25 research outputs found
Modified Vaccinia Virus Ankara-Based Vaccine Vectors Induce Apoptosis in Dendritic Cells Draining from the Skin via both the Extrinsic and Intrinsic Caspase Pathways, Preventing Efficient Antigen Presentation
Dendritic cells (DC) are potent antigen-presenting cells and central to the induction of immune responses following infection or vaccination. The collection of DC migrating from peripheral tissues by cannulation of the afferent lymphatic vessels provides DC which can be used directly ex vivo without extensive in vitro manipulations. We have previously used bovine migrating DC to show that recombinant human adenovirus 5 vectors efficiently transduce afferent lymph migrating DEC-205(+) CD11c(+) CD8(-) DC (ALDC). We have also shown that recombinant modified vaccinia virus Ankara (MVA) infects ALDC in vitro, causing downregulation of costimulatory molecules, apoptosis, and cell death. We now show that in the bovine system, modified vaccinia virus Ankara-induced apoptosis in DC draining from the skin occurs soon after virus binding via the caspase 8 pathway and is not associated with viral gene expression. We also show that after virus entry, the caspase 9 pathway cascade is initiated. The magnitude of T cell responses to mycobacterial antigen 85A (Ag85A) expressed by recombinant MVA-infected ALDC is increased by blocking caspase-induced apoptosis. Apoptotic bodies generated by recombinant MVA (rMVA)-Ag85A-infected ALDC and containing Ag85A were phagocytosed by noninfected migrating ALDC expressing SIRPα via actin-dependent phagocytosis, and these ALDC in turn presented antigen. However, the addition of fresh ALDC to MVA-infected cultures did not improve on the magnitude of the T cell responses; in contrast, these noninfected DC showed downregulation of major histocompatibility complex class II (MHC-II), CD40, CD80, and CD86. We also observed that MVA-infected ALDC promoted migration of DEC-205(+) SIRPα(+) CD21(+) DC as well as CD4(+) and CD8(+) T cells independently of caspase activation. These in vitro studies show that induction of apoptosis in DC by MVA vectors is detrimental to the subsequent induction of T cell responses
Translocation of a Bak C-Terminus Mutant from Cytosol to Mitochondria to Mediate Cytochrome c Release: Implications for Bak and Bax Apoptotic Function
One of two proapoptotic Bcl-2 proteins, Bak or Bax, is required to permeabilize the mitochondrial outer membrane during apoptosis. While Bax is mostly cytosolic and translocates to mitochondria following an apoptotic stimulus, Bak is constitutively integrated within the outer membrane. Membrane anchorage occurs via a C-terminal transmembrane domain that has been studied in Bax but not in Bak, therefore what governs their distinct subcellular distribution is uncertain. In addition, whether the distinct subcellular distributions of Bak and Bax contributes to their differential regulation during apoptosis remains unclear.To gain insight into Bak and Bax targeting to mitochondria, elements of the Bak C-terminus were mutated, or swapped with those of Bax. Truncation of the C-terminal six residues (C-segment) or substitution of three basic residues within the C-segment destabilized Bak. Replacing the Bak C-segment with that from Bax rescued stability and function, but unexpectedly resulted in a semi-cytosolic protein, termed Bak/BaxCS. When in the cytosol, both Bax and Bak/BaxCS sequestered their hydrophobic transmembrane domains in their hydrophobic surface groove. Upon apoptotic signalling, Bak/BaxCS translocated to the mitochondrial outer membrane, inserted its transmembrane domain, oligomerized, and released cytochrome c. Despite this Bax-like subcellular distribution, Bak/BaxCS retained Bak-like regulation following targeting of Mcl-1.Residues in the C-segment of Bak and of Bax contribute to their distinct subcellular localizations. That a semi-cytosolic form of Bak, Bak/BaxCS, could translocate to mitochondria and release cytochrome c indicates that Bak and Bax share a conserved mode of activation. In addition, the differential regulation of Bak and Bax by Mcl-1 is predominantly independent of the initial subcellular localizations of Bak and Bax
Induction of Noxa-Mediated Apoptosis by Modified Vaccinia Virus Ankara Depends on Viral Recognition by Cytosolic Helicases, Leading to IRF-3/IFN-β-Dependent Induction of Pro-Apoptotic Noxa
Viral infection is a stimulus for apoptosis, and in order to sustain viral replication many viruses are known to carry genes encoding apoptosis inhibitors. F1L, encoded by the orthopoxvirus modified vaccinia virus Ankara (MVA) has a Bcl-2-like structure. An MVA mutant lacking F1L (MVAΔF1L) induces apoptosis, indicating that MVA infection activates and F1L functions to inhibit the apoptotic pathway. In this study we investigated the events leading to apoptosis upon infection by MVAΔF1L. Apoptosis largely proceeded through the pro-apoptotic Bcl-2 family protein Bak with some contribution from Bax. Of the family of pro-apoptotic BH3-only proteins, only the loss of Noxa provided substantial protection, while the loss of Bim had a minor effect. In mice, MVA preferentially infected macrophages and DCs in vivo. In both cell types wt MVA induced apoptosis albeit more weakly than MVAΔF1L. The loss of Noxa had a significant protective effect in macrophages, DC and primary lymphocytes, and the combined loss of Bim and Noxa provided strong protection. Noxa protein was induced during infection, and the induction of Noxa protein and apoptosis induction required transcription factor IRF3 and type I interferon signalling. We further observed that helicases RIG-I and MDA5 and their signalling adapter MAVS contribute to Noxa induction and apoptosis in response to MVA infection. RNA isolated from MVA-infected cells induced Noxa expression and apoptosis when transfected in the absence of viral infection. We thus here describe a pathway leading from the detection of viral RNA during MVA infection by the cytosolic helicase-pathway, to the up-regulation of Noxa and apoptosis via IRF3 and type I IFN signalling
A TM:groove interaction forms in cytosolic Bax and in cytosolic Bak/BaxCS.
