmetric recruitment gives rise to directed sliding away from the DNA linker closest to the ADP-ribosylation site. Our data suggest a mechanism by which ALC1 slides nucleosomes away from a DNA break to render it more accessible to repair factors.


Assuntos
Nucleossomos , Poli ADP Ribosilação , Nucleossomos/genética , Microscopia Crioeletrônica , Poli(ADP-Ribose) Polimerase-1/metabolismo , Cromatina , Reparo do DNA , Quebras de DNA
4.
Mol Cell ; 84(3): 429-446.e17, 2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38215753

RESUMO

Nucleosomes, the basic structural units of chromatin, hinder recruitment and activity of various DNA repair proteins, necessitating modifications that enhance DNA accessibility. Poly(ADP-ribosyl)ation (PARylation) of proteins near damage sites is an essential initiation step in several DNA-repair pathways; however, its effects on nucleosome structural dynamics and organization are unclear. Using NMR, cryoelectron microscopy (cryo-EM), and biochemical assays, we show that PARylation enhances motions of the histone H3 tail and DNA, leaving the configuration of the core intact while also stimulating nuclease digestion and ligation of nicked nucleosomal DNA by LIG3. PARylation disrupted interactions between nucleosomes, preventing self-association. Addition of LIG3 and XRCC1 to PARylated nucleosomes generated condensates that selectively partition DNA repair-associated proteins in a PAR- and phosphorylation-dependent manner in vitro. Our results establish that PARylation influences nucleosomes across different length scales, extending from the atom-level motions of histone tails to the mesoscale formation of condensates with selective compositions.


Assuntos
Nucleossomos , Poli ADP Ribosilação , Nucleossomos/genética , Poli ADP Ribosilação/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Microscopia Crioeletrônica , Condensados Biomoleculares , Reparo do DNA , Histonas/genética , Histonas/metabolismo , DNA/genética , DNA/metabolismo , Dano ao DNA , Poli(ADP-Ribose) Polimerase-1/metabolismo
5.
Nat Commun ; 15(1): 184, 2024 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-38167803

RESUMO

The intracellular ATP-ribosyltransferases PARP1 and PARP2, contribute to DNA base excision repair (BER) and DNA demethylation and have been implicated in epigenetic programming in early mammalian development. Recently, proteomic analyses identified BER proteins to be covalently poly-ADP-ribosylated by PARPs. The role of this posttranslational modification in the BER process is unknown. Here, we show that PARP1 senses AP-sites and SSBs generated during TET-TDG mediated active DNA demethylation and covalently attaches PAR to each BER protein engaged. Covalent PARylation dissociates BER proteins from DNA, which accelerates the completion of the repair process. Consistently, inhibition of PARylation in mESC resulted both in reduced locus-specific TET-TDG-targeted DNA demethylation, and in reduced general repair of random DNA damage. Our findings establish a critical function of covalent protein PARylation in coordinating molecular processes associated with dynamic DNA methylation.


Assuntos
Reparo do DNA , Animais , Poli ADP Ribosilação , Desmetilação do DNA , Proteômica , Poli(ADP-Ribose) Polimerase-1/metabolismo , Dano ao DNA , DNA/genética , DNA/metabolismo , Mamíferos/genética
6.
DNA Repair (Amst) ; 133: 103593, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38029688

RESUMO

To maintain tissue homeostasis, cell proliferation is balanced by cell death. PARP1 is an important protein involved in both processes. Upon sensing DNA damage, PARP1 forms poly(ADP-ribose) (PAR) chains to recruit the repair proteins, ensuring genome integrity and faithful cell proliferation. In addition, PAR also regulates the activity of PARP1. Persistent DNA damage can signal the cell to progress toward programmed cell death, apoptosis. During apoptosis, proteolytic cleavage of PARP1 generates an N-terminal, ZnF1-2PARP1 (DNA binding or regulatory fragment), and C-terminal, PARP1ΔZnF1-2 (catalytic or PAR carrier fragment), which exhibits a basal activity. Regulation of the apoptotic fragments by PAR has not been studied. Here, we report that PAR inhibits the basal level activity of PARP1ΔZnF1-2, and ZnF1-2PARP1 interacts with PARP1ΔZnF1-2 to exhibit DNA-dependent stimulation and partially restores the PAR-dependent stimulation. Interestingly, along with the auto-modification domain of PARP1, the DNA-binding domains, ZnF1-2PARP1, also acts as an acceptor of PARylation; therefore, ZnF1-2PARP1 exhibits a reduced affinity for DNA upon PARylation. Furthermore, we show that ZnF1-2PARP1 shows trans-dominant inhibition of DNA-dependent stimulation of PARP2. Altogether, our study explores the regulation of the catalytic activity of PARP1ΔZnF1-2 and PARP2 by the regulatory apoptotic fragment of PARP1.


