Publications (110) View all
-
Article: Intermolecular disulfide bonds among nucleoporins regulate karyopherin-dependent nuclear transport.
[show abstract] [hide abstract]
ABSTRACT: Disulfide (S-S) bonds play important roles in the regulation of protein function and cellular stress responses. In this study, we demonstrate that distinct sets of nucleoporins (Nups), components of the nuclear pore complex (NPC), form S-S bonds and regulate nuclear transport through the NPC. Kinetic analysis of importin β demonstrated that the permeability of the NPC was increased by dithiothreitol treatment and reduced by oxidative stress. The permeability of small proteins such as GFP was not affected by either oxidative stress or a reducing reagent. Immunoblot analysis revealed that the oxidative stress significantly induced S-S bond formation in Nups358, 155, 153, and 62 but not 88 and 160. The direct involvement of cysteine residues in the formation of S-S bonds was confirmed by mutating conserved cysteine residues in Nup62, which abolished the formation of S-S bonds and enhanced the permeability of the NPC. Knocking down Nup62 reduced the stress-inducible S-S bonds of Nup155, suggesting that Nups62 and 155 are covalently coupled via S-S bonds. From these results, we propose that the inner channel of the NPC is somehow insulated from the cytoplasm, and is more sensitive than the cytoplasm to the intracellular redox state.Journal of Cell Science 05/2013; · 6.11 Impact Factor -
Article: Early steps of double-strand break repair in Bacillus subtilis.
[show abstract] [hide abstract]
ABSTRACT: All organisms rely on integrated networks to repair DNA double-strand breaks (DSBs) in order to preserve the integrity of the genetic information, to re-establish replication, and to ensure proper chromosomal segregation. Genetic, cytological, biochemical and structural approaches have been used to analyze how Bacillus subtilis senses DNA damage and responds to DSBs. RecN, which is among the first responders to DNA DSBs, promotes the ordered recruitment of repair proteins to the site of a lesion. Cells have evolved different mechanisms for efficient end processing to create a 3'-tailed duplex DNA, the substrate for RecA binding, in the repair of one- and two-ended DSBs. Strand continuity is re-established via homologous recombination (HR), utilizing an intact homologous DNA molecule as a template. In the absence of transient diploidy or of HR, however, two-ended DSBs can be directly re-ligated via error-prone non-homologous end-joining. Here we review recent findings that shed light on the early stages of DSB repair in Firmicutes.DNA repair 02/2013; · 4.20 Impact Factor -
Article: Core Histone Charge and Linker Histone H1 Effects on the Chromatin Structure of Schizosaccharomyces pombe.
[show abstract] [hide abstract]
ABSTRACT: Histones are highly conserved proteins among eukaryotes. However, yeast histones are more divergent in their sequences. In particular, the histone tail regions of the fission yeast, Schizosaccharomyces pombe, have fewer lysine residues, making their charges less positive than those of higher eukaryotes. In addition, the S. pombe chromatin lacks linker histones. How these factors affected yeast chromatin folding was analysed by biochemical reconstitution in combination with atomic force microscopy. Reconstitution of a nucleosome array showed that S. pombe chromatin has a more open structure similar to reconstituted human acetylated chromatin. The S. pombe nucleosomal array formed thinner fibers than those of the human nucleosomal array in the presence of mammalian linker histone H1. Such S. pombe fibers were more comparable to human acetylated fibers. These findings suggest that the core histone charges would determine the intrinsic characteristics of S. pombe chromatin and affect inter-nucleosomal interactions.Bioscience Biotechnology and Biochemistry 12/2012; · 1.28 Impact Factor -
Article: Visualization of structural changes accompanying activation of NMDA receptors using fast-scan AFM imaging.
[show abstract] [hide abstract]
ABSTRACT: NMDA receptors are widely expressed in the central nervous system, and play a major role in excitatory synaptic transmission and plasticity. Here, we used atomic force microscopy (AFM) imaging to visualize activation-induced structural changes in the GluN1/GluN2A NMDA receptor reconstituted into a lipid bilayer. In the absence of agonist, AFM imaging revealed two populations of particles, with heights above the bilayer surface of 8.6 nm and 3.4 nm. The taller, but not the shorter, particles could be specifically decorated by an anti-GluN1 antibody, which recognizes the S2 segment of the agonist-binding domain, indicating that the two populations represent the extracellular and intracellular regions of the receptor, respectively. In the presence of glycine and glutamate, there was a reduction in the height of the extracellular region to 7.3 nm. In contrast, the height of the intracellular domain was unaffected. Fast-scan AFM imaging, combined with UV photolysis of caged glutamate, permitted the detection of a rapid reduction in the height of individual NMDA receptors. The reduction in height did not occur in the absence of the co-agonist glycine or in the presence of the selective NMDA receptor antagonist D-(-)-2-amino-5-phosphonopentanoic acid (D-AP5), indicating that the observed structural change was caused by receptor activation. These results represent the first demonstration of an activation-induced effect on the structure of the NMDA receptor at the single-molecule level. A change in receptor size following activation could have important functional implications, in particular by affecting interactions between the NMDA receptor and its extracellular synaptic partners.Journal of Biological Chemistry 12/2012; · 4.77 Impact Factor -
Article: Lamin B receptor recognizes specific modifications of histone H4 in heterochromatin formation.
[show abstract] [hide abstract]
ABSTRACT: Inner nuclear membrane (INM) proteins provide a structural framework for chromatin, modulating transcription beneath the nuclear envelope (NE). Lamin B receptor (LBR) is a classical INM protein that associates with heterochromatin, and its mutations are known to cause Pelger-Huet anomaly in humans. However, the mechanisms by which LBR organizes heterochromatin remain to be elucidated. Here, we show that LBR represses transcription by binding to chromatin regions that are marked by specific histone modifications. The tudor domain (residues 1-62) of LBR primarily recognizes histone H4 lysine 20 dimethylation and is essential for chromatin compaction, while the whole nucleoplasmic region (residues 1-211) is required for transcriptional repression. We propose a model in which the nucleoplasmic domain of LBR tethers epigenetically-marked chromatin to the NE and transcriptional repressors are loaded onto the chromatin through their interaction with LBR.Journal of Biological Chemistry 10/2012; · 4.77 Impact Factor
