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 HEK293RESUMO
ADP-ribosylation is a reversible reaction with ADP-ribosyltransferases catalyzing the forward reaction and ADP-ribose-acceptor hydrolases (ARHs) hydrolyzing the ADP-ribose acceptor bond. ARH2 is a member of the 39-kDa ARH family (ARH1-3), which is expressed in heart and skeletal muscle. ARH2 failed to exhibit any in vitro enzymatic activity. To determine its possible in vivo activities, Arh2 -knockout (KO) and - heterozygous (Het) mice were generated using CRISPR-Cas9. Arh2 -KO mice exhibited decreased cardiac contractility by MRI, echocardiography and dobutamine stress with cardiomegaly and abnormal motor function. Arh2 -Het mice showed results similar to those seen in Arh2 -KO mice except for cardiomegaly. Arh2 -KO and -Het mice and mouse embryonic fibroblasts (MEFs) developed spontaneous tumors and subcutaneous tumors in nude mice. We identified 13 mutations in Arh2 -Het MEFs and heterozygous tumors, corresponding to human ARH2 mutations in cancers obtained from COSMIC. Of interest, the L116R mutation in Arh2 gene plays a critical role in aggressive tumorigenesis in nude mice, corresponding to human ARH2 mutations in stomach adenocarcinoma. Both genders of Arh2 -KO and -Het mice showed increased unexpectedly deaths and decreased survival rate during a 24-month observation, caused by tumor, inflammation, non-inflammation (e.g., cardiomegaly, dental dysplasia), and congenital diseases. Thus, Arh2 plays a pivotal role in cardiac function, tumorigenesis, inflammation, and overall survival.
RESUMO
The novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2 or COVID-19) has caused a global pandemic. The SARS-CoV-2 RNA genome is replicated by a conserved "core" replication-transcription complex (RTC) containing an error-prone RNA-dependent RNA polymerase holoenzyme (holo-RdRp, nsp12-nsp7-nsp8) and a RNA proofreading nuclease (nsp14-nsp10). Although structures and functions of SARS-CoV-2 holo-RdRp have been extensively studied and ribonucleotide-analog inhibitors, such as Remdesivir, have been treated for COVID-19 patients, the substrate and nucleotide specificity of SARS-CoV-2 holo-RdRp remain unknown. Here, our biochemical analysis of SARS-CoV-2 holo-RdRp reveals that it has a robust DNA-dependent RNA polymerase activity, in addition to its intrinsic RNA-dependent RNA polymerase activity. Strikingly, SARS-CoV-2 holo-RdRp fully extends RNAs with a low-fidelity even when only ATP and pyrimidine nucleotides, in particular CTP, are provided. This ATP-dependent error-prone ribonucleotide incorporation by SARS-CoV-2 holo-RdRp resists excision by the RNA proofreading nuclease in vitro. Our collective results suggest that a physiological concentration of ATP likely contributes to promoting the error-prone incorporation of ribonucleotides and ribonucleotide-analogs by SARS-CoV-2 holo-RdRp and provide a useful foundation to develop ribonucleotide analogs as an effective therapeutic strategy to combat coronavirus-mediated outbreak.
