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1.
Nucleic Acids Res ; 52(1): 259-273, 2024 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-37994723

RESUMO

R-loops are three-stranded nucleic acid structures that can cause replication stress by blocking replication fork progression. However, the detailed mechanism underlying the collision of DNA replication forks and R-loops remains elusive. To investigate how R-loops induce replication stress, we use single-molecule fluorescence imaging to directly visualize the collision of replicating Phi29 DNA polymerase (Phi29 DNAp), the simplest replication system, and R-loops. We demonstrate that a single R-loop can block replication, and the blockage is more pronounced when an RNA-DNA hybrid is on the non-template strand. We show that this asymmetry results from secondary structure formation on the non-template strand, which impedes the progression of Phi29 DNAp. We also show that G-quadruplex formation on the displaced single-stranded DNA in an R-loop enhances the replication stalling. Moreover, we observe the collision between Phi29 DNAp and RNA transcripts synthesized by T7 RNA polymerase (T7 RNAp). RNA transcripts cause more stalling because of the presence of T7 RNAp. Our work provides insights into how R-loops impede DNA replication at single-molecule resolution.


Assuntos
Replicação do DNA , Estruturas R-Loop , Imagem Individual de Molécula , RNA/química , DNA Polimerase Dirigida por DNA/metabolismo
2.
Nucleic Acids Res ; 51(15): 7936-7950, 2023 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-37378431

RESUMO

Replication protein A (RPA), a eukaryotic single-stranded DNA (ssDNA) binding protein, dynamically interacts with ssDNA in different binding modes and plays essential roles in DNA metabolism such as replication, repair, and recombination. RPA accumulation on ssDNA due to replication stress triggers the DNA damage response (DDR) by activating the ataxia telangiectasia and RAD3-related (ATR) kinase, which phosphorylates itself and downstream DDR factors, including RPA. We recently reported that the N-methyl-D-aspartate receptor synaptonuclear signaling and neuronal migration factor (NSMF), a neuronal protein associated with Kallmann syndrome, promotes RPA32 phosphorylation via ATR upon replication stress. However, how NSMF enhances ATR-mediated RPA32 phosphorylation remains elusive. Here, we demonstrate that NSMF colocalizes and physically interacts with RPA at DNA damage sites in vivo and in vitro. Using purified RPA and NSMF in biochemical and single-molecule assays, we find that NSMF selectively displaces RPA in the more weakly bound 8- and 20-nucleotide binding modes from ssDNA, allowing the retention of more stable RPA molecules in the 30-nt binding mode. The 30-nt binding mode of RPA enhances RPA32 phosphorylation by ATR, and phosphorylated RPA becomes stabilized on ssDNA. Our findings provide new mechanistic insight into how NSMF facilitates the role of RPA in the ATR pathway.


Assuntos
Proteínas Serina-Treonina Quinases , Proteína de Replicação A , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quinase 1 do Ponto de Checagem/metabolismo , Dano ao DNA , Replicação do DNA , DNA de Cadeia Simples , Proteínas de Ligação a DNA/genética , Fosforilação , Ligação Proteica , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteína de Replicação A/metabolismo , Humanos
3.
Nucleic Acids Res ; 51(2): 631-649, 2023 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-36594163

RESUMO

TRAIP is a key factor involved in the DNA damage response (DDR), homologous recombination (HR) and DNA interstrand crosslink (ICL) repair. However, the exact functions of TRAIP in these processes in mammalian cells are not fully understood. Here we identify the zinc finger protein 212, ZNF212, as a novel binding partner for TRAIP and find that ZNF212 colocalizes with sites of DNA damage. The recruitment of TRAIP or ZNF212 to sites of DNA damage is mutually interdependent. We show that depletion of ZNF212 causes defects in the DDR and HR-mediated repair in a manner epistatic to TRAIP. In addition, an epistatic analysis of Zfp212, the mouse homolog of human ZNF212, in mouse embryonic stem cells (mESCs), shows that it appears to act upstream of both the Neil3 and Fanconi anemia (FA) pathways of ICLs repair. We find that human ZNF212 interacted directly with NEIL3 and promotes its recruitment to ICL lesions. Collectively, our findings identify ZNF212 as a new factor involved in the DDR, HR-mediated repair and ICL repair though direct interaction with TRAIP.