<p>(<b>A</b>) Schematic of Bax converting from a TM:groove to a TM:membrane conformation during apoptosis. Bax is shown as surface representation (<i>orange</i>) with the hydrophobic surface groove highlighted (<i>white</i>). The Bax TM domain is represented (<i>blue</i>) and the C-segment (KKMG) has the sidechains shown. Residues proposed to interact during TM:groove interaction are highlighted (S184 and D98; <i>red</i>). Images were generated in Pymol using the RCSB Protein Data Bank file 1F16 for Bax <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031510#pone.0031510-Suzuki1" target="_blank">[38]</a>. (<b>B</b>) Mutations in both the TM domain and the groove alter mitochondrial targeting. Cytosolic and membrane fractions from <i>bak<sup>−/−</sup>bax<sup>−/−</sup></i> MEFs expressing the indicated Bax or Bak/BaxCS cysteine mutants were immunoblotted for Bax, Bak/BaxCS, or HSP70. (<b>C</b>) A TM:groove interaction forms in cytosolic Bax and in cytosolic Bak/BaxCS. Cytosolic and membrane fractions from cells in (B) were incubated with oxidant (CuPhe) and electrophoresed under non-reducing conditions (<i>upper</i>) or reducing conditions (+2ME, <i>lower</i>) and immunoblotted for Bax or Bak/BaxCS. Intramolecular cysteine linkage (D98C:S184C in Bax; S117C:Q202C in Bak/BaxCS) results in faster migration under non-reducing conditions (<i>X-link</i>). (<b>D</b>) Etoposide treatment decreases TM:groove interaction in membrane-associated Bak/BaxCS. Cells expressing the Bak/BaxCS S117C/Q202C variant were incubated with or without etoposide for 24 h. Cytosolic and membrane fractions were incubated with CuPhe, electrophoresed under non-reducing conditions, and immunoblotted for Bak. (<b>E</b>) tBid treatment decreases the TM:groove interaction in membrane-associated Bak/BaxCS. Cells in (D) were permeabilized and incubated with or without tBid (100 nM). Cytosolic and membrane fractions were separated and then incubated with CuPhe, electrophoresed under non-reducing conditions, and immunoblotted for Bak. (<b>F</b>) The TM domain of Bak/BaxCS inserts into membranes following tBid. Permeabilized cells were treated with or without tBid as in (E), then incubated with or without IASD. Membrane fractions were electrophoresed under reducing conditions and immunoblotted for Bak. Results are representative of two or more independent experiments.</p
Bak regulation is independent of initial subcellular localization.
<p>(<b>A</b>) Schematic of Bak-mediated apoptosis initiated by Noxa-Mcl-1 signalling in MEFs. The four prosurvival proteins expressed in MEF (Mcl-1, Bcl-x<sub>L</sub>, Bcl-w and Bcl-2) are depicted, together with their preferential binding of BH3-only proteins (Noxa, Bim and Bad) and of Bak and Bax <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031510#pone.0031510-Willis1" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031510#pone.0031510-Chen1" target="_blank">[46]</a>. The Noxa-Mcl-1-Bak pathway to apoptosis is indicated (<i>bold</i>). (<b>B</b>) Expression of Bim<sub>S</sub><sup>NOXA</sup> preferentially mediates apoptosis via either Bak or Bak/BaxCS. <i>bak<sup>−/−</sup>bax<sup>−/−</sup></i>MEFs expressing the indicated Bak and Bax variants were retrovirally infected with Bim<sub>S</sub> or with Bim<sub>S</sub> containing the Bad or Noxa BH3 domains (Bim<sub>S</sub><sup>BAD</sup> and Bim<sub>S</sub><sup>NOXA</sup>). Percentage cell death at 36 h (normalized to the efficiency of infection) is expressed as the mean ± SEM of three independent experiments. Statistical significance for treatment when compared to Bak; **p<0.01.</p
Basic residues in the C-segment are necessary for Bak stability and function.
