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1.
Nat Commun ; 15(1): 7076, 2024 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-39152113

RESUMO

During the repair of interstrand crosslinks (ICLs) a DNA double-strand break (DSB) is generated. The Fanconi anemia (FA) core complex, which is recruited to ICLs, promotes high-fidelity repair of this DSB by homologous recombination (HR). However, whether the FA core complex also promotes HR at ICL-independent DSBs, for example induced by ionizing irradiation or nucleases, remains controversial. Here, we identified the FA core complex members FANCL and Ube2T as HR-promoting factors in a CRISPR/Cas9-based screen. Using isogenic cell line models, we further demonstrated an HR-promoting function of FANCL and Ube2T, and of their ubiquitination substrate FANCD2. We show that FANCL and Ube2T localize at DSBs in a FANCM-dependent manner, and are required for the DSB accumulation of FANCD2. Mechanistically, we demonstrate that FANCL ubiquitin ligase activity is required for the accumulation of CtIP at DSBs, thereby promoting end resection and Rad51 loading. Together, these data demonstrate a dual genome maintenance function of the FA core complex and FANCD2 in promoting repair of both ICLs and DSBs.


Assuntos
Quebras de DNA de Cadeia Dupla , Proteína do Grupo de Complementação D2 da Anemia de Fanconi , Proteína do Grupo de Complementação L da Anemia de Fanconi , Recombinação Homóloga , Enzimas de Conjugação de Ubiquitina , Humanos , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação L da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação L da Anemia de Fanconi/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Sistemas CRISPR-Cas , Ubiquitinação , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/genética , Células HEK293 , Reparo de DNA por Recombinação , Reparo do DNA , Reparo do DNA por Junção de Extremidades , DNA Helicases
2.
Biosci Rep ; 40(6)2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32420600

RESUMO

Fanconi Anemia (FA) is a rare genetic disorder characterized by developmental defects, bone marrow failure and high predisposition to cancer. The FA DNA repair pathway is required in humans to coordinate repair of DNA interstrand cross-links. The central event in the activation of the pathway is the monoubiquitination of FANCD2 and FANCI by the E2-E3 pair, Ube2T-FANCL, with the central UBC-RWD (URD) domain of FANCL recognizing the substrates. Whole genome sequencing studies of cancer cells from patients identified point mutations in the FANCL URD domain. We analysed 17 such variants of FANCL, including known substrate binding mutants (W212A, W214A and L248A, F252A, L254A, I265A), a FA mutation (R221C) and 14 cancer-associated mutations (F110S, I136V, L149V, L154S, A192G, E215Q, E217K, R221W, T224K, M247V, F252L, N270K, V287G, E289Q) through recombinant expression analysis, thermal shift assay, interaction with FANCD2, in vitro ubiquitination activity, and cellular sensitivity to an interstrand cross-linking agent. We find that the FANCL mutations I136V, L154S, W212A and L214A, R221W, R221C, and V287G are destabilizing, with N270K and E289Q destabilizing the C-terminal helices of the URD domain. The hydrophobic patch mutant (L248A, F252A, L254A, I265A), along with mutations E217K, T224K, and M247V, cause defects in the catalytic function of FANCL. This highlights the C-terminal lobe of the FANCL URD domain as important for the activity and function of FANCL. These mutations which affect the fold and activity of FANCL may contribute to tumorigenesis in these non-FA cancer patients, and this implicates FA genes in general cancer progression.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias Ósseas/genética , Proteína do Grupo de Complementação L da Anemia de Fanconi/genética , Anemia de Fanconi/genética , Mutação , Osteossarcoma/genética , Antineoplásicos Alquilantes/farmacologia , Biomarcadores Tumorais/metabolismo , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Análise Mutacional de DNA , Anemia de Fanconi/diagnóstico , Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação L da Anemia de Fanconi/metabolismo , Predisposição Genética para Doença , Humanos , Mitomicina/farmacologia , Osteossarcoma/tratamento farmacológico , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Conformação Proteica em alfa-Hélice , Dobramento de Proteína , Estabilidade Proteica , Proteólise , Ubiquitinação
3.
Hum Mutat ; 41(5): 1033-1041, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32048394

