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1.
Nature ; 571(7766): 521-527, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31270457

RESUMO

The integrity of genomes is constantly threatened by problems encountered by the replication fork. BRCA1, BRCA2 and a subset of Fanconi anaemia proteins protect stalled replication forks from degradation by nucleases, through pathways that involve RAD51. The contribution and regulation of BRCA1 in replication fork protection, and how this role relates to its role in homologous recombination, is unclear. Here we show that BRCA1 in complex with BARD1, and not the canonical BRCA1-PALB2 interaction, is required for fork protection. BRCA1-BARD1 is regulated by a conformational change mediated by the phosphorylation-directed prolyl isomerase PIN1. PIN1 activity enhances BRCA1-BARD1 interaction with RAD51, thereby increasing the presence of RAD51 at stalled replication structures. We identify genetic variants of BRCA1-BARD1 in patients with cancer that exhibit poor protection of nascent strands but retain homologous recombination proficiency, thus defining domains of BRCA1-BARD1 that are required for fork protection and associated with cancer development. Together, these findings reveal a BRCA1-mediated pathway that governs replication fork protection.


Assuntos
Proteína BRCA1/química , Proteína BRCA1/metabolismo , Replicação do DNA , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteína BRCA1/genética , Linhagem Celular Tumoral , Replicação do DNA/genética , Instabilidade Genômica/genética , Humanos , Isomerismo , Mutação , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Fosforilação , Fosfosserina/metabolismo , Ligação Proteica , Rad51 Recombinase/metabolismo
2.
J Biol Chem ; 294(15): 5980-5992, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30765603

RESUMO

Genetic testing for BRCA1, a DNA repair protein, can identify carriers of pathogenic variants associated with a substantially increased risk for breast and ovarian cancers. However, an association with increased risk is unclear for a large fraction of BRCA1 variants present in the human population. Most of these variants of uncertain clinical significance lead to amino acid changes in the BRCA1 protein. Functional assays are valuable tools to assess the potential pathogenicity of these variants. Here, we systematically probed the effects of substitutions in the C terminus of BRCA1: the N- and C-terminal borders of its tandem BRCT domain, the BRCT-[N-C] linker region, and the α1 and α'1 helices in BRCT-[N] and -[C]. Using a validated transcriptional assay based on a fusion of the GAL4 DNA-binding domain to the BRCA1 C terminus (amino acids 1396-1863), we assessed the functional impact of 99 missense variants of BRCA1. We include the data obtained for these 99 missense variants in a joint analysis to generate the likelihood of pathogenicity for 347 missense variants in BRCA1 using VarCall, a Bayesian integrative statistical model. The results from this analysis increase our understanding of BRCA1 regions less tolerant to changes, identify functional borders of structural domains, and predict the likelihood of pathogenicity for 98% of all BRCA1 missense variants in this region recorded in the population. This knowledge will be critical for improving risk assessment and clinical treatment of carriers of BRCA1 variants.


Assuntos
Proteína BRCA1 , Neoplasias da Mama , Modelos Moleculares , Mutação de Sentido Incorreto , Neoplasias Ovarianas , Substituição de Aminoácidos , Proteína BRCA1/química , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Feminino , Células HEK293 , Humanos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Domínios Proteicos , Relação Estrutura-Atividade
3.
Spectrochim Acta A Mol Biomol Spectrosc ; 209: 109-117, 2019 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-30384016

RESUMO

SISLL-TAT and TAT-SISLL were synthesized by modifying the N- or C-termini of cell-penetrating peptides as transacting activator of transcription TAT (47-57) by attaching BRCA1 (782-786) (SISLL). The novel peptides were synthesized through Fmoc solid-phase synthesis procedures and characterized by LCQ Fleet MS, 1H NMR and 13C NMR. SISLL-TAT and TAT-SISLL displayed forceful antibacterial activities against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Salmonella typhimurium with low hemolysis. SISLL-TAT showed better antibacterial activity than TAT-SISLL, with the minimum inhibitory concentration (MIC) values of 10-33 µg·mL-1. The results of the DNA-binding activities showed that both SISLL-TAT and TAT-SISLL could interact with DNA via the minor groove mode, and the binding constants were 4.97 × 105 L·mol-1 and 4.42 × 105 L·mol-1 at 310 K, respectively. Circular dichroism analysis showed slight transformation of the lysozyme secondary structure caused by SISLL-TAT and TAT-SISLL. We also found that the novel peptides SISLL-TAT and TAT-SISLL targeted bacterial DNA resulting in cell death. This explains the antibacterial mechanism of SISLL-TAT and TAT-SISLL, and is a solid theoretical basis for further designing novel and highly effective antibiotics for clinical application.