Assuntos
Reparo do DNA , Anemia de Fanconi , Animais , Camundongos , Humanos , Reparo do DNA/genética , Dano ao DNA , Replicação do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genômica , Anemia de Fanconi/genética , Mamíferos/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas do Tecido Nervoso/genética
4.
Nucleic Acids Res ; 51(11): 5584-5602, 2023 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-37140056

RESUMO

DNA double-strand break (DSB) repair via homologous recombination is initiated by end resection. The extent of DNA end resection determines the choice of the DSB repair pathway. Nucleases for end resection have been extensively studied. However, it is still unclear how the potential DNA structures generated by the initial short resection by MRE11-RAD50-NBS1 are recognized and recruit proteins, such as EXO1, to DSB sites to facilitate long-range resection. We found that the MSH2-MSH3 mismatch repair complex is recruited to DSB sites through interaction with the chromatin remodeling protein SMARCAD1. MSH2-MSH3 facilitates the recruitment of EXO1 for long-range resection and enhances its enzymatic activity. MSH2-MSH3 also inhibits access of POLθ, which promotes polymerase theta-mediated end-joining (TMEJ). Collectively, we present a direct role of MSH2-MSH3 in the initial stages of DSB repair by promoting end resection and influencing the DSB repair pathway by favoring homologous recombination over TMEJ.


Assuntos
Reparo do DNA , Exodesoxirribonucleases , Proteína 2 Homóloga a MutS , Proteína 3 Homóloga a MutS , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Exodesoxirribonucleases/metabolismo , Recombinação Homóloga , Proteína 2 Homóloga a MutS/metabolismo , Humanos , Linhagem Celular , DNA Helicases/metabolismo , Proteína 3 Homóloga a MutS/metabolismo
5.
EMBO Rep ; 23(7): e53492, 2022 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-35582821

RESUMO

Genome instability is one of the leading causes of gastric cancers. However, the mutational landscape of driver genes in gastric cancer is poorly understood. Here, we investigate somatic mutations in 25 Korean gastric adenocarcinoma patients using whole-exome sequencing and show that PWWP2B is one of the most frequently mutated genes. PWWP2B mutation correlates with lower cancer patient survival. We find that PWWP2B has a role in DNA double-strand break repair. As a nuclear protein, PWWP2B moves to sites of DNA damage through its interaction with UHRF1. Depletion of PWWP2B enhances cellular sensitivity to ionizing radiation (IR) and impairs IR-induced foci formation of RAD51. PWWP2B interacts with MRE11 and participates in homologous recombination via promoting DNA end-resection. Taken together, our data show that PWWP2B facilitates the recruitment of DNA repair machinery to sites of DNA damage and promotes HR-mediated DNA double-strand break repair. Impaired PWWP2B function might thus cause genome instability and promote gastric cancer development.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Neoplasias Gástricas , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Reparo do DNA , Instabilidade Genômica , Recombinação Homóloga , Humanos , Rad51 Recombinase/metabolismo , Reparo de DNA por Recombinação , Neoplasias Gástricas/genética , Ubiquitina-Proteína Ligases/metabolismo
6.
Aesthet Surg J ; 2024 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-39116358