Assuntos
DNA , Poli Adenosina Difosfato Ribose , Poli(ADP-Ribose) Polimerase-1/metabolismo , DNA/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Poli ADP Ribosilação , Reparo do DNA , Dano ao DNA
7.
Genes Genomics ; 46(1): 27-36, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37713069

RESUMO

BACKGROUND: Tendon stem/progenitor cells (TSPCs) play a vital role in tendon repair, regeneration and homeostasis. However, the specific mechanism of TSPCs aging is still unclear. OBJECTIVE: This study aims to explore the role and molecular mechanism of HPF1 in the aging of TSPCs. METHODS: Young and aged TSPCs (Y-TSPCs and A-TSPCs) were acquired from 3 to 4 and 24-26-month-old Sprague-Dawley male rats, TSPCs (Y-TSPCs and A-TSPCs) were subjected to senescence-associated ß-galactosidase (SA-ß-Gal))staining and telomerase activity detection, p16, p21, Scx, Tnmd, Col1, Col3HPF1 and PAPR1 expression levels were detected by Western blot or Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR), Reciprocal co-immunoprecipitation (co-IP) was used to explore the interaction between HPF1 and PARP1. Ribonucleoprotein immunoprecipitation (RNP-IP) was used to analyze the binding of HuR to the senescence marker gene mRNAs, IP was used to perform HPF1 to the PARylation of HuR, and the half-life of p16 and p21 were detected. Finally, we established an in vivo model, and the tendon tissue was used to perform hematoxylin and eosin (HE) and masson's trichrome staining, as well as the immunohistochemical analysis of Col I and TNMD. RESULTS: Compared with Y-TSPCs, A-TSPCs had significantly enhanced cell senescence and significantly reduced tendon differentiation ability, and significantly increased the expression of HPF1 and PARP1. In addition, HPF1 and PARP1 interacted and coordinated the senescence and differentiation of TSPCs, HPF1 could also regulate the expression of p21 and p21, the interaction of p16 or p21 with HuR, and the poly-ADP ribosylation of PARP1 to HuR. HPF1 overexpression and siHuR co-transfection significantly reduced the half-life of p16 and p21, and HPF1 and PARP1 regulated the mRNA levels of p16 and p21 through HuR. Finally, in vivo experiments have shown that HPF1 or PARP1 overexpression could both inhibit the ability of tendon differentiation and promote cell senescence. CONCLUSIONS: HPF1 promoted the senescence of TSPCs and inhibits the tendon differentiation of TSPCs through PARP1-mediated poly-ADP ribosylation of HuR.


Assuntos
Senescência Celular , Poli ADP Ribosilação , Ratos , Animais , Masculino , Ratos Sprague-Dawley , Tendões/metabolismo , Células-Tronco/metabolismo
8.
Int J Mol Sci ; 24(23)2023 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-38069287

RESUMO

Tankyrases, a versatile protein group within the poly(ADP-ribose) polymerase family, are essential for post-translational poly(ADP-ribosyl)ation, influencing various cellular functions and contributing to diseases, particularly cancer. Consequently, tankyrases have become important targets for anti-cancer drug development. Emerging approaches in drug discovery aim to disrupt interactions between tankyrases and their binding partners, which hinge on tankyrase-binding motifs (TBMs) within partner proteins and ankyrin repeat cluster domains within tankyrases. Our study addresses the challenge of identifying and ranking TBMs. We have conducted a comprehensive review of the existing literature, classifying TBMs into three distinct groups, each with its own scoring system. To facilitate this process, we introduce TBM Hunter-an accessible, web-based tool. This user-friendly platform provides a cost-free and efficient means to screen and assess potential TBMs within any given protein. TBM Hunter can handle individual proteins or lists of proteins simultaneously. Notably, our results demonstrate that TBM Hunter not only identifies known TBMs but also uncovers novel ones. In summary, our study offers an all-encompassing perspective on TBMs and presents an easy-to-use, precise, and free tool for identifying and evaluating potential TBMs in any protein, thereby enhancing research and drug development efforts focused on tankyrases.


