Novel function of Bcl-2/Bcl-XL proteins as regulators of ryanodine receptors

Summary thesis Intracellular Ca2+ signalling controls a wide array of cellular processes such as cell death, muscle contraction, memory formation, autophagy, the cell cycle… The endoplasmic reticulum (ER) is one of the main organelles involved in these Ca2+ signals. At the level of the ER two families of Ca2+ release channels are present: the inositol 1,4,5-trisphosphate receptor (IP3R) and the ryanodine receptor (RyR) family. Ca2+ release from each of these families needs to be finely tuned in order provide the right Ca2+ signal at the right cellular location (spatio-temporal aspect of Ca2+ signalling). Several regulatory mechanisms of these channels exist. Both channels can be regulated by small molecules and can be altered by posttranslational modifications such as phosphorylation. In addition, several proteins are known to interact with these Ca2+ channels thereby modulating their function. The B-cell lymphoma (Bcl)-2 protein family is known to play an important role in Ca2+ signalling. This protein family is a critical regulator of apoptosis and consists of both pro- and anti-apoptotic family members. All Bcl-2 family members are characterized by the presence of at least one Bcl-2 homology (BH) domain. The anti-apoptotic Bcl-2 family members (such as Bcl-2 and Bcl-XL) have four of these domains. The BH1, BH2 and BH3 domain form a hydrophobic cleft by which Bcl-2 targets the BH3 domains of the pro-apoptotic family members, thereby inhibiting their function. In addition to this, BH4 domain of Bcl-2, but not of Bcl-XL, is shown to bind to and inhibit the IP3R, thereby inhibiting pro-apoptotic Ca2+ signals. In contrast to the IP3R, which is present in every cell type, RyR are expressed at high levels in certain cell types including muscle cells, heart cells and the brain. As RyR expression is more restricted, the Ca2+ signals originating from the RyR are involved in more cell specific functions such as muscle contraction, secretion and learning. Up until now it was unknown whether Bcl-2 proteins also targeted RyRs. Sequence alignment of the known Bcl-2-binding site on the IP3R revealed a striking similarity with a stretch of 22 amino acids located in the central part of the RyR. During my PhD we were able to show for the first time that Bcl-2 via its BH4 domain binds to the RyR. This interaction inhibited RyR-mediated Ca2+ release in cell models and dissociated hippocampal neurons. Both chemical inhibition (ABT-199) and mutational disruption of the hydrophobic cleft revealed that the hydrophobic cleft of Bcl-2 is not involved in binding to and regulating RyRs. In contrast to the IP3R, also Bcl-XL showed a great similarity in its ability to regulate RyRs. We could show that Bcl-XL, also via its BH4 domain bound, to the same regulatory region of the RyR thereby inhibiting the channel. One difference between Bcl-2 and Bcl-XL was that full size Bcl-XL also depended on Lys87, located in the BH3 domain of the protein, for its interaction with the RyR. This suggests that in contrast to Bcl-2, Bcl-XL may also need the hydrophobic cleft for targeting the RyR. Finally, we showed that RyRs are also involved in regulating autophagy, a cellular degradation pathway responsible for maintaining proper cell homeostasis and known to be modulated by Ca2+. Chemical inhibition of RyRs utilizing dantrolene or ryanodine resulted in an increased autophagic flux. Studying this process further suggested that RyR inhibition had no effect on the proximal stages of autophagy such as the formation of autophagosomes but increased the turn over rate of autophagosomal markers at the level of the lysosomes, thus regulating the later stages in autophagy. In conclusion we showed for the first time that anti-apoptotic Bcl-2 targets, a centrally located region on the RyR, via its BH4 domain. This interaction inhibits RyR-mediated Ca2+ release. Bcl-XL also targets this region on the RyR. In contrast to Bcl-2, fullsize Bcl-XL requires in addition to its BH4 domain Lys87 in the BH3 domain to bind to and inhibit the RyR. Finally we also showed that chemical inhibition of RyRs increases the autophagic flux at the level of the lysosomes.nrpages: 104status: publishe