Fig 1.
Structure and sequence analysis of Ipr1 protein.
(A) Domain structure of Ipr1, Sp100 and Sp140 proteins. Ipr1 protein consists of a conserved Sp100-like domain and a SAND domain containing a DNA-binding motif. (B) Comparison of the amino acid sequences of Sp100-like and SAND domains among Ipr1, Sp100, and Sp140. Identical amino acids are indicated by an asterisk, and strongly or weakly similar amino acids are indicated by a colon or a period. Some of the conserved amino acid residues are marked by shaded boxes. The percentage of identical residues and similarity between domains of Ipr1, Sp100 and Sp140 is calculated in the followed tables.
Fig 2.
Assessment of the Sp100-like and SAND domain necessities for the dimerization and ND-targeting of Ipr1.
(A) Lysates of 293FT cells expressing p3×FLAG-Ipr1 with HA-Ipr1, HA-Ipr1-ΔSp100, or HA-Ipr1-ΔSAND were immunoprecipitated (IP) with anti-FLAG antibodyand detected by western blot (WB) with anti-HA antibody. Input represents 10% of the starting material. (B) 293FT cells were transfected with p3×FLAG-Ipr1. After 24 h, the cells were collected and the resulting cell extracts were subjected to chemical cross-linking by using DSS in DMSO or to DMSO alone as a control. The protein samples were then analyzed by western blot assays using anti-FLAG antibody. (C) RAW264.7 cells were transfected with EGFP-fused Ipr1-WT (top), Ipr1-ΔSAND (middle), or Ipr1-ΔSp100 (bottom). Immunofluorescence was performed using rat anti-PML antibody (red). Merged images show the co-localization of these proteins in yellow.
Fig 3.
Functional validation of the Ipr1 cNLSs.
(A) Schematic representation of Ipr1 protein. The positions of two cNLSs (black rectangles) identified by web-based program analyses are indicated, and the sequence of each cNLS is presented below. (B) Schematic representation of the EGFP-GST construct used in the nuclear import assessment. Each Ipr1 cNLS sequence was cloned into downstream of the EGFP-GST. The fluorescence images show representative samples of NIH3T3 cells transfected with each of the expression plasmids for 24 h. The cell nuclei were counterstained with DAPI. (C) Schematic representation of Ipr1 mutants bearing deletions of cNLS1, cNLS2, or both. The fluorescence images show representative results from the nucleocytoplasmic localization of these Ipr1 mutants in NIH3T3 cells. The cell nuclei were counterstained with DAPI.
Fig 4.
Assessment of the Ipr1 arginine/lysine-rich element as a non-classical NLS of Ipr1.
(A) Schematic illustrations of the different EGFP-fused constructs with different C-terminus deletion constructs of Ipr1-ΔNLS1/2. The minimal motif thought to contribute to nuclear import is shaded. The double substitutions (red) of key basic residues (green), Arg-424 and Lys-429, were shown at the bottom. (B) The fluorescence images show representative results of NIH3T3 cells transfected with each plasmid shown in (A) for 24 h. The cell nuclei were counterstained with DAPI. (C) An NPI-1 interaction test was performed with lysates from transiently co-transfected 293FT cells expressing p3×FLAG-NPI-1 with ΔcNLS1/2 or ΔcNLS1/2-R424A-K429A. These lysates were immunoprecipitated (IP) with anti-FLAG antibody (Sigma), and the interactions were detected by performing western blots (WB) with anti-HA antibody. Input represents 10% of the starting material. (D) A space-filling representation of the Ipr1 monomer SAND domain was obtained by using the program RASMOL (Rutgers Protein Data Bank accession number: 1ufn). The KDWK motif is shown in dark grey, DNA in blue, and the amino acids involved in the arginine/lysine rich element in red. The enlargement shows the amino acids comprising the arginine/lysine-rich elements. (E) Multiple sequence alignment of the mouse Ipr1 protein sequence with homologous proteins from humans, cattle, goats, horses, and camels. Secondary structure elements are shown on top of the alignment. The KDWK motif is indicated in a white box, and the arginine/lysine residues adjacent to the KWDK motif are shown in shaded boxes. (F) The dendrogram shows the evolutionary relationships of the Ipr1 SAND domains between different species.
Fig 5.
Functional activity of wildtype and mutant Ipr1 in regulation assays.
(A) A representative blot from a western blot analysis demonstrating the expression of the different mutant proteins. (B) Results from an assay measuring the gene repression induced by Ipr1. The expressions of Il10, Pmp2, and Ccl2 were determined by qPCR after the transient transfection of RAW264.7 cells with wildtype Ipr1 or various Ipr1 mutants. (C) Results from an assay measuring the gene activation induced by Ipr1. The expressions of Il6, Ccnd2, and Pdcd2 were determined by qPCR after the transient transfection of RAW264.7 cells with wildtype Ipr1 or various Ipr1 mutants. (D) RAW 264.7 cells were transfected with empty vector as control, wildtype Ipr1, or Ipr1-R424A-K429A. After 12 h, each group of transfected cells was incubated in the absence or presence of H37Ra. Apoptotic cells were evaluated by Annexin-V staining followed by flow cytometric analysis. The apoptotic cell rate, which is presented in the right panel, was quantified by the following algorithm: percentage of Annexin-V+ and PI− cells in the presence of H37Ra minus the percentage of Annexin-V+ and PI− cells in the absence of H37Ra. Data represent the mean ± SD of three independent experiments. Two asterisks, p < 0.01.
Fig 6.
Schematic representation of the potential mechanism for Ipr1 nuclear import and transcriptional regulation.
(A) A model illustrating the ability of Ipr1 forms homo/hetero dimers and transports into the nuclear by binding with importin protein. (B) The hypothesized regulation mode of Ipr1 on both transcriptional inhibition and activation.