Assuntos
Tanquirases , Tanquirases/metabolismo , Repetição de Anquirina , Poli ADP Ribosilação
9.
Nucleic Acids Res ; 51(20): 11056-11079, 2023 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-37823600

RESUMO

Zinc finger (ZNF) motifs are some of the most frequently occurring domains in the human genome. It was only recently that ZNF proteins emerged as key regulators of genome integrity in mammalian cells. In this study, we report a new role for the Krüppel-type ZNF-containing protein ZNF432 as a novel poly(ADP-ribose) (PAR) reader that regulates the DNA damage response. We show that ZNF432 is recruited to DNA lesions via DNA- and PAR-dependent mechanisms. Remarkably, ZNF432 stimulates PARP-1 activity in vitro and in cellulo. Knockdown of ZNF432 inhibits phospho-DNA-PKcs and increases RAD51 foci formation following irradiation. Moreover, purified ZNF432 preferentially binds single-stranded DNA and impairs EXO1-mediated DNA resection. Consequently, the loss of ZNF432 in a cellular system leads to resistance to PARP inhibitors while its overexpression results in sensitivity. Taken together, our results support the emerging concept that ZNF-containing proteins can modulate PARylation, which can be embodied by the pivotal role of ZNF432 to finely balance the outcome of PARPi response by regulating homologous recombination.


Assuntos
Poli ADP Ribosilação , Poli Adenosina Difosfato Ribose , Humanos , DNA/genética , DNA/metabolismo , Dano ao DNA , Reparo do DNA , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo
10.
SLAS Discov ; 28(8): 394-401, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37844763

RESUMO

PARP1/2 inhibitors (PARPi) are effective clinically used drugs for the treatment of cancers with BRCA deficiencies. PARPi have had limited success and applicability beyond BRCA deficient cancers, and their effect is diminished by resistance mechanisms. The recent discovery of Histone PARylation Factor (HPF1) and the role it plays in the PARylation reaction by forming a shared active site with PARP1 raises the possibility that novel inhibitors that target the PARP1-HPF1 complex can be identified. Herein we describe a simple and cost-effective high-throughput screening (HTS) method aimed at discovering inhibitors of the PARP1-HPF1 complex. Upon HTS validation, we first applied this method to screen a small PARP-focused library of compounds and then scale up our approach using robotic automation to conduct a pilot screen of 10,000 compounds and validating >100 hits. This work demonstrates for the first time the capacity to discover potent inhibitors of the PARP1-HPF1 complex, which may have utility as probes to better understand the DNA damage response and as therapeutics for cancer.


Assuntos
Histonas , Neoplasias , Inibidores de Poli(ADP-Ribose) Polimerases , Humanos , Domínio Catalítico , Histonas/metabolismo , Neoplasias/tratamento farmacológico , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Poli ADP Ribosilação , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico
11.
Cell Death Dis ; 14(8): 524, 2023 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-37582914

RESUMO

Although only a small number of primordial follicles are known to be selectively activated during female reproductive cycles, the mechanisms that trigger this recruitment remain largely uncharacterized. Misregulated activation of primordial follicles may lead to the exhaustion of the non-renewable pool of primordial follicles, resulting in premature ovarian insufficiency. Here, we found that poly(ADP-ribose) polymerase 1 (PARP1) enzymatic activity in the surrounding granulosa cells (GCs) in follicles determines the subpopulation of the dormant primordial follicles to be awakened. Conversely, specifically inhibiting PARP1 in oocytes in an in vitro mouse follicle reconstitution model does not affect primordial follicle activation. Further analysis revealed that PARP1-catalyzed transcription factor YY1 PARylation at Y185 residue facilitates YY1 occupancy at Grp78 promoter, a key molecular chaperone of endoplasmic reticulum stress (ERS), and promotes Grp78 transcription in GCs, which is required for GCs maintaining proper ERS during primordial follicle activation. Inhibiting PARP1 prevents the loss of primordial follicle pool by attenuating the excessive ERS in GCs under fetal bisphenol A exposure. Together, we demonstrate that PARP1 in GCs acts as a pivotal modulator to determine the fate of the primordial follicles and may represent a novel therapeutic target for the retention of primordial follicle pool in females.