RESUMO

BACKGROUND: Understanding the attachment patterns of the corrugator supercilii muscle (CS) is vital for treatments designed to improve facial symmetry and functionality. OBJECTIVES: The aim of this research was to elucidate the anatomical trajectory and intricate relationships of the CS within the upper face and midface, specifically focusing on its connections with the frontalis (FT) and orbicularis oculi (OOc) muscles. METHODS: The CS was examined in 41 specimens of embalmed adult Korean cadavers using microdissection, histological analyses, and microcomputed tomography, an in-depth exploration of its anatomical positioning and intricate interactions with adjacent muscles. RESULTS: Some lower fibers of the CS extended to the upper orbital part of the OOc in 59.5% of cases, while the CS interdigitated or blended exclusively with the FT in 40.5% of cases. The fibers of the CS demonstrated diverse extensions towards the upper face and midface, exhibiting varied trajectories and lengths. Additionally, lower fibers of the CS extended to significant anatomical landmarks such as the OOc, malaris muscle, and the superficial musculoaponeurotic system (SMAS). CONCLUSIONS: This study demonstrates that precise understanding of the CS and its relationship with the FT and OOc is crucial for optimizing invasive or non-invasive treatment like botulinum toxin injection, SMAS lifting and browplasty surgery. The extension of lower fibers of the CS to significant anatomical landmarks indicates complex interactions with adjacent facial structures, highlighting the necessity of detailed anatomical knowledge for clinical applications.

7.
Mol Cancer ; 22(1): 177, 2023 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-37932786

RESUMO

BACKGROUND: Although the development of BCR::ABL1 tyrosine kinase inhibitors (TKIs) rendered chronic myeloid leukemia (CML) a manageable condition, acquisition of drug resistance during blast phase (BP) progression remains a critical challenge. Here, we reposition FLT3, one of the most frequently mutated drivers of acute myeloid leukemia (AML), as a prognostic marker and therapeutic target of BP-CML. METHODS: We generated FLT3 expressing BCR::ABL1 TKI-resistant CML cells and enrolled phase-specific CML patient cohort to obtain unpaired and paired serial specimens and verify the role of FLT3 signaling in BP-CML patients. We performed multi-omics approaches in animal and patient studies to demonstrate the clinical feasibility of FLT3 as a viable target of BP-CML by establishing the (1) molecular mechanisms of FLT3-driven drug resistance, (2) diagnostic methods of FLT3 protein expression and localization, (3) association between FLT3 signaling and CML prognosis, and (4) therapeutic strategies to tackle FLT3+ CML patients. RESULTS: We reposition the significance of FLT3 in the acquisition of drug resistance in BP-CML, thereby, newly classify a FLT3+ BP-CML subgroup. Mechanistically, FLT3 expression in CML cells activated the FLT3-JAK-STAT3-TAZ-TEAD-CD36 signaling pathway, which conferred resistance to a wide range of BCR::ABL1 TKIs that was independent of recurrent BCR::ABL1 mutations. Notably, FLT3+ BP-CML patients had significantly less favorable prognosis than FLT3- patients. Remarkably, we demonstrate that repurposing FLT3 inhibitors combined with BCR::ABL1 targeted therapies or the single treatment with ponatinib alone can overcome drug resistance and promote BP-CML cell death in patient-derived FLT3+ BCR::ABL1 cells and mouse xenograft models. CONCLUSION: Here, we reposition FLT3 as a critical determinant of CML progression via FLT3-JAK-STAT3-TAZ-TEAD-CD36 signaling pathway that promotes TKI resistance and predicts worse prognosis in BP-CML patients. Our findings open novel therapeutic opportunities that exploit the undescribed link between distinct types of malignancies.


Assuntos
Crise Blástica , Leucemia Mielogênica Crônica BCR-ABL Positiva , Animais , Camundongos , Humanos , Crise Blástica/tratamento farmacológico , Crise Blástica/genética , Crise Blástica/patologia , Proteínas de Fusão bcr-abl/genética , Resistencia a Medicamentos Antineoplásicos/genética , Transdução de Sinais , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Inibidores de Proteínas Quinases/farmacologia , Tirosina Quinase 3 Semelhante a fms/metabolismo
8.
Nucleic Acids Res ; 49(10): 5605-5622, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-33963872

