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1.
J Med Chem ; 66(20): 14133-14149, 2023 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-37782247

RESUMO

Methyl-lysine reader p53 binding protein 1 (53BP1) is a central mediator of DNA break repair and is associated with various human diseases, including cancer. Thus, high-quality 53BP1 chemical probes can aid in further understanding the role of 53BP1 in genome repair pathways. Herein, we utilized focused DNA-encoded library screening to identify the novel hit compound UNC8531, which binds the 53BP1 tandem Tudor domain (TTD) with an IC50 of 0.47 ± 0.09 µM in a TR-FRET assay and Kd values of 0.85 ± 0.17 and 0.79 ± 0.52 µM in ITC and SPR, respectively. UNC8531 was cocrystallized with the 53BP1 TTD to guide further optimization efforts, leading to UNC9512. NanoBRET and 53BP1-dependent foci formation experiments confirmed cellular target engagement. These results show that UNC9512 is a best-in-class small molecule 53BP1 antagonist that can aid further studies investigating the role of 53BP1 in DNA repair, gene editing, and oncogenesis.


Assuntos
Reparo do DNA , Peptídeos e Proteínas de Sinalização Intracelular , Humanos , DNA , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/química , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Domínio Tudor
2.
Nat Commun ; 13(1): 360, 2022 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-35042897

RESUMO

Human 53BP1 is primarily known as a key player in regulating DNA double strand break (DSB) repair choice; however, its involvement in other biological process is less well understood. Here, we report a previously uncharacterized function of 53BP1 at heterochromatin, where it undergoes liquid-liquid phase separation (LLPS) with the heterochromatin protein HP1α in a mutually dependent manner. Deletion of 53BP1 results in a reduction in heterochromatin centers and the de-repression of heterochromatic tandem repetitive DNA. We identify domains and residues of 53BP1 required for its LLPS, which overlap with, but are distinct from, those involved in DSB repair. Further, 53BP1 mutants deficient in DSB repair, but proficient in LLPS, rescue heterochromatin de-repression and protect cells from stress-induced DNA damage and senescence. Our study suggests that in addition to DSB repair modulation, 53BP1 contributes to the maintenance of heterochromatin integrity and genome stability through LLPS.


Assuntos
Heterocromatina/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Homólogo 5 da Proteína Cromobox/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Knockout , Mutação/genética , Domínios Proteicos , Estresse Fisiológico , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/química
3.
Cell Rep ; 35(13): 109306, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34192545

RESUMO

53BP1 is recruited to chromatin in the vicinity of DNA double-strand breaks (DSBs). We identify the nuclear kinesin, KIF18B, as a 53BP1-interacting protein and define its role in 53BP1-mediated DSB repair. KIF18B is a molecular motor protein involved in destabilizing astral microtubules during mitosis. It is primarily nuclear throughout the interphase and is constitutively chromatin bound. Our observations indicate a nuclear function during the interphase for a kinesin previously implicated in mitosis. We identify a central motif in KIF18B, which we term the Tudor-interacting motif (TIM), because of its interaction with the Tudor domain of 53BP1. TIM enhances the interaction between the 53BP1 Tudor domain and dimethylated lysine 20 of histone H4. TIM and the motor function of KIF18B are both required for efficient 53BP1 focal recruitment in response to damage and for fusion of dysfunctional telomeres. Our data suggest a role for KIF18B in efficient 53BP1-mediated end-joining of DSBs.