Assuntos
COVID-19 , SARS-CoV-2 , Trifosfato de Adenosina , Antivirais/química , RNA Polimerases Dirigidas por DNA , Humanos , RNA Viral/química , RNA Viral/genética , RNA Polimerase Dependente de RNA , Ribonucleotídeos , SARS-CoV-2/genética , Proteínas não Estruturais Virais/químicaRESUMO
We present a Chemistry and Structure Screen Integrated Efficiently (CASSIE) approach (named for Greek prophet Cassandra) to design inhibitors for cancer biology and pathogenesis. CASSIE provides an effective path to target master keys to control the repair-replication interface for cancer cells and SARS CoV-2 pathogenesis as exemplified here by specific targeting of Poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribose glycohydrolase ARH3 macrodomains plus SARS CoV-2 nonstructural protein 3 (Nsp3) Macrodomain 1 (Mac1) and Nsp15 nuclease. As opposed to the classical massive effort employing libraries with large numbers of compounds against single proteins, we make inhibitor design for multiple targets efficient. Our compact, chemically diverse, 5000 compound Goldilocks (GL) library has an intermediate number of compounds sized between fragments and drugs with predicted favorable ADME (absorption, distribution, metabolism, and excretion) and toxicological profiles. Amalgamating our core GL library with an approved drug (AD) library, we employ a combined GLAD library virtual screen, enabling an effective and efficient design cycle of ranked computer docking, top hit biophysical and cell validations, and defined bound structures using human proteins or their avatars. As new drug design is increasingly pathway directed as well as molecular and mechanism based, our CASSIE approach facilitates testing multiple related targets by efficiently turning a set of interacting drug discovery problems into a tractable medicinal chemistry engineering problem of optimizing affinity and ADME properties based upon early co-crystal structures. Optimization efforts are made efficient by a computationally-focused iterative chemistry and structure screen. Thus, we herein describe and apply CASSIE to define prototypic, specific inhibitors for PARG vs distinct inhibitors for the related macrodomains of ARH3 and SARS CoV-2 Nsp3 plus the SARS CoV-2 Nsp15 RNA nuclease.
Assuntos
COVID-19 , Ácidos Nucleicos , Síndrome Respiratória Aguda Grave , Reparo do DNA , Humanos , Simulação de Acoplamento Molecular , SARS-CoV-2RESUMO
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/metabolismoRESUMO
Androgen signaling through the androgen receptor (AR) directs gene expression in both normal and prostate cancer cells. Androgen regulates multiple aspects of the AR life cycle, including its localization and post-translational modification, but understanding how modifications are read and integrated with AR activity has been difficult. Here, we show that ADP-ribosylation regulates AR through a nuclear pathway mediated by Parp7. We show that Parp7 mono-ADP-ribosylates agonist-bound AR, and that ADP-ribosyl-cysteines within the N-terminal domain mediate recruitment of the E3 ligase Dtx3L/Parp9. Molecular recognition of ADP-ribosyl-cysteine is provided by tandem macrodomains in Parp9, and Dtx3L/Parp9 modulates expression of a subset of AR-regulated genes. Parp7, ADP-ribosylation of AR, and AR-Dtx3L/Parp9 complex assembly are inhibited by Olaparib, a compound used clinically to inhibit poly-ADP-ribosyltransferases Parp1/2. Our study reveals the components of an androgen signaling axis that uses a writer and reader of ADP-ribosylation to regulate protein-protein interactions and AR activity.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/genética , Poli(ADP-Ribose) Polimerases/genética , Neoplasias da Próstata/genética , Processamento de Proteína Pós-Traducional , Receptores Androgênicos/genética , ADP-Ribosilação/efeitos dos fármacos , Adenocarcinoma , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Humanos , Masculino , Metribolona/farmacologia , Proteínas de Neoplasias/metabolismo , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores Androgênicos/metabolismo , Transdução de Sinais , Análise de SobrevidaRESUMO
ADP-ribosylation is a reversible and site-specific post-translational modification that regulates a wide array of cellular signaling pathways. Regulation of ADP-ribosylation is vital for maintaining genomic integrity, and uncontrolled accumulation of poly(ADP-ribosyl)ation triggers a poly(ADP-ribose) (PAR)-dependent release of apoptosis-inducing factor from mitochondria, leading to cell death. ADP-ribosyl-acceptor hydrolase 3 (ARH3) cleaves PAR and mono(ADP-ribosyl)ation at serine following DNA damage. ARH3 is also a metalloenzyme with strong metal selectivity. While coordination of two magnesium ions (MgA and MgB) significantly enhances its catalytic efficiency, calcium binding suppresses its function. However, how the coordination of different metal ions affects its catalysis has not been defined. Here, we report a new crystal structure of ARH3 complexed with its product ADP-ribose and calcium. This structure shows that calcium coordination significantly distorts the binuclear metal center of ARH3, which results in decreased binding affinity to ADP-ribose, and suboptimal substrate alignment, leading to impaired hydrolysis of PAR and mono(ADP-ribosyl)ated serines. Furthermore, combined structural and mutational analysis of the metal-coordinating acidic residues revealed that MgA is crucial for optimal substrate positioning for catalysis, whereas MgB plays a key role in substrate binding. Our collective data provide novel insights into the different roles of these metal ions and the basis of metal selectivity of ARH3 and contribute to understanding the dynamic regulation of cellular ADP-ribosylations during the DNA damage response.