RESUMO

Proper activation of DNA repair pathways in response to DNA replication stress is critical for maintaining genomic integrity. Due to the complex nature of the replication fork (RF), problems at the RF require multiple proteins, some of which remain unidentified, for resolution. In this study, we identified the N-methyl-D-aspartate receptor synaptonuclear signaling and neuronal migration factor (NSMF) as a key replication stress response factor that is important for ataxia telangiectasia and Rad3-related protein (ATR) activation. NSMF localizes rapidly to stalled RFs and acts as a scaffold to modulate replication protein A (RPA) complex formation with cell division cycle 5-like (CDC5L) and ATR/ATR-interacting protein (ATRIP). Depletion of NSMF compromised phosphorylation and ubiquitination of RPA2 and the ATR signaling cascade, resulting in genomic instability at RFs under DNA replication stress. Consistently, NSMF knockout mice exhibited increased genomic instability and hypersensitivity to genotoxic stress. NSMF deficiency in human and mouse cells also caused increased chromosomal instability. Collectively, these findings demonstrate that NSMF regulates the ATR pathway and the replication stress response network for genome maintenance and cell survival.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a RNA/metabolismo , Proteína de Replicação A/metabolismo , Fatores de Transcrição/fisiologia , Animais , Replicação do DNA , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Knockout
9.
Nucleic Acids Res ; 49(19): 11083-11102, 2021 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-34614178

RESUMO

Mutual crosstalk among poly(ADP-ribose) (PAR), activated PAR polymerase 1 (PARP1) metabolites, and DNA repair machinery has emerged as a key regulatory mechanism of the DNA damage response (DDR). However, there is no conclusive evidence of how PAR precisely controls DDR. Herein, six deubiquitinating enzymes (DUBs) associated with PAR-coupled DDR were identified, and the role of USP39, an inactive DUB involved in spliceosome assembly, was characterized. USP39 rapidly localizes to DNA lesions in a PAR-dependent manner, where it regulates non-homologous end-joining (NHEJ) via a tripartite RG motif located in the N-terminus comprising 46 amino acids (N46). Furthermore, USP39 acts as a molecular trigger for liquid demixing in a PAR-coupled N46-dependent manner, thereby directly interacting with the XRCC4/LIG4 complex during NHEJ. In parallel, the USP39-associated spliceosome complex controls homologous recombination repair in a PAR-independent manner. These findings provide mechanistic insights into how PAR chains precisely control DNA repair processes in the DDR.


Assuntos
Reparo do DNA por Junção de Extremidades , DNA Ligase Dependente de ATP/genética , Proteínas de Ligação a DNA/genética , DNA/genética , Poli(ADP-Ribose) Polimerases/genética , Proteases Específicas de Ubiquitina/genética , Motivos de Aminoácidos , Ciclo Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Ligase Dependente de ATP/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endopeptidases/genética , Endopeptidases/metabolismo , Fator de Iniciação 3 em Eucariotos/genética , Fator de Iniciação 3 em Eucariotos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Osteoblastos/citologia , Osteoblastos/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Reparo de DNA por Recombinação , Transdução de Sinais , Spliceossomos , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Proteases Específicas de Ubiquitina/metabolismo
10.
Nucleic Acids Res ; 49(1): 269-284, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33313823

RESUMO

R-loops are three-stranded, RNA-DNA hybrid, nucleic acid structures produced due to inappropriate processing of newly transcribed RNA or transcription-replication collision (TRC). Although R-loops are important for many cellular processes, their accumulation causes genomic instability and malignant diseases, so these structures are tightly regulated. It was recently reported that R-loop accumulation is resolved by methyltransferase-like 3 (METTL3)-mediated m6A RNA methylation under physiological conditions. However, it remains unclear how R-loops in the genome are recognized and induce resolution signals. Here, we demonstrate that tonicity-responsive enhancer binding protein (TonEBP) recognizes R-loops generated by DNA damaging agents such as ultraviolet (UV) or camptothecin (CPT). Single-molecule imaging and biochemical assays reveal that TonEBP preferentially binds a R-loop via both 3D collision and 1D diffusion along DNA in vitro. In addition, we find that TonEBP recruits METTL3 to R-loops through the Rel homology domain (RHD) for m6A RNA methylation. We also show that TonEBP recruits RNaseH1 to R-loops through a METTL3 interaction. Consistent with this, TonEBP or METTL3 depletion increases R-loops and reduces cell survival in the presence of UV or CPT. Collectively, our results reveal an R-loop resolution pathway by TonEBP and m6A RNA methylation by METTL3 and provide new insights into R-loop resolution processes.


