Bcl-2 protein regulation by conformational flexibility
Final Report Abstract
Mitochondrial apoptosis is the most common form of programmed cell death and involves proteins of the B-cell lymphoma-2 (BCL-2) family. Activation of the functionally redundant pro-apoptotic BCL-2 proteins BAX and BAK leads to the permeabilization of the outer mitochondrial membrane (OMM) and subsequent release of intermembrane space (IMS) proteins into the cytoplasm. Release of IMS proteins initiates the caspase cascade and therefore the complete dismantling of the cell. Activation of BAX or BAK is the first irreversible step in intrinsic apoptosis signaling and commits the cell to apoptosis. In healthy cells, pro-survival BCL-2 proteins (e.g. BCL-2, BCL- xL or MCL-1) antagonize BAX and BAK by retrotranslocation from the mitochondria into the cytosol after major conformational changes. Translocation and retrotranslocation of BAX establish an equilibrium between cytosolic and mitochondrial localization. This project aimed at exploring the intricate process that controls BAX and possibly BAK in our cells and characterize the influence of pro-survival BCL-2 proteins, BH3-only proteins and chaperones in the major conformational changes and dynamic translocation processes regulating the inhibition of mitochondrial apoptosis or the commitment to programmed cell suicide. Employing various assays for membrane segment interactions and transient interactions in the context of living cells, we identified regulatory interactions between C-terminal membrane segments of BCL-2 proteins. The membrane anchors are sufficient and required for dimerization or oligomerization of BCL-2 proteins or between pro-apoptotic and pro-survival BCL-2 proteins. The membrane anchor interactions modulate apoptosis regulation and can inhibit OMM permeabilization. A major effort to establish an in organelle BAX retrotranslocation assay also demonstrated retrotranslocation of BAX with at least one OMM-integral segment into the cytosol. Based on BAX shuttling dependent on the transport of nucleotides and divalent cations across the OMM, we identified the highly abundant VDAC2 protein as receptor for BAX binding on the OMM. Further experiments support the role of VDAC2 as platform in BAX retrotranslocation dependent on transient interactions with pro-apoptotic and pro-survival BCL- 2 proteins. The analysis of a BAX variant containing an intramolecular disulfide tether between two central helices shows that separation of both helices is essential for BAX association with the mitochondria. However, the apoptotic activity of this variant shows that major conformational changes in this part of the molecule are not required for BAX activation, contrasting the interpretation of a previous study. Specific targeting of this BAX variant to the OMM resulted in BAX degradation, rather than BAX accumulation and commitment to apoptosis. Our analysis uncovered that misregulated BAX is ubiquitinated at the mitochondria by the E3 ligase Parkin and degraded in a proteasome-dependent manner. This previously unappreciated mechanism can provide an additional layer of apoptosis inhibition in the presence of Parkin. Next, we wanted to understand whether retrotranslocation controls only BAX or also the highly redundant BAK that is found largely at the OMM of healthy cells. Strikingly, different human tissues contain BAK in the cytosolic fraction and mitochondrial BAK is retrotranslocated into the cytosol by BCL-xL, in parallel to BAX but at lower rates. An in debt analysis of the contribution of the BAX and BAK membrane anchors to the shuttling process revealed that these segments determine retrotranslocation rates, protein localization, mitochondrial dwell time and commitment to apoptosis. Our experiments could separate mitochondrial BAX accumulation and OMM permeabilization and demonstrated that the mitochondrial pool prior apoptosis induction correlated with the cellular predisposition to apoptosis. In order to investigate, whether BAX/BAK localization could predict tumor response to chemotoxic stress, we analyzed the relative localization of BAX and BAK in pre-treatment bone marrow samples of two patient cohorts of human AML. The analysis reveals a strikingly better survival probability among patients with mitochondrial BAX localization. Genetic analysis of the patients indicated that different BAX localizations might be determined by diverse molecular mechanisms and cell stress rather than common genetic alterations. Furthermore, we have contributed to the establishment of a cellular system to rapidly and synchronously induce apoptosis and allow the analysis of BH3 mimetics in different cellular settings.