Assuntos
Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Magnésio/metabolismo , Dano ao DNA , Humanos , Hidrólise , Modelos Moleculares , Poli Adenosina Difosfato Ribose/metabolismo , Conformação Proteica , Especificidade por SubstratoRESUMO
The XRCC1-DNA ligase IIIα complex (XL) is critical for DNA single-strand break repair, a key target for PARP inhibitors in cancer cells deficient in homologous recombination. Here, we combined biophysical approaches to gain insights into the shape and conformational flexibility of the XL as well as XRCC1 and DNA ligase IIIα (LigIIIα) alone. Structurally-guided mutational analyses based on the crystal structure of the human BRCT-BRCT heterodimer identified the network of salt bridges that together with the N-terminal extension of the XRCC1 C-terminal BRCT domain constitute the XL molecular interface. Coupling size exclusion chromatography with small angle X-ray scattering and multiangle light scattering (SEC-SAXS-MALS), we determined that the XL is more compact than either XRCC1 or LigIIIα, both of which form transient homodimers and are highly disordered. The reduced disorder and flexibility allowed us to build models of XL particles visualized by negative stain electron microscopy that predict close spatial organization between the LigIIIα catalytic core and both BRCT domains of XRCC1. Together our results identify an atypical BRCT-BRCT interaction as the stable nucleating core of the XL that links the flexible nick sensing and catalytic domains of LigIIIα to other protein partners of the flexible XRCC1 scaffold.
Assuntos
DNA Ligase Dependente de ATP/metabolismo , Reparo do DNA , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/metabolismo , Cromatografia em Gel , Cristalografia por Raios X , DNA Ligase Dependente de ATP/química , Dimerização , Humanos , Microscopia Eletrônica , Modelos Moleculares , Complexos Multiproteicos , Mutação , Mutação de Sentido Incorreto , Coloração Negativa , Mutação Puntual , Conformação Proteica , Domínios Proteicos , Mapeamento de Interação de Proteínas , Proteínas Recombinantes/metabolismo , Espalhamento a Baixo Ângulo , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/química , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genéticaRESUMO
Poly(ADP-ribosyl)ation (PARylation) regulates DNA damage response, chromatin structure, and cell-fate. Dynamic regulation of cellular PAR levels is crucial for the maintenance of genomic integrity and excessive cellular PAR activates a PAR-dependent cell death pathway. Thus, PAR serves as a cell-death signal; however, it has been debated how the protein-free PAR is generated. Here, we demonstrate that PAR glycohydrolases (PARGs) from mammals to bacteria have a robust endo-glycohydrolase activity, releasing protein-free PAR chains longer than three ADP-ribose units as early reaction products. Released PAR chains are transient and rapidly degraded to monomeric ADP-ribose, which is consistent with a short half-life of PAR during DNA damage responses. Computational simulations using a tri-ADP-ribose further support that PARG can efficiently bind to internal sites of PAR for the endo-glycosidic cleavage. Our collective results suggest PARG as a key player in producing protein-free PAR during DNA damage signaling and establish bacterial PARG as a useful tool to enrich short PAR chains that emerge as important reagents for biomedical research.