Assuntos
Núcleo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Cinesinas/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Linhagem Celular Tumoral , Células HEK293 , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilação , Ligação Proteica , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/química
4.
Cell ; 184(4): 1081-1097.e19, 2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33606978

RESUMO

Mutations in DNA damage response (DDR) genes endanger genome integrity and predispose to cancer and genetic disorders. Here, using CRISPR-dependent cytosine base editing screens, we identify > 2,000 sgRNAs that generate nucleotide variants in 86 DDR genes, resulting in altered cellular fitness upon DNA damage. Among those variants, we discover loss- and gain-of-function mutants in the Tudor domain of the DDR regulator 53BP1 that define a non-canonical surface required for binding the deubiquitinase USP28. Moreover, we characterize variants of the TRAIP ubiquitin ligase that define a domain, whose loss renders cells resistant to topoisomerase I inhibition. Finally, we identify mutations in the ATM kinase with opposing genome stability phenotypes and loss-of-function mutations in the CHK2 kinase previously categorized as variants of uncertain significance for breast cancer. We anticipate that this resource will enable the discovery of additional DDR gene functions and expedite studies of DDR variants in human disease.


Assuntos
Dano ao DNA , Edição de Genes , Testes Genéticos , Sequência de Aminoácidos , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Sequência de Bases , Sistemas CRISPR-Cas/genética , Camptotecina/farmacologia , Linhagem Celular , Dano ao DNA/genética , Reparo do DNA/genética , Feminino , Humanos , Mutação/genética , Fenótipo , Ligação Proteica , Domínios Proteicos , RNA Guia de Cinetoplastídeos/genética , Inibidores da Topoisomerase/farmacologia , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/química , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Ubiquitina Tiolesterase/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
5.
J Zhejiang Univ Sci B ; 22(1): 38-46, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33448186

RESUMO

Maintenance of cellular homeostasis and genome integrity is a critical responsibility of DNA double-strand break (DSB) signaling. P53-binding protein 1 (53BP1) plays a critical role in coordinating the DSB repair pathway choice and promotes the non-homologous end-joining (NHEJ)-mediated DSB repair pathway that rejoins DSB ends. New insights have been gained into a basic molecular mechanism that is involved in 53BP1 recruitment to the DNA lesion and how 53BP1 then recruits the DNA break-responsive effectors that promote NHEJ-mediated DSB repair while inhibiting homologous recombination (HR) signaling. This review focuses on the up- and downstream pathways of 53BP1 and how 53BP1 promotes NHEJ-mediated DSB repair, which in turn promotes the sensitivity of poly(ADP-ribose) polymerase inhibitor (PARPi) in BRCA1-deficient cancers and consequently provides an avenue for improving cancer therapy strategies.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA/fisiologia , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Proteína BRCA1/metabolismo , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Humanos , Proteínas Mad2/metabolismo , Modelos Biológicos , Poli ADP Ribosilação , Inibidores de Poli(ADP-Ribose) Polimerases/metabolismo , Domínios e Motivos de Interação entre Proteínas , Pirofosfatases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Proteínas de Ligação a Telômeros/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/química
6.
Biochem J ; 478(1): 135-156, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33439989

RESUMO

Genomic integrity is most threatened by double-strand breaks, which, if left unrepaired, lead to carcinogenesis or cell death. The cell generates a network of protein-protein signaling interactions that emanate from the DNA damage which are now recognized as a rich basis for anti-cancer therapy development. Deciphering the structures of signaling proteins has been an uphill task owing to their large size and complex domain organization. Recent advances in mammalian protein expression/purification and cryo-EM-based structure determination have led to significant progress in our understanding of these large multidomain proteins. This review is an overview of the structural principles that underlie some of the key signaling proteins that function at the double-strand break site. We also discuss some plausible ideas that could be considered for future structural approaches to visualize and build a more complete understanding of protein dynamics at the break site.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Transdução de Sinais/genética , Hidrolases Anidrido Ácido/química , Hidrolases Anidrido Ácido/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/química , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteína Homóloga a MRE11/química , Proteína Homóloga a MRE11/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Processamento de Proteína Pós-Traducional/genética , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/química , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo
7.
Cancer Lett ; 501: 43-54, 2021 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-33359708

