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1.
Biochim Biophys Acta Mol Basis Dis ; 1868(9): 166453, 2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-35644338

RESUMO

Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. The FA proteins have functions in genome maintenance and in the cytoplasmic process of selective autophagy, beyond their canonical roles of repairing DNA interstrand cross-links. FA core complex proteins FANCC, FANCF, FANCL, FANCA, FANCD2, BRCA1 and BRCA2, which previously had no known direct functions outside the nucleus, have recently been implicated in mitophagy. Although mutations in FANCL account for only a very small number of cases in FA families, it plays a key role in the FA pathophysiology and might drive carcinogenesis. Here, we demonstrate that FANCL protein is present in mitochondria in the control and Oligomycin and Antimycin (OA)-treated cells and its ubiquitin ligase activity is not required for its localization to mitochondria. CRISPR/Cas9-mediated knockout of FANCL in HeLa cells overexpressing parkin results in increased sensitivity to mitochondrial stress and defective clearing of damaged mitochondria upon OA treatment. This defect was reversed by the reintroduction of either wild-type FANCL or FANCL(C307A), a mutant lacking ubiquitin ligase activity. To summarize, FANCL protects from mitochondrial stress and supports Parkin-mediated mitophagy in a ubiquitin ligase-independent manner.


Assuntos
Anemia de Fanconi , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação L da Anemia de Fanconi , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Células HeLa , Humanos , Mitofagia , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética
2.
Mol Genet Genomic Med ; 9(7): e1693, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33960719

RESUMO

BACKGROUND: Fanconi anemia (FA) is an inherited bone marrow failure syndrome associated with characteristic dysmorphology primarily caused by biallelic pathogenic germline variants in any of 22 different DNA repair genes. There are limited data on the specific molecular causes of FA in different ethnic groups. METHODS: We performed exome sequencing and copy number variant analyses on 19 patients with FA from 17 families undergoing hematopoietic cell transplantation evaluation in Pakistan. The scientific literature was reviewed, and we curated germline variants reported in patients with FA from South Asia and the Middle East. RESULTS: The genetic causes of FA were identified in 14 of the 17 families: seven FANCA, two FANCC, one FANCF, two FANCG, and two FANCL. Homozygous and compound heterozygous variants were present in 12 and two families, respectively. Nine families carried variants previously reported as pathogenic, including two families with the South Asian FANCL founder variant. We also identified five novel likely deleterious variants in FANCA, FANCF, and FANCG in affected patients. CONCLUSIONS: Our study supports the importance of determining the genomic landscape of FA in diverse populations, in order to improve understanding of FA etiology and assist in the counseling of families.


Assuntos
Anemia de Fanconi/genética , Frequência do Gene , Adolescente , Ásia , Criança , Pré-Escolar , Variações do Número de Cópias de DNA , Exoma , Anemia de Fanconi/diagnóstico , Proteína do Grupo de Complementação F da Anemia de Fanconi/genética , Proteína do Grupo de Complementação G da Anemia de Fanconi/genética , Proteína do Grupo de Complementação L da Anemia de Fanconi/genética , Feminino , Efeito Fundador , Humanos , Masculino , Oriente Médio , Mutação
3.
Mol Biol Rep ; 48(1): 585-593, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33394227

RESUMO

Fanconi Anemia (FA) is a rare genetic disease with the incidence of 1 in 360,000 and is characterised by bone marrow failure, physical abnormalities, pancytopenia, and high frequency of chromosomal breakage and increased risk of evolving into malignancy. Telomere plays an important role in genomic stability, ageing process and cancers. Telomere shortening has been reported in FA. We studied telomere length in FA subjects and compared with complementation groups. Chromosomal breakage analysis from PHA stimulated, MMC induced peripheral blood culture was carried out in 37 clinically diagnosed FA. Molecular study of FANCA, G, and L was done through Sanger sequencing and next generation sequencing. Telomere length was estimated using real time quantitative polymerase chain reaction (qPCR) method. Student t-test was applied to test the significance. A high frequency of chromosomal breakage was observed in all the patients compared to healthy controls. We found significantly shorter telomere length in all the three complementation groups compare to age matched healthy controls. Among all complementation groups, FANCL showed severe telomere shortening (P value 0.0001). A negative correlation was observed between telomere length and chromosomal breakage frequency (R = -0.3116). Telomere shortening is not uncommon in FA subjects. However the telomere length shortening is different in complementation groups as FANCL showed severe telomere shortening in FA subjects. Though BM transplantation is essential for the management of the FA subjects, the telomere length can be considered as biological marker to understand the prognosis of the disease as FA subjects primarily treated with androgens.