Assuntos
Glicosídeo Hidrolases/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Glicosídeo Hidrolases/química , Humanos , Modelos Moleculares , Poli Adenosina Difosfato Ribose/química , Ligação Proteica , Conformação ProteicaRESUMO
Poly(ADP-ribose)ylation (PARylation) by PAR polymerase 1 (PARP1) and PARylation removal by poly(ADP-ribose) glycohydrolase (PARG) critically regulate DNA damage responses; yet, conflicting reports obscure PARG biology and its impact on cancer cell resistance to PARP1 inhibitors. Here, we found that PARG expression is upregulated in many cancers. We employed chemical library screening to identify and optimize methylxanthine derivatives as selective bioavailable PARG inhibitors. Multiple crystal structures reveal how substituent positions on the methylxanthine core dictate binding modes and inducible-complementarity with a PARG-specific tyrosine clasp and arginine switch, supporting inhibitor specificity and a competitive inhibition mechanism. Cell-based assays show selective PARG inhibition and PARP1 hyperPARylation. Moreover, our PARG inhibitor sensitizes cells to radiation-induced DNA damage, suppresses replication fork progression and impedes cancer cell survival. In PARP inhibitor-resistant A172 glioblastoma cells, our PARG inhibitor shows comparable killing to Nedaplatin, providing further proof-of-concept that selectively inhibiting PARG can impair cancer cell survival.
Assuntos
Replicação do DNA/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Glicosídeo Hidrolases/antagonistas & inibidores , Neoplasias/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Inibidores Enzimáticos/química , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Humanos , Neoplasias/enzimologia , Neoplasias/metabolismo , Neoplasias/fisiopatologia , Poli ADP Ribosilação/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Bibliotecas de Moléculas Pequenas/químicaRESUMO
ADP-ribosyl-acceptor hydrolase 3 (ARH3) plays important roles in regulation of poly(ADP-ribosyl)ation, a reversible post-translational modification, and in maintenance of genomic integrity. ARH3 degrades poly(ADP-ribose) to protect cells from poly(ADP-ribose)-dependent cell death, reverses serine mono(ADP-ribosyl)ation, and hydrolyzes O-acetyl-ADP-ribose, a product of Sirtuin-catalyzed histone deacetylation. ARH3 preferentially hydrolyzes O-linkages attached to the anomeric C1â³ of ADP-ribose; however, how ARH3 specifically recognizes and cleaves structurally diverse substrates remains unknown. Here, structures of full-length human ARH3 bound to ADP-ribose and Mg2+, coupled with computational modeling, reveal a dramatic conformational switch from closed to open states that enables specific substrate recognition. The glutamate flap, which blocks substrate entrance to Mg2+ in the unliganded closed state, is ejected from the active site when substrate is bound. This closed-to-open transition significantly widens the substrate-binding channel and precisely positions the scissile 1â³-O-linkage for cleavage while securing tightly 2â³- and 3â³-hydroxyls of ADP-ribose. Our collective data uncover an unprecedented structural plasticity of ARH3 that supports its specificity for the 1â³-O-linkage in substrates and Mg2+-dependent catalysis.
Assuntos
Adenosina Difosfato Ribose/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Conformação Proteica , Adenosina Difosfato Ribose/química , Sequência de Aminoácidos , Catálise , Domínio Catalítico , Cristalografia por Raios X , Humanos , Hidrólise , Modelos Moleculares , Homologia de Sequência , Especificidade por SubstratoRESUMO
BACKGROUND: It has been reported that 4,4'-diamino-diphenyl sulfone (DDS), the longtime treatment of choice for leprosy, prolongs the lifespan and increases mobility in animal models by reducing the levels of reactive oxygen species and inhibiting muscle pyruvate kinase activity. This study aimed to investigate whether sarcopenic status in leprosy survivors was influenced by recent history of DDS medication. METHODS: Forty-one elderly female leprosy survivors were recruited. The DDS group was defined as survivors who had been taking the drug for the past year or more. Body composition measured by dual energy X-ray absorptiometry, limb muscle strength, short physical performance battery, and International Physical Activity Questionnaire in Korean were compared. RESULTS: The DDS group tended to have higher skeletal muscle mass index (24.4 ± 2.7 vs. 22.6 ± 2.2%, P = 0.066) and regional skeletal muscle mass index in non-dominant leg (8.9 ± 1.0 vs. 7.9 ± 0.9%, P = 0.018) than those of the control group although they had significantly worse leprosy disability than the control group (P = 0.027). The DDS group had greater strength than the control group in non-dominant shoulder abductor, elbow flexor, hip flexor, and knee extensor (P = 0.005, P = 0.029, P = 0.021, and P = 0.002, respectively). Weekly walking amount was significantly longer (P = 0.020) in the DDS group than the control group. The total lifetime DDS exposure significantly correlated with skeletal muscle mass of the lower extremity in non-dominant leg (r = 0.379, P = 0.015). CONCLUSIONS: DDS-taking leprosy survivors had larger skeletal muscle mass and greater muscle strength over non-taking survivors. There was a dose-response relationship between total lifetime DDS exposure and skeletal muscle mass of lower extremity. These findings might suggest potential anti-sarcopenic effects of DDS.