Assuntos
Antibacterianos/farmacologia , Proteína BRCA1/química , Bactérias/efeitos dos fármacos , DNA Bacteriano/metabolismo , Muramidase/metabolismo , Fragmentos de Peptídeos/farmacologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Antibacterianos/química , Proteína BRCA1/metabolismo , Dicroísmo Circular , Humanos , Técnicas In Vitro , Testes de Sensibilidade Microbiana , Fragmentos de Peptídeos/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo
4.
Biosens Bioelectron ; 112: 72-78, 2018 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-29698810

RESUMO

A novel electrochemical DNA (E-DNA) biosensing strategy was designed and used for the detection of breast cancer susceptibility gene (BRCA-1). The biosensor was based on gold nanoparticles-reduced graphene oxide (AuNPs-GO) modified glass carbon electrode (GCE) covered with the layer of molecularly imprinted polymers (MIPs) synthesized with rhodamine B (RhB) as template, methacrylic acid (MAA) as the monomer, and Nafion as additive. The signal amplification tracing tag SiO2@Ag NPs were prepared by covering AgNPs on the surface of SiO2 nanoparticles in situ, and then DNA probes were modified on AgNPs by Ag-S bond, forming the composites SiO2@Ag/DNA. In presence of target DNA (T-DNA), homogeneous hybridization was performed with SiO2@Ag/DNA and RhB labeled DNA, and the resulting SiO2@Ag/dsDNA/RhB was specifically recognized by MIPs via the interaction between imprinting cavities and RhB. Under optimal conditions, the proposed biosensor exhibited wide linear range from 10 fM to 100 nM, low detection limit of 2.53 fM (S/N = 3), excellent selectivity, reproducibility, stability, and feasibility in serum analysis. Overall, these findings suggest the promising prospects of the proposed biosensing strategy in clinical diagnostics.


Assuntos
Proteína BRCA1/isolamento & purificação , Técnicas Biossensoriais , Neoplasias da Mama/diagnóstico , DNA de Neoplasias/isolamento & purificação , Proteína BRCA1/química , Proteína BRCA1/genética , DNA de Neoplasias/química , DNA de Neoplasias/genética , Técnicas Eletroquímicas/métodos , Feminino , Ouro/química , Grafite/química , Humanos , Limite de Detecção , Impressão Molecular , Dióxido de Silício/química
5.
Int J Clin Oncol ; 23(1): 36-44, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28884397

RESUMO

The BRCA1 protein, a hereditary breast and ovarian cancer-causing gene product, is known as a multifunctional protein that performs various functions in cells. It is well known, along with BRCA 2, to cause hereditary breast and ovarian cancer, but here we will specifically focus on BRCA1. We introduce the mechanism and the latest report on homologous recombination repair, replication, involvement in checkpoint regulation, transcription, chromatin remodeling, and cytoplasmic function (centrosome regulation, apoptosis, selective autophagy), and consider the possibility of carcinogenesis from inhibition of the intracellular functions in each. We also consider the possibility of drug development based on each function. Finally, we will explain, from data obtained through basic research, that an appropriate regimen is important for raising the response rate for poly (ADP)-ribose polymerase inhibitors, in the case of low susceptibility, iatrogenic toxicity, tolerance, etc.