<p>(<b>A</b>) C-terminal sequence of Bak variants indicating which basic residues in the C-segment that were substituted with serine. (<b>B</b>) Substitution of basic residues in the C-segment reduces Bak proapoptotic function. <i>bak<sup>−/−</sup>bax<sup>−/−</sup></i> MEFs expressing Bak, BakRRS, BakRSS or BakSSS were left untreated, or treated with UV or etoposide for 24 h. Percentage cell death is expressed as the mean ± SEM from three independent experiments. Statistical significance for treatment when compared to Bak; *p<0.05, **p<0.01. Upper panel is a western blot of cell lysates immunoblotted for Bak, and for β-actin as a loading control. (<b>C</b>) Substitution of basic residues in the C-segment destabilizes Bak. Cells from (B) were incubated with cycloheximide for up to 24 h and cell lysates immunoblotted for Bak, and for β-actin as a loading control. Note that due to low expression of BakSSS, 4-fold total protein was loaded onto the gels. (<b>D</b>) Substitution of basic residues does not prevent targeting to membranes. Cells from (B) were left untreated or treated with UV, separated into cytosolic and membrane fractions, and immunoblotted for Bak and for the cytosolic marker HSP70. Results are representative of two or more independent experiments.</p
The Bak C-segment is essential for stability, membrane targeting and proapoptotic function.
<p>(<b>A</b>) C-terminal sequence of Bak variants truncated at the C-terminus. The C-terminus (CT) contains a hydrophobic transmembrane (TM) domain and a hydrophilic C-segment (CS). (<b>B</b>) Truncation of the Bak C-terminus or C-segment blocks proapoptotic function. <i>bak<sup>−/−</sup>bax<sup>−/−</sup></i>MEFs expressing Bak, BakΔCT and BakΔCS were left untreated, or treated with UV or etoposide for 24 h. Percentage cell death is expressed as the mean ± SEM from three independent experiments. Statistical significance for treatment when compared to Bak; ***p<0.001. Upper panel is a western blot of cell lysates immunoblotted for Bak, and for β-actin as a loading control. (<b>C</b>) Truncation of the C-segment reduces half-life. Cells from (B) were incubated with cycloheximide for up to 24 h and cell lysates immunoblotted for Bak, and for β-actin as a loading control. Note that due to low expression of BakΔCS, 4-fold total protein was loaded onto the gels. (<b>D</b>) Truncation of the Bak C-terminus prevents membrane targeting. Cells were left untreated or treated with UV, separated into cytosolic and membrane fractions, and immunoblotted for Bak and the cytosolic marker HSP70. (<b>E</b>) Bak lacking the C-terminus fails to undergo conformation change and oligomerization in response to UV. Cells treated as in (D) were exposed to oxidant (CuPhe), separated into cytosolic and membrane fractions, run on non-reducing SDS-PAGE and immunoblotted for Bak. M<sub>X</sub>, non-activated intramolecular cross-linked monomer; M, non-crosslinked monomer; D, intermolecular crosslinked dimers. Results are representative of two or more independent experiments.</p
Bak/BaxCS translocates to mitochondria and following tBid releases cytochrome <i>c</i>.
<p>(<b>A</b>) Cytosolic and mitochondrial Bak/BaxCS both contribute to permeabilization of MEF mitochondria. Cytosol and membrane fractions from <i>bak<sup>−/−</sup>bax<sup>−/−</sup></i> MEFs or from <i>bak<sup>−/−</sup>bax<sup>−/−</sup></i> MEFs expressing either Bak or Bak/BaxCS were combined as shown, and incubated at 30°C in the presence of 100 nM tBid for 0, 30 or 60 mins, or without tBid for 60 mins (60-). Supernatant (<i>Cyt</i>) and membrane (<i>Memb</i>) fractions were immunoblotted for cytochrome <i>c</i> and for Bak. (<b>B</b>) Cytosolic Bak/BaxCS is sufficient to permeabilize mouse liver mitochondria. MEF cytosol fractions derived as in (A) were combined with mitochondria isolated from wild-type (<i>bak<sup>+/+</sup></i>) or <i>bak<sup>−/−</sup></i> mouse liver, and incubated at 37°C with or without 100 nM tBid for 60 min. Supernatant (<i>Cyt</i>) and membrane (<i>Memb</i>) fractions were immunoblotted for cytochrome <i>c</i> and for Bak. Note that Bak/BaxCS levels appear high compared to the endogenous mouse Bak, possibly due to different recognition by the anti-Bak antibody. (<b>C</b>) Mcl-1 is higher in MEF membranes than in mouse liver mitochondria. MEF membranes and mouse liver mitochondria at the concentrations used in (A) and (B) were examined for levels of Mcl-1, Bcl-x<sub>L</sub> and VDAC1 by immunoblotting. Results are representative of two or more independent experiments.</p