Assuntos
Adenosina/análogos & derivados , Replicação do DNA/genética , Metiltransferases/fisiologia , Estruturas R-Loop/genética , Fatores de Transcrição/fisiologia , Adenosina/metabolismo , Linhagem Celular Tumoral , DNA/genética , DNA/metabolismo , Adutos de DNA/metabolismo , Dano ao DNA , Difusão , Células HEK293 , Humanos , Metilação , Ligação Proteica , Mapeamento de Interação de Proteínas , Estruturas R-Loop/efeitos da radiação , Ribonuclease H/fisiologia , Raios Ultravioleta
11.
Surg Radiol Anat ; 45(4): 461-468, 2023 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-36792669

RESUMO

PURPOSE: The aim of this study was to determine the width of the fibers that extend from the orbicularis oculi muscle (OOc) to the upper lip, and the lateral and inferior lengths of the OOc at the lateral canthus level. METHODS: The OOc was investigated in the 40 hemifaces of 20 Korean cadavers. The lateral fibers of the OOc (OOc lat) were traced to determine whether or not these fibers extended to the upper lip. RESULTS: The OOc lat extended to the upper lip at the lateral canthus level in 31 of the 40 specimens (77.5%), whereas some inferolateral fibers of the OOc that extended to the upper lip were observed near the level of the lower margin of the OOc in the other 9 specimens (22.5%). The mean ± SD and maximum widths of the OOc lat that extended to the upper lip at the lateral canthus level were 6.9 ± 3.3 mm and 14.3 mm, respectively. CONCLUSION: The obtained data will be helpful to distinguish the muscles that underly the wrinkles around the lateral canthus for safer and more-efficient BoNT-A treatments for crow's feet.


Assuntos
Toxinas Botulínicas Tipo A , Aparelho Lacrimal , Envelhecimento da Pele , Humanos , Lábio , Músculos Faciais
12.
Am J Hum Genet ; 104(3): 439-453, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30773278

RESUMO

SPONASTRIME dysplasia is a rare, recessive skeletal dysplasia characterized by short stature, facial dysmorphism, and aberrant radiographic findings of the spine and long bone metaphysis. No causative genetic alterations for SPONASTRIME dysplasia have yet been determined. Using whole-exome sequencing (WES), we identified bi-allelic TONSL mutations in 10 of 13 individuals with SPONASTRIME dysplasia. TONSL is a multi-domain scaffold protein that interacts with DNA replication and repair factors and which plays critical roles in resistance to replication stress and the maintenance of genome integrity. We show here that cellular defects in dermal fibroblasts from affected individuals are complemented by the expression of wild-type TONSL. In addition, in vitro cell-based assays and in silico analyses of TONSL structure support the pathogenicity of those TONSL variants. Intriguingly, a knock-in (KI) Tonsl mouse model leads to embryonic lethality, implying the physiological importance of TONSL. Overall, these findings indicate that genetic variants resulting in reduced function of TONSL cause SPONASTRIME dysplasia and highlight the importance of TONSL in embryonic development and postnatal growth.