Assuntos
Estresse do Retículo Endoplasmático , Células da Granulosa , Poli(ADP-Ribose) Polimerase-1 , Poli ADP Ribosilação , Animais , Feminino , Camundongos , Catálise , Chaperona BiP do Retículo Endoplasmático , Células da Granulosa/metabolismo , Oócitos/metabolismo , Folículo Ovariano/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo
12.
Biochemistry ; 62(16): 2382-2390, 2023 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-37531469

RESUMO

PARP1, upon binding to damaged DNA, is activated to perform poly ADP-ribosylation (PARylation) on itself and other proteins, which leads to relaxation of chromatin and recruitment of DNA repair factors. HPF1 was recently discovered as a protein cofactor of PARP1 that directs preferential PARylation of histones over other targets by contributing to and altering the PARP1 active site. Inhibitors of PARP1 (PARPi) are used in the treatment of BRCA-/- cancers, but the basis for their potency in cells, especially in the context of HPF1, is not fully understood. Here, we demonstrate the simple one-step association for eight different PARPi to PARP1 with measured rates of association (kon) of 0.8-6 µM-1 s-1. We find only minor differences in these on rates when comparing PARP1 with the PARP1-HPF1 complex. By characterizing the rates of dissociation (koff) and the binding constants (KD) for two more recently discovered PARPi, we find, for example, that saruparib has a half-life for dissociation of 22.5 h and fluzoparib has higher affinity for PARP1 in the presence of HPF1, just like the structurally related compound olaparib. By using the measured KD and kon to calculate koff, we find that the potency of PARPi in cells correlates best with the koff from the PARP1-HPF1 complex. Our data suggest that dissociation of a drug compound from the PARP1-HPF1 complex should be the parameter of choice for guiding the development of next-generation PARPi.


Assuntos
Dano ao DNA , Histonas , Poli(ADP-Ribose) Polimerase-1/metabolismo , Histonas/metabolismo , Reparo do DNA , Poli ADP Ribosilação
13.
J Biol Chem ; 299(8): 105032, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37437887

RESUMO

Radiotherapy is one of the mainstay treatments for hepatocellular carcinoma (HCC). However, a substantial number of patients with HCC develop radioresistance and eventually suffer from tumor progression or relapse, which is a major impediment to the use of radiotherapy. Therefore, elucidating the mechanisms underlying radioresistance and identifying novel therapeutic targets to improve patient prognosis are important in HCC management. In this study, using in vitro and in vivo models, laser microirradiation and live cell imaging methods, and coimmunoprecipitation assays, we report that a DNA repair enhancer, human positive cofactor 4 (PC4), promotes nonhomologous end joining-based DNA repair and renders HCC cells resistant to radiation. Mechanistically, PC4 interacts with poly (ADP-ribose) polymerase 1 and directs Ku complex PARylation, resulting in the successful recruitment of the Ku complex to damaged chromatin and increasing the efficiency of nonhomologous end joining repair. Clinically, PC4 is highly expressed in tumor tissues and is correlated with poor prognosis in patients with HCC. Taken together, our data suggest that PC4 is a DNA repair driver that can be targeted to radiosensitize HCC cells.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/radioterapia , Dano ao DNA , Reparo do DNA por Junção de Extremidades , Reparo do DNA , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo , Neoplasias Hepáticas/genética , Recidiva Local de Neoplasia , Poli ADP Ribosilação , Tolerância a Radiação
14.
Int J Mol Sci ; 24(9)2023 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-37175698

RESUMO

Atopic dermatitis (AD) is a chronic inflammatory skin disease of very high prevalence, especially in childhood, with no specific treatment or cure. As its pathogenesis is complex, multifactorial and not fully understood, further research is needed to increase knowledge and develop new targeted therapies. We have recently demonstrated the critical role of NAD+ and poly (ADP-ribose) (PAR) metabolism in oxidative stress and skin inflammation. Specifically, we found that hyperactivation of PARP1 in response to DNA damage induced by reactive oxygen species, and fueled by NAMPT-derived NAD+, mediated inflammation through parthanatos cell death in zebrafish and human organotypic 3D skin models of psoriasis. Furthermore, the aberrant induction of NAMPT and PARP activity was observed in the lesional skin of psoriasis patients, supporting the role of these signaling pathways in psoriasis and pointing to NAMPT and PARP1 as potential novel therapeutic targets in treating skin inflammatory disorders. In the present work, we report, for the first time, altered NAD+ and PAR metabolism in the skin of AD patients and a strong correlation between NAMPT and PARP1 expression and the lesional status of AD. Furthermore, using a human 3D organotypic skin model of AD, we demonstrate that the pharmacological inhibition of NAMPT and PARP reduces pathology-associated biomarkers. These results help to understand the complexity of AD and reveal new potential treatments for AD patients.


