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 , HumanosRESUMO
Imatinib is the first molecularly targeted compound for chronic myeloid leukemia (CML) capable to inhibit BCR-ABL kinase activity. However, recent clinical evidence indicates that a substantial proportion of CML patients exhibit BCR-ABL-dependent or independent resistance to imatinib. Despite the importance of imatinib resistance in CML, the underlying molecular mechanisms of this resistance are largely unknown. Here, we identified GCA (grancalcin) as a critical regulator of imatinib resistance in chronic phase CML via activation of autophagy. Mechanistically, we demonstrated that GCA activates TRAF6 ubiquitin ligase activity to induce Lys63 ubiquitination of ULK1, a crucial regulator of autophagy, resulting in its stabilization and activation. We also highlighted the role of GCA-TRAF6-ULK1 autophagy regulatory axis in imatinib resistance. Our findings represent the basis for novel therapeutic strategies against CML.Abbreviation: ACTB/ß-actin: actin beta; ADM: adrenomedullin; AMBRA1: autophagy and beclin 1 regulator 1; AMPK: AMP-activated protein kinase; ANXA5: annexin A5; CP: cytogenetic response; CML: chronic myeloid leukemia; CUL3: cullin 3; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GCA: grancalcin; Dx: at diagnosis; E-64-d: (2S,3S)-trans-Epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester; IMres: Imatinib resistance; KLHL20: Kelch-like protein 20; LRMP: lymphoid-restricted membrane protein; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MMR: major molecular response; NH4Cl: ammonium chloride; PBMCs: peripheral blood mononuclear cells; PTPRC: protein tyrosine phosphatase, receptor type, C; SQSTM1/p62: sequestosome 1; SYK: spleen associated tyrosine kinase; TAP1: transporter 1, ATP binding cassette subfamily B member; TKIs: ABL-specific tyrosine kinase inhibitors; TLR9: toll- like receptor 9; TRAF6: TNF receptor associated factor 6; ULK1: unc-51 like autophagy activating kinase 1.
Assuntos
Antineoplásicos/farmacologia , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Autofagia/genética , Proteínas de Ligação ao Cálcio/metabolismo , Mesilato de Imatinib/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagia/efeitos dos fármacos , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/química , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteínas de Ligação ao Cálcio/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucócitos Mononucleares/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transcriptoma/genética , Transplante Heterólogo , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/genéticaRESUMO
Histone H2B monoubiquitination has been shown to play critical roles in diverse cellular processes including DNA damage response. Although recent data indicate that H2B monoubiquitination is strongly connected with tumor progression and regulation, the implications of this modification in lung adenocarcinoma are relatively unknown. In the present study, we demonstrated the clinical implication of H2B monoubiquitination and the potential role of tumor necrosis factor receptorassociated factorinteracting protein (TRAIP) in regulating its modification in lung adenocarcinoma. Immunohistochemical analysis showed that H2B monoubiquitination was significantly downregulated in 68 human lung adenocarcinoma patient samples compared to their normal adjacent tissues. Depletion of TRAIP by specific siRNA treatment markedly decreased ionizing radiation (IR)induced H2B monoubiquitination. In addition, deletion mutants without RING domain or Cterminus of TRAIP diminished the ability to induce H2B monoubiquitination at lysine 120. Notably, the nuclear expression of TRAIP was positively related with H2B monoubiquitination levels in patients with lung adenocarcinoma. Furthermore, statistical analysis indicated that low levels of both TRAIP and H2B monoubiquitination, not each alone, in patients with lung adenocarcinoma were strongly correlated with poor survival. Taken together, these results suggest that TRAIP is a novel regulator of H2B monoubiquitination in DNA damage response and cancer development in lung adenocarcinoma.
Assuntos
Adenocarcinoma de Pulmão/genética , Histonas/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genética , Adenocarcinoma de Pulmão/patologia , Adenocarcinoma de Pulmão/radioterapia , Dano ao DNA/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Lisina/genética , Masculino , Domínios Proteicos/efeitos da radiação , RNA Interferente Pequeno/genética , Radiação Ionizante , Deleção de Sequência/genéticaRESUMO
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.