RESUMO

TP53 binding protein 1 (53BP1) plays an important role in DNA damage repair and maintaining genomic stability. However, the mutations of 53BP1 in human cancers have not been systematically examined. Here, we have analyzed 541 somatic mutations of 53BP1 across 34 types of human cancer from databases of The Cancer Genome Atlas, International Cancer Genome Consortium and Catalogue of Somatic Mutations in Cancer. Among these cancer-associated 53BP1 mutations, truncation mutations disrupt the nuclear localization of 53BP1 thus abolish its biological functions in DNA damage repair. Moreover, with biochemical analyses and structural modeling, we have examined the detailed molecular mechanism by which missense mutations in the key domains causes the DNA damage repair defects. Taken together, our results reveal the functional defects of a set of cancer-associated 53BP1 mutations.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Mutação de Sentido Incorreto , Neoplasias/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Linhagem Celular Tumoral , Biologia Computacional , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Modelos Moleculares , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/química
8.
Biochem Soc Trans ; 48(5): 2317-2333, 2020 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-33084906

RESUMO

Lamins are type V intermediate filament proteins which are ubiquitously present in all metazoan cells providing a platform for binding of chromatin and related proteins, thereby serving a wide range of nuclear functions including DNA damage repair. Altered expression of lamins in different subtypes of cancer is evident from researches worldwide. But whether cancer is a consequence of this change or this change is a consequence of cancer is a matter of future investigation. However changes in the expression levels of lamins is reported to have direct or indirect association with cancer progression or have regulatory roles in common neoplastic symptoms like higher nuclear deformability, increased genomic instability and reduced susceptibility to DNA damaging agents. It has already been proved that loss of A type lamin positively regulates cathepsin L, eventually leading to degradation of several DNA damage repair proteins, hence impairing DNA damage repair pathways and increasing genomic instability. It is established in ovarian cancer, that the extent of alteration in nuclear morphology can determine the degree of genetic changes and thus can be utilized to detect low to high form of serous carcinoma. In this review, we have focused on ovarian cancer which is largely caused by genomic alterations in the DNA damage response pathways utilizing proteins like RAD51, BRCA1, 53BP1 which are regulated by lamins. We have elucidated the current understanding of lamin expression in ovarian cancer and its implications in the regulation of DNA damage response pathways that ultimately result in telomere deformation and genomic instability.


Assuntos
Dano ao DNA , Reparo do DNA , Regulação Neoplásica da Expressão Gênica , Laminina/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Animais , Proteína BRCA1/química , Catepsina L/metabolismo , Núcleo Celular/metabolismo , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Instabilidade Genômica , Genômica , Humanos , Laminas/metabolismo , Camundongos , Domínios Proteicos , Rad51 Recombinase/química , Telômero/metabolismo , Resultado do Tratamento , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/química
9.
Phys Chem Chem Phys ; 22(11): 6136-6144, 2020 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-32124883

RESUMO

Histone lysine methylation regulates the recruitment of mammalian DNA repair factor 53BP1 to the histone H4 lysine 20 (H4K20), through specific recognition of the tandem Tudor domain of 53BP1. The di- and mono-methylated H4K20 bind to 53BP1 with high affinity, but the non- and tri-methylated H4K20 do not. Here, we develop a new approach to carry out computational study to unravel the binding mechanism of methylated H4K20 by 53BP1 and to compute relative binding affinities of different methylations of H4K20 by 53BP1. First, hot spots in 53BP1 were predicted by computational alanine scanning and aromatic cages formed by W1495, Y1500, Y1502, and Y1523 are found to provide the dominant binding to di- and mono-methylated H4K20 in addition to D1521. Secondly, a de-methylation method is proposed to predict relative binding free energies between 53BP1 and different methylated states of H4K20. Finally, the tri-methylated and non-methylated H4K20/53BP1 complexes are found to be dynamically unstable, explaining the experimental finding that neither can bind to 53BP1. The present work provides an important theoretical basis for our understanding of histone methylations of H4K20 and their recognition mechanism by DNA repair factor 53BP1.