Assuntos
Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Neoplasias da Mama/genética , Neoplasias Ovarianas/genética , Apoptose/genética , Proteína BRCA1/química , Neoplasias da Mama/tratamento farmacológico , Pontos de Checagem do Ciclo Celular/genética , Montagem e Desmontagem da Cromatina , Reparo do DNA/genética , Feminino , Genes BRCA1 , Predisposição Genética para Doença , Recombinação Homóloga , Humanos , Mutação , Neoplasias Ovarianas/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia
6.
Cancer Genomics Proteomics ; 14(5): 293-298, 2017 Sep-Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28870997

RESUMO

BRCA1 and BRCA2 are both tumor suppressors whose mutations are the cause of most hereditary breast cancers. Both genes are highly involved in ensuring genome stability. BRCA1 homologs are found in the plant and animal kingdoms while BRCA2 homologs are additionally found in the fungi kingdom. The initial origin of both genes remains unknown, however it is expected that the common ancestors originated around 1.6 billion years ago prior to the kingdoms diverging. There has been a great amount of divergence between homologs that is not observed in other tumor suppressors with only functionally important domains conserved. This divergence continues today with evidence of primate BRCA1/2 evolution. Cancer-associated mutations have been found to occur at conserved sites, indicating that conserved sites are important for function. In this study, we present a review on the phylogenesis of BRCA1 and BRCA2.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/genética , Evolução Molecular , Proteína BRCA1/química , Proteína BRCA1/metabolismo , Proteína BRCA2/química , Proteína BRCA2/metabolismo , Feminino , Humanos , Filogenia , Domínios Proteicos
7.
J Mol Med (Berl) ; 95(8): 799-807, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28681078

RESUMO

A decade has passed since the first reported connection between RAP80 and BRCA1 in DNA double-strand break repair. Despite the initial identification of RAP80 as a factor localizing BRCA1 to DNA double-strand breaks and potentially promoting homologous recombination, there is increasing evidence that RAP80 instead suppresses homologous recombination to fine-tune the balance of competing DNA repair processes during the S/G2 phase of the cell cycle. RAP80 opposes homologous recombination by inhibiting DNA end-resection and sequestering BRCA1 into the BRCA1-A complex. Ubiquitin and SUMO modifications of chromatin at DNA double-strand breaks recruit RAP80, which contains distinct sequence motifs that recognize ubiquitin and SUMO. Here, we review RAP80's role in repressing homologous recombination at DNA double-strand breaks and how this role is facilitated by its ability to bind ubiquitin and SUMO modifications.


Assuntos
Proteínas de Transporte/genética , Dano ao DNA , Proteínas Nucleares/genética , Proteína SUMO-1/genética , Ubiquitina/genética , Animais , Proteína BRCA1/química , Proteína BRCA1/genética , Proteínas de Transporte/química , Recombinação Homóloga , Humanos , Proteínas Nucleares/química , Estrutura Secundária de Proteína , Proteína SUMO-1/química , Ubiquitina/química
8.
PLoS One ; 12(7): e0181062, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28704513

RESUMO

Current PCR-based target enrichment methods for next generation sequencing (NGS) of overlapping amplicons often requires separate PCR reactions and subsequent pooling of amplicons from the different reactions. The study presents a novel method, deemed stem-loop inhibition mediated amplification (SLIMamp), for amplifying overlapping or tiled amplicons in a single multiplex PCR reaction. During a SLIMamp PCR reaction, a stem loop structure formed by the overlapping amplicon suppresses additional amplification of itself by preventing the annealing of the primers. Using the SLIMamp strategy, we designed a next-generation sequencing (NGS) assay to enrich the exon regions of BRCA1 and BRCA2 for sequencing on an Illumina MiSeq system. We used 35 cell line DNAs and 6 patient blood DNAs in the study to evaluate the assay performance. For each sample, all targeted regions were successfully amplified and sequenced with excellent coverage uniformity and specificity. >99% of the total sequencing reads were mapped to the human reference genome (hg19) and >99% of the mapped reads were on the targeted exons. >98% of bases were covered at >0.20x of the mean coverage and >99% are covered at >0.15x of the mean depth. Among 34 independently sequenced samples, all variants were reliably detected with no false positives or false negatives. SLIMamp provides a robust method for single-tube multiplex PCR amplification of numerous, overlapping amplicons that tile for targeted next-generation sequencing.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Análise de Sequência de DNA/métodos , Proteína BRCA1/química , Proteína BRCA2/química , Linhagem Celular Tumoral , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , Técnicas de Diagnóstico Molecular/normas , Reação em Cadeia da Polimerase Multiplex/normas , Sensibilidade e Especificidade , Análise de Sequência de DNA/normas
9.
Biochem Biophys Res Commun ; 488(2): 355-361, 2017 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-28501617

