Still embedded together binding to membranes regulates Bcl-2 protein interactions

Abstract

The dysregulation of apoptosis is a key step in developing tumours, and mediates resistance to cancer therapy. Many different signals for cell death converge on permeabilization of the outer mitochondrial membrane, which is controlled by the Bcl-2 family of proteins. The importance of this step is becoming increasingly relevant as the first generation of small molecules that inhibit the interaction of Bcl-2 family proteins enters clinical trials as anticancer agents. The Bcl-2 family can be divided into three classes: BH3-only proteins that are activated by various forms of cellular stress, Bax and Bak proteins that mediate mitochondrial membrane permeabilization, and inhibitory proteins such as Bcl-2 and Bcl-XL. The recently proposed embedded together model emphasizes the fact that many of the regulatory interactions between different classes of Bcl-2 family members occur at intracellular membranes, and binding to membranes causes conformational changes in the proteins that dictate functions in a dynamic manner. Within this context, recent results indicate that Bcl-XL functions as a dominant-negative Bax, a concept that resolves the paradox of similar structures but opposite functions of Bcl-XL and Bax. We have also shown that the conformational change that allows Bax to insert into the outer mitochondrial membrane is the rate-limiting step in the multistep process of Bax activation. Nevertheless, investigating the structure of activated Bax or Bak as monomers and as components of the oligomeric structures that mediate membrane permeabilization is the focus of ongoing research (and controversy) at many laboratories worldwide.

Figure 1

Models of regulation of apoptosis by Bcl-2 family proteins. Simplified versions of the three models of apoptosis regulation to emphasize the different roles postulated for interactions between the three different classes of Bcl-2 proteins (BH3-only proteins, antiapoptotic members such as Bcl-XL and membrane-permeabilizing proteins such as Bax). These diagrams neglect the role of the membrane in permitting a comparison of the direct activation and derepression models (no role for membrane) with the protein interactions of the embedded together model. The direct activation and derepression models postulate different functional states for Bax or Bak; in the former, these proteins are inactive and must be activated by BH3-only proteins, whereas in the latter they are constitutively active and must be repressed by antiapoptotic proteins such as Bcl-XL. Thus, they propose different mechanisms of action for Bcl-XL inhibition of apoptosis. The embedded together model recognizes both of these interactions. In the derepression model, BH3-only proteins are distinguished by their differential affinity to the varying antiapoptotic Bcl-2 family members, but in the direct activation model they are divided into two classes: those that directly activate Bax or Bak (activators) and those that prevent Bcl-XL from binding to activators (sensitizers). The embedded together model also recognizes these two different classes, although it considers the fact that both classes have a role in ‘activating’ Bcl-XL by causing its membrane insertion. The consequence of the subsequent interaction in the membrane differs: BH3-only activator proteins are sequestered by Bcl-XL and therefore cannot activate Bax or Bak, whereas BH3-only sensitizer proteins prevent Bcl-XL from binding to activator proteins, thereby inhibiting Bcl-XL.

Figure 2

Mitochondrial outer membrane permeabilization by tBid-activated Bax is a multistep process. Using fluorescently labelled recombinant proteins and membranes to monitor reactions in real time, our results (Lovell et al., 2008) indicate that Bax activation is a multistep process whereby tBid binds to membranes (step I), then recruits Bax (step II), which causes a conformational change that allows Bax to insert into membranes (step III—the rate-limiting step in the process); membrane associated Bax recruits other cytosolic Bax (step IV), which then oligomerizes in membranes (step V). Membrane permeabilization by oligomerized Bax (step VI–left panel) can be conveniently and reproducibly measured by the release of a fluorescent molecule encapsulated within the liposome that is quenched by a co-encapsulated quencher. Thus, this step can also be measured in real time (step VI—right panel).