12 research outputs found
Etude de la SUMOylation des différentes isoformes de PML en réponse à l'arsenic et leur capacité à protéger de l'infection par le virus de la rage
PARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF
Resistance to Rabies Virus Infection Conferred by the PMLIV Isoform â–¿
Various reports implicate PML and PML nuclear bodies (NBs) in an intrinsic antiviral response targeting diverse cytoplasmic replicating RNA viruses. PML conjugation to the small ubiquitin-like modifier (SUMO) is required for its localization within NBs. PML displays antiviral effects in vivo, as PML deficiency renders mice more susceptible to infection with the rhabdovirus vesicular stomatitis virus (VSV). Cells derived from these mice are also more sensitive to infection with rabies virus, another member of the rhabdovirus family. Alternative splicing from a single gene results in the synthesis of several PML isoforms, and these are classified into seven groups, designated PMLI to -VII. We report here that expression of PMLIV or PMLIVa, which is missing exon 5, inhibited viral mRNA and protein synthesis, leading to a reduction in viral replication. However, the expression of other nuclear isoforms (PMLI to -VI) and cytoplasmic PMLVIIb failed to impair viral production. This antiviral effect required PMLIV SUMOylation, as it was not observed with PMLIV 3KR, in which the lysines involved in SUMO conjugation were mutated. Thus, PMLIV and PMLIVa may exert this isoform-specific function through interaction with specific NB protein partners via their common C-terminal region
Requirement of PML SUMO interacting motif for RNF4- or arsenic trioxide-induced degradation of nuclear PML isoforms.
PML, the organizer of nuclear bodies (NBs), is expressed in several isoforms designated PMLI to VII which differ in their C-terminal region due to alternative splicing of a single gene. This variability is important for the function of the different PML isoforms. PML NB formation requires the covalent linkage of SUMO to PML. Arsenic trioxide (As₂O₃) enhances PML SUMOylation leading to an increase in PML NB size and promotes its interaction with RNF4, a poly-SUMO-dependent ubiquitin E3 ligase responsible for proteasome-mediated PML degradation. Furthermore, the presence of a bona fide SUMO Interacting Motif (SIM) within the C-terminal region of PML seems to be required for recruitment of other SUMOylated proteins within PML NBs. This motif is present in all PML isoforms, except in the nuclear PMLVI and in the cytoplasmic PMLVII. Using a bioluminescence resonance energy transfer (BRET) assay in living cells, we found that As₂O₃ enhanced the SUMOylation and interaction with RNF4 of nuclear PML isoforms (I to VI). In addition, among the nuclear PML isoforms, only the one lacking the SIM sequence, PMLVI, was resistant to As₂O₃-induced PML degradation. Similarly, mutation of the SIM in PMLIII abrogated its sensitivity to As₂O₃-induced degradation. PMLVI and PMLIII-SIM mutant still interacted with RNF4. However, their resistance to the degradation process was due to their inability to be polyubiquitinated and to recruit efficiently the 20S core and the β regulatory subunit of the 11S complex of the proteasome in PML NBs. Such resistance of PMLVI to As₂O₃-induced degradation was alleviated by overexpression of RNF4. Our results demonstrate that the SIM of PML is dispensable for PML SUMOylation and interaction with RNF4 but is required for efficient PML ubiquitination, recruitment of proteasome components within NBs and proteasome-dependent degradation of PML in response to As₂O₃
Interaction by BRET of RNF4 with SUMOylated PMLIII or PMLVI.
<p>HEK293T cells transiently transfected with a Luc-PML fusion (PMLIII or VI) and increasing amounts of RNF4-YFP, were untreated or treated with As<sub>2</sub>O<sub>3</sub> (5 µM, 4 h) and used for BRET titration assays. BRET saturation curves are presented for each PML isoform in the absence (open square) or presence of As<sub>2</sub>O<sub>3</sub> (closed square). Bar graphs are for presenting the BRET signal (BRET), the luciferase expression and YFP expression of individual untreated (gray bars) or treated (black bars) samples. A strong increase in the YFP/Luc expression ratios upon As<sub>2</sub>O<sub>3</sub> treatment was observed for the BRET pair Luc-PMLIII/RNF4-YFP when compared to the YFP/Luc ratios obtained with the same untreated samples. This increase in the YFP/Luc expression ratios was accounted by a significant decrease in the level of Luc-PMLIII in the presence of As<sub>2</sub>O<sub>3</sub> whereas the level of RNF4-YFP remained essentially unaffected by the treatment; this is evidenced by the bar graphs of the treated and untreated samples presented below the BRET curves. The shift toward higher YFP/Luc ratios resulting from Luc-PML degradation was much more limited for PMLVI than for the other nuclear PML isoforms such as PMLIII (this figure) and PMLI to PMLV (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044949#pone.0044949.s004" target="_blank">Figs. S4A and S4B</a>). Noticeably, in response to As<sub>2</sub>O<sub>3</sub>, the Luc-PML reduced expression was not found to be dependent on the concentration of RNF4 added. Since this assay is conducted in conditions of overexpression of both PML and RNF4 in transiently transfected cells, we believe that this is possibly due to the fact that, in comparison with PML and RNF4, the ubiquitin and/or proteasomal machineries were limiting in the cells.</p
Requirement of PML SUMO Interacting Motif for RNF4- or Arsenic Trioxide-Induced Degradation of Nuclear PML Isoforms
Detection by BRET of the increase in the SUMOylation of nuclear PML isoforms by SUMO1 in response to As<sub>2</sub>O<sub>3</sub> in living cells.