Assuntos
Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Proteína do Grupo de Complementação G da Anemia de Fanconi/genética , Proteína do Grupo de Complementação L da Anemia de Fanconi/genética , Anemia de Fanconi/genética , Encurtamento do Telômero/genética , Adolescente , Adulto , Criança , Pré-Escolar , Quebra Cromossômica , Proteínas de Ligação a DNA/genética , Anemia de Fanconi/patologia , Feminino , Regulação da Expressão Gênica/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Telômero/genética , Adulto Jovem
6.
PLoS One ; 15(8): e0237825, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32822435

RESUMO

Cattle temperament is a complex and economically relevant trait. The objective of this study was to identify genomic regions and genes associated with cattle temperament. From a Brahman cattle population of 1,370 animals evaluated for temperament traits (Exit velocity-EV, Pen Score-PS, Temperament Score-TS), two groups of temperament-contrasting animals were identified based on their EV-average values ±1/2 standard deviation (SD). To be considered in the calm group, the EV of females ranged between 0.16-1.82 m/s (n = 50) and the EV of males ranged between 0.4-1.56 m/s (n = 48). Females were classified as temperamental if their EV ranged between 3.13-7.66 m/s (n = 46) and males were classified as temperamental if their EV ranged between 3.05-10.83 m/s (n = 45). Selected animals were genotyped using a total of 139,376 SNPs (GGP-HD-150K), evaluated for their association with EV. The Genome-Wide Association analysis (GWAS) identified fourteen SNPs: rs135340276, rs134895560, rs110190635, rs42949831, rs135982573, rs109393235, rs109531929, rs135087545, rs41839733, rs42486577, rs136661522, rs110882543, rs110864071, rs109722627, (P<8.1E-05), nine of them were located on intergenic regions, harboring seventeen genes, of which only ACER3, VRK2, FANCL and SLCO3A1 were considered candidate associated with bovine temperament due to their reported biological functions. Five SNPs were located at introns of the NRXN3, EXOC4, CACNG4 and SLC9A4 genes. The indicated candidate genes are implicated in a wide range of behavioural phenotypes and complex cognitive functions. The association of the fourteen SNPs on bovine temperament traits (EV, PS and TS) was evaluated; all these SNPs were significant for EV; only some were associated with PS and TS. Fourteen SNPs were associated with EV which allowed the identification of twenty-one candidate genes for Brahman temperament. From a functional point of view, the five intronic SNPs identified in this study, are candidates to address control of bovine temperament, further investigation will probe their role in expression of this trait.


Assuntos
Comportamento Animal , Bovinos/genética , Bovinos/psicologia , Emoções , Temperamento , Ceramidase Alcalina/genética , Animais , Proteína do Grupo de Complementação L da Anemia de Fanconi/genética , Feminino , Estudos de Associação Genética , Estudo de Associação Genômica Ampla , Genótipo , Masculino , Transportadores de Ânions Orgânicos/genética , Fenótipo , Polimorfismo de Nucleotídeo Único , Proteínas Serina-Treonina Quinases/genética , Trocadores de Sódio-Hidrogênio/genética , Proteínas de Transporte Vesicular/genética
7.
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
8.
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
9.
Hum Mutat ; 41(1): 122-128, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31513304

RESUMO

Fanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure, predisposition to cancer, and congenital abnormalities. FA is caused by pathogenic variants in any of 22 genes involved in the DNA repair pathway responsible for removing interstrand crosslinks. FANCL, an E3 ubiquitin ligase, is an integral component of the pathway, but patients affected by disease-causing FANCL variants are rare, with only nine cases reported worldwide. We report here a FANCL founder variant, anticipated to be synonymous, c.1092G>A;p.K364=, but demonstrated to induce aberrant splicing, c.1021_1092del;p.W341_K364del, that accounts for the onset of FA in 13 cases from South Asia, 12 from India and one from Pakistan. We comprehensively illustrate the pathogenic nature of the variant, provide evidence for a founder effect, and propose including this variant in genetic screening of suspected FA patients in India and Pakistan, as well as those with ancestry from these regions of South Asia.