Assuntos
Fibroblastos/patologia , Genes Letais , Mutação , NF-kappa B/genética , Osteocondrodisplasias/patologia , Adolescente , Adulto , Animais , Células Cultivadas , Criança , Pré-Escolar , Dano ao DNA , Derme/metabolismo , Derme/patologia , Feminino , Fibroblastos/metabolismo , Humanos , Lactente , Recém-Nascido , Camundongos , Camundongos Endogâmicos C57BL , Osteocondrodisplasias/genética , Sequenciamento do Exoma/métodos , Adulto Jovem
13.
Biosci Biotechnol Biochem ; 85(12): 2420-2428, 2021 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-34643674

RESUMO

Ubiquitination and deubiquitination of signaling molecules are critical regulatory mechanisms in various biological contexts such as inflammatory signaling and the DNA damage response. Thus, finely tuned regulation of protein ubiquitination is essential for maintaining cellular homeostasis. Here, we showed that the RING finger protein RNF126 interacts with TRAF3 and promotes its K63-linked polyubiquitination, which is a crucial step in the TRAF3-dependent antiviral response. We found that RNF126 also interacts with OTUB1, a deubiquitinating enzyme that negatively regulates K63-linked ubiquitination of TRAF3. RNF126 promotes ubiquitination of OTUB1, leading to reduced deubiquitinating activity toward TRAF3. Moreover, RNF126 promotes ubiquitination of OTUB1 on cysteine 91, which is reportedly required for its catalytic activity. Taken together, our results suggest that RNF126 positively regulates the antiviral response by directly promoting K63-linked polyubiquitination of TRAF3 and by reducing OTUB1 activity.


Assuntos
Fator 3 Associado a Receptor de TNF
14.
Sensors (Basel) ; 20(7)2020 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-32244381

RESUMO

As a piezoelectric material, (Bi,Sc)O3-(Pb,Ti)O3 ceramics have been tested and analyzed for sensors and energy harvester applications owing to their relatively high Curie temperature and high piezoelectric coefficient. In this work, we prepared optimized (Bi,Sc)O3-(Pb,Ti)O3 piezoelectric materials through the conventional ceramic process. To increase the output energy, a multilayered structure was proposed and designed, and to obtain the maximum output energy, impedance matching techniques were considered and tested. By varying and measuring the energy harvesting system, we confirmed that the output energies were optimized by varying the load resistance. As the load resistance increased, the output voltage became saturated. Then, we calculated the optimized output power using the electric energy formula. Consequently, we identified the highest output energy of 5.93 µW/cm2 at 3 MΩ for the quadruple-layer harvester and load resistor using the impedance matching system. We characterized and improved the electrical properties of the piezoelectric energy harvesters by introducing impedance matching and performing the modeling of the energy harvesting component. Modeling was conducted for the piezoelectric generator component by introducing the mechanical force dependent voltage sources and load resistors and piezoelectric capacitor connected in parallel. Moreover, the generated output voltages were simulated by introducing an impedance matching technique. This work is designed to explain the modeling of piezoelectric energy harvesters. In this model, the relationship between applied mechanical force and output energy was discussed by employing experimental results and simulation.

15.
J Biol Chem ; 293(2): 588-598, 2018 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-29167269

RESUMO

Cells have evolved sophisticated mechanisms to maintain genomic integrity in response to DNA damage. Ionizing radiation (IR)-induced DNA damage results in the formation of IR-induced foci (iRIF) in the nucleus. The iRIF formation is part of the DNA damage response (DDR), which is an essential signaling cascade that must be strictly regulated because either the loss of or an augmented DDR leads to loss of genome integrity. Accordingly, negative regulation of the DDR is as critical as its activation. In this study, we have identified ring finger protein 126 (RNF126) as a negative regulator of the DDR from a screen of iRIF containing 53BP1. RNF126 overexpression abolishes not only the formation of 53BP1 iRIF but also of RNF168, FK2, RAP80, and BRCA1. However, the iRIF formation of γH2AX, MDC1, and RNF8 is maintained, indicating that RNF126 acts between RNF8 and RNF168 during the DDR. In addition, RNF126 overexpression consistently results in the loss of RNF168-mediated H2A monoubiquitination at lysine 13/15 and inhibition of the non-homologous end joining capability. Taken together, our findings reveal that RNF126 is a novel factor involved in the negative regulation of DDR, which is important for sustaining genomic integrity.


