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      Bax/Bak activation in the absence of Bid, Bim, Puma, and p53

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          Abstract

          How BH3-only proteins activate Bax/Bak, the two gateway proteins of the mitochondria-dependent apoptotic pathway, remains incompletely understood. Although all pro-apoptotic BH3-only proteins are known to bind/neutralize the anti-apoptotic Bcl-2 proteins, the three most potent ones, Bid (tBid), Bim, and Puma, possess an additional activity of directly activating Bax/Bak in vitro. This latter activity has been proposed to be responsible for triggering Bax/Bak activation following apoptotic stimulation. To test this hypothesis, we generated Bid −/ Bim −/ Puma −/ (TKO), TKO /Bax −/ /Bak −/ (PentaKO), and PentaKO /Mcl-1 −/− (HexaKO) HCT116 cells through gene editing. Surprisingly, although the TKO cells were resistant to several apoptotic stimuli, robust apoptosis was induced upon the simultaneous inactivation of Bcl-xL and Mcl-1, two anti-apoptotic Bcl-2 proteins known to suppress Bax/Bak activation and activity. Importantly, such apoptotic activity was completely abolished in the PentaKO cells. In addition, ABT-737, a BH3 mimetic that inhibits Bcl-xL/Bcl-w/Bcl-2, induced Bax activation in HexaKO cells reconstituted with endogenous level of GFP-Bax. Further, by generating TKO /p53 −/− (QKO) cells, we demonstrated that p53, a tumor suppressor postulated to directly activate Bax, is not required for Bid/Bim/Puma-independent Bax/Bak activation. Together, these results strongly suggest that the direct activation activities of Bid (tBid), Bim, Puma, and p53 are not essential for activating Bax/Bak once the anti-apoptotic Bcl-2 proteins are neutralized.

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          Most cited references43

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          Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting

          TALENs are important new tools for genome engineering. Fusions of transcription activator-like (TAL) effectors of plant pathogenic Xanthomonas spp. to the FokI nuclease, TALENs bind and cleave DNA in pairs. Binding specificity is determined by customizable arrays of polymorphic amino acid repeats in the TAL effectors. We present a method and reagents for efficiently assembling TALEN constructs with custom repeat arrays. We also describe design guidelines based on naturally occurring TAL effectors and their binding sites. Using software that applies these guidelines, in nine genes from plants, animals and protists, we found candidate cleavage sites on average every 35 bp. Each of 15 sites selected from this set was cleaved in a yeast-based assay with TALEN pairs constructed with our reagents. We used two of the TALEN pairs to mutate HPRT1 in human cells and ADH1 in Arabidopsis thaliana protoplasts. Our reagents include a plasmid construct for making custom TAL effectors and one for TAL effector fusions to additional proteins of interest. Using the former, we constructed de novo a functional analog of AvrHah1 of Xanthomonas gardneri. The complete plasmid set is available through the non-profit repository AddGene and a web-based version of our software is freely accessible online.
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            Cell death: critical control points.

            Programmed cell death is a distinct genetic and biochemical pathway essential to metazoans. An intact death pathway is required for successful embryonic development and the maintenance of normal tissue homeostasis. Apoptosis has proven to be tightly interwoven with other essential cell pathways. The identification of critical control points in the cell death pathway has yielded fundamental insights for basic biology, as well as provided rational targets for new therapeutics.
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              Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis.

              We report here that BID, a BH3 domain-containing proapoptotic Bcl2 family member, is a specific proximal substrate of Casp8 in the Fas apoptotic signaling pathway. While full-length BID is localized in cytosol, truncated BID (tBID) translocates to mitochondria and thus transduces apoptotic signals from cytoplasmic membrane to mitochondria. tBID induces first the clustering of mitochondria around the nuclei and release of cytochrome c independent of caspase activity, and then the loss of mitochondrial membrane potential, cell shrinkage, and nuclear condensation in a caspase-dependent fashion. Coexpression of BclxL inhibits all the apoptotic changes induced by tBID. Our results indicate that BID is a mediator of mitochondrial damage induced by Casp8.
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                Author and article information

                Journal
                Cell Death Dis
                Cell Death Dis
                Cell Death & Disease
                Nature Publishing Group
                2041-4889
                June 2016
                16 June 2016
                1 June 2016
                : 7
                : 6
                : e2266
                Affiliations
                [1 ]Eppley Institute for Research in Cancer and Allied Diseases, Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center , Omaha, NE 68198-7696, USA
                [2 ]Xiangya Hospital, Central South University , Changsha 410008, China
                Author notes
                [* ]Eppley Institute for Research in Cancer and Allied Diseases, Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center , Omaha, NE 68198-7696, USA. Tel: +1 402 559 4643; Fax: +1 402 559 3739; E-mail: xuluo@ 123456unmc.edu
                [3]

                Current address: Zhongshan Hospital, Xiamen University, Xiamen 361004, China.

                [4]

                These authors contributed equally to this work.

                Article
                cddis2016167
                10.1038/cddis.2016.167
                5143395
                27310874
                6a0e3a71-8b2b-4327-84f9-9bb0cf26629c
                Copyright © 2016 Macmillan Publishers Limited

                Cell Death and Disease is an open-access journal published by Nature Publishing Group. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

                History
                : 16 March 2016
                : 20 April 2016
                : 05 May 2016
                Categories
                Original Article

                Cell biology
                Cell biology

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