Switch from type II to I Fas/CD95 death signaling on in vitro culturing of primary hepatocytes

Walter, Dorothee; Schmich, Kathrin; Vogel, Sandra; Pick, Robert; Kaufmann, Thomas; Hochmuth, Florian Christoph; Haber, Angelika; Neubert, Karin; McNelly, Sabine; Weizsäcker, Fritz von; Merfort, Irmgard; Maurer, Ulrich; Strasser, Andreas; Borner, Christoph

doi:10.1002/hep.22541

Cited by 52 publications

(67 citation statements)

References 39 publications

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“…Cell Culture and Apoptosis Induction-Primary mouse hepatocytes, SW480hBax (overexpressing human Bax), FDM, FDC-P1, and MEF cells were prepared and cultured as previously described (43,44). Bax/Bak DKO MEFs stably co-expressing FLAG-Bax and myc-Bax were generated as described (34).…”

Section: Methodsmentioning

confidence: 99%

Cytosolic Bax

Vogel

Raulf

Bregenhorn

et al. 2012

Journal of Biological Chemistry

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Bax is kept inactive in the cytosol by refolding its C-terminal transmembrane domain into the hydrophobic binding pocket. Although energetic calculations predicted this conformation to be stable, numerous Bax binding proteins were reported and suggested to further stabilize inactive Bax. Unfortunately, most of them have not been validated in a physiological context on the endogenous level. Here we use gel filtration analysis of the cytosol of primary and established cells to show that endogenous, inactive Bax runs 20 -30 kDa higher than recombinant Bax, suggesting Bax dimerization or the binding of a small protein. Dimerization was excluded by a lack of interaction of differentially tagged Bax proteins and by comparing the sizes of dimerized recombinant Bax with cytosolic Bax on blue native gels. Surprisingly, when analyzing cytosolic Bax complexes by high sensitivity mass spectrometry after anti-Bax immunoprecipitation or consecutive purification by gel filtration and blue native gel electrophoresis, we detected only one protein, called p23 hsp90 co-chaperone, which consistently and specifically copurified with Bax. However, this protein could not be validated as a crucial inhibitory Bax binding partner as its over-or underexpression did not show any apoptosis defects. By contrast, cytosolic Bax exhibits a slight molecular mass shift on SDS-PAGE as compared with recombinant Bax, which suggests a posttranslational modification and/or a structural difference between the two proteins. We propose that in most healthy cells, cytosolic endogenous Bax is a monomeric protein that does not necessarily need a binding partner to keep its pro-apoptotic activity in check.It has become widely accepted that the pro-apoptotic members of the Bcl-2 family, Bax and Bak, are crucial for the permeabilization of the outer mitochondrial membrane (MOMP), 4 a prerequisite of the release of apoptogenic factors such as cytochrome c from the intermembrane space and a point-of-no-return for caspase-dependent and -independent apoptotic processes (1). Whereas Bak is an integral MOM protein inhibited by pro-survival Bcl-2 proteins (2, 3) and/or voltage-dependent anion channel-2 (4, 5), Bax is soluble and resides inactive in the cytosol or loosely attached to mitochondria (6, 7). In response to various apoptotic stimuli, both Bax and Bak undergo conformational changes (8 -10), oligomerize (11, 12), and form proteinaceous (13,14) or lipidic pores (15, 16) through yet unknown mechanisms (1). These conformational changes occur in the MOM and require a tight cooperation between Bax or Bak, BH3-only proteins, and the lipid bilayer. The current model postulates that some BH3-only proteins such as Bid, Bim, and Puma rapidly translocate to the MOM after their synthesis and/or posttranslational modification (17-19) and then serve as activators of Bax and Bak (20 -25). As Bak is already an inserted protein, the role of Bid, Bim, and Puma is probably to disengage Bak from its inhibitors (2, 20), allowing its conformational change and oligomerization. How...

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Section: Methodsmentioning

confidence: 99%

Cytosolic Bax

Vogel

Raulf

Bregenhorn

et al. 2012

Journal of Biological Chemistry

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“…Alternatively, FasL was used as a multimerized, exosomal form (N2A FasL) secreted from Neuro2A cells stably transfected with a mouse FasL expression vector and provided by A. Fontana (University Clinic of Zurich, Zurich, Switzerland) (37). It was quantified and used for apoptosis assays as described previously (38). The mouse monoclonal anti-FADD Ab (clone 7A2) was provided by A. Strasser (Walter and Eliza Hall Institute, Parkville, VIC, Australia).…”

Section: Reagents and Absmentioning

confidence: 99%

A Novel Mitochondrial MAVS/Caspase-8 Platform Links RNA Virus–Induced Innate Antiviral Signaling to Bax/Bak-Independent Apoptosis

