Description
Artur P. G. Dingeldein1, Jörgen Ådén1, Tobias Sparrman1, Hanna Wacklin2, Luke Clifton3, Radek Sachl4, Sarka Pokorna4, Martin Hof4, Gerhard Gröbner1
1Department of Chemistry, University of Umeå,Sweden, 2ESS, Lund, Sweden, 3 ISIS, Rutherford Appleton Laboratory, Harwell, UK, 4J. Heyrovský Institute of Physical Chemistry, Prague, Czech Republic.
Programmed cell death (apoptosis) is an essential mechanism in life. Key regulators of the intrinsic mitochondrial apoptotic pathway are pro- and anti-apoptotic members of the Bcl-2 family who meet at the mitochondrion’s surface - as defined by its outer membrane system - where they arbitrate a life or death decision. Our main objective was to address this molecular regulation mechanism occurring at this mitochondrial outer membrane (MOM). For this purpose we use the anti-apoptotic Bcl-2 protein itself which is an integral membrane protein, and its counterpart, the pro-apoptotic Bax protein.
The MOM has a unique, spatially heterogenous lipid composition. Elevated oxidative stress levels which initiate apoptosis, severely alter the mitochondrial membranes due to the generation of oxidized lipids (OxPls). By developing a realistic, MOM-like lipid system, we could show that damaged mitochondrial outer membranes – generated upon oxidative stress - increase the affinity and therefore the translocation of Bax towards these membranes dramatically, and even promote partial penetration of full-length Bax [1,2]. This process was characterized by a combination of solid state NMR spectroscopy, differential scanning calorimetry and CD spectroscopy [2]. More recently, we found that Bax under these conditions is already able to induce MOM pore formation; albeit creating a pore size not sufficient for cytochrome c release [2]. Surprisingly, the Bax-induced leakage increased with the amount of oxidized lipid species PazePC present, without any detectable threshold neither for PazePC nor Bax. But this process was, highly unexpected, inhibited if a very similar OxPl, namely PoxnoPC, was present (1). Based on these findings and our work on Bax-Bcl-2 affinities, we developed a mechanistic concept of their regulative action at the membrane:
Fig. 1: Our concept: Membrane-mediated recognition of Bcl-2 by Bax and its consequences is based on the “embedded together model” which includes the membrane as an active partner; (left) In a normal cell Bcl-2 inhibits the few primed Bax molecules upon membrane binding; (middle) ROS induced production of OxPls changes membrane organization with sub-sequent higher affinity for Bax and presumably changes in Bax location and organization in the membrane. Excess membrane-bound Bax will neutralize Bcl-2 and cause final membrane permeabilization and cell death. Hower the penetration depth of Bax upon its partial insertion into MOM membrane and its overall arrangement are not really known. To address these questions we have developed a neutron reflectometry based research strategy.
[1] A. Dingeldein….. G. Gröbner. Apoptotic Bax at oxidatively stressed mitochondrial membranes: Lipid dynamics and permeabilization. Biophysical Journal (2017) in press.
[2] M. Lidman, Š. Pokorná, A.P.G. Dingeldein, T. Sparrman, .. G. Gröbner. The oxidized phospholipid PazePC promotes the formation of Bax pores in mitochondrial membranes. BBA Biomembranes 1858 (2016) 1288 - 1297.
Primary author
Prof.
Gerhard Gröbner
(Umeå University)