Assuntos
Dermatite Atópica , Psoríase , Animais , Humanos , Inflamação , NAD/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Poli ADP Ribosilação , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Psoríase/etiologia , Peixe-Zebra/metabolismo
15.
Sci Rep ; 13(1): 7869, 2023 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-37188809

RESUMO

PARP2 is a member of the PARP enzyme family. Although, PARP2 plays role in DNA repair, it has regulatory roles in mitochondrial and lipid metabolism, it has pivotal role in bringing about the adverse effects of pharmacological PARP inhibitors. Previously, we showed that the ablation of PARP2 induces oxidative stress and, consequently, mitochondrial fragmentation. In attempt to identify the source of the reactive species we assessed the possible role of a central regulator of cellular antioxidant defense, nuclear factor erythroid 2-related factor 2 (NRF2). The silencing of PARP2 did not alter either the mRNA or the protein expression of NRF2, but changed its subcellular localization, decreasing the proportion of nuclear, active fraction of NRF2. Pharmacological inhibition of PARP2 partially restored the normal localization pattern of NRF2 and in line with that, we showed that NRF2 is PARylated that is absent in the cells in which PARP2 was silenced. Apparently, the PARylation of NRF2 by PARP2 has pivotal role in regulating the subcellular (nuclear) localization of NRF2. The silencing of PARP2 rearranged the expression of genes encoding proteins with antioxidant function, among these a subset of NRF2-dependent genes.


Assuntos
Antioxidantes , Fator 2 Relacionado a NF-E2 , Núcleo Celular , Reparo do DNA , Fator 2 Relacionado a NF-E2/genética , Poli ADP Ribosilação , Animais , Camundongos
16.
Nucleic Acids Res ; 51(12): 6461-6478, 2023 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-37224531

RESUMO

In light of the numerous studies identifying post-transcriptional regulators on the surface of the endoplasmic reticulum (ER), we asked whether there are factors that regulate compartment specific mRNA translation in human cells. Using a proteomic survey of spatially regulated polysome interacting proteins, we identified the glycolytic enzyme Pyruvate Kinase M (PKM) as a cytosolic (i.e. ER-excluded) polysome interactor and investigated how it influences mRNA translation. We discovered that the PKM-polysome interaction is directly regulated by ADP levels-providing a link between carbohydrate metabolism and mRNA translation. By performing enhanced crosslinking immunoprecipitation-sequencing (eCLIP-seq), we found that PKM crosslinks to mRNA sequences that are immediately downstream of regions that encode lysine- and glutamate-enriched tracts. Using ribosome footprint protection sequencing, we found that PKM binding to ribosomes causes translational stalling near lysine and glutamate encoding sequences. Lastly, we observed that PKM recruitment to polysomes is dependent on poly-ADP ribosylation activity (PARylation)-and may depend on co-translational PARylation of lysine and glutamate residues of nascent polypeptide chains. Overall, our study uncovers a novel role for PKM in post-transcriptional gene regulation, linking cellular metabolism and mRNA translation.


Assuntos
Poli ADP Ribosilação , Biossíntese de Proteínas , Piruvato Quinase , Humanos , Glutamatos/análise , Glutamatos/genética , Glutamatos/metabolismo , Lisina/metabolismo , Proteômica , Piruvato Quinase/genética , Piruvato Quinase/análise , Piruvato Quinase/metabolismo , Ribossomos/metabolismo
17.
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
18.
Commun Biol ; 6(1): 429, 2023 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-37076532

RESUMO

Poly(ADP-ribosyl)ation (PARylation) is a reversible post-translational protein modification that has profound regulatory functions in metabolism, development and immunity, and is conserved throughout the eukaryotic lineage. Contrary to metazoa, many components and mechanistic details of PARylation have remained unidentified in plants. Here we present the transcriptional co-regulator RADICAL-INDUCED CELL DEATH1 (RCD1) as a plant PAR-reader. RCD1 is a multidomain protein with intrinsically disordered regions (IDRs) separating its domains. We have reported earlier that RCD1 regulates plant development and stress-tolerance by interacting with numerous transcription factors (TFs) through its C-terminal RST domain. This study suggests that the N-terminal WWE and PARP-like domains, as well as the connecting IDR play an important regulatory role for RCD1 function. We show that RCD1 binds PAR in vitro via its WWE domain and that PAR-binding determines RCD1 localization to nuclear bodies (NBs) in vivo. Additionally, we found that RCD1 function and stability is controlled by Photoregulatory Protein Kinases (PPKs). PPKs localize with RCD1 in NBs and phosphorylate RCD1 at multiple sites affecting its stability. This work proposes a mechanism for negative transcriptional regulation in plants, in which RCD1 localizes to NBs, binds TFs with its RST domain and is degraded after phosphorylation by PPKs.


