High-yield expression and purification of soluble forms of the anti-apoptotic Bcl-x(L) and Bcl-2 as TolAIII-fusion proteins.

A method is presented to produce large amounts of Bcl-2 and Bcl-x(L), two anti-apoptotic proteins of considerable biomedical interest. Expression constructs were prepared in which the Escherichia coli protein TolAIII, known to promote over expression of soluble product, was added to the N-terminus of Bcl-2 or Bcl-x(L) proteins, which had their C-terminal hydrophobic anchors deleted. Here the expression of these TolAIII-fusion constructs, followed by a two-step metal-affinity based purification protocol is described. The method delivers at least 20 and 10mg of more than 90% pure TolAIII-Bcl-x(L)DeltaC and TolAIII-Bcl-2(2)DeltaC proteins, respectively, per liter of E. coli cell culture. The proteins are released by proteolysis with thrombin providing > 12 mg of Bcl-x(L)DeltaC or > 6 mg of Bcl-2(2)DeltaC per liter of E. coli cell culture with a purity of more than 95%. Whereas Bcl-x(L)DeltaC is soluble both before and after TolAIII removal, Triton X-100 can significantly increase the extraction of TolAIII- Bcl-2(2)DeltaC from the bacterial cells and its subsequent solubility. Far-UV CD spectroscopy demonstrated that they both have an alpha-helical structure. Fluorescence spectroscopy was used to quantitatively analyze the binding of the respiratory inhibitor antimycin A to recombinant Bcl-2 and Bcl-x(L) proteins as well as the displacement of this ligand from the hydrophobic pocket with BH3 Bad-derived peptide. Purified Bcl-x(L)DeltaC and Bcl-2(2)DeltaC both protect isolated mitochondria from Bax-induced release of cytochrome c. The ensemble of data shows that the expressed proteins are correctly folded and functional. Therefore, the TolAIII-fusion system provides a convenient tool for functional characterization and structural studies of anti-apoptotic proteins.

Helix orientations in membrane-associated Bcl-X(L) determined by 15N-solid-state NMR spectroscopy.

Controlled cell death is fundamental to tissue hemostasis and apoptosis malfunctions can lead to a wide range of diseases. Bcl-x(L) is an anti-apoptotic protein the function of which is linked to its reversible interaction with mitochondrial outer membranes. Its interfacial and intermittent bilayer association makes prediction of its bound structure difficult without using methods able to extract data from dynamic systems. Here we investigate Bcl-x(L) associated with oriented lipid bilayers at physiological pH using solid-state NMR spectroscopy. The data are consistent with a C-terminal transmembrane anchoring sequence and an average alignment of the remaining helices, i.e. including helices 5 and 6, approximately parallel to the membrane surface. Data from several biophysical approaches confirm that after removal of the C-terminus from Bcl-x(L) its membrane interactions are weak. In the presence of membranes Bcl-x(L) can still interact with a Bak BH3 domain peptide suggesting a model where the hydrophobic C-terminus of the protein unfolds and inserts into the membrane. During this conformational change the Bcl-x(L) hydrophobic binding pocket becomes accessible for protein-protein interactions whilst the structure of the N-terminal region remains intact.

Bax activation and mitochondrial insertion during apoptosis.

The mitochondrial apoptotic pathway is a highly regulated biological mechanism which determines cell fate. It is defined as a cascade of events, going from an apoptotic stimulus to the MOM permeabilization, resulting in the activation of the so-called executive phase. This pathway is very often altered in cancer cells. The mitochondrial permeabilization is under the control of the Bcl-2 family of proteins (pBcls). These proteins share one to four homology domains (designed BH1-4) with Bcl-2, and are susceptible of homo- and/or hetero-dimerization. In spite of a poor amino-acid sequence homology, these proteins exhibit very similar tertiary structures. Strikingly, while some of these proteins are anti-apoptotic, the others are pro-apoptotic. Pro-apoptotic proteins are further divided in two sub-classes: multi-domains proteins, among which Bax and Bak, which exhibit BH1-3 domains, and BH3-only proteins (or BOPs). Schematically, BOPs and anti-apoptotic proteins antagonistically regulate the activation of the multi-domain proteins Bax and Bak and their oligomerization in the MOM, the latter process being responsible for the apoptotic mitochondrial permeabilization. Considering the critical role of Bax in cancer cells apoptosis, we focus in this review on the molecular events of Bax activation through its interaction with the other proteins from the Bcl-2 family. The mechanism by which Bax triggers the MOM permeabilization once activated will be discussed in some other reviews in this special issue.