RESUMO
Substantial evidence has shown that overexpression of the inhibitor of apoptosis protein (IAP) survivin in human tumors correlates significantly with treatment resistance and poor patient prognosis. Survivin serves as a radiation resistance factor that impacts the DNA damage response by interacting with DNA-dependent protein kinase (DNA-PKcs). However, the complexity, molecular determinants, and functional consequences of this interrelationship remain largely unknown. By applying coimmunoprecipitation and flow cytometry-based Förster resonance energy transfer assays, we demonstrated a direct involvement of the survivin baculovirus IAP repeat domain in the regulation of radiation survival and DNA repair. This survivin-mediated activity required an interaction of residues S20 and W67 with the phosphoinositide 3-kinase (PI3K) domain of DNA-PKcs. In silico molecular docking and dynamics simulation analyses, in vitro kinase assays, and large-scale mass spectrometry suggested a heterotetrameric survivin-DNA-PKcs complex that results in a conformational change within the DNA-PKcs PI3K domain. Overexpression of survivin resulted in enhanced PI3K enzymatic activity and detection of differentially abundant phosphopeptides and proteins implicated in the DNA damage response. The survivin-DNA-PKcs interaction altered the S/T-hydrophobic motif substrate specificity of DNA-PKcs with a predominant usage of S/T-P phosphorylation sites and an increase of DNA-PKcs substrates including Foxo3. These data demonstrate that survivin differentially regulates DNA-PKcs-dependent radiation survival and DNA double-strand break repair via formation of a survivin-DNA-PKcs heterotetrameric complex. SIGNIFICANCE: These findings provide insight into survivin-mediated regulation of DNA-PKcs kinase and broaden our knowledge of the impact of survivin in modulating the cellular radiation response.See related commentary by Iliakis, p. 2270 GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/9/2304/F1.large.jpg.
Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Dano ao DNA , Proteína Quinase Ativada por DNA/metabolismo , Complexos Multiproteicos/metabolismo , Transdução de Sinais/genética , Survivina/metabolismo , Domínio Catalítico/genética , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Proteína Quinase Ativada por DNA/genética , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Complexos Multiproteicos/genética , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/genética , Especificidade por Substrato/genética , Survivina/genética , TransfecçãoRESUMO
Gromacs is one of the most popular molecular simulation suites currently available. In this contribution we present streaMD, the first interface between Gromacs trajectory files and the statistical language R. The amount of data created due to ever increasing computational power renders fast and efficient analysis of trajectories into a challenge. Especially as standard approaches such as root-mean square fluctuations and the like provide only limited physical insight. In our streaMD package integration of the Gromacs I/O libraries with advanced, graph-based analysis methods as the java library Stream leads to both: improved speed and analysis depth. We benchmark our results and highlight the applicability of the package by an interesting problem in RNA design, namely the interaction of tetracycline with an aptamer. © 2018 Wiley Periodicals, Inc.