Assuntos
Biologia Computacional , Histonas/metabolismo , Modelos Moleculares , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Histonas/química , Lisina/química , Metilação , Ligação Proteica , Estabilidade Proteica , Estrutura Quaternária de Proteína , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/química
10.
Chem Commun (Camb) ; 55(84): 12639-12642, 2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31580339

RESUMO

Polyubiquitination with diverse linkages on histones provides another layer of accuracy and complexity for epigenetic regulation, which is rarely studied. Herein, K27 or K48-diubiquitin modified H2A analogues were chemically synthesized using thiirane linkers. These permitted in vitro binding studies suggested the plasticity of ubiquitin chains in 53BP1 recognition.


Assuntos
Histonas/química , Poliubiquitina/química , Sulfetos/química , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/química , Ligação Proteica , Proteínas Recombinantes/química , Ubiquitinação
11.
Cell Death Dis ; 10(8): 550, 2019 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-31320608

RESUMO

Although oxaliplatin is an effective chemotherapeutic drug for treatment of colorectal cancer (CRC), tumor cells can develop mechanisms to evade oxaliplatin-induced cell death and show high tolerance and acquired resistance to this drug. Heterogeneous nuclear ribonucleoprotein L (hnRNP L) has been proved to play a critical role in DNA repair during IgH class switch recombination (CSR) in B lymphocytes, while, its role in CRC and chemotherapeutic resistance remain unknown. Our study aims to uncover an unidentified mechanism of regulating DNA double-strand breaks (DSBs) by hnRNP L in CRC cells treated by oxaliplatin. In present study, we observed that knockdown of hnRNP L enhanced the level of DNA breakage and sensitivity of CRC cells to oxaliplatin. The expression of key DNA repair factors (BRCA1, 53BP1, and ATM) was unaffected by hnRNP L knockdown, thereby excluding the likelihood of hnRNP L mediation via mRNA regulation. Moreover, we observed that phosphorylation level of ATM changed oppositely to 53BP1 and BRCA1 in the CRC cells (SW620 and HCT116) which exhibit synergistic effect by oxaliplatin plus hnRNP L impairment. And similar phenomenon was observed in the foci formation of these critical repair factors. We also found that hnRNP L binds directly with these DNA repair factors through its RNA-recognition motifs (RRMs). Analysis of cell death indicated that the RRMs of hnRNP L are required for cell survival under incubation with oxaliplatin. In conclusion, hnRNP L is critical for the recruitment of the DNA repair factors in oxaliplatin-induced DSBs. Targeting hnRNP L is a promising new clinical approach that could enhance the effectiveness of current chemotherapeutic treatment in patients with resistance to oxaliplatin.


Assuntos
Antineoplásicos/farmacologia , Proteína BRCA1/metabolismo , Neoplasias Colorretais/metabolismo , Quebras de DNA de Cadeia Dupla , Ribonucleoproteínas Nucleares Heterogêneas Grupo L/metabolismo , Oxaliplatina/farmacologia , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas Mutadas de Ataxia Telangiectasia/química , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/química , Proteína BRCA1/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Neoplasias Colorretais/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Células HCT116 , Ribonucleoproteínas Nucleares Heterogêneas Grupo L/genética , Humanos , Fosforilação , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/química , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética
12.
Artif Cells Nanomed Biotechnol ; 47(1): 2196-2204, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31159605