RESUMO
Exonuclease 1 (Exo1) has important roles in DNA metabolic transactions that are essential for genome maintenance, telomere regulation and cancer suppression. However, the mechanisms for regulating Exo1 activity in these processes remain incompletely understood. Here, we report that Exo1 activity is regulated by a direct interaction with poly(ADP-ribose) (PAR), a prominent posttranslational modification at the sites of DNA damage. This PAR-binding activity promotes the early recruitment of Exo1 to sites of DNA damage, where it is retained through an interaction with PCNA, which interacts with the C-terminus of Exo1. The effects of both PAR and PCNA on Exo1 damage association are antagonized by the 14-3-3 adaptor proteins, which interact with the central domain of Exo1. Although PAR binding inhibits both the exonuclease activity and the 5' flap endonuclease activity of purified Exo1, the pharmacological blockade of PAR synthesis does not overtly affect DNA double-strand break end resection in a cell free Xenopus egg extract. Thus, the counteracting effects of PAR on Exo1 recruitment and enzymatic activity may enable appropriate resection of DNA ends while preventing unscheduled or improper processing of DNA breaks in cells.
Assuntos
Dano ao DNA , Reparo do DNA , Exodesoxirribonucleases/metabolismo , Endonucleases Flap/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas 14-3-3/metabolismo , Animais , Extratos Celulares , Núcleo Celular , Glicosídeo Hidrolases/metabolismo , Células HEK293 , Humanos , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases , Processamento de Proteína Pós-Traducional , XenopusRESUMO
Mammalian DNA ligase III (LigIII) functions in both nuclear and mitochondrial DNA metabolism. In the nucleus, LigIII has functional redundancy with DNA ligase I whereas LigIII is the only mitochondrial DNA ligase and is essential for the survival of cells dependent upon oxidative respiration. The unique LigIII zinc finger (ZnF) domain is not required for catalytic activity but senses DNA strand breaks and stimulates intermolecular ligation of two DNAs by an unknown mechanism. Consistent with this activity, LigIII acts in an alternative pathway of DNA double strand break repair that buttresses canonical non-homologous end joining (NHEJ) and is manifest in NHEJ-defective cancer cells, but how LigIII acts in joining intermolecular DNA ends versus nick ligation is unclear. To investigate how LigIII efficiently joins two DNAs, we developed a real-time, fluorescence-based assay of DNA bridging suitable for high-throughput screening. On a nicked duplex DNA substrate, the results reveal binding competition between the ZnF and the oligonucleotide/oligosaccharide-binding domain, one of three domains constituting the LigIII catalytic core. In contrast, these domains collaborate and are essential for formation of a DNA-bridging intermediate by adenylated LigIII that positions a pair of blunt-ended duplex DNAs for efficient and specific intermolecular ligation.
