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Bim and Bmf synergize to induce apoptosis in Neisseria gonorrhoeae infection.

Kepp O, Gottschalk K, Churin Y, Rajalingam K, Brinkmann V, Machuy N, Kroemer G, Rudel T - PLoS Pathog. (2009)

Bottom Line: Depletion and inhibition of Rac-1, JNK-1, Bim, or Bmf prevented the activation of Bak and Bax and the subsequent activation of caspases.Apoptosis could be reconstituted in Bim-depleted and Bmf-depleted cells by additional silencing of antiapoptotic Mcl-1 and Bcl-X(L), respectively.Our data indicate a synergistic role for both cytoskeletal-associated BH3-only proteins, Bim, and Bmf, in an apoptotic pathway leading to the clearance of Ngo-infected cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Max Planck Institute for Infection Biology, Berlin, Germany.

ABSTRACT
Bcl-2 family proteins including the pro-apoptotic BH3-only proteins are central regulators of apoptotic cell death. Here we show by a focused siRNA miniscreen that the synergistic action of the BH3-only proteins Bim and Bmf is required for apoptosis induced by infection with Neisseria gonorrhoeae (Ngo). While Bim and Bmf were associated with the cytoskeleton of healthy cells, they both were released upon Ngo infection. Loss of Bim and Bmf from the cytoskeleton fraction required the activation of Jun-N-terminal kinase-1 (JNK-1), which in turn depended on Rac-1. Depletion and inhibition of Rac-1, JNK-1, Bim, or Bmf prevented the activation of Bak and Bax and the subsequent activation of caspases. Apoptosis could be reconstituted in Bim-depleted and Bmf-depleted cells by additional silencing of antiapoptotic Mcl-1 and Bcl-X(L), respectively. Our data indicate a synergistic role for both cytoskeletal-associated BH3-only proteins, Bim, and Bmf, in an apoptotic pathway leading to the clearance of Ngo-infected cells.

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Related in: MedlinePlus

Bim-specific and Bmf-specific targeting of Mcl-1 and Bcl-XL.(A,B) The activation of Bak and Bax upon siRNA-mediated knockdown was visualized 15 h post-infection by immunoprecipitation with conformation-specific antibodies followed by SDS-PAGE and immunodetection with the indicated antibodies. (C) The network of Bcl-2 family proteins was analyzed by caspase activation assays after single or double knockdowns, 15 h post-infection. Shown are the means±SD of three independent experiments. (D,E) Knockdowns were validated by Western blot using the indicated antibodies (D) and qRT-PCR (E).
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ppat-1000348-g005: Bim-specific and Bmf-specific targeting of Mcl-1 and Bcl-XL.(A,B) The activation of Bak and Bax upon siRNA-mediated knockdown was visualized 15 h post-infection by immunoprecipitation with conformation-specific antibodies followed by SDS-PAGE and immunodetection with the indicated antibodies. (C) The network of Bcl-2 family proteins was analyzed by caspase activation assays after single or double knockdowns, 15 h post-infection. Shown are the means±SD of three independent experiments. (D,E) Knockdowns were validated by Western blot using the indicated antibodies (D) and qRT-PCR (E).

Mentions: The role of Bim and Bmf in the activation of Bak and Bax – direct activation or deinhibition? - is still a matter of controversy [8],[15],[17]. Irrespective of their exact of mode of action the function of Bim and Bmf in Ngo-induced apoptosis cannot be redundant because depletion of either of them prevented the induction of apoptosis by Ngo infection. To unravel the mechanisms of this non-redundancy, we assessed the activation of Bak and Bax by means of antibodies that recognize their exposed N-termini and hence their activated conformation. SiRNA- and shRNA-mediated knockdown of Bim as well as siRNA-mediated knockdown of Bmf prior to Ngo infection prevented the activation of both Bak and Bax (Figure 5A and 5B and Figure S4), underlining the essential need of both Bim and Bmf in this pathway.