RESUMO

RAPTA compounds, ([Ru(η6-arene)(PTA)Cl2], PTA = 1,3,5-triaza-7-phosphaadamantane), have been reported to overcome drug resistance in cisplatin resistant cells. However, the exact mechanism of these complexes is still largely unexplored. In this study, the interaction of some RAPTA compounds with the N-terminal fragment of the BRCA1 RING domain protein was investigated. The binding of the RAPTA compounds to the BRCA1 protein resulted in a release of Zn2+ ions in a dose and time dependent manner, as well as thermal alteration of ruthenated-BRCA1 proteins. Electron Transfer Dissociation (ETD) fragmentation mass spectrometry revealed the preferential binding sites of the RAPTA complexes on the BRCA1 zinc finger RING domain at a similar short peptide stretch, Cys24Lys25Phe26Cys27Met28Leu29 and Lys35 (residues 44-49 and 55 on full length BRCA1). Changes in the conformation and binding constants of ruthenium-BRCA1 adducts were established, resulting in inactivation of the RING heterodimer BRCA1/BARD1-mediated E3 ubiquitin ligase function. These findings could provide mechanistic insight into the mode of action of RAPTA complexes for on tested BRCA1 model protein.


Assuntos
Adamantano/análogos & derivados , Proteína BRCA1/metabolismo , Compostos Organofosforados/farmacologia , Domínios RING Finger/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo , Adamantano/química , Adamantano/farmacologia , Proteína BRCA1/antagonistas & inibidores , Proteína BRCA1/química , Sítios de Ligação/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Compostos Organofosforados/química , Relação Estrutura-Atividade , Ubiquitina-Proteína Ligases/antagonistas & inibidores
10.
Funct Integr Genomics ; 17(4): 375-385, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28470340

RESUMO

The proneness of diseases and susceptibility towards drugs vary from person to person. At present, there is a strong demand for the personalization of drugs. The genetic signature behind proneness of the disease has been studied through a comprehensive 'octopodial approach'. All the genetic variants included in the approach have been introduced. The breast cancer associated with BRCA1 mutation has been taken as the illustrative example to introduce all these factors. The genetic variants associated with the drug action of tamoxifen have been fully illustrated in the manuscript. The design of a new personalized anti-breast cancer drug has been explained in the third phase. For the design of new personalized drugs, a metabolite of anti-cancer drug chlorambucil has been taken as the template. The design of drug has been made with respect to the protein 1T15 of BRCA1 gene corresponding to the genetic signature of rs28897696.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Desenho de Drogas , Predisposição Genética para Doença , Farmacogenética/métodos , Medicina de Precisão/métodos , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proteína BRCA1/química , Proteína BRCA1/genética , Neoplasias da Mama/genética , Clorambucila/química , Clorambucila/farmacologia , Feminino , Humanos , Polimorfismo de Nucleotídeo Único
11.
Sci Rep ; 7: 43435, 2017 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-28262780

RESUMO

The precise manner in which physical changes to the breast cancer susceptibility protein (BRCA1) affect its role in DNA repair events remain unclear. Indeed, cancer cells harboring mutations in BRCA1 suffer from genomic instability and increased DNA lesions. Here, we used a combination of molecular imaging and biochemical tools to study the properties of the BRCA1 in human cancer cells. Our results reveal new information for the manner in which full-length BRCA1 engages its binding partner, the BRCA1-associated Ring Domain protein (BARD1) under oxidative stress conditions. We also show how physical differences between wild type and mutated BRCA15382insC impact the cell's response to oxidative damage. Overall, we demonstrate how clinically relevant changes to BRCA1 affect its structure-function relationship in hereditary breast cancer.