RESUMO

Double-strand break (DSB) repair foci are important therapeutic targets. Here we describe platforms for delivery of macromolecules, nanomaterials and nanomedicines to repair foci. The strategy is based on the high affinity of the human 53BP1 protein for modified chromatin present at sites of DNA damage. As proof of concept, we created, expressed, and purified an engineered fragment of 53BP1 and coupled it to fluorescent streptavidin, a model cargo with no intrinsic affinity for repair foci. This binary complex was in turn coupled to the iron carrier protein, transferrin, which engages a high-affinity cell surface receptor. In a different version of the complex, transferrin was omitted and a protein transduction domain was incorporated directly into the primary structure of the 53BP1. These complexes were efficiently taken up into human osteosarcoma cells and synchronously released from endocytic vesicles by brief exposure to far-red light in the presence of the photosensitizer, disulfonated aluminum phthalocyanine. Upon release, the streptavidin cargo entered the nucleus and was recruited to repair foci. 53BP1-based platforms provide a method for targeted, temporally controlled delivery of macromolecular agents to sites of double-strand break repair. With the delivery platforms, we are capable to visualize, modify and redirect DSB repair pathways by coupling various nanomaterials to study machinery or manipulate for therapy purpose in the future.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Portadores de Fármacos/química , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/química , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Citoplasma/metabolismo , Corantes Fluorescentes/química , Humanos , Transporte Proteico
13.
Elife ; 82019 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-31135337

RESUMO

Coordination of the cellular response to DNA damage is organised by multi-domain 'scaffold' proteins, including 53BP1 and TOPBP1, which recognise post-translational modifications such as phosphorylation, methylation and ubiquitylation on other proteins, and are themselves carriers of such regulatory signals. Here we show that the DNA damage checkpoint regulating S-phase entry is controlled by a phosphorylation-dependent interaction of 53BP1 and TOPBP1. BRCT domains of TOPBP1 selectively bind conserved phosphorylation sites in the N-terminus of 53BP1. Mutation of these sites does not affect formation of 53BP1 or ATM foci following DNA damage, but abolishes recruitment of TOPBP1, ATR and CHK1 to 53BP1 damage foci, abrogating cell cycle arrest and permitting progression into S-phase. TOPBP1 interaction with 53BP1 is structurally complimentary to its interaction with RAD9-RAD1-HUS1, allowing these damage recognition factors to bind simultaneously to the same TOPBP1 molecule and cooperate in ATR activation in the G1 DNA damage checkpoint.


Assuntos
Proteínas de Transporte/química , Dano ao DNA/genética , Proteínas de Ligação a DNA/química , Complexos Multiproteicos/química , Proteínas Nucleares/química , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/química , Proteínas Mutadas de Ataxia Telangiectasia/química , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas de Transporte/genética , Pontos de Checagem do Ciclo Celular/genética , Quinase 1 do Ponto de Checagem/química , Quinase 1 do Ponto de Checagem/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Células HeLa , Humanos , Metilação , Complexos Multiproteicos/genética , Proteínas Nucleares/genética , Fosforilação , Ligação Proteica/genética , Conformação Proteica , Domínios Proteicos/genética , Processamento de Proteína Pós-Traducional/genética , Fase S/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Ubiquitinação/genética
14.
Biochem Biophys Res Commun ; 510(2): 236-241, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30685087

RESUMO

53BP1 (TP53-binding protein 1) plays a key role in DNA double-strand break repair by promoting non-homologous end joining (NHEJ) especially during G1 phase of the cell cycle. Nuclear import of 53BP1 is required for proper localization of 53BP1 and maintenance of genome integrity. 53BP1 has a classical bipartite nuclear localization signal (NLS) of sequence 1666-GKRKLITSEEERSPAKRGRKS-1686. Ser1678 within the 53BP1 NLS can be phosphorylated by CDK1/cyclin B, and a phosphomimetic substitution of Ser1678 with aspartate has been shown to negatively regulate nuclear import of 53BP1. Here, the X-ray crystal structures of the nuclear import adaptor importin-α1 bound to the wild-type 53BP1 NLS and the S1678D mutant of 53BP1 NLS are reported at resolutions of 1.9 and 1.7 Å, respectively. In the wild-type structure, not only the two basic clusters of the 53BP1 NLS but also the linker region between the basic clusters made extensive interactions with importin-α1. In the mutant structure, the linker region between the basic clusters in the 53BP1 NLS made fewer interactions with importin-α1 than those observed in the wild-type structure. However, biochemical binding assays using purified proteins showed that the 53BP1 mutation S1678D reduces the binding affinity to importin-α1 only to a modest extent. Implications of these findings for regulatory mechanism of 53BP1 nuclear import are discussed.