Assuntos
Radiação Ionizante , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Dano ao DNA/efeitos da radiação , Células HeLa , Histonas/metabolismo , Histonas/efeitos da radiação , Humanos , Imunoprecipitação , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/efeitos da radiação
16.
J Biol Chem ; 291(34): 17579-92, 2016 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-27325694

RESUMO

The error-free segregation of chromosomes, which requires the precisely timed search and capture of chromosomes by spindles during early mitotic and meiotic cell division, is responsible for genomic stability and is achieved by the spindle assembly checkpoint in the metaphase-anaphase transition. Mitotic kinases orchestrate M phase events, such as the reorganization of cell architecture and kinetochore (KT) composition with the exquisite phosphorylation of mitotic regulators, to ensure timely and temporal progression. However, the molecular mechanisms underlying the changes of KT composition for stable spindle attachment during mitosis are poorly understood. Here, we show that the sequential action of the kinase Cdk1 and the phosphatase Cdc14A control spindle attachment to KTs. During prophase, the mitotic spindle protein Spag5/Astrin is transported into centrosomes by Kinastrin and phosphorylated at Ser-135 and Ser-249 by Cdk1, which, in prometaphase, is loaded onto the spindle and targeted to KTs. We also demonstrate that Cdc14A dephosphorylates Astrin, and therefore the overexpression of Cdc14A sequesters Astrin in the centrosome and results in aberrant chromosome alignment. Mechanistically, Plk1 acts as an upstream kinase for Astrin phosphorylation by Cdk1 and targeting phospho-Astrin to KTs, leading to the recruitment of outer KT components, such as Cenp-E, and the stable attachment of spindles to KTs. These comprehensive findings reveal a regulatory circuit for protein targeting to KTs that controls the KT composition change of stable spindle attachment and chromosome integrity.


Assuntos
Anáfase/fisiologia , Proteínas de Ciclo Celular/metabolismo , Cinetocoros/metabolismo , Metáfase/fisiologia , Proteína Quinase CDC2 , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Células HeLa , Humanos , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Fosfatases , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Quinase 1 Polo-Like
17.
Biochem Biophys Res Commun ; 492(3): 441-446, 2017 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-28842250

RESUMO

RAP80, a member of the BRCA1-A complex, is a well-known crucial regulator of cell cycle checkpoint and DNA damage repair in the nucleus. However, it is still unclear whether Rap80 localizes to another region outside the nucleus and plays different roles with its partners. Here, we found mitochondrial p32 as a novel binding partner of RAP80 by using yeast two-hybrid screening. RAP80 directly binds the internal region of p32 through its arginine rich C-terminal domain. Based on the interaction, we showed that a subset of RAP80 localizes to mitochondria where p32 exists. Loss of function study revealed that RAP80 deficiency reduces the protein level of p32 and p32 dependent mitochondrial translating proteins such as Rieske and COX1. As a result, mitochondrial membrane potential and oxygen consumption are reduced in RAP80 knockdown cells, indicating mitochondrial dysfunction. Our study identifies a novel interaction between RAP80 and p32, which is important for preserving intact mitochondrial function.


Assuntos
Proteínas de Transporte/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Transporte/genética , Células Cultivadas , Proteínas de Ligação a DNA , Chaperonas de Histonas , Humanos , Proteínas Mitocondriais/genética , Mutação , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética
18.
Cancer Sci ; 107(3): 267-73, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26748910

