No interaction of barrier-to-autointegration factor (BAF) with HIV-1 MA, cone-rod homeobox (Crx) or MAN1-C in absence of DNA.

Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
PLoS ONE (Impact Factor: 3.53). 09/2011; 6(9):e25123. DOI: 10.1371/journal.pone.0025123
Source: PubMed

ABSTRACT Barrier-to-autointegration factor is a cellular protein that protects retroviral DNA from autointegration. Its cellular role is not well understood, but genetic studies show that it is essential and depletion or knockout results in lethal nuclear defects. In addition to binding DNA, BAF interacts with the LEM domain, a domain shared among a family of lamin-associated polypeptides. BAF has also been reported to interact with several other viral and cellular proteins suggesting that these interactions may be functionally relevant. We find that, contrary to previous reports, BAF does not interact with HIV-1 MA, cone-rod homeobox (Crx) or MAN1-C. The reported interactions can be explained by indirect association through DNA binding and are unlikely to be biologically relevant. A mutation that causes a premature aging syndrome lies on the previously reported MAN1-C binding surface of BAF. The absence of direct binding of BAF to MAN1-C eliminates disruption of this interaction as the cause of the premature aging phenotype.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Barrier-to-autointegration factor (BAF or BANF1) is highly conserved in multicellular eukaryotes and was first identified for its role in retroviral DNA integration. Homozygous BAF mutants are lethal and depletion of BAF results in defects in chromatin segregation during mitosis and subsequent nuclear envelope assembly. BAF exists both in phosphorylated and unphosphorylated forms with phosphorylation sites Thr2, Thr3, and Ser4, near the N-terminus. Vaccinia related kinase 1 (VRK1) is the major kinase responsible for phosphorylation of BAF. We have identified the major phosphatase responsible for dephosphorylation of Ser4 to be PP4C. By examining the cellular distribution of phosphorylated BAF (pBAF) and total BAF (tBAF) through the cell cycle, we found that pBAF is associated with the core region of telophase chromosomes. Depletion of BAF or perturbing its phosphorylation state results not only in nuclear envelope defects including mislocalization of LEM domain proteins and extensive invaginations into the nuclear interior, but also impaired cell cycle progression. This phenotype is strikingly similar to that seen in cells from patients with progeroid syndrome resulting from a point mutation in BAF.
    Journal of Biological Chemistry 11/2013; 289(2). DOI:10.1074/jbc.M113.492777 · 4.60 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: In order to understand the binding mechanism between Barrier-to-autointegration factor (BAF) and DNA, two DNA:BAF complexes with wild type (WT) BAF and its Gly25Glu point mutate type (MT) were generated by molecular docking on the basis of the crystal structures of BAF (PDB code: 2ODG, chain A) and DNA (PDB code: 2BZF, chain B and C). Then, molecular dynamics (MD) simulations were performed on the two docked structures, as well as BAF (WT) and BAF (MT). The results show that monomer BAF is more flexible than BAF in DNA:BAF complex, suggesting that DNA is effective to stabilize conformation of BAF, which is in good agreement with the experimental results. Besides, the mutated Glu25 in DNA:BAF (MT) can change the BAF conformation to some extent. With deeper investigation on the DNA:BAF structures, the hydrogen bonds are found to make great contribution to the interaction between DNA and BAF. The hydrogen bonds in DNA:BAF (MT) are fewer than those in DNA:BAF (WT), indicating that the Gly25Glu mutation in BAF has an important effect on the hydrogen bonds in the DNA:BAF complex. Besides, the binding free energy in DNA:BAF (MT) is also higher than that in DNA:BAF (WT). It results from the influence of Glu25 side chain on the orientation of Lys6 and Lys33 in the interface between DNA and BAF. The binding free energy of Lys72, another key residue, decreases a lot in DNA:BAF (MT) anomalously. The decreasing energy causes the destruction of hydrophobic pocket in the binding site between DNA and BAF (MT). Our results are helpful for further experimental investigations.
    Journal of Molecular Modeling 05/2014; 20(5):2246. DOI:10.1007/s00894-014-2246-0 · 1.87 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Barrier to autointegration factor (BAF) is an essential component of the nuclear lamina that binds lamins, LEM-domain proteins, histones and DNA. Under normal conditions, BAF protein is highly mobile when assayed by Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss in Photobleaching (FLIP). We report that C. elegans BAF-1 mobility is regulated by caloric restriction, food deprivation and heat shock. This was not a general response of chromatin-associated proteins, since food deprivation did not affect the mobility of Heterochromatin Protein HPL-1 or HPL-2. Heat shock also increased the level of BAF-1 Ser4 phosphorylation. By using missense mutations that affect BAF-1 binding to different partners we find that overall, the ability of BAF-1 mutants to be immobilized by heat shock in intestine cells correlated with normal or increased affinity for emerin in vitro. These results show BAF-1 localization and mobility at the nuclear lamina are regulated by stress, and unexpectedly reveal BAF-1 immobilization as a specific response to caloric restriction in C. elegans intestinal cells.
    Molecular biology of the cell 02/2014; 25(7). DOI:10.1091/mbc.E13-08-0477 · 5.98 Impact Factor