Bim and Bmf synergize to induce apoptosis in Neisseria gonorrhoeae infection.

Kepp O, Gottschalk K, Churin Y, Rajalingam K, Brinkmann V, Machuy N, Kroemer G, Rudel T - PLoS Pathog. (2009)

Bim-specific and Bmf-specific targeting of Mcl-1 and Bcl-XL.(A,B) The activation of Bak and Bax upon siRNA-mediated knockdown was visualized 15 h post-infection by immunoprecipitation with conformation-specific antibodies followed by SDS-PAGE and immunodetection with the indicated antibodies. (C) The network of Bcl-2 family proteins was analyzed by caspase activation assays after single or double knockdowns, 15 h post-infection. Shown are the means±SD of three independent experiments. (D,E) Knockdowns were validated by Western blot using the indicated antibodies (D) and qRT-PCR (E).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654407&req=5

ppat-1000348-g005: Bim-specific and Bmf-specific targeting of Mcl-1 and Bcl-XL.(A,B) The activation of Bak and Bax upon siRNA-mediated knockdown was visualized 15 h post-infection by immunoprecipitation with conformation-specific antibodies followed by SDS-PAGE and immunodetection with the indicated antibodies. (C) The network of Bcl-2 family proteins was analyzed by caspase activation assays after single or double knockdowns, 15 h post-infection. Shown are the means±SD of three independent experiments. (D,E) Knockdowns were validated by Western blot using the indicated antibodies (D) and qRT-PCR (E).
Mentions: The role of Bim and Bmf in the activation of Bak and Bax – direct activation or deinhibition? - is still a matter of controversy [8],[15],[17]. Irrespective of their exact of mode of action the function of Bim and Bmf in Ngo-induced apoptosis cannot be redundant because depletion of either of them prevented the induction of apoptosis by Ngo infection. To unravel the mechanisms of this non-redundancy, we assessed the activation of Bak and Bax by means of antibodies that recognize their exposed N-termini and hence their activated conformation. SiRNA- and shRNA-mediated knockdown of Bim as well as siRNA-mediated knockdown of Bmf prior to Ngo infection prevented the activation of both Bak and Bax (Figure 5A and 5B and Figure S4), underlining the essential need of both Bim and Bmf in this pathway.

Bottom Line: Depletion and inhibition of Rac-1, JNK-1, Bim, or Bmf prevented the activation of Bak and Bax and the subsequent activation of caspases.Apoptosis could be reconstituted in Bim-depleted and Bmf-depleted cells by additional silencing of antiapoptotic Mcl-1 and Bcl-X(L), respectively.Our data indicate a synergistic role for both cytoskeletal-associated BH3-only proteins, Bim, and Bmf, in an apoptotic pathway leading to the clearance of Ngo-infected cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Max Planck Institute for Infection Biology, Berlin, Germany.

ABSTRACT
Bcl-2 family proteins including the pro-apoptotic BH3-only proteins are central regulators of apoptotic cell death. Here we show by a focused siRNA miniscreen that the synergistic action of the BH3-only proteins Bim and Bmf is required for apoptosis induced by infection with Neisseria gonorrhoeae (Ngo). While Bim and Bmf were associated with the cytoskeleton of healthy cells, they both were released upon Ngo infection. Loss of Bim and Bmf from the cytoskeleton fraction required the activation of Jun-N-terminal kinase-1 (JNK-1), which in turn depended on Rac-1. Depletion and inhibition of Rac-1, JNK-1, Bim, or Bmf prevented the activation of Bak and Bax and the subsequent activation of caspases. Apoptosis could be reconstituted in Bim-depleted and Bmf-depleted cells by additional silencing of antiapoptotic Mcl-1 and Bcl-X(L), respectively. Our data indicate a synergistic role for both cytoskeletal-associated BH3-only proteins, Bim, and Bmf, in an apoptotic pathway leading to the clearance of Ngo-infected cells.

Show MeSH
Related in: MedlinePlus