Oncogenic KRAS mutations modulate BAX-mediated cell death.

Kirsten rat sarcoma viral oncogene homolog (KRAS) belongs to the GTPase RAS superfamily, which regulates several cell-signaling pathways involved in the control of important cellular functions, including apoptosis. Oncogenic mutations in KRAS are considered the most common gain-of-function mutations, affecting 30-50 % of colorectal cancer (CRC) patients. While RAS proteins usually play an anti-apoptotic role, little is known about the involvement of KRAS mutations in apoptosis regulation.

The membrane insertion of the pro-apoptotic protein Bax is a Tom22-dependent multi-step process: a study in nanodiscs.

Membrane insertion of the pro-apoptotic protein Bax was investigated by setting up cell-free synthesis of full-length Bax in the presence of pre-formed nanodiscs. While Bax was spontaneously poorly inserted in nanodiscs, co-synthesis with the mitochondrial receptor Tom22 stimulated Bax membrane insertion. The initial interaction of Bax with the lipid bilayer exposed the hydrophobic GALLL motif in Hα1 leading to Bax precipitation through hydrophobic interactions.

Bcl-xL Is Spontaneously Inserted into Preassembled Nanodiscs and Stimulates Bax Insertion in a Cell-Free Protein Synthesis System.

The antiapoptotic protein Bcl-xL is a major regulator of cell death and survival, but many aspects of its functions remain elusive. It is mostly localized in the mitochondrial outer membrane (MOM) owing to its C-terminal hydrophobic α-helix. In order to gain further information about its membrane organization, we set up a model system combining cell-free protein synthesis and nanodisc insertion.

Non-phosphorylatable mutants of Ser184 lead to incomplete activation of Bax.

The S184 residue of Bax is the target of several protein kinases regulating cell fate, including AKT. It is well-established that, , the substitution of S184 by a non-phosphorylatable residue stimulates both the mitochondrial localization of Bax, cytochrome c release, and apoptosis. However, in experiments, substituted mutants did not exhibit any increase in their binding capacity to isolated mitochondria or liposomes.

Cell-free synthesis and reconstitution of Bax in nanodiscs: Comparison between wild-type Bax and a constitutively active mutant.

Bax is a major player in the mitochondrial pathway of apoptosis, by making the Outer Mitochondrial Membrane (OMM) permeable to various apoptogenic factors, including cytochrome c. In order to get further insight into the structure and function of Bax when it is inserted in the OMM, we attempted to reconstitute Bax in nanodiscs. Cell-free protein synthesis in the presence of nanodiscs did not yield Bax-containing nanodiscs, but it provided a simple way to purify full-length Bax without any tag.

Contribution of Yeast Studies to the Understanding of BCL-2 Family Intracellular Trafficking.

BCL-2 family members are major regulators of apoptotic cell death in mammals. They form an intricate regulatory network that ultimately regulates the release of apoptogenic factors from mitochondria to the cytosol. The ectopic expression of mammalian BCL-2 family members in the yeast which lacks BCL-2 homologs, has been long established as a useful addition to the available models to study their function and regulation.