Assuntos
Reparo do DNA por Junção de Extremidades , DNA Ligases/metabolismo , DNA/metabolismo , DNA Ligase Dependente de ATP , DNA Ligases/química , Humanos , Modelos Moleculares , Proteínas de Ligação a Poli-ADP-Ribose , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas de Xenopus , Dedos de ZincoRESUMO
The DNA end resection process dictates the cellular response to DNA double strand break damage and is essential for genome maintenance. Although insufficient DNA resection hinders homology-directed repair and ATR (ataxia telangiectasia and Rad3 related)-dependent checkpoint activation, overresection produces excessive single-stranded DNA that could lead to genomic instability. However, the mechanisms controlling DNA end resection are poorly understood. Here we show that the major resection nuclease Exo1 is regulated both positively and negatively by protein-protein interactions to ensure a proper level of DNA resection. We have shown previously that the sliding DNA clamp proliferating cell nuclear antigen (PCNA) associates with the C-terminal domain of Exo1 and promotes Exo1 damage association and DNA resection. In this report, we show that 14-3-3 proteins interact with a central region of Exo1 and negatively regulate Exo1 damage recruitment and subsequent resection. 14-3-3s limit Exo1 damage association, at least in part, by suppressing its association with PCNA. Disruption of the Exo1 interaction with 14-3-3 proteins results in elevated sensitivity of cells to DNA damage. Unlike Exo1, the Dna2 resection pathway is apparently not regulated by PCNA and 14-3-3s. Our results provide critical insights into the mechanism and regulation of the DNA end resection process and may have implications for cancer treatment.
Assuntos
Proteínas 14-3-3/metabolismo , Quebras de DNA de Cadeia Dupla , Exodesoxirribonucleases/metabolismo , Regulação da Expressão Gênica , Antígeno Nuclear de Célula em Proliferação/metabolismo , Animais , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Sobrevivência Celular , DNA/genética , Reparo do DNA , Glutationa Transferase/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Microscopia de Fluorescência , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , XenopusRESUMO
The posttranslational modification of proteins with poly(ADP-ribose) (PAR) regulates protein-protein interactions in DNA repair, gene expression, chromatin structure, and cell fate determination. The PAR polymerase PARP1 binds to damaged chromatin and synthesizes PAR chains to signal DNA damage and recruit the DNA repair scaffold, XRCC1. Pharmacological blockade of PARP1 enzymatic activity impairs XRCC1-dependent repair of DNA damage and selectively kills cancer cells lacking other DNA repair functions. As such, PARP inhibitors are promising new therapies for repair-deficient tumors such as BRCA mutated breast cancers. Although the XRCC1-PARP1 complex is relevant to the proposed therapeutic mechanism of PARP inhibitors, the physical makeup and dynamics of this complex are not well characterized at the molecular level. Here we describe a fluorescence-based, real-time assay that quantitatively monitors interactions between PARylated PARP1 and XRCC1. Using this assay, we show that the PAR posttranslational modification by itself is a high affinity ligand for XRCC1, requiring a minimum chain length of 7 ADP-ribose units in the oligo(ADP-ribose) ligand for a stable interaction with XRCC1. This discrete binding interface enables the PAR glycohydrolase (PARG) to completely disassemble the PARP1-XRCC1 complex without assistance from a mono(ADP-ribose) glycohydrolase. Our quantitative, real-time assay of PAR-dependent protein-protein interactions and PAR turnover by PARG is an excellent tool for high-throughput screening to identify pharmacological modulators of PAR metabolism that may be useful therapeutic alternatives to PARP inhibitors.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Glicosídeo Hidrolases/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Processamento de Proteína Pós-Traducional , Adenosina Difosfato Ribose/metabolismo , Sítios de Ligação , Proteínas de Ligação a DNA/química , Humanos , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/química , Ligação Proteica , Especificidade por Substrato , Proteína 1 Complementadora Cruzada de Reparo de Raio-XRESUMO
Because of chronicity and poor environments, elderly leprosy survivors might be at greater risk of developing obesity and sarcopenia than healthy individuals. This study aimed to investigate whether body composition and the prevalence of obesity and sarcopenia among elderly leprosy survivors with no or mild physical impairment differ from those of the general population. A total of 36 leprosy survivors aged 65-90 years with no or mild physical impairment were recruited. Individuals matched for sex, age, and height were selected as a control group from the Fourth Korea National Health and Nutrition Examination Survey. Anthropometric characteristics, body composition, appendicular skeletal muscle mass (ASM), modified skeletal muscle mass index (SMI), and the prevalence of obesity and sarcopenia were compared between the leprosy survivors and the control group. Compared to the control group, the leprosy survivors had higher body weight, BMI, total fat mass, and total fat percentage. The leprosy survivor group also had lower ASM (P=0.035) and SMI (P<0.001) values. Comparison of the composition of regional body parts showed that the lean body mass of the legs was lower in the leprosy survivor group even though this group had higher body weight. The leprosy survivor group also had a significantly higher prevalence of sarcopenia than the control group (38.7% vs. 5.6%; P=0.002). These findings suggest that leprosy survivors are at greater risk of developing obesity and sarcopenia than healthy individuals. Further researches are required to investigate causes and mechanisms of sarcopenia in leprosy survivors.
