Methylene blue (MB) disrupts the structure of TRIM32-containing CBs in HEK293 cells.

<p>(<b>A</b>) MB chemical structure. (<b>B</b>) Fluorescence microscopy images of HEK293 cells with preformed TRIM32 CBs that were exposed to either DMSO or 50 μM MB dissolved in DMSO for 2 and 4 h. (<b>C</b>) FLAG-TRIM32-transfected HEK293 cells with preformed TRIM32 CBs were exposed to 50 μM MB dissolved in DMSO (+) or DMSO only (-) for 2 and 4 h. Whole proteins were extracted with 1% SDS and western-blotted with anti-FLAG to monitor total TRIM32 levels. (<b>D</b>) HEK293 cells with preformed TRIM32 CBs were treated with DMSO and MB, then homogenized in PBS. Western blots of soluble (S) and insoluble (P) proteins with anti-FLAG (upper panel) and anti-PAN 14-3-3 antibodies (lower panel). The cytosolic marker 14-3-3 was used as a loading control for soluble and insoluble fractions. (<b>E</b>) Fluorescence microscopy images of HEK293 cells with preformed TRIM32 CBs that were exposed to DMSO, AlaC (50 μM), or SW02 (50 μM) for 4 h. (<b>F</b>) The percentage (out of ~400 cells) of CB-containing HEK293 cells exposed to DMSO, AlaC or SW02. Bars in (B) and (E), 10 μm.</p

HSP70 stabilizes TRIM63-containing CBs in HEK293 cells.

<p>(<b>A</b>) HEK293 cells were transiently transfected with FLAG-TRIM63 (+) or vector alone (-). The association of TRIM63 with endogenous HSP70 was analyzed with co-immunoprecipitation followed by western blotting (WB). (<b>B</b>) HEK293 cells transfected with FLAG-TRIM32 (upper panels) or FLAG-TRIM63 (lower panels) were incubated for 24 h in the presence or absence of MB. Total TRIM32/63 levels were analyzed with western blotting. (<b>C</b>) Fluorescence microscopy images of HEK293 cells with preformed TRIM63-containing CBs, exposed to either DMSO or MB for 2 or 4 h. Bars in (C), 10 μm.</p

TRIM32-Cytoplasmic-Body Formation Is an ATP-Consuming Process Stimulated by HSP70 in Cells

<div><p>The spontaneous and energy-releasing reaction of protein aggregation is typically prevented by cellular quality control machinery (QC). TRIM32 is a member of the TRIM (tripartite motif-containing) ubiquitin E3 ligases, and when overexpressed in cultured cells, readily forms spherical inclusions designated as cytoplasmic bodies (CBs) even without proteasome inhibition. Here, we show that HSP70, a central QC component, is a primary binding factor of overexpressed TRIM32. Contrary to expectation, however, we find that this molecular chaperone facilitates and stabilizes CB assembly depending on intrinsic ATPase activity, rather than preventing CB formation. We also show that the HSP70-TRIM32 complex is biochemically distinct from the previously characterized 14-3-3-TRIM32 phospho-complex. Moreover, the two complexes have opposing roles, with HSP70 stimulating CB formation and 14-3-3 retaining TRIM32 in a diffuse form throughout the cytosol. Our results suggest that CB inclusion formation is actively controlled by cellular QC and requires ATP, similar to protein folding and degradation reactions.</p></div

The HSP70-TRIM32 complex is biochemically separate from the 14-3-3-TRIM32 phospho-complex.

