| Tez Künye | Durumu | ||
| Dissecting the regulation of the pro-apoptotic Bcl-2 protein Bax in mammalian cells / Yazar:ZEYNEP ÇAKIR Danışman: Dr. FRANK EDLICH Yer Bilgisi: Albert-Ludwigs-Universität Freiburg im Breisgau / Yurtdışı Enstitü Konu:Biyoloji = Biology Dizin: | Onaylandı Doktora İngilizce 2016 145 s. | ||
| Bcl-2 family proteins are the main regulators of the mitochondrial apoptosis pathway. In response to cellular stress, the pro-apoptotic Bcl-2 family members Bax and Bak undergo major conformational changes on the mitochondria and form oligomers. Bax/Bak activation leads to the permeabilization of the outer mitochondrial membrane and the release of mitochondrial intermembrane space proteins, such as cytochrome c into the cytosol. Cytosolic cytochrome c induces the caspase cascade and thereby, the cell removes itself in a coordinated manner. Bax and Bak are tightly regulated by other Bcl-2 proteins to prevent spontaneous cell death. Anti-apoptotic Bcl-2 proteins, for example, block Bax and Bak activation by the transient interactions on the mitochondria. Bax resides in the cytosol and constantly translocates to the mitochondria. Anti-apoptotic Bcl-2 proteins with the transient interactions retrotranslocate Bax into the cytosol and thus, inactive Bax is stabilized in the cytosol. We showed that Bax retrotranslocation is not only dependent on interactions between Bax BH3 domain and the hydrophobic grooves, but also requires the Bcl-xL C-terminus. Deletions or substitutions in the Bcl-xL membrane anchor cause the deceleration of Bax retrotranslocation measured by fluorescence loss in photobleaching (FLIP). Moreover, the Bax population on the mitochondria is increased when the Bax retrotranslocation is diminished. However, mitochondrial Bax is not per se active at reduced Bax retrotranslocation. Only in response to apoptotic stimuli, cells with increased mitochondrial Bax pools are sensitized to death. We discovered that mitochondrial Bak is also retrotranslocated, but Bak retrotranslocation is slower than Bax shuttling. Therefore, the increased mitochondrial Bak localization is likely caused by the lower Bak retrotranslocation rate. Generation of chimeras with exchanged C-terminal domains of Bax and Bak cause inverse subcellular localization of these proteins. The mitochondrial Bax with the Bak membrane anchor has lower shuttling rates and shows more toxicity in the absence of apoptotic stimuli compared to wildtype Bax. In contrast, Bak with the Bax membrane anchor has great similarities in terms of the localization patterns and retrotranslocation rates compared to wildtype Bax. In other words, interchanging the membrane anchors between Bax and Bak shows that retrotranslocation rates are highly dependent on the membrane anchor hydrophobicities. Ultimately, this determines the Bax and Bak localization and their shuttling rates. Bax undergoes conformational changes during translocation to the mitochondria where Bax association is mediated by the Bax C-terminus. Recent structural evidence suggests that in order to expose Bax C-terminus residing within the Bax globular protein, the latch (helices 6-8) domain has to separate from the core (helices 1- 5) domain. However, we found that the Bax helix 6 is important for the interaction with VDAC2 and thus, Bax and Bcl-xL could transiently interact on VDAC2 to induce Bax retrotranslocation. However, the failure of Bax retrotranslocation from the mitochondria could lead to Bax oligomerization and cell death. Hindering the conformational changes involving helix 5 and helix 6 in Bax, generating Bax 5-6, enabled us to analyze the consequences of failed Bax retrotranslocation in cell. Bax 5-6 does not participate to VDAC2 complex on the mitochondria, as shown by bluenative PAGE. Furthermore, under stress conditions Bax 5-6 has higher apoptotic activities in cells. We discovered misregulated Bax can be ubiquitinated in the cell by the E3 ligase Parkin leading to proteasomal degradation of targeted Bax to protect cells from stimulus-independent cell death. Proteasome-dependent Bax degradation could represent a quality control mechanism for Bax retrotranslocation. | |||