Publications
- (2017) Mitochondrial BAX Determines the Predisposition to Apoptosis in Human AML. Clinical cancer research : an official journal of the American Association for Cancer Research 23 (16)4805–4816
Reichenbach, Frank; Wiedenmann, Cornelius; Schalk, Enrico; Becker, Diana; Funk, Kathrin; Scholz-Kreisel, Peter; Todt, Franziska; Wolleschak, Denise; Döhner, Konstanze; Marquardt, Jens U.; Heidel, Florian; Edlich, Frank
(See online at https://doi.org/10.1158/1078-0432.ccr-16-1941) - (2017) Parkin promotes proteasomal degradation of misregulated BAX. Journal of cell science 130 (17)2903–2913
Cakir, Zeynep; Funk, Kathrin; Lauterwasser, Joachim; Todt, Franziska; Zerbes, Ralf M.; Oelgeklaus, Aline; Tanaka, Atsushi; van der Laan, Martin; Edlich, Frank
(See online at https://doi.org/10.1242/jcs.200162) - (2018) BCL-2 proteins and apoptosis: Recent insights and unknowns. Biochemical and biophysical research communications 500 (1)26–34
Edlich, Frank
(See online at https://doi.org/10.1016/j.bbrc.2017.06.190) - (2013) The C-terminal helix of BCL-x(L) mediates BAX retrotranslocation from the mitochondria. Cell Death Differ 20, 333-42
Todt, F., Cakir, Z., Reichenbach, F., Youle, R.J., Edlich, F.
(See online at https://doi.org/10.1038/cdd.2012.131) - (2015) Platelets induce apoptosis via membrane-bound FasL. Blood 126, 1483-93
Schleicher, R., Reichenbach, F., Kraft, P., Kumar, A., Lescan, M., Todt, F., Göbel, K., Hilgendorf, I., Geisler, T., Bauer, A., Olbrich, M., Schaller, M., Wesselborg, S., O'Reilly, L., Meuth, S., Schulze- Osthoff, K., Gawaz, M., Li, X., Kleinschnitz, C., Edlich, F., Langer, H.F.
(See online at https://doi.org/10.1182/blood-2013-12-544445) - (2015). Differential retrotranslocation of mitochondrial BAX and BAK. EMBO J 34, 67-80
Todt, F., Cakir, Z., Reichenbach, F., Emschermann, F., Lauterwasser, J., Kaiser, A., Ichim, G., Tait, S.W.G., Frank, S., Langer, H.F. and Edlich, F.
(See online at https://doi.org/10.15252/embj.201488806) - The great migration of BAX and BAK. (2015) Mol. Cell. Oncol. 2, e995029
Edlich, F.
(See online at https://doi.org/10.4161/23723556.2014.995029) - (2016) The porin VDAC2 is the mitochondrial platform for BAX retrotranslocation. Sci Rep 6:32994
Lauterwasser, J., Todt, F., Zerbes, R.M., Nguyen, T., Craigen, W., Lazarou, M., van der Laan, M. and Edlich, F.
(See online at https://doi.org/10.1038/srep32994) - (2016). Mito-priming as a method of engineered BCL-2 addiction. Nat Comm 7:10538
Lopez, J., Bessou, M., Riley, J.S., Giampazolias, E., Todt, F., Rochegüe, T., Oberst, A., Green, D.R., Edlich, F., Ichim, G., Tait, S.W.G.
(See online at https://doi.org/10.1038/ncomms10538) - (2017) The BAX transmembrane domain interacts with BCL-2 proteins in nonapoptotic cells. Proc Natl Acad Sci USA 114,310-5
Andreu-Fernández, V., Genovés, A., Todt, F., Lauterwasser, J., Jahreis, G., Pérez-Payá, E., Mingarro, I., Edlich, F., Orzáez, M.
(See online at https://doi.org/10.1073/pnas.1612322114)