<p>(<b>A</b>) HEK293 cells were transiently transfected with FLAG-TRIM32 or its truncated mutants, immunoprecipitated with anti-FLAG, then western-blotted with anti-HSP70 and anti-FLAG. The 14-3-3 binding data are from our previous study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169436#pone.0169436.ref008" target="_blank">8</a>]. “Full,” “C-140,” “C-265,” “C-365,” and “N-361” indicate the full TRIM32 protein and its truncated mutants. (<b>B</b>) HEK293 cells were transfected with FLAG-TRIM32, the PKA catalytic subunit, and increasing amounts of myc-14-3-3η (indicated by the shaded triangle above the western blot [WB]). Transfection was followed by immunoprecipitation with anti-FLAG, and then western blots, as described in (A). (<b>C</b>) The FLAG-TRIM32 complex shown in lane 5 of (B) was further subjected to immunoprecipitation with anti-myc Sepharose beads to purify the myc-14-3-3-FLAG-TRIM32 complex, and western-blotted with specific anti-HSP70 antibody to monitor endogenous HSP70 presence (left panel). The right panel shows the myc-HSP70-FLAG-TRIM32 complex with a western blot using anti-14-3-3 antibodies to monitor endogenous 14-3-3 presence.</p

Schematic illustration of the proposed role for HSP70 in TRIM32-containing-CB formation.

<p>(<b>A</b>) Intracellular localization of TRIM32 is differentially controlled by 14-3-3 and HSP70. (<b>B</b>) HSP70 binds folding intermediates of TRIM32 and accelerates their accumulation into CBs.</p

HSP70 promotes the formation of TRIM32-containing CBs depending on ATPase activity.

<p>(<b>A</b>) HEK293 cells were transfected with FLAG-TRIM32 and the constructs (names indicated in yellow) were fixed and stained with monoclonal mouse anti-FLAG (green) and polyclonal rabbit anti-myc (red). Representative images of cells from at least three independent experiments are shown. Bars, 10 μm. (<b>B–D</b>) HEK293 cells were stained as described in (A); the size (B) and volume (C) of TRIM32-containing CBs, as well as the number of CB-containing HEK293 cells (D) were quantified from 100–500 cells in at least three experimental repeats. Volumes were determined with the assumption that CBs are round. Data are means ± SD. * indicates P < 0.0001 and ** indicates P < 0.0005. (<b>E</b>) Three-dimensional image of TRIM32 CBs in myc-HSP70-expressing HEK293 cells. (<b>F</b>) Whole proteins from HEK293 cells were extracted with 1% SDS after 24 h (lanes 1–3) or 48 h (lanes 4–6) of transfection with HSP70 or K71S plasmids, and analyzed with western blotting. Tubulin was used as an internal control.</p

Identification of HSP70 as a major binding partner of the overexpressed TRIM32 polypeptide.

<p>(<b>A</b>) Schematic representation of the human TRIM32 protein (upper) and its MEF-fused version (lower). (<b>B</b>) Association of TRIM32 and HSP70 as assessed with western blotting (WB). HEK293 cells were transfected with the FLAG-TRIM32 construct or the vector alone, with (lanes 1–3) or without (lanes 4–6) myc-HSP70. The lysates were separated with SDS-PAGE and western-blotted with anti-FLAG (for FLAG-TRIM32), anti-myc (for myc-HSP70), or anti-HSP70 (for endogenous HSP70) (lower panels, marked as “Ext”). FLAG-TRIM32 proteins were also immunoprecipitated from the lysates with nonimmune IgG (c-IgG) or anti-FLAG Sepharose beads (anti-FLAG). The immunoprecipitates were western-blotted with the aforesaid antibodies (upper panels, marked as “IP”). Lane 6′ represents lane 6 at a higher exposure. (<b>C</b>) HSP70 directly binds to TRIM32 depending on the specific nucleotide-binding state. Purified GST-TRIM32 or GST alone immobilized on agarose beads was incubated with purified HSP70 (lanes 1–3) or the K71S mutant protein (lanes 4–6) in the presence or absence of ATP or ADP, and then western-blotted with anti-HSP70 (for HSP70 and K71S) or anti-GST (for GST-TRIM32 and GST). “+” and “-” respectively indicate presence and absence of ATP/ADP. (<b>D</b>) FLAG-TRIM32-myc-HSP70/K71S-transfected HEK293 cells were co-immunoprecipitated to analyze the association between TRMI32 and K71S, as described in (B).</p

Analysis of NS3 expression, serine protease activity, effects on IFN-β promoter activity and RNA helicase activity.