<p>HEK293T cells, transiently transfected with a fixed amount of a Luc-PML fusion (PMLI to VII) and increasing amounts of YFP-SUMO1, were treated in the presence or absence of As<sub>2</sub>O<sub>3</sub> (5 µM, 4 h) and used for BRET titration assays. BRET saturation curves are presented for each PML isoform in the absence (open square) or presence of As<sub>2</sub>O<sub>3</sub> (closed square).The BRET donor of energy (luciferase fused to a PML isoform) and acceptor of energy (YFP fused to SUMO) are illustrated; upon addition of the membrane permeant luciferase substrate (coelenterazine Deep Blue), the bioluminescent signal resulting from the degradation of the substrate (emission 480 nm) is transferred to the YFP acceptor when the acceptor is within close proximity (50–100A°). The transferred energy results in a fluorescent signal emitted by the YFP acceptor (emission 530 nm). The BRET signal corresponds to the acceptor fluorescence/donor bioluminescence ratio (y axis) and is plotted as a function of the YFP/Luc fusion protein expression (x axis). ‘K’ represents a lysine to which SUMO can be covalently coupled.</p
The SIM of PML is not required for the interaction with RNF4 but is necessary for PML ubiquitination in response to As<sub>2</sub>O<sub>3</sub>.
<p>(A) Co-immunoprecipitation of RNF4 and PML isoforms and mutant. HEK293 cells were co-tranfected with a construct encoding Flag-RNF4 and PMLIII, PMLVI or PMLIII-SIM. As controls, cells were transfected with PMLIII or RNF4 alone. Two days later, the different cell extracts were immunoprecipitated with an anti-Flag antibody as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044949#s4" target="_blank">Materials and Methods</a> and samples were analyzed by Western blot using anti-PML and anti-Flag antibodies. (B) Ubiquitination of PML in response to As<sub>2</sub>O<sub>3</sub>. PML<sup>−/−</sup> MEFs were transiently transfected with the empty vector (EV) or PMLIII (left panel) or contructs encoding PMLIII, PMLIII-SIM or PMLVI (right panel). Two days later, they were treated with As<sub>2</sub>O<sub>3</sub> for 1 h. The cell extracts were immunoprecipitated with rabbit anti-PML antibody and the immunoprecipitate was analyzed by Western blot using anti-ubiquitin and anti-PML antibodies.</p
Differential degradation of PML isoforms in response to As<sub>2</sub>O<sub>3</sub>.
<p>(A) U373MG cells stably expressing each PML isoform were untreated (C) or treated with 5 µM of As<sub>2</sub>O<sub>3</sub> for the indicated time (24 h or 48 h). (B) U373MG cells stably expressing PMLVI, PMLVII or PMLIII-SIM were untreated (C) or treated from 24 to 48 h with 5 μM of As<sub>2</sub>O<sub>3</sub>. The different cell extracts were analyzed by Western blot with anti-PML or anti-actin antibodies. (C) U373MG cells were untreated (C, lane 1) or treated with 1000 units/ml of IFNγ (lanes 2 and 4) to increase the expression of the endogenous forms of PML. One day later, As<sub>2</sub>O<sub>3</sub> was added at the concentration of 5 μM (lanes 3 and 4) for 24 h. In A, B and C, cell extracts were analyzed by Western blot with anti-PML (top) or anti-actin antibodies (bottom). Unmodified PML isoforms are indicated by arrowheads and PML forms resistant to As<sub>2</sub>O<sub>3</sub>-induced degradation in IFN-treated cells by stars.</p
The β regulatory subunit of the 11S proteasome was recruited to PML NBs in response to As<sub>2</sub>O<sub>3</sub> in cells stably expressing PMLI, PMLII, PMLIV and PMLV.
<p>Confocal immunofluorescence analysis of PML and 11Sβ were performed on U373MG cells stably expressing PMLI, PMLII, PMLIV or PMLV, treated or not with 5 μM of As<sub>2</sub>O<sub>3</sub> for 1 h. PML and 11Sβ were detected with a mouse anti-PML and a rabbit anti-11Sβ antibodies followed by the corresponding anti-IgG antibody conjugated to Alexa 594 (red) and 488 (green), respectively. The merged images revealed co-localization of PML and endogenous 11Sβ for PMLI, PMLII, PMLIV and PMLV in the presence of As<sub>2</sub>O<sub>3</sub> in nearly all cells.</p
Requirement of the SIM for the recruitment of theβ regulatory subunit of the 11S proteasome to PML NBs in response to As<sub>2</sub>O<sub>3</sub>.
<p>Confocal immunofluorescence analysis of PML and of the β regulatory subunit of the 11S proteasome (11Sβ) were performed on U373MG cells stably expressing PMLIII (A) or PMLIII-SIM (B), treated or not with 5 μM of As<sub>2</sub>O<sub>3</sub> for 1 h, 24 h or 48 h. PML and 11Sβ were detected with a mouse anti-PML and a rabbit anti-11Sβ antibodies followed by the corresponding anti-IgG antibody conjugated to Alexa 594 (red) and 488 (green), respectively. The merged images at 1 h of As<sub>2</sub>O<sub>3</sub> treatment revealed in all cells a co-localization of endogenous 11Sβ with PMLIII but not with PMLIII-SIM mutant.</p