Assuntos
Poli ADP Ribosilação , Poli Adenosina Difosfato Ribose , Poli Adenosina Difosfato Ribose/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação da Expressão Gênica
19.
Cell Oncol (Dordr) ; 46(3): 761-776, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-36913068

RESUMO

Accumulating evidence indicates that long noncoding RNAs (lncRNAs) are abnormal expression in various malignant tumors. Our previous research demonstrated that focally amplified long non-coding RNA (lncRNA) on chromosome 1 (FALEC) is an oncogenic lncRNA in prostate cancer (PCa). However, the role of FALEC in castration-resistant prostate cancer (CRPC) is poorly understood. In this study, we showed FALEC was upregulated in post-castration tissues and CRPC cells, and increased FALEC expression was associated with poor survival in post-castration PCa patients. RNA FISH demonstrated FALEC was translocated into nucleus in CRPC cells. RNA pulldown and followed Mass Spectrometry (MS) assay demonstrated FALEC directly interacted with PARP1 and loss of function assay showed FALEC depletion sensitized CRPC cells to castration treatment and restored NAD+. Specific PARP1 inhibitor AG14361 and NAD+ endogenous competitor NADP+ sensitized FALEC-deleted CRPC cells to castration treatment. FALEC increasing PARP1 meditated self PARylation through recruiting ART5 and down regulation of ART5 decreased CRPC cell viability and restored NAD+ through inhibiting PARP1meditated self PARylation in vitro. Furthermore, ART5 was indispensable for FALEC directly interaction and regulation of PARP1, loss of ART5 impaired FALEC and PARP1 associated self PARylation. In vivo, FALEC depleted combined with PARP1 inhibitor decreased CRPC cell derived tumor growth and metastasis in a model of castration treatment NOD/SCID mice. Together, these results established that FALEC may be a novel diagnostic marker for PCa progression and provides a potential new therapeutic strategy to target the FALEC/ART5/PARP1 complex in CRPC patients.


Assuntos
Neoplasias de Próstata Resistentes à Castração , RNA Longo não Codificante , Humanos , Masculino , Animais , Camundongos , Neoplasias de Próstata Resistentes à Castração/patologia , RNA Longo não Codificante/genética , NAD/metabolismo , Poli ADP Ribosilação , Camundongos Endogâmicos NOD , Camundongos SCID , Poli(ADP-Ribose) Polimerase-1/genética
20.
Mol Biol (Mosk) ; 57(2): 254-268, 2023.
Artigo em Russo | MEDLINE | ID: mdl-37000654

RESUMO

Poly(ADP-ribose) (PAR) is a negatively charged polymer, linear or branched, that consists of ADP-ribose monomers. PAR is synthesized by poly(ADP-ribose)polymerase (PARP) enzymes, which are activated upon DNA damage and use nicotinamide adenine dinucleotide (NAD^(+)) as a substrate. The best-studied members of the PARP family, PARP1 and PARP2, are the most important nuclear proteins involved in many cell processes, including the regulation of DNA repair. PARP1 and PARP2 catalyze PAR synthesis and transfer to amino acid residues of target proteins, including autoPARylation. PARP1 and PARP2 are promising targets for chemotherapy in view of their key role in regulating DNA repair. A novel histone PARylation factor (HPF1) was recently discovered to modulate PARP1/2 activity by forming a transient joint active site with PARP1/2. Histones are modified at serine residues in the presence of HPF1. The general mechanism of the interaction between HPF1 and PARP1/2 is a subject of intense research now. The review considers the discovery and classical mechanism of PARylation in higher eukaryotes and the role of HPF1 in the process.


Assuntos
Histonas , Poli ADP Ribosilação , Histonas/genética , Histonas/metabolismo , Poli ADP Ribosilação/genética , Inibidores de Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/química , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Reparo do DNA , Dano ao DNA , Adenosina Difosfato Ribose/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...