<p>(A) Immunofluorescence analysis of NS3wt, various NS3 mutants and NS3/4A in Huh-7.5 cells transfected with the DNA vaccine candidates using anti-NS3 monoclonal antibody. (B) Serine protease analysis of NS3wt, various NS3 mutants and NS3/4A. Huh-7.5 cells were transiently transfected with each of the NS3 expression plasmids together with pNS5A/5BΔC (as a substrate). Cell lysates were subjected to immunoblot analysis using anti-NS3 and anti-NS5A monoclonal antibodies to detect NS3 (top panel) and NS5A/5BΔC and NS5A (middle panel), respectively. The amounts of GAPDH (bottom panel) were measured as an internal control to verify equal amounts of sample loading. (C) Effects of NS3wt or NS3 mutants on RIG-I-mediated IFN-β promoter activity. Huh-7 cells were transfected with a plasmid expressing NS3wt or each NS3 mutant together with pSG5-NS4A, pEF1A/N-RIG-I-FLAG, pIFN-β-luc and pRL-TK. Firefly luciferase activity was measured 48 h post transfection and normalized to Renilla luciferase activity. Data represent mean ± SEM of the data from three independent experiments. *, <i>p</i><0.01; †, <i>p</i><0.05, compared with NS3wt. (D) RNA helicase analysis of NS3wt and its mutant. NS3 helicase assay was performed using GST-NS3wt, GST-NS3(K210N) and GST as a negative control, as described in the Materials and methods section. The mean activity obtained with the GST control was subtracted from those obtained with test samples. The mean activity of GST-NS3wt was arbitrarily expressed as 100%. *, <i>p</i><0.05, compared with NS3wt.</p

NS3-specific CTL activity induced by DNA vaccination.

<p>BALB/c mice (2 mice/group) were immunized with each of the DNA vaccines expressing NS3wt, various NS3 mutants or NS3/4A. Splenocytes obtained from the immunized mice were stimulated in vitro for 5 days with P815-NS3 cells and GST-NS3wt (5 µg/ml). Effectors and targets (P815-NS3) were cocultured for 4 h with the ratios of 50∶1, 25∶1, and 12.5∶1. Released LDH was measured and the percentage of specific killing was calculated. Specific CTL activity of splenocytes obtained from NS3-immunized mice and the mock-immunized control are shown with solid and dashed lines, respectively. Data represent mean ± SEM of the data from three independent experiments. *, <i>p</i><0.01; †, <i>p</i><0.05, compared with the mock-immunized control.</p

IFN-γ production induced by NS3 DNA vaccination.

<p>(A) IFN-γ production by splenocytes obtained from immunized mice. BALB/c mice (2 mice/group) were immunized with each of the DNA vaccines expressing NS3wt, various NS3 mutants or NS3/4A. Splenocytes obtained from the immunized mice were cultured in the presence of GST-NS3 (5 µg/ml) for 72 h. The amounts of IFN-γ in culture supernatants were measured with ELISA. Data represent mean ± SEM of the data from three independent experiments. *, <i>p</i><0.01 compared with the mock-immunized control. (B) IFN-γ mRNA expression. Splenocytes obtained from immunized mice were cultured in the presence of GST-NS3 (5 µg/ml) for 24 h. The amounts of IFN-γ mRNA were determined by real-time quantitative RT-PCR analysis and normalized to GAPDH mRNA expression levels. Data represent mean ± SEM of the data from three independent experiments. The value for splenocytes from the mock-immunized control was arbitrarily expressed as 1.0. *, <i>p</i><0.01; †, <i>p</i><0.05, compared with the control.</p