Assuntos
Proteína do Grupo de Complementação L da Anemia de Fanconi/genética , Anemia de Fanconi/epidemiologia , Anemia de Fanconi/genética , Efeito Fundador , Variação Genética , Alelos , Ásia/epidemiologia , Aberrações Cromossômicas , Consanguinidade , Feminino , Genótipo , Humanos , Índia/epidemiologia , Masculino , Mutação , Prevalência
10.
Nature ; 575(7781): 234-237, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31666700

RESUMO

The Fanconi anaemia (FA) pathway repairs DNA damage caused by endogenous and chemotherapy-induced DNA crosslinks, and responds to replication stress1,2. Genetic inactivation of this pathway by mutation of genes encoding FA complementation group (FANC) proteins impairs development, prevents blood production and promotes cancer1,3. The key molecular step in the FA pathway is the monoubiquitination of a pseudosymmetric heterodimer of FANCD2-FANCI4,5 by the FA core complex-a megadalton multiprotein E3 ubiquitin ligase6,7. Monoubiquitinated FANCD2 then recruits additional protein factors to remove the DNA crosslink or to stabilize the stalled replication fork. A molecular structure of the FA core complex would explain how it acts to maintain genome stability. Here we reconstituted an active, recombinant FA core complex, and used cryo-electron microscopy and mass spectrometry to determine its structure. The FA core complex comprises two central dimers of the FANCB and FA-associated protein of 100 kDa (FAAP100) subunits, flanked by two copies of the RING finger subunit, FANCL. These two heterotrimers act as a scaffold to assemble the remaining five subunits, resulting in an extended asymmetric structure. Destabilization of the scaffold would disrupt the entire complex, resulting in a non-functional FA pathway. Thus, the structure provides a mechanistic basis for the low numbers of patients with mutations in FANCB, FANCL and FAAP100. Despite a lack of sequence homology, FANCB and FAAP100 adopt similar structures. The two FANCL subunits are in different conformations at opposite ends of the complex, suggesting that each FANCL has a distinct role. This structural and functional asymmetry of dimeric RING finger domains may be a general feature of E3 ligases. The cryo-electron microscopy structure of the FA core complex provides a foundation for a detailed understanding of its E3 ubiquitin ligase activity and DNA interstrand crosslink repair.


Assuntos
Microscopia Crioeletrônica , Proteínas de Grupos de Complementação da Anemia de Fanconi/química , Proteínas de Grupos de Complementação da Anemia de Fanconi/ultraestrutura , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , Subunidades Proteicas/química , Animais , Galinhas , Anemia de Fanconi/enzimologia , Proteína do Grupo de Complementação L da Anemia de Fanconi/química , Proteína do Grupo de Complementação L da Anemia de Fanconi/ultraestrutura , Espectrometria de Massas , Modelos Moleculares , Domínios Proteicos , Multimerização Proteica , Relação Estrutura-Atividade , Ubiquitinação
11.
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
12.
Endocrinology ; 159(11): 3699-3722, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30184072

RESUMO

Sex determination and differentiation are complex processes. As a juvenile hermaphrodite or undifferentiated gonochorist, zebrafish undergo a special juvenile ovarian phase during sex differentiation, making it an excellent model for studying early oogenesis and folliculogenesis. We provide lines of evidence at morphological, molecular, and genetic levels for roles of factor in the germline α (Figla), an oocyte-specific transcription factor, in early zebrafish gonadogenesis. As in mammals, Figla/figla was also expressed in the gonads and its expression in the ovary was also restricted to early oocytes. Disruption of figla gene by CRISPR/Cas9 led to an all-male phenotype in the mutant. Detailed analysis of early gonadal development showed that the germ cells in the mutant were clustered in cysts and underwent meiosis, forming oocytes at prefollicular chromatin nucleolar (CN) stage (stage IA). However, the subsequent transition from cystic CN oocytes to individual follicular perinucleolar oocytes (stage IB) was blocked, resulting in an all-male phenotype in the mutant. The phenotype of figla mutant could not be rescued by estrogen treatment, in contrast to cyp19a1a mutant, and introduction of tp53 mutation also had no effect, unlike in fancd1 and fancl mutants. Transcriptome analysis revealed that many biological processes and pathways related to germ cell development, especially oogenesis, were upregulated in the presence of Figla and that the regulation of figla expression may involve heat shock proteins. Our results strongly suggest important roles for Figla in juvenile ovary development, especially the formation of individual follicles from cystic oocytes.