Maadidi

Faletti

Berg

et al. 2014

The Journal of Immunology

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Semliki Forest virus (SFV) requires RNA replication and Bax/Bak for efficient apoptosis induction. However, cells lacking Bax/Bak continue to die in a caspase-dependent manner. In this study, we show in both mouse and human cells that this Bax/Bak-independent pathway involves dsRNA-induced innate immune signaling via mitochondrial antiviral signaling (MAVS) and caspase-8. Bax/Bak-deficient or Bcl-2– or Bcl-xL–overexpressing cells lacking MAVS or caspase-8 expression are resistant to SFV-induced apoptosis. The signaling pathway triggered by SFV does neither involve death receptors nor the classical MAVS effectors TNFR-associated factor-2, IRF-3/7, or IFN-β but the physical interaction of MAVS with caspase-8 on mitochondria in a FADD-independent manner. Consistently, caspase-8 and -3 activation are reduced in MAVS-deficient cells. Thus, after RNA virus infection MAVS does not only elicit a type I antiviral response but also recruits caspase-8 to mitochondria to mediate caspase-3 activation and apoptosis in a Bax/Bak-independent manner.

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“…Incorporation of these caspases into the receptorassociated death-inducing signaling complex causes their autoactivation and leads to ensuing activation of effector caspases-3 and -7. In most cell types (type II cells), amplification of extrinsic pathway signaling through caspase-8-mediated activation of the BH3-only protein Bid is critical for efficient execution of apoptosis (8,9); in type I cells direct activation of effector caspases by caspase-8 is sufficient. Bid plays an important role in a number of cellular pathways including regulation of Fas-and TNFR1-mediated hepatocellular injury (9 -13).…”

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X Chromosome-linked Inhibitor of Apoptosis Regulates Cell Death Induction by Proapoptotic Receptor Agonists

Varfolomeev¹,

Alicke²,

Elliott³

et al. 2009

Journal of Biological Chemistry

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Apoptosis, or programmed cell death, is a genetically regulated process with critical roles in development and homeostasis in metazoans (1). Deficient apoptosis leads to the absence of normal cell death and contributes to the development and progression of human cancers (2). Apoptotic cell death can be initiated through the engagement of cell surface proapoptotic receptors by their specific ligands or by changes in internal cellular integrity (3, 4). Both of these pathways converge at the activation of caspases, cysteine-dependent aspartyl-specific proteases that comprise the effector arm of apoptotic cell death (5, 6). The intrinsic or mitochondrial pathway is initiated by developmental cues or cellular stress signals. These signals activate Bcl-2 homology 3 (BH3) 3 proteins, leading to neutralization of the antiapoptotic proteins, such as Bcl-2, Bcl-x L , or Mcl-1, activation of proapoptotic proteins Bax and Bak, and subsequent disruption of mitochondrial membrane potential (7). The resulting release of cytochrome c from the mitochondria into the cytoplasm leads to Apaf-1-mediated caspase-9 activation and consequent activation of effector caspases-3 and -7 and culminates in cell death.The extrinsic apoptotic pathway is triggered when proapoptotic receptors such as Fas or death receptor 5 (DR5) are engaged by their respective ligands, resulting in recruitment of the adaptor protein FADD and the apical caspases 8-or -10 (3). Incorporation of these caspases into the receptorassociated death-inducing signaling complex causes their autoactivation and leads to ensuing activation of effector caspases-3 and -7. In most cell types (type II cells), amplification of extrinsic pathway signaling through caspase-8-mediated activation of the BH3-only protein Bid is critical for efficient execution of apoptosis (8, 9); in type I cells direct activation of effector caspases by caspase-8 is sufficient. Bid plays an important role in a number of cellular pathways including regulation of Fas-and TNFR1-mediated hepatocellular injury (9 -13). In addition to stimulation by their respective ligands, proapoptotic receptors can be engaged by agonistic antibodies (14). DR5 agonist antibody (PRO95780) binds DR5 tightly and selectively, triggering apoptosis in various types of cancer cells and inhibiting tumor xenograft growth in vivo (15,16).IAP proteins represent the ultimate line of defense against cellular suicide by regulating caspase activity and preventing caspase activation (17). c-IAP1 and c-IAP2 are components of TNF receptor (TNFR) complexes where they modulate apoptotic signaling and caspase-8 activation (18 -20). X chromosome-linked IAP (XIAP) is the only true endogenous inhibitor of caspases because other IAP proteins exhibit weak binding to and inhibition of caspases (21). XIAP inhibits caspases-3 and -7 using the linker region between its baculoviral IAP-repeat (BIR) domain 1 (BIR1) and BIR2 as well as the BIR2 domain, whereas inhibition of caspase-9 relies on the binding of the BIR3 domain to an N-terminal

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Switch from type II to I Fas/CD95 death signaling on in vitro culturing of primary hepatocytes

Cited by 52 publications

References 39 publications

Cytosolic Bax

Cytosolic Bax

A Novel Mitochondrial MAVS/Caspase-8 Platform Links RNA Virus–Induced Innate Antiviral Signaling to Bax/Bak-Independent Apoptosis

X Chromosome-linked Inhibitor of Apoptosis Regulates Cell Death Induction by Proapoptotic Receptor Agonists

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