Stoichiometry of homo- and hetero-complexes of Bcl-2 proteins at the single molecule level

Abstract

The proteins of the Bcl-2 family form a complex interaction network that determines the permeabilization of the mitochondrial outer membrane (MOM) in apoptosis. However, the molecular mechanism how the multiple interactions between Bcl-2 proteins are integrated to control MOM permeabilization remains one of the key questions in the field.

The main objective of this project is to uncover the stoichiometry of homo- and hetero-complexes of Bcl-2 proteins in the membrane environment and to examine how protein concentration, phosphorylation, membrane curvature or other family members, regulate these complexes. We aim to achieve unprecedented detail thanks to the use of tailored single molecule techniques, which will provide quantitative information about binding affinity, structural organization, temporal and population heterogeneities, and direct visualization of individual complexes. Extending on our previous single molecule studies of Bax, we will address the following concrete questions:

  1. Role of protein concentration and membrane curvature in the distribution of Bax oligomeric species.

  2. Stoichiometry and lifetime of cBid/Bax and Bcl-xL/Bax complexes in the membrane.

  3. Relative contribution of Bax auto-activation vs. activation by cBid in the final distribution of Bax oligomeric species. Effect of Bcl-xL on the process.

  4. Stoichiometry of Bok oligomers in the membrane and regulation by cBid and Bcl-xL, as well as the inhibitory mechanism of Bfl1.

  5. Affinity between Bcl-2 and PUMA, Bim or tBid, and role of phosphorylation on the interaction between these proteins and in apoptosis regulation.

  6. Structural conformation of Bim alone and in complex with DLC1, Bax or Bcl-xL in the membrane environment.

The outcome of this research will provide a detailed molecular description of the homo- and heterocomplexes of Bcl-2 proteins (including their regulation) that improves current mechanistic models and brings us closer to a functional understanding of how mitochondrial apoptosis is orchestrated. Given the implication of the Bcl-2 family in several diseases, the outcome of our research will also be important to design new ways of therapeutic intervention targeting the Bcl-2 family and improve the existing ones.

Scientific Goals

The main objective of this project is to uncover the stoichiometry of homo- and hetero-complexes of Bcl-2 proteins in the membrane environment and to examine how these complexes are regulated in space and time. We aim to achieve unprecedented detail thanks to the use of tailored single molecule techniques, which will provide quantitative information about binding affinity, structural organization, temporal and population heterogeneities, and direct visualization of individual complexes. We will specifically address the following questions:

  1. Role of protein concentration and membrane curvature in the distribution of Bax oligomeric species.

  2. Stoichiometry and lifetime of cBid/Bax and Bcl-xL/Bax complexes in the membrane.

  3. Relative role of Bax auto-activation vs. activation by cBid in the final distribution of Bax oligomeric species. Effect of Bcl-xL on the process.

  4. Stoichiometry of Bok oligomers in the membrane and regulation by cBid and Bcl-xL, as well as the inhibitory mechanism of Bfl1.

  5. Affinity between Bcl-2 and PUMA, Bim or tBid, and role of phosphorylation on the interaction between these proteins and in apoptosis regulation.

  6. Structural conformation of Bim alone and in complex with DLC1, Bax or Bcl-xL in the membrane environment.

The outcome of this research will dissect the molecular steps involved in the coordination of the Bcl-2 network during the regulation of MOM permeabilization and continue to provide key quantitative data for the elaboration of sophisticated mathematical models of apoptosis. Altogether, our findings will allow creating detailed models of Bcl-2 signaling that enhance therapeutic intervention.

A)Bax oligomers in SLBs are imaged with TIRF microscopy. B) Individual Bax particles detected in the images obtained. C) The histogram with the brightness of the individual particles is fitted to calculate the fraction of each oligomeric species (D). Adapted from Subburaj et al. (2)

People

Ana J. Garcia Saez (PI, Professor)

Raed Shalaby (PhD student)

Hector Flores Romero (Postdoc)

Cooperation within FOR2036

Georg Häcker, Markus Morrison, Thomas Kaufmann, Thomas Brunner, Christoph Borner, Andreas Villunger


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  • Ana Garcia Saez
  • Hector Flores Romero
  • Raed Shalaby