RESUMO

Epithelial-mesenchymal transition (EMT) has been closely related with invasive and metastatic properties of cancer. Recently, the convergence of DNA damage response and EMT in cancer development has received a great amount of scientific attention. Here, we showed that EMT is induced by the downregulation of RAP80, a well-known regulator for DNA damage response. The knockdown of RAP80 leads to EMT-like morphological changes and the increase of tumor sphere formation in non-adhesive culture. Mechanistically, RAP80 controls a reciprocal regulatory axis of ZEB1 (for EMT activation) and miR200c (for EMT inhibition). The downregulation of RAP80 increases ZEB1 protein and decreases miR200c expression to activate EMT signaling in the form of drastic inhibitions of E-cadherin, p16 and p21 expression. Using in vivo metastasis analysis, RAP80 knockdown cells are shown to dramatically metastasize into the lung and generate more malignant phenotype compared to controls. Interestingly, the expression level of RAP80 was positively correlated with the survival rate in lung adenocarcinoma and breast cancer patients. These findings indicate that RAP80 is a critical gatekeeper in impeding EMT-induced metastasis and malignant phenotypes of cancer as well as preserving DNA integrity.


Assuntos
Proteínas de Transporte/fisiologia , Transição Epitelial-Mesenquimal , Proteínas de Homeodomínio/metabolismo , Neoplasias Pulmonares/secundário , Proteínas Nucleares/fisiologia , Fatores de Transcrição/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Proteínas de Ligação a DNA , Feminino , Técnicas de Silenciamento de Genes , Células HeLa , Chaperonas de Histonas , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Homeobox 1 de Ligação a E-box em Dedo de Zinco
19.
Biochem Biophys Res Commun ; 470(4): 881-7, 2016 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-26820530

RESUMO

TRAIP/RNF206 plays diverse roles in cell cycle progression, DNA damage response, and DNA repair pathways. Physiological importance of TRAIP is highlighted by the identification of pathogenic mutations of TRAIP gene in patients diagnosed with primordial dwarfism. Although the diverse functions of TRAIP in the nucleus have been well characterized, molecular mechanism of TRAIP retention in the nucleus has not been determined. Here, we discovered that TRAIP is post-translationally modified by the small ubiquitin-like protein (SUMO). In addition, we identified five SUMOylation sites in TRAIP, and successfully generated SUMOylation deficient mutant of TRAIP. In an attempt to define the functional roles of TRAIP SUMOylation, we discovered that SUMOylation deficient TRAIP is not retained in the nucleus. In addition, protein stability of SUMOylation deficient TRAIP is lower than wild type TRAIP, demonstrating that SUMOylation is critical for both proper subcellular localization and protein stability of TRAIP. Taken together, these findings improve the understanding clinical implication of TRAIP in various diseases including primordial dwarfism and cancers.


Assuntos
Núcleo Celular/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Células HEK293 , Células HeLa , Humanos , Estabilidade Proteica
20.
Acta Biochim Biophys Sin (Shanghai) ; 48(7): 658-64, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27325824

RESUMO

Sustaining genomic integrity is essential for preventing onset of cancers. Therefore, human cells evolve to have refined biological pathways to defend genetic materials from various genomic insults. DNA damage response and DNA repair pathways essential for genome maintenance are accomplished by cooperative executions of multiple factors including breast cancer type 1 susceptibility protein (BRCA1). BRCA1 is initially identified as an altered gene in the hereditary breast cancer patients. Since then, tremendous efforts to understand the functions of BRAC1 reveal that BRCA1 is found in distinct complexes, including BRCA1-A, BRCA1-B, BRCA1-C, and the BRCA1/PALB2/BRCA2 complex, and plays diverse roles in a context-dependent manner. Among the complexes, BRCA1-A is critical for BRCA1 recruitment to the sites of DNA damage. Factors comprising the BRCA1-A include RAP80, CCDC98/Abraxas, BRCC36, BRCC45, BARD1, BRCA1, and MERIT40, a RAP80-associated factor. In this review, we summarize recent findings of the factors that form the BRCA1-A complex.


Assuntos
Proteína BRCA1/metabolismo , Dano ao DNA , Proteína BRCA1/química , Proteína BRCA1/genética , Humanos
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