Assuntos
Transporte Ativo do Núcleo Celular , Sinais de Localização Nuclear/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/química , alfa Carioferinas/química , Núcleo Celular/metabolismo , Humanos , Cinética , Mutação , Fosforilação , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/química
15.
DNA Repair (Amst) ; 73: 110-119, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30497961

RESUMO

Maintenance of genome integrity and stability is a critical responsibility of the DNA damage response (DDR) within cells, such that any disruption in this kinase-based signaling pathway leads to development of various disorders, particularly cancer. The tumor suppressor P53-binding protein 1 (53BP1), as one of the main mediators of DDR, plays a pivotal role in orchestrating the choice of double-strand break (DSB) repair pathway and contains interaction surfaces for numerous DSB-responsive proteins. It has been extensively demonstrated that aberrant expression of 53BP1 contributes to tumor occurrence and development. 53BP1 loss of function in tumor tissues is also related to tumor progression and poor prognosis in human malignancies. Due to undeniable importance of this protein in various aspects of cancer initiation/progression, angiogenesis, metastasis and development of drug resistance, as well as its targeting in the treatment of cancer, this review focused on explaining the structure and function of 53BP1 and its contribution to cancer.


Assuntos
Dano ao DNA , Neoplasias/genética , Neoplasias/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Genoma Humano/genética , Humanos , Neoplasias/patologia , Transdução de Sinais , Transcrição Gênica , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/química , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética
16.
Proc Natl Acad Sci U S A ; 115(40): 10028-10033, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30224481

RESUMO

The KAT5 (Tip60/Esa1) histone acetyltransferase is part of NuA4, a large multifunctional complex highly conserved from yeast to mammals that targets lysines on H4 and H2A (X/Z) tails for acetylation. It is essential for cell viability, being a key regulator of gene expression, cell proliferation, and stem cell renewal and an important factor for genome stability. The NuA4 complex is directly recruited near DNA double-strand breaks (DSBs) to facilitate repair, in part through local chromatin modification and interplay with 53BP1 during the DNA damage response. While NuA4 is detected early after appearance of the lesion, its precise mechanism of recruitment remains to be defined. Here, we report a stepwise recruitment of yeast NuA4 to DSBs first by a DNA damage-induced phosphorylation-dependent interaction with the Xrs2 subunit of the Mre11-Rad50-Xrs2 (MRX) complex bound to DNA ends. This is followed by a DNA resection-dependent spreading of NuA4 on each side of the break along with the ssDNA-binding replication protein A (RPA). Finally, we show that NuA4 can acetylate RPA and regulate the dynamics of its binding to DNA, hence targeting locally both histone and nonhistone proteins for lysine acetylation to coordinate repair.


Assuntos
Quebras de DNA de Cadeia Dupla , DNA Fúngico , Histona Acetiltransferases , Proteínas de Saccharomyces cerevisiae , Acetilação , DNA Fúngico/química , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/química , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/química , Exodesoxirribonucleases/metabolismo , Histona Acetiltransferases/química , Histona Acetiltransferases/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/química , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
18.
Nat Commun ; 9(1): 2689, 2018 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-30002377

RESUMO

53BP1 performs essential functions in DNA double-strand break (DSB) repair and it was recently reported that Tudor interacting repair regulator (TIRR) negatively regulates 53BP1 during DSB repair. Here, we present the crystal structure of the 53BP1 tandem Tudor domain (TTD) in complex with TIRR. Our results show that three loops from TIRR interact with 53BP1 TTD and mask the methylated lysine-binding pocket in TTD. Thus, TIRR competes with histone H4K20 methylation for 53BP1 binding. We map key interaction residues in 53BP1 TTD and TIRR, whose mutation abolishes complex formation. Moreover, TIRR suppresses the relocation of 53BP1 to DNA lesions and 53BP1-dependent DNA damage repair. Finally, despite the high-sequence homology between TIRR and NUDT16, NUDT16 does not directly interact with 53BP1 due to the absence of key residues required for binding. Taken together, our study provides insights into the molecular mechanism underlying TIRR-mediated suppression of 53BP1-dependent DNA damage repair.