Assuntos
Proteína BRCA1/química , Reparo do DNA , Regulação Neoplásica da Expressão Gênica , Proteínas Supressoras de Tumor/química , Ubiquitina-Proteína Ligases/química , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Dano ao DNA , Feminino , Guanina/análogos & derivados , Guanina/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Glândulas Mamárias Humanas/efeitos dos fármacos , Glândulas Mamárias Humanas/metabolismo , Glândulas Mamárias Humanas/patologia , Modelos Moleculares , Imagem Molecular , Mutação , Estresse Oxidativo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Homologia Estrutural de Proteína , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
12.
Science ; 355(6324): 520-524, 2017 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-28154079

RESUMO

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a central component of nonhomologous end joining (NHEJ), repairing DNA double-strand breaks that would otherwise lead to apoptosis or cancer. We have solved its structure in complex with the C-terminal peptide of Ku80 at 4.3 angstrom resolution using x-ray crystallography. We show that the 4128-amino acid structure comprises three large structural units: the N-terminal unit, the Circular Cradle, and the Head. Conformational differences between the two molecules in the asymmetric unit are correlated with changes in accessibility of the kinase active site, which are consistent with an allosteric mechanism to bring about kinase activation. The location of KU80ct194 in the vicinity of the breast cancer 1 (BRCA1) binding site suggests competition with BRCA1, leading to pathway selection between NHEJ and homologous recombination.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Proteína Quinase Ativada por DNA/química , Proteína Quinase Ativada por DNA/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteína BRCA1/química , Domínio Catalítico , Cristalografia por Raios X , DNA/química , DNA/ultraestrutura , Células HeLa , Humanos , Autoantígeno Ku/química , Peptídeos/química , Ligação Proteica , Conformação Proteica
13.
J Biomol Struct Dyn ; 35(1): 1-7, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26727155

RESUMO

Familial inheritance of breast and ovarian cancer is attributed to mutations discovered in functional domains of BRCA1 gene. BRCA1 is a multifunctional protein responsible for maintaining the genomic integrity and has transcriptional regulatory function encoded in its C-terminal region. The different amino-terminal e extensions to BRCA1 BRCT domain are responsible for transcription activation. However, only BRCA1 BRCT (1649-1859) amino acids have been explored for its structural characteristics. Noting the importance of extended region to the N-terminus of BRCT different regions of BRCA1 which demonstrates maximum transactivation activity has been explored for their structure and functional activity. Secondary and tertiary structural analysis revealed a limited alpha-helical content with well-folded tertiary structure. In silico tools were used to corroborate the in vitro results. Amino acids composition and sequence analysis display a propensity for intrinsic disorder and coiled-coil formation in BRCA1 (1396-1863) (BRCA1-TAD). The results presented in this paper suggest the extreme flexibility in coiled-coil motif might be an important requirement in the establishment of protein-protein interaction networks for BRCA1.


Assuntos
Proteína BRCA1/química , Modelos Moleculares , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Sequência de Aminoácidos , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Humanos , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes , Relação Estrutura-Atividade , Ativação Transcricional
14.
Protein Sci ; 26(3): 475-483, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27977889

RESUMO

The tumor-suppressor protein BRCA1 works with BARD1 to catalyze the transfer of ubiquitin onto protein substrates. The N-terminal regions of BRCA1 and BARD1 that contain their RING domains are responsible for dimerization and ubiquitin ligase activity. This activity is a common feature among hundreds of human RING domain-containing proteins. RING domains bind and activate E2 ubiquitin-conjugating enzymes to promote ubiquitin transfer to substrates. We show that the identity of residues at specific positions in the RING domain can tune activity levels up or down. We report substitutions that create a structurally intact BRCA1/BARD1 heterodimer that is inactive in vitro with all E2 enzymes. Other substitutions in BRCA1 or BARD1 RING domains result in hyperactivity, revealing that both proteins have evolved attenuated activity. Loss of attenuation results in decreased product specificity, providing a rationale for why nature has tuned BRCA1 activity. The ability to tune BRCA1 provides powerful tools for understanding its biological functions and provides a basis to assess mechanisms for rescuing the activity of cancer-associated variations. Beyond the applicability to BRCA1, we show the identity of residues at tuning positions that can be used to predict and modulate the activity of an unrelated RING E3 ligase. These findings provide valuable insights into understanding the mechanism and function of RING E3 ligases like BRCA1.