Assuntos
Composição Corporal/fisiologia , Hanseníase/complicações , Atividade Motora/fisiologia , Inquéritos Nutricionais , Sarcopenia/etiologia , Idoso , Antropometria , Estudos Transversais , Feminino , Humanos , Incidência , Hanseníase/epidemiologia , Masculino , Obesidade/epidemiologia , Obesidade/etiologia , Obesidade/fisiopatologia , Prevalência , República da Coreia/epidemiologia , Fatores de Risco , Sarcopenia/epidemiologia , Sarcopenia/fisiopatologia , SobreviventesRESUMO
Mutations in the DJ-1 gene have been implicated in the autosomal recessive early onset parkinsonism. DJ-1 is a soluble dimeric protein with critical roles in response to oxidative stress and in neuronal maintenance. However, several lines of evidence suggest the existence of a nonfunctional aggregated form of DJ-1 in the brain of patients with some neurodegenerative diseases. Here, we show that inorganic phosphate, an important anion that exhibits elevated levels in patients with Parkinson disease, transforms DJ-1 into filamentous aggregates. According to the 2.4-A crystal structure, DJ-1 dimers are linearly stacked through P(i)-mediated interactions to form protofilaments, which are then bundled into a filamentous assembly.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas Oncogênicas/química , Fosfatos/química , Encéfalo/metabolismo , Cristalização , Cristalografia por Raios X/métodos , Dimerização , Humanos , Luz , Microscopia Eletrônica , Conformação Molecular , Doença de Parkinson/metabolismo , Ligação Proteica , Conformação Proteica , Proteína Desglicase DJ-1 , Estrutura Terciária de Proteína , Espalhamento de RadiaçãoRESUMO
To find the accessory proteins participating in expression and maturation of nickel-containing superoxide dismutase (NiSOD), a metal-binding protein (CbiXhp) homologous to cobaltochelatase (CbiX) of Bacillus megaterium was isolated by nickel-nitrilotriacetic acid resin from Streptomyces seoulensis. The deduced amino acid sequence of cbiXhp showed 87% and 39% identity to CbiX of Streptomyces coelicolor and that of B. megaterium, respectively. Overexpression of CbiXhp increased the activity and the expression of NiSOD in the presence and absence of nickel, but to a lesser extent in its absence. This result indicates that CbiXhp is involved in the expression of NiSOD.
Assuntos
Proteínas de Bactérias , Liases/genética , Liases/metabolismo , Níquel/metabolismo , Streptomyces/enzimologia , Streptomyces/genética , Superóxido Dismutase/genética , Sequência de Aminoácidos , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Dados de Sequência Molecular , Ligação Proteica , Superóxido Dismutase/metabolismoRESUMO
Human DJ-1 and Escherichia coli Hsp31 belong to ThiJ/PfpI family, whose members contain a conserved domain. DJ-1 is associated with autosomal recessive early onset parkinsonism and Hsp31 is a molecular chaperone. Structural comparisons between DJ-1, Hsp31, and an Archaea protease, a member of ThiJ/PfpI family, lead to the identification of the chaperone activity of DJ-1 and the proteolytic activity of Hsp31. Moreover, the comparisons provide insights into how the functional diversity is realized in proteins that share an evolutionarily conserved domain. On the basis of the chaperone activity the possible role of DJ-1 in the pathogenesis of Parkinson's disease is discussed.