Assuntos
Proteínas de Transporte/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Ligação Competitiva , Proteínas de Transporte/química , Proteínas de Transporte/genética , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , Dano ao DNA , Células HEK293 , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilação , Mutação , Ligação Proteica , Proteínas de Ligação a RNA , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/química , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética
19.
Nat Struct Mol Biol ; 25(7): 591-600, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29967538

RESUMO

Dynamic protein interaction networks such as DNA double-strand break (DSB) signaling are modulated by post-translational modifications. The DNA repair factor 53BP1 is a rare example of a protein whose post-translational modification-binding function can be switched on and off. 53BP1 is recruited to DSBs by recognizing histone lysine methylation within chromatin, an activity directly inhibited by the 53BP1-binding protein TIRR. X-ray crystal structures of TIRR and a designer protein bound to 53BP1 now reveal a unique regulatory mechanism in which an intricate binding area centered on an essential TIRR arginine residue blocks the methylated-chromatin-binding surface of 53BP1. A 53BP1 separation-of-function mutation that abolishes TIRR-mediated regulation in cells renders 53BP1 hyperactive in response to DSBs, highlighting the key inhibitory function of TIRR. This 53BP1 inhibition is relieved by TIRR-interacting RNA molecules, providing proof-of-principle of RNA-triggered 53BP1 recruitment to DSBs.


Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/química , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Proteínas de Transporte/genética , Cristalografia por Raios X , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Histonas/química , Histonas/metabolismo , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica , Engenharia de Proteínas , Mapas de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Pirofosfatases/química , Pirofosfatases/genética , Pirofosfatases/metabolismo , Proteínas de Ligação a RNA/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética
20.
FEBS J ; 285(11): 2091-2103, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29645362

RESUMO

As a reader of di-methylated arginine on various proteins, such as histone, RNA polymerase II, PIWI and Fragile X mental retardation protein, the Tudor domain of Tudor domain-containing protein 3 (TDRD3) mediates transcriptional activation in nucleus and formation of stress granules in the cytoplasm. Despite the TDRD3 implication in cancer cell proliferation and invasion, warheads to block the di-methylated arginine recognition pocket of the TDRD3 Tudor domain have not yet been uncovered. Here we identified 14 small molecule hits against the TDRD3 Tudor domain through NMR fragment-based screening. These hits were further cross-validated by using competitive fluorescence polarization and isothermal titration calorimetry experiments. The crystal structure of the TDRD3 Tudor domain in complex with hit 1 reveals a distinct binding mode from the nature substrate. Hit 1 protrudes into the aromatic cage of the TDRD3 Tudor domain, where the aromatic residues are tilted to accommodate a sandwich-like π-π interaction. The side chain of the conserved residue N596 swings away 3.1 Å to form a direct hydrogen bond with hit 1. Moreover, this compound shows a decreased affinity against the single Tudor domain of survival motor neuron protein, but no detectable binding to neither the tandem Tudor domain of TP53-binding protein 1 nor the extended Tudor domain of staphylococcal nuclease domain-containing protein 1. Our work depicts the structural plasticity of the TDRD3 Tudor domain and paves the way for the subsequent structure-guided discovery of selective inhibitors targeting Tudor domains. DATABASE: Structural data are available in the PDB under the accession number 5YJ8.


Assuntos
Conformação Proteica , Proteínas/química , Bibliotecas de Moléculas Pequenas/química , Domínio Tudor , Sequência de Aminoácidos/genética , Proliferação de Células/genética , Cristalografia por Raios X , Endonucleases , Humanos , Ligação de Hidrogênio , Ressonância Magnética Nuclear Biomolecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas/genética , Ativação Transcricional/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/química , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética
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