Assuntos
Proteína BRCA1/química , Multimerização Proteica , Proteínas Supressoras de Tumor/química , Ubiquitina-Proteína Ligases/química , Ubiquitina/química , Ubiquitinação , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Humanos , Domínios Proteicos , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
15.
Nucleus ; 8(2): 116-125, 2017 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-28032817

RESUMO

The protein product of the breast and ovarian cancer gene, BRCA1, is part of an obligate heterodimer with BARD1. Together these RING bearing proteins act as an E3 ubiquitin ligase. Several functions have been attributed to BRCA1 that contribute to genome integrity but which of these, if any, require this enzymatic function was unclear. Here we review recent studies clarifying the role of BRCA1 E3 ubiquitin ligase in DNA repair. Perhaps the most surprising finding is the narrow range of BRCA1 functions this activity relates to. Remarkably ligase activity promotes chromatin remodelling and 53BP1 positioning through the remodeller SMARCAD1, but the activity is dispensable for the cellular survival in response to cisplatin or replication stressing agents. Implications for therapy response and tumor susceptibility are discussed.


Assuntos
Proteína BRCA1/metabolismo , Reparo do DNA , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteína BRCA1/química , Cromatina/metabolismo , Inativação Gênica , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia
16.
Methods Mol Biol ; 1518: 139-156, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27873205

RESUMO

Microarray screening technology has transformed the life sciences arena over the last decade. The platform is widely used in the area of mapping interaction networks, to molecular fingerprinting and small molecular inhibitor discovery. The technique has significantly impacted both basic and applied research. The microarray platform can likewise enable high-throughput screening and discovery of protein-protein interaction (PPI) inhibitors. Herein we demonstrate the application of microarray-guided PPI inhibitor discovery, using human BRCA1 as an example. Mutations in BRCA1 have been implicated in ~50 % of hereditary breast cancers. By targeting the (BRCT)2 domain, we showed compound 15a and its prodrug 15b inhibited BRCA1 activities in tumor cells. Unlike previously reported peptide-based PPI inhibitors of BRCA1, the compounds identified could be directly administered to tumor cells, thus making them useful in targeting BRCA1/PARP-related pathways involved in DNA damage and repair response, for cancer therapy.


Assuntos
Proteína BRCA1/metabolismo , Análise em Microsséries/métodos , Bibliotecas de Moléculas Pequenas/análise , Apoptose , Proteína BRCA1/química , Calorimetria , Caspase 3/metabolismo , Proliferação de Células , Cristalografia por Raios X , Polarização de Fluorescência , Células HeLa , Recombinação Homóloga , Humanos , Peptídeos/química , Ligação Proteica/efeitos dos fármacos , Coloração e Rotulagem
18.
Cell Rep ; 17(12): 3099-3106, 2016 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-28009280

RESUMO

BRCA1 is a tumor suppressor found to be mutated in hereditary breast and ovarian cancer and plays key roles in the maintenance of genomic stability by homologous recombination repair. It is recruited to damaged chromatin as a component of the BRCA1-A deubiquitinase, which cleaves K63-linked ubiquitin chains attached to histone H2A and H2AX. BRCA1-A contributes to checkpoint regulation, repair pathway choice, and HR repair efficiency through molecular mechanisms that remain largely obscure. The structure of an active core complex comprising two Abraxas/BRCC36/BRCC45/MERIT40 tetramers determined by negative-stain electron microscopy (EM) reveals a distorted V-shape architecture in which a dimer of Abraxas/BRCC36 heterodimers sits at the base, with BRCC45/Merit40 pairs occupying each arm. The location and ubiquitin-binding activity of BRCC45 suggest that it may provide accessory interactions with nucleosome-linked ubiquitin chains that contribute to their efficient processing. Our data also suggest how ataxia telangiectasia mutated (ATM)-dependent BRCA1 dimerization may stabilize self-association of the entire BRCA1-A complex.


Assuntos
Proteína BRCA1/química , Proteínas de Transporte/química , Enzimas Desubiquitinantes/química , Histonas/química , Complexos Multiproteicos/química , Proteínas Mutadas de Ataxia Telangiectasia/química , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteína BRCA1/genética , Proteína BRCA1/ultraestrutura , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteínas de Transporte/genética , Proteínas de Transporte/ultraestrutura , Cromatina/química , Cromatina/genética , Dano ao DNA/genética , Reparo do DNA/genética , Enzimas Desubiquitinantes/genética , Enzimas Desubiquitinantes/ultraestrutura , Instabilidade Genômica , Histonas/genética , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/ultraestrutura , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Reparo de DNA por Recombinação/genética , Ubiquitina/genética
19.
J Cell Sci ; 129(23): 4366-4378, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27802165

RESUMO

Understanding the effect of an ever-growing number of human variants detected by genome sequencing is a medical challenge. The yeast Saccharomyces cerevisiae model has held attention for its capacity to monitor the functional impact of missense mutations found in human genes, including the BRCA1 breast and ovarian cancer susceptibility gene. When expressed in yeast, the wild-type full-length BRCA1 protein forms a single nuclear aggregate and induces a growth inhibition. Both events are modified by pathogenic mutations of BRCA1. However, the biological processes behind these events in yeast remain to be determined. Here, we show that the BRCA1 nuclear aggregation and the growth inhibition are sensitive to misfolding effects induced by missense mutations. Moreover, misfolding mutations impair the nuclear targeting of BRCA1 in yeast cells and in a human cell line. In conclusion, we establish a connection between misfolding and nuclear transport impairment, and we illustrate that yeast is a suitable model to decipher the effect of misfolding mutations.


Assuntos
Proteína BRCA1/química , Proteína BRCA1/metabolismo , Dobramento de Proteína , Saccharomyces cerevisiae/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Fluorescência , Humanos , Modelos Biológicos , Mutação/genética , Sinais de Localização Nuclear , Agregados Proteicos , Domínios Proteicos , Estabilidade Proteica , Transporte Proteico , Saccharomyces cerevisiae/crescimento & desenvolvimento
20.
PLoS Comput Biol ; 12(8): e1005057, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27560145

RESUMO

Inhibition of the protein-protein interaction (PPI) mediated by breast-cancer-gene 1 C-terminal (BRCT) is an attractive strategy to sensitize breast and ovarian cancers to chemotherapeutic agents that induce DNA damage. Such inhibitors could also be used for studies to understand the role of this PPI in DNA damage response. However, design of BRCT inhibitors is challenging because of the inherent flexibility associated with this domain. Several studies identified short phosphopeptides as tight BRCT binders. Here we investigated the thermodynamic properties of 18 phosphopeptides or peptide with phosphate mimic and three compounds with phosphate groups binding to BRCT to understand promiscuous molecular recognition and guide inhibitor design. We performed molecular dynamics (MD) simulations to investigate the interactions between inhibitors and BRCT and their dynamic behavior in the free and bound states. MD simulations revealed the key role of loops in altering the shape and size of the binding site to fit various ligands. The mining minima (M2) method was used for calculating binding free energy to explore the driving forces and the fine balance between configuration entropy loss and enthalpy gain. We designed a rigidified ligand, which showed unfavorable experimental binding affinity due to weakened enthalpy. This was because it lacked the ability to rearrange itself upon binding. Investigation of another phosphate group containing compound, C1, suggested that the entropy loss can be reduced by preventing significant narrowing of the energy well and introducing multiple new compound conformations in the bound states. From our computations, we designed an analog of C1 that introduced new intermolecular interactions to strengthen attractions while maintaining small entropic penalty. This study shows that flexible compounds do not always encounter larger entropy penalty, compared with other more rigid binders, and highlights a new strategy for inhibitor design.


Assuntos
Proteína BRCA1 , Simulação de Dinâmica Molecular , Fosfopeptídeos , Antineoplásicos/análise , Antineoplásicos/química , Antineoplásicos/metabolismo , Proteína BRCA1/antagonistas & inibidores , Proteína BRCA1/química , Proteína BRCA1/metabolismo , Entropia , Humanos , Ligantes , Fosfopeptídeos/análise , Fosfopeptídeos/química , Fosfopeptídeos/metabolismo , Ligação Proteica , Termodinâmica
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