RESUMO

The Fanconi anemia (FA) pathway is mainly involved in DNA interstrand crosslinks (ICLs) repair in the genome. Several FA genes, including FANCD1/BRCA2, FANCM, and FANCU/XRCC2, have been identified as causative genes for premature ovary insufficiency (POI). Fanconi anemia group L protein (FANCL) cooperates with FANCT/UBE2T to ubiquitinate the FANCI-D2 dimer, which is a crucial event in the process of ICLs repair. Fancl-knockout mice phenocopy human POI, but the role of FANCL mutations in POI pathogenesis has not been confirmed. In the present work, potentially pathogenic mutations in the FANCL gene were screened in 200 Chinese patients with idiopathic POI and in 200 matched controls. Two novel heterozygous frameshift mutations, c.1048_1051delGTCT (p.Gln350Valfs*18) and c.739dupA (p.Met247Asnfs*4), were identified in the FANCL gene in POI patients but not in controls. Wild-type FANCL protein was predominantly localized in the nuclei, while both mutant FANCL proteins were retained in the cytoplasm. In addition, the FANCL variants exhibited impaired ubiquitin-ligase activity and compromised DNA repair ability after mitomycin C treatment. Furthermore, the FANCL variants were deleterious and might be associated with haploinsufficiency. Our results show that FANCL mutations are potentially causative for POI by disrupting DNA damage repair processes.


Assuntos
Proteína do Grupo de Complementação L da Anemia de Fanconi/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Mutação , Insuficiência Ovariana Primária/diagnóstico , Insuficiência Ovariana Primária/genética , Adulto , Biomarcadores , Linhagem Celular , Dano ao DNA , Reparo do DNA , Proteína do Grupo de Complementação L da Anemia de Fanconi/metabolismo , Feminino , Mutação da Fase de Leitura , Técnicas de Silenciamento de Genes , Estudos de Associação Genética/métodos , Heterozigoto , Humanos , Insuficiência Ovariana Primária/metabolismo , Transporte Proteico , Interferência de RNA , Fatores de Risco , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Adulto Jovem
4.
Nat Chem Biol ; 16(3): 291-301, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31873223

RESUMO

DNA-damage repair is implemented by proteins that are coordinated by specialized molecular signals. One such signal in the Fanconi anemia (FA) pathway for the repair of DNA interstrand crosslinks is the site-specific monoubiquitination of FANCD2 and FANCI. The signal is mediated by a multiprotein FA core complex (FA-CC) however, the mechanics for precise ubiquitination remain elusive. We show that FANCL, the RING-bearing module in FA-CC, allosterically activates its cognate ubiqutin-conjugating enzyme E2 UBE2T to drive site-specific FANCD2 ubiquitination. Unlike typical RING E3 ligases, FANCL catalyzes ubiquitination by rewiring the intraresidue network of UBE2T to influence the active site. Consequently, a basic triad unique to UBE2T engages a structured acidic patch near the target lysine on FANCD2. This three-dimensional complementarity, between the E2 active site and substrate surface, induced by FANCL is central to site-specific monoubiquitination in the FA pathway. Furthermore, the allosteric network of UBE2T can be engineered to enhance FANCL-catalyzed FANCD2-FANCI di-monoubiquitination without compromising site specificity.


Assuntos
Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação L da Anemia de Fanconi/metabolismo , Regulação Alostérica/fisiologia , Sequência de Aminoácidos , Dano ao DNA , Reparo do DNA , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/fisiologia , Proteína do Grupo de Complementação L da Anemia de Fanconi/fisiologia , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/fisiologia , Humanos , Ligação Proteica , Especificidade por Substrato , Enzimas de Conjugação de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/fisiologia , Ubiquitinação
5.
ACS Chem Biol ; 14(10): 2148-2154, 2019 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-31525021

RESUMO

The Fanconi anemia pathway orchestrates the repair of DNA interstrand cross-links and stalled replication forks. A key step in this pathway is UBE2T and FANCL-dependent monoubiquitylation of the FANCD2-FANCI complex. The Fanconi anemia pathway represents an attractive therapeutic target, because activation of this pathway has been linked to chemotherapy resistance in several cancers. However, to date, very few selective inhibitors of ubiquitin conjugation pathways are known. By using a high-throughput screen-compatible assay, we have identified a small-molecule inhibitor of UBE2T/FANCL-mediated FANCD2 monoubiquitylation that sensitizes cells to the DNA cross-linking agent, carboplatin.


Assuntos
Proteína do Grupo de Complementação L da Anemia de Fanconi/antagonistas & inibidores , Anemia de Fanconi/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores , Linhagem Celular Tumoral , Proteína do Grupo de Complementação L da Anemia de Fanconi/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação
6.
ACS Chem Biol ; 12(7): 1858-1866, 2017 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-28535027

RESUMO

Human exposure to arsenic in drinking water is known to be associated with the development of bladder, lung, kidney, and skin cancers. The molecular mechanisms underlying the carcinogenic effects of arsenic species remain incompletely understood. DNA interstrand cross-links (ICLs) are among the most cytotoxic type of DNA lesions that block DNA replication and transcription, and these lesions can be induced by endogenous metabolism and by exposure to exogenous agents. Fanconi anemia (FA) is a congenital disorder manifested with elevated sensitivity toward DNA interstrand cross-linking agents, and monoubiquitination of FANCD2 by FANCL is a crucial step in FA-mediated DNA repair. Here, we demonstrated that As3+ could bind to the PHD/RING finger domain of FANCL in vitro and in cells. This binding led to compromised ubiquitination of FANCD2 in cells and diminished recruitment of FANCD2 to chromatin and DNA damage sites induced by 4,5',8-trimethylpsoralen plus UVA irradiation. Furthermore, clonogenic survival assay results showed that arsenite coexposure rendered cells more sensitive toward DNA interstrand cross-linking agents. Together, our study suggested that arsenite may compromise genomic stability via perturbation of the Fanconi anemia pathway, thereby conferring its carcinogenic effect.


Assuntos
Arsenitos/metabolismo , Arsenitos/toxicidade , Reparo do DNA , Proteína do Grupo de Complementação L da Anemia de Fanconi/metabolismo , Instabilidade Genômica/efeitos dos fármacos , Domínios RING Finger , Ubiquitina-Proteína Ligases/química , Arsenitos/química , Sítios de Ligação , Linhagem Celular Tumoral , Dano ao DNA , Proteína do Grupo de Complementação L da Anemia de Fanconi/química , Humanos , Ubiquitinação
7.
Mol Cell ; 65(2): 247-259, 2017 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-27986371

RESUMO

Monoubiquitination and deubiquitination of FANCD2:FANCI heterodimer is central to DNA repair in a pathway that is defective in the cancer predisposition syndrome Fanconi anemia (FA). The "FA core complex" contains the RING-E3 ligase FANCL and seven other essential proteins that are mutated in various FA subtypes. Here, we purified recombinant FA core complex to reveal the function of these other proteins. The complex contains two spatially separate FANCL molecules that are dimerized by FANCB and FAAP100. FANCC and FANCE act as substrate receptors and restrict monoubiquitination to the FANCD2:FANCI heterodimer in only a DNA-bound form. FANCA and FANCG are dispensable for maximal in vitro ubiquitination. Finally, we show that the reversal of this reaction by the USP1:UAF1 deubiquitinase only occurs when DNA is disengaged. Our work reveals the mechanistic basis for temporal and spatial control of FANCD2:FANCI monoubiquitination that is critical for chemotherapy responses and prevention of Fanconi anemia.


Assuntos
Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Anemia de Fanconi/metabolismo , Ubiquitinação , Linhagem Celular , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Anemia de Fanconi/genética , Proteína do Grupo de Complementação A da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação C da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação E da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação G da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação L da Anemia de Fanconi/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Humanos , Proteína 2 Inibidora de Diferenciação/metabolismo , Complexos Multiproteicos , Proteínas Nucleares/metabolismo , Ligação Proteica , Multimerização Proteica , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Fatores de Tempo , Transfecção , Proteases Específicas de Ubiquitina/metabolismo
8.
Oncogene ; 35(47): 6087-6095, 2016 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-27132514

RESUMO

SALL4 is aberrantly expressed in human myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). We have generated a SALL4 transgenic (SALL4B Tg) mouse model with pre-leukemic MDS-like symptoms that transform to AML over time. This makes our mouse model applicable for studying human MDS/AML diseases. Characterization of the leukemic initiation population in this model leads to the discovery that Fancl (Fanconi anemia, complementation group L) is downregulated in SALL4B Tg leukemic and pre-leukemic cells. Similar to the reported Fanconi anemia (FA) mouse model, chromosomal instability with radial changes can be detected in pre-leukemic SALL4B Tg bone marrow (BM) cells after DNA damage challenge. Results from additional studies using DNA damage repair reporter assays support a role of SALL4 in inhibiting the homologous recombination pathway. Intriguingly, unlike the FA mouse model, after DNA damage challenge, SALL4B Tg BM cells can survive and generate hematopoietic colonies. We further elucidated that the mechanism by which SALL4 promotes cell survival is through Bcl2 activation. Overall, our studies demonstrate for the first time that SALL4 has a negative impact in DNA damage repair, and support the model of dual functional properties of SALL4 in leukemogenesis through inhibiting DNA damage repair and promoting cell survival.


Assuntos
Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Animais , Células da Medula Óssea/metabolismo , Transplante de Medula Óssea , Instabilidade Cromossômica/efeitos dos fármacos , Análise por Conglomerados , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação L da Anemia de Fanconi/genética , Proteína do Grupo de Complementação L da Anemia de Fanconi/metabolismo , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Genes bcl-2 , Células-Tronco Hematopoéticas/metabolismo , Recombinação Homóloga , Humanos , Cariótipo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Mitomicina/farmacologia , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Fatores de Transcrição/genética
9.
Oncotarget ; 7(22): 32351-61, 2016 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-27083049

RESUMO

Mutations in the human RecQ helicase, BLM, causes Bloom Syndrome, which is a rare autosomal recessive disorder and characterized by genomic instability and an increased risk of cancer. Fanconi Anemia (FA), resulting from mutations in any of the 19 known FA genes and those yet to be known, is also characterized by chromosomal instability and a high incidence of cancer. BLM helicase and FA proteins, therefore, may work in a common tumor-suppressor signaling pathway. To date, it remains largely unclear as to how BLM and FA proteins work concurrently in the maintenance of genome stability. Here we report that BLM is involved in the early activation of FA group D2 protein (FANCD2). We found that FANCD2 activation is substantially delayed and attenuated in crosslinking agent-treated cells harboring deficient Blm compared to similarly treated control cells with sufficient BLM. We also identified that the domain VI of BLM plays an essential role in promoting FANCD2 activation in cells treated with DNA crosslinking agents, especially ultraviolet B. The similar biological effects performed by ΔVI-BLM and inactivated FANCD2 further confirm the relationship between BLM and FANCD2. Mutations within the domain VI of BLM detected in human cancer samples demonstrate the functional importance of this domain, suggesting human tumorigenicity resulting from mtBLM may be at least partly attributed to mitigated FANCD2 activation. Collectively, our data show a previously unknown regulatory liaison in advancing our understanding of how the cancer susceptibility gene products act in concert to maintain genome stability.


Assuntos
Síndrome de Bloom/enzimologia , Proteína do Grupo de Complementação L da Anemia de Fanconi/metabolismo , Anemia de Fanconi/enzimologia , Neoplasias/enzimologia , RecQ Helicases/metabolismo , Transdução de Sinais , Síndrome de Bloom/genética , Síndrome de Bloom/patologia , Neoplasias Ósseas/enzimologia , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular , Reagentes de Ligações Cruzadas/farmacologia , Anemia de Fanconi/genética , Anemia de Fanconi/patologia , Proteína do Grupo de Complementação L da Anemia de Fanconi/química , Proteína do Grupo de Complementação L da Anemia de Fanconi/genética , Feminino , Humanos , Mutação , Neoplasias/genética , Neoplasias/patologia , Osteossarcoma/enzimologia , Osteossarcoma/genética , Osteossarcoma/patologia , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Interferência de RNA , RecQ Helicases/química , RecQ Helicases/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Transfecção , Raios Ultravioleta
10.
J Proteome Res ; 15(4): 1333-41, 2016 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-26956768

RESUMO

Metabolic profiling has great potential to help the diagnosis and prognosis of cancer patients. Fanconi Anemia (FA) tumor-suppressor signaling has been instrumental in understanding human tumorigenesis. However, this instrumental understanding has never been demonstrated at the metabolic level. Here, we show that impaired FA signaling can lead cells to exhibit metabolic signatures of tumorigenesis. This is consistent with our original studies of the roles of FA signaling in suppressing non-FA tumorigenesis at functional and genetic levels. Using ultraperformance liquid chromatography-mass spectroscopy and gas chromatography-mass spectrometry, we characterized metabolic alterations in bladder cancer cells carrying an intact or impaired FA pathway. The latter was obtained by ectopically expressing FAVL (FAVL-high), which we previously found to be capable of inactivating FA signaling. A total of 18 metabolites, end products of cell proliferation or apoptosis, were significantly different between FAVL-high and -low cells. Methionine, phenylalanine, and threonine, resulting from a tumorigenic process, were substantially increased in FAVL-high cells. With this study, we achieved genomic, functional, and metabolomic characterization of the roles of FA signaling in the development of human cancer. Furthermore, this study provides novel insights into how to translate FA basic research into strategies for producing effective biomarkers in human cancer diagnosis and prognosis.


Assuntos
Transformação Celular Neoplásica/metabolismo , Células Epiteliais/metabolismo , Proteína do Grupo de Complementação L da Anemia de Fanconi/genética , Metaboloma , Transdução de Sinais , Processamento Alternativo , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Cromatografia Líquida , Células Epiteliais/patologia , Proteína do Grupo de Complementação L da Anemia de Fanconi/metabolismo , Expressão Gênica , Humanos , Espectrometria de Massas , Metionina/metabolismo , Fenilalanina/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Treonina/metabolismo , Transgenes
11.
J Biomed Sci ; 22: 77, 2015 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-26385482

RESUMO

BACKGROUND: Cisplatin is one of the most commonly used chemotherapy agent for lung cancer. The therapeutic efficacy of cisplatin is limited by the development of resistance. In this study, we test the effect of RNA interference (RNAi) targeting Fanconi anemia (FA)/BRCA pathway upstream genes on the sensitivity of cisplatin-sensitive (A549 and SK-MES-1) and -resistant (A549/DDP) lung cancer cells to cisplatin. RESULT: Using small interfering RNA (siRNA), knockdown of FANCF, FANCL, or FANCD2 inhibited function of the FA/BRCA pathway in A549, A549/DDP and SK-MES-1 cells, and potentiated sensitivity of the three cells to cisplatin. The extent of proliferation inhibition induced by cisplatin after knockdown of FANCF and/or FANCL in A549/DDP cells was significantly greater than in A549 and SK-MES-1 cells, suggesting that depletion of FANCF and/or FANCL can reverse resistance of cisplatin-resistant lung cancer cells to cisplatin. Furthermore, knockdown of FANCL resulted in higher cisplatin sensitivity and dramatically elevated apoptosis rates compared with knockdown of FANCF in A549/DDP cells, indicating that FANCL play an important role in the repair of cisplatin-induced DNA damage. CONCLUSION: Knockdown of FANCF, FANCL, or FANCD2 by RNAi could synergize the effect of cisplatin on suppressing cell proliferation in cisplatin-resistant lung cancer cells through inhibition of FA/BRCA pathway.


Assuntos
Proteína BRCA1 , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Proteína do Grupo de Complementação D2 da Anemia de Fanconi , Proteína do Grupo de Complementação F da Anemia de Fanconi , Proteína do Grupo de Complementação L da Anemia de Fanconi , Neoplasias Pulmonares , Interferência de RNA , Transdução de Sinais , Proteína BRCA1/antagonistas & inibidores , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/antagonistas & inibidores , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação F da Anemia de Fanconi/antagonistas & inibidores , Proteína do Grupo de Complementação F da Anemia de Fanconi/genética , Proteína do Grupo de Complementação F da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação L da Anemia de Fanconi/antagonistas & inibidores , Proteína do Grupo de Complementação L da Anemia de Fanconi/genética , Proteína do Grupo de Complementação L da Anemia de Fanconi/metabolismo , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
12.
J Biol Chem ; 290(34): 20995-21006, 2015 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-26149689

RESUMO

The Fanconi Anemia (FA) DNA repair pathway is essential for the recognition and repair of DNA interstrand crosslinks (ICL). Inefficient repair of these ICL can lead to leukemia and bone marrow failure. A critical step in the pathway is the monoubiquitination of FANCD2 by the RING E3 ligase FANCL. FANCL comprises 3 domains, a RING domain that interacts with E2 conjugating enzymes, a central domain required for substrate interaction, and an N-terminal E2-like fold (ELF) domain. The ELF domain is found in all FANCL homologues, yet the function of the domain remains unknown. We report here that the ELF domain of FANCL is required to mediate a non-covalent interaction between FANCL and ubiquitin. The interaction involves the canonical Ile44 patch on ubiquitin, and a functionally conserved patch on FANCL. We show that the interaction is not necessary for the recognition of the core complex, it does not enhance the interaction between FANCL and Ube2T, and is not required for FANCD2 monoubiquitination in vitro. However, we demonstrate that the ELF domain is required to promote efficient DNA damage-induced FANCD2 monoubiquitination in vertebrate cells, suggesting an important function of ubiquitin binding by FANCL in vivo.


Assuntos
Reparo do DNA , Proteínas de Drosophila/química , Proteína do Grupo de Complementação L da Anemia de Fanconi/química , Proteínas de Grupos de Complementação da Anemia de Fanconi/química , Ubiquitina/química , Proteínas de Xenopus/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Anemia de Fanconi/genética , Proteína do Grupo de Complementação L da Anemia de Fanconi/genética , Proteína do Grupo de Complementação L da Anemia de Fanconi/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Regulação da Expressão Gênica , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Transdução de Sinais , Ubiquitina/genética , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis
13.
Cell Rep ; 12(1): 35-41, 2015 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-26119737

RESUMO

Fanconi anemia (FA) is a rare bone marrow failure and cancer predisposition syndrome resulting from pathogenic mutations in genes encoding proteins participating in the repair of DNA interstrand crosslinks (ICLs). Mutations in 17 genes (FANCA-FANCS) have been identified in FA patients, defining 17 complementation groups. Here, we describe an individual presenting with typical FA features who is deficient for the ubiquitin-conjugating enzyme (E2), UBE2T. UBE2T is known to interact with FANCL, the E3 ubiquitin-ligase component of the multiprotein FA core complex, and is necessary for the monoubiquitination of FANCD2 and FANCI. Proband fibroblasts do not display FANCD2 and FANCI monoubiquitination, do not form FANCD2 foci following treatment with mitomycin C, and are hypersensitive to crosslinking agents. These cellular defects are complemented by expression of wild-type UBE2T, demonstrating that deficiency of the protein UBE2T can lead to Fanconi anemia. UBE2T gene gains an alias of FANCT.


Assuntos
Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação L da Anemia de Fanconi/metabolismo , Anemia de Fanconi/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação L da Anemia de Fanconi/genética , Fibroblastos/metabolismo , Deleção de Genes , Células HEK293 , Humanos , Ligação Proteica , Enzimas de Conjugação de Ubiquitina/deficiência , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitinação
14.
Am J Hum Genet ; 96(6): 1001-7, 2015 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-26046368

RESUMO

Fanconi anemia (FA) is a rare genetic disorder characterized by genome instability, increased cancer susceptibility, progressive bone marrow failure (BMF), and various developmental abnormalities resulting from the defective FA pathway. FA is caused by mutations in genes that mediate repair processes of interstrand crosslinks and/or DNA adducts generated by endogenous aldehydes. The UBE2T E2 ubiquitin conjugating enzyme acts in FANCD2/FANCI monoubiquitination, a critical event in the pathway. Here we identified two unrelated FA-affected individuals, each harboring biallelic mutations in UBE2T. They both produced a defective UBE2T protein with the same missense alteration (p.Gln2Glu) that abolished FANCD2 monoubiquitination and interaction with FANCL. We suggest this FA complementation group be named FA-T.


Assuntos
Anemia de Fanconi/genética , Anemia de Fanconi/patologia , Modelos Moleculares , Mutação de Sentido Incorreto/genética , Enzimas de Conjugação de Ubiquitina/genética , Sequência de Aminoácidos , Sequência de Bases , Criança , Pré-Escolar , Proteína do Grupo de Complementação L da Anemia de Fanconi/metabolismo , Feminino , Componentes do Gene , Genótipo , Humanos , Japão , Masculino , Dados de Sequência Molecular , Linhagem , Conformação Proteica , Alinhamento de Sequência , Análise de Sequência de DNA , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação/genética
15.
Mol Cell ; 54(5): 858-69, 2014 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-24905007

RESUMO

Fanconi anaemia (FA) is a cancer predisposition syndrome characterized by cellular sensitivity to DNA interstrand crosslinkers. The molecular defect in FA is an impaired DNA repair pathway. The critical event in activating this pathway is monoubiquitination of FANCD2. In vivo, a multisubunit FA core complex catalyzes this step, but its mechanism is unclear. Here, we report purification of a native avian FA core complex and biochemical reconstitution of FANCD2 monoubiquitination. This demonstrates that the catalytic FANCL E3 ligase subunit must be embedded within the complex for maximal activity and site specificity. We genetically and biochemically define a minimal subcomplex comprising just three proteins (FANCB, FANCL, and FAAP100) that functions as the monoubiquitination module. Residual FANCD2 monoubiquitination activity is retained in cells defective for other FA core complex subunits. This work describes the in vitro reconstitution and characterization of this multisubunit monoubiquitin E3 ligase, providing key insight into the conserved FA DNA repair pathway.


Assuntos
Proteínas Aviárias/metabolismo , Galinhas/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Ubiquitinação , Animais , Proteínas Aviárias/química , Proteínas Aviárias/genética , Linhagem Celular , Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/química , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação L da Anemia de Fanconi/química , Proteína do Grupo de Complementação L da Anemia de Fanconi/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/química , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Humanos , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismo
16.
J Biol Chem ; 289(10): 7003-7010, 2014 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-24451376

RESUMO

Fanconi anemia (FA) is a genome instability syndrome characterized by bone marrow failure and cellular hypersensitivity to DNA cross-linking agents. In response to DNA damage, the FA pathway is activated through the cooperation of 16 FA proteins. A central player in the pathway is a multisubunit E3 ubiquitin ligase complex or the FA core complex, which monoubiquitinates its substrates FANCD2 and FANCI. FANCE, a subunit of the FA core complex, plays an essential role by promoting the integrity of the complex and by directly recognizing FANCD2. To delineate its role in substrate ubiquitination from the core complex assembly, we analyzed a series of mutations within FANCE. We report that a phenylalanine located at the highly conserved extreme C terminus, referred to as Phe-522, is a critical residue for mediating the monoubiquitination of the FANCD2-FANCI complex. Using the FANCE mutant that specifically disrupts the FANCE-FANCD2 interaction as a tool, we found that the interaction-deficient mutant conferred cellular sensitivity in reconstituted FANCE-deficient cells to a similar degree as FANCE null cells, suggesting the significance of the FANCE-FANCD2 interaction in promoting cisplatin resistance. Intriguingly, ectopic expression of the FANCE C terminus fragment alone in FA normal cells disrupts DNA repair, consolidating the importance of the FANCE-FANCD2 interaction in the DNA cross-link repair.


Assuntos
Reparo do DNA , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação E da Anemia de Fanconi/metabolismo , Anemia de Fanconi/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Anemia de Fanconi/genética , Proteína do Grupo de Complementação E da Anemia de Fanconi/química , Proteína do Grupo de Complementação E da Anemia de Fanconi/genética , Proteína do Grupo de Complementação L da Anemia de Fanconi/metabolismo , Células HEK293 , Células HeLa , Humanos , Dados de Sequência Molecular , Fenilalanina/química , Fenilalanina/genética , Fenilalanina/metabolismo , Estrutura Terciária de Proteína , Ubiquitinação
17.
Mol Biol Cell ; 24(16): 2582-92, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23783032

RESUMO

Fanconi anemia hematopoietic stem cells display poor self-renewal capacity when subjected to a variety of cellular stress. This phenotype raises the question of whether the Fanconi anemia proteins are stabilized or recruited as part of a stress response and protect against stem cell loss. Here we provide evidence that FANCL, the E3 ubiquitin ligase of the Fanconi anemia pathway, is constitutively targeted for degradation by the proteasome. We confirm biochemically that FANCL is polyubiquitinated with Lys-48-linked chains. Evaluation of a series of N-terminal-deletion mutants showed that FANCL's E2-like fold may direct ubiquitination. In addition, our studies showed that FANCL is stabilized in a complex with axin1 when glycogen synthase kinase-3ß is overexpressed. This result leads us to investigate the potential regulation of FANCL by upstream signaling pathways known to regulate glycogen synthase kinase-3ß. We report that constitutively active, myristoylated-Akt increases FANCL protein level by reducing polyubiquitination of FANCL. Two-dimensional PAGE analysis shows that acidic forms of FANCL, some of which are phospho-FANCL, are not subject to polyubiquitination. These results indicate that a signal transduction pathway involved in self-renewal and survival of hematopoietic stem cells also functions to stabilize FANCL and suggests that FANCL participates directly in support of stem cell function.


Assuntos
Proteína do Grupo de Complementação L da Anemia de Fanconi/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Axina/genética , Proteína Axina/metabolismo , Linhagem Celular , Ativação Enzimática , Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação L da Anemia de Fanconi/genética , Expressão Gênica , Quinase 3 da Glicogênio Sintase/biossíntese , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Células HEK293 , Células HeLa , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Dobramento de Proteína , Proteínas Proto-Oncogênicas c-akt/genética , Interferência de RNA , RNA Interferente Pequeno , Transdução de Sinais , Ubiquitinação
19.
Cell Cycle ; 11(15): 2947-55, 2012 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-22828653

RESUMO

Effectiveness of DNA cross-linking drugs in the treatment of bladder cancer suggests that bladder cancer cells may have harbored an insufficient cellular response to DNA cross-link damage, which will sensitize cells to DNA cross-linking agents. Cell sensitivity benefits from deficient DNA damage responses, which, on the other hand, can cause cancer. Many changed cellular signaling pathways are known to be involved in bladder tumorigenesis; however, DNA cross-link damage response pathway [Fanconi anemia (FA) pathway], whose alterations appear to be a plausible cause of the development of bladder cancer, remains an under-investigated area in bladder cancer research. In this study, we found FAVL (variant of FA protein L--FANCL) was elevated substantially in bladder cancer tissues examined. Ectopic expression of FAVL in bladder cancer cells as well as normal human cells confer an impaired FA pathway and hypersensitivity to Mitomycin C, similar to those found in FA cells, indicating that FAVL elevation may possess the same tumor promotion potential as an impaired FA pathway harbored in FA cells. Indeed, a higher level of FAVL expression can promote the growth of bladder cancer cells in vitro and in vivo, which, at least partly, results from FAVL perturbation of FANCL expression, an essential factor for the activation of the FA pathway. Moreover, a higher level of FAVL expression was found to be associated with chromosomal instability and the invasiveness of bladder cancer cells. Collectively, FAVL elevation can increase the tumorigenic potential of bladder cancer cells, including the invasive potential that confers the development of advanced bladder cancer. These results enhance our understanding the pathogenesis of human bladder cancer, holding a promise to develop additional effective tools to fight human bladder cancer.


Assuntos
Proteína do Grupo de Complementação L da Anemia de Fanconi/metabolismo , Anemia de Fanconi/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Dano ao DNA , Reparo do DNA , Anemia de Fanconi/genética , Anemia de Fanconi/patologia , Proteína do Grupo de Complementação L da Anemia de Fanconi/genética , Humanos , Masculino , Camundongos , Camundongos Nus , Mitomicina/farmacologia , Invasividade Neoplásica , Interferência de RNA , RNA Interferente Pequeno , Transdução de Sinais/genética , Transplante Heterólogo , Neoplasias da Bexiga Urinária/genética
20.
Blood ; 120(2): 323-34, 2012 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-22653977

RESUMO

Bone marrow failure is a nearly universal complication of Fanconi anemia. The proteins encoded by FANC genes are involved in DNA damage responses through the formation of a multisubunit nuclear complex that facilitates the E3 ubiquitin ligase activity of FANCL. However, it is not known whether loss of E3 ubiquitin ligase activity accounts for the hematopoietic stem cell defects characteristic of Fanconi anemia. Here we provide evidence that FANCL increases the activity and expression of ß-catenin, a key pluripotency factor in hematopoietic stem cells. We show that FANCL ubiquitinates ß-catenin with atypical ubiquitin chain extension known to have nonproteolytic functions. Specifically, ß-catenin modified with lysine-11 ubiquitin chain extension efficiently activates a lymphocyte enhancer-binding factor-T cell factor reporter. We also show that FANCL-deficient cells display diminished capacity to activate ß-catenin leading to reduced transcription of Wnt-responsive targets c-Myc and Cyclin D1. Suppression of FANCL expression in normal human CD34(+) stem and progenitor cells results in fewer ß-catenin active cells and inhibits expansion of multilineage progenitors. Together, these results suggest that diminished Wnt/ß-catenin signaling may be an underlying molecular defect in FANCL-deficient hematopoietic stem cells leading to their accelerated loss.


Assuntos
Proteína do Grupo de Complementação L da Anemia de Fanconi/metabolismo , beta Catenina/metabolismo , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Ciclina D1/metabolismo , Anemia de Fanconi/etiologia , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Anemia de Fanconi/patologia , Proteína do Grupo de Complementação C da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação C da Anemia de Fanconi/genética , Proteína do Grupo de Complementação C da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação L da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação L da Anemia de Fanconi/genética , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Células HEK293 , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Camundongos , Camundongos Knockout , Modelos Biológicos , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/patologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Fatores de Transcrição TCF/metabolismo , Ubiquitinação , beta Catenina/química
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