Assuntos
Oogênese/genética , Organogênese/genética , Folículo Ovariano/crescimento & desenvolvimento , Ovário/crescimento & desenvolvimento , Processos de Determinação Sexual/genética , Proteínas de Peixe-Zebra/genética , Animais , Aromatase/genética , Proteína BRCA2/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Estradiol/farmacologia , Estrogênios/farmacologia , Proteína do Grupo de Complementação L da Anemia de Fanconi/genética , Feminino , Perfilação da Expressão Gênica , Proteínas de Choque Térmico/metabolismo , Masculino , Meiose , Mutação , Oogênese/efeitos dos fármacos , Organogênese/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Ovário/efeitos dos fármacos , Fenótipo , Proteína Supressora de Tumor p53/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo
13.
Cancer ; 123(20): 3943-3954, 2017 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-28678401

RESUMO

BACKGROUND: Patients with Fanconi anemia (FA) have an increased risk for head and neck squamous cell carcinoma (HNSCC). The authors sought to determine the prevalence of undiagnosed FA and FA carriers among patients with HNSCC as well as an age cutoff for FA genetic screening. METHODS: Germline DNA samples from 417 patients with HNSCC aged <50 years were screened for sequence variants by targeted next-generation sequencing of the entire length of 16 FA genes. RESULTS: The sequence revealed 194 FA gene variants in 185 patients (44%). The variant spectrum was comprised of 183 nonsynonymous point mutations, 9 indels, 1 large deletion, and 1 synonymous variant that was predicted to effect splicing. One hundred eight patients (26%) had at least 1 rare variant that was predicted to be damaging, and 57 (14%) had at least 1 rare variant that was predicted to be damaging and had been previously reported. Fifteen patients carried 2 rare variants or an X-linked variant in an FA gene. Overall, an age cutoff for FA screening was not identified among young patients with HNSCC, because there were no significant differences in mutation rates when patients were stratified by age, tumor site, ethnicity, smoking status, or human papillomavirus status. However, an increased burden, or mutation load, of FA gene variants was observed in carriers of the genes FA complementation group D2 (FANCD2), FANCE, and FANCL in the HNSCC patient cohort relative to the 1000 Genomes population. CONCLUSIONS: FA germline functional variants offer a novel area of study in HNSCC tumorigenesis. FANCE and FANCL, which are components of the core complex, are known to be responsible for the recruitment and ubiquitination, respectively, of FANCD2, a critical step in the FA DNA repair pathway. In the current cohort, the increased mutation load of FANCD2, FANCE, and FANCL variants among younger patients with HNSCC indicates the importance of the FA pathway in HNSCC. Cancer 2017;123:3943-54. © 2017 American Cancer Society.


Assuntos
Carcinoma de Células Escamosas/genética , Anemia de Fanconi/genética , Neoplasias de Cabeça e Pescoço/genética , Adulto , Idade de Início , Proteína BRCA2/genética , Análise Mutacional de DNA , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação E da Anemia de Fanconi/genética , Proteína do Grupo de Complementação L da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Feminino , Mutação em Linhagem Germinativa , Heterozigoto , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Recombinases/genética , Análise de Sequência de DNA , Carcinoma de Células Escamosas de Cabeça e Pescoço
14.
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
15.
Eur J Med Genet ; 60(7): 369-373, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28419882

RESUMO

Fanconi Anemia (FA) is a rare genetically heterogeneous disorder with 17 known complement groups caused by mutations in different genes. FA complementation group L (FA-L, OMIM #608111) occurred in 0.2% of all FA and only eight mutant variants in the FANCL gene were documented. Phenotype and genotype correlation in FANCL associated FA is still obscure. Here we describe a Chinese girl with FA-L caused by a novel homozygous mutation c.822_823insCTTTCAGG (p.Asp275LeufsX13) in the FANCL gene. The patient's clinical course was typical for FA with progression to bone marrow failure, and death from acute myelomonocytic leukemia (AML-M4) at 9 years of age. Mutation analysis also detected a likely somatic c.2608G > A (p.Gly870Ser) in the SETBP1 gene. Consistent copy number losses of 7q and 18p and gains of 3q and 21q and accumulated non-clonal single cell chromosomal abnormalities were detected in blood leukocytes as her FA progressed. This is the first Chinese FA-L case caused by a novel FANCL mutation. The somatic gene mutation and copy number aberrations could be used to monitor disease progression and the clinical findings provide further information for genotype-phenotype correlation for FA-L.


Assuntos
Proteínas de Transporte/genética , Proteína do Grupo de Complementação L da Anemia de Fanconi/genética , Anemia de Fanconi/genética , Proteínas Nucleares/genética , Adulto , Biomarcadores/sangue , Pré-Escolar , Aberrações Cromossômicas , Variações do Número de Cópias de DNA , Anemia de Fanconi/sangue , Anemia de Fanconi/diagnóstico , Feminino , Heterozigoto , Homozigoto , Humanos
16.